**Part 5**

**Pulmonary Oedema: Cardiogenic and Non-Cardiogenic** 

522 Lung Diseases – Selected State of the Art Reviews

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## **Non-Cardiogenic Pulmonary Edema**

J. Gonzales and A. Verin

*Georgia Health Sciences University, Augusta, Georgia United States* 

#### **1. Introduction**

Pulmonary edema (PE) remains one of the more common reasons for admission to the hospital. Pulmonary edema is either cardiac or non-cardiac. The cardiac causes of pulmonary edema occur because the cardiac pump function has failed and there is increased capillary hydrostatic pressure secondary to elevated pulmonary venous pressure. Cardiogenic pulmonary edema is the accumulation of fluid with a low-protein content in the lung interstitium and alveoli and occurs when the pulmonary venous and left atrial venous return exceeds left ventricular output. Most often this is due to left heart failure, cardiac valve disease, volume overload, kidney failure or cardiac tamponade. Non cardiogenic pulmonary edema (NCPE) is a condition that is associated with high morbidity and mortality. Pulmonary edema fluid accumulates in the lungs through damaged capillary endothelial cells and this leads to impaired gas exchange (oxygen and carbon dioxide) with hypoxia and respiratory failure. The best example of non-cardiogenic pulmonary edema is acute respiratory distress syndrome (ARDS) (Sartori et al, 2010). ARDS is a serious condition of hypoxia, bilateral lung infiltrates on chest roentgenogram with subsequent respiratory failure. The hallmark of ALI (acute lung injury)/ARDS on the cellular level is pulmonary capillary endothelial cell permeability and fluid leakage into the pulmonary parenchyma, followed by neutrophils, cytokines and an acute inflammatory response. It is associated with a high morbidity and 30-50% mortality. ARDS has multiple causes with the most common being sepsis or pneumonia, less common causes of ARDS with ensuing pulmonary edema are trauma or pancreatitis (Ware & Mathay, 2005). Other causes of NCPE in hospitalized patients are intravenous fluid with volume overload, neurogenic pulmonary edema, reperfusion pulmonary edema, re-expansion pulmonary edema, opiate overdose, salicylate toxicity. Less common, forms of NCPE are high altitude pulmonary edema (HAPE), immersion pulmonary edema and negative pressure pulmonary edema (NPPE).

#### **2. Increased permeability pulmonary edema**

NCPE causes direct injury to the lungs in several forms. Under normal conditions fluid outflow that occurs from the lung capillaries through tiny gaps in the vascular endothelial cell (EC) junction is removed from the interstitial space and returned to the circulation by the lymphatic system. Physiologically, the main forces regulating fluid balance in the lungs are the microvascular pressure of the capillaries. Fluid leaves the capillaries and enters the pulmonary interstitium in proportion to the net capillary hydrostatic pressure minus the net osmotic pressure across the vessel wall. The formula for filtration across the pulmonary capillary semi-permeable membrane is

#### Q=K-[CPmv-Ppmv)-(πmv-πpmv)]

which is different from the systemic capillary fluid exchange. Q=net transvascular flow of fluid, K=membrane permeability, Pmv=hydrostatic pressure in the microvessels, Ppmv=hydrostatic pressure in the peri-microvascular interstitium, πmv=plasma protein osmotic pressure in the circulation, πpmv=protein osmotic pressure in the perimicrovascular interstitium. This equation only reflects the known hydrostatic pressures without including the lymph hydrostatic pressure which has been studied but is unknown in relation to the Starling forces equation (Sartori et al, 2010). It is known that one of the main functions of the lymphatic system is to return plasma proteins from the interstitial tissue space to the bloodstream. Analyses of blood plasma and lymph have shown that all of the proteins that are found in the plasma are also found in the lymph although in lower concentrations (Ono et al, 2005). In normal circumstances, low pulmonary capillary hydrostatic pressure provides a "safety" factor against pulmonary edema across the lung tissue.

Fig. 1. Representation of the pulmonary arterial and venous capillary barrier and lymphatic flow.

Although hydrostatic pressures in the pulmonary arterial and venous system are known, the hydrostatic pressures of the interrelated pulmonary lymphatic system is unknown and largely unstudied. The illustration generously permitted by Dr. Davide Brunelli of Medars.it.

In some forms of NCPE such as ARDS disturbances of pulmonary capillary fluid balance and pulmonary permeability occur as a direct result of endotoxins and inflammation that cause disruption in the capillary EC barrier with barrier disruption and subsequent pulmonary venous congestion. As the volume excess initially enters the interstitium it is taken up by the lymphatic system to be returned to the vascular system. In normal circumstances the interstitial space can increase its volume by as much as 40% without resulting in pulmonary edema (Sartori et al, 2010). In pulmonary injury due to toxins or

osmotic pressure across the vessel wall. The formula for filtration across the pulmonary

Q=K-[CPmv-Ppmv)-(πmv-πpmv)] which is different from the systemic capillary fluid exchange. Q=net transvascular flow of fluid, K=membrane permeability, Pmv=hydrostatic pressure in the microvessels, Ppmv=hydrostatic pressure in the peri-microvascular interstitium, πmv=plasma protein osmotic pressure in the circulation, πpmv=protein osmotic pressure in the perimicrovascular interstitium. This equation only reflects the known hydrostatic pressures without including the lymph hydrostatic pressure which has been studied but is unknown in relation to the Starling forces equation (Sartori et al, 2010). It is known that one of the main functions of the lymphatic system is to return plasma proteins from the interstitial tissue space to the bloodstream. Analyses of blood plasma and lymph have shown that all of the proteins that are found in the plasma are also found in the lymph although in lower concentrations (Ono et al, 2005). In normal circumstances, low pulmonary capillary hydrostatic pressure provides a "safety" factor against pulmonary edema across the lung

Fig. 1. Representation of the pulmonary arterial and venous capillary barrier and lymphatic

Although hydrostatic pressures in the pulmonary arterial and venous system are known, the hydrostatic pressures of the interrelated pulmonary lymphatic system is unknown and largely unstudied. The illustration generously permitted by Dr. Davide Brunelli of Med-

In some forms of NCPE such as ARDS disturbances of pulmonary capillary fluid balance and pulmonary permeability occur as a direct result of endotoxins and inflammation that cause disruption in the capillary EC barrier with barrier disruption and subsequent pulmonary venous congestion. As the volume excess initially enters the interstitium it is taken up by the lymphatic system to be returned to the vascular system. In normal circumstances the interstitial space can increase its volume by as much as 40% without resulting in pulmonary edema (Sartori et al, 2010). In pulmonary injury due to toxins or

capillary semi-permeable membrane is

tissue.

flow.

ars.it.

inflammation the fluid volume increase overwhelms the lymphatic drainage system and the hydrostatic forces become altered resulting in further injury to the pulmonary capillary endothelium. The result is persistent fluid accumulation that overwhelms the lymphatic drainage and tissue edema results. The final step occurs as the fluid volume overwhelms the hydrostatic forces and the excess fluid flows into the alveoli. The edema that is caused by the increasing fluid and vascular permeability is a hallmark of inflammation and tissue injury (Zimmerman & McIntyre, 2004). The edema formation can have severe consequences because the fluid and protein components in the edematous tissues and alveoli increase the diffusion barrier for oxygen and carbon dioxide with subsequent disruption of gas exchange thus precipitating hypoxia and respiratory failure. The rate of volume expansion is also a factor in pulmonary edema.

Other forms of NCPE such as HAPE, immersion pulmonary edema and NPPE may occur due to greatly altered thoracic pressures and are thought to have a largely hydrostatic noninflammatory component (Fremont et al, 2007). The accelerated pulmonary edema that occurs with HAPE, immersion PE or NPPE is thought to be due to significant fluid shifts that come secondary to changes in intrathoracic pressure, and in the case of HAPE or immersion PE, possibly due to diminished atmospheric or oxygen inhalation pressures. Physiologically negative intrathoracic pressure is generated in the chest when a patient inspires against an obstructed airway. The rapid pressure change in the pulmonary venous circulation alters the transpulmonary fluid gradient. The increase in capillary hydrostatic pressure most likely causes capillary gap formation as well as transcellular fluid shifts. The fluid gradient is thus going from a high gradient to a low gradient. Although considered a second mechanism of the EC barrier disruption it is part of the capillary hydrostatic pressure. While it is unlikely that the rapidly resolved post obstructive PE and HAPE are of a different nature, it is more likely that all of the non-cardiogenic PE have the same hydrostatic mechanisms initially (Sartori et al, 2010 & Gantor et al, 2006). The NCPE in ARDS is ultimately a result of capillary permeability secondary to cellular damage, inflammatory cascades, and over inflation by mechanical ventilation resulting in endothelial permeability. The permeability from HAPE and scuba diving is not initiated by inflammation but rather stress failure occurring due to the increased pressures that occur at the capillary level in healthy subjects (Slade et al, 2001). The pulmonary capillary endothelial cell barrier is a semi-permeable membrane and is known to be an active biological interface between the blood and the surrounding tissue. The EC is a single layer of continuous endothelium lining the pulmonary capillaries and forms a single layer between blood and the pulmonary interstitium. The pulmonary capillaries have extremely thin walls to allow rapid exchange of respiratory gases across them (Costello et al, 1992).The endothelium modulates tone, growth, homeostasis and inflammation in the lungs and throughout the circulatory system (Ware & Matthay, 2005 & Umapathy et al, 2010). In NCPE due to diseases such as ARDS, the ensuing endotoxins and inflammatory markers induce capillary endothelial disarray and gap formations. This is followed by neutrophil chemotaxis, diapedesis and protein rich edema fluid leakage into the interstitial spaces. Inflammatory cascades are triggered and also enter into the interstitial milieu. As the capillary pressure increases there is damage of the capillary EC causing larger molecules such as proteins from the vascular space to flow into the interstitial space (Slade et al, 2001). NCPE in an ill patient with ARDS is a high protein pulmonary edema. The fluid/plasma ratio may be used to differentiate the etiology of pulmonary edema in NCPE and CPE. It is a measurement of the alveolar fluid, obtained by broncho- alveolar lavage (BAL), to the serum plasma during acute pulmonary edema and has been shown in studies to be sensitive enough to differentiate a low protein fluid that results from CPE or NCPE in a patient without illness compared to a high protein ratio NCPE such as occurs with illness such as endotoxin induced ARDS (Fremont et al, 2001; Ware et al, 2010). The protein concentration in the pulmonary interstitium of ARDS exceeds 60% of the plasma value whereas the protein concentration in HAPE, reperfusion PE, neurogenic PE and other non ARDS causes is less than 45% (Fein et al, 1079 & Staub et al, 1967). The result is capillary injury with gap formation and high permeability of the EC barrier with an increase of protein rich edema fluid into the interstitial space. The resulting pulmonary interstitial protein remains elevated compared to circulating blood plasma. At this point there is no longer a "quick" resolution possible.

Fig. 2. Schematic representation of the endothelial barrier in inflammatory pulmonary edema.

NCPE causes direct injury to the lungs. The endothelial barrier is normally single layer of continuous endothelium lining the pulmonary capillaries. In ALI/ARDS the endotoxins recruited by the macrophages and neutrophils induce capillary endothelial disarray and gap formations. This is followed by neutrophil chemotaxis, diapedesis, proteases, cytokines and protein rich fluid into the interstitial spaces.

## **3. Neutrophils and molecular mechanisms in the endothelial and epithelial cell barrier in ALI/ARDS**

Neutrophils play an important role in the development of pulmonary edema associated with ALI/ARDS. It is well known that neutrophils are prevalent in ARDS pulmonary edema and are central to the pathogenesis (Bdeir et al, 2010). Initially pulmonary macrophages are activated and recruit circulating neutrophils to the pulmonary microvascular system and the injured capillaries. The interaction of the neutrophils and the endothelium is initiated by adhesion receptors and by soluble or membrane bound chemoattractants. Intercellular adhesion molecules-1 (ICAM-1) and other proteins are known to be present but the specific role of these molecules and proteins is unclear (Downey et al, 1999). Once the neutrophils become activated and move subsequently into the pulmonary parenchymal interstitium, they sequester and initiate an important component of the inflammatory response in endotoxin induced ALI/ARDS. This activation

acute pulmonary edema and has been shown in studies to be sensitive enough to differentiate a low protein fluid that results from CPE or NCPE in a patient without illness compared to a high protein ratio NCPE such as occurs with illness such as endotoxin induced ARDS (Fremont et al, 2001; Ware et al, 2010). The protein concentration in the pulmonary interstitium of ARDS exceeds 60% of the plasma value whereas the protein concentration in HAPE, reperfusion PE, neurogenic PE and other non ARDS causes is less than 45% (Fein et al, 1079 & Staub et al, 1967). The result is capillary injury with gap formation and high permeability of the EC barrier with an increase of protein rich edema fluid into the interstitial space. The resulting pulmonary interstitial protein remains elevated compared to circulating blood plasma. At this point there is no longer a "quick" resolution

Fig. 2. Schematic representation of the endothelial barrier in inflammatory pulmonary

NCPE causes direct injury to the lungs. The endothelial barrier is normally single layer of continuous endothelium lining the pulmonary capillaries. In ALI/ARDS the endotoxins recruited by the macrophages and neutrophils induce capillary endothelial disarray and gap formations. This is followed by neutrophil chemotaxis, diapedesis, proteases, cytokines and

**3. Neutrophils and molecular mechanisms in the endothelial and epithelial** 

Neutrophils play an important role in the development of pulmonary edema associated with ALI/ARDS. It is well known that neutrophils are prevalent in ARDS pulmonary edema and are central to the pathogenesis (Bdeir et al, 2010). Initially pulmonary macrophages are activated and recruit circulating neutrophils to the pulmonary microvascular system and the injured capillaries. The interaction of the neutrophils and the endothelium is initiated by adhesion receptors and by soluble or membrane bound chemoattractants. Intercellular adhesion molecules-1 (ICAM-1) and other proteins are known to be present but the specific role of these molecules and proteins is unclear (Downey et al, 1999). Once the neutrophils become activated and move subsequently into the pulmonary parenchymal interstitium, they sequester and initiate an important component of the inflammatory response in endotoxin induced ALI/ARDS. This activation

possible.

edema.

protein rich fluid into the interstitial spaces.

**cell barrier in ALI/ARDS** 

and migration of neutrophils is a characteristic event in the progression of ALI and ARDS. Animal studies have shown that endothelial injury appears within minutes to hours after ALI initiation with resulting intercellular gaps of the EC. The EC gaps allow for permeability of fluid, neutrophils and cytokines into the pulmonary parenchymal space (Grommes & Soehnlein, 2011).

The neutrophils that infiltrate the lungs and migrate into the airways express proinflammatory cytokines such as TNF-α, IL-1β, IL-6 and contribute to both the endothelial and epithelial integrity disruption of the barriers (Bdeir et al, 2010; Grommes & Soehnlein, 2011; Abraham, 2003). It has also been well documented that the percentage of neutrophils correlates directly with the alveolar-arterial PO2 difference in ALI/ARDS pulmonary edema (Weiland et al, 1986). Neutrophil sequestration is aided by chemotactic factors and by the adhesion molecules on both the neutrophils and capillary endothelial cells (Hasko et al, 2006; Steinberg, 1994; Geerts et al, 2001). The activated neutrophils expressing IL-1β produce other pro-inflammatory cytokines after endotoxin administration. In fact, the removal of neutrophils after endotoxin administration almost entirely prevents an increase of IL-1β expression and attenuates endotoxin induced TNF-α. Neutrophils are the major source of IL-1β in murine models of the lung in ALI (Abraham, 2003). Another feature of the neutrophils that accumulate in the lung of murine models is increased activation of the transcriptional regulatory factor NF-кB. NF-кB is a protein complex that controls transcription of DNA and is involved in cellular responses to stimuli such as pulmonary edema due to ALI/ARDS. It is key in regulating the endotoxin induced immune response in neutrophils and produces increased amounts of pro-inflammatory cytokines whose transcription is dependent on NF-кB (Blackwell et al, 1996). Neutrophils also become an increasing liability in the edematous pulmonary interstitium as they release free radicals.

The alveolar-capillary barrier is a very thin membrane allowing oxygen CO2 exchange for normal respiration. The major consequence of pulmonary edema is impaired gas exchange that interrupts the normal fluid exchange balance. The alveoli epithelium removes fluid by molecular mechanisms of sodium transport, however, the capillary endothelial barrier function has only incompletely defined pathways affecting the concurrent barrier disruption. Permeability of the EC in the capillaries with concurrent alveolar-capillary membrane damage and with leakage of fluid, neutrophils, proteases, cytokines and free radicals that all contribute to the ensuing pulmonary edema is a prominent feature of permeability edema and ALI/ARDS (Holter et al, 1986). The alveolar liquid clearance from the alveolus into the interstitium is based on active sodium transport largely through the highly regulated apical amiloride sensitive epithelial sodium channel complex (ENaC) with concomitant passive water transport and the Na+, K+ ATPase exchange (Elia et al, 2003; Folkesson & Matthay, 2006). The Na+, K+ ATPase exchange transports the alveolar liquid into the interstitium and ultimately into the lymphatic and blood vessels (Hamacher et al, 2010; Lucas et al, 2009). However, these transport processes are often impaired in ALI or ARDS.

It is likely that the induction of increased permeability of the pulmonary capillary bed is directly linked to reversible physical modifications of the pulmonary capillary endothelium (Kaner et al, 2000). The capillary endothelial regulation of endothelial permeability involves various pathways such as those involving reactive oxygen species (ROS), Rho GTPases, and tyrosine phosphorylation of junctional proteins all converge to regulate junctional permeability. They either affect the stability of junctional proteins or modulate their interactions (Lucas et al, 2009). The regulation of permeability at the junctions is mediated by active communication between the proteins of the adherens junctions and the actin cytoskeleton. Actin mediated endothelial cell contraction is the result of myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) in Ca2+/calmodulin- dependent manner. RhoA also potentiates MLC phosphorylation by inhibiting MLC phosphatase activity through its downstream effector Rho kinase (ROCK). As the actin/myosin driven contraction generates a contractile force it pulls VE-cadherin inward. This contraction will force VE-cadherin to dissociate from its adjacent partner causing endothelial gaps - the basic pathology in permeability pulmonary edema (Lucas et al, 2009).

## **4. Serum Biomarkers in permeability edema and ARDS**

A complex progression of events is recognized in the development of permeability edema and ARDS but the exact nature of events is still an area of active study. A large variety of inflammatory mediators have been found to be elevated in ARDS including lung specific proteins, endotoxin binding proteins, tumor necrosis factor alpha (TNF-α), interleukins (ILs), chemokines and markers of endothelium activation such as adhesion molecules and von Willebrand factor antigen (VWF) (Tzouvelekis et al, 2005). A comprehensive review is beyond the scope of this article but some of the most widely known are discussed. There is an increased expression of the vascular endothelial growth factor VEGF gene in pulmonary edema. Although VEGF is widely expressed in the body, the highest level of expression in normal tissues is in the lung. Normally increased expression and angiogenesis is associated with lung tumors and has been studied as a target for therapy in lung cancer however VEGF also is known to stimulate actin stress fiber formation and new focal adhesions in endothelial cells suggesting a regulatory role in endothelial morphology (Kaner et al, 2000). A study by Kaner et al demonstrated that excess expression of VEGF within the murine lung was associated with increased permeability of pulmonary edema (Kaner et al, 2000). In addition to endotoxin effects on endothelial permeability there is strong evidence that cytokines such as tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 are associated with pulmonary edema. Shutte measured cytokine levels in patients with ARDS, severe pneumonia and cardiogenic pulmonary edema for comparison and found consistently higher levels of IL-8, IL-6 and TNF-α in the bronchoalveolar lavage fluid (BALF) and the serum of patients with ARDS and pneumonia compared to cardiogenic pulmonary edema (Schutte et al, 1996). Cytokines have various effects on activating endothelium inducing endothelial expression of adhesion molecules and leukocyte chemotaxis leading to a local inflammatory response in the lung. The cytokine, TNF-α induces macrophages and TH1 cells and activates ECs and macrophages (Braun et al, 2005). Studies have been conflicting as far as elevation of TNF-α in patients with ARDS pulmonary edema, for example, Bauer demonstrated that TNF-α concentrations were significantly higher in patients with ARDS than those of pneumonia or of the control subjects (Bauer et al, 2000). Others, such as Hyers have found variability in the elevation of TNF-α, however, there is speculation that TNF-α is an acute phase cytokine and the timing of studies is important for its evaluation (Hyers, 1991). Although Bauer found that the TNF-α levels were significantly elevated it was also speculated that the elevation was more related to the severity of the lung disease and could not be extrapolated as a possible diagnostic marker for ARDS (Bauer et al, 2000). TNF-α is a known acute phase reactant and is also a cytokine involved in systemic inflammation. It is able to induce apoptotic cell death, inflammation, and inhibit tumorgenesis and viral replication. TNF-α is produced mainly by macrophages

by active communication between the proteins of the adherens junctions and the actin cytoskeleton. Actin mediated endothelial cell contraction is the result of myosin light chain (MLC) phosphorylation by MLC kinase (MLCK) in Ca2+/calmodulin- dependent manner. RhoA also potentiates MLC phosphorylation by inhibiting MLC phosphatase activity through its downstream effector Rho kinase (ROCK). As the actin/myosin driven contraction generates a contractile force it pulls VE-cadherin inward. This contraction will force VE-cadherin to dissociate from its adjacent partner causing endothelial gaps - the basic

A complex progression of events is recognized in the development of permeability edema and ARDS but the exact nature of events is still an area of active study. A large variety of inflammatory mediators have been found to be elevated in ARDS including lung specific proteins, endotoxin binding proteins, tumor necrosis factor alpha (TNF-α), interleukins (ILs), chemokines and markers of endothelium activation such as adhesion molecules and von Willebrand factor antigen (VWF) (Tzouvelekis et al, 2005). A comprehensive review is beyond the scope of this article but some of the most widely known are discussed. There is an increased expression of the vascular endothelial growth factor VEGF gene in pulmonary edema. Although VEGF is widely expressed in the body, the highest level of expression in normal tissues is in the lung. Normally increased expression and angiogenesis is associated with lung tumors and has been studied as a target for therapy in lung cancer however VEGF also is known to stimulate actin stress fiber formation and new focal adhesions in endothelial cells suggesting a regulatory role in endothelial morphology (Kaner et al, 2000). A study by Kaner et al demonstrated that excess expression of VEGF within the murine lung was associated with increased permeability of pulmonary edema (Kaner et al, 2000). In addition to endotoxin effects on endothelial permeability there is strong evidence that cytokines such as tumor necrosis factor (TNF-α), interleukin (IL)-1β, IL-6, and IL-8 are associated with pulmonary edema. Shutte measured cytokine levels in patients with ARDS, severe pneumonia and cardiogenic pulmonary edema for comparison and found consistently higher levels of IL-8, IL-6 and TNF-α in the bronchoalveolar lavage fluid (BALF) and the serum of patients with ARDS and pneumonia compared to cardiogenic pulmonary edema (Schutte et al, 1996). Cytokines have various effects on activating endothelium inducing endothelial expression of adhesion molecules and leukocyte chemotaxis leading to a local inflammatory response in the lung. The cytokine, TNF-α induces macrophages and TH1 cells and activates ECs and macrophages (Braun et al, 2005). Studies have been conflicting as far as elevation of TNF-α in patients with ARDS pulmonary edema, for example, Bauer demonstrated that TNF-α concentrations were significantly higher in patients with ARDS than those of pneumonia or of the control subjects (Bauer et al, 2000). Others, such as Hyers have found variability in the elevation of TNF-α, however, there is speculation that TNF-α is an acute phase cytokine and the timing of studies is important for its evaluation (Hyers, 1991). Although Bauer found that the TNF-α levels were significantly elevated it was also speculated that the elevation was more related to the severity of the lung disease and could not be extrapolated as a possible diagnostic marker for ARDS (Bauer et al, 2000). TNF-α is a known acute phase reactant and is also a cytokine involved in systemic inflammation. It is able to induce apoptotic cell death, inflammation, and inhibit tumorgenesis and viral replication. TNF-α is produced mainly by macrophages

pathology in permeability pulmonary edema (Lucas et al, 2009).

**4. Serum Biomarkers in permeability edema and ARDS** 

but can also be produced by other cells including endothelial cells (Schutte et al, 1996). Large amounts of TNF-α are released in response to LPS endotoxin, bacterial products and IL-1. TNF- α works with IL-1 and IL-6 to produce actions on various organ systems (Schutte et al, 1996; Braun et al, 2005; Bauer et al, 2000). TNF-α also induces EC activation and barrier dysfunction both of which occur in the pathogenesis of pulmonary edema and ALI/ARDS. It can promote edema by TNF receptor dependent chemokine production and adhesion molecule expression and leads to neutrophil chemotaxis (Braun et al, 2005; Ward, 1996). It also causes a decrease in transendothelial electrical resistance across human pulmonary artery EC (HPAEC) (Petrache et al 2003). The pulmonary capillary EC have a balanced system between contracting and tethering forces that normally act to protect the EC barrier from paracellular gaps. The balancing forces depend on cytoskeletal components such as actin based microfilaments, intermediate filaments and microtubules. TNF-α causes contraction via the actin filaments and this results in the formation of gaps and EC permeability (Braun 2005). In pulmonary edema associated with ARDS, TNF-α can mediate acute inflammation and edema formation. It can also have a beneficial effect by increasing alveolar fluid clearance via an amiloride sensitive, cAMP independent mechanism to enhance alveolar fluid clearance. This is accomplished by binding to its receptors or activating Na+ channels in the epithelium (Fukuda et al, 2001).

Other networks of cytokines regulate lung inflammation in lung injury and edema. Complement, C5a and or the membrane attack complex, C5b-9 can directly activate EC to up-regulate adhesion molecules (P-selectin) or act synergistically with TNF-α to up regulate I-CAM and E Selectin (Shutte et al, 1996 & Ward, 1996). IL-4 and IL-10 suppress TNF-α and can have a strong attenuating effect on TNF-α. Studies have shown that blocking IL-10 increased TNF-α production, neutrophil recruitment and the intensity of lung inflammation (Ward, 1996). Although the role of cytokines in the pathogenesis of ARDS has been widely recognized, their importance in the clinical diagnosis has not been clearly defined.

Recent studies have identified that the receptor for advanced glycation end products (RAGE) is activated by its ligands in many disorders including ALI/ARDS. RAGE and interaction with the high mobility group box-1 (HMGB-1)- one of its ligands- promotes local lung endothelial inflammation and evokes both local and systemic inflammation (Nakamura et al, 2011 & Wolfson et al, 2010). In vitro studies determined that RAGE is the primary receptor signaling HMGB-1 induced endothelial barrier disruption and endothelial gap formation in human pulmonary artery endothelial cells (Wolfson et al, 2010). Soluable RAGE (sRAGE) has also been noted in the plasma of patients with ARDS and it was investigated as a biomarker of severity and clinical outcomes in patients with ARDS. In addition sRAGE and HMGB-1 levels were elevated in non- survivors compared to survivors in ARDS (Nakamura et al, 2011). An analysis of biomarker levels in two randomized controlled trials of ventilator therapy for ALI was done and the biomarkers that were elevated were ICAM-1, von Willebrand factor, IL-8, soluble TNF receptor-1, and surfactant protein-D. It was concluded that combining three or more biomarkers may be useful for selecting a high risk ALI group of patients (Calfee et al, 2011). Biomarker identification of risk remains an area of intense research.

#### **5. Non-inflammatory NCPE**

Categories of NCPE that resolve more quickly than ARDS and NCPE due to infection, trauma or other medical illness are high altitude pulmonary edema (HAPE), Immersion pulmonary edema (SCUBA diving and swimming) and negative pressure pulmonary edema (NPPE). Most commonly the non-inflammatory forms of NCPE occur in baseline healthy people although there may be a predilection to development of pulmonary edema in some people. However, there is no known way to predict who will develop pulmonary edema and who will not in the same circumstances.

Fig. 3. Schematic representation of the endothelial barrier in Non-inflammatory Pulmonary Edema

Non- inflammatory pulmonary edema may have an element of hemorrhage leading to a pink frothy tint but will not initially have an inflammatory secretion of cells. The pulmonary edema resolves quickly and the endothelial barrier is thought to accommodate hydrostatic changes that resolve quickly compared to toxin mediated changes that heal slowly and may develop fibrinous changes.

## **6. HAPE**

HAPE is a life threatening form of NCPE that has a rapid onset in healthy people who venture to elevations above 8200feet. This occurs when the lower barometric pressures result in hypoxia, usually less than 90% SPO2 or 60 PaO2. Studies done at high altitude on patients who developed HAPE and compared to those who did not, have shown that pulmonary artery pressures are elevated and lead to a protein rich and mildly hemorrhagic edema. Leukocytes, cytokines, nitric oxide metabolites and eicosanoids are normal when compared to control subjects who did not develop HAPE (Swenson et al, 2002). The mechanism of the increased pulmonary vasoconstriction and resulting increased pulmonary pressures is not resolved. The mechanism is thought to be increased pulmonary and arterial capillary pressures second to hypoxic pulmonary vasoconstriction. Another effect is a noninflammatory increase in the permeability of the vascular endothelium. Some people are more susceptible to HAPE than others but the differentiating factors are not known. The most important treatment is to descend as soon as possible and provide oxygen therapy. Other treatments are Dexamethasone and nifedipine. Phosphodiesterase inhibitors are effective but the side effects worsen mountain sickness headaches (Maggiorini, 2010).

## **7. Immersion PE**

Immersion PE that occurs in diving with self contained underwater breathing apparatus (SCUBA), and some triathlon athletes, combat swimmers (such as found in military missions) and breath hold divers is another type of non-inflammatory NCPE. Only about 1- 2% of immersion divers develop PE and studies have not shown clearly what makes some people susceptible. One study did show that women, hypertension, fish oil and asthma may

pulmonary edema (SCUBA diving and swimming) and negative pressure pulmonary edema (NPPE). Most commonly the non-inflammatory forms of NCPE occur in baseline healthy people although there may be a predilection to development of pulmonary edema in some people. However, there is no known way to predict who will develop pulmonary

Fig. 3. Schematic representation of the endothelial barrier in Non-inflammatory Pulmonary

Non- inflammatory pulmonary edema may have an element of hemorrhage leading to a pink frothy tint but will not initially have an inflammatory secretion of cells. The pulmonary edema resolves quickly and the endothelial barrier is thought to accommodate hydrostatic changes that resolve quickly compared to toxin mediated changes that heal slowly and may

HAPE is a life threatening form of NCPE that has a rapid onset in healthy people who venture to elevations above 8200feet. This occurs when the lower barometric pressures result in hypoxia, usually less than 90% SPO2 or 60 PaO2. Studies done at high altitude on patients who developed HAPE and compared to those who did not, have shown that pulmonary artery pressures are elevated and lead to a protein rich and mildly hemorrhagic edema. Leukocytes, cytokines, nitric oxide metabolites and eicosanoids are normal when compared to control subjects who did not develop HAPE (Swenson et al, 2002). The mechanism of the increased pulmonary vasoconstriction and resulting increased pulmonary pressures is not resolved. The mechanism is thought to be increased pulmonary and arterial capillary pressures second to hypoxic pulmonary vasoconstriction. Another effect is a noninflammatory increase in the permeability of the vascular endothelium. Some people are more susceptible to HAPE than others but the differentiating factors are not known. The most important treatment is to descend as soon as possible and provide oxygen therapy. Other treatments are Dexamethasone and nifedipine. Phosphodiesterase inhibitors are effective but the side effects worsen mountain sickness headaches (Maggiorini, 2010).

Immersion PE that occurs in diving with self contained underwater breathing apparatus (SCUBA), and some triathlon athletes, combat swimmers (such as found in military missions) and breath hold divers is another type of non-inflammatory NCPE. Only about 1- 2% of immersion divers develop PE and studies have not shown clearly what makes some people susceptible. One study did show that women, hypertension, fish oil and asthma may

edema and who will not in the same circumstances.

Edema

**6. HAPE** 

**7. Immersion PE** 

develop fibrinous changes.

be factors that predispose to this condition (Miller III et al, 2010). The mechanism is poorly understood but reviews of cases have shown that the onset is rapid and that rapid improvement is also seen, similar to patients who develop HAPE (Slade et al, 2001). This observation lends credence to the probability that this is an acute but transient increase in pressure. The stress from the increased transalveolar pressure gradient that occurs with immersion is such that the non-cardiogenic capillary endothelium layer cell develops leaks (Slade et al, 2001). It has been shown that even asymptomatic dives result in an increased accumulation of extravascular lung water (Marinovic et al, 2010). They further demonstrated an increase in lung water, pulmonary artery pressure, NT-proBNP levels and decreased left ventricular contractility in healthy study volunteers who did not develop pulmonary edema. Immersion is known to increase preload and cold exposure increases both preload and afterload by vasoconstriction; it is possible that the changes could lead to damage in the pulmonary endothelium and lead to intrapulmonary redistribution of blood with regional overperfusion of some pulmonary capillaries and stress failure increasing capillary damage and permeability (Marinovic et al, 2010 & Pons et al, 1995). Hyperoxia and low tank pressures are other possible mechanisms that may damage the pulmonary endothelium and increase endothelial permeability the development of pulmonary edema in immersion PE.

### **8. Negative pressure pulmonary edema**

Negative pressure pulmonary edema (NPPE) is pulmonary edema that occurs following an acute upper airway obstruction and may also be referred to as a post obstructive pulmonary edema. It often occurs in otherwise healthy patients. The most common cause is laryngospasm soon after extubation from an endo-tracheal intubation in about 0.1% in post anesthesia patients (Pathak et al, 2011). There are also case reports of NPPE after other causes such as foreign body, epiglottitis, tracheal secretions, upper airway tumors, obesity, obstructive sleep apnea, tumors (Fremont et al, 2007). Following an episode of obstruction of the airway there is a marked increase in negative intrathoracic pressure against the obstructed upper airway. The rapid increase in intrathoracic pressure causes a rise in venous return to the right heart, which increases pulmonary venous volume and pulmonary venous pressure which then increases the pulmonary capillary transmural pressure. Systemic pressures also rise due to catecholamine induced veno-constriction from anxiety, hypoxia and hypercarbia (Schwartz et al, 1999). Fremont et al have shown that the mechanism is due to hydrostatic changes with the fluid moving from high pressure in the pulmonary venous system to low pressure into the pulmonary interstitium and airspaces. Studies confirmed that the pulmonary edema fluid to plasma protein ratio were consistent with hydrostatic causes of acute fluid shifts and pulmonary edema. This causes an increase of blood flow (venous return) to the right heart increasing pulmonary venous pressure (Fremont et al, 2007). The treatment is resolving the obstruction, oxygen and either reintubation or if the patient is stable CPAP may be used. The issue is not volume overload and diuretics are not indicated (Kapoor, 2011).

#### **9. Clinical manifestation of non cardiogenic pulmonary edema**

Clinically non-cardiogenic pulmonary edema is a permeability edema. The Starling equation predicts that a change in permeability of the microvascular membrane will result in an increase in the amount of fluid and protein that leaves the vascular space and enters the interstitial space. When the interstitial fluid increases in the interstitium then the outward movement continues and fluid enters the alveolar spaces through the tight junctions of the epithelium. Many entities are associated with permeability edema manifested as ARDS, the most common entities are sepsis, pneumonia, multiple blood transfusions, gastric aspiration, trauma, drug overdose and pancreatitis. and others. Clinically NCPE prototype ALI/ARDS presents with progressive hypoxia and respiratory failure. Multi-organ failure is frequent and one of the reasons the mortality is so high. ARDS affects 200,000 people per year and has a mortality rate of 30-50%. This was initially described in the 1960s' by Petty and Ashbaugh (Ashbaugh et al, 1967). The diagnosis is made by four clinical criteria, acute onset of bilateral chest infiltrates, hypoxia, no evidence of left atrial hypertension (or clinical manifestations indicating left heart failure) and a ratio of arterial oxygen to fraction of inspired oxygen (PaO2/FIO2 ratio) of 201-300 for ALI and less than 200 for ARDS (Bernard et al, 1994 & The Acute Respiratory Distress Syndrome Network, 2000). Due to the profound respiratory failure most patients require mechanical ventilation and care in the intensive care unit (ICU). The best therapeutic approach for ARDS permeability edema is to find and treat the cause. There is no pharmacological treatment for ARDS, the only "treatment" that has emerged since it was first described in 1971 is the ARDS-net trial protocol of low tidal volume mechanical ventilation. This is a strategy that is called "lung-protective ventilation". The main benefit of lung-protective ventilation is to avoid further injury to the lungs from high tidal volumes often used for patients in the ICU as these large tidal volumes increase the injury and subsequently cause an increase in the permeability pulmonary edema in the injured lung. The standard tidal volumes used in ICU have been 10-15mg/kg for all mechanically ventilated patients, however, low tidal volumes of 6-8mg/kg are recommended for patients with ARDS. A mortality benefit was one of the primary outcomes demonstrated in the ARDS-net trial for patients with ARDS who are mechanically ventilated at these low tidal volumes (Matthay et al, 2002 & Marino 2007). Another aspect of NCPE and ARDS is heterogeneity of the lung parenchyma and the alveoli. The chest roentgenogram shows bilateral infiltrates that are difficult to distinguish from cardiogenic pulmonary edema and sometimes from pneumonia. The computed tomographic (CT) images are not necessarily diagnostic but in a patient with known ARDS shows that the lung edema and consolidation is not homogeneous but involves various lung regions, while other regions appear normal (Rouby et al, 2003). Alveolar over-distension from mechanical ventilation as well as repeated opening and collapse of the alveoli has been shown to cause lung injury initiating an increase in capillary stress and an increase in pro-inflammatory cytokine cascades (Matthay et al, 2002 & Slutsky &Tremblay, 1998). Hemodynamic evaluation of NCPE and ARDS requires careful ongoing evaluation, and may require monitoring cardiac filling pressures and cardiac output using a pulmonary artery catheter or currently, a noninvasive evaluation is more likely to be used. Diuretic therapy is then tailored to achieve the lowest cardiac filling pressures that do not compromise cardiac output and systemic oxygen transport. The nature of the lung interstitial infiltration is an inflammatory process, so that diuretic therapy to remove fluid does not remove the inflammation. Fluid treatment versus diuresis is not necessarily the goal of treatment in NCPE secondary to ARDS (Marino, 2007). Ultimately the treatment is supportive care using the appropriate ventilator strategies, promoting oxygenation, and treating the multi organ failure that is often contributes to the mortality. Histologically in this acute phase of lung injury there is widespread interstitial and alveolar edema with an abundance of neutrophils, erythrocytes, macrophages, cell

increase in the amount of fluid and protein that leaves the vascular space and enters the interstitial space. When the interstitial fluid increases in the interstitium then the outward movement continues and fluid enters the alveolar spaces through the tight junctions of the epithelium. Many entities are associated with permeability edema manifested as ARDS, the most common entities are sepsis, pneumonia, multiple blood transfusions, gastric aspiration, trauma, drug overdose and pancreatitis. and others. Clinically NCPE prototype ALI/ARDS presents with progressive hypoxia and respiratory failure. Multi-organ failure is frequent and one of the reasons the mortality is so high. ARDS affects 200,000 people per year and has a mortality rate of 30-50%. This was initially described in the 1960s' by Petty and Ashbaugh (Ashbaugh et al, 1967). The diagnosis is made by four clinical criteria, acute onset of bilateral chest infiltrates, hypoxia, no evidence of left atrial hypertension (or clinical manifestations indicating left heart failure) and a ratio of arterial oxygen to fraction of inspired oxygen (PaO2/FIO2 ratio) of 201-300 for ALI and less than 200 for ARDS (Bernard et al, 1994 & The Acute Respiratory Distress Syndrome Network, 2000). Due to the profound respiratory failure most patients require mechanical ventilation and care in the intensive care unit (ICU). The best therapeutic approach for ARDS permeability edema is to find and treat the cause. There is no pharmacological treatment for ARDS, the only "treatment" that has emerged since it was first described in 1971 is the ARDS-net trial protocol of low tidal volume mechanical ventilation. This is a strategy that is called "lung-protective ventilation". The main benefit of lung-protective ventilation is to avoid further injury to the lungs from high tidal volumes often used for patients in the ICU as these large tidal volumes increase the injury and subsequently cause an increase in the permeability pulmonary edema in the injured lung. The standard tidal volumes used in ICU have been 10-15mg/kg for all mechanically ventilated patients, however, low tidal volumes of 6-8mg/kg are recommended for patients with ARDS. A mortality benefit was one of the primary outcomes demonstrated in the ARDS-net trial for patients with ARDS who are mechanically ventilated at these low tidal volumes (Matthay et al, 2002 & Marino 2007). Another aspect of NCPE and ARDS is heterogeneity of the lung parenchyma and the alveoli. The chest roentgenogram shows bilateral infiltrates that are difficult to distinguish from cardiogenic pulmonary edema and sometimes from pneumonia. The computed tomographic (CT) images are not necessarily diagnostic but in a patient with known ARDS shows that the lung edema and consolidation is not homogeneous but involves various lung regions, while other regions appear normal (Rouby et al, 2003). Alveolar over-distension from mechanical ventilation as well as repeated opening and collapse of the alveoli has been shown to cause lung injury initiating an increase in capillary stress and an increase in pro-inflammatory cytokine cascades (Matthay et al, 2002 & Slutsky &Tremblay, 1998). Hemodynamic evaluation of NCPE and ARDS requires careful ongoing evaluation, and may require monitoring cardiac filling pressures and cardiac output using a pulmonary artery catheter or currently, a noninvasive evaluation is more likely to be used. Diuretic therapy is then tailored to achieve the lowest cardiac filling pressures that do not compromise cardiac output and systemic oxygen transport. The nature of the lung interstitial infiltration is an inflammatory process, so that diuretic therapy to remove fluid does not remove the inflammation. Fluid treatment versus diuresis is not necessarily the goal of treatment in NCPE secondary to ARDS (Marino, 2007). Ultimately the treatment is supportive care using the appropriate ventilator strategies, promoting oxygenation, and treating the multi organ failure that is often contributes to the mortality. Histologically in this acute phase of lung injury there is widespread interstitial and alveolar edema with an abundance of neutrophils, erythrocytes, macrophages, cell debris, plasma proteins and strands of fibrin. At this phase there is injury to the capillary endothelium and denuding of the alveolar epithelium. If the patient survives this phase the pulmonary edema may completely resolve within a few months. Other patients with ALI/ARDS progress to a subacute phase over one to two weeks and may continue to have respiratory failure with hypoxia requiring mechanical ventilation. These patients develop fibrosis and capillary obliteration in the lungs, a condition called fibrosing alveolitis. These patients continue to progress with respiratory failure although they may recover from the initial event.

Progress from bench to bedside is being made; the mortality from NCPE and ALI/ARDS has improved from the initial 60% or more to 30-50%. The establishment of the NIH ARDS Network for clinical trials has improved the quantity and quality of large multicenter clinical trials for ALI/ARDS patients. New research is continuing and tools such as genetic analysis, genomics and proteomics may offer more value for patients in the future.

#### **10. References**

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## **High Altitude Pulmonary Edema**

## Zhou Qiquan and Luo Yongjun

*Department of High Altitude Diseases, College of High Altitude Military Medicine, Third Military Medical University, Key Laboratory of High Altitude Medicine, Ministry of Education and Key Laboratory of High Altitude Medicine of PLA, Chongqing, China*

## **1. Introduction**

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High altitude pulmonary edema (HAPE) is a form of high altitude idiopathy that occurs in a minority of people upon either the first or subsequent exposure to high altitudes. It is triggered by a shortage of oxygen and certain other predisposing factors, all of which lead to a sudden increase in pulmonary arterial pressure, increase in lung blood volume, disturbance of pulmonary circulation, and leakage of fluid in microcirculation into the pulmonary interstitium and alveoli. The clinical symptoms of this condition include dyspnea and hacking cough.

HAPE is a severe type of acute high altitude disease, typically occurring at altitudes above 4,000 meters. However, cases have been reported at altitudes as low as 2,261 meters in Xining, China. HAPE is a rapid-onset condition that can progress and change quickly, especially during the first stage of high altitude exposure, usually within a week, peaking within three days. According to a report based on 332 cases, 63% of HAPE patients presented symptoms within three days, and the fastest onset was after only a few hours. HAPE occurs mostly in unacclimatized sea-level residents when they first ascend to high altitudes or acclimatized individuals ascending from lower to higher altitudes. It can also occur in long-term high altitude residents or high altitude natives undertaking excessive physical activities or in those who return to high altitudes after living in low-altitude areas for a period of time. HAPE patients can recover after short-term treatment and continue to stay at high altitudes. However, improper treatment may lead to negative effects. The incidence rate is closely related to the altitude, rapidity of exposure, season, the individual's physical condition, and the intensity of activity.

#### **2. Epidemiology**

#### **2.1 Incidence rate**

#### 1. Population Incidence

The incidence rate of HAPE varies significantly between China and the others country reports, for example it ranges between 0.15% and 9.9% in Chinese report. The incidence rate is higher in kids and teenagers than in adults. For instance, data collected from the Andes Mountain area of Peru reports that the incidence rate was 10% in children aged 2-12, 17% in teens aged 13-20, and 3% in adults over 21.

#### 2. Hospitalization rate

Due to the differences in location and population served, different treatment facilities have reported rather different hospitalization rate. The Menon hospital, at an altitude of 3,450 meters, treated 101 HAPE patients over two years, accounting for 5-10% of all inpatients in the department of internal medicine; a hospital in Changdu (Tibetan), at an altitude of 3,200 meters, treated 33 HAPE patients over 11 years, accounting for 0.37% of all inpatients in the internal medicine department and 6.28% of all inpatients with high altitude diseases; the General Hospital of the Tibet Military Region, at an altitude of 3,658 meters, treated 2,853 HAPE patients from 1956 to 2005, accounting for 89.6% of all inpatients with high altitude diseases during the same time period.

#### 3. Age Distribution

Reports from the Andes and Rocky Mountain areas show higher incidence in children, but occurrences in children below 2 years old are rare and the maximum age of all HAPE patients covered in these reports was found to be 53 years old. In the General Hospital of the Tibet Military Region, the youngest HAPE patient was 1 year old and the oldest one was 63 years old. We think that HAPE can occur at any age, but children and young adults are more susceptible.

#### 4. Gender Differences

HAPE can occur in both males and females. Many reports show higher incidence in males, mainly because more males travel to high altitude areas. In mountaineering and high altitude medical research teams, male participants are more common, hence a higher reported incidence of HAPE in males. Horrobin et al. mentioned that in Kenyan mountain areas there were many female mountaineers, none of whom developed HAPE. However Hultgren et al. reported that among 97 lifelong mountain residents, HAPE occurred mainly in females.

#### 5. Racial Differences

It has been shown that there is no significant difference in HAPE incidence between Peruvian Indians and Caucasians in the plateau areas of Peru. However, some have suggested that the Sherpa of Nepal have a lower HAPE incidence than the Indians of Peru. They point out that the Indians have only inhabited the Andes for about 10,000 years, whereas the Sherpa, originally from the Tibetan Plateau, have lived in high altitude areas for several tens of thousands of years. Therefore, the Sherpa may be more adapted to high altitudes environment than Peruvian Indians. Our investigation found that, on the Tibetan Plateau, HAPE incidence in native Tibetans was lower than in Han immigrants. In the 923 HAPE cases treated by the General Hospital of the Tibet Military Region over the past ten years, there was only one Tibetan patient. Recently we have retrospectively analyzed for severe acute mountain sickness of 3184 Inpatients cases in General Hospital of Tibet Military region Hospitalization from June 1956 to June 2005, and in which the incidence and clinical characteristics of native Tibetan plateau were analyzed.The results detected 24 cases of high altitude native Tibetan suffering severe cases of acute mountain sickness, high altitude pulmonary edema 21 cases and high altitude cerebral edema 3 cases in them, incidence of severe acute mountain sickness in native Tibetan population was 0.75% (24/3184).

6. First and Repeated Exposure to High Altitudes

Whether triggered by the patient's first or a subsequent exposure to high altitude, HAPE onset usually takes place between one and seven days, but it can start as early as three hours or as

Due to the differences in location and population served, different treatment facilities have reported rather different hospitalization rate. The Menon hospital, at an altitude of 3,450 meters, treated 101 HAPE patients over two years, accounting for 5-10% of all inpatients in the department of internal medicine; a hospital in Changdu (Tibetan), at an altitude of 3,200 meters, treated 33 HAPE patients over 11 years, accounting for 0.37% of all inpatients in the internal medicine department and 6.28% of all inpatients with high altitude diseases; the General Hospital of the Tibet Military Region, at an altitude of 3,658 meters, treated 2,853 HAPE patients from 1956 to 2005, accounting for 89.6% of all inpatients with high altitude

Reports from the Andes and Rocky Mountain areas show higher incidence in children, but occurrences in children below 2 years old are rare and the maximum age of all HAPE patients covered in these reports was found to be 53 years old. In the General Hospital of the Tibet Military Region, the youngest HAPE patient was 1 year old and the oldest one was 63 years old. We think that HAPE can occur at any age, but children and young adults are

HAPE can occur in both males and females. Many reports show higher incidence in males, mainly because more males travel to high altitude areas. In mountaineering and high altitude medical research teams, male participants are more common, hence a higher reported incidence of HAPE in males. Horrobin et al. mentioned that in Kenyan mountain areas there were many female mountaineers, none of whom developed HAPE. However Hultgren et al. reported that among 97 lifelong mountain residents, HAPE occurred mainly in females.

It has been shown that there is no significant difference in HAPE incidence between Peruvian Indians and Caucasians in the plateau areas of Peru. However, some have suggested that the Sherpa of Nepal have a lower HAPE incidence than the Indians of Peru. They point out that the Indians have only inhabited the Andes for about 10,000 years, whereas the Sherpa, originally from the Tibetan Plateau, have lived in high altitude areas for several tens of thousands of years. Therefore, the Sherpa may be more adapted to high altitudes environment than Peruvian Indians. Our investigation found that, on the Tibetan Plateau, HAPE incidence in native Tibetans was lower than in Han immigrants. In the 923 HAPE cases treated by the General Hospital of the Tibet Military Region over the past ten years, there was only one Tibetan patient. Recently we have retrospectively analyzed for severe acute mountain sickness of 3184 Inpatients cases in General Hospital of Tibet Military region Hospitalization from June 1956 to June 2005, and in which the incidence and clinical characteristics of native Tibetan plateau were analyzed.The results detected 24 cases of high altitude native Tibetan suffering severe cases of acute mountain sickness, high altitude pulmonary edema 21 cases and high altitude cerebral edema 3 cases in them, incidence of

severe acute mountain sickness in native Tibetan population was 0.75% (24/3184).

Whether triggered by the patient's first or a subsequent exposure to high altitude, HAPE onset usually takes place between one and seven days, but it can start as early as three hours or as

6. First and Repeated Exposure to High Altitudes

2. Hospitalization rate

3. Age Distribution

more susceptible. 4. Gender Differences

5. Racial Differences

diseases during the same time period.

late as ten days after exposure. Occasionally, HAPE is triggered in high altitude residents by such factors as fatigue. For those who travel to high altitudes by plane, the typical time of HAPE onset is within three days. Data collected from Peru and the U.S. suggest that high altitude residents are likely to develop HAPE when returning to the plateau after spending typically one to three weeks on the plains. In Tibetan areas, HAPE tends to be triggered when residents spend typically three to six weeks on the plains before returning.

#### 7. Mode of Transportation to High Altitude Area

Traveling to high altitude areas, whether on foot, by ground vehicle, or by airplane, can trigger HAPE. In recent years, as more people take airplane flights to the Tibetan Plateau, the number of HAPE patients who have arrived by plane has substantially increased. Among the 2,853 HAPE patients treated by the General Hospital of the Tibet Military Region between 1956 and 2005, 2,054 patients were traveled to Tibet by airplane.

8. Occupation and Labor Intensity

HAPE can occur in individuals of all occupations when they are exposed to high altitudes, but it is more common in those engaged in heavy physical labor. For instance, during the Qinghai-Tibet Highway, construction workers have a higher HAPE incidence than drivers, who in turn have a higher HAPE incidence than travelers taking rides. Those who are rapidly exposed to high altitudes and extreme fatigue are particularly susceptible to HAPE. Therefore, individuals engaged in physical activities at high altitudes, such as plateau mountaineering, alpine skiing, or traveling to high altitudes are all at risk for HAPE. In addition, HAPE is more likely to occur in young boys, as they tend to be more active and less willing to rest than young girls and adults.

9. Season of Onset and Changes in Climate

HAPE can occur in any season, but in general it is more common in the winter and spring. Earlier statistics from domestic show the onset of HAPE to be distributed mostly from November to March of the next year. More recent data suggest an increase in the prevalence of HAPE between January and October, mainly because there have been more people traveling to and from the plateau or engaging in high altitude activities during this period.

10. Upper Respiratory Tract Infection and Acute mild mountain sickness

Upper respiratory tract infection can also trigger HAPE. Among the 865 HAPE patients treated by a Tibetan hospital in Lhasa, 30% had already had a previous upper respiratory tract infection at the time of onset. It is suggested that upper respiratory tract infection may trigger HAPE because contracting an upper respiratory tract infection after reaching a high altitude substantially worsens the shortage of oxygen.

A minority of those who show Acute mild mountain sickness (AMMS) may develop HAPE without prompt treatment. Among the 230 HAPE cases reported by a hospital in southern Xinjiang, 112 (47%) started out with AMMS. From this it can be concluded that, when developing acute or even minor AMMS or upper respiratory tract infection, one should rest and receive immediate treatment to prevent the condition from progressing to HAPE.

#### 11. Individual and Familial Susceptibility

It has been reported that there are patients who develop severe HAPE more than two times, up to four times. Analysis on the 923 HAPE cases treated by a hospital in Lhasa showed that 27% of the patients returning to high altitudes developed HAPE two or more times, two of them even seven times. In our clinical experience we have seen one worker developing HAPE eight times. It is worth pointing out that some HAPE patients, when returning to high altitudes from the plain, may still develop HAPE again even if they take active measures to prevent it, such as bed rest, oxygen inhalation, and medicine.

Some reports have shown that HAPE can co-occur in fathers and sons, brothers, and mothers and daughters. In Tibetan, Zhang reported HAPE occurring in three generations of one family, suggesting that familial and individual factors are involved in patients' susceptibility to HAPE. Animal studies have also shown species and individual differences in susceptibility to HAPE.

#### **2.2 Fatality rate and cause of death**

#### 1. Fatality Rate

A collection of data showed that among 160 HAPE cases that occurred between 1958 and 1965 in China, the fatality rate was 9.4%. In recent years, due to substantial improvements in medical conditions and the promptness of treatment, the fatality rate has decreased significantly. For instance, among the 923 cases of HAPE treated by the General Hospital of the Tibet Military Region in the northern Tibetan plateau, the fatality rate was 0.33%.

#### 2. Cause of Deaths

When treated promptly, most HAPE patients can recover in three to five days. However, death still occurs occasionally, and the causes can be generally summarized as belonging to one of the following categories: 1) Delay in diagnosis or treatment due to poor transportation and substandard medical conditions in remote areas. 2) Deterioration of the patient's general condition caused by undiagnosed and untreated complications such as heart failure, shock, massive pulmonary embolism, severe lung infection, and cerebral encephaledema/encephalorrhagia. 3) Severe HAPE may cause instant death, and cooccurring pulmonary embolisms or encephalorrhagia can also lead to death.

In summary, the incidence of HAPE is closely related to factors such as the mode of transportation to high altitude regions, the rapidity of exposure to high altitudes, the altitude itself, the medical support received, and individual susceptibility. Fatigue, upper respiratory tract infection, and excessive mental stress are all important triggers of HAPE. HAPE mostly occurs at altitudes above 3,000 meters because there are more immigrants at these altitudes. HAPE is not clearly correlated with age or gender. With the increasing of knowledge on HAPE and improvements to medical conditions, the fatality rate of HAPE has become extremely low.

#### **3. Causes and predisposing factors**

HAPE is a disease that occurs at high altitudes, its main cause being scarcity of oxygen. All factors that aggravate oxygen shortage in the body lower the body's tolerance to low oxygen levels, or add load to pulmonary circulation can trigger HAPE. The most common predisposing factors include cold, fatigue, and upper respiratory tract infection.

#### 1. Cold

Cold is a basic feature of plateau weather. The temperature on the Tibetan Plateau is on average more than 20°C lower than the temperature at sea level on the same latitude. At night, wind blows downwards from the snow-covered mountaintops, further accelerating the drop in body surface temperature and making the night at high altitudes especially cold. Under external conditions, extreme coldness accompanied by wind and snow, the body will speed up its metabolism and consume more oxygen. At the same time, sympathetic excitability will become elevated, increasing the venous blood return from the peripheral veins, especially those on the surface of the skin, increasing the load of pulmonary circulation. In addition, pulmonary arterioles contract, inducing or aggravating pulmonary hypertension and eventually triggering HAPE.

#### 2. Fatigue

542 Lung Diseases – Selected State of the Art Reviews

them even seven times. In our clinical experience we have seen one worker developing HAPE eight times. It is worth pointing out that some HAPE patients, when returning to high altitudes from the plain, may still develop HAPE again even if they take active

Some reports have shown that HAPE can co-occur in fathers and sons, brothers, and mothers and daughters. In Tibetan, Zhang reported HAPE occurring in three generations of one family, suggesting that familial and individual factors are involved in patients' susceptibility to HAPE. Animal studies have also shown species and individual differences

A collection of data showed that among 160 HAPE cases that occurred between 1958 and 1965 in China, the fatality rate was 9.4%. In recent years, due to substantial improvements in medical conditions and the promptness of treatment, the fatality rate has decreased significantly. For instance, among the 923 cases of HAPE treated by the General Hospital of

When treated promptly, most HAPE patients can recover in three to five days. However, death still occurs occasionally, and the causes can be generally summarized as belonging to one of the following categories: 1) Delay in diagnosis or treatment due to poor transportation and substandard medical conditions in remote areas. 2) Deterioration of the patient's general condition caused by undiagnosed and untreated complications such as heart failure, shock, massive pulmonary embolism, severe lung infection, and cerebral encephaledema/encephalorrhagia. 3) Severe HAPE may cause instant death, and co-

In summary, the incidence of HAPE is closely related to factors such as the mode of transportation to high altitude regions, the rapidity of exposure to high altitudes, the altitude itself, the medical support received, and individual susceptibility. Fatigue, upper respiratory tract infection, and excessive mental stress are all important triggers of HAPE. HAPE mostly occurs at altitudes above 3,000 meters because there are more immigrants at these altitudes. HAPE is not clearly correlated with age or gender. With the increasing of knowledge on HAPE and improvements to medical conditions, the fatality rate of HAPE

HAPE is a disease that occurs at high altitudes, its main cause being scarcity of oxygen. All factors that aggravate oxygen shortage in the body lower the body's tolerance to low oxygen levels, or add load to pulmonary circulation can trigger HAPE. The most common

Cold is a basic feature of plateau weather. The temperature on the Tibetan Plateau is on average more than 20°C lower than the temperature at sea level on the same latitude. At night, wind blows downwards from the snow-covered mountaintops, further accelerating

predisposing factors include cold, fatigue, and upper respiratory tract infection.

the Tibet Military Region in the northern Tibetan plateau, the fatality rate was 0.33%.

occurring pulmonary embolisms or encephalorrhagia can also lead to death.

measures to prevent it, such as bed rest, oxygen inhalation, and medicine.

in susceptibility to HAPE.

1. Fatality Rate

2. Cause of Deaths

has become extremely low.

1. Cold

**3. Causes and predisposing factors** 

**2.2 Fatality rate and cause of death** 

Physical labor can increase oxygen demand ten times. Physical labor further aggravates oxygen shortage. In addition, physical labor at high altitudes increases the release of catecholamine, and hyperventilation can cause respiratory alkalosis, decrease the concentration of PaCO2, and lead to venoconstriction in the systemic circulation and subsequent increases both in the cardiac output of the right side of the heart and in pulmonary blood volume. Physical labor may further increase the pulmonary arterial pressure and decrease the concentration of PaCO2. Those who engage in excessive mental labor may also be susceptible to HAPE due to bodily fatigue.

#### 3. Upper respiratory tract infection

Upper respiratory tract infection often causes fever, which increases oxygen consumption. If complicated by bronchitis, causing coughing and an increase in bronchial secretions, it will affect pulmonary ventilation and cause damage to the alveolar epithelia, impeding the generation of surface-active substances. According to statistics from Lhasa, 30% of HAPE cases were triggered by upper respiratory tract infections; data from southern Xinjiang showed 29%.

#### 4. Excessive mental stress

Mental stress, anxiety, and fear can increase the release of catecholamine, which in turn increases pulmonary arterial pressure and triggers HAPE.

#### 5. Rapidity of exposure to high altitudes

Among those who quickly enter the plateau areas by ground vehicle or by plane without adaptation trainings, the incidence of HAPE is substantially higher than in those who come by slower means. This is because rapid exposure to high altitudes leads to acute oxygen shortage, in which the body does not have enough time to adapt and shows extremely poor tolerance to low oxygen levels.

#### 6. Sleep and hypnotic drugs

During sleep, the horizontal position of the body increases pulmonary blood volume (500 ml more than an upright position). The shallow breathing that takes place during sleep, especially the periodic or irregular breathing accompanied by temporary apnea can aggravate the oxygen shortage.

The incidence of HAPE mainly depends on the altitude, temperature, and adaptability of the body. High altitudes, low temperatures, and failure to acclimate are three basic factors that trigger HAPE. Individuals may not develop HAPE when only one factor is present. With two factors, the incidence rate is still not high. However, when all three factors are present, one is much more likely to develop HAPE. The incidence rate will increase further if there are other coexisting conditions. Those who are exposed to high altitudes without a thorough physical examination may have undetected organic cardiovascular diseases, organic diseases of the respiratory tract, liver, brain or kidney, malnutrition, or hypoproteinemia.

## **4. Pathogenesis**

The incidence of HAPE is closely correlated with oxygen shortage at high altitudes. Currently, it is believed that the following processes are important: an excessive increase in the pulmonary arterial pressure, an increase in the permeability of the pulmonary capillaries, and impairment in alveolar epithelium water clearance. Among these three, an excessive increase in the pulmonary arterial pressure is the key link.

#### **4.1 Excessive increase in the pulmonary arterial pressure**

In 1904, Plumier et al. observed that low oxygen levels could lead to pulmonary hypertension. In 1964, Hultgnen performed right cardiac catheterization on four acute HAPE patients and found that in both the clinical and the recovery periods their pulmonary arterial pressures were significantly higher than that of control subjects at the same altitude, but their right arterial pressures, pulmonary venous pressures, and pulmonary capillary pressures were all essentially normal. There are several ways in which hypoxia that occurs at high altitudes can lead to pulmonary hypertension.

1. Pulmonary vasoconstriction caused by hypoxia

Hypoxic pulmonary vasoconstriction can redirect blood flow from low-oxygen alveolar regions to alveoli with higher oxygen content, which improves the ventilation/perfusion ratio and gaseous exchange, allows sufficient oxygenation of the blood, and increases arterial partial pressure of oxygen. However, pulmonary hypertension over a long period of time can also cause a series of pathophysiological changes and become an important pathologic basis of the initiation and development of HAPE.

Studies on the mechanisms of hypoxic pulmonary vasoconstriction have demonstrated that, at low oxygen levels, the calcium concentration and transmembrane inflow of calcium ions in the pulmonary arterial smooth muscle cells are both significantly increased. As hypoxia the distribution of ions across the membranes of the pulmonary arterial smooth muscle cells, calcium and sodium ions flow into the cells and potassium ions flow out, causing the resting membrane potential to decrease, approaching its excitation threshold. This depolarization of the pulmonary arterial smooth muscle cells leads to increased reactivity of the pulmonary vessels and increased tension in the pulmonary arterioles and pulmonary arteriolar contraction. Some investigators believe that hypoxia directly changes the transmembrane potential of the pulmonary artery smooth muscle cells, leading to calcium ion inflow and decrease of excitation threshold of these cells, which then causes the smooth muscles of the arterioles to contract. In addition, vasoactive substances such as prostaglandin, thromboxane A2, angiotonin, and histamine may serve as regulators or synergists in hypoxic pulmonary vasoconstriction. However, persistent pulmonary hypertension caused by hypoxia may involve many vasoactive substances, the importance of each varying depending on the specific conditions.

Hypoxic pulmonary vasoconstriction doubtlessly leads to pulmonary hypertension. As to how the pulmonary hypertension causes HAPE, currently there are three hypotheses.

a. Regional maldistribution of blood flow. It has been proposed that when those who are sensitive to hypoxia at high altitudes are exposed to low-oxygen environments, their

physical examination may have undetected organic cardiovascular diseases, organic diseases of the respiratory tract, liver, brain or kidney, malnutrition, or hypoproteinemia.

The incidence of HAPE is closely correlated with oxygen shortage at high altitudes. Currently, it is believed that the following processes are important: an excessive increase in the pulmonary arterial pressure, an increase in the permeability of the pulmonary capillaries, and impairment in alveolar epithelium water clearance. Among these three, an

In 1904, Plumier et al. observed that low oxygen levels could lead to pulmonary hypertension. In 1964, Hultgnen performed right cardiac catheterization on four acute HAPE patients and found that in both the clinical and the recovery periods their pulmonary arterial pressures were significantly higher than that of control subjects at the same altitude, but their right arterial pressures, pulmonary venous pressures, and pulmonary capillary pressures were all essentially normal. There are several ways in which hypoxia that occurs

Hypoxic pulmonary vasoconstriction can redirect blood flow from low-oxygen alveolar regions to alveoli with higher oxygen content, which improves the ventilation/perfusion ratio and gaseous exchange, allows sufficient oxygenation of the blood, and increases arterial partial pressure of oxygen. However, pulmonary hypertension over a long period of time can also cause a series of pathophysiological changes and become an important

Studies on the mechanisms of hypoxic pulmonary vasoconstriction have demonstrated that, at low oxygen levels, the calcium concentration and transmembrane inflow of calcium ions in the pulmonary arterial smooth muscle cells are both significantly increased. As hypoxia the distribution of ions across the membranes of the pulmonary arterial smooth muscle cells, calcium and sodium ions flow into the cells and potassium ions flow out, causing the resting membrane potential to decrease, approaching its excitation threshold. This depolarization of the pulmonary arterial smooth muscle cells leads to increased reactivity of the pulmonary vessels and increased tension in the pulmonary arterioles and pulmonary arteriolar contraction. Some investigators believe that hypoxia directly changes the transmembrane potential of the pulmonary artery smooth muscle cells, leading to calcium ion inflow and decrease of excitation threshold of these cells, which then causes the smooth muscles of the arterioles to contract. In addition, vasoactive substances such as prostaglandin, thromboxane A2, angiotonin, and histamine may serve as regulators or synergists in hypoxic pulmonary vasoconstriction. However, persistent pulmonary hypertension caused by hypoxia may involve many vasoactive substances, the importance of each varying

Hypoxic pulmonary vasoconstriction doubtlessly leads to pulmonary hypertension. As to how the pulmonary hypertension causes HAPE, currently there are three hypotheses. a. Regional maldistribution of blood flow. It has been proposed that when those who are sensitive to hypoxia at high altitudes are exposed to low-oxygen environments, their

excessive increase in the pulmonary arterial pressure is the key link.

**4.1 Excessive increase in the pulmonary arterial pressure** 

at high altitudes can lead to pulmonary hypertension. 1. Pulmonary vasoconstriction caused by hypoxia

pathologic basis of the initiation and development of HAPE.

depending on the specific conditions.

**4. Pathogenesis** 

muscle arterioles contract rigorously and precapillary resistance increases, while their non-muscle arterioles expand under pulmonary hypertension. The sudden increase in the hydrostatic pressure in the afflicted areas, plus other factors, can cause HAPE. As the changes described above occur in some but not all pulmonary arterioles and capillaries, the pathological changes manifested in the HAPE are usually regional, in accordance with the patchy distributions of edema in HAPE observed via X-ray.


The left ventricle is a relatively large, muscular organ that needs to overcome the high pressure and high resistance of systemic circulation to pump blood. At high altitudes, when oxygen shortage is minor, the body can employ a series of adaptive mechanisms on levels ranging from the systemic to the cellular to alleviate the damage to myocardia caused by hypoxia. However, when oxygen shortage is severe (such as may occur in cases of rapid ascent, excessive physical activity, and severe cold), there is no time for the body to set up these adaptive mechanisms of antihypoxia on the cellular level. In this case, hypoxia will cause direct damage to myocardia, especially those on the left side of the heart. Although left heart failure is not the main cause of HAPE, in clinical situations, cardiac agents have proven to be effective to a certain extent. Animal studies have also shown that compensation in heart function can significantly affect the progress of hypoxic pulmonary hypertension.

When the myocardial damage induced by oxygen shortage exceeds a certain degree, the function of the left heart decompensates, causing an increase in the left ventricular end diastolic pressure (LVEDP) and in the left atrial pressure (LAP). This in turn boosts pulmonary hypertension and contributes further to the development of pulmonary edema. Pulmonary edema impedes gaseous exchange and oxygenation, and the resulting decrease in arterial partial pressure of oxygen further aggravates myocardial anoxia and damage. Increases in high altitude pulmonary blood volume cause an increase in the pulmonary venous resistance.

3. Increases in pulmonary circulation blood volume

Increases in pulmonary blood volume are another important factor that can elevate pressure in pulmonary circulation. It has been shown that 48-72 hours after healthy soldiers were airlifted from sea level to 3,658 meters, their pulmonary blood volumes increased by 82%. This is because oxygen shortage acts on the central nervous system and causes increased sympathetic excitability. Increased sympathetic excitability results in the large-scale release of catecholamines. At the same time, due to the increase in resistance in the peripheral vessels, the left ventricular load increases and cardiac output decreases while left atrial pressure increases. Pulmonary venous return is impeded, causing blood to fill the blood vessels of the lungs. The increased blood supply and reduced blood output in the pulmonary circulation increase pulmonary blood volume. The compensatory erythrocytosis and increase in both water and sodium retention caused by hypoxia increase blood volume in general, including pulmonary blood volume, which also leads to pulmonary edema.

#### 4. Formation of micro-thrombi in the pulmonary vessels

The formation of micro-thrombi in the pulmonary circulation is a pathological feature of HAPE. Autopsies of HAPE patients have found extensive blockages by thrombi in the pulmonary capillaries and some in the branches of the pulmonary veins and arteries, demonstrating that the formation of thrombi in the pulmonary vessels is relatively important to the pathogenesis of HAPE. Due to extensive micro-thrombus formation in the pulmonary capillaries and in other organs such as the brain, the liver, the spleen, the kidney, and the intestine, it has been suggested that hypoxic pulmonary hypertension results from abnormal coagulation and general thrombus blockage of the pulmonary capillaries.

As to the mechanism by which micro-thrombi form in the pulmonary vessels, it is generally believed that as the levels of fibrinogen and anti-fibrinoclase released by the liver increase and levels of fibrinoclase activators released by the lung decrease, the resulting abnormal fibrinolysis is an important pathophysiological basis of the formation of micro-thrombi in the pulmonary vessels. Singh et al. proposed that hypoxia could lead to damage to the fibrinoclase system and that this damage might disturb the dynamic balance between the formation and dissolution of fibrin, causing fibrin to build up in the pulmonary vessels and hence cause the formation of micro-thrombi.

Studies have shown that during the initial stages of HAPE, there are substantial increases in the level of platelet factor 3, and in the release of ADP, resulting in decreased platelet mobility. They have also found that, in HAPE patients, the levels of plasma immunoglobulins, including IgG, IgA, and IgM, all increase significantly. IgG and IgM can adhere to the surface of platelets and change their electrophoretic mobility, increasing platelet adhesivity and release of ADP. ADP can promote the utilizing of platelet factor 3, which further speeds up the coagulation process. Imbalances in cellular immune function cause immune complexes to build up. As the immune complexes activate blood coagulation factors, they further aggravate blood clotting in the blood vessels. The aforementioned weakening of the cellular immune function co-occurs with a decrease in the dissolution activity of fibrin. When the dissolution activity of fibrin improves, cellular immune function also recovers. In addition, recent studies have found that the magnesium content of erythrocytes and leukocytes increases significantly in healthy individuals who adapt well to high altitudes, but in HAPE patients it decreases significantly. Magnesium can alleviate coagulation by expanding blood vessels, stabilizing fibrinogen and platelets, and accelerating the dissolution of fibrin.

#### **4.2 Increases in the permeability of the pulmonary capillaries**

It has been reported that when dogs were placed at a simulated altitude of 6,401 meters, the flow of lymph in the right lymphatic duct increased. After inhaling pure oxygen, lymph flow decreased, the lymphatic duct expanded, but there was no sign of blockage. The causes of lymph flow increase included increased pressure and increased permeability resulting from pores opening in the walls of the lymphatic ducts in a low-pressure, low-oxygen environment. When the dogs' arterial oxygen saturation dropped to 75% (corresponding to an altitude of 5,200 meters), the lymph flow began to increase. When the arterial oxygen saturation dropped to 52.5% (corresponding to an altitude of 6,100 meters), the lymph flow increased substantially. Once red blood cells enter the lymph, the capillaries are considered damaged. Schoene collected bronchoalveolar lavage fluid from HAPE patients in a lab at an altitude of 4,400 meters by branchofiberoscope. Component analysis showed elevated

The formation of micro-thrombi in the pulmonary circulation is a pathological feature of HAPE. Autopsies of HAPE patients have found extensive blockages by thrombi in the pulmonary capillaries and some in the branches of the pulmonary veins and arteries, demonstrating that the formation of thrombi in the pulmonary vessels is relatively important to the pathogenesis of HAPE. Due to extensive micro-thrombus formation in the pulmonary capillaries and in other organs such as the brain, the liver, the spleen, the kidney, and the intestine, it has been suggested that hypoxic pulmonary hypertension results from

As to the mechanism by which micro-thrombi form in the pulmonary vessels, it is generally believed that as the levels of fibrinogen and anti-fibrinoclase released by the liver increase and levels of fibrinoclase activators released by the lung decrease, the resulting abnormal fibrinolysis is an important pathophysiological basis of the formation of micro-thrombi in the pulmonary vessels. Singh et al. proposed that hypoxia could lead to damage to the fibrinoclase system and that this damage might disturb the dynamic balance between the formation and dissolution of fibrin, causing fibrin to build up in the pulmonary vessels and

Studies have shown that during the initial stages of HAPE, there are substantial increases in the level of platelet factor 3, and in the release of ADP, resulting in decreased platelet mobility. They have also found that, in HAPE patients, the levels of plasma immunoglobulins, including IgG, IgA, and IgM, all increase significantly. IgG and IgM can adhere to the surface of platelets and change their electrophoretic mobility, increasing platelet adhesivity and release of ADP. ADP can promote the utilizing of platelet factor 3, which further speeds up the coagulation process. Imbalances in cellular immune function cause immune complexes to build up. As the immune complexes activate blood coagulation factors, they further aggravate blood clotting in the blood vessels. The aforementioned weakening of the cellular immune function co-occurs with a decrease in the dissolution activity of fibrin. When the dissolution activity of fibrin improves, cellular immune function also recovers. In addition, recent studies have found that the magnesium content of erythrocytes and leukocytes increases significantly in healthy individuals who adapt well to high altitudes, but in HAPE patients it decreases significantly. Magnesium can alleviate coagulation by expanding blood vessels, stabilizing fibrinogen and platelets, and

It has been reported that when dogs were placed at a simulated altitude of 6,401 meters, the flow of lymph in the right lymphatic duct increased. After inhaling pure oxygen, lymph flow decreased, the lymphatic duct expanded, but there was no sign of blockage. The causes of lymph flow increase included increased pressure and increased permeability resulting from pores opening in the walls of the lymphatic ducts in a low-pressure, low-oxygen environment. When the dogs' arterial oxygen saturation dropped to 75% (corresponding to an altitude of 5,200 meters), the lymph flow began to increase. When the arterial oxygen saturation dropped to 52.5% (corresponding to an altitude of 6,100 meters), the lymph flow increased substantially. Once red blood cells enter the lymph, the capillaries are considered damaged. Schoene collected bronchoalveolar lavage fluid from HAPE patients in a lab at an altitude of 4,400 meters by branchofiberoscope. Component analysis showed elevated

abnormal coagulation and general thrombus blockage of the pulmonary capillaries.

4. Formation of micro-thrombi in the pulmonary vessels

hence cause the formation of micro-thrombi.

accelerating the dissolution of fibrin.

**4.2 Increases in the permeability of the pulmonary capillaries** 

protein content, even higher than that in the edema fluid collected from adult respiratory distress syndrome (ARDS) patients. All these directly demonstrate that HAPE results from the leakage of fluid, protein, and even blood formed elements caused by the increased permeability of the pulmonary capillaries, suggesting that HAPE is a type of protein-rich, high-permeability pulmonary edema. In 1991, West JB et al. successfully simulated the pathophysiological process of HAPE in the laboratory by increasing the pulmonary arterial pressure of laboratory animals. He observed that when the rabbits' pulmonary capillary pressure reached 40 mmHg, the endothelia of the pulmonary capillaries and alveoli and sometimes even all the linings of the alveoli started to rupture.

The mechanisms by which the pulmonary capillary permeability increases may include the following factors.

1. Direct damage to the respiratory membrane structure

According to studies, under emergency conditions involving hypoxia, the endochylema of the pulmonary capillary and alveolar endothelial cells become condensed, causing the cells to shrink, which in turn expands the intercellular space of the alveolar capillary membranes and increases their permeability.

2. Decrease in secretion of alveolar surfactants

The alveolar surfactant is a type of phospholipin secreted by alveolar epithelial type II cells. When the lung tissues experience shortages of oxygen and blood, the normal metabolism of the alveolar epithelial type II cells is disturbed, and the secretion of this substance decreases. In high altitude, secretion of this substance may decrease, causing the permeability of the barrier between the capillaries and lung tissues to increase, leading to HAPE.

3. Increases in levels of acidic metabolites in local tissues

The anaerobic metabolism increases and the acidic metabolites accumulate, causing the physicochemical properties of the adhesion substance between the capillary endothelial cells to change and the basilar membrane to denature, which eventually leads to increased capillary permeability.

4. Respiratory tract infection

Any respiratory tract infection can directly damage the pulmonary capillaries and tissues through inflammatory metabolites and bacterial toxins, increasing their permeability. It is already known that hypoxia can induce inflammatory reactions involving immunocytes and epithelial cells, and therefore it can be deduced that high altitude hypoxia may induce the secretion of inflammatory cytokines, causing HAPE by producing pulmonary extravasation. To understand the relationship between inflammation and the pathogenesis of HAPE, researchers measured the levels of several inflammation mediators under hypoxia, including interleukin-6 (IL-6), interleukin -1 receptor antibody (IL-1ra), and C reactive protein (CRP). The moderate increase of these inflammatory markers reflects the presence of general local inflammation and suggests that inflammation may be involved in the pathogenesis of HAPE. Another study measured continuously the level of NO, a marker of respiratory inflammation, in the expiratory air of human subjects, and found that at 4,359 meters, the 13 subjects who were also HAPE patients among 28 subjects did not show trend of elevated NO levels during the clinical period, found the NO content in the expiratory air of subjects during their stay at high altitudes was 30% lower, indicating that respiratory tract infection does not precede HAPE.

5. Increases in plasma fibrinogen and the decreases in the dissolution activity of fibrin cause fibrin to accumulate in the pulmonary capillaries, forming thrombi that block them. This increases the permeability of the capillary walls.

## **4.3 Impairment of water clearance by the alveolar epithelium**

HAPE results from unbalanced fluid secretion and reabsorption in the alveoli. In particular, the amiloride-sensitive epithelial sodium channel (ENaC) is involved in 40-60% of the reabsorption processes. It has also been found that 2 antagonist, which activates epithelial sodium channels (ENaC), also promotes water clearance in the lungs. In addition, *in vitro* studies have found that hypoxia inhibits Na+/K+-ATPase activity in alveolar epithelial type II cells as well as the co-transportation of Na, K, and Cl. This all suggests that hypoxia impedes the transmembrane re-absorption of water and sodium in epithelial cells.

## **4.4 Increases in sympathetic excitability**

Studies have found that sympathectomy or blockage of cervical sympathetic nerves can eliminate HAPE. This suggests that the pathogenesis of HAPE is clearly related to sympathetic excitability. It has been found that the subjects' sympathetic excitability instantly increased at 3,500 meters, reaching a peak value within 24-72 hours, which is exactly the period of time during which HAPE is most likely to occur.

## **4.5 Individual susceptibility**

Given the same conditions, the incidence of HAPE shows differences along racial, individual, and age lines. A study reported that among the 43 patients with recurrent HAPE, thirty-two developed HAPE 2 times, seven 3 times, three 4 times, and one 6 times. This shows that individual susceptibility is one of the factors for the incidence of HAPE. When comparing HAPE patients to normal subjects, researchers have found that the chest circumferences and chest anteroposterior diameters of acute HAPE patients are both longer than those of controls. In addition, there are more circular muscle fibers on the pulmonary arteriolar walls of the acute HAPE patients. Individual differences in internal factors such as the tissue structure of the pulmonary vessels and immune function status may lead to individual differences in sensitivity, tolerance to hypoxia, and other steps of the pathogenesis of HAPE.

In summary, the pathogenesis of HAPE is complex, involving many steps. Currently wellaccepted factors include the following:


## **5. Pathological changes**

The pathological changes involved in HAPE mainly occur in the lungs. Visual inspections reveal increases in the volume, weight, and surface moisture of the two lungs and that the

HAPE results from unbalanced fluid secretion and reabsorption in the alveoli. In particular, the amiloride-sensitive epithelial sodium channel (ENaC) is involved in 40-60% of the reabsorption processes. It has also been found that 2 antagonist, which activates epithelial sodium channels (ENaC), also promotes water clearance in the lungs. In addition, *in vitro* studies have found that hypoxia inhibits Na+/K+-ATPase activity in alveolar epithelial type II cells as well as the co-transportation of Na, K, and Cl. This all suggests that hypoxia

Studies have found that sympathectomy or blockage of cervical sympathetic nerves can eliminate HAPE. This suggests that the pathogenesis of HAPE is clearly related to sympathetic excitability. It has been found that the subjects' sympathetic excitability instantly increased at 3,500 meters, reaching a peak value within 24-72 hours, which is

Given the same conditions, the incidence of HAPE shows differences along racial, individual, and age lines. A study reported that among the 43 patients with recurrent HAPE, thirty-two developed HAPE 2 times, seven 3 times, three 4 times, and one 6 times. This shows that individual susceptibility is one of the factors for the incidence of HAPE. When comparing HAPE patients to normal subjects, researchers have found that the chest circumferences and chest anteroposterior diameters of acute HAPE patients are both longer than those of controls. In addition, there are more circular muscle fibers on the pulmonary arteriolar walls of the acute HAPE patients. Individual differences in internal factors such as the tissue structure of the pulmonary vessels and immune function status may lead to individual differences in

In summary, the pathogenesis of HAPE is complex, involving many steps. Currently well-

a. Hypoxia directly damages pulmonary capillary endothelial cells and expands or destroys the intercellular space, leading to general damage to the structure of the airblood barrier, which causes blood plasma to leak directly into the alveolar space. b. Hypoxia damages alveolar epithelial type II cells, diminishing their ability to secrete surfactants, leading to a decrease in alveolar surface tension, which causes the fluid and

c. Hypoxia prompts the alveoli to release cytokines such as histamine, serotonin, interleukin-1, interleukin-6, leukotriene E4, tumor necrosis factor, and C-reactive

d. Hypoxia prompts pulmonary arteriole constriction and an increase in vascular resistance, resulting in pulmonary hypertension. At the same time, sympathetic excitability increases and blood is redistributed, leading to an increase in pulmonary blood volume and body fluid retention, which causes capillary hydrostatic pressure to

The pathological changes involved in HAPE mainly occur in the lungs. Visual inspections reveal increases in the volume, weight, and surface moisture of the two lungs and that the

increase and fluid components to leak out, further aggravating HAPE.

impedes the transmembrane re-absorption of water and sodium in epithelial cells.

exactly the period of time during which HAPE is most likely to occur.

sensitivity, tolerance to hypoxia, and other steps of the pathogenesis of HAPE.

proteins in the pulmonary capillaries likely to leak out.

protein, causing capillary permeability to increase.

**4.3 Impairment of water clearance by the alveolar epithelium** 

**4.4 Increases in sympathetic excitability** 

**4.5 Individual susceptibility** 

accepted factors include the following:

**5. Pathological changes** 

peripulmonary membrane has become stretched, rubbery, and dark red in color. When pressed, pink spumous fluid streams to the surface. Under optical microscope, expansion of the pulmonary capillaries is observed, with red blood cells accumulating inside and bleeding around the vessels. The middle layers of the muscular pulmonary arterioles thicken, the elastic layers appear serrated, and the arterioles muscularize. The pulmonary arterioles are expanded and interrupted and capillary thrombi are formed. The bronchioles and alveoli present hyaline membranous edema, and the alveolar ducts become filled with blended protein fluid or cluttered red blood cells. The capillaries in the alveolar septum expand, with blood accumulating inside and forming thrombi. Under electron microscope, unformed edema fluid with low electron concentration is observed inside the pulmonary alveolar space. Red blood cells accumulate, the capillaries in the alveolar septum expand, the intercellular space between endothelial cells enlarges, and the endothelial cells swell. The number of pinocytotic vesicles and some vacuolated were increased. The alveolar epithelial type I and type II cells swell, shedding surface microvilli. The perinuclear space of the alveolar epithelial type II cells expands, and the laminated bodies in their cytoplasts increase in number and show vacuolization.

## **6. Clinical manifestation**

Like those of other types of acute pulmonary edema, the clinical manifestation of HAPE includes dyspnea, cyanopathy, cough, the production of large amounts of white or pink spumous phlegm when coughing, and moist rales in one or both lungs.

#### **6.1 Symptoms**

At the early stage, the most common symptoms of HAPE include severe headache, dyspnea, palpitations, shortness of breath, chest tightness, chest pain, panic, extreme fatigue, weakness, persistent dry cough, worsened nighttime insomnia, pale complexion, and moist, cold skin. As the condition progresses, the above symptoms worsen, and patients may experience severe dyspnea, an inability to lie flat, and coughing out spumous phlegm, which is initially white or faint yellow in color and later turns pink, in some worse cases gushing out of the mouth and nose. Most patients display dysphoria while a minority experience hypersomnia, in some cases accompanied by altitude coma.

#### **6.2 Signs**

One distinctive sign of HAPE is moist pulmonary rales. In severe cases, moist rales can be heard in all regions of both lungs along with wheezing and phlegm. The cardiac sound is often masked. In mild cases, moist rales can be heard at the base of one or both lungs. Most patients display cyanosis in the lips, nail beds, and parts of the face. Due to dyspnea, patients often take bed rest in a semi-reclining position. About two thirds of patients experience fever, usually at 37.5-39oC. If body temperature persists over 38.5oC, it usually indicates complicating upper respiratory tract infection. Signs also include increased heart rate, loud or splitting P2, and grade 2-3 systolic murmur in the apex region of the heart. Some patients exhibit sulcus terminalis cordis expansion, possibly with diastolic gallop, jugular vein distention, hepatosplenomegaly, and edema complicated in some cases by cardiac insufficiency. Compared to other acute pulmonary edemas, HAPE has the following distinctive clinical features:


## **6.3 Diagnostic examination**


## **7. Clinical classification**

Using clinical signs and symptoms and the results of diagnostic examination, HAPE can be classified into three types: mild, moderate, and severe.

a. At early stages, HAPE patients only show symptoms of mild AMS, such as headache, dizziness, palpitations, insomnia, anorexia, and nausea. As these can be early symptoms of HAPE, they must be addressed with caution. Pulmonary auscultation may show normal results, but X-ray examination will reveal typical infiltrated shadows. b. In some patients, HAPE progresses very rapidly, with acute onset and severe symptoms. Patients experience extreme dyspnea, asphyxia, and rales in all lung regions, quickly reaching impending death status. They may exhibit bloody pleural

c. Patients tend to have high blood pressure, fine pulse, tachycardia, loud or splitting P2, and either a mild systolic murmur or diastolic gallop in the apex region of the heart.

d. Some patients mainly show neurological and psychiatric symptoms, which often include headache, vertigo, diplopia, vomiting, phrenitis, and irritation. A minority of patients exhibit symptoms of derangement, meningeal irritation, or even coma. Further examination typically reveals increased pressure in the cerebrospinal fluid and edema in the optic papilla. In these cases, encephaledema often co-occurs, as can be detected

e. At the onset of HAPE, there is usually no fever, but some patients show low fever and

a. Hemogram: The leukocyte count is typically normal or mildly increased; about 40% patients have a count over 10000/mm3. The highest count recorded in our study was 64000/mm3. The neutrophilic granulocyte count also increases mildly. If the leukocyte and neutrophilic granulocyte counts continue to rise, it usually indicates concurrent

b. X-rays: HAPE patients often show unilateral or bilateral flake-like or cloudy shadows centered at the porta pulmonis, mostly in the right lung. A minority of patients show large patchy or butterfly/batwing shadows. The apex regions of the lung are usually clear. At early stages, there is only thickening of the lung markings, also called

c. Electrocardiography: Manifestations of HAPE on the electrocardiogram include nodal tachycardia, right axis deviation, right bundle branch blockage, sharply tented P waves

d. Blood gas: HAPE patients show substantial decreases in levels of PaO2 and SaO2, which are not only lower than those of healthy controls but also significantly lower than those

e. Pulmonary function: HAPE mainly features decreases in the expiratory flow rate or

f. Hemodynamics: The pulmonary arterial pressure and resistance to the pulmonary artery are significantly increased. Left atrial pressure remains normal. The pulmonary

Using clinical signs and symptoms and the results of diagnostic examination, HAPE can be

capillary wedge pressure and cardiac index remain normal or decrease slightly.

or P pulmonale, T wave inversion, and ST segment depression.

fluid in one or both lungs before death.

by encephalic CT or MRI examination.

intolerance to coldness.

pulmonary interstitial edema.

of mild AMS patients.

diffusion capacity.

**7. Clinical classification** 

classified into three types: mild, moderate, and severe.

**6.3 Diagnostic examination** 

infection.

Only a few also experience right heart failure.


## **8. Diagnosis and differential diagnosis**

#### **8.1 Diagnosis and diagnostic criteria**

#### 1. Clinical diagnostic criteria

In China, the clinical diagnosis of HAPE is mostly based on the clinical diagnostic criteria recommended in 1995 by the Chinese Medical Association's third national medicine academic seminar. These criteria have been approved by the International Society for Mountain Medicine. The criteria include the following:


As we gain a deeper knowledge of HAPE and accordingly improve both protective and therapeutic measures, clinically typical HAPE cases have become rare. If we continue to refer to the previous diagnostic criteria, it will be difficult to identify HAPE patients. For this reason, we carried out a study funded by the National Sci-Tech Support Plan and proposed the following as the criteria for early diagnosis of HAPE:


It should be noted that criteria a-c must be met first. Conditions in d-h are then evaluated. The criteria are then combined collectively to produce an accurate early diagnosis. In clinical practice under aforementioned criteria, if we also refer to the severities of the condition and of the signs, the features and sizes of the shadows on the chest film, make proper diagnoses, and give prompt, effective, on-site treatment, it is completely possible that we will be able to keep early-stage HAPE under control.

## **8.2 Differential diagnosis**

1. Adult respiratory distress syndrome


a. Upon recent exposure to high altitudes (usually considered to be 3,000 meters or more above sea level), the at-rest subject experiences palpitations, chest tightening, dyspnea,

b. There is local, unilateral or bilateral coarse breathing with or without focal moist rales. There is also central cyanosis, tachycardia (100 beats/minute), and tachypnea (24

c. Early routine X-ray examination shows a decrease in transmittance of the lungs, blurred or increased lung markings, and the presence of ground-glass opacity or small patchy shadows in the lung. CT scan shows increased number and thickening of lung markings, ground-glass opacity, nodular shadows, scattered or isolated alveolar edema

d. Routine blood examination shows increases in the leukocyte count and the proportion

e. Arterial blood gas examination shows persistent hypoxemia accompanied by mild

f. Electrocardiographic examination shows nodal tachycardia, clockwise rotation, sharply

It should be noted that criteria a-c must be met first. Conditions in d-h are then evaluated. The criteria are then combined collectively to produce an accurate early diagnosis. In clinical practice under aforementioned criteria, if we also refer to the severities of the condition and of the signs, the features and sizes of the shadows on the chest film, make proper diagnoses, and give prompt, effective, on-site treatment, it is completely possible that we will be able to

a. Differences in the cause of disease: The fundamental cause of HAPE is oxygen shortage at high altitudes and low pressure leading to disturbed pulmonary circulation and body fluid maldistribution, of which oxygen shortage is the cause. ARDS is the secondary lesion of the lung tissues directly or indirectly caused by trauma or severe infection, in

b. Differences in pathological changes: Both show high levels of permeability edema. However, HAPE features short duration, fibrosis of the interstitial tissues and alveolar walls, thickening of the interstitium, only mild hyperplasia in the epithelial cells of alveolar walls, and a full recovery without sequelae. ARDS may evolve into subacute and chronic conditions, such as alveolar wall fibrosis. Chronic patients may exhibit pathological changes in general bronchopneumonia, which can eventually lead to prominent fibrosis of the interstitium and the alveoli and the pathological changes of

c. Differences in reaction to oxygen inhalation: After treatments such as oxygen inhalation and measures that decrease pulmonary arterial pressure, most HAPE patients can quickly improve and recover in 2-7 days. Only in rare cases will patients die, usually due to a long delay before treatment, extreme severity of the condition, or complication

g. Ultrasonic cardiogram shows early, prominent, persistent pulmonary hypertension. h. All symptoms quickly improve after treatment such as bed rest, oxygen inhalation,

and coughing with or without small amounts of white spumous phlegm.

on terminal bronchioles, and fine reticular shadows.

decreasing pulmonary arterial pressure, and diuresis.

breaths/minute).

of neutrophilic granulocytes.

tented P waves, and so on.

keep early-stage HAPE under control.

1. Adult respiratory distress syndrome

which oxygen shortage is the consequence.

**8.2 Differential diagnosis** 

emphysema.

respiratory alkalosis.

by adult respiratory distress syndrome. ARDS usually features more severity and a longer duration, often with hemosputum or hemorrhagic sputum and unilateral or focal tubular sound with few moist rales. Oxygen inhalation, even high-pressure oxygen inhalation and assisted respiration, is often not effective. The fatality rate is relatively high, usually between 40% and 70%.

d. Differences in X-ray manifestations: HAPE often shows flake-like or cloudy shadows spreading outward from the porta pulmonis, mostly scattered at the middle and lower fields and rarely fused into large patch. The X-ray film of ARDS patients typically shows patchy shadow at the edges of the lungs, in server cases fused into a large patch. At the terminal stage, "white lung" is manifested, and the pulmonary shadows gradually disappear.

#### 2. High altitude pneumonia

High altitude pneumonia and upper respiratory tract infection can trigger HAPE, and HAPE tends to be complicated by high altitude pneumonia. Therefore, we should carefully differentiate the two in both diagnosis and treatment.


## **9. Complications**

#### **9.1 High Altitude Cerebral Edema**

High altitude cerebral edema (HACE) is a common complication of HAPE, as verified by clinical examinations, lab examinations, and autopsies. Because patients may have one or both of these two diseases, we should take great caution in diagnosis and treatment. HAPE patients often show signs and symptoms such as headache, vomiting, hypersomnia, and coma, which relate to increased intracranial pressure and cerebral edema. Fundus examination sometimes shows papilledema and fundus hemorrhage, and lumbar puncture often shows increased cerebrospinal fluid pressure. Such cases merit special attention during diagnosis and treatment.

Shortly after rapid exposure to a high altitude environment, blood within the body is redistributed via neural, fluid regulation. The vasomotion of the blood vessels in some organs undergo prominent changes resulting in large amounts blood moving to important organs, such as the lung, heart, and brain, ensuring their oxygen supply and normal function. However, if the blood flow volume, rate, and pressure in these organs become too high, disruption to microcirculation may occur. In particular, in the lung and brain, where low-pressure space is normally present, it can easily cause fluid to leak out into neighboring tissues, leading to edema. HAPE and HACE share some common pathogenesis, for the most part in the pathological changes in hemodynamics. HACE and HAPE may occur separately or jointly, sometimes in succession. Severe HAPE and large amounts of extravasation from the alveoli seriously impair oxygen uptake from the external environment, aggravating hypoxemia, which promotes the hemangiectasis of the cerebral blood-vessels via neuroendocrine regulation induced by severe hypoxia of the brain tissue. The resulting increase in cerebral blood flow and blood volume then further aggravates cerebral edema. However, HACE escalates the extravasation of pulmonary tissue and worsens HAPE via neural, fluid regulation. When HACE extends to the respiratory and cardiovascular centers of the medulla oblongata, respiration is inhibited and blood pressure drops, in severe cases leading to cerebral hernia, which can cause respiratory circulation failure or even sudden cardiac arrest,resulting in vicious circle. This is an important cause of death among patients with HAPE complicated by cerebral edema.

#### **9.2 Cerebral infarction**

Cerebral infarction is another common complication of HAPE, possibly induced by the following: 1) Acute erythrocytosis occurs due to excessive erythropoiesis triggered when the body is exposed to altitudes above a certain elevation. The compensatory erythrocytosis in the plasma leads to a significant increase in blood viscosity. Blood flow rate decreases and blood cells cluster together, resulting in increased contact between the platelets and the blood vessel walls, rendering the blood more likely to coagulate. 2) Wade et al. proposed that disturbances in cerebral circulation might play a major role in generating cerebral thrombosis. HAPE complicated with HACE is an important cause of disturbances in cerebral microcirculation, which slows down the blood flow and increases the blood viscosity of the patient, leading to cerebral thrombosis. 3) At high altitudes, the generation of blood coagulation factor in the plasma increases, creating a hypercoagulative condition. The blood of High altitude polycythemia (HAPC) patients is already in a hypercoagulative state, and second hyperfibrinolysis can easily occur. 4) HAPE patients have severe hypoxemia, which may result in damage to blood vessel endothelial cells. The above changes caused severe damage to the pulmonary capillary endothelial cells of HAPE patients, boosting platelet adhesion and activating the blood coagulation system, finally leading to cerebral thrombosis.

both of these two diseases, we should take great caution in diagnosis and treatment. HAPE patients often show signs and symptoms such as headache, vomiting, hypersomnia, and coma, which relate to increased intracranial pressure and cerebral edema. Fundus examination sometimes shows papilledema and fundus hemorrhage, and lumbar puncture often shows increased cerebrospinal fluid pressure. Such cases merit special attention

Shortly after rapid exposure to a high altitude environment, blood within the body is redistributed via neural, fluid regulation. The vasomotion of the blood vessels in some organs undergo prominent changes resulting in large amounts blood moving to important organs, such as the lung, heart, and brain, ensuring their oxygen supply and normal function. However, if the blood flow volume, rate, and pressure in these organs become too high, disruption to microcirculation may occur. In particular, in the lung and brain, where low-pressure space is normally present, it can easily cause fluid to leak out into neighboring tissues, leading to edema. HAPE and HACE share some common pathogenesis, for the most part in the pathological changes in hemodynamics. HACE and HAPE may occur separately or jointly, sometimes in succession. Severe HAPE and large amounts of extravasation from the alveoli seriously impair oxygen uptake from the external environment, aggravating hypoxemia, which promotes the hemangiectasis of the cerebral blood-vessels via neuroendocrine regulation induced by severe hypoxia of the brain tissue. The resulting increase in cerebral blood flow and blood volume then further aggravates cerebral edema. However, HACE escalates the extravasation of pulmonary tissue and worsens HAPE via neural, fluid regulation. When HACE extends to the respiratory and cardiovascular centers of the medulla oblongata, respiration is inhibited and blood pressure drops, in severe cases leading to cerebral hernia, which can cause respiratory circulation failure or even sudden cardiac arrest,resulting in vicious circle. This is an important cause of death among

Cerebral infarction is another common complication of HAPE, possibly induced by the following: 1) Acute erythrocytosis occurs due to excessive erythropoiesis triggered when the body is exposed to altitudes above a certain elevation. The compensatory erythrocytosis in the plasma leads to a significant increase in blood viscosity. Blood flow rate decreases and blood cells cluster together, resulting in increased contact between the platelets and the blood vessel walls, rendering the blood more likely to coagulate. 2) Wade et al. proposed that disturbances in cerebral circulation might play a major role in generating cerebral thrombosis. HAPE complicated with HACE is an important cause of disturbances in cerebral microcirculation, which slows down the blood flow and increases the blood viscosity of the patient, leading to cerebral thrombosis. 3) At high altitudes, the generation of blood coagulation factor in the plasma increases, creating a hypercoagulative condition. The blood of High altitude polycythemia (HAPC) patients is already in a hypercoagulative state, and second hyperfibrinolysis can easily occur. 4) HAPE patients have severe hypoxemia, which may result in damage to blood vessel endothelial cells. The above changes caused severe damage to the pulmonary capillary endothelial cells of HAPE patients, boosting platelet adhesion and activating the blood coagulation system, finally

during diagnosis and treatment.

patients with HAPE complicated by cerebral edema.

**9.2 Cerebral infarction** 

leading to cerebral thrombosis.

## **9.3 Multiple Organ Dysfunction Syndrome**

Patients with HAPE complicated by cerebral edema are prone to multiple organ dysfunction syndrome (MODS). Patients show symptoms including headache, chest tightness, shortness of breath, nausea, aggravated vomiting, and abnormal psychological behavior. Auscultation can reveal aggravated pulmonary rales; fundus examination may show spotting or patchy bleeding in the retina and papilledema; there is gastrointestinal hemorrhage or fecal occult blood, hematuria, or proteinuria; chest X ray of most patients show enlarged hilar shadows, unilateral or bilateral cloudy shadows of uniform density in the lung field, in some cases fused into large, dense, patchy unilateral or bilateral shadows of uniform density; encephalic CT reveals decreased brain parenchymal density, narrowed bilateral cerebral ventricles, and the shallower, narrower sulci; laboratory examination will reveal increased leukocyte counts, often above 13.0×109/L, increased bleeding and clotting time, increased fibrinolytic activity, and increases in thromboxane B2, vWF, fibrinogen, tissue-type plasminogen activator and inhibitor in the plasma, increased levels of D-dimers, increased levels of alpha-granular membrane protein, significantly decreased levels of 6-keto-PGF1a and antithrombin III, and severe dysfunction of the coagulation and fibrolysis systems.

AMS complicated by MODS has been underemphasized and the diagnostic yield has been low. Our investigation shows that 2.5% of AMS cases are complicated by MODS, which is considerable. We need to improve early diagnosis and early detection because early treatment is crucial in reducing the fatality rate of AMS complicated by MODS.

## **10. Prevention and treatment**

#### **10.1 Prevention**

1. Protection of susceptible populations from exposure to high altitudes

Physical examinations, especially inspections of cardiac and pulmonary functions, should be performed on individuals who are about to travel to high altitude areas. Those with heart and/or lung ailments should be advised against exposure to high altitudes.

2. Prevention of respiratory tract infection

Individuals with respiratory tract infections are more susceptible to HAPE at high altitudes. Those who catch upper respiratory tract infections before planned trips to high altitude areas should first seek treatment and only make the trip after full recovery. Prior to high altitude exposure, one should perform cold resistance exercises. After arrival, one should take active measures to keep warm and prevent respiratory tract infection.

#### 3. Acclimatization to hypoxia

Prior to high altitude exposure, one should receive hypoxic training using masks or hypoxic respirators to increase the body's tolerance to hypoxia so as to promote high altitude acclimatization.

When possible, one should ascend to higher altitudes gradually and multisteply rather than rapidly ascend to higher altitudes to avoid body damage. Before traveling to high altitude regions, individuals should familiarize themselves with the climate characteristics and geological environment of the area and familiarize themselves with preventative treatments for high altitude diseases.

4. Reduction and control of activity level

During the first week of high-altitude exposure, one should take proper rest, reduce or avoid intense physical activity, avoid fatigue, and minimize oxygen consumption. Normal physical activity should be resumed only after the body is acclimatized to the hypoxic environment.


There are four courses of Chinese traditional medicines that may serve to prevent HAPE:

Compound codonopsis tablets: Take orally. Start 3 days before hypoxia exposure, 3-5 tablets/dose, 3 doses each day. Continue for 5-7 days after hypoxia exposure.

Ginseng and astragalus pollen tablets: Take orally. Start 3 days before high altitude exposure, 5 tablets/dose, 3 doses/day. Continue for 5-7 days after high altitude exposure.

Rhodiola rosea oral solution: Take orally. Start 3 days before high altitude exposure, 10 ml/dose, 3 doses/day. Continue for 5-7 days after high altitude exposure.

Compound rhodiola capsules: Take orally. Start 3 days before high altitude exposure, 2 capsules/dose, 3 doses/day, continue for 5-7 days after high altitude exposure.


#### **10.2 Treatment**

Accurate, effective early-stage treatment usually quickly improves symptoms. Therefore, early diagnosis and timely treatment are crucial to controlling the course of the disease and prognosis.

1. Oxygen inhalation or hyperbaric oxygen therapy

Oxygen inhalation can substantially decrease pulmonary arterial pressure in HAPE patients and quickly alleviate hypoxia and the series of clinical symptoms that it causes. HAPE

During the first week of high-altitude exposure, one should take proper rest, reduce or avoid intense physical activity, avoid fatigue, and minimize oxygen consumption. Normal physical activity should be resumed only after the body is acclimatized to the hypoxic

There are four courses of Chinese traditional medicines that may serve to prevent

Compound codonopsis tablets: Take orally. Start 3 days before hypoxia exposure, 3-5

Ginseng and astragalus pollen tablets: Take orally. Start 3 days before high altitude exposure, 5 tablets/dose, 3 doses/day. Continue for 5-7 days after high altitude

Rhodiola rosea oral solution: Take orally. Start 3 days before high altitude exposure, 10

Compound rhodiola capsules: Take orally. Start 3 days before high altitude exposure, 2

Dexamethasone: Take orally. Start 1 day before high altitude exposure, 5 mg/dose, 3

Nephramid (a.k.a. acetazolamide): Take orally. Start 1 day before high altitude

Methazolamide (a.k.a. Ni Mu Ke Si): Take orally. Start 1 day before high altitude

Nifedipine: Take orally. Start 1 day before high altitude exposure, 10 mg/dose, 2 doses/day or sublingual administration 10 mg/dose, 3 doses/day. Take for 1-3 days.

Salbutamol: Inhale. Start 1 day before high altitude exposure, 125 g/dose, 2

Sildenafil: Take orally. Start 1 day before high altitude exposure, 50 mg/dose, 3

Tadalafil: Take orally. Start 1 day before high altitude exposure, 10 mg/dose, 1

Accurate, effective early-stage treatment usually quickly improves symptoms. Therefore, early diagnosis and timely treatment are crucial to controlling the course of the disease and

Oxygen inhalation can substantially decrease pulmonary arterial pressure in HAPE patients and quickly alleviate hypoxia and the series of clinical symptoms that it causes. HAPE

tablets/dose, 3 doses each day. Continue for 5-7 days after hypoxia exposure.

ml/dose, 3 doses/day. Continue for 5-7 days after high altitude exposure.

doses/day. Continue for 2 days after high altitude exposure.

exposure, 250 mg/dose, 3 doses/day. Take for 2-3 days.

exposure, 25-50 mg/dose, 3 doses/day. Take for 2-3 days.

doses/day. Continue for 2-3 days after high altitude exposure.

doses/day. Continue for 2-3 days after high altitude exposure.

dose/day. Continue for 2-3 days after high altitude exposure.

1. Oxygen inhalation or hyperbaric oxygen therapy

capsules/dose, 3 doses/day, continue for 5-7 days after high altitude exposure.

4. Reduction and control of activity level

5. Administration of prophylactic medicine

a. Chinese traditional medicine

b. Glucocorticoid preparations

c. Carbonic anhydrase inhibitors

d. Calcium antagonists

**10.2 Treatment** 

prognosis.

e. Beta2- receptor stimulants

f. Phosphodiesterase inhibitors

environment.

HAPE:

exposure.

patients should in general use continuous administration of low-flow oxygen (4-6 L/minute). For patients with severe hypoxia, high-flow continuous oxygen may be administrated (8-10 L/minute) but for no longer than 24 hours in order to avoid oxygen toxicity. If the symptoms include excessive spumous phlegm, an appropriate amount of alcohol may be added to the oxygen humidifying containers for froth suppression.

Hyperbaric oxygen treatment can temporarily remove hypoxia for HAPE patients. Most patients show symptom improvement after 1-2 treatments and achieve recovery after 2-3 treatments. However, for a minority of patients, HAPE signs and symptoms worsen after departure from the hyperbaric oxygen chamber, which can be associated with disease severity variance and individual difference. Therefore, when treating HAPE patients with hyperbaric oxygen therapy, caution should be taken to acknowledge individual variability and monitor the severity of the condition.

2. Nitric oxide inhalation

Inhalation of low-concentration nitric oxide can quickly, selectively alleviate the pulmonary hypertension caused by hypoxia. The inhalation method is as follows: Mix 10 ppm nitric oxide with pure air and inhale through a nasogastric feeding tube at 3-5 L/minute, 30-60 minutes/treatment and 2-3 treatments/day. Patients with mild or moderate HAPE typically recover after 2-3 days. For severe HAPE patients, the duration and daily frequency of the inhalation treatments should be increased accordingly.

Inhaled nitric oxide can be oxidized into NO2- and NO3- in high-oxygen environments and can accumulate in the blood, which will damage the blood cells. For this reason, when treating HAPE patients with nitric oxide, simultaneous inhalation of high concentration oxygen should be avoided. Animal studies have shown that the effects of inhaling concentrations of nitric oxide ranging from 5-80 ppm on decreasing pulmonary arterial pressure are statistically the same.

#### 3. Aminophylline

Aminophylline is the drug of choice for standard HAPE treatment. It can quickly diminish pulmonary arterial and vena cava pressure and decrease right atrial venous return volume. It can also be cardiotonic, diuretic and a smooth muscle relaxant and can reduce resistance in the systemic circulation, improving the heart function.

Regular dose: 0.25g diluted to 20ml 10-50% glucose, intravenously injected at an even speed. It can be repeated after 4-6 hours. For mild HAPE patients, administer 2 times/day. For severe patients, upgrade to 0.5g/administration, and increase the frequency of administration according to the severity.

#### 4. Anticholinergic agents

Atropine and anisodamine can treat pulmonary vasospasms. They also decrease resistance in the pulmonary blood vessels, improve pulmonary microcirculation, keep pulmonary blood flow unimpeded, and prevent blood clotting and pulmonary thrombosis inside the blood vessels.

Regular dose: Atropine 2-5 mg/0.5hour. Anisodamine (654-2) 20-40 mg/0.5 hour, intravenous drip.

#### 5. Dexamethasone

Dexamethasone can be used in both treating and preventing HAPE. Regular dose: 10 mg, IV injection, 2 times/day for no more than 3 days. For patients with comorbid conditions such as epilepsy, peptic ulcer, high blood pressure, or diabetes mellitus, dexamethasone should be taken with caution or contraindicated.

6. Diuretics

Nicorol: 10 mg IV injection, 1 dose/day; Nephramid: Take orally.250 mg/dose, 3 doses/day; Drugs that alleviate pulmonary hypertension: nifedipine: 10-20 mg/dose orally or sublingually, 2 doses/day. Sodium nitroprusside: IV drip, 10-20 mg/dose.

#### 7. Cardiotonics

Cedilanid: IV injection, 0.4-0.8 mg. Strophanthin K: IV injection, 0.125-0.5 mg.

8. Sedatives

Morphine hydrochloride: 5-10 mg, subcutaneous injection. In severe cases, dilute 5 mg to 20 ml 10% glucose and administer IV injection. For some patients with anxiety symptoms, Valium may be used with caution, but its inhibitory effects on breathing should be monitored.

9. Antibiotics

HAPE is very likely to be complicated by pulmonary infection. When the two diseases cooccur, each aggravates the other and the situation becomes difficult to control. In treating HAPE, broad-spectrum antibiotics are usually used to prevent and treat infection. Patients with mild symptoms should take broad-spectrum antibiotics (e.g. amoxicillin, norfloxacin, trimethoprim and sulphame-thoxazole etc.) orally for anti-infection purposes, and control the intake of sodium chloride to avoid worsening the HAPE. The commonly used antibiotics include the following:


#### 10. Decent to lower altitude

When possible, patients should be quickly transferred to lower altitudes (below 3,000 meters) for further treatment. After leaving the hypoxic environment, the elevated pulmonary arterial pressure can quickly return to normal levels and the series of symptoms caused by hypoxia quickly disappear. However, descent treatment is only applicable in less remote areas within a relatively short amount of time. In remote mountain areas where transportation conditions are extremely poor and continuous oxygen supplies cannot be guaranteed in transit, it is for the best to administer on-site treatment.

as epilepsy, peptic ulcer, high blood pressure, or diabetes mellitus, dexamethasone should

Nicorol: 10 mg IV injection, 1 dose/day; Nephramid: Take orally.250 mg/dose, 3 doses/day; Drugs that alleviate pulmonary hypertension: nifedipine: 10-20 mg/dose orally

Morphine hydrochloride: 5-10 mg, subcutaneous injection. In severe cases, dilute 5 mg to 20 ml 10% glucose and administer IV injection. For some patients with anxiety symptoms, Valium

HAPE is very likely to be complicated by pulmonary infection. When the two diseases cooccur, each aggravates the other and the situation becomes difficult to control. In treating HAPE, broad-spectrum antibiotics are usually used to prevent and treat infection. Patients with mild symptoms should take broad-spectrum antibiotics (e.g. amoxicillin, norfloxacin, trimethoprim and sulphame-thoxazole etc.) orally for anti-infection purposes, and control the intake of sodium chloride to avoid worsening the HAPE. The commonly used antibiotics

c. Trimethoprim and sulphame-thoxazole: Take orally, 1-2 tablets/dose, 2 doses/day, take

e. Penicillin (still the first choice): 4,800,000-6,400,000 units diluted in 250-500 ml glucose or saline, IV drip, 1-2 times/day. Contraindicated in patients clinically significant

f. Ampicillin sodium/sulbactam sodium: For mild infections, 1.5g/day in 2-3 IM injections. For moderate infections, 4-9g/day in 3-4 IM injections. For severe infections

When possible, patients should be quickly transferred to lower altitudes (below 3,000 meters) for further treatment. After leaving the hypoxic environment, the elevated pulmonary arterial pressure can quickly return to normal levels and the series of symptoms caused by hypoxia quickly disappear. However, descent treatment is only applicable in less remote areas within a relatively short amount of time. In remote mountain areas where transportation conditions are extremely poor and continuous oxygen supplies cannot be

may be used with caution, but its inhibitory effects on breathing should be monitored.

or sublingually, 2 doses/day. Sodium nitroprusside: IV drip, 10-20 mg/dose.

Cedilanid: IV injection, 0.4-0.8 mg. Strophanthin K: IV injection, 0.125-0.5 mg.

be taken with caution or contraindicated.

6. Diuretics

7. Cardiotonics

8. Sedatives

9. Antibiotics

include the following:

allergy to penicillin.

10. Decent to lower altitude

9-12g/day in 2-3 IV drips.

g. Cefamezin: 2g/dose, 3 doses/day, IV drip. h. Cefradine: 100-150 mg/kg/day, IV drip.

orally.

a. Amoxicillin: Take orally, 1g/dose, 3 doses/day. b. Norfloxacin: Take orally, 0.2g/dose, 3 doses/day.

d. Levofloxacin: Take orally, 0.1-0.2 g/dose, 2 doses/day.

i. Lincomycin hydrochloride: 0.6g/dose, 1-2 doses/day, IV drip. j. Ciprofloxacin Lactate: 0.2g/dose, 2 doses/day, IV drip.

guaranteed in transit, it is for the best to administer on-site treatment.

When transferring patients to lower altitudes, the following should be noted: 1) Transportation utilities: fast, stable transportation utilities are preferable, e.g. helicopter, truck, heavy medical vehicle, small ambulance. 2) Accompanying crew: there should be one doctor and one nurse, or at least one medical professional who can perform effective treatment through the transfer. 3) The patient in should assume a semireclining position during descent to keep the airway clear. 4) Patients with high altitude coma should assume a semireclining position. Head movement and vehicle pitching should be minimized to prevent cerebral hernia. 5) The driver should proceed slowly when road conditions are rough so that the patient's position can remain relatively stable. 6) The accompanying crew should read and record vital signs and give effective treatment when needed. 7) If a patient passes away, the time and order of vital sign loss should be recorded and body position should respectfully be kept unchanged.

#### **11. References**


## **Mechanical Forces Impair Alveolar Ion Transport Processes – A Putative Mechanism Contributing to the Formation of Pulmonary Edema**

Martin Fronius *Institute of Animal Physiology, Justus-Liebig-University Giessen Germany* 

## **1. Introduction**

560 Lung Diseases – Selected State of the Art Reviews

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a patient managed successfully with non-invasive positive pressure ventilation in

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Out of 5000 pilgrims, 228 were randomly chosen. Sixty-eight percent had AMS,

The aim of this chapter is to highlight the importance of transepithelial ion transport processes for lung function in general and to focus on the impact of mechanical forces on pulmonary ion transport in particular. Linking mechanical forces with pulmonary ion transport derives from the fact that the lung is a dynamic organ as well as from several studies providing evidence that the amount of mechanical forces as used during artificial ventilation correlates with mortality rates in patients with respiratory failure such as ALI (acute lung injury) and ARDS (acute respiratory distress syndrome) (ARDS Network Investigators, 2000). In these patients the formation of pulmonary edema is a characteristic symptom (Frank and Matthay, 2003; Ricard et al., 2003) and the basic rationale behind this is, that mechanical perturbations cause epithelial leakage in response to mechanically induced damage of the epithelial layer. This damage is suggested to be a major cause for the formation of pulmonary edema as well as the inability to reabsorb the edema fluid. However, little is known whether or not mechanical forces may directly interfere with pulmonary ion transport processes and this represents a putative mechanism that facilitates the formation of pulmonary edema – in addition to damages of the epithelial layer.

## **2. Air breathing and pulmonary ion transport**

The water land transition of tetrapods represents a fundamental process within vertebrate evolution that was accompanied by the development of lungs as gas exchanging structures. The major advantage of air breathing is the almost infinite access to oxygen, although this bears also some risks. The gas-exchanging structures must be moistened to facilitate oxygen solubility and the architecture of these structures must be adapted to the requirement of efficient gas exchange by diffusion. These problems were basically fixed by invagination of the gas-exchanging surface, protecting them from desiccation and from mechanical damage. Therefore, invagination could be considered as a basic improvement allowing the development of a highly conserved architecture of the air blood barrier that can be found within all air-breathing vertebrates. This architecture is referred to as the "three ply design" (Maina and West, 2005). The three layers of the air-blood barrier are represented by the pulmonary epithelium, the basal lamina and the endothelium that forms the pulmonary capillaries. Although invagination and the development of the three ply design fixed a lot of problems, other challenges arose. To ensure a constant supply of oxygen as well as a continuous replacement of the breathing medium, ventilation mechanisms were needed. This was achieved by different strategies within the different vertebrate classes. Early tetrapods and amphibians ventilate their lungs by a buccal pump (Brainerd and Owerkowicz, 2006). In higher vertebrates ventilation occurs by a costal pump (Roux, 2002) where the movement of the ribs ensures aspiration of air. But independent of the strategy how the exchange of the breathing medium is accomplished, the gas-exchanging surface is permanently exposed to pollutions and pathogens that are omnipresent in the air supplied to the lungs.

It is also well known that the entire pulmonary epithelium is covered by a thin fluid layer (PLL: pulmonary liquid layer) (Daniels and Orgeig, 2003), that is of particular importance for the function of the lung. The PLL extends from the distal parts of the lung to the upper airways and consists of mucus, surfactant and periciliary fluid (Rubin, 2002) (Fig. 1). On the one hand the PLL is the first physical border that is exposed to the environment and the first line of host defense that absorbs pathogens. This is of particular importance in the airways, where the PLL is part of the innate immune system and crucial for protecting the host from the permanent exposure to pollutions and pathogens due to their removal by the mucociliary clearance (Welsh, 1987; Davis and Lazarowski, 2008).

Fig. 1. Schematic drawing of the distal lung region. The distal lung is represented by respiratory bronchioles and terminal alveoli that are lined by a continuous layer of epithelial cells consisting of different cell types. The entire epithelium is further covered with a liquid film (PLL: pulmonary liquid layer) consisting of different layers. In the airways the PLL is composed from a liquid layer (periciliary fluid) and a layer of mucus and surfactant. In the alveolar regions the PLL consists of a liquid layer (alveolar fluid) and a surfactant layer.

pulmonary epithelium, the basal lamina and the endothelium that forms the pulmonary capillaries. Although invagination and the development of the three ply design fixed a lot of problems, other challenges arose. To ensure a constant supply of oxygen as well as a continuous replacement of the breathing medium, ventilation mechanisms were needed. This was achieved by different strategies within the different vertebrate classes. Early tetrapods and amphibians ventilate their lungs by a buccal pump (Brainerd and Owerkowicz, 2006). In higher vertebrates ventilation occurs by a costal pump (Roux, 2002) where the movement of the ribs ensures aspiration of air. But independent of the strategy how the exchange of the breathing medium is accomplished, the gas-exchanging surface is permanently exposed to

It is also well known that the entire pulmonary epithelium is covered by a thin fluid layer (PLL: pulmonary liquid layer) (Daniels and Orgeig, 2003), that is of particular importance for the function of the lung. The PLL extends from the distal parts of the lung to the upper airways and consists of mucus, surfactant and periciliary fluid (Rubin, 2002) (Fig. 1). On the one hand the PLL is the first physical border that is exposed to the environment and the first line of host defense that absorbs pathogens. This is of particular importance in the airways, where the PLL is part of the innate immune system and crucial for protecting the host from the permanent exposure to pollutions and pathogens due to their removal by the

bronchiole

alveolar type I cell

red blood cell

alveolar type II cell

liquid surfactant mucus

pollutions and pathogens that are omnipresent in the air supplied to the lungs.

mucociliary clearance (Welsh, 1987; Davis and Lazarowski, 2008).

alveolar airspace

Fig. 1. Schematic drawing of the distal lung region. The distal lung is represented by

respiratory bronchioles and terminal alveoli that are lined by a continuous layer of epithelial cells consisting of different cell types. The entire epithelium is further covered with a liquid film (PLL: pulmonary liquid layer) consisting of different layers. In the airways the PLL is composed from a liquid layer (periciliary fluid) and a layer of mucus and surfactant. In the alveolar regions the PLL consists of a liquid layer (alveolar fluid) and a surfactant layer.

airway epithelial cells

pulmonary capillary

On the other hand the height and volume of the PLL in the respiratory zone – primarily represented by the alveolar fluid – determines the distance of diffusion for the gases that is a limiting rate for the diffusion efficiency as defined by Fick's law of diffusion. Thus, increased fluid content in the alveolar region decreases oxygenation of the blood (Matalon and O'Brodovich, 1999; Matthay et al., 2000).

Therefore, the consistency as well as the volume of the PLL layer must be tightly regulated and controlled – in the airways as well as in the gas-exchanging region – to ensure effective host defense as well as effective gas exchange.

This principle becomes evident regarding pulmonary diseases that are associated with inappropriate fluid balance in the lung. For example hyperabsorption of water from the airspace increases the viscosity of the PLL and this interferes with the removal of inhaled pathogens due to impaired mucociliary clearance as observed in patients with cystic fibrosis (Fig. 2A) (Widdicombe et al., 1985; Riordan et al., 1989; Matsui et al., 1998). In contrast, too much fluid in the lung impairs gas diffusion and this can be observed in patients with pulmonary edema (Figure 2B) (Sznajder, 2001; Hoschele and Mairbaurl, 2003).

Thus, a defined content of water covering the pulmonary epithelia is a basic requirement for proper lung function and this depends on the development of appropriate ion transport processes accomplished by the pulmonary epithelia.

Fig. 2. Impaired epithelial fluid transport affects basic lung functions. A) Increased water reabsorption in the airways results in dehydration of the periciliary liquid layer (PCL) leading to an impaired mucociliary clearance and mucus accumulation within the airways. B) In the alveolar region an imbalance between fluid reabsorption and fluid infiltration leads to the formation of pulmonary edema that impairs the exchange of the breathing gases.

#### **2.1 Transepithelial Na<sup>+</sup> and Cl– transport are the main pathways to control the water content in the lung**

Studying ion transport processes across pulmonary epithelia within the last decades improved our understanding how the fluid content in the lung is sustained. The basic principle of pulmonary water transport is ubiquitous – ions are transported across the epithelial layer and this generates transepithelial osmotic gradients that cause water diffusion across the epithelium (Sackin and Boulpaep, 1975). It is well accepted that pulmonary epithelia are Na+ reabsorptive epithelia where active Na+ reabsorption represents the bulk of transepithelial ion transport. Na+ transport occurs via two steps: 1) Na+ ions are taken up through epithelial Na+ channels (ENaC) at the luminal side of the epithelial cells and are pumped out from the cells at the basolateral side by the Na+/K+ATPase. This process is suggested to be the principle mechanism for water reabsorption from the airspace into the body (Matthay et al., 2002). Another major component with significant impact on transepithelial water movement is represented by the transepithelial transport of Cl–. In the airways it is well accepted that Cl– is secreted via luminal Cl– channels (Smith et al., 1982; Willumsen et al., 1989; Chambers et al., 2007). However the particular role of Cl– channels Cl– in alveolar epithelial cells remains unclear since there is evidence that Cl– is secreted (McCray et al., 1993; Tizzano et al., 1994; Lazrak et al., 2002; Sommer et al., 2007), as well as absorbed (Fang et al., 2002; Fang et al., 2006). There are at least two different Cl– channels identified in the apical membrane of pulmonary epithelial cells – the Ca2+ dependent Cl– channel (TMEM16a) (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008) and the cAMP dependent CFTR Cl– channel (Riordan et al., 1989; Welsh and Smith, 1993). In addition it might be noted that alveolar epithelial cells are characterized by a high water permeability (Folkesson et al., 1994; Dobbs et al., 1998) although the role of these proteins in pulmonary fluid handling is uncertain since no significant impact of aquaporins on alveolar clearance has been detected in transgenic (aquaporin deficient) animals (Verkman et al., 2000; Verkman, 2007).

However, the crucial importance of defined and regulated ion transport processes in the lung to control the water content is beyond dispute, since this was impressively confirmed by several studies using transgenic animal models with impaired ion channel functions. For example deletion of the ENaC subunit in mice leads to early death due to the inability to reabsorb the alveolar fluid from the lungs after birth (Hummler et al., 1996). Interestingly, rescuing ENaC expression in transgenic mice that were derived from ENaC deficient mice demonstrated that these animals expressed decreased levels of ENaC mRNA and that this resulted in an increased susceptibility to the formation of pulmonary edema (Olivier et al., 2002). In addition overexpression of the �ENaC subunit and hyperabsorption of Na+ is associated with impaired mucociliary clearance resulting in a phenotype that is characteristic for cystic fibrosis (Mall et al., 2004). Recent studies established that mutations in the CFTR gene of pigs resulted in a cystic fibrosis like lung disease (Rogers et al., 2008; Stoltz et al., 2010).

#### **3. Mechanical forces and breathing**

Ventilation of the lungs due to the movement of the chest is associated with the appearance of physical forces. These forces are pressure (force per area), strain (deformation e.g. reasoned by the impact of pressure) and shear stress (movement of fluid at the cellular surface) (Fig. 3) (Wirtz and Dobbs, 2000). It might also be considered that due to the complex anatomy of the gas exchanging area it is difficult to estimate the distinct forces acting on individual cells (Liu et al., 1999) and that the local appearance of forces can be influenced by parameters like surfactants, focal adhesion molecules, the contractile machinery of the cells as well as the activity of molecular motor proteins within the cells (Fredberg and Kamm, 2006).

Although more or less all cell types within the lung are exposed to these physical stimuli the following section will focus on epithelial cells. Especially, the appearance and the reason for the stimuli as well as their impact on the pulmonary epithelial cells will be discussed.

pulmonary epithelia are Na+ reabsorptive epithelia where active Na+ reabsorption represents the bulk of transepithelial ion transport. Na+ transport occurs via two steps: 1) Na+ ions are taken up through epithelial Na+ channels (ENaC) at the luminal side of the epithelial cells and are pumped out from the cells at the basolateral side by the Na+/K+ATPase. This process is suggested to be the principle mechanism for water reabsorption from the airspace into the body (Matthay et al., 2002). Another major component with significant impact on transepithelial water movement is represented by the transepithelial transport of Cl–. In the airways it is well accepted that Cl– is secreted via luminal Cl– channels (Smith et al., 1982; Willumsen et al., 1989; Chambers et al., 2007). However the particular role of Cl– channels Cl– in alveolar epithelial cells remains unclear since there is evidence that Cl– is secreted (McCray et al., 1993; Tizzano et al., 1994; Lazrak et al., 2002; Sommer et al., 2007), as well as absorbed (Fang et al., 2002; Fang et al., 2006). There are at least two different Cl– channels identified in the apical membrane of pulmonary epithelial cells – the Ca2+ dependent Cl– channel (TMEM16a) (Caputo et al., 2008; Schroeder et al., 2008; Yang et al., 2008) and the cAMP dependent CFTR Cl– channel (Riordan et al., 1989; Welsh and Smith, 1993). In addition it might be noted that alveolar epithelial cells are characterized by a high water permeability (Folkesson et al., 1994; Dobbs et al., 1998) although the role of these proteins in pulmonary fluid handling is uncertain since no significant impact of aquaporins on alveolar clearance has been detected in transgenic

However, the crucial importance of defined and regulated ion transport processes in the lung to control the water content is beyond dispute, since this was impressively confirmed by several studies using transgenic animal models with impaired ion channel functions. For example deletion of the ENaC subunit in mice leads to early death due to the inability to reabsorb the alveolar fluid from the lungs after birth (Hummler et al., 1996). Interestingly, rescuing ENaC expression in transgenic mice that were derived from ENaC deficient mice demonstrated that these animals expressed decreased levels of ENaC mRNA and that this resulted in an increased susceptibility to the formation of pulmonary edema (Olivier et al., 2002). In addition overexpression of the �ENaC subunit and hyperabsorption of Na+ is associated with impaired mucociliary clearance resulting in a phenotype that is characteristic for cystic fibrosis (Mall et al., 2004). Recent studies established that mutations in the CFTR gene of pigs resulted in a cystic fibrosis like lung disease (Rogers et al., 2008; Stoltz et al., 2010).

Ventilation of the lungs due to the movement of the chest is associated with the appearance of physical forces. These forces are pressure (force per area), strain (deformation e.g. reasoned by the impact of pressure) and shear stress (movement of fluid at the cellular surface) (Fig. 3) (Wirtz and Dobbs, 2000). It might also be considered that due to the complex anatomy of the gas exchanging area it is difficult to estimate the distinct forces acting on individual cells (Liu et al., 1999) and that the local appearance of forces can be influenced by parameters like surfactants, focal adhesion molecules, the contractile machinery of the cells as well as the activity of molecular motor proteins within the cells

Although more or less all cell types within the lung are exposed to these physical stimuli the following section will focus on epithelial cells. Especially, the appearance and the reason for the stimuli as well as their impact on the pulmonary epithelial cells will be discussed.

(aquaporin deficient) animals (Verkman et al., 2000; Verkman, 2007).

**3. Mechanical forces and breathing** 

(Fredberg and Kamm, 2006).

Fig. 3. Breathing is associated with the appearance of physical forces. Airway epithelial cells are primarily exposed to shear forces that are reasoned by the airstream flowing passing the surface of the airways. In the alveolar region the main physical force is strain due to the positive and negative pressure that are reasoned by the movement of the chest.

#### **3.1 Pressure and strain in the lung**

The appearance of pressure is the consequence of the movement of the chest. During inspiration and expiration epithelial cells are exposed to negative and positive pressures. This pressure is defined as transpulmonary pressure and resembles the pressure difference between the pressure in the pleural space and the atmospheric pressure (Fredberg and Kamm, 2006). During inspiration at rest the inflating pressure is approx. 5 cm H2O and this can increase to approx. 30 cm H2O at deep inspirations (Fredberg and Kamm, 2006). In the lung the appearance of pressure will always cause deformation of the cells due to the delicate anatomy and morphology of the alveolar structure. Therefore, increasing pressure is always associated with the appearance of strain. From this point of view, it seems obvious to consider that strain rather than pressure is the adequate stimulus acting on epithelial cells as a consequence of the breathing movements (Liu et al., 1999).

#### **3.2 Shear stress in the lung**

In addition to strain, the luminal surface of the epithelial cells is exposed to shear stress. Shear stress is defined by the tangential movement of particles (e.g. air or fluid) at the surface between different physical compartments. In the airways shear stress is primarily caused by the oscillating airflow passing the surface of the airway epithelial cells (Tarran et al., 2006). In the distal lung regions shear stress is a consequence of the movement due to distention, where the epithelial cell represents one compartment that is distended and the fluid of the PLL represents a static component of another compartment. Thus a relative movement of the epithelial cell (with respect to the fluid) will cause shear stress at the luminal surface of the cells.

The next sections will focus on the impact of pressure and strain in particular on lung functions.

#### **3.3 Strain is crucial for several functions of epithelial cells**

It is well known that physical forces are important stimuli for distinct cellular functions of pulmonary epithelial cells (Fig. 4). An important indication for this is represented by the fact that development of the mammalian lung within the last third of gestation depends on breathing movements although the lungs are fluid filled (Kitterman, 1996). In animal models prevention of these breathing movements by spinal nerve sections resulted in retarded lung growth and development (Fewell et al., 1981). Further, *in vitro* studies demonstrated that distention of alveolar epithelial cells is an important trigger that affects alveolar cell differentiation. Mechanical distention of isolated and cultivated fetal alveolar cells promotes their differentiation to AT I cells whereas that lack of distention promotes the differentiation to ATII cells (Edwards, 2001).

Fig. 4. Under normal breathing conditions mechanical stimuli are important for several functions of the lung.

Besides affecting cell differentiation and lung development there is also evidence for the contribution of physical stress as a factor that mediates production and secretion of surfactant proteins by ATII cells (Edwards et al., 1999) as well as by cultured H441 cells (Sanchez-Esteban et al., 1998). There is also evidence that mechanical forces induce apoptosis in ATII cells (Edwards et al., 1999; Hammerschmidt et al., 2007). In addition, different signaling mediators and ATP in particular are released in response to mechanical stress (Homolya et al., 2000; Okada et al., 2006; Button et al., 2007). The release of ATP offers the possibility to initiate a variety of different cellular reactions by acting on multiple purinergic receptors – including P2X, P2Y and P1 receptors (Leipziger, 2003; Bucheimer and Linden, 2004; Barth and Kasper, 2009). The relevance of purinergic signaling has been implicated by a study identifying alterations of transepithelial ion transport processes in airway epithelia in response to adenosine as a factor contributing to cystic fibrosis lung disease (Tarran et al., 2005; Tarran et al., 2006).

### **3.4 Deleterious effects of mechanical forces in the lung**

566 Lung Diseases – Selected State of the Art Reviews

movement of the epithelial cell (with respect to the fluid) will cause shear stress at the

The next sections will focus on the impact of pressure and strain in particular on lung

It is well known that physical forces are important stimuli for distinct cellular functions of pulmonary epithelial cells (Fig. 4). An important indication for this is represented by the fact that development of the mammalian lung within the last third of gestation depends on breathing movements although the lungs are fluid filled (Kitterman, 1996). In animal models prevention of these breathing movements by spinal nerve sections resulted in retarded lung growth and development (Fewell et al., 1981). Further, *in vitro* studies demonstrated that distention of alveolar epithelial cells is an important trigger that affects alveolar cell differentiation. Mechanical distention of isolated and cultivated fetal alveolar cells promotes their differentiation to AT I cells whereas that lack of distention promotes the differentiation

> fetal growth and development

> > mechanical stimulus

Fig. 4. Under normal breathing conditions mechanical stimuli are important for several

Besides affecting cell differentiation and lung development there is also evidence for the contribution of physical stress as a factor that mediates production and secretion of surfactant proteins by ATII cells (Edwards et al., 1999) as well as by cultured H441 cells (Sanchez-Esteban et al., 1998). There is also evidence that mechanical forces induce apoptosis in ATII cells (Edwards et al., 1999; Hammerschmidt et al., 2007). In addition, different signaling mediators and ATP in particular are released in response to mechanical stress (Homolya et al., 2000; Okada et al., 2006; Button et al., 2007). The release of ATP offers the possibility to initiate a variety of different cellular reactions by acting on multiple purinergic receptors – including P2X, P2Y and P1 receptors (Leipziger, 2003; Bucheimer and Linden, 2004; Barth and Kasper, 2009). The relevance of purinergic signaling has been

ATP release

apoptosis of AT II cells

**3.3 Strain is crucial for several functions of epithelial cells** 

secretion of surfactant

luminal surface of the cells.

to ATII cells (Edwards, 2001).

cell differentiation ATI or ATII

functions of the lung.

functions.

Although, there is more than sufficient evidence that identifies mechanical stimuli as an important mediator for normal lung functions, a main reason for studying their effect in the lung arises from the hazardous effects that are caused by mechanical forces during artificial ventilation (Frank and Matthay, 2003; Ricard et al., 2003) (Fig. 5).

Fig. 5. Effect of deleterious physical forces related to ventilator induced lung injury. This scheme was modified from (Frank and Matthay, 2003) and extended by the potential interference of mechanical stimuli with the activity of epithelial ion channels. This represents a yet unidentified mechanism to improve the understanding how mechanical forces contribute to the formation and probably maintenance of pulmonary edema.

Artificial ventilation is a remedy of first choice for the treatment of patients with respiratory failure (e.g. acute lung injury (ALI); acute respiratory distress syndrome (ARDS)) (Ware and Matthay, 2000). However, the development of pulmonary edema due to fluid influxes into the airspace as well as the inability to resolve the edema fluid by decreased fluid reabsorption, is a major reason for the morbidity and mortality in these patients (Morty et al., 2007). The inability of the patients to resolve the edema fluid correlates with the mechanical ventilation duration times and mortality. High fluid clearance was associated with shorter durations of mechanical ventilation as well as with significantly decreased mortality rates (Sznajder, 2001; Ware and Matthay, 2001).

Considering the important role of pulmonary ion transport processes as discussed above, it is obvious that pulmonary edema are somehow related to alterations of pulmonary ion transport processes (Matthay, 2002). Although changes of ion transport processes do not have to be causative for the development of pulmonary edema – there is sufficient evidence demonstrating that artificial ventilation worsens the situation in ALI/ARDS patients. This phenomenon has been termed ventilator induced lung injury (VILI) (Ricard et al., 2003) and is attributed to inappropriate ventilation strategies and thus the appearance of extensive physical forces (Fig. 5) (Plataki and Hubmayr, 2010).

This indication arises from the outcome of a multicenter trail study demonstrating a significantly reduced mortality in a group of patients that was ventilated with decreased tidal volumes (6 ml/kg) compared with high tidal volumes (12 ml/kg) (ARDS Network Investigators, 2000). Ventilation with decreased tidal volumes was also accompanied by the appearance of lower plateau pressures (ARDS Network Investigators, 2000) and this is associated with decreased mechanical forces.

However, regarding the symptoms observed in patients with ALI, ARDS and VILI there is an obvious connection between artificial ventilation and the development of pulmonary edema. There are two possible explanations reasonable for the development of pulmonary edema related to artificial ventilation:


The first point has been extensively studied and there is no doubt that mechanical strain induced by artificial ventilation is a major reason for the development of pulmonary edema (Frank and Matthay, 2003). This is clearly indicated by the appearance of proteins in the edema fluid (Ware and Matthay, 2000). The second point is also obvious since a correlation between the ability to reabsorb alveolar fluid and the outcome of patients has been identified (Ware and Matthay, 2001). But for this instance, the mechanisms how artificial ventilation and thus mechanical forces interfere with ion transport processes are unknown.

#### **3.5 Tools for studying the impact of mechanical forces on pulmonary ion transport**

The first choice for studying functional epithelial ion transport is represented by electrophysiological Ussing chamber recordings. This technique was established by Hans Ussing during his studies on ion transport processes across the amphibian skin (Ussing and Zerahn, 1951). A major advantage as well as a prerequisite of this technique is the use of an intact epithelial layer consisting of differentiated polar cells. Although this technique is applicable for many freshly dissected epithelia including airway epithelia from mammals (Olver et al., 1975; Widdicombe and Welsh, 1980), the use of mammalian alveolar epithelium for elaborating this technique is not possible. This is due to the complex anatomy reasoned by miniaturization of the gas-exchanging region. Improved protocols and procedures for the isolation and cultivation of alveolar cells generally fixed this problem, but the use of isolated cells bears the risk of using cells with an artificial non-physiological phenotype. There is a bunch of studies published demonstrating that in isolated and cultivated cells the expression of ion transport proteins as well as their phenotype in terms of ion transport properties vary in dependence of the cultivation conditions used (Kunzelmann et al., 1996; Jain et al., 2001; Leroy et al., 2004; Dvorak et al., 2011). Further, these cells do not have their native surrounding (neighboring cells of different cell type, basal lamina etc.) that is important for the detection and transmission of forces. It remains also a challenge to expose these cultivated cells to defined physical forces (e.g. strain) to determine immediate changes

transport processes (Matthay, 2002). Although changes of ion transport processes do not have to be causative for the development of pulmonary edema – there is sufficient evidence demonstrating that artificial ventilation worsens the situation in ALI/ARDS patients. This phenomenon has been termed ventilator induced lung injury (VILI) (Ricard et al., 2003) and is attributed to inappropriate ventilation strategies and thus the appearance of extensive

This indication arises from the outcome of a multicenter trail study demonstrating a significantly reduced mortality in a group of patients that was ventilated with decreased tidal volumes (6 ml/kg) compared with high tidal volumes (12 ml/kg) (ARDS Network Investigators, 2000). Ventilation with decreased tidal volumes was also accompanied by the appearance of lower plateau pressures (ARDS Network Investigators, 2000) and this is

However, regarding the symptoms observed in patients with ALI, ARDS and VILI there is an obvious connection between artificial ventilation and the development of pulmonary edema. There are two possible explanations reasonable for the development of pulmonary

1. Artificial ventilation causes ruptures and damages of the epithelial layer – enabling an

2. Mechanical forces as appearing during ventilation directly interfere with pulmonary

The first point has been extensively studied and there is no doubt that mechanical strain induced by artificial ventilation is a major reason for the development of pulmonary edema (Frank and Matthay, 2003). This is clearly indicated by the appearance of proteins in the edema fluid (Ware and Matthay, 2000). The second point is also obvious since a correlation between the ability to reabsorb alveolar fluid and the outcome of patients has been identified (Ware and Matthay, 2001). But for this instance, the mechanisms how artificial ventilation and thus mechanical forces interfere with ion transport processes are unknown.

**3.5 Tools for studying the impact of mechanical forces on pulmonary ion transport**  The first choice for studying functional epithelial ion transport is represented by electrophysiological Ussing chamber recordings. This technique was established by Hans Ussing during his studies on ion transport processes across the amphibian skin (Ussing and Zerahn, 1951). A major advantage as well as a prerequisite of this technique is the use of an intact epithelial layer consisting of differentiated polar cells. Although this technique is applicable for many freshly dissected epithelia including airway epithelia from mammals (Olver et al., 1975; Widdicombe and Welsh, 1980), the use of mammalian alveolar epithelium for elaborating this technique is not possible. This is due to the complex anatomy reasoned by miniaturization of the gas-exchanging region. Improved protocols and procedures for the isolation and cultivation of alveolar cells generally fixed this problem, but the use of isolated cells bears the risk of using cells with an artificial non-physiological phenotype. There is a bunch of studies published demonstrating that in isolated and cultivated cells the expression of ion transport proteins as well as their phenotype in terms of ion transport properties vary in dependence of the cultivation conditions used (Kunzelmann et al., 1996; Jain et al., 2001; Leroy et al., 2004; Dvorak et al., 2011). Further, these cells do not have their native surrounding (neighboring cells of different cell type, basal lamina etc.) that is important for the detection and transmission of forces. It remains also a challenge to expose these cultivated cells to defined physical forces (e.g. strain) to determine immediate changes

uncontrolled influx of protein rich fluid into the airspace.

physical forces (Fig. 5) (Plataki and Hubmayr, 2010).

associated with decreased mechanical forces.

edema related to artificial ventilation:

epithelial ion transport processes

of ion transport processes because usually the cells are cultivated on rigid polyester or polycarbonate membranes.

On the other hand pioneer studies addressing the function and relevance of pulmonary ion transport were performed on anaesthetized animals (Egan et al., 1976; Matthay et al., 1982). These studies identified the basic mechanisms of water and electrolyte transport by the alveolar epithelium. However, from such studies it is difficult to disentangle the particular ion conductances and to identify the specific ion transporting molecules that are involved in those processes.

#### **3.5.1 Using native lung preparations for studying the impact of strain on pulmonary epithelial ion transport**

Our lab has therefore established a native model for investigations of pulmonary ion transport. In accordance to Krogh's principle (Krebs, 1975) we decided to use lung preparations derived from the South African Clawed Frog *Xenopus laevis*. The alveolar epithelium of the *Xenopus* lung consists of one cell type referred to as pneumocytes (Meban, 1973). The anatomy and morphology of these cells is reminiscent to that of alveolar type I cells (Fischer et al., 1989), but they expose functional properties of alveolar type II cells as represented by the presence of lamellar bodies (Fischer et al., 1989). Most important, the *Xenopus* pneumocytes expose a Na+ reabsorptive phenotype (Fischer et al., 1989; Kim, 1990; Fronius et al., 2003). In addition, expression and function of the CFTR Cl– channel has been detected (Sommer et al., 2007). Recent studies identified the presence of the Na+/K+/2Cl– cotransporter, the function of a HCO3–/Cl– exchanger (Berger et al., 2010) and evidence for basolateral Cl– channels (Berger et al., 2011). Thus, the *Xenopus* pneumocytes exhibit the basic repertoire of ion channels and transporters that are supposed to be important in mediating the volume of the PLL.

The main advantage of this amphibian organ is its relatively simple sac-like structure. This feature is a prerequisite for dissecting a preparation that is suitable for Ussing chamber recordings. Comparable studies with other native lung preparations from higher vertebrates – and mammals in particular - are not possible, because of the growing complexity and miniaturization of the gas-exchanging region. In addition to establishing the use of *Xenopus*  lung preparations for electrophysiological recordings, an Ussing chamber was developed that enables the exposure of the mounted pulmonary epithelium to mechanical forces. Mechanical forces are applied via an increased hydrostatic pressure and this is achieved by changing the outflow-height from the compartments Fig. 6.

## **4. Impact of strain on pulmonary ion transport in the** *Xenopus* **lung**

Although the connection between high tidal volumes and pulmonary edema has been well established, little is known whether or not the underlying mechanisms can be attributed – at least partly – to a direct interaction of the mechanical forces with ion transport processes. Studies have been published providing evidence that high volume ventilation resulted in a decreased Na+ transport due to a decreased Na+/K+-ATPase activity (Lecuona et al., 1999). Other studies demonstrated an increased Na+/K+-ATPase activity in response to cyclic stretch (Fisher and Margulies, 2002). Although these studies identified interference of strain with Na+/K+-ATPase activity, changes were observed hours after exposure to mechanical forces. So far little is known about a direct short-term effect of mechanical forces on ion transport processes in the lung.

## **customized Ussing chamber**

Fig. 6. Drawing of the Ussing chamber used to study the effect of mechanical forces on pulmonary epithelial ion transport (modified from (Bogdan et al., 2008)). Mechanical forces were applied by increasing the water column (5 cm water column) of the outflow. Both chamber compartments (apical, basolateral) were continuously perfused and the transepithelial short-circuit current (ISC) and potential (VT) was permanently monitored (ti: mounted tissue).

Using *Xenopus* lung preparations in combination with a customized Ussing chamber (Fig. 6) 5 cm hydrostatic pressure was applied from the apical side to mechanically challenge the tissue. Application of hydrostatic pressure was accompanied by immediate changes of the measured transepithelial short-circuit current (ISC). The net effect induced by 5 cm H2O was characterized by a decreased transepithelial current (Bogdan et al., 2008). Interestingly, the application of 5 cm H2O from the basolateral side induced exactly the same response whereas the application of 5 cm H2O synchronously from the apical and basolateral side did not cause any effect of the ISC (Bogdan et al., 2008). These observations clearly demonstrate that the effective mechanical stimulus is strain and that it does not matter from which side the tissue is deflected.

In this setup a decreased transepithelial ion current is an indication for a decreased net reabsorption of ions from the apical to the basolateral side of the epithelium. Further, a reduced ion reabsorption reasoned by changes in ion transport processes will also reduce the osmoticaly driven water reabsorption from the airspace. This means that the changes of ion transport in the pulmonary epithelium observed with hydrostatic pressure decrease water reabsorption from the airspace and this represents a mechanism to facilitate the development of pulmonary edema – without affecting the integrity of the epithelial barrier that was assessed by determining the transepithelial electrical resistance.

#### **4.1 Strain induces short-term activation of Na+ , K+ and Cl– channels**

Further investigations using different ion channel inhibitors and substituting different ions from the perfusion solution revealed that the observed effect is reasoned by the activation of different ion channels and ion conductances resembled by:


#### 3. activation of an apical K+ secretion

570 Lung Diseases – Selected State of the Art Reviews

**customized Ussing chamber**

out

plugg

**0 cm water column 5 cm water column**

Fig. 6. Drawing of the Ussing chamber used to study the effect of mechanical forces on pulmonary epithelial ion transport (modified from (Bogdan et al., 2008)). Mechanical forces were applied by increasing the water column (5 cm water column) of the outflow. Both chamber compartments (apical, basolateral) were continuously perfused and the

transepithelial short-circuit current (ISC) and potential (VT) was permanently monitored (ti:

Using *Xenopus* lung preparations in combination with a customized Ussing chamber (Fig. 6) 5 cm hydrostatic pressure was applied from the apical side to mechanically challenge the tissue. Application of hydrostatic pressure was accompanied by immediate changes of the measured transepithelial short-circuit current (ISC). The net effect induced by 5 cm H2O was characterized by a decreased transepithelial current (Bogdan et al., 2008). Interestingly, the application of 5 cm H2O from the basolateral side induced exactly the same response whereas the application of 5 cm H2O synchronously from the apical and basolateral side did not cause any effect of the ISC (Bogdan et al., 2008). These observations clearly demonstrate that the effective mechanical stimulus is strain and that it does not matter from which side

In this setup a decreased transepithelial ion current is an indication for a decreased net reabsorption of ions from the apical to the basolateral side of the epithelium. Further, a reduced ion reabsorption reasoned by changes in ion transport processes will also reduce the osmoticaly driven water reabsorption from the airspace. This means that the changes of ion transport in the pulmonary epithelium observed with hydrostatic pressure decrease water reabsorption from the airspace and this represents a mechanism to facilitate the development of pulmonary edema – without affecting the integrity of the epithelial barrier

**, K+**

Further investigations using different ion channel inhibitors and substituting different ions from the perfusion solution revealed that the observed effect is reasoned by the activation of

 **and Cl–**

 **channels** 

that was assessed by determining the transepithelial electrical resistance.

**4.1 Strain induces short-term activation of Na+**

2. activation of an apical Cl– secretion

different ion channels and ion conductances resembled by: 1. activation of amiloride-sensitive Na+ reabsorption

**apical basol.**

in in

**ti VT ISC** out

**apical basol.**

in in

**ti VT ISC**

out out

**5 cm**

mounted tissue).

the tissue is deflected.

Therefore, the inhibitory effect observed in the absence of drugs is an overlay of these three conductances (Fig. 7). Activation of Na+ reabsorption as well as an increase of Cl– secretion will produce an increase of the ISC. In contrast to this, activation of apical K+ channels will cause a decrease of the ISC. Since the net pressure effect is an inhibition of the ISC, the major response observed by the application of hydrostatic pressure is due to an activation of apical K+ channels. Accordingly, the inhibitory effect of hydrostatic pressure should be prevented (or reversed activation of the ISC) following pre-incubation with K+ channel inhibitors. And indeed this is exactly what we observed (Bogdan et al., 2008). Further, it has been found that the pressure-induced effect was largely prevented by glibenclamide (Bogdan et al., 2008), a compound that is a high affinity inhibitor of ATP-sensitive K+ channels (KATP) (Nichols, 2006). Among other activating mechanisms, the activity of KATP channels is dependent on intracellular cyclic nucleotide levels and ATP in particular (Nichols, 2006). Interestingly, increased extracellular ATP concentrations were observed in response to the application of hydrostatic pressure and this represents a likely mechanism to explain our observations.

KATP channels are octameric complexes consisting of four pore forming Kir (inward rectifying K+ channels) subunits and four associated SUR (sulfonylurea receptor) subunits (Nichols, 2006). In particular Kir channels are supposed to be involved in controlling the

Fig. 7. Scheme illustrating the putative mechanism how hydrostatic pressure acts on epithelial ion transport processes in *Xenopus* lung epithelium (modified from Bogdan et al., 2008). The entire process could be initiated by the release of ATP via a yet unknown mechanism (1). The decrease of intracellular ATP levels ([ATP]i) is likely to activate KATP channels (2). This will then cause the cell membrane potential to hyperpolarize (3) and subsequently facilitates the uptake of Na+ via apical Na+ channels (4) as well as the secretion of Cl– via apical Cl– channels.

membrane potential (Nichols, 2006) and this in turn enables the possibility to influence the driving forces for other conductances such as Na+ and Cl–. From this point of view it might be suggested that strain is primarily transduced in activation of KATP channels and that this in addition influences secondarily Na+ and Cl– transport as we observe by the application of hydrostatic pressure on *Xenopus* lung epithelia. Interestingly, increased ATP levels are also observed in rats ventilated with injurious ventilation parameters. In those experiments the increase of extracellular ATP concentrations was not reasoned by cell damage or cell lysis (Rich et al., 2003). These observations together with our findings indicate that the release of ATP (by a yet unidentified mechanism) might play a key role concerning the activation of ion channels in response to mechanical forces.

Last but not least it might be highlighted that the effect that was observed in response to the application of hydrostatic pressure (inhibition of the ISC) represents a mechanism that impairs ion reabsorption from the alveolar airspace. This will result in a decrease osmotic gradient across the epithelial layer and will subsequently cause a reduction of fluid reabsorption from the airspace. Thus, this is a likely mechanism that impairs the resolution of pulmonary edema or maybe represents a mechanism that – among other incidents – initiates the formation of pulmonary edema in response to strain as induced by artificial ventilation.

#### **5. Conclusions**

The fact that mechanical forces directly affect pulmonary epithelial ion transport is important for future therapeutic options. On the one hand it further confirms the observations that modified ventilation strategies with low pressures and reduced volumes are beneficial for the outcome of patients with respiratory failure that are admitted to artificial ventilation. Therefore, the development of new ventilation strategies should be considered with the background that a minimum of mechanical stress should be used because this decreases interference with ion transport processes and this will preserve the ability of the epithelium for effective ion and water reabsorption.

On the other hand, it offers new therapeutic targets since we have evidence that K+ channels and KATP channels in particular play a major role in the response observed by increased hydrostatic pressure. In our experiments, the inhibition of these channels has been identified to largely abolish the mechanically induced activation of K+ channels and this would be beneficial to prevent the reduced ion transport absorption as that correlates with a decreased water reabsorption from the airspace. Another possibility in order to prevent the effects of strain on ion channels might be represented by the possibility to target the release of ATP, although the mechanisms of ATP release are still under debated.

## **6. Acknowledgements**

The work was supported by the Deutsche Forschungsgemeinschaft (grant FR 2124 and the graduate program 455).

#### **7. References**

ARDS Network Investigators. (2000). Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory

membrane potential (Nichols, 2006) and this in turn enables the possibility to influence the driving forces for other conductances such as Na+ and Cl–. From this point of view it might be suggested that strain is primarily transduced in activation of KATP channels and that this in addition influences secondarily Na+ and Cl– transport as we observe by the application of hydrostatic pressure on *Xenopus* lung epithelia. Interestingly, increased ATP levels are also observed in rats ventilated with injurious ventilation parameters. In those experiments the increase of extracellular ATP concentrations was not reasoned by cell damage or cell lysis (Rich et al., 2003). These observations together with our findings indicate that the release of ATP (by a yet unidentified mechanism) might play a key role concerning the activation of

Last but not least it might be highlighted that the effect that was observed in response to the application of hydrostatic pressure (inhibition of the ISC) represents a mechanism that impairs ion reabsorption from the alveolar airspace. This will result in a decrease osmotic gradient across the epithelial layer and will subsequently cause a reduction of fluid reabsorption from the airspace. Thus, this is a likely mechanism that impairs the resolution of pulmonary edema or maybe represents a mechanism that – among other incidents – initiates the formation of pulmonary edema in response to strain as induced by artificial

The fact that mechanical forces directly affect pulmonary epithelial ion transport is important for future therapeutic options. On the one hand it further confirms the observations that modified ventilation strategies with low pressures and reduced volumes are beneficial for the outcome of patients with respiratory failure that are admitted to artificial ventilation. Therefore, the development of new ventilation strategies should be considered with the background that a minimum of mechanical stress should be used because this decreases interference with ion transport processes and this will preserve the

On the other hand, it offers new therapeutic targets since we have evidence that K+ channels and KATP channels in particular play a major role in the response observed by increased hydrostatic pressure. In our experiments, the inhibition of these channels has been identified to largely abolish the mechanically induced activation of K+ channels and this would be beneficial to prevent the reduced ion transport absorption as that correlates with a decreased water reabsorption from the airspace. Another possibility in order to prevent the effects of strain on ion channels might be represented by the possibility to target the release

The work was supported by the Deutsche Forschungsgemeinschaft (grant FR 2124 and the

ARDS Network Investigators. (2000). Ventilation with lower tidal volumes as compared

with traditional tidal volumes for acute lung injury and the acute respiratory

ability of the epithelium for effective ion and water reabsorption.

of ATP, although the mechanisms of ATP release are still under debated.

ion channels in response to mechanical forces.

ventilation.

**5. Conclusions** 

**6. Acknowledgements** 

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578 Lung Diseases – Selected State of the Art Reviews

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## **Functional Evaluation in Respiratory Disorders**

Cirelene Grobler, David M. Maree and Elvis M. Irusen

*Pulmonology Division, University of Stellenbosch & Tygerberg Academic Hospital, Cape Town, South Africa* 

## **1. Introduction**

In evaluating the respiratory system, many different tests are used and these can be divided into different categories based on the aspect of lung function they measure. Depending on availability and need, the tests can be complementary and yield results that allow deeper insight into respiratory function to understand disease processes and therapeutic interventions - both medical and surgical.

Table 1. The categories of pulmonary function testing

## **1.1 Spirometry & other related tests**

Spirometry is a physiological test that measures how an individual inhales or exhales volumes of air as a function of time. The primary signal measured may be volume or time. Spirometry is the pulmonary function test performed most often due to the large number of indications. "It is most often performed as a screening procedure because it may be the first test to indicate the presence of pulmonary disease"(Ruppel, 2009).

## **Indications for Spirometry**

	- 1. History of pulmonary symptoms (dyspnoea, wheezing, cough, phlegm production, orthopnoea)
	- 2. Physical indicators ( decreased breath sounds, chest wall abnormalities)
	- 3. Abnormal Laboratory findings ( Chest x-ray or CT studies)
	- 1. Pulmonary disease (COPD, Asthma)
	- 2. Cardiac disease (Cardiac Failure)
	- 3. Neuromuscular disease (e.g. Guillain-Barrè syndrome)
	- 1. Lung resection
	- 2. Thoracic procedures
	- 3. Pulmonary rehabilitation

Table 2. List of indications for spirometry (Ruppel, 2009)

"Spirometry is recommended as the "gold standard" for the diagnosis of obstructive lung disease. However spirometry alone may not be sufficient enough to completely define the extent of disease, therapy response, preoperative risk, or level of impairment (Ruppel,2009)".

In view of the importance of spirometry in aiding an accurate diagnosis and monitoring changes that can be extremely subtle, a good quality spirometer is essential. As the machines become increasingly sophisticated and computerised, it is imperative that they meet the technical specifications so that are accurate and precise. These criteria are quite complex but are well laid out by the American Thoracic Society (ATS- Standardization of Spirometry, 1994 Update). Thus in purchasing or utilising such a device it is crucial that one obtains the manufacturer's guarantee that a reputable testing facility has checked that the spirometer meets and conforms with the ATS recommendations for accuracy and precision. Equally, a well trained pulmonary function technologist who understands the calibration and pitfalls of the testing can be invaluable.

Spirometry is a physiological test that measures how an individual inhales or exhales volumes of air as a function of time. The primary signal measured may be volume or time. Spirometry is the pulmonary function test performed most often due to the large number of indications. "It is most often performed as a screening procedure because it may be the first

**Indications for Spirometry** 

2. Physical indicators ( decreased breath sounds, chest wall

3. Abnormal Laboratory findings ( Chest x-ray or CT studies)

3. Neuromuscular disease (e.g. Guillain-Barrè syndrome) c. Measure effects of occupational / environmental exposures

1. History of pulmonary symptoms (dyspnoea, wheezing, cough,

"Spirometry is recommended as the "gold standard" for the diagnosis of obstructive lung disease. However spirometry alone may not be sufficient enough to completely define the extent of disease, therapy response, preoperative risk, or level of impairment

g. Epidemiologic or clinical research involving lung health or disease.

In view of the importance of spirometry in aiding an accurate diagnosis and monitoring changes that can be extremely subtle, a good quality spirometer is essential. As the machines become increasingly sophisticated and computerised, it is imperative that they meet the technical specifications so that are accurate and precise. These criteria are quite complex but are well laid out by the American Thoracic Society (ATS- Standardization of Spirometry, 1994 Update). Thus in purchasing or utilising such a device it is crucial that one obtains the manufacturer's guarantee that a reputable testing facility has checked that the spirometer meets and conforms with the ATS recommendations for accuracy and precision. Equally, a well trained pulmonary function technologist who understands the calibration and pitfalls

test to indicate the presence of pulmonary disease"(Ruppel, 2009).

a. Diagnose the presence / absence of lung disease.

phlegm production, orthopnoea)

1. Pulmonary disease (COPD, Asthma) 2. Cardiac disease (Cardiac Failure)

d. Determine beneficial / negative effects of therapy

b. Quantify the extent of known disease on lung function

Table 2. List of indications for spirometry (Ruppel, 2009)

e. Assess risk for surgical procedures

1. Lung resection 2. Thoracic procedures 3. Pulmonary rehabilitation f. Evaluate disability or impairment

(Ruppel,2009)".

of the testing can be invaluable.

**1.1 Spirometry & other related tests** 

abnormalities)

"The two most important measurements of spirometry are (1) **forced vital capacity (FVC)**, which is the volume delivered during an expiration made as forcefully and completely as possible starting from full inspiration, and the (2) **forced expiratory volume** in one second **(FEV1)** of an FVC manoeuvre (ATS/ERS,2005)".

Another variable derived from spirometry is the slow **vital capacity (VC)**, which is the volume of gas measured from a slow, complete expiration after a maximal inspiration, without forced or rapid effort. The **Inspiratory capacity (IC)** and **expiratory reserve volume (ERV)** are subdivisions of the VC. The IC is the largest volume of gas that can be inspired from a resting expiratory level. ERV is the largest volume of gas that can be expired from the resting end-expiratory level. IC and ERV are used in the calculation of the **residual volume (RV)** and **total lung capacity (TLC)**. The RV is the volume of gas remaining in the lungs at the end of maximal expiration regardless of the lung volume at which exhalation was started. The TLC is the volume of gas contained in the lungs after maximal inspiration (ATS/ERS,2005).

Fig. 1. A schematic presentation of the lung volumes and capacities.


Table 3. A list of contraindications to spirometry (Ruppel,2009).

## **2. Flow-volume loop**

This procedure is used to measure the FVC, FEV1 and other forced expiratory flow volumes. This test is dependent on patient effort.

## **2.1 Significance and pathophysiology**

#### **2.1.1 Forced Vital Capacity**

The FVC usually equals VC in healthy individuals and should be within 150ml of each other. The FVC and VC may differ if the patient's effort is variable or if significant airway obstruction is present (FEV1 / FVC is less than 70%). The FVC may be lower than VC in patients with obstructive diseases as forced expiration can cause airway collapse. In these situations a slow VC (SVC) may be more accurate.

Healthy adults can expire their FVC within 4-6 seconds. Healthy children and adolescents may exhale their FVC in less than 4 seconds. Patients with severe obstruction may require 15 seconds or more to exhale completely.

## **2.1.2 Forced expiratory volume in the first second (FEV1)**

FEV1 is reported as a volume, although it measures flow over a specific interval. FEV1 may be reduced in either obstructive or restrictive patterns. The FEV1 and FEV1 / FVC ratio are the most standardized indices of obstructive diseases. An obstructive defect is defined best by a reduced ratio.

The severity of an obstructive disease may be gauged by the extent to which FEV1 is reduced. The ATS / ERS 2005 Task force suggests the following classifications of severity (Ruppel, 2005):


Once the VC is below normal, a concomitant restrictive defect may also be present, and this can be determined by further measurement of volumes, in particular TLC. Restrictive processes such as fibrosis, oedema, and obesity may all cause a decrease in FEV1. Unlike the pattern seen in obstructive diseases, in which VC is preserved and FEV1 reduced, in restriction VC and FEV1 values are proportionally decreased.

The FEV1 is the most widely used spirometric parameter, particularly for the assessment of airway obstruction. It is also used in conjunction with VC for simple screening, assessment of response to bronchodilators, and detection of exercise-induced bronchospasm.

## **2.1.3 FEV1 / FVC ratio**

The normal ratio expressed as a percentage for healthy adults is between 75% - 85%. This value can decrease with age, presumably because of gradual loss of lung elasticity. Diagnosis of an obstructive pattern based on spirometry should focus on three primary variables: FVC, FEV1, and FEV1 / FVC.

Examples of patterns seen in flow volume loops follow: The actual curve (with asterisks) is usually superimposed on the predicted as derived by the computer based on age, gender, height and ethnicity.

This procedure is used to measure the FVC, FEV1 and other forced expiratory flow volumes.

The FVC usually equals VC in healthy individuals and should be within 150ml of each other. The FVC and VC may differ if the patient's effort is variable or if significant airway obstruction is present (FEV1 / FVC is less than 70%). The FVC may be lower than VC in patients with obstructive diseases as forced expiration can cause airway collapse. In these

Healthy adults can expire their FVC within 4-6 seconds. Healthy children and adolescents may exhale their FVC in less than 4 seconds. Patients with severe obstruction may require 15

FEV1 is reported as a volume, although it measures flow over a specific interval. FEV1 may be reduced in either obstructive or restrictive patterns. The FEV1 and FEV1 / FVC ratio are the most standardized indices of obstructive diseases. An obstructive defect is defined best

The severity of an obstructive disease may be gauged by the extent to which FEV1 is reduced. The ATS / ERS 2005 Task force suggests the following classifications of severity

Once the VC is below normal, a concomitant restrictive defect may also be present, and this can be determined by further measurement of volumes, in particular TLC. Restrictive processes such as fibrosis, oedema, and obesity may all cause a decrease in FEV1. Unlike the pattern seen in obstructive diseases, in which VC is preserved and FEV1 reduced, in

The FEV1 is the most widely used spirometric parameter, particularly for the assessment of airway obstruction. It is also used in conjunction with VC for simple screening, assessment

The normal ratio expressed as a percentage for healthy adults is between 75% - 85%. This value can decrease with age, presumably because of gradual loss of lung elasticity. Diagnosis of an obstructive pattern based on spirometry should focus on three primary

Examples of patterns seen in flow volume loops follow: The actual curve (with asterisks) is usually superimposed on the predicted as derived by the computer based on age, gender,

of response to bronchodilators, and detection of exercise-induced bronchospasm.

**2. Flow-volume loop** 

**2.1.1 Forced Vital Capacity** 

by a reduced ratio.

**2.1.3 FEV1 / FVC ratio** 

height and ethnicity.

variables: FVC, FEV1, and FEV1 / FVC.

(Ruppel, 2005):

This test is dependent on patient effort.

**2.1 Significance and pathophysiology** 

seconds or more to exhale completely.

situations a slow VC (SVC) may be more accurate.

Mild FEV1 > 70% predicted Moderate FEV1 = 60% - 69% predicted Moderately severe FEV1 = 50% -59% predicted Severe FEV1 = 35% - 49% predicted Very severe FEV1 < 35% of predicted

**2.1.2 Forced expiratory volume in the first second (FEV1)** 

restriction VC and FEV1 values are proportionally decreased.

#### **2.2 Reversibility testing**

This is the determination of reversibility in airflow-limitation with drug administration and is commonly undertaken as part of lung function testing. The choice of drug, dose and mode of delivery is a clinical decision depending on what the clinician wishes to learn from the test. The aim is to determine whether the patient's lung function can be improved with therapy.

The subject first undergoes baseline lung function testing, preferably with no prior drug therapy. According to the ATS/ERS 2005 guidelines, short-acting inhaled drugs should not be used 4hr prior to testing and long-acting β-agonist bronchodilators or oral aminophylline should be stopped 12hr prior to testing. Smoking should be avoided for an hour or more prior to testing as well as throughout the duration of the test procedure.

#### **2.2.1 Procedure (ATS/ERS, 2005)**


#### **2.2.2 Determination of reversibility**

A positive response to bronchodilator therapy is when either the FVC or the FEV1 of the post attempt improves by 12% and 200ml from the pre attempt.

$$1.\text{ BD response} = \frac{\text{FEV}\_1(\text{post}) - \text{FEV}\_1(\text{pre})}{1} \times \frac{100}{1} \text{s}$$

$$\text{2. BD response} = \frac{\text{FVC (post)} - \text{FVC (pre)}}{1} \times \frac{100}{1}$$

## **2.2.3 Clinical significance**

Reversibility of airway obstruction is considered significant for increases of greater than 12% and 200ml for either the FEV1 or FVC. If the sGaw is assessed, an increase of 30%-40% is usually considered as significant. Some patients may show little or even no improvement in FEV1, but have a significant improvement in their sGaw.

Increases greater than 50% in the FEV1 may occur in patients with asthma. Patients with chronic obstructive pulmonary diseases may show little improvement in their flows. Failure to show a significant improvement after inhaled bronchodilator therapy does not exclude a response.

It has been erroneously extrapolated that reversibility testing can define a disease; this is not true ( Richter & Irusen, 2008). Asthma can be irreversible on spirometry ( especially when there is uncontrolled inflammation impairing bronchodilatation) and COPD can be spirometrically reversible in up to 50% of patients. ( In this respect, the absolute volume of improvement is more important as it is easy to get a significant percentage change when one starts with a low baseline.)

## **3. Maximal inspiratory / expiratory pressure ( MIP & MEP)**

Forced manoeuvres during spirometry require the patient to give a maximal effort, yet it also requires that the patient should have normal muscle function. Muscle function is best assessed by measurement of maximal inspiratory and expiratory pressures. **Maximal inspiratory pressure (MIP)** is the lowest pressure developed during a forceful inspiration against an occluded airway. **Maximal expiratory pressure (MEP)** is the highest pressure that can be developed during a forceful expiratory effort against an occluded airway.

## **3.1 Significance and pathophysiology**

MIP primarily measures inspiratory muscle strength. Healthy adults can generate inspiratory pressures greater than -50cmH2O in women, and -75 cmH2O in men (Ruppel, 2009). Decreased MIP is seen in patients with neuromuscular disease or diseases involving the diaphragm, intercostals or accessory muscles. MIP may also be decreased in patients with hyperinflation as in emphysema. MIP is sometimes used to assess patient response to strength training of respiratory muscles. It is also used in the assessment of respiratory muscle function in patients who need ventilatory support.

MEP measures the pressures generated during maximal expiration. Healthy adults can generate MEP values greater than 80 cmH2O in women and greater than 100 cmH2O in men (Ruppel, 2009). MEP may be decreased in patients with neuromuscular disorders, particularly those resulting in generalized muscle weakness.

Reduced MEP often accompanies increased RV as seen in emphysema. A low MEP is associated with inability to cough effectively.

Accurate measurement of MIP & MEP depends largely on patient effort. The best efforts should be reproducible within 20% or 10 cmH2O, whichever is greater. Widely varying pressures for either MIP or MEP should be assessed carefully before interpretation.

## **4. Body plethysmography**

586 Lung Diseases – Selected State of the Art Reviews

Reversibility of airway obstruction is considered significant for increases of greater than 12% and 200ml for either the FEV1 or FVC. If the sGaw is assessed, an increase of 30%-40% is usually considered as significant. Some patients may show little or even no improvement in

Increases greater than 50% in the FEV1 may occur in patients with asthma. Patients with chronic obstructive pulmonary diseases may show little improvement in their flows. Failure to show a significant improvement after inhaled bronchodilator therapy does not exclude a

It has been erroneously extrapolated that reversibility testing can define a disease; this is not true ( Richter & Irusen, 2008). Asthma can be irreversible on spirometry ( especially when there is uncontrolled inflammation impairing bronchodilatation) and COPD can be spirometrically reversible in up to 50% of patients. ( In this respect, the absolute volume of improvement is more important as it is easy to get a significant percentage change when one

Forced manoeuvres during spirometry require the patient to give a maximal effort, yet it also requires that the patient should have normal muscle function. Muscle function is best assessed by measurement of maximal inspiratory and expiratory pressures. **Maximal inspiratory pressure (MIP)** is the lowest pressure developed during a forceful inspiration against an occluded airway. **Maximal expiratory pressure (MEP)** is the highest pressure

MIP primarily measures inspiratory muscle strength. Healthy adults can generate inspiratory pressures greater than -50cmH2O in women, and -75 cmH2O in men (Ruppel, 2009). Decreased MIP is seen in patients with neuromuscular disease or diseases involving the diaphragm, intercostals or accessory muscles. MIP may also be decreased in patients with hyperinflation as in emphysema. MIP is sometimes used to assess patient response to strength training of respiratory muscles. It is also used in the assessment of respiratory

MEP measures the pressures generated during maximal expiration. Healthy adults can generate MEP values greater than 80 cmH2O in women and greater than 100 cmH2O in men (Ruppel, 2009). MEP may be decreased in patients with neuromuscular disorders,

Reduced MEP often accompanies increased RV as seen in emphysema. A low MEP is

Accurate measurement of MIP & MEP depends largely on patient effort. The best efforts should be reproducible within 20% or 10 cmH2O, whichever is greater. Widely varying

pressures for either MIP or MEP should be assessed carefully before interpretation.

that can be developed during a forceful expiratory effort against an occluded airway.

1 1

2. BD response = FVC post – FVC pre <sup>100</sup>

FEV1, but have a significant improvement in their sGaw.

**3. Maximal inspiratory / expiratory pressure ( MIP & MEP)** 

**2.2.3 Clinical significance** 

starts with a low baseline.)

**3.1 Significance and pathophysiology** 

muscle function in patients who need ventilatory support.

particularly those resulting in generalized muscle weakness.

associated with inability to cough effectively.

response.

The forces governing maximal airflow are the elastic recoil pressure of the lung and airway resistance upstream from the equal pressure point. **Airway resistance (Raw)** is the pressure difference per unit flow as gas flows into or out of the lungs. Raw is the difference between the mouth pressure and alveolar pressure, divided by flow at the mouth.

**Airway conductance (Gaw)** is the flow generated per unit of pressure drop across the airways. Gaw is not commonly reported as it changes with lung volume. Instead, specific airway conductance (sGaw), which is Gaw divided by the lung volume at which the measurement was made, is usually reported (Ruppel, 2009).

Spirometry may be performed with the patient in the plethysmograph. The pneumotachometer must be capable of accurately measuring the entire range of gas flows required.

Spirometry, lung volumes, and airway resistance can all be obtained in a single sitting using plethysmography.

## **4.1 The most common measurements made using a body plethysmograph are:**

	- VC= Volume measured from a maximal inspiration followed by a complete slow expiration.
	- FRC= It is the volume of air left in the lungs at the end of a quiet exhalation
	- IC = Maximal volume of air inspired from a resting expiratory level.
	- ERV= Maximal volume of air expired from a resting expiratory level
	- TGV= Is the absolute volume of gas in the thorax at any point in time and at any level of alveolar pressure
	- RV= Volume of air remaining in the lungs at the end of a maximal expiration.
	- TLC= Volume of gas that the lungs contain after maximal inspiration.
	- RV/TLC (must be in the range 20 35% in order to be normal)

## **4.2 Important derivatives**

## **4.2.1 Thoracic Gas Volume (TGV)**

The TGV is a quick and accurate means of measuring lung volumes. It can be used in combination with simple spirometry to derive all lung volume compartments. The plethysmograph's primary advantage is that it measures all gas in the thorax, whether in ventilatory communication with the atmosphere or not.

#### **4.2.1.1 Clinical Significance**

Normative data for TGV and pulmonary subdivisions allow definition of restrictive lung disease as distinct from obstructive, in the presence of a reduced VC. Definition of abnormally increased lung volumes in obstructive lung disease is a further appropriate clinical use of whole-body plethysmography. While lung volumes can be measured by gas dilution techniques, it is well known that dilution techniques measure only the volume of ventilated airspaces. Accordingly, when whole-body plethysmography is combined with dilution measures of lung volumes, the volume of trapped gas is estimated by the difference between FRCBox and dilutional FRCGas.

The ratio of FRCBox/FRCGas can be used as an index of gas trapping. This ratio is usually near 1.0 in patients with normal lungs, or even with restriction.

Values greater than 1 indicate gas volumes detectable by the plethysmograph but hidden to the gas techniques. Care must be taken that lung volumes determined by the 2 methods are reliable before the values can be expressed as a ratio. This ratio has been used to evaluate candidates for lung volume reduction surgery (LVRS). Lung volume reduction attempts to directly reduce gas trapping by removal of unperfused lung tissue. Patients with bullous emphysema may have a litre or more difference in TLC (Total Lung Capacity) when the methods are compared.

Some evidence suggests that in severe airway obstruction, FRC may actually be overestimated when the plethysmographic technique is used. This occurs primarily because PMouth may not equal alveolar pressure if the airways are severely obstructed. Rapid panting rates aggravate this inaccuracy.

## **4.2.2 RAW**

Airway resistance (Raw) is the pressure difference per unit flow as gas flows into or out of the lungs. Raw is the difference between mouth pressure and alveolar pressure, divided by flow at the mouth. This pressure difference is caused primarily by the friction of gas molecules in contact with the airways.

#### **4.2.2.1 Clinical Significance**

The tracing labelled "(a)" displays a schematic sRaw loop in a normal patient during tidal breathing, which is shown after numerical software compensations to close the sRaw loop. Normal patients manifest a steep linear loop during tidal breathing without hysteresis. In contrast, during voluntary panting efforts, the upper and lower end portions of the loop may become slightly curvilinear.

The curvilinearity is in the form of a very slight "S" shape, analogous to that shown in tracing "(d)", but much less exaggerated. In normal patients during voluntary panting, the flattening of the sRaw loop at the upper right extremity (mid-inspiration) and at the lower left extremity (mid-expiration) of the loop are only barely visible, depending on the absolute value of flow rates achieved.

Tracing "(b)" is typical of patients with large (central) airway constriction that is relatively uniform (and not a localized stenosis) and without significant small airway obstruction. This might be seen in a patient with mild asthma.

It is well known that expiratory flow limitation and dynamic airway compression may occur during tidal breathing in COPD, and this contributes to the characteristic shape of the sRaw loop in tracing "(c)".

Tracing "(d)" shows the influence of a fixed or functional stenosis of the upper airways, for example laryngeal abnormality, or paralysis of one vocal cord. This type of "orifice" constriction manifests flow limitation during inspiration, such that, at sufficiently high flows, further increases in driving pressure do not result in any increase in airflow. This reflects localized upper airway obstruction, analogous to that which pertains in the maximal expiratory flow–volume curve. Thus, during forced expiration, when a critical driving pressure for expiratory airflow (intra-pleural pressure for forced expiration) is achieved, further increases in driving pressure do not cause any further increases in flow rate. A similar flow limitation may occur in the extra-thoracic airway during inspiration, as shown in the upper right portion of tracing "(d)".

The ratio of FRCBox/FRCGas can be used as an index of gas trapping. This ratio is usually

Values greater than 1 indicate gas volumes detectable by the plethysmograph but hidden to the gas techniques. Care must be taken that lung volumes determined by the 2 methods are reliable before the values can be expressed as a ratio. This ratio has been used to evaluate candidates for lung volume reduction surgery (LVRS). Lung volume reduction attempts to directly reduce gas trapping by removal of unperfused lung tissue. Patients with bullous emphysema may have a litre or more difference in TLC (Total Lung Capacity) when the

Some evidence suggests that in severe airway obstruction, FRC may actually be overestimated when the plethysmographic technique is used. This occurs primarily because PMouth may not equal alveolar pressure if the airways are severely obstructed. Rapid

Airway resistance (Raw) is the pressure difference per unit flow as gas flows into or out of the lungs. Raw is the difference between mouth pressure and alveolar pressure, divided by flow at the mouth. This pressure difference is caused primarily by the friction of gas

The tracing labelled "(a)" displays a schematic sRaw loop in a normal patient during tidal breathing, which is shown after numerical software compensations to close the sRaw loop. Normal patients manifest a steep linear loop during tidal breathing without hysteresis. In contrast, during voluntary panting efforts, the upper and lower end portions of the loop

The curvilinearity is in the form of a very slight "S" shape, analogous to that shown in tracing "(d)", but much less exaggerated. In normal patients during voluntary panting, the flattening of the sRaw loop at the upper right extremity (mid-inspiration) and at the lower left extremity (mid-expiration) of the loop are only barely visible, depending on the absolute

Tracing "(b)" is typical of patients with large (central) airway constriction that is relatively uniform (and not a localized stenosis) and without significant small airway obstruction. This

It is well known that expiratory flow limitation and dynamic airway compression may occur during tidal breathing in COPD, and this contributes to the characteristic shape of the sRaw

Tracing "(d)" shows the influence of a fixed or functional stenosis of the upper airways, for example laryngeal abnormality, or paralysis of one vocal cord. This type of "orifice" constriction manifests flow limitation during inspiration, such that, at sufficiently high flows, further increases in driving pressure do not result in any increase in airflow. This reflects localized upper airway obstruction, analogous to that which pertains in the maximal expiratory flow–volume curve. Thus, during forced expiration, when a critical driving pressure for expiratory airflow (intra-pleural pressure for forced expiration) is achieved, further increases in driving pressure do not cause any further increases in flow rate. A similar flow limitation may occur in the extra-thoracic airway during inspiration, as shown

near 1.0 in patients with normal lungs, or even with restriction.

methods are compared.

**4.2.2 RAW** 

panting rates aggravate this inaccuracy.

molecules in contact with the airways.

**4.2.2.1 Clinical Significance** 

may become slightly curvilinear.

value of flow rates achieved.

loop in tracing "(c)".

might be seen in a patient with mild asthma.

in the upper right portion of tracing "(d)".

Fig. 2. A schematic presentation of the flow / volume shift measuring Raw

## **5. Functional Residual Capacity (FRC)**

The FRC is the volume of air left in the lungs at the end of a quiet exhalation. There are 2 methods of measuring FRC which are (1) helium dilution and (2) the Nitrogen washout technique.

#### **5.1 Helium dilution**

The method for measuring lung volumes is based on the equilibration of gas in the lung with a known volume of gas containing helium. The test gas consists of air with added oxygen of 25–30%, but higher concentrations are acceptable.

#### **5.1.1 Measurement technique (ATS / ERS, 2005)**

Specific details of procedures will vary with different types of equipment and degrees of automation, but the basic procedure is as follows.


### **5.2 Nitrogen washout technique (ATS / ERS, 2005)**

This technique is based on washing out the N2 from the lungs, while the patient breathes 100% O2. The initial alveolar N2 concentration and the amount of N2 washed out can then be used to calculate the lung volume at the start of washout. The technique originally utilized gas collections for a 7-min period, a period deemed adequate for washout of N2 from the lungs of healthy subjects. The measurement technique should adhere to the following steps:


Fig. 3. A schematic presentation of the single breath DLCO manoeuvre

## **6. Diffusing capacity (DLCO)**

## **6.1 Definition**

590 Lung Diseases – Selected State of the Art Reviews

10. Helium equilibration is considered to be complete when the change in helium concentration is, 0.02% for 30 s. The test rarely exceeds 10 min, even in patients with

11. Once the helium equilibration is complete, the patient is turned ''out'' (i.e. disconnected from the test gas) of the system. If the measurements of ERV and IC are to be linked to the FRC measured, it should be ensured that the spirometer has an adequate volume for

12. At least one technically satisfactory measurement should be obtained. Due to the extra costs and time in making multiple measurements, and the relatively good inter-day variability in adults, two or more measurements of FRCHe need to be made only when necessitated by clinical or research need . If only one measurement of FRCHe is made, caution should be used in the interpretation. For younger children, however, it is recommended that at least two technically satisfactory measurements be performed. If more than one measurement of FRCHe is carried out, the value reported for FRCHe

This technique is based on washing out the N2 from the lungs, while the patient breathes 100% O2. The initial alveolar N2 concentration and the amount of N2 washed out can then be used to calculate the lung volume at the start of washout. The technique originally utilized gas collections for a 7-min period, a period deemed adequate for washout of N2 from the lungs of healthy subjects. The measurement technique should adhere to the following steps: 1. The equipment should be turned on and allowed an adequate warm-up time, with

2. The patient should be asked if he/she has a perforated eardrum (if so, an earplug

3. The patient is seated comfortably, with no need to remove dentures. The procedure is explained, emphasizing the need to avoid leaks around the mouthpiece during the

4. The patient breathes on the mouthpiece for 30–60 s to become accustomed to the

5. When breathing is stable and consistent with the end-tidal volume being at FRC, the patient is switched into the circuit so that 100% O2 is inspired instead of room air. 6. The N2 concentration is monitored during the washout. A change in inspired N2 of .1% or sudden large increases in expiratory N2 concentrations indicate a leak; hence, the test

8. At least one technically satisfactory measurement should be obtained. If additional washouts are performed, a waiting period of ≥ 15 min is recommended between trials. In patients with severe obstructive or bullous disease, the time between trials should be ≥ 1 h, if more than one measurement of FRCN2 is made, the value reported for FRCN2 should be the mean of technically acceptable results that agree within 10%. If only one measurement of FRCN2 is made, caution should be used in the

should be stopped and repeated after a 15-min period of breathing room air. 7. The washout is considered to be complete when the N2 concentration is, 1.5% for at

should be the mean of technically acceptable results that agree within 10%.

9. The helium concentration is noted every 15 s.

severe gas-exchange abnormalities [9].

the full ERV and IVC manoeuvres (fig. 5).

**5.2 Nitrogen washout technique (ATS / ERS, 2005)** 

calibration as instructed by the manufacturer.

apparatus, and to assure a stable end-tidal expiratory level.

should be used).

washout and using a nose clip.

least three successive breaths.

interpretation.

DLCO measures the transfer of a diffusion-limited gas (CO) across the alveoli capillary membranes. DLCO is reported in millilitres of CO/minute/ml of Mercury at STPD.

## **6.2 Technique**

CO combines with Haemoglobin (Hb) approximately 210 times more readily than O2. In the presence of normal amounts of Hb and normal ventilator function, the primary limiting factor to diffusion of CO is the status of alveolocapillary membranes.

Diffusing capacity can be affected by factors that change the membrane component, as well as by alterations in Hb and in the capillary blood volume. DLCO is used to assess the gas exchange ability of the lungs, specifically oxygenation of mixed venous blood.

DLCO is used to evaluate pulmonary involvement in systemic diseases such as rheumatoid arthritis. DLCO measurements are often included in the evaluation of patients with obstructive lung disease such as emphysema.

DLCO may be indicated to monitor changes in lung function induced by drugs used to treat cardiac arrhythmias as well as changes caused by chemo and radiation therapy for lung cancer.


Table 4. A list of DLCO indications (Ruppel, 2009)

The most commonly used method is the single-breath or breath-hold technique. The singlebreath method is also the most widely standardized.

## **6.3 Significance and pathophysiology**

The expected DLCO value in a healthy patient varies directly with the patient's lung volume. Women have slightly lower normal values, presumably because of smaller normal lung volumes. DLCO values can increase 2-3 times in healthy individuals during exercise in response to increased pulmonary capillary blood flow.

DLCO is often decreased in restrictive lung diseases, particularly those associated with pulmonary fibrosis. Fibrotic changes in the lung parenchyma are associated with asbestosis, berylliosis, and silicosis. Idiopathic pulmonary fibrosis, sarcoidosis, SLE, scleroderma are associated with a decreased DLCO. Inhalation of toxic gases causes alveolitis and a decrease in DLCO values.

A decrease in DLCO is more likely to be related to the loss of lung volume, alveolar surface area, or capillary bed than to thickening of the alveolocapillary membrane. DLCO also decrease when there is a loss of lung tissue or replacement of normal parenchyma by space occupying lesions such as tumours. DLCO may also be reduced in the presence of pulmonary oedema.

Low resting DLCO (less than 50-60 % of predicted) may indicate the need for assessment of oxygenation during exercise. DLCO is directly related to lung volume (VA) in healthy individuals. DL / VA relationship can be useful to differentiate whether decreased DLCO is the result of loss of lung volume or from some other causes.

In obstruction, low DLCO without reduction in VA results in a low ratio. In a purely restrictive process, a decrease in DLCO reflects loss of VA and the DL / VA ratio is preserved.

Numerous other physiologic factors can influence the observed DLCO:


#### **6.4 Interpretive strategies (Ruppel, 2009)**


## **7. Blood gases**

592 Lung Diseases – Selected State of the Art Reviews

The most commonly used method is the single-breath or breath-hold technique. The single-

The expected DLCO value in a healthy patient varies directly with the patient's lung volume. Women have slightly lower normal values, presumably because of smaller normal lung volumes. DLCO values can increase 2-3 times in healthy individuals during exercise in

DLCO is often decreased in restrictive lung diseases, particularly those associated with pulmonary fibrosis. Fibrotic changes in the lung parenchyma are associated with asbestosis, berylliosis, and silicosis. Idiopathic pulmonary fibrosis, sarcoidosis, SLE, scleroderma are associated with a decreased DLCO. Inhalation of toxic gases causes alveolitis and a decrease

A decrease in DLCO is more likely to be related to the loss of lung volume, alveolar surface area, or capillary bed than to thickening of the alveolocapillary membrane. DLCO also decrease when there is a loss of lung tissue or replacement of normal parenchyma by space occupying lesions such as tumours. DLCO may also be reduced in the presence of

Low resting DLCO (less than 50-60 % of predicted) may indicate the need for assessment of oxygenation during exercise. DLCO is directly related to lung volume (VA) in healthy individuals. DL / VA relationship can be useful to differentiate whether decreased DLCO is

In obstruction, low DLCO without reduction in VA results in a low ratio. In a purely restrictive






process, a decrease in DLCO reflects loss of VA and the DL / VA ratio is preserved.

Numerous other physiologic factors can influence the observed DLCO:

likely that a gas exchange abnormality exists. Evaluate DL / VA

breath method is also the most widely standardized.

response to increased pulmonary capillary blood flow.

the result of loss of lung volume or from some other causes.

**6.3 Significance and pathophysiology** 

in DLCO values.

pulmonary oedema.





**6.4 Interpretive strategies (Ruppel, 2009)** 

uneven distribution of ventilation.

consider undiagnosed asthma.

exercise desaturation study.

hypertension. Consider clinical correlation.

Blood gas is the most basic test of lung function. Blood gas analysis is often done in conjunction with pulmonary function studies. Blood is drawn from a peripheral artery without being exposed to air (anaerobically). Blood gas analysis includes measurement of ph, pCO2, and pO2.

The same specimen may be used for blood oximetry to measure total Hb, oxyhaemoglobin (O2Hb), carboxyhaemoglobin (COHb) and methaemoglobin (MetHb). Blood gas is the ideal measurement of pulmonary function because is assesses the two primary functions of the lung – oxygenation and carbon dioxide removal.

## **7.1 Indications**


## **7.2 3 Most important variables in a blood gas result:**


### **7.3 Significance and pathophysiology**

pH < 7.35 = Acidemia

pH > 7.45 = Alkalemia

Acid-base disorders arising from lung disease are often related to PCO2 and its transport as carbonic acid.


#### **- Acid-base disorders -**

Table 5. Three helpful parameters in interpreting a blood-gas result (in the simple uncompensated state- Ruppel, 2009)

pO2 is the pressure of O2 dissolved in blood. The amount of Hb and whether it is capable of binding O2 has only a minimal effect on pO2. Hypoxemia commonly results from inadequate or abnormal Hb. The severity of impaired oxygenation is indicated by the PaO2 at rest. PaO2 is a good index of the lungs 'ability to match pulmonary capillary blood flow with adequate ventilation.

Delivery of O2 to the tissues however depends on Hb concentration and cardiac output as well as adequate gas transfer in the lungs. Because most O2 transported is bound to Hb, there must be an adequate supply (12-15 g/dl) of functional Hb.

## **8. Pulse oximetry**

SpO2 estimates SaO2 by analyzing absorption of light passing through a capillary bed, either by transmission or reflectance. Pulse oximetry is non-invasive.

## **8.1 Interfering factors**

Motion artefact, shivering, bright ambient lightning, hypotension, low perfusion, hypothermia, vasoconstrictor drugs and dark skin pigmentation can confound.

## **8.2 Significance and pathophysiology**

Most pulse oximeters are capable of accuracy of +/- 2% of actual saturation when SaO2 is above 90%. Other uses of pulse oximetry are:

	-

Pulse oximetry may not be appropriate in all situations e.g. to evaluate hyperoxemia or acidbase status in a patient, a blood gas analysis is required. Measurement of O2 delivery, which depends on Hb concentration, cannot be adequately assessed by pulse oximetry.

## **9. Six minute walk test (6MWT)**

This is a simple exercise test used to assess the response to a medical or surgical intervention, but also been used to assess functional capacity as well as to estimate morbidity and mortality. This test doesn't require any sophisticated equipment.


Table 6. A list of indications for a six minute walk test (Ruppel, 2009)


Table 7. A list of the contraindications of a six minute walk test (Ruppel, 2009)

Reasons for immediately stopping a 6MWT include the following:

1. chest pain,

594 Lung Diseases – Selected State of the Art Reviews

Delivery of O2 to the tissues however depends on Hb concentration and cardiac output as well as adequate gas transfer in the lungs. Because most O2 transported is bound to Hb,

SpO2 estimates SaO2 by analyzing absorption of light passing through a capillary bed, either

Motion artefact, shivering, bright ambient lightning, hypotension, low perfusion,

Most pulse oximeters are capable of accuracy of +/- 2% of actual saturation when SaO2 is

Pulse oximetry may not be appropriate in all situations e.g. to evaluate hyperoxemia or acidbase status in a patient, a blood gas analysis is required. Measurement of O2 delivery, which

This is a simple exercise test used to assess the response to a medical or surgical intervention, but also been used to assess functional capacity as well as to estimate

INDICATIONS FOR THE SIX-MINUTE WALK TEST

hypothermia, vasoconstrictor drugs and dark skin pigmentation can confound.


depends on Hb concentration, cannot be adequately assessed by pulse oximetry.

morbidity and mortality. This test doesn't require any sophisticated equipment.

there must be an adequate supply (12-15 g/dl) of functional Hb.

by transmission or reflectance. Pulse oximetry is non-invasive.


**8. Pulse oximetry** 

**8.1 Interfering factors** 


**8.2 Significance and pathophysiology** 

**9. Six minute walk test (6MWT)** 

Lung transplantation / Resection Lung volume reduction surgery

Pulmonary rehabilitation

Pulmonary hypertension

Peripheral vascular disease

Predictor of morbidity and mortality

Table 6. A list of indications for a six minute walk test (Ruppel, 2009)

COPD

Heart failure Cystic fibrosis Heart failure

Pre-treatment and post treatment comparisons

above 90%. Other uses of pulse oximetry are:


Technicians must be trained to recognize these problems and the appropriate responses. If a test is stopped for any of these reasons, the patient should sit or lie supine as appropriate depending on the severity or the event and the technician's assessment of the severity of the event and the risk of syncope.The following should be obtained based on the judgment of the technician: blood pressure, pulse rate, oxygen saturation, and a physician evaluation. Oxygen should be administered as appropriate.

### **9.1 Equipment required**


### **9.2 Measurements (ATS / ERS 2002)**


Note pulse regularity and whether the oximeter signal quality is acceptable. The rationale for measuring oxygen saturation is that although the distance is the primary outcome measure, improvement during serial evaluations may be manifest either by an increased distance or by reduced symptoms with the same distance walked. The SpO2 should not be used for constant monitoring during the exercise. The technician must not walk with the patient to observe the SpO2.

If worn during the walk, the pulse oximeter must be lightweight (less than 2 pounds), battery powered, and held in place (perhaps by a "fanny pack") so that the patient does not have to hold or stabilize it and so that stride is not affected.


"The object of this test is to walk as far as possible for 6 minutes. You will walk back and forth in this hallway. Six minutes is a long time to walk, so you will be exerting yourself. You will probably get out of breath or become exhausted. You are permitted to slow down, to stop, and to rest as necessary. You may lean against the wall while resting, but resume walking as soon as you are able. You will be walking back and forth around the cones. You should pivot briskly around the cones and continue back the other way without hesitation. Now I'm going to show you. Please watch the way I turn without hesitation." Demonstrate by walking one lap yourself. Walk and pivot around a cone briskly. "Are you ready to do that? I am going to use this counter to keep track of the number of laps you complete. I will click it each time you turn around at this starting line. Remember that the object is to walk AS FAR AS POSSIBLE for 6 minutes, but don't run or jog. Start now or whenever you are ready."


Table 8. The Borg scale

not walk with the patient to observe the SpO2.

Borg scale

0 Nothing at all

1 Very slight 2 Slight (light) 3 Moderate

7 Very severe

6

8 9

4 Somewhat severe 5 Severe (heavy)

Table 8. The Borg scale

0.5 Very, very slight (just noticeable)

10 Very, very severe (maximal)

7. Instruct the patient as follows:

not have to hold or stabilize it and so that stride is not affected.

but don't run or jog. Start now or whenever you are ready."

Note pulse regularity and whether the oximeter signal quality is acceptable. The rationale for measuring oxygen saturation is that although the distance is the primary outcome measure, improvement during serial evaluations may be manifest either by an increased distance or by reduced symptoms with the same distance walked. The SpO2 should not be used for constant monitoring during the exercise. The technician must

If worn during the walk, the pulse oximeter must be lightweight (less than 2 pounds), battery powered, and held in place (perhaps by a "fanny pack") so that the patient does

5. Have the patient stand and rate their baseline dyspnoea and overall fatigue using the

6. Set the lap counter to zero and the timer to 6 minutes. Assemble all necessary equipment (lap counter, timer, clipboard, Borg Scale, worksheet) and move to the starting point.

**THE BORG SCALE** 

"The object of this test is to walk as far as possible for 6 minutes. You will walk back and forth in this hallway. Six minutes is a long time to walk, so you will be exerting yourself. You will probably get out of breath or become exhausted. You are permitted to slow down, to stop, and to rest as necessary. You may lean against the wall while resting, but resume walking as soon as you are able. You will be walking back and forth around the cones. You should pivot briskly around the cones and continue back the other way without hesitation. Now I'm going to show you. Please watch the way I turn without hesitation." Demonstrate by walking one lap yourself. Walk and pivot around a cone briskly. "Are you ready to do that? I am going to use this counter to keep track of the number of laps you complete. I will click it each time you turn around at this starting line. Remember that the object is to walk AS FAR AS POSSIBLE for 6 minutes, At the beginning of the 6-minute exercise, show the scale to the patient and ask the patient this: "Please grade your level of shortness of breath using this scale." Then ask this: "Please grade your level of fatigue using this scale." At the end of the exercise, remind the patient of the breathing number that they chose before the exercise and ask the patient to grade their breathing level again. Then ask the patient to grade their level of fatigue, after reminding them of their grade before the exercise.


## **9.3 Interpretation**

Most 6MWTs will be done before and after intervention, and the primary question to be answered after both tests have been completed is whether the patient has experienced a clinically significant improvement. With a good quality-assurance program, with patients tested by the same technician, and after one or two practice tests, short-term reproducibility of the 6MWD is excellent. It is not known whether it is best for clinical purposes to express change in 6MWD as


Until further research is available, we recommend that change in 6MWD be expressed as an absolute value (e.g., the patient walked 50 m farther).

## **10. Stair climbing**

In a setting where corridor length is limited, but a few flights of stairs are available, the stair climb is an ideal test as a primary screening test to decide if a patient can undergo thoracic surgery or needs additional testing e.g. Cardio Pulmonary Exercise Testing.

The stair climb as a test is easy to perform and easy to understand by patients as well as being safe. Minimal equipment and personnel is required. To do the test one instructs the patient to climb the flights of stairs at their fastest pace to a minimum ascent of +/- 20 metre. This climb is timed so as to calculate the speed of ascent, which correlates well with VO2max measured during cycle ergometry. It was shown that climbing at a speed of ascent of ≥15m/min to an elevation of 20 metres accurately predicted a VO2max ≥ 20ml/kg/min. (Koegeleneberg, 2009)

## **11. Cardiopulmonary Exercise Testing (CPET)**

Cardiopulmonary exercise testing is used to define work limitations. Cardiopulmonary variables are assessed in relation to the workload. The patterns of change in any particular variable are then compared with the expected normal response. The primary indications for performing this test are dyspnoea and exertion, pain (especially angina) and fatigue. Exercise induces airway narrowing in the majority of patients with asthma. Exerciseinduced airway narrowing is called exercise- induced asthma (EIA) and exercise-induced bronchoconstriction (EIB).

Other indications include:


Exercise testing can detect the following:


The preferred modes of exercise are the motor-driven treadmill with adjustable speed and grade or the electromagnetically braked cycle ergometer. Heart rate should be monitored from a three-lead electrocardiographic configuration as a minimum.

Alternatively, a pulse oximeter or other device able to reliably determine heart rate may be used. For those at higher risk for coronary artery disease, a 12- lead ECG configuration is advisable.

## **11.1 Treadmill protocol (ATS / ERS 1999)**

The treadmill speed and gradient are chosen to produce 4–6 min of exercise at nearmaximum targets with a total duration of exercise of 6–8 min. For children less than 12 yr of age, the time is usually 6 min; for older children and adults the time is usually 8 min. starting at a low speed and gradient, both are progressively advanced during the first 2–3 min of exercise until the heart rate is 80–90% of the predicted maximum. Ventilation rather than heart rate can be used to monitor exercise intensity. Ventilation should reach 40–60% of the predicted maximum voluntary ventilation (MVV, estimated as FEV1 x 35). The degree of physical fitness and body weight will strongly influence the grade and speed necessary to obtain the desired heart rate. A reasonable procedure is to quickly advance to a rapid, but comfortable, speed and then raise the treadmill slope until the desired heart rate or ventilation is obtained.

≥15m/min to an elevation of 20 metres accurately predicted a VO2max ≥ 20ml/kg/min.

Cardiopulmonary exercise testing is used to define work limitations. Cardiopulmonary variables are assessed in relation to the workload. The patterns of change in any particular variable are then compared with the expected normal response. The primary indications for performing this test are dyspnoea and exertion, pain (especially angina) and fatigue. Exercise induces airway narrowing in the majority of patients with asthma. Exerciseinduced airway narrowing is called exercise- induced asthma (EIA) and exercise-induced

(Koegeleneberg, 2009)

bronchoconstriction (EIB). Other indications include:

advisable.

ventilation is obtained.

**11. Cardiopulmonary Exercise Testing (CPET)** 



Exercise testing can detect the following:




**11.1 Treadmill protocol (ATS / ERS 1999)** 




from a three-lead electrocardiographic configuration as a minimum.

The preferred modes of exercise are the motor-driven treadmill with adjustable speed and grade or the electromagnetically braked cycle ergometer. Heart rate should be monitored

Alternatively, a pulse oximeter or other device able to reliably determine heart rate may be used. For those at higher risk for coronary artery disease, a 12- lead ECG configuration is

The treadmill speed and gradient are chosen to produce 4–6 min of exercise at nearmaximum targets with a total duration of exercise of 6–8 min. For children less than 12 yr of age, the time is usually 6 min; for older children and adults the time is usually 8 min. starting at a low speed and gradient, both are progressively advanced during the first 2–3 min of exercise until the heart rate is 80–90% of the predicted maximum. Ventilation rather than heart rate can be used to monitor exercise intensity. Ventilation should reach 40–60% of the predicted maximum voluntary ventilation (MVV, estimated as FEV1 x 35). The degree of physical fitness and body weight will strongly influence the grade and speed necessary to obtain the desired heart rate. A reasonable procedure is to quickly advance to a rapid, but comfortable, speed and then raise the treadmill slope until the desired heart rate or For older children and adults 8 min of exercise is usually required to elicit EIB when dry air temperature is inhaled. A treadmill speed greater than 3 mph (about 4.5 km/h) and a gradient greater than 15% or an oxygen consumption of 35 ml/min/kg or greater will usually achieve the target ventilation or heart rate in young healthy subjects. Nomograms have been proposed to predict speed and grade that will elicit the desired heart rate, but they have not been extensively validated. It may be preferable to use nomograms relating oxygen consumption per kilogram to speed and slope of the treadmill.

The test ends when the patient has exercised at the target ventilation or heart rate for at least 4 min. This usually requires a total of 6–8 min of exercise. The test may be terminated by the patient at any time.

#### **11.1.1 Assessing the response**

Forced expiratory volume in 1 s (FEV1) is the primary outcome variable. Spirometry should be performed and evaluated as described earlier. One exception to ATS-recommended techniques for spirometry is allowed. If the only outcome variable to be used is the FEV1, the duration of the expiration may be limited to 2–3 s. In all cases it is important to vigorously coach the patient to inhale fully even in the presence of chest tightness. Incomplete inhalations will result in false reductions in FEV1.

If vocal cord dysfunction or other possible causes of central airway obstruction are suspected, full inspiratory and expiratory flow–volume loops should be obtained.

An appropriate post-exercise testing schedule is 5, 10, 15, 20, and 30 min after cessation of exercise. Some investigators include earlier measurements (1 and 3 min post-exercise) because severe EIB can sometimes be present at the cessation of exercise. Early recognition allows it to be dealt with promptly. If the FEV1 has returned from its nadir to the baseline level or greater, spirometry testing may be terminated at 20 min post exercise. A β-agonist bronchodilator may be administered at any time to reverse the bronchoconstrictive response if the patient experiences appreciable dyspnoea, or if the FEV1 has not recovered to within 10% of baseline when the patient is ready to leave the laboratory.

The presence of exercise-induced bronchoconstriction is defined by plotting FEV1 as a percentage of the pre-exercise baseline FEV1 at each post-exercise interval.

A decrease below 90% of the baseline FEV1 (i.e., a 10% decrease) is a generally accepted abnormal response. Some authors suggest a value of 15% is more diagnostic of EIB, particularly if exercise has been performed in the field.

#### **12. Bronchial provocation testing**

Bronchial challenge testing is used to identify and characterize airway hyperresponsiveness. Challenge test may be performed in patients with symptoms of bronchospasm who have normal pulmonary function studies or uncertain results of bronchodilator studies. It can also be used to assess changes in hyper-reactivity of the airways or to quantify its severity.

Several commonly used provocative agents can be used to assess airway hyper reactivity. These include the following:


#### - Exercise

FEV1 is the variable most commonly used, although airway resistance and specific conductance can also be measured before and after testing.

## **12.1 Dosing protocols (ATS /ERS 1999)**

	- a. Set up and check the dosimeter.
	- b. Prepare the following five concentrations of methacholine in sterile vials; place them in a holder; and store them in a refrigerator.
	- c. Remove the vials from the refrigerator 30 min before testing, so that the contents warm to room temperature before use. Insert 2.0 ml of the first concentration into the nebulizer, using a sterile syringe. The patient is seated throughout the test.
	- d. Perform baseline spirometry.
	- e. Briefly open the dosimeter solenoid to make sure the nebulizer is nebulising.
	- f. Ask the patient to hold the nebulizer upright with the mouthpiece in his/her mouth. Watch the patient during the breathing manoeuvres to ensure that the inhalation and breath hold are correct and that the nebulizer is not tipped.
	- g. At end exhalation during tidal breathing (functional residual capacity), instruct the patient to inhale slowly and deeply from the nebulizer. Trigger the dosimeter soon after the inhalation begins; dosimeters may do this automatically. Encourage the patient to continue inhaling slowly (about 5 s to complete the inhalation) and to hold the breath (at total lung capacity, TLC) for another 5 s.
	- h. Repeat step g for a total of five inspiratory capacity inhalations. Take no more than a total of 2 min to perform these five inhalations.
	- i. Measure the FEV1 at about 30 and 90 s after the fifth inhalation from the nebulizer. Obtain an acceptable-quality FEV1 at each time point. This may require repeated attempts. Perform no more than three or four manoeuvres after each dose. It should take no more than 3 min to perform these manoeuvres. To keep the cumulative effect of methacholine relatively constant, the time interval between the commencements of two subsequent concentrations should be kept to 5 min.
	- j. At each dose, report the highest FEV1 from acceptable manoeuvres.
	- k. If the FEV1 falls less than 20%, empty the nebulizer, shake it dry, and trigger the dosimeter once to dry the nebulizer nozzle. Add 2.0 ml of the next higher concentration, and repeat steps g–j.
	- l. If the FEV1 falls more than 20% from baseline (or the highest concentration has been given), give no further methacholine, note signs and symptoms, administer inhaled albuterol, wait 10 min, and repeat the spirometry.

#### **12.2 Interpretive strategies**


#### **13. Conclusion**

600 Lung Diseases – Selected State of the Art Reviews

FEV1 is the variable most commonly used, although airway resistance and specific

b. Prepare the following five concentrations of methacholine in sterile vials; place

c. Remove the vials from the refrigerator 30 min before testing, so that the contents warm to room temperature before use. Insert 2.0 ml of the first concentration into the nebulizer, using a sterile syringe. The patient is seated throughout the test.

e. Briefly open the dosimeter solenoid to make sure the nebulizer is nebulising. f. Ask the patient to hold the nebulizer upright with the mouthpiece in his/her mouth. Watch the patient during the breathing manoeuvres to ensure that the

inhalation and breath hold are correct and that the nebulizer is not tipped. g. At end exhalation during tidal breathing (functional residual capacity), instruct the patient to inhale slowly and deeply from the nebulizer. Trigger the dosimeter soon after the inhalation begins; dosimeters may do this automatically. Encourage the patient to continue inhaling slowly (about 5 s to complete the inhalation) and to

h. Repeat step g for a total of five inspiratory capacity inhalations. Take no more than

i. Measure the FEV1 at about 30 and 90 s after the fifth inhalation from the nebulizer. Obtain an acceptable-quality FEV1 at each time point. This may require repeated attempts. Perform no more than three or four manoeuvres after each dose. It should take no more than 3 min to perform these manoeuvres. To keep the cumulative effect of methacholine relatively constant, the time interval between the

commencements of two subsequent concentrations should be kept to 5 min.

k. If the FEV1 falls less than 20%, empty the nebulizer, shake it dry, and trigger the dosimeter once to dry the nebulizer nozzle. Add 2.0 ml of the next higher

l. If the FEV1 falls more than 20% from baseline (or the highest concentration has been given), give no further methacholine, note signs and symptoms, administer

1. Were there any factors present that could influence the result e.g. failure to withhold

3. Were spirometric efforts repeatable and acceptable before and during the challenge? If

j. At each dose, report the highest FEV1 from acceptable manoeuvres.

inhaled albuterol, wait 10 min, and repeat the spirometry.

2. Respiratory infection? If so interpret cautiously or not at all.

hold the breath (at total lung capacity, TLC) for another 5 s.

a total of 2 min to perform these five inhalations.

concentration, and repeat steps g–j.

not, interpret very cautiously or not at all.

**12.2 Interpretive strategies** 

bronchodilators ?

conductance can also be measured before and after testing.

them in a holder; and store them in a refrigerator.

**12.1 Dosing protocols (ATS /ERS 1999)**  1. Two-minute tidal breathing dosing protocol.

a. Set up and check the dosimeter.

d. Perform baseline spirometry.

2. Five-breath dosimeter protocol.


There are a variety of tests, both simple and sophisticated, that allow greater insights into respiratory disease, functional impairment and suitability for operative intervention. Some of the tests are more complicated to perform and both improvement and deterioration in respiratory status can be subtle. For these reasons strict quality control, in terms of appropriately validated equipment, standardisation and trained and experienced personnel are mandatory. To ensure excellent quality results good patient co-operation is required as well. Disregard for these precautions can lead to diagnostic confusion, misclassification of improvement and deterioration or unnecessary further interventions. These can be costly and also result in needless morbidity and mortality. Thus a full understanding of the tests, their appropriateness and pitfalls can complement patient evaluation and result in greater diagnostic certainty and patient management.

#### **14. References**


## **Novel Methods for Diagnosis of Pulmonary Microangiopathy in Diabetes Mellitus**

Kalicka Renata and Kuziemski Krzysztof

*Gdansk University of Technology, Department of Biomedical Engineering, Medical University of Gdansk, Department of Allergology, Poland* 

#### **1. Introduction**

602 Lung Diseases – Selected State of the Art Reviews

Ruppel, G.L. 2009. Manual of Pulmonary Function Testing. Ruppel. G.L (ed.). 9th ed. St

Wanger J, Clausen JL, Coates A. Standardisation of the Measurement of Lung Volumes*. Eur* 

172,291

*Respir J* 2005; 26: 511-522.

Louis Missouri: Mosby Elsevier. p.p.3,7-10,68-70,134,144-146,158,159,162-168,170-

Lung microangiopathy is a little known negative influence of diabetes mellitus on the functioning of the lungs. In current medical practice lung microangiopathy is diagnosed by comparing two measurements of lung diffusing capacity – one with the subject standing and one with the subject lying. The necessity to take two measurements is inconvenient.

In lung microangiopathy we observe a reduction of diffusing capacity, lung flow and volume. Diabetes is a chronic illness that can lead to diabetic angiopathy, a pathology of the blood vessels (arteries, veins and capillaries). There are two types of diabetic angiopathy: macroangiopathy (disease of the larger blood vessels) and microangiopathy (microvascular pathology). The examples of angiopathy include: neuropathy, nephropathy and retinopathy.

Current knowledge regarding diabetic lung microangiopathy is limited. Histopathological examination of lung biopsy samples is not a conclusive test of the consequences of diabetes (Dalquen, 1999). Animal experiments and post-mortem examinations have disclosed the influence of diabetes on the lung capillaries and alveolar-capillary membranes (Kida et al, 1993), (Kodolova et al, 1982) and (Popov& Simionescu, 1997). Histopathological tests have revealed the thickening of the alveolar and venous capillary walls (Matsubara& Hara, 1991) and (Weynand et al, 1999).

Lung diffusing capacity measurements illustrate the state of alveolar-capillary barrier (Goldman, 2003). These are measurements of diffusion across the alveolar-capillary membrane. For diagnosing microangiopathy, the lung diffusing capacity is measured in two body positions: standing ( standing *DL* ) and lying on the back ( lying *DL* ), (Strojek et al, 1993) and (Kuziemski et al, 2008). On account of the human anatomic structure, diffusing capacity depends on the body's position. For healthy subjects the diffusing capacity increases in the reclined position, lying standing *D D L L* . The opposite is observed in the case of microangiopathic patients: the diffusing capacity decreases when the subject is lying lying standing *D D L L* . This is the result of blood vessel damage and alveolar thickening caused by diabetes (Kuziemski et al, 2009). Only the fact that diffusing capacity increases or decreases in a given position is important as far as microangiopathy diagnosis is concerned.

Spirometry (measurement of the volume and flow of inhaled and exhaled air) is the most common pulmonary function test. It is helpful in diagnosing asthma, pulmonary fibrosis, cystic fibrosis, and COPD (Chronic Obstructive Pulmonary Disease).

Research (Goldman, 2003), (Kaminski, 2004) has shown that a single spirometry test does not provide sufficient information for a diagnosis of lung flow limitation and lung volume reduction caused by diabetes mellitus. Instead we need long-term observations of spirometry results to diagnose lung microangiopathy. Microangiopathy lung impairment is characterised by a decrease in spirometric parameters *FVC* (the maximum volume of air that can be exhaled or inspired during a forced manoeuvre) and *FEV*1 (the forced expiratory volume during the first second of expiration) (Davis et al, 2004), (Litonjua et al, 2005), (Yeh et al, 2008).

A later study (Kuziemski et al, 2009) proved that a single spirometric test is insufficient to identify lung microangiopathy on account of functional reserve breathing. The reserves compensate, to some extent, the lung dysfunction, and thus the negative effect of diabetes is concealed until the effect reaches a higher level of impairment.

Since the direct measurements of pulmonary function are not conclusive and post-mortem autopsies do not serve the given patient, the diagnostics have to be based on such indirect results as spirometric measurements and modelling. It is probably also capable of revealing lung microangiopathy if the results are very carefully analysed.

Perfusion computed tomography (pCT) is a diagnostic method that enables the imaging of the organ and tissue. This is a powerful tool for diagnosing perfusion of internal organs such as the brain, liver, pancreas, spleen, kidneys and lungs (Alonzi & Hoskin, 2006), (Blomley et al, 1993), (Cao, 2011), (Eichinger et al, 2010), (Kuziemski et al, 2011). The method enables quantitative evaluation of circulation by determining changes in tissue during the flow of a contrast agent in the blood vessels. Changes, when compared to a normal tissue, can indicate tissue pathology. Lung pCT measurements can help in the diagnosis of cystic fibrosis (Eichinger et al, 2010) and diabetes (Kuziemski et al, 2011). It can also help to differentiate benign pulmonary nodules from lung cancer (Alonsi & Hoskin, 2006).

The technique of lung pCT involves the intravenous injection of a non-iodinated contrast agent and the sequential scanning of the diagnosed region of the chest. There are a number of pulmonary perfusion parameters, calculated pixel by pixel on the basis of raw CT data, which are next analysed in order to reveal differences between the normal and altered tissue. The most useful perfusion parameters are: blood volume (BV), blood flow (BF), mean transit time (MTT), time to peak (TTP) and permeability surface (PS) (Kuziemski et al, 2011). The diagnosis of pulmonary function has to be based on indirect results, such as diffusing

capacity, spirometry and pCT because direct assessment is difficult.

#### **2. Materials**

The tests were performed on a group of 72 diabetics. People with cardiovascular disease and smokers were not included in this group.

First the diabetics were tested for microangiopathy, by comparing diffusing capacity in standing and lying position, and on this basis they were split into two groups: 44 *angiop M* and 28 *non angiop M* , with and without microangiopathy respectively.

The spirometric tests were carried out on all the patients ( *angiop no an* n- *giop MM M* ) to obtain the following spirometric parameters: *FEV*1 (forced expiratory volume during the first second of expiration), *FVC* (the maximum volume of air that can be exhaled or inspired during a forced manoeuvre), *PEF* (peak expiratory flow), *MEF*50 (maximal instantaneous forced expiratory flow where 50% of the *FVC* remains to be expired), *MEF*75/25 (maximal mid-expiratory flow), *IC* (inspiratory capacity) and *FEV FVC* 1% (percentage relation of *FEV*1 to *FVC* ).

The pCT test was performed on a group of 18 subjects: 10 diabetics and 8 non-diabetic volunteers. The local perfusion parameters *BF* (blood flow), *BV* (blood volume), *MTT* (mean transit time) and *PS* (permeability surface) were obtained in selected regions of interest (ROI) in the artery and parenchyma.

The patients classified as suffering from microangiopathy had breathing impairment symptoms cause only by diabetes. They were all non-smokers and had not been diagnosed with any other acute or chronic respiratory disease.

## **3. Diffusing capacity**

604 Lung Diseases – Selected State of the Art Reviews

Research (Goldman, 2003), (Kaminski, 2004) has shown that a single spirometry test does not provide sufficient information for a diagnosis of lung flow limitation and lung volume reduction caused by diabetes mellitus. Instead we need long-term observations of spirometry results to diagnose lung microangiopathy. Microangiopathy lung impairment is characterised by a decrease in spirometric parameters *FVC* (the maximum volume of air that can be exhaled or inspired during a forced manoeuvre) and *FEV*1 (the forced expiratory volume during the first second of expiration) (Davis et al, 2004), (Litonjua et al,

A later study (Kuziemski et al, 2009) proved that a single spirometric test is insufficient to identify lung microangiopathy on account of functional reserve breathing. The reserves compensate, to some extent, the lung dysfunction, and thus the negative effect of diabetes is

Since the direct measurements of pulmonary function are not conclusive and post-mortem autopsies do not serve the given patient, the diagnostics have to be based on such indirect results as spirometric measurements and modelling. It is probably also capable of revealing

Perfusion computed tomography (pCT) is a diagnostic method that enables the imaging of the organ and tissue. This is a powerful tool for diagnosing perfusion of internal organs such as the brain, liver, pancreas, spleen, kidneys and lungs (Alonzi & Hoskin, 2006), (Blomley et al, 1993), (Cao, 2011), (Eichinger et al, 2010), (Kuziemski et al, 2011). The method enables quantitative evaluation of circulation by determining changes in tissue during the flow of a contrast agent in the blood vessels. Changes, when compared to a normal tissue, can indicate tissue pathology. Lung pCT measurements can help in the diagnosis of cystic fibrosis (Eichinger et al, 2010) and diabetes (Kuziemski et al, 2011). It can also help to

The technique of lung pCT involves the intravenous injection of a non-iodinated contrast agent and the sequential scanning of the diagnosed region of the chest. There are a number of pulmonary perfusion parameters, calculated pixel by pixel on the basis of raw CT data, which are next analysed in order to reveal differences between the normal and altered tissue. The most useful perfusion parameters are: blood volume (BV), blood flow (BF), mean transit time (MTT), time to peak (TTP) and permeability surface (PS) (Kuziemski et al, 2011). The diagnosis of pulmonary function has to be based on indirect results, such as diffusing

The tests were performed on a group of 72 diabetics. People with cardiovascular disease and

First the diabetics were tested for microangiopathy, by comparing diffusing capacity in standing and lying position, and on this basis they were split into two groups: 44 *angiop M*

The spirometric tests were carried out on all the patients ( *angiop no an* n- *giop MM M* ) to obtain the following spirometric parameters: *FEV*1 (forced expiratory volume during the first second of expiration), *FVC* (the maximum volume of air that can be exhaled or inspired during a forced manoeuvre), *PEF* (peak expiratory flow), *MEF*50 (maximal instantaneous forced expiratory flow where 50% of the *FVC* remains to be expired), *MEF*75/25 (maximal

differentiate benign pulmonary nodules from lung cancer (Alonsi & Hoskin, 2006).

concealed until the effect reaches a higher level of impairment.

lung microangiopathy if the results are very carefully analysed.

capacity, spirometry and pCT because direct assessment is difficult.

and 28 *non angiop M* , with and without microangiopathy respectively.

2005), (Yeh et al, 2008).

**2. Materials** 

smokers were not included in this group.

The quality of gas exchange in the lungs depends on the diffusing capacity <sup>1</sup> [ ] *D mol s kPa <sup>L</sup>* . During the measurement of *DL* (American Thoracic Society, 1995) a person takes a full inhalation of air mixed with small amounts of carbon monoxide and helium. The mixture is held in the lungs for a few seconds and then exhaled. The first part of the expired gas is discarded. The next portion, which includes gas from the alveoli, is collected. The *DL* is determined by analyzing and comparing the concentrations of carbon monoxide and helium in the samples of the inhaled gas and the exhaled gas. The alveolar volume *VA* is also determined in this test by using the single-breath helium dilution technique.

#### **3.1 The oxygen pathway model**

Fig. 1 shows oxygen diffusion. Oxygen transportation from the alveoli to erythrocytes, through the alveoli-capillary barrier, is presented as the flow <sup>1</sup> <sup>21</sup> [ ] *- f g s* . The blood saturation *S*(0,1) , (also given as *S*(0,100%) ) shows what part (percentage) of oxygen capacity (maximum amount of oxygen transported by the erythrocytes) is currently being transported in the blood. Poorly oxygenated blood enters the pulmonary artery and then, enriched in oxygen, flows out of the lung through the pulmonary vein. Lung arterial blood saturation *SA* differs from lung venous blood saturation *SV* , *S S <sup>A</sup> <sup>V</sup>* .

Flows <sup>1</sup> <sup>20</sup> *f g*[ ] *s* and <sup>1</sup> <sup>02</sup> *f g*[ ] *s* represent the amount of oxygen in the blood flowing into the lung and flowing out of the lung. The oxygen diffusion model is:

$$\begin{cases} \dot{m}\_1(t) = -f\_{21}(t) + u(t), & m\_1(0) \\ \dot{m}\_2(t) = f\_{21}(t) + f\_{20}(t) - f\_{02}(t), & m\_2(0) \end{cases} \tag{1}$$

where the 1 *mtg* ( )[ ] and 2 *mtg* ( )[ ] are the *O*2 masses in the alveoli and in the blood vessels respectively. The initial states 1 *m g* (0)[ ] and 2 *m g* (0)[ ] depend on oxygen partial pressure <sup>1</sup>*P P* (0) 13.32 [k ] *a* , 2*P k* (0) 12.63 [ ] *Pa* and region volume *V V* <sup>1</sup> *<sup>A</sup>* , 4 3 <sup>2</sup> *V m* 10 [ ] , (Dalquen, 1999) ,West, 2008) and (Taylor et al, 1989). The alveolar volume *V V* <sup>1</sup> *<sup>A</sup>* is measured during the diffusing capacity test. The relationship between the *O*2 mass and the pressure is: 2 () () *P t RTm t M V i i Oi* , *<sup>i</sup>* 1,2 , where 1 1 [ ] *- - R N m K mol* is gas constant, *T* is absolute temperature and 2 <sup>1</sup> *MO* [ ] *g mol* is the molecular mass. The mass diffusion, i.e. flow <sup>21</sup> *f* ( )*t* via the membrane is caused by the concentration gradient 1 2 *cc c* 0 , where () () *i ii ct mt V* , 1,2 *i* :

Fig. 1. The passage of oxygen from the airways to the lung vessels. The diffusing capacity *DL* describes the condition of the alveoli-capillary barrier. The oxygen is bound in reversible bonds 8 [ ] *HbO* with the haemoglobin in the lung vessels

$$f\_{21}(t) = D\_L \cdot R \cdot T \cdot \left(\frac{m\_1(t)}{V\_1} - \frac{m\_2(t)}{V\_2}\right) \tag{2}$$

The signal *u t*( ) represents respiratory flow with the period *Tp* , the delay time 0*t* [s] (the time the air passes through the airway to the alveoli) and the duty cycle *d* . The input amplitude <sup>1</sup> [ ] *g s* depends on the organism's metabolic rate ( *MR* ), i.e. the organism's need for oxygen.

In medical practice, blood saturation *S* gives the basic information concerning the quantity of oxygen transported from the lungs to all the other organs. The kinetics of oxygen association with haemoglobin is described in Hill's equation (Khee-Shing, 2007):

$$S\_V(t) = \frac{K \cdot \left(P\_2(t)\right)^n}{1 + K \cdot \left(P\_2(t)\right)^n} \tag{3}$$

where 2*P t*( ) is oxygen partial pressure in the blood, 2.8 *n* is the Hill's constant and <sup>10</sup> 1.2256 10 [1 ( ) ] *<sup>n</sup> K Pa* is the association constant. The relationship between the endogenous inflow 20 *f* , the outflow 02 *f* and the organism's need for oxygen *MR* is as follows:

$$f\_{20}(t) = f\_{02}(t) - MR\tag{4}$$

while the outflow 02 *f* depends on the blood velocity 3 1 [ ] *m s* , maximum erythrocyte oxygen capacity <sup>3</sup> [ ] *mol m* and venous blood saturation ( ) *S t <sup>V</sup>*

$$f\_{02}\left(t\right) = S\_V\left(t\right) \cdot \boldsymbol{\varphi} \cdot \Phi \cdot \boldsymbol{M}\_{O\_2} \tag{5}$$

The elimination flow 02 *f* , according to Hill's equation, is:

606 Lung Diseases – Selected State of the Art Reviews

Fig. 1. The passage of oxygen from the airways to the lung vessels. The diffusing capacity *DL* describes the condition of the alveoli-capillary barrier. The oxygen is bound in reversible

> () () ( ) *<sup>L</sup> mt mt f t D RT V V*

The signal *u t*( ) represents respiratory flow with the period *Tp* , the delay time 0*t* [s] (the time the air passes through the airway to the alveoli) and the duty cycle *d* . The input

In medical practice, blood saturation *S* gives the basic information concerning the quantity of oxygen transported from the lungs to all the other organs. The kinetics of oxygen

 

*V n K Pt*

where 2*P t*( ) is oxygen partial pressure in the blood, 2.8 *n* is the Hill's constant and <sup>10</sup> 1.2256 10 [1 ( ) ] *<sup>n</sup> K Pa* is the association constant. The relationship between the endogenous inflow 20 *f* , the outflow 02 *f* and the organism's need for oxygen *MR* is as

association with haemoglobin is described in Hill's equation (Khee-Shing, 2007):

*S t*

1 2

1 2

(2)

(3)

20 02 *f* () () *t f t MR* (4)

depends on the organism's metabolic rate ( *MR* ), i.e. the organism's need

 2 <sup>2</sup> 1

*K Pt*

*n*

bonds 8 [ ] *HbO* with the haemoglobin in the lung vessels

amplitude <sup>1</sup> [ ] *g s*

for oxygen.

follows:

21

$$c\_{f02}\left(t\right) = \frac{c\_{Hill}}{1 + c\_{Hill}} \cdot \boldsymbol{\varphi} \cdot \boldsymbol{\Phi} \cdot \boldsymbol{M}\_{O\_2 \text{ } \prime} \quad c\_{Hill} = \boldsymbol{K} \cdot \boldsymbol{m}\_2^n\left(t\right) \cdot \left(\frac{\boldsymbol{R} \cdot \boldsymbol{T}}{\boldsymbol{M}\_{O\_2} \cdot \boldsymbol{V}\_2}\right)^n \tag{6}$$

Taking into account (4), (5) and (6), the oxygen diffusion model is:

$$\begin{cases} \dot{m}\_1(t) = -p\_1 \cdot m\_1(t) + p\_2 \cdot m\_2(t) + u\_1(t), & m\_1(0) \\ \dot{m}\_2(t) = p\_1 \cdot m\_1(t) - p\_2 \cdot m\_2(t) - MR, & m\_2(0) \end{cases} \tag{7}$$

where 1 0 0 0 *p pp i MR u t iT t iT t T d d* **1 1** .

The model parameters 1 1 *<sup>L</sup> p D RTV* and 2 2 *<sup>L</sup> p D RTV* can be estimated with the use of measurements 1 , *D VL* and physiological constants 2 *RTV* , , .

The example measurements (lying body position), 3 3 5.42 10 [ ] *non angiop V m <sup>A</sup>* , 7 1 1.54 10 [ ] *non angiop DL mol s Pa* , 3 3 5.71 10 [ ] *angiop V m <sup>A</sup>* and 7 1 1.59 10 [ ] *angiop DL mol s Pa* together with the constants: 3 3 <sup>2</sup> *V m* 0.10 10 [ ] (blood volume in lung capillary vessels), 293 [ ] *T K* (absolute temperature) and 1 1 *R N m mol K* 8.314 [ ] (gas constant), allow calculation of the example model parameter estimates:

$$\begin{aligned} \mathbf{p}^{angiop} &= [p\_1^{angiop}, p\_2^{angiop}] = [5.6315 \cdot 10^{-2}, \,\,\, 3.3271] \\ \mathbf{p}^{non-angiop} &= [p\_1^{non-angiop}, p\_2^{non-angiop}] = [6.2128 \cdot 10^{-2}, \,\, 3.9621] \end{aligned}$$

The model parameter estimates have been calculated for all the 72 *M* patients.

#### **3.2 Statistical comparison of measurement and modelling results for microangiopathic and non- microangiopathic patients**

Measurements were made for two groups of diabetic patients: ones with diagnosed microangiopathy and others with no microangiopathy. Lung microangiopathy was identified when standing lying *D D L L* . This examination also gives the alveoli volume *V V <sup>A</sup>* <sup>1</sup> . The null hypothesis *H*0 assumes that the mean values in both groups of patients are the same. Calculated *ex post* significance level *p* is compared with *ex ante* significance level (test-T). If a test of statistical significance gives *ex post* significance level *p* , which is lower than the , the null hypothesis is rejected, alternatively we no grounds to reject this hypothesis. Table 1 and Table 2 show a statistical comparison of spirometry measurements and model parameters obtained from microangiopathic and non-microangiopathic patients. The results presented in Table 1 ( lying lying <sup>0</sup> : *angiop non angiop HD D L L* is rejected and the conclusion is: lying*angiop* lying*non angiop D D L L* ) suggest the possibility of diagnosing microangiopathy on the basis of lying *DL* only, instead of lying *DL* and standing *DL* . The lack of statistical significance for standing *DL* means that it is not useful as an individual value for microangiopathy diagnosis.


Table 1. Statistical comparison of *DL* and *VA* . For lying *DL ex post* significance level is *<sup>p</sup>* , 0.05 . The null hypothesis lying lying <sup>0</sup> : *angiop non angiop HD D L L* concerning equality of mean diffusing capacity values in both groups was rejected. lying *DL* allows us to distinguish between patients with and without microangiopathy, while the rest do not.


Table 2. Statistical comparison of 1 *p* and 2 *p* . In the lying body position the ex post significance level is *p* , 0.01 and the *H*<sup>0</sup> , concerning the equality of mean parameter values, is rejected. These parameters allow for a distinction to be made between patients with and without microangiopathy.

Next, the null hypothesis 0 *angiop non angiop Hp p i i* , 1,2 *i* was tested (Table 2). The Kolmogorov-Smirnov test accepted the normality hypothesis concerning 1 *p* and 2 *p* at the significance level of *p* 0.01 . Therefore the mean values and standard deviations were calculated and the null hypothesis 0 *angiop non angiop Hp p i i* was tested.

For 1 *p* and 2 *p* , in lying body position, the *ex post* significance level *p* , 0.01 and so the *H*0 was rejected. These parameters enable a distinction to be made between patients with and without microangiopathy.

In a standing body position ( *p* 0.05 ) the parameters 1 *p* and 2 *p* are not useful for microangiopathy diagnosis.

microangiopathy on the basis of lying *DL* only, instead of lying *DL* and standing *DL* . The lack of statistical significance for standing *DL* means that it is not useful as an individual value for

standing *DL* 1.64 10-7 0.14 10-7 1.48 10-7 0.11 10-7 *<sup>p</sup>* 0.05 standing *VA* 5.95 10-3 0.21 10-3 5.65 10-3 0.21 10-3 *<sup>p</sup>* 0.05 lying *DL* 1.35 10-7 0.06 10-7 1.63 10-7 0.14 10-7 *<sup>p</sup>* 0.05 lying *VA* 6.04 10-3 0.27 10-3 5.90 10-3 0.40 10-3 *<sup>p</sup>* 0.05

Table 1. Statistical comparison of *DL* and *VA* . For lying *DL ex post* significance level is *<sup>p</sup>*

diffusing capacity values in both groups was rejected. lying *DL* allows us to distinguish

<sup>1</sup> *p* <sup>2</sup> 6.6176 10 <sup>2</sup> 0.4720 10 <sup>2</sup> 6.1900 10 <sup>2</sup> 0.2792 10 *p* 0.05 <sup>2</sup> *p* 3.9330 0.3447 3.5301 0.2730 *p* 0.05

<sup>1</sup> *p* 5.4339 10-2 0.2233 10-2 6.5672 10-2 0.3139 10-2 *p* 0.01 <sup>2</sup> *p* 3.2427 0.1551 4.0981 0.3518 *p* 0.01

parameter values, is rejected. These parameters allow for a distinction to be made between

Kolmogorov-Smirnov test accepted the normality hypothesis concerning 1 *p* and 2 *p* at the significance level of *p* 0.01 . Therefore the mean values and standard deviations were

*angiop non angiop Hp p i i*

the *H*0 was rejected. These parameters enable a distinction to be made between patients

In a standing body position ( *p* 0.05 ) the parameters 1 *p* and 2 *p* are not useful for

Table 2. Statistical comparison of 1 *p* and 2 *p* . In the lying body position the ex post

*angiop non angiop Hp p i i*

For 1 *p* and 2 *p* , in lying body position, the *ex post* significance level *p*

between patients with and without microangiopathy, while the rest do not.

Standard

<sup>0</sup> : *angiop non angiop HD D L L*

Standard

0.05 . The null hypothesis lying lying

Measurements taken in standing body position

Measurements taken in lying body position

patients with and without microangiopathy.

 , 0.01 

significance level is *p*

Next, the null hypothesis 0

calculated and the null hypothesis 0

with and without microangiopathy.

microangiopathy diagnosis.

Microangiopathic Non-microangiopathic Statistical

deviation Mean value Standard

Microangiopathic Non-microangiopathic Statistical

deviation Mean value Standard

and the *H*<sup>0</sup> , concerning the equality of mean

was tested.

, 1,2 *i* was tested (Table 2). The

 , 0.01 

and so

Mean value level *p*

deviation

concerning equality of mean

deviation

Mean value level *p*

significance

,

significance

microangiopathy diagnosis.

The results in Table 1 and Table 2 indicate the possibility of using a diagnostic test based on a single measurement lying *DL* , and also on the basis of modelling results 1 *<sup>p</sup>* .and 2 *<sup>p</sup>* .

#### **3.3 Binary classification on the basis of diffusing capacity measurement and modelling**

Statistical analyses show that a single measurement lying *DL* and the model parameters 1 *<sup>p</sup>* , <sup>2</sup> *p* contain information concerning lung microangiopathy. The question remains as to whether or not such values can be used for binary classification. Binary classification is the classifying of the members of mixed group *angiop non an* - *giop MM M* into two subgroups, *angiop M* and *non angiop M* , on the basis of whether or not they have microangiopathy. For this purpose an appropriate classification algorithm has to be chosen.

To select the best potential classification algorithm, statistical measures, sensitivity and specificity (Panzer, Blach & Griner, 1991) were considered. Sensitivity *Sens* is the ability of a test to detect a disease when it is really present. Specificity *Spec* is the ability to confirm the absence of a disease in patients when it is really absent.

$$Sens = \frac{True\ Positive}{True\ Positive + False\ Negative} \tag{8}$$

$$Spec = \frac{True \text{ Negative}}{True \text{ Negative} + False \text{ Positive}} \tag{9}$$

The theoretically optimal prediction is: 100% *Sens* ( all the sick patients were identified as sick) and *Spec* 100% (none of the healthy patients was identified as sick).

Fig. 2. Classification algorithms based on 1 *p* and 2 *p* ; I) 2 *Mp* are classified as microangiopathic and 2 *M Mp* are classified as non-microangiopathic, II) 1 2 *M M p p* are classified as microangiopathic and 1 2 ( ) *MM M p p* are classified as non-microangiopathic

For binary classification the discrimination levels (boundaries, diagnostic thresholds) *DL H* , <sup>1</sup> *Hp* and 2 *Hp* have to be calculated, respectively for *DL* , 1 *p* and 2 *p* . The discrimination levels classify the test result as positive or as negative. The parameter value where *Sens Spec* max was chosen as the parameter's diagnostic threshold.

Among different classification algorithms using 1 *p* , 2 *p* , 1 *Hp* and 2 *Hp* the best are the two shown in Fig. 2.


Table 3. Classification results obtained for entire group of 72 *M* subjects and for the group of 1 *M* 22 women, over 50 y, under 1.65 m. The best results are underlined.

Therefore the diagnostic procedure shown in Fig. 3 is recommend.

Fig. 3. Diagnostic procedure with the use of lying *DL* , lying *VA* measurements, constants *R*, *T*, *V*2 and calculated 1 2 *p* , *p* , <sup>1</sup> *Hp* and 2 *Hp* . For binary classification the more conclusive algorithms I) (with respect to *Spec* ) and II) (with respect to *Sens* ) are recommended.

The binary classification was performed for the 72 *M* subjects. The results were compared with already known medical diagnoses of 44 microangiopathic patients and 18 patients with no microangiopathy. Then the sensitivity and the specificity were calculated according to equations (8) and (9), (Table 3). The larger the *Sens* and *Spec* , the better. It seems reasonable to assume that results larger than 75% are satisfactory. Therefore an algorithm should be selected to fulfil this requirement, and not every entry in Table 3 does.

Diagnostic thresholds, calculated on the basis of 72 *M* measurement data, are: 1 <sup>2</sup> 5.80 10 *Hp* , 2 3.50 *Hp* and -7 1.57 10 *DL <sup>H</sup>* . The best statistical measures obtained are: 76% *Sens* (algorithm II) and *Spec* 84% (algorithm I). The results for *lying DL* , *Sens* 33% and *Spec* 61% , are less than 75% and quite inadequate.

The thresholds established as common for the entire group did no take into account such important factors as age, height and gender. Therefore a subgroup 1 *M* 22 was selected (women, over 50 years old and less than 1.65 m tall). Then the new diagnostic thresholds 1 <sup>2</sup> 5.42 10 *Hp* , 2 3.06 *Hp* , -7 1.30 10 *DL <sup>H</sup>* and new *Sens* and *Spec* were calculated. As expected, both the new *Sens* and *Spec* were noticeably larger.

The diagnostic procedure in Fig. 3 uses lying *DL* and lying *VA* measurements, and *<sup>R</sup>* , *T* and *V*<sup>2</sup> physiological constants to calculate the 1 *p* and 2 *p* in a patient. Then algorithm I (with the most conclusive *Sens* ) and algorithm II (with the most conclusive *Spec* ) are applied together with 1 *Hp* and 2 *Hp* diagnostic thresholds to obtain a binary classification result.

This procedure produces one of two possible results: 1) high probability of lung microangiopathy or 2) high probability of no lung microangiopathy. This can serve as useful confirmation in a doctor's diagnosis.

The final decision is made by the doctor conducting the diagnosis, who can take into account this classification result along with other diagnostic data.

#### **4. Spirometry**

610 Lung Diseases – Selected State of the Art Reviews

I. 1 2 *p* , *p* 50 84 62 87 II. 2 1 *p* , *p* 76 50 79 80 lying *DL* 33 61 62 71

Table 3. Classification results obtained for entire group of 72 *M* subjects and for the group

Fig. 3. Diagnostic procedure with the use of lying *DL* , lying *VA* measurements, constants *R*, *T*, *V*2 and calculated 1 2 *p* , *p* , <sup>1</sup> *Hp* and 2 *Hp* . For binary classification the more conclusive algorithms I) (with respect to *Spec* ) and II) (with respect to *Sens* ) are recommended.

The binary classification was performed for the 72 *M* subjects. The results were compared with already known medical diagnoses of 44 microangiopathic patients and 18 patients with no microangiopathy. Then the sensitivity and the specificity were calculated according to equations (8) and (9), (Table 3). The larger the *Sens* and *Spec* , the better. It seems reasonable to assume that results larger than 75% are satisfactory. Therefore an algorithm

Diagnostic thresholds, calculated on the basis of 72 *M* measurement data, are:

 , 2 3.50 *Hp* and -7 1.57 10 *DL <sup>H</sup>* . The best statistical measures obtained are: 76% *Sens* (algorithm II) and *Spec* 84% (algorithm I). The results for *lying DL* ,

should be selected to fulfil this requirement, and not every entry in Table 3 does.

*Sens* 33% and *Spec* 61% , are less than 75% and quite inadequate.

*Sens*[%] *Spec*[%] *Sens*[%] *Spec*[%]

Narrowed range of anthropometric features <sup>1</sup> *M* 22 (women, over 50 y, under 1.65 m)

Wide range of anthropometric features, 72 *M* (men and women, 21-69 y, 1.50-1.95m)

of 1 *M* 22 women, over 50 y, under 1.65 m. The best results are underlined.

Therefore the diagnostic procedure shown in Fig. 3 is recommend.

Classification algorithm

1

<sup>2</sup> 5.80 10 *Hp*

The most popular spirometry method is a dynamic one. The pneumotachometer (Miller, Harkinson&Brusasco, 2005) defines the volume *Vt l* ( ) [ ] and airflow <sup>1</sup> *Q t ls* ( )[ ] during the inhalation and exhalation. The flow-volume curve *Q V*( ) is received on the basis of the spirometry measurements of *V t*( ) and *Q t*( ) . Specific ventilation manoeuvres are required in the spirometric test. The measurement is preceded by a period of quiet breathing–in and out (Fry, Hyatt, 1960). Next the maximal breath–in and the maximal forced breath–out manoeuvres are performed as the important parts of the spirometric test.

The respiration parameters are defined on the basis of the volume–time curve *V t*( ) and the flow–volume curves *Q V*( ) (see Fig. 4 and Fig. 5), (Miller, Harkinson&Brusasco, 2005).

In medical practice two terms, volume and capacity*,* are used. The difference between them is based on the assumption that volume refers to lung volumes, while capacity refers to different combinations of lung volumes, usually in relation to inhalation and exhalation.

The relations between volumes and capacities are presented in equations (10).

$$\begin{aligned} \text{TLC} &= \text{VC} + \text{RV} \\ \text{TLC} &= \text{IC} + \text{FRC} \end{aligned} \tag{10}$$

The following respiratory parameters have been defined: *TLC l*[ ] (Total Lung Capacity) is the volume of air in the lungs at the end of maximal inhalation; *VC l*[ ] (Vital Capacity) is the maximum volume of air that can be exhaled or inhaled during either a forced ( *FVC* ) or a slow (*VC* ) manoeuvre; *RV l*[ ] (Residual Volume) is the volume of air remaining in the lungs after the maximal exhalation; *IC l*[ ] (Inspiratory Capacity) is the maximal volume of air that can be inhaled from the resting expiratory level; *FRC l*[ ] (Functional Residual Capacity) is the volume of air in the lungs at the resting end-expiration.

Also defined are the following respiratory parameters: 1 *FEV l*[ ] the forced expiratory volume during the first second of expiration; *FET s* the forced expiratory time; [ ] *FIT s* the forced inspiratory time; <sup>1</sup> *PEF l s* [ ] (Peak Expiratory Flow) and <sup>1</sup> *PIF l s* [ ] (Peak Inspiratory Flow) are respectively the maximal expiratory and the maximal inspiratory flow

Fig. 4. The spirometry parameters defined on the basis of volume-time curve *V t* .

Fig. 5. The spirometric parameters defined on the basis of flow–volume curve *Q V* .

Fig. 4. The spirometry parameters defined on the basis of volume-time curve *V t* .

Fig. 5. The spirometric parameters defined on the basis of flow–volume curve *Q V* .

rates achieved; *TPEF s* the time of maximal expiratory flow; <sup>1</sup> 75 50 25 , , [] *<sup>M</sup> - EF MEF MEF l s* maximal instantaneous forced expiratory flow where 75, 50, 25% of the *FVC* remains to be expired; <sup>1</sup> 75/25 [ ] *M - EF l s* is the maximal mid-expiratory flow and *FEV FVC* 1% is the percentage relation of *FEV*1 to *FVC* .

The European Respiratory Society (Miller, Harkinson & Brusasco, 2005) has published the spirometric norms and the reference values for these spirometric parameters. The reference values of the respiration parameters, obtained for a representative healthy population, are used for the interpreting the spirometry data and making the diagnosis. The reference values depend on the patient's anthropometric data, such as age, height and gender. In some medical cases (asthma, pulmonary fibrosis, cystic fibrosis and COPD) the spirometric parameter boundary values have already been defined. However, as far as lung microangiopathy is concerned, this has not yet been done.

According to medical practice experience, lung microangiopathy is diagnosed on the basis of the following spirometric parameters: *FEV*<sup>1</sup> , *FVC* , *PEF* , *MEF*<sup>50</sup> , *MEF*75/25 , *IC* and *FEV FVC* 1% . These spirometric parameters were collected from the 72 *M* diabetic patients. However, a single spirometric test is not sufficient to diagnose the lung flow and volume reduction caused by microangiopathy; long term observation is necessary. Therefore one should consider an alternative method. By using the spirometric results in a statistical significance test, we can find the spirometric parameters that are most responsive to lung microangiopathy.

#### **4.1 Spirometric parameters. Statistical comparison of results for microangiopathic and non- microangiopathic patients**

Traditional spirometric parameters were tested for their ability to detect lung microangiopathy. The mean, standard deviation and the statistical significance level *p* for spirometric parameters are presented in Table 4.


Table 4. Statistical comparison of spirometric parameters for microangiopathic and nonmicroangiopathic patients. In every case the *ex post* significance level *p* , 0.05 , which means that *H*0 concerning mean parameter value equality is not rejected. The parameters do not allow for a distinction to be made between patients with and without microangiopathy.

The parameters do not show statistical significance in detecting microangiopathy: *ex post* significance level *p* is larger than *ex ante* significance level 0.05 , which confirms the null hypothesis concerning equality of mean values for both groups. Therefore, none of spirometric parameters are useful in diagnosing microangiopathy.

#### **4.2 Spirometry modelling**

The maximal breathing-in flow was modelled by means of the sine function. The forced breathing-out flow was finally mimicked using the gamma variate function (Askey R.A.& Roy R,2007) – after testing a large group of prospective regression functions, such as exponentials, polynomials, etc (Kalicka et al, 2007).

The maximal inflow ( ) *Q V in* is modelled as follows:

$$Q\_{in}(V) = A\_{in} \cdot \sin\left(\varpi \cdot V\right), \ 0 \le V \le FV\mathbb{C} \tag{11}$$

where 1 [ ] *A ls in* and <sup>1</sup> [ ] *l* are model parameters and *FVC* is the forced vital capacity. For modelling ( ) *Q V out* during the maximal outflow, the following gamma variate function is implemented:

$$Q\_{\rm out} \left( V \right) = \mathcal{K} \left( V \right)^{\flat} e^{-V \cdot \mathcal{a}}, \; FV \ge V \ge 0 \tag{12}$$

where <sup>1</sup> *Kls* [ ] , *b* and <sup>1</sup> *a l*[ ] are model parameters.

The model consists of the equations (11) and (12). The model parameters are arranged into vector [ ] [ , , ,,] *i in* **p** *p A Kba* , *i* 1 5 . The parameter estimates were obtained using the least square procedure, according to the following:

$$\mathbf{p} = \arg\left[\min\_{\mathbf{p}} \sum\_{V=0}^{V\_{\text{max}}} \left( Q^{\text{model}} \left( V, \mathbf{p} \right) - Q^{\text{meas}} \left( V, \mathbf{y}^{\text{meas}} \right) \right)^2 \right] \tag{13}$$

where meas meas <sup>1</sup> 50 25/50 [ ] [ , , , , , , % ]; 1 7 *<sup>j</sup>* **y** *y FEV FVC PEF MEF MEF IC FEV FVC j* is vector of measurements and *V FVC* max . The fitting procedure was executed separately to obtain , *Ain* and *Kba* , , based on the equations (11) and (12) respectively.

The parameter vector [ , , , , ] [3.780 1.030 20.580 0.772 1.507] **p** *A K b a , , , , in* is an example of the model identification results. This was obtained from a 59-year-old woman suffering from lung microangiopathy. The spirometric parameters obtained from the patient were: <sup>1</sup> *FEV* 2.25 , 2.84 *FVC* , *PEF l s* 5.79 , 50 *MEF l s* 3.47 , 75/25 *MEF l s* 2.63 , 2.81 *IC* and *FEV FVC* 1% 78.95% .

Calculations of [ , , ,,] **p** *A Kba in* were performed for 72 *M* diabetic patients. Fig. 6 shows an example of the model regression functions ( ) *Q V in* and ( ) *Q V out* drawn for the [ , , ,,] **p** *A Kba in* [3.780 1.030 20.580 0.772 1.507] *, , , ,* . In Fig. 6 the *Q V*( ) spirometric test results have been added, for comparison.

#### **4.3 Statistical comparison of modelling results for microangiopathic and nonmicroangiopathic patients**

The mean parameter value and standard deviation were calculated separately for microangiopathic and non-microangiopathic patients (Table 5). The aim was to find out

The parameters do not show statistical significance in detecting microangiopathy: *ex post*

null hypothesis concerning equality of mean values for both groups. Therefore, none of

The maximal breathing-in flow was modelled by means of the sine function. The forced breathing-out flow was finally mimicked using the gamma variate function (Askey R.A.& Roy R,2007) – after testing a large group of prospective regression functions, such as

> *Q V A V V FVC in in* ( ) sin , 0

For modelling ( ) *Q V out* during the maximal outflow, the following gamma variate function

, 0 *<sup>b</sup> V a Q V K V e FVC V out*

The model consists of the equations (11) and (12). The model parameters are arranged into

arg min , ,

0

 and *Kba* , , based on the equations (11) and (12) respectively. The parameter vector [ , , , , ] [3.780 1.030 20.580 0.772 1.507] **p** *A K b a , , , , in*

**p p**

*V*

*V*

(11)

(12)

are model parameters and *FVC* is the forced vital capacity.

, *i* 1 5 . The parameter estimates were obtained using the

**<sup>p</sup> <sup>y</sup>** (13)

is an example of

were performed for 72 *M* diabetic patients. Fig. 6

 max <sup>2</sup> model meas meas

*Q V QV*

<sup>1</sup> 50 25/50 [ ] [ , , , , , , % ]; 1 7 *<sup>j</sup>* **y** *y FEV FVC PEF MEF MEF IC FEV FVC j* is vector

of measurements and *V FVC* max . The fitting procedure was executed separately to obtain

the model identification results. This was obtained from a 59-year-old woman suffering from lung microangiopathy. The spirometric parameters obtained from the patient were: <sup>1</sup> *FEV* 2.25 , 2.84 *FVC* , *PEF l s* 5.79 , 50 *MEF l s* 3.47 , 75/25 *MEF l s* 2.63 , 2.81 *IC*

shows an example of the model regression functions ( ) *Q V in* and ( ) *Q V out* drawn for the

The mean parameter value and standard deviation were calculated separately for microangiopathic and non-microangiopathic patients (Table 5). The aim was to find out

**4.3 Statistical comparison of modelling results for microangiopathic and non-**

[3.780 1.030 20.580 0.772 1.507] *, , , ,* . In Fig. 6 the *Q V*( ) spirometric test

, which confirms the

significance level *p* is larger than *ex ante* significance level 0.05

spirometric parameters are useful in diagnosing microangiopathy.

exponentials, polynomials, etc (Kalicka et al, 2007). The maximal inflow ( ) *Q V in* is modelled as follows:

where <sup>1</sup> *Kls* [ ] , *b* and <sup>1</sup> *a l*[ ] are model parameters.

least square procedure, according to the following:

 and <sup>1</sup> [ ] *l*

**4.2 Spirometry modelling** 

where 1 [ ] *A ls in*

is implemented:

vector [ ] [ , , ,,] *i in* **p** *p A Kba*

where meas meas

and *FEV FVC* 1% 78.95% .

**microangiopathic patients** 

[ , , ,,] **p** *A Kba in* 

Calculations of [ , , ,,] **p** *A Kba in*

results have been added, for comparison.

, *Ain* 

Fig. 6. An example result of the spirometric measurement and modelling of the flow– volume curve *Q V*( ) .


Table 5. Statistical comparison of model parameters for microangiopathic and nonmicroangiopathic patients. For *Ain* , and *b* , the *ex post* significance level was *p* , 0.05 , and thus the *H*<sup>0</sup> , concerning mean parameter values equality, was not rejected – i.e. the parameters are not useful for diagnosis. However, parameters *K* and *a* are useful in distinguishing between patients with and without microangiopathy because the *ex post* significance level is *p* , 0.05 .

whether or not the model parameters are helpful in distinguishing between microangiopathic and non-microangiopathic patients. For this purpose the null hypothesis 0 *angiop non angiop Hp p i i* , *i* 1,..,5 was tested.

At first the normality hypothesis concerning the parameter estimates *<sup>i</sup> p* was investigated, and the hypothesis was accepted (Kolmogorov-Smirnov test) at the significance level of *p* 0.05 . Then the mean values and standard deviations (Table 5) were calculated and statistical test-T was performed. For *K* and *a* the *ex post* significance level *p* , 0.05 and *H*0 is rejected. The two model parameters *K* and *a* allow for a distinction to be made between patients with and without microangiopathy.

The remaining model parameters *Ain* , and *a* do not reveal the occurrence or absence of lung microangiopathy.

#### **4.3 Binary classification on the basis of spirometric measurements and modelling**

Binary classification algorithms, based on the model parameters *K* and *a* , were tested with discrimination levels *HK* and *Ha* . The discrimination levels were first established for all *M* 72 subjects as 50.18 *HK* and 1.72 *Ha* . These discrimination levels correspond to the *K* and *a* values for which *Sens Spec* max . The 72 *M* subjects were now divided into two groups on the basis of these discrimination thresholds.

Two algorithms, I and II (which gave the best results), were applied for binary classification. The algorithms are presented in Fig. 7. The binary classification decision was compared with previously known medical diagnoses, and then the *Sens* and *Spec* were calculated according to equations (8) and (9), (see Table 6).

None of the tested algorithms could produce simultaneously a large *Sens* and a large *Spec* . Algorithm II gave the best 82% *Sens* and algorithm I gave the best *Spec* 62% . Algorithm II accurately detected the majority of the microangiopathy cases ( 82% *Sens* ), and only missed out on a few. Yet *Spec* 50% means algorithm II identified only 50% of the nonmicroangiopathic patients as non-microangiopathic and gave too many false alarms. This is an unacceptable result. Somewhat better *Spec* 62% was obtained with algorithm I but this was still not satisfactory. The most likely explanation for these poor results is the fact that the thresholds *HK* and *Ha* are too general and do not take into consideration important anthropometric features.

Therefore the range of anthropometric features was made more specific ( <sup>1</sup> *M* 22 women, over 50 years old and under 1.65 m tall) and new diagnostic thresholds were calculated: 48.11 *HK* and 1.68 *Ha* . The new classification results are presented in Table 6.

This more accurate choice of the diagnostic thresholds gave much better results. The *Sens* 100% means that algorithm II identified all the sick patients as sick. This excellent result has been obtained for the sample 1 *M* 22 selected from the general population and therefore should be treated with care. An another sample may give a somewhat different result but the great improvement is obvious.

The diagnostic procedure, utilising the algorithms I and II, is shown in Fig. 8.

Fig. 7. Classification algorithms based on *a* and *K* ; I) *MK* are classified as microangiopathic and *M MK* are classified as non-microangiopathic, II) *Ma K M* are classified as microangiopathic and ( ) *MMM a K* are classified as non-microangiopathic

Two algorithms, I and II (which gave the best results), were applied for binary classification. The algorithms are presented in Fig. 7. The binary classification decision was compared with previously known medical diagnoses, and then the *Sens* and *Spec* were calculated

None of the tested algorithms could produce simultaneously a large *Sens* and a large *Spec* . Algorithm II gave the best 82% *Sens* and algorithm I gave the best *Spec* 62% . Algorithm II accurately detected the majority of the microangiopathy cases ( 82% *Sens* ), and only missed out on a few. Yet *Spec* 50% means algorithm II identified only 50% of the nonmicroangiopathic patients as non-microangiopathic and gave too many false alarms. This is an unacceptable result. Somewhat better *Spec* 62% was obtained with algorithm I but this was still not satisfactory. The most likely explanation for these poor results is the fact that the thresholds *HK* and *Ha* are too general and do not take into consideration important

Therefore the range of anthropometric features was made more specific ( <sup>1</sup> *M* 22 women, over 50 years old and under 1.65 m tall) and new diagnostic thresholds were calculated:

This more accurate choice of the diagnostic thresholds gave much better results. The *Sens* 100% means that algorithm II identified all the sick patients as sick. This excellent result has been obtained for the sample 1 *M* 22 selected from the general population and therefore should be treated with care. An another sample may give a somewhat different

48.11 *HK* and 1.68 *Ha* . The new classification results are presented in Table 6.

The diagnostic procedure, utilising the algorithms I and II, is shown in Fig. 8.

Fig. 7. Classification algorithms based on *a* and *K* ; I) *MK* are classified as

microangiopathic and *M MK* are classified as non-microangiopathic, II) *Ma K M* are classified as microangiopathic and ( ) *MMM a K* are classified as non-microangiopathic

**4.3 Binary classification on the basis of spirometric measurements and modelling**  Binary classification algorithms, based on the model parameters *K* and *a* , were tested with discrimination levels *HK* and *Ha* . The discrimination levels were first established for all *M* 72 subjects as 50.18 *HK* and 1.72 *Ha* . These discrimination levels correspond to the *K* and *a* values for which *Sens Spec* max . The 72 *M* subjects were now divided into

two groups on the basis of these discrimination thresholds.

according to equations (8) and (9), (see Table 6).

result but the great improvement is obvious.

anthropometric features.


Table 6. Classification results obtained for entire group of 72 *M* subjects and for the subgroup 1 *M* 22 of women over 50 years old and under 1.65 m. The best results are underlined.

Fig. 8. Classification algorithms: I) *MK* are classified as microangiopathic and *M MK* as non-microangiopathic, II) *Ma K M* are classified as microangiopathic and ( ) *MMM a K* as non-microangiopathic

Applied with *Ha* and *HK* , algorithm I gives the best (most conclusive) *Sens* classification result, while the algorithm II gives the best (most conclusive) *Spec* result.

This procedure gives one of two alternative results: 1) high probability of lung microangiopathy or 2) high probability of no lung microangiopathy. The final decision is made by the doctor.

## **5. Perfusion computed tomography (pCT)**

Tests commonly used in clinical practice such as spirometry and lung diffusion capacity measurements are not considered precise enough to identify lung microangiopathy. There are very few publications in this field and most of them concern the decrease of diffusion capacity in diabetics (Villa et al, 2004), (Goldman, 2003). Autopsies of patients who died of diabetes complications revealed a thickening in the basement membrane of alveolar capillaries and arterioles in the lungs as well as of the entire walls of pulmonary arterioles together with fibroblast proliferation (Weynand et al, 1999).

Impaired gas exchange in pulmonary alveoli results in the progressive reduction of reserves in small pulmonary vessels. For this reason lung perfusion in regions of lung microangiopathy may be diminished. Computed tomography of chest perfusion allows imaging of pulmonary vessels and parenchyma and may be useful in diagnosing pulmonary microangiopathy (Kuziemski et al, 2011).

## **5.1 Patients and study protocol**

A group of 10 never-smoking diabetic adults and a control group of 8 non-diabetic volunteers were chosen. All participants had a similar body mass index and none of them suffered any disease that affected pulmonary function. The mean time from when diabetes mellitus was first diagnosed was 15.5 years. Nephropathy was diagnosed in 3 diabetics, retinopathy in 6 and polyneuropathy in 4. The question was whether or not they also suffered from lung microangiopathy. In the control group lung and cardio-vascular diseases were not diagnosed.

For all patients, spirometry and diffusing capacity tests were performed.

For the pCT tests a 64-row CT scanner GE - Light Speed VCT (GE Healthcare USA) was used. Pulmonary perfusion was evaluated after the intravenous administration of 40 ml of contrast at the rate of 4 ml/s. CT images were taken with 1 s resolution in the period of 40 s of a selected lung section (4 cm thick layer situated 2 cm below carina). Within this 4 cm thick segment, three cross-sections, anterior, medium and posterior, 5 mm thick each, were selected at uniform distances from each other. 6 ROIs were chosen in each of the crosssections (18 ROIs in all, numbered ROI 3-ROI 20). The ROIs 3–20 were situated in the upper, medial and lower parts of the left and right lungs. The most vascularised areas of the lungs were left beyond the ROIs so as not to distort the evaluation of lung parenchyma.

The pCT measurements were performed using standard CT Perfusion 4 (GE Healthcare USA). The following perfusion parameters were calculated:


#### **5.2 pCT results**

The example results of pCT scans, obtained from a 62 year old diabetic male, are shown in Fig. 9 a), b), c) and d). Perfusion parameters *BF* , *BV* , *MTT* and *PS* were calculated and mapped out on the lung cross-section. Analysis was made of the pulmonary artery - ROI 1, which represents the arterial input function, i.e. the flow blood into the pulmonary system.

In Fig. 9 the ROI 1 is marked with a circle. The calculated values of perfusion parameters (mean value for all pixels in the region and standard deviation of the mean) are displayed in the right lower corner of the scan.

Table 7 shows mean perfusion parameters *Avg* and standard deviation *Dev* in the ROI 1.

capacity in diabetics (Villa et al, 2004), (Goldman, 2003). Autopsies of patients who died of diabetes complications revealed a thickening in the basement membrane of alveolar capillaries and arterioles in the lungs as well as of the entire walls of pulmonary arterioles

Impaired gas exchange in pulmonary alveoli results in the progressive reduction of reserves in small pulmonary vessels. For this reason lung perfusion in regions of lung microangiopathy may be diminished. Computed tomography of chest perfusion allows imaging of pulmonary vessels and parenchyma and may be useful in diagnosing

A group of 10 never-smoking diabetic adults and a control group of 8 non-diabetic volunteers were chosen. All participants had a similar body mass index and none of them suffered any disease that affected pulmonary function. The mean time from when diabetes mellitus was first diagnosed was 15.5 years. Nephropathy was diagnosed in 3 diabetics, retinopathy in 6 and polyneuropathy in 4. The question was whether or not they also suffered from lung microangiopathy. In the control group lung and cardio-vascular diseases

For the pCT tests a 64-row CT scanner GE - Light Speed VCT (GE Healthcare USA) was used. Pulmonary perfusion was evaluated after the intravenous administration of 40 ml of contrast at the rate of 4 ml/s. CT images were taken with 1 s resolution in the period of 40 s of a selected lung section (4 cm thick layer situated 2 cm below carina). Within this 4 cm thick segment, three cross-sections, anterior, medium and posterior, 5 mm thick each, were selected at uniform distances from each other. 6 ROIs were chosen in each of the crosssections (18 ROIs in all, numbered ROI 3-ROI 20). The ROIs 3–20 were situated in the upper, medial and lower parts of the left and right lungs. The most vascularised areas of the lungs

The pCT measurements were performed using standard CT Perfusion 4 (GE Healthcare


The example results of pCT scans, obtained from a 62 year old diabetic male, are shown in Fig. 9 a), b), c) and d). Perfusion parameters *BF* , *BV* , *MTT* and *PS* were calculated and mapped out on the lung cross-section. Analysis was made of the pulmonary artery - ROI 1, which represents the arterial input function, i.e. the flow blood into the pulmonary system. In Fig. 9 the ROI 1 is marked with a circle. The calculated values of perfusion parameters (mean value for all pixels in the region and standard deviation of the mean) are displayed in

Table 7 shows mean perfusion parameters *Avg* and standard deviation *Dev* in the ROI 1.

For all patients, spirometry and diffusing capacity tests were performed.

were left beyond the ROIs so as not to distort the evaluation of lung parenchyma.


to extravascular space, observed in 100g of lung tissue during 1 min.

USA). The following perfusion parameters were calculated: - *BV ml g* [ 100 ] - blood volume in 100g of lung tissue.

together with fibroblast proliferation (Weynand et al, 1999).

pulmonary microangiopathy (Kuziemski et al, 2011).

**5.1 Patients and study protocol** 

were not diagnosed.

**5.2 pCT results** 

the right lower corner of the scan.

As the table shows, the obtained permeability surface *PS* for the impermeable pulmonary artery is equal to zero. The *Dev*, i.e. the disperse of parameter values within the ROI 1, is low, 4-7%.

Fig. 9. The pCT scans (a) blood flow *BF* , (b) blood volume *BV* , (c) mean transit time *MTT* and (d) permeability surface *PS* for a 62 year old diabetic male.


Table 7. Example results of perfusion parameters calculated as the mean values in the ROI 1 marked in Fig. 9. The calculated standard deviations *Dev* illustrates the distribution of test results within ROI 1.

Fig. 10. The *PS* maps for (a) healthy subject and (b) diabetic subject. For example, in ROI 13 (right lower circle) 3.31 *PS* for the healthy subject and 79.80 *PS* for the diabetic subject. The measurements were obtained in the same location of the lung cross-section.

Fig. 10 shows *PS* maps obtained from a healthy patient and a diabetic patient. The qualitative comparison (amount of the red, permeable area) as well as the quantitative comparison (healthy patient 3.31 *PS* and diabetic patient 79.80 *PS* ) indicate damage to capillaries and pulmonary arterioles of the diabetic patient.

All perfusion parameters were calculated in 18 ROIs (3-20) in the parenchyma to discover whether parameter values depend on the location of tested lung cross-section. Statistical analysis revealed no correlation between perfusion parameter value and ROI location in the upper central or lower part of the lungs (Kuziemski et al, 2011). Therefore mean perfusion parameters were calculated for all 18 ROIs. The results are shown in Table 8 (Kuziemski et al, 2011).

Here a noticeable increase was observed in the parenchyma perfusion parameter values of the diabetics with respect to control group, which may be due to diabetic mellitus.


Table 8. Mean parameters *BF* , *BV* and *PS* in the artery and parenchyma of the diabetics and of the control group. The calculated standard deviation *Dev* illustrates the distribution of test results in 18 ROIs (ROI 3 – 20, in upper, central and lower part of lungs) with respect to the mean of the eighteen results. No significant *MTT* differences were found.

Chest perfusion CT allows imaging of lung vessels and parenchyma, which is essential in diagnosing pulmonary angiopathy. The analyzed results are very new and currently not all that numerous. While this does not allow for a complex statistical analysis, the above preliminary results indicate the great potential of pCT tests in diagnosing lung microangiopathy.

#### **5.3 Lung area extraction**

Each pCT image (see Fig. 9) consists of the body cross-section, including the lungs, essential for medical diagnosis, the rest of the body and the background, which is unnecessary for lung diagnostics but was nevertheless included in the perfusion map calculations.

As computations of perfusion parameters involve complicated methods of signal processing, it is desirable that the distinction between diagnostically important regions (the lung crosssection) and other areas (background and the rest of body cross-section) is conducted before the process starts. This shortens the time needed to obtain parametric maps by omitting data processing from the area outside the lungs. It can also help avoiding false lung malfunction diagnoses, which can occur when the automatic methods of pathological changes wrongly interpret signals from areas outside the lungs as abnormal lung signals.

Literature describes many methods of lung area extraction from CT chest scans (Homma et al, 2009), (de Nunzio et al, 2011), (Vinhais & Campilho, 2006), (Zhou et al, 2004). Most methods are devoted to extracting the lung area from a single image. Some of these methods allow for neighbouring (upper and lower) slices to be analysed (Homma et al, 2009).

The lung extraction presented here was obtained using the two-step procedure. The objective of the preliminary processing was to define the rough mask, i.e. the border within which the lung contour is always contained during the pCT test while the lungs are breathing and the heart is beating. The lung and heart region contour pixels are subjected to the greatest changes. This behaviour can be utilised to detect the contours. It can be achieved by using a variability factor *VF* , which is defined as follows:

$$VF = \frac{\mathcal{X}\_{\text{max}} - \mathcal{X}\_{\text{min}}}{\mathcal{X}\_{\text{median}}} \tag{14}$$

where max *x* , min *x* and *median x* are respectively each pixel's maximum, minimum and median value. *VF* can be used to create a variability map, *MVF* . Such a map is shown in Fig. 11.

Fig. 11. Map *MVF* of variability factor *VF* . The white areas are the most variable due to the breathing lungs and beating heart.

Then the final lung mask was obtained with the use of morphological operators (dilation, erosion, closing and opening) to extract lung cross-sections from the chest pCT. Fig. 12 shows the *BF* **,** *BV* **,** *MTT* and *PS* maps of just the lungs, extracted using the mask. The lung mask was superimposed onto the calculated maps in Fig. 9.

Fig. 12. Lung extraction. The pCT lung maps obtained for the scans in Fig. 9 - (a) blood flow *BF* , (b) blood volume *BV* , (c) mean transit time *MTT* and (d) permeability surface *PS* .

Visual comparison of Fig. 9 and Fig. 12 shows just how much unnecessary computations are usually performed in areas outside the lungs. What is more, a clearly defined lung area allows for a much more accurate and reliable medical diagnosis.

## **6. Conclusion**

620 Lung Diseases – Selected State of the Art Reviews

parameters were calculated for all 18 ROIs. The results are shown in Table 8 (Kuziemski et

Here a noticeable increase was observed in the parenchyma perfusion parameter values of

*BF ml g Dev* [ /100 /min] *BV ml g Dev* [ /100 ] *PS ml g Dev* [ /100 /min] Artery Parenchyma Artery Parenchyma Parenchyma

> Diabetes 16.26.4

Control 12.01.8

Diabetes 35.626.1

Control 8.64.8

the diabetics with respect to control group, which may be due to diabetic mellitus.

Diabetes 62.825.2

Control 61.05.9

to the mean of the eighteen results. No significant *MTT* differences were found.

Table 8. Mean parameters *BF* , *BV* and *PS* in the artery and parenchyma of the diabetics and of the control group. The calculated standard deviation *Dev* illustrates the distribution of test results in 18 ROIs (ROI 3 – 20, in upper, central and lower part of lungs) with respect

Chest perfusion CT allows imaging of lung vessels and parenchyma, which is essential in diagnosing pulmonary angiopathy. The analyzed results are very new and currently not all that numerous. While this does not allow for a complex statistical analysis, the above preliminary results indicate the great potential of pCT tests in diagnosing lung

Each pCT image (see Fig. 9) consists of the body cross-section, including the lungs, essential for medical diagnosis, the rest of the body and the background, which is unnecessary for

As computations of perfusion parameters involve complicated methods of signal processing, it is desirable that the distinction between diagnostically important regions (the lung crosssection) and other areas (background and the rest of body cross-section) is conducted before the process starts. This shortens the time needed to obtain parametric maps by omitting data processing from the area outside the lungs. It can also help avoiding false lung malfunction diagnoses, which can occur when the automatic methods of pathological changes wrongly

Literature describes many methods of lung area extraction from CT chest scans (Homma et al, 2009), (de Nunzio et al, 2011), (Vinhais & Campilho, 2006), (Zhou et al, 2004). Most methods are devoted to extracting the lung area from a single image. Some of these methods

The lung extraction presented here was obtained using the two-step procedure. The objective of the preliminary processing was to define the rough mask, i.e. the border within which the lung contour is always contained during the pCT test while the lungs are breathing and the heart is beating. The lung and heart region contour pixels are subjected to the greatest changes. This behaviour can be utilised to detect the contours. It can be achieved

> max min *median*

(14)

*x x VF x*

allow for neighbouring (upper and lower) slices to be analysed (Homma et al, 2009).

lung diagnostics but was nevertheless included in the perfusion map calculations.

interpret signals from areas outside the lungs as abnormal lung signals.

by using a variability factor *VF* , which is defined as follows:

al, 2011).

Diabetes 722.5301.8

Control 681.7133.2

microangiopathy.

**5.3 Lung area extraction** 

Diabetes 282.2115.0

Control 207.653.4

> Pulmonary diabetic microangiopathy has not yet been sufficiently studied and is difficult to diagnose. In clinical practice, lung microangiopathy is diagnosed by comparing two diffusing capacity measurements, taken with the patient standing up and lying down. Other methods to assist diagnosis of lung microangiopathy are needed.

The three methods presented here seem good enough to be competitive options to complement those currently used in medicine today.

Diffusing capacity measurement and modelling allows for the development of a procedure to enhance the diagnostic process. This procedure is based on a single diffusing capacity measurement taken from a patient lying down, together with a model of oxygen diffusion from the alveoli to the blood. The model parameters, calculated using routine medical test results and physiological constants, turned out to be useful for binary classification in lung microangiopathy diagnosis. Diagnostic thresholds were calculated. Next binary classification was performed, based on model parameters, and, for comparison, on the diffusing capacity test result. These results were compared with already known medical diagnoses. This allowed for the sensitivity and specificity to be calculated. The best binary classification results were 79% *Sens* and *Spec* 87% , when based on model parameters, and, when based on diffusing capacity measurements, 62% *Sens* and *Spec* 71% . These results show that for microangiopathy diagnosis the model parameters were more sensitive and more specific than the diffusing capacity measurements.

The next method to be examined for usefulness in lung microangiopathy diagnosis was spirometry. Statistical comparison of the spirometry parameters of microangiopathic and non-microangiopathic patients, revealed that such parameters do not allow for a distinction to be made between patients with and without microangiopathy. Therefore a spirometry model was developed and its parameters were tested for their ability to distinguish between microangiopathic and non-microangiopathic subjects. Two, out of five, model parameters were statistically significant and helpful in lung microangiopathy diagnosis. Diagnostic thresholds were calculated and binary classification was performed, based on the statistically significant model parameters. The best results were 100% *Sens* and *Spec* 83% .

Both diagnostic procedures, the one based on the diffusing capacity and the one based on spirometric measurements and modelling, produce one of two possible results: 1) high probability of lung microangiopathy or 2) high probability of no lung microangiopathy.

Perfusion CT measurement seems to have considerable diagnostic potential with regard to lung microangiopathy. A study of lung tissue perfusion in diabetic patients in comparison with a group of healthy subjects showed an increase in the value of some parameters. These increased values may result from pulmonary microangiopathy. Further studies on the application of pCT in the detection of diabetes mellitus may not only improve diagnosis but also help us better understand pulmonary microangiopathy.

## **7. References**


The three methods presented here seem good enough to be competitive options to

Diffusing capacity measurement and modelling allows for the development of a procedure to enhance the diagnostic process. This procedure is based on a single diffusing capacity measurement taken from a patient lying down, together with a model of oxygen diffusion from the alveoli to the blood. The model parameters, calculated using routine medical test results and physiological constants, turned out to be useful for binary classification in lung microangiopathy diagnosis. Diagnostic thresholds were calculated. Next binary classification was performed, based on model parameters, and, for comparison, on the diffusing capacity test result. These results were compared with already known medical diagnoses. This allowed for the sensitivity and specificity to be calculated. The best binary classification results were 79% *Sens* and *Spec* 87% , when based on model parameters, and, when based on diffusing capacity measurements, 62% *Sens* and *Spec* 71% . These results show that for microangiopathy diagnosis the model parameters were more sensitive

The next method to be examined for usefulness in lung microangiopathy diagnosis was spirometry. Statistical comparison of the spirometry parameters of microangiopathic and non-microangiopathic patients, revealed that such parameters do not allow for a distinction to be made between patients with and without microangiopathy. Therefore a spirometry model was developed and its parameters were tested for their ability to distinguish between microangiopathic and non-microangiopathic subjects. Two, out of five, model parameters were statistically significant and helpful in lung microangiopathy diagnosis. Diagnostic thresholds were calculated and binary classification was performed, based on the statistically significant model parameters. The best results were 100% *Sens* and

Both diagnostic procedures, the one based on the diffusing capacity and the one based on spirometric measurements and modelling, produce one of two possible results: 1) high probability of lung microangiopathy or 2) high probability of no lung microangiopathy. Perfusion CT measurement seems to have considerable diagnostic potential with regard to lung microangiopathy. A study of lung tissue perfusion in diabetic patients in comparison with a group of healthy subjects showed an increase in the value of some parameters. These increased values may result from pulmonary microangiopathy. Further studies on the application of pCT in the detection of diabetes mellitus may not only improve diagnosis but

Alonzi R. & Hoskin P. Functional Imaging in Clinical Oncology: Magnetic Resonance

American Thoracic Society: Single-breath carbon monoxide diffusing capacity (transfer

Askey R.A., Roy R. Gamma function. In: *Digital Library of Mathematical Functions*. Edited by

Imaging- and Computerised Tomography-based Techniques, *Clin. Oncol*., 18, 7, pp.

factor): recommendations for a standard technique - 1995 update. *Am. J. Respir. Crit.* 

complement those currently used in medicine today.

and more specific than the diffusing capacity measurements.

also help us better understand pulmonary microangiopathy.

Olver FWJ, Lozier DM, Boisvert RF., N.I.S.T., 2007

*Spec* 83% .

**7. References** 

555-570, 2006

*Care Med*.;152:2185-2198, 1995


## **The Pneumoconioses**

Nlandu Roger Ngatu1, Ntumba Jean-Marie Kayembe2, Benjamin Longo-Mbenza2,3 and Narufumi Suganuma1

> *1Kochi University Medical School, Kochi, 2University of Kinshasa, Kinshasa, 3Walter Sisulu University, Mtata, 1Japan 2Democratic Republic of Congo 3Republic of South Africa*

## **1. Introduction**

624 Lung Diseases – Selected State of the Art Reviews

Popov D, Simionescu M. Alterations of lung structure in experimental diabetes, and

Strojek K., Ziora D., Sroczyński J.& Oklek K. Late lung symptoms of diabetes. *Pneunomol.* 

Taylor AE, Rehder K, Hyatt RE & Parker JC. Clinical Respiratory Physiology. Emeryville:

Villa M.P., Montesano M., Barreto M., Pagani J., Stegagno M. & Multari G. Diffusing

West JB., Respiratory physiology - the essentials. Philadelphia: Lippincott Williams &

Weynand B., Jonckheere A. Frans &, Rahier J. Diabetes mellitus induces a thickening of the

Yeh H., Punjabi N.M. & Wang N. Cross-sectional and prospective study of lung function in

Homma, N., Shimoyama, S., Ishibashi, T., and Yoshizawa, M.: Lung Area Extraction from X-

Active Contour Model, WSEAS Trans. Inf. Sci. Appl., 5, 6, pp. 746-755, 2009 de Nunzio, G., Tommasi, E., Agrusti, A., Cataldo, R., de Mitri, I., Favetta, M., Maglio, S.,

Vinhais, C., and Campilho, A.: Lung Parenchyma Segmentation from CT Images Based on Material Decomposition, *Lect. Notes Comput*. Sc., 4142, pp. 624-635, 2006 Zhou, X., Hayashi, T., Hara, T., Fujita, H., Yokoyama, R., Kiryu, T., and Hoshi, H.:

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47:1931–5, 2004

Wilkins, 2008

19, 1999

*Alergol. Pol.* 61:166, 1993.

W.B.Saunders Company, 1989

Study. *Diabetes Care*; 31:741–746, 2008

*Imaging*, 24, 1, pp. 11-2, 2011

*Proceedings of SPIE*, 5370, pp. 1629-1633, 2004.

diabetes associated with hyperlipidaemia in hamsters. *Eur. Respir. Journal*.;10:1850–

capacity for carbon monoxide in children with type 1 diabetes. *Diabetologia*;

pulmonary basal lamina. *Respiration, International Journal of Thoracic Medicine*. 66:14–

adults with type 2 diabetes: The Atherosclerosis Risk in Communities (ARIC)

ray CT Images for Computer-aided Diagnosis of Pulmonary Nodules by using

Massafra, A., Quarta, M., Torsello, M., Zecca, I., Bellotti, R., Tangaro, S., Calvini, P., Camarlinghi, N., Falaschi, F., Cerello, P., and Oliva, P.: Automatic Lung Segmentation in CT Images with Accurate Handling of the Hilar Region, *J. Digit.* 

Automatic recognition of lung lobes and fissures from multi-slice CT images,

#### **1.1 Overview on pneumoconioses**

Pneumoconiosis is an occupational lung disease caused by the exposure to dust. This section summarizes the generalities on pneumoconioses, including definitions, epidemiology and clinical manifestations of those occupational and environmental lung diseases. The most important step in the diagnosis of pneumoconiosis in to question the subject regarding specifics of the actual job and the minerals or materials involved in case of history of dust exposure. It is very important to seek a detailed account of workers' past employment, too; as some pneumoconioses develop after only a brief but intense dust exposure.

 Lately, many cases have been linked to environmental exposure to dust. This suggests that pneumoconiosis is no more a pathology exclusively related to work. Clinicians and epidemiologists should keep in mind the fact that cigarette smoking has a devastating impact of the health of dust-exposed individuals. Rates of cigarette smoking as high as 80% have been recorded among miners and other dust-exposed populations (Baum et al, 1998; Hammond et al, 1979). Asbestos-related diseases, silicosis and coal worker's pneumoconiosis (CWP) are most predominant and widely investigated pneumoconioses.

Pneumoconioses have relatively specific radiologic features that are not well-known to most physicians. Radiological imaging plays an important role in the diagnosis of those occupational lung diseases, including asbestos-related diseases, Silicosis and coal worker's pneumoconiosis (Ngatu et al, 2010; Blum et al, 2008). This review is aimed at providing health care workers, especially clinicians, with basic and accurate knowledge on principal radiologic features often found on a pneumoconiotic chest radiograph that characterize each of the above mentioned lung diseases related to occupational or environmental exposure to dust.

#### **1.2 Asbestosis and other asbestos-related diseases (ARDs)**

#### **1.2.1 Definitions and epidemiology**

**Asbestosis** refers to the pneumoconiosis caused by inhalation of asbestos fibers. It is characterized by a slowly progressive, diffuse pulmonary fibrosis. Asbestos is a fibrous mineral whose specific properties have encouraged its use since ancient times, in particular for industrial applications since the 19th century (Tarres et al, 2009; American Thoracic Society [ATS], 2001). Few natural materials used in industry have been the subject of more epidemiological and pathological research than the fibrous mineral, asbestos. Asbestos fibers are divided into two categories based on fiber's shape:


Of the different asbestos fibers used in industries, crocidolite (blue asbestos) is generally considered to be less toxic. The amphibole and amosite are reported to carry the greater risk of causing other asbestos-related diseases such as pleural plaque, mesothelioma and lung cancer. Exposed workers who have developed asbestosis are at risk of fatal complications (Wagner, 1997; Sichletidis et al, 2009; Mossman et al, 1990).

In patients with asbestosis, the chest radiograph usually shows small bilateral parenchymal opacities with a multinodular or reticular pattern that may be associated with pleural abnormalities. However, in some individuals with histopathologic evidence of pulmonary fibrosis, no interstitial abnormalities are found on the chest radiographs (Larson et al, 2010; Kipen et al, 1987). Other asbestos-related disorders are: pleural plaques, mesothelioma, diffuse pleural fibrosis, rounded atelectasia, benign asbestos-related pleural effusion and chronic bronchitis. In the course of asbestosis development, other radiographic features such as honeycombing changes and ground-glass opacities can be seen on the radiograph; they are well visualized on computed tomography (CT) and high resolution computed tomography (HRCT) scans.

It is estimated that approximately 27 millions workers in the United States received a significant exposure to asbestos during the middle of the last four decades of the twentieth century.

**Pleural disease** is one of the characteristics of asbestos exposure. Pleural plaques are present in about 50 percent of asbestos exposed individuals and most commonly involve the parietal pleura, while involvement of the visceral pleura is scarce. The histological examination of pleural plaque specimens shows the presence of predominant acellular bandles of collagen (Yang et al, 2010).

The prevalence of both pleural plaques and asbestosis is associated with "*time since the first exposure*" (TSFE) to asbestos, the "exposure intensity level", the "duration or cumulative exposure" to asbestos. Meanwhile, *time since the first exposure* seems to be the best predictor for pleural plaques related to asbestos exposure, whereas *cumulative exposure* to asbestos is reported to be a major determinant for asbestosis (American Thoracic Society [ATS], 2004; Schart, 1991). In a study conducted in Finish construction, shipyards and asbestos industry workers by Koskinen and coworkers (1998), a strong relationship was found between pleural plaques and *time since the first exposure* to asbestos. A recent study by Paris et al. (2009) showed for the first time, in a multivariate analysis, strong and independent correlations between *time since first exposure* and pleural plaques, and between *cumulative or level of exposure* with both pleural plaques and asbestosis, indicating that time and dose parameters should be included in the definition of high-risk populations in screening programs.

**Malignant mesothelioma** is an aggressive tumor that develops from the mesothelial cell of the pleura; it may also develop from the peritoneum, pericardium, or testicular tunica vaginalis.

mineral whose specific properties have encouraged its use since ancient times, in particular for industrial applications since the 19th century (Tarres et al, 2009; American Thoracic Society [ATS], 2001). Few natural materials used in industry have been the subject of more epidemiological and pathological research than the fibrous mineral, asbestos. Asbestos



Of the different asbestos fibers used in industries, crocidolite (blue asbestos) is generally considered to be less toxic. The amphibole and amosite are reported to carry the greater risk of causing other asbestos-related diseases such as pleural plaque, mesothelioma and lung cancer. Exposed workers who have developed asbestosis are at risk of fatal complications

In patients with asbestosis, the chest radiograph usually shows small bilateral parenchymal opacities with a multinodular or reticular pattern that may be associated with pleural abnormalities. However, in some individuals with histopathologic evidence of pulmonary fibrosis, no interstitial abnormalities are found on the chest radiographs (Larson et al, 2010; Kipen et al, 1987). Other asbestos-related disorders are: pleural plaques, mesothelioma, diffuse pleural fibrosis, rounded atelectasia, benign asbestos-related pleural effusion and chronic bronchitis. In the course of asbestosis development, other radiographic features such as honeycombing changes and ground-glass opacities can be seen on the radiograph; they are well visualized on computed tomography (CT) and high resolution computed

It is estimated that approximately 27 millions workers in the United States received a significant exposure to asbestos during the middle of the last four decades of the twentieth

**Pleural disease** is one of the characteristics of asbestos exposure. Pleural plaques are present in about 50 percent of asbestos exposed individuals and most commonly involve the parietal pleura, while involvement of the visceral pleura is scarce. The histological examination of pleural plaque specimens shows the presence of predominant acellular bandles of collagen

The prevalence of both pleural plaques and asbestosis is associated with "*time since the first exposure*" (TSFE) to asbestos, the "exposure intensity level", the "duration or cumulative exposure" to asbestos. Meanwhile, *time since the first exposure* seems to be the best predictor for pleural plaques related to asbestos exposure, whereas *cumulative exposure* to asbestos is reported to be a major determinant for asbestosis (American Thoracic Society [ATS], 2004; Schart, 1991). In a study conducted in Finish construction, shipyards and asbestos industry workers by Koskinen and coworkers (1998), a strong relationship was found between pleural plaques and *time since the first exposure* to asbestos. A recent study by Paris et al. (2009) showed for the first time, in a multivariate analysis, strong and independent correlations between *time since first exposure* and pleural plaques, and between *cumulative or level of exposure* with both pleural plaques and asbestosis, indicating that time and dose parameters should be included

**Malignant mesothelioma** is an aggressive tumor that develops from the mesothelial cell of the pleura; it may also develop from the peritoneum, pericardium, or testicular tunica vaginalis.

fibers are divided into two categories based on fiber's shape:

(Wagner, 1997; Sichletidis et al, 2009; Mossman et al, 1990).

in the definition of high-risk populations in screening programs.

crocidolite and other types of asbestos.

United States;

tomography (HRCT) scans.

century.

(Yang et al, 2010).

The association between malignant mesothelioma and asbestos exposure has been well-known worldwide since the 1950s. Approximately, 90% of malignant mesothelioma cases are related to ealier exposure to asbestos and the risk of developing the disease is greater in case of exposure to the amphibole fibers in crocidolite and amosite, but serpentine fibers in chrysotile can also cause the disease with a relatively long latency period (30 years or more) (Fujimoto et al, 2010; Snashall et al, 2001). In Japan, one of the biggest consumers of asbestos in the last four decades of the twentieth century, the number of cases of malignant mesothelioma is shown to correlate with the amount of asbestos consumption and the country has been expected to confront an epidemic of asbestos-related malignancy (Suganuma et al, 2001; Takahashi, 1999).

 The incidence of malignant pleural mesothelioma, an asbestos-related tumor, is increasing, with a median survival of seven to ten months; its clinical pattern usually involves substantial pain and dyspnea. The disease causes approximately 15,000 to 20,000 deaths per year worldwide (Pass et al, 2008).

**Lung cancer** can be consecutive to asbestos exposure. Clinician's search during risk assessment in patients with possible dust exposure may be hindered by the long latency period between the inhalation and the appearance clinical manifestions. In addition to its fibrogenic properties, asbestos is a first-level carcinogen and the most accepted oncological model is the dose-response without safety level (Rosell-Murphy et al, 2010; Goldberg et al, 1999). According to some previous studies, smoking increases the risk of lung cancer in asbestos exposed individuals by 50 – 90 times more than non-smokers (Valavanidis et al, 1996; Hartly et al, 2000).

A variety of occupational and nonoccupational settings can be source of exposure to asbestos:


Since a while, there is an increasing number of reports on cases of asbestos-related diseases in workers whose occupations have not been traditionally on the lists of occupational health specialists. Asbestosis and pleural plaques have been found in dentists and mechanics in Europe and the United States. And in some cases, these asbestos-related abnormalities are of unusual source of exposure.

Torbica and Krstev (2006) suggested that asbestos-related diseases in dentists might be linked to exposure to inorganic dust in the manufacturing of cobalt-chromiummolybdenum- based dental protheses. With the use of white asbestos-made lining material for casting rings in dentistry, dental technicians and dentists are potentially exposed to asbestos; and some cases of pleural plaques and malignant mesothelioma have been reported in a number of dentists after a relatively long period of work (35 to 45 years) (Choel et al, 2001; Reid et al, 1991; Sichletidis et at, 2009; Radi et al, 2002).

Similarly, pneumoconiosis may occur in mechanics. Most motor vehicles, from passenger cars to heavy duty trucks, have disc brakes on the front wheels and drum brakes on the rear wheels which help control their movement. Asbestos containing brake lining is generally found in those vehicles, ranging from 35% to 65% of chrysotile contents. Their repair or replacement can be a source of exposure to asbestos (Gilles, 2005; Paustnebach et al, 2004; Kakooei et al, 2004).

According to previously published reports, the incidence of lung cancer and malignant mesothelioma in auto mechanics is higher than in the general population. In the Unites States, concentrations of airborne asbestos more than 300 times higher than the current permissible exposure limit (Occupational Safety and Health Agency, OSHA) of 0.1 fiber/cc were found (Levin et al, 1999; Lorimer et al, 1976; Rohl et al, 1976).

#### **1.2.2 Clinical manifestations of asbestos-related diseases**

For asbestosis, in general, the latency period between exposure and the appearance of symptoms is inversely proportional to the intensity of asbestos exposure. And the majority of patients remain asymptomatic for 20- 30 years after the first exposure, even though pleural plaque is present. The first symptom of asbestosis to appear is usually breathlessness with exertion. Dyspnea will follow as the disease progresses; however, cough, wheezing, sputum production are often present when the patient has a history of cigarette smoking. With the progression of the disease, the patient will develop bibasilar crackles heard mostly at the end of inspiration, and clubbing. Pleural disease occurs earlier mostly within 15 years since the initial exposure to asbestos (Epler et al, 1982; Paris et al, 2009).

## **1.3 Silicosis**

#### **1.3.1 Definition and epidemiology**

**Silicosis** is an interstitial pulmonary disease secondary to the inhalation of crystalline silica (silicon dioxide), usually in the form of quartz, and less commonly as cristobalite and tridymite. It is one of the world's oldest known occupational diseases characterized by irreversible, progressive lung disease. Crystalline silica has long been considered as the toxic form of silica; however, very little was known about the toxicity of amorphous silica until the very recent toxicological and mechanistic study in animals by Constantini and cowokers that revealed the fact that both crystalline and amorphous (which has been regarded as nonfibrogenic) silica are phagocytosed and are both toxic to alveolar macrophages and have similar pathway that lead to apoptosis.

Silica, the second most abundant element that forms the quarter part of the earth's crust, is an ubiquitous mineral in human environment. Thus, exposure to silicon dioxide and salts of silicic acid is a fact of life. However, intense exposure can lead to silicosis (Pascual et al, 2011; Wagner, 1995; Constantini et al, 2011; Baum et al, 1998). Silica is also bound to other minerals; it is then called silicate. Silicates are used in some industries; they include asbestos, talc (Mg3S4O10 (OH)2), and kaoline (Al2Si2O5(OH)4) [Gamble, 1986].

New cases of Silicosis are annually recognized worldwide. In the United States, up to 200,000 miners and 1.7 million non-mining workers have experienced significant occupational exposure to inhaled silica; and every year the country register between 3600 and 7300 new cases of Silicosis. The overall mortality attributable to Silicosis has decreased in the United states thanks to the improvement of workplace protection, compared to that of 30 years ago (3000 deaths a year) [Rosenman et al, 2003; CDC, 2008; NIOSH, 1999].

Silicosis can occur in many industries and work settings with evident dust exposure such as:

Surface mining, underground mining.

Quarrying

628 Lung Diseases – Selected State of the Art Reviews

wheels which help control their movement. Asbestos containing brake lining is generally found in those vehicles, ranging from 35% to 65% of chrysotile contents. Their repair or replacement can be a source of exposure to asbestos (Gilles, 2005; Paustnebach et al, 2004;

According to previously published reports, the incidence of lung cancer and malignant mesothelioma in auto mechanics is higher than in the general population. In the Unites States, concentrations of airborne asbestos more than 300 times higher than the current permissible exposure limit (Occupational Safety and Health Agency, OSHA) of 0.1 fiber/cc

For asbestosis, in general, the latency period between exposure and the appearance of symptoms is inversely proportional to the intensity of asbestos exposure. And the majority of patients remain asymptomatic for 20- 30 years after the first exposure, even though pleural plaque is present. The first symptom of asbestosis to appear is usually breathlessness with exertion. Dyspnea will follow as the disease progresses; however, cough, wheezing, sputum production are often present when the patient has a history of cigarette smoking. With the progression of the disease, the patient will develop bibasilar crackles heard mostly at the end of inspiration, and clubbing. Pleural disease occurs earlier mostly within 15 years

**Silicosis** is an interstitial pulmonary disease secondary to the inhalation of crystalline silica (silicon dioxide), usually in the form of quartz, and less commonly as cristobalite and tridymite. It is one of the world's oldest known occupational diseases characterized by irreversible, progressive lung disease. Crystalline silica has long been considered as the toxic form of silica; however, very little was known about the toxicity of amorphous silica until the very recent toxicological and mechanistic study in animals by Constantini and cowokers that revealed the fact that both crystalline and amorphous (which has been regarded as nonfibrogenic) silica are phagocytosed and are both toxic to alveolar macrophages and have

Silica, the second most abundant element that forms the quarter part of the earth's crust, is an ubiquitous mineral in human environment. Thus, exposure to silicon dioxide and salts of silicic acid is a fact of life. However, intense exposure can lead to silicosis (Pascual et al, 2011; Wagner, 1995; Constantini et al, 2011; Baum et al, 1998). Silica is also bound to other minerals; it is then called silicate. Silicates are used in some industries; they include asbestos,

New cases of Silicosis are annually recognized worldwide. In the United States, up to 200,000 miners and 1.7 million non-mining workers have experienced significant occupational exposure to inhaled silica; and every year the country register between 3600 and 7300 new cases of Silicosis. The overall mortality attributable to Silicosis has decreased in the United states thanks to the improvement of workplace protection, compared to that

of 30 years ago (3000 deaths a year) [Rosenman et al, 2003; CDC, 2008; NIOSH, 1999]. Silicosis can occur in many industries and work settings with evident dust exposure such as:

were found (Levin et al, 1999; Lorimer et al, 1976; Rohl et al, 1976).

since the initial exposure to asbestos (Epler et al, 1982; Paris et al, 2009).

talc (Mg3S4O10 (OH)2), and kaoline (Al2Si2O5(OH)4) [Gamble, 1986].

**1.2.2 Clinical manifestations of asbestos-related diseases** 

Kakooei et al, 2004).

**1.3 Silicosis** 

**1.3.1 Definition and epidemiology** 

similar pathway that lead to apoptosis.

Surface mining, underground mining.


The list is not complete; there are numerous other work sites with silica exposure, as free silica can be found anywhere. Mining and construction work that involve drilling, cutting, grinding or crushing the earth's crust or rocks are associated with high level of silica exposure. In several developing countries, in Africa and Asia particularly, mining and minerals processing industries represent the main source of income. It is common to find employers who fail to provide necessary personal protective materials to workers, putting them at high risk of occupational lung disease such as silicosis.

It is also obvious that, in some occupational settings, workers may be exposed to both silica and asbestos or other dust and develop what is called a mixed-dust pneumoconiosis (MDP). MDP is pathologically defined as an occupational lung disease that shows dust macules or mixed-dust fibrotic nodules, with or without silicotic nodules, in an individual with a history of exposure to mixed dust [Honma et al, 2004]. Radiologic features of MDP may include those related to exposure to silica and other fibrogenic duts such as silicates, metals, carbon.

#### **1.3.2 Clinical manifestations of Silicosis**

Clinical forms of Silicosis are described according to both their clinical and radiological manifestations. They are divided into:


*Simple Silicosis* is characterized by the presence of small (less than 10 mm in diameter) opacities that are rounded in general, on the chest radiograph, and mostly without symptoms.

*Acute Silicosis* often occurs after weeks or a few years of an intense exposure to silica. The presence of silicotic nodules on the chest radiograph or computed tomography scan is the main radiologic manifestation.

*Chronic silicosis* usually appears 10 – 30 years after the initial exposure to silica and develops slowly. This clinical form often share the same radiographic feature with simple Silicosis; but in some cases silicotic nodules may coalesce and form a large opacity which represent a progressive massive fibrosis (PMF). Both accelerated and chronic silicosis have similar radiographic features. The only trait that differentiates them is the interval between the exposure to silica dust and the development of the disease symptoms.

*Accelerated Silicosis* occurs within ten years after initial exposure and is associated to highlevel exposure to silica. Both chronic and accelerated Silicosis may be asymptomatic with the patient having only an abnormal chest radiograph consistent with Silicosis. However, symptomatic patients would present with a chronic cough and dyspnea on exertion. Later, symptoms will become severe with the worsening of the radiographic abnormalities. Crackles, rhonchi and or wheeze may be present in some patients.

The presence of large opacity or progressive massive fibrosis is always accompanied by the aggravation of patient's status and respiratory impairment. The presence of emphysema, air trapping on chest radiograph may be observed (Munakata et al, 1985). Progressive massive fibrosis (PMF) occurs when small rounded opacities enlarge progressively and coalesce to generate larger opacities (more than 10 mm in diameter). PMF, when it is present, its location is either the upper or the middle zone of the lung.

*Malignancy* represents one of the complications of Silicosis. The international Agency for Research on Cancer (IARC) determined in 1997 that there was evidence of carcinogenicity of crystalline silica (Scarselli et al, 2011). The relationship between silica exposure and lung cancer risk have been demonstrated in several studies. More than ten cohort studies have shown a standardized mortality ratio (SMR) between 1.37 and 3.70, and few reports showed stronger associations with highest risks and excess mortality from lung cancer among silicotics (Scarselli et al, 2011; Lacasse et al, 2005; Erren et a, 2009;Pelucchi et al, 2006).

This suggests that clinicians should keep in mind the relationship between a workplace with exposure to silica dust and the development of lung cancer. Ignoring this fact may mistakenly lead the clinician towards a wrong diagnosis of the patient's condition.

#### **1.4 Coal worker's pneumoconiosis (CWP)**

#### **1.4.1 Definition and epidemiology**

**Coal worker's pneumoconiosis** is defined as an occupational lung disease caused by the deposition of coal mine dust in the lung parenchyma and the reaction of tissues to its presence. Coal mining has been used as a source of fuel for hundreds of years, and it still remains a major industry in countries such as the United States, France, Germany, Australia, China, India and South Africa.

Recent researches suggest changes in terms of the epidemiology and clinical features of pneumoconiosis among underground coal miners that are characterized by an increase in the severity and rapid disease progression. Factors such as over-exposure to silica dust in coal mines, the increase in coal production and increasing hours of work are thought to be responsible of the current high prevalence and increased severity of the coal worker's pneumoconiosis (CDC, 2007; Laney, 2009; Antao, 2005).

Laney and Attfield's more recent work, conducted in the Unites States, demonstrated the role of size of mine as another factor that contribute to the high prevalence and fatality of disease observed the last decades. In their study, workers from small mines had a greater risk of coal worker's pneumoconiosis and progressive massive fibrosis than their large mines counterparts. Smaller mining operations often have limited capital to upgrade safety equipment, and dedicated safety and personnel are less likely to be available to workers. This influences the implementation of dust monitoring and control activities in the mines (Laney & Attfield, 2010].

#### **1.4.2 Clinical forms of CWP**

CWP comprises two clinical forms: the *simple* and *complicated* Coal worker's pneumoconiosis. Generally, simple CWP is not associated with symptoms. It is often characterized by a history of underground exposure of more than 10 years. As for silicosis, the early radiological features are small rounded opacities that first appear on upper lobes of the lung.

In contrast, complicated CWP is associated with symptoms and a marked impairment of lung function. Shortness of breath, cough and sputum production are common symptoms (Caplan, 1953). The pattern and severity of respiratory impairment in coal worker's pneumoconiosis are related to the levels of coal mine dust exposure, geologic factors, exposure to other respiratory hazards, and the immunologic response to dust (Shen et al, 2004).

fibrosis (PMF) occurs when small rounded opacities enlarge progressively and coalesce to generate larger opacities (more than 10 mm in diameter). PMF, when it is present, its

*Malignancy* represents one of the complications of Silicosis. The international Agency for Research on Cancer (IARC) determined in 1997 that there was evidence of carcinogenicity of crystalline silica (Scarselli et al, 2011). The relationship between silica exposure and lung cancer risk have been demonstrated in several studies. More than ten cohort studies have shown a standardized mortality ratio (SMR) between 1.37 and 3.70, and few reports showed stronger associations with highest risks and excess mortality from lung cancer among

This suggests that clinicians should keep in mind the relationship between a workplace with exposure to silica dust and the development of lung cancer. Ignoring this fact may

**Coal worker's pneumoconiosis** is defined as an occupational lung disease caused by the deposition of coal mine dust in the lung parenchyma and the reaction of tissues to its presence. Coal mining has been used as a source of fuel for hundreds of years, and it still remains a major industry in countries such as the United States, France, Germany, Australia,

Recent researches suggest changes in terms of the epidemiology and clinical features of pneumoconiosis among underground coal miners that are characterized by an increase in the severity and rapid disease progression. Factors such as over-exposure to silica dust in coal mines, the increase in coal production and increasing hours of work are thought to be responsible of the current high prevalence and increased severity of the coal worker's

Laney and Attfield's more recent work, conducted in the Unites States, demonstrated the role of size of mine as another factor that contribute to the high prevalence and fatality of disease observed the last decades. In their study, workers from small mines had a greater risk of coal worker's pneumoconiosis and progressive massive fibrosis than their large mines counterparts. Smaller mining operations often have limited capital to upgrade safety equipment, and dedicated safety and personnel are less likely to be available to workers. This influences the implementation of dust monitoring and control activities in the mines (Laney & Attfield, 2010].

CWP comprises two clinical forms: the *simple* and *complicated* Coal worker's pneumoconiosis. Generally, simple CWP is not associated with symptoms. It is often characterized by a history of underground exposure of more than 10 years. As for silicosis, the early radiological features are small rounded opacities that first appear on upper lobes

In contrast, complicated CWP is associated with symptoms and a marked impairment of lung function. Shortness of breath, cough and sputum production are common symptoms (Caplan, 1953). The pattern and severity of respiratory impairment in coal worker's pneumoconiosis are related to the levels of coal mine dust exposure, geologic factors, exposure to other respiratory

silicotics (Scarselli et al, 2011; Lacasse et al, 2005; Erren et a, 2009;Pelucchi et al, 2006).

mistakenly lead the clinician towards a wrong diagnosis of the patient's condition.

location is either the upper or the middle zone of the lung.

**1.4 Coal worker's pneumoconiosis (CWP)** 

pneumoconiosis (CDC, 2007; Laney, 2009; Antao, 2005).

hazards, and the immunologic response to dust (Shen et al, 2004).

**1.4.1 Definition and epidemiology** 

China, India and South Africa.

**1.4.2 Clinical forms of CWP** 

of the lung.

#### **2. Classification of High Resolution Computed Tomography (HRCT) scans and chest radiographs of pneumoconioses**

#### **2.1 Overview on the classification of pneumoconiotic HRCT scans**

The use of Computed Tomography (CT) has revolutionized the radiologic diagnosis of chest diseases as compared with conventional radiography. However, the ability of CT scanners to evaluate pulmonary parenchymal abnormalities is limited because of their resolving power. With the introduction of High-Resolution Computed Tomography (HRCT) techniques, it became possible to perform images of the lung with excellent spatial resolution, providing anatomic detail similar to that available from gross pathologic specimens (Kusaka et al., 2005). Thus, HRCT increases the specificity in the diagnosis of lung diseases, including pneumoconioses. In addition, the early diagnosis of these chronic diseases allows an early care and avoidance of further exposure to the hazardous dusts. For that reason, the proposed International Classification of HRCT of pneumoconiosis is used in some developed countries (such as Japan, Germany and Finland) in the screening and surveillance programs; however, its acceptance by international health institutions might take a time.

The chest radiograph, which is easily accessible and cheaper, presently remains the unique internationally accepted diagnostic tool for pneumoconiosis. For this practical reason, in this chapter, we only present different parameters that are taken into account in the international classification of HRCT of pneumoconiosis.

This section summarizes briefly the information on the proposed international classification of HRCT of pneumoconiosis. Though it is not yet accepted by the International Labor Office (ILO), clinicians should have in mind the existence of this tool which, sooner or later could be of use worldwide.

The purpose of the international HRCT classification is to describe and code parenchymal and pleural manifestations of diffuse non-malignant occupational and environmental respiratory diseases. It is used for the screening (for early detection of pneumoconiosis) and surveillance and follow-up of the exposed individuals) (Kusaka et al., 2005).

In *asbestosis*, the most common HRCT findings are centrilobular nodules on branching areas of high attenuation, and the thickened interlobular and intralobular lines, subpleural dot-like or curvilinear opacities and honeycombing predominantly distributed in the bases of the lungs. The interstitial fibrosis may also be manifested as traction bronchiectasis, honeycombing.

On the other hand, in case of *silicosis or CWP*, the common characteristic radiologic features on HRCT scan are small opacities predominantly distributed in the upper zones of the lung and, sometimes, images of progressive massive fibrosis (large opacity) (Aberle et al., 1988; Akira et al., 2002).

The reading sheet used in the coding system for CT or HRCT films of pneumoconiosis has the following main parameters to be reported: film quality, parenchymal abnormalities (small opacities, large opacity, emphysema, honeycombing, inhomogenous attenuation of lung parenchyma, pleural abnormalities) ( Kusaka et al., 2005).


0 = no definite opacities;

1=mild (present but only few small opacities);

2=moderate (numerous small opacities);

3=severe (very numerous small opacities and normal anatomical lung structure are poorly.

A number of additional parenchymal abnormalities have to be checked:


Detailed information on the international classification of HRCT of pneumoconioses is available elsewhere (Kusaka et al., 2005).

#### **2.2 The ILO International Classification of chest Radiographs of Pneumoconiosis (ILO/ICRP) and ILO/WHO global program for the elimination of Silicosis (ILO/WHO GPES)**

The ILO 2000 International Classification of Radiographs of Pneumoconiosis (ILO/ ICRP) is used worldwide by experts to categorize conditions that are consistent with pneumoconiosis, with the use of a set of ILO standard films.

This international classification system was created with the purpose of coding anterioposterior chest radiographs in a simple and reproducible manner; it provides a means for a systematic description and recording of radiographic abnormalities in the chest caused by the inhalation of dusts. It is useful in epidemiologic research, surveillance and medical checks of dust-exposed workers (Suganuma, 2001; International Labor Office [ILO], 2002). Chest radiographs of patients with silicosis, coal worker's pneumoconiosis asbestosis and other asbestos-related diseases can be interpreted and classified the same way anywhere in the world using the ILO/ICRP system. In this section, basics on the principal radiologic features of pneumoconioses and their characteristics are summarized. In order to fully understand the system, a special training is of utmost importance.

To classify a chest radiograph of a dust-exposed individual, there are four main points to be considered:


3=severe (very numerous small opacities and normal anatomical lung structure are poorly.

a. *inhomogenous attenuation* (absence [No] or presence [Yes] which may be due to the presence of *ground glass opacities* (GGO). When present on HRCT scan, the inhomogenous attenuation should be graded for each side and each zone for its extent

c. *emphysema*: absence (No) or presence (Yes) of emphysema has to be reported, and graded for right (R) and left (L) side, and for all zones as 1 (mild [up to 15% of area of



Detailed information on the international classification of HRCT of pneumoconioses is

The ILO 2000 International Classification of Radiographs of Pneumoconiosis (ILO/ ICRP) is used worldwide by experts to categorize conditions that are consistent with

This international classification system was created with the purpose of coding anterioposterior chest radiographs in a simple and reproducible manner; it provides a means for a systematic description and recording of radiographic abnormalities in the chest caused by the inhalation of dusts. It is useful in epidemiologic research, surveillance and medical checks of dust-exposed workers (Suganuma, 2001; International Labor Office [ILO], 2002). Chest radiographs of patients with silicosis, coal worker's pneumoconiosis asbestosis and other asbestos-related diseases can be interpreted and classified the same way anywhere in the world using the ILO/ICRP system. In this section, basics on the principal radiologic features of pneumoconioses and their characteristics are summarized. In order to fully

To classify a chest radiograph of a dust-exposed individual, there are four main points to be



**2.2 The ILO International Classification of chest Radiographs of Pneumoconiosis (ILO/ICRP) and ILO/WHO global program for the elimination of Silicosis (ILO/WHO** 

A number of additional parenchymal abnormalities have to be checked:

one zone]), 2(moderate [15-30%]), 3(severe [more than 30%]).

1=mild (present but only few small opacities); 2=moderate (numerous small opacities);

as follows: 1=focal; 2=patchy; 3=diffuse. b. *honeycombing* (HC): absence (No) or presence (Yes);

d. *large opacity*: absence (No) or presence (Yes).

diaphragmatic,..) are also reported.

available elsewhere (Kusaka et al., 2005).

pneumoconiosis, with the use of a set of ILO standard films.

understand the system, a special training is of utmost importance.


reading sheet.

**GPES)** 

considered:

sheet).


*Technical quality* There are four grades:


A technical defect may be the presence of artifact, with poor contrast, under (light) or overexposed (dark) film or due to an improper position of the subject (overlapping of scapula, for example) or other defect. Artifacts can be interpreted as pathological abnormalities in case they mimic opacities, while the overlap of scapula (Fig. 1) can easily be interpreted as inprofile or face on plaque. An abnormal feature related to the lung parenchyma or pleura may be present on the chest radiograph, but hidden by the overlapping scapula. When a chest radiograph is qualified as unreadable, it is necessary that another one be indicated.

Fig. 1. Chest radiograph of poor quality showing overlapping scapula (white arrows) looking like pleural plaques on both right and left lung fields.

*[Courtesy of Prof. Dr. Yukinori Kusaka, Asian Intensive Reading of Pneumoconiosis radiographs (AIR PNEUMO)]*

#### **2.2.1 Parenchymal abnormalities**

They include two types of opacities: small and large opacities.

**Small opacities** are described by *profusion, affected lung zones,* the *shape* (rounded or irregular) and *size.*


To classify the dust-exposed individual's chest radiograph, it has to be compared with the standard radiographs from ILO (known as ILO standard radiographs) which profusion seems to be closer to that in the worker's film. Standard radiographs provide examples of appearances classifiable as subcategory 0/0; 1/1; 2/2; 3/3.


**Large opacities** are opacities whose longest diameter exceeds 10 mm. There are three categories of large opacities:


Fig. 2. Silicotic chest radiograph showing a large opacity of category B (black arrows) on the right upper zone. Numerous rounded opacities (q type) are present in all zones of the lung field. *(Courtesy of Dr Hisao Shida)* 

seems to be closer to that in the worker's film. Standard radiographs provide examples of



**Large opacities** are opacities whose longest diameter exceeds 10 mm. There are three



Fig. 2. Silicotic chest radiograph showing a large opacity of category B (black arrows) on the right upper zone. Numerous rounded opacities (q type) are present in all zones of the lung field.

divided into three zones (upper, middle, lower) by horizontal lines.

appearances classifiable as subcategory 0/0; 1/1; 2/2; 3/3.

asbestos, for example (mixed-dust pneumoconiosis).

not exceeding the equivalent of the right upper zone (Fig. 2);

categories of large opacities:

upper zone.

*(Courtesy of Dr Hisao Shida)* 

Fig. 3. Chest radiograph showing calcified diaphragmatic pleural plaques (white arrows) and face-on plaques (blue arrows) bilaterally. In this film, pleural plaques can also be seen in other site (mediastinal region bilaterally).

*(Courtesy of Prof. Dr Yukinori Kusaka, AIR PNEUMO)* 

Fig. 4. Chest radiograph showing an in-profile pleural plaque (arrows) in asbestos-exposed worker.

*(Courtesy of Dr Hisao Shida)* 

#### **2.2.2 Pleural abnormalities**

They comprise *pleural plaques* (localized pleural thickening), *costophrenic angle obliteration* and *diffuse pleural thickening* (DPT).

Pleural plaques may be located on the diaphragm, on the chest wall (in-profile or face-on plaques) and at other sites; they generally represent a thickening of the parietal pleura and are recoded as present or absent.

Other details regarding the site (right or left lung), presence or absence of calcification, width and extent of the plaque are recorded.

#### **2.2.3 Costophrenic angle obliteration and diffuse pleural thickening**

*Costophrenic angle obliteration* is recoded as present or absent. However, with the ILO classification*, diffuse pleural thickening* extending up to the lateral chest wall is recorded only in case of an obliterated costophrenic angle. All details are summarized in a single chest radiograph reading sheet (ILO, 2002).

The global program for the elimination of Silicosis (GPES) was adopted in 2003, Geneva, by a panel of experts in occupational health. It is aimed at establishing a wide international cooperation so as to eliminate Silicosis by the year 2030 (World Health Organization [WHO], 2003). GPES was established following recommendations of the twelfth session of the joint ILO/WHO Committee on occupational health in 1995, which indentified the global elimination of silicosis as a priority area for action in occupational health. The committee wanted countries to place it high on their agendas, with the belief that the experience gained through implementation of this program would provide a prevention model for other pneumoconioses and a proven system for the management of exposure to mineral dust.

The GEPS has two different approaches; the primary prevention of Silicosis that emphasizes the control of silica hazard at source using engineered methods of dust control, while the secondary prevention includes surveillance of the work environment to assess the efficacy of the implemented measures for dust exposure control, exposure evaluation by assessing health risk for workers and surveillance of workers' health for the early diagnosis of the disease.

In most industrialized countries, following recommendation of the ILO/WHO Global Program for the Elimination of Silicosis, pneumoconiosis prevention programs have been initiated to protect workers from dust exposure and the actual trend of show a decrease in number of new cases. On the other hand, in the majority of developing countries millions of workers continue to be exposure to dust. In order to reach the goal by 2030, each country should have its own national program. Unfortunately, the reality shows that there are still obstacles that might make it difficult to reach the GPES goal by 2030: one is the non adherence of some countries to the ILO/WHO program and, the insufficiency of qualified individuals in the interpretation of pneumoconiotic chest radiographs.

Under the ILO/WHO GPES, experts focused manly on secondary prevention, upgrading skills of occupational physicians in developing countries in using the ILO 2000 Classification of Radiographs of Pneumoconiosis (ILO 2000 ICRP) and strengthening national Silicosis elimination programs (Luton, 2007).

### **3. Short training on pneumoconiosis chest radiographs reading**

#### **3.1 Objective**

The ILO classification system is an important tool for training occupational physicians in the diagnosis of pneumoconioses. Based on this ILO system, some countries have developed their own national program and produced necessary teaching materials. In the United States, the National Institute for Occupational Health (NIOSH) provides certification tests physicians, radiologists and other professionals in the field of occupational health through

Pleural plaques may be located on the diaphragm, on the chest wall (in-profile or face-on plaques) and at other sites; they generally represent a thickening of the parietal pleura and

Other details regarding the site (right or left lung), presence or absence of calcification,

*Costophrenic angle obliteration* is recoded as present or absent. However, with the ILO classification*, diffuse pleural thickening* extending up to the lateral chest wall is recorded only in case of an obliterated costophrenic angle. All details are summarized in a single chest

The global program for the elimination of Silicosis (GPES) was adopted in 2003, Geneva, by a panel of experts in occupational health. It is aimed at establishing a wide international cooperation so as to eliminate Silicosis by the year 2030 (World Health Organization [WHO], 2003). GPES was established following recommendations of the twelfth session of the joint ILO/WHO Committee on occupational health in 1995, which indentified the global elimination of silicosis as a priority area for action in occupational health. The committee wanted countries to place it high on their agendas, with the belief that the experience gained through implementation of this program would provide a prevention model for other pneumoconioses and a proven system for the management of exposure to mineral dust. The GEPS has two different approaches; the primary prevention of Silicosis that emphasizes the control of silica hazard at source using engineered methods of dust control, while the secondary prevention includes surveillance of the work environment to assess the efficacy of the implemented measures for dust exposure control, exposure evaluation by assessing health risk for workers and surveillance of workers' health for the early diagnosis of the disease. In most industrialized countries, following recommendation of the ILO/WHO Global Program for the Elimination of Silicosis, pneumoconiosis prevention programs have been initiated to protect workers from dust exposure and the actual trend of show a decrease in number of new cases. On the other hand, in the majority of developing countries millions of workers continue to be exposure to dust. In order to reach the goal by 2030, each country should have its own national program. Unfortunately, the reality shows that there are still obstacles that might make it difficult to reach the GPES goal by 2030: one is the non adherence of some countries to the ILO/WHO program and, the insufficiency of qualified

**2.2.3 Costophrenic angle obliteration and diffuse pleural thickening** 

individuals in the interpretation of pneumoconiotic chest radiographs.

**3. Short training on pneumoconiosis chest radiographs reading** 

Under the ILO/WHO GPES, experts focused manly on secondary prevention, upgrading skills of occupational physicians in developing countries in using the ILO 2000 Classification of Radiographs of Pneumoconiosis (ILO 2000 ICRP) and strengthening national Silicosis

The ILO classification system is an important tool for training occupational physicians in the diagnosis of pneumoconioses. Based on this ILO system, some countries have developed their own national program and produced necessary teaching materials. In the United States, the National Institute for Occupational Health (NIOSH) provides certification tests physicians, radiologists and other professionals in the field of occupational health through

are recoded as present or absent.

radiograph reading sheet (ILO, 2002).

elimination programs (Luton, 2007).

**3.1 Objective** 

width and extent of the plaque are recorded.

the B-readers system. And, since the year 2008, the Asian Intensive Reading of Pnemoconioses chest radiographs (AIR-PNEUMO) program started, organizing annual workshops and certification test for Asian countries and also Brazil. These programs contribute a lot for the achievement of the ILO/WHO Global Program for the Elimination of Silicosis, and also those dedicated to the elimination of asbestosis.

However, the number of physicians who have required skills to interpret pneumoconiotic radiographs remains insignificant in many regions of the world, as participation to ILO training course by physicians is quite costly and, moreover, the absence of local trainers constitutes a real obstacle. Countries that do not yet get involved in the ILO/WHO program are numerous. Despite the existence of institution such Air-pneumo, NIOSH and others, the elimination of silicosis may not be achieved by the year 2030 without involvement of governments and health authorities of all nations. Providing basic knowledge on pneumoconiosis to clinicians and those who are about to graduate from medical school would also be helpful for the diagnosis of pneumoconiosis, particularly in regions where occupational or environmental exposure to dust represent a real public health issue. Here is presented a short training on pneumocnioses and the evaluation of its potential to improve physicians' ability to detect main pneumoconiotic radiographic features on chest radiographs of dust-exposed individuals.

#### **3.2 Differentiating normal and pneumoconiotic radiographs**

Radiological imaging plays an important role in the diagnosis of pneumoconiosis. Although chest radiography has some limitations in terms of detecting earliest parenchymal and pleural abnormalities, it remains the most useful and universally accepted method for assessing pneumoconiosis; it is an easily accessible, cheap diagnostic tool (Takashima et al., 2007).

On a normal chest radiograph (Fig. 5), there are anatomical structures that resemble abnormal features and may lead the reader to wrong interpretation. The presence of lymph

Fig. 5. Normal chest radiograph showing normal anatomical features. Note the presence of opacities that represent lymph nodes and blood vessels. *(Courtesy of Dr Hisao Shida)*

Fig. 6. Silicotic chest radiograph showing small rounded opacities of q type mostly, with involvement of all lung zones (upper, middle and lower zones). Coalescence of small rounded opacities is seen on the right upper zone. *(Courtesy of Dr Hisao Shida)* 

nodes, muscles (serratus anterior, oblique externus abdominis...), pleural fat's shadow and normal cross-section or tangential views of blood vessels can be interpreted as opacities or pleural plaques related to pneumoconiosis, for example. This poses a challenge during chest radiograph reading process and may lead to misclassification of conditions consistent with pneumoconioses (Ngatu et al, 2010; Jinkai et al, 2008).

For individuals who present with pneumoconiotic-like radiographic features, establishing the history of dust exposure is an important step towards the confirmation of the diagnosis. A silicotic or coal worker's pneumoconiosis' chest radiograph will often present small rounded opacities (opacities having a diameter up to 10 mm) as parenchymal abnormalities (Fig. 6). When they coalesce, small opacities form a large opacity (having a diameter > 10 mm). In addition, a silicotic radiograph may also show calcified lymph nodes (eggshell calcifications). In silicosis, small opacities are first localized on the upper zone of the lung, then will progress towards the middle and lower zones.

In contrast, a chest radiograph from an asbestosis patient will often present small irregular opacities (opacities having a longest dimension up to 10 mm) as parenchymal abnormalities (Fig. 7) and/or pleural plaque (s). Asbestosis small opacities appear on the lower zone of the lung, then later progress towards the middle and the upper lung zones. Other features may also be present such as diffuse pleural thickening, honeycombing changes.

The table below summarizes frequently found pneumoconiotic radiographic features for mineral dust-exposed subjects: their localization according to the type of pneumoconiosis, and their shape and size for small opacities (Table 1). It is important to know that individuals exposed to both silica and asbestos may develop a "Mixed Dust Pneumoconiosis (MDP); the radiograph may show a combination of features of silica, asbestos or other fibrogenic dusts related diseases.

Fig. 6. Silicotic chest radiograph showing small rounded opacities of q type mostly, with involvement of all lung zones (upper, middle and lower zones). Coalescence of small

nodes, muscles (serratus anterior, oblique externus abdominis...), pleural fat's shadow and normal cross-section or tangential views of blood vessels can be interpreted as opacities or pleural plaques related to pneumoconiosis, for example. This poses a challenge during chest radiograph reading process and may lead to misclassification of conditions consistent with

For individuals who present with pneumoconiotic-like radiographic features, establishing the history of dust exposure is an important step towards the confirmation of the diagnosis. A silicotic or coal worker's pneumoconiosis' chest radiograph will often present small rounded opacities (opacities having a diameter up to 10 mm) as parenchymal abnormalities (Fig. 6). When they coalesce, small opacities form a large opacity (having a diameter > 10 mm). In addition, a silicotic radiograph may also show calcified lymph nodes (eggshell calcifications). In silicosis, small opacities are first localized on the upper zone of the lung,

In contrast, a chest radiograph from an asbestosis patient will often present small irregular opacities (opacities having a longest dimension up to 10 mm) as parenchymal abnormalities (Fig. 7) and/or pleural plaque (s). Asbestosis small opacities appear on the lower zone of the lung, then later progress towards the middle and the upper lung zones. Other features may

The table below summarizes frequently found pneumoconiotic radiographic features for mineral dust-exposed subjects: their localization according to the type of pneumoconiosis, and their shape and size for small opacities (Table 1). It is important to know that individuals exposed to both silica and asbestos may develop a "Mixed Dust Pneumoconiosis (MDP); the radiograph may show a combination of features of silica, asbestos or other

also be present such as diffuse pleural thickening, honeycombing changes.

rounded opacities is seen on the right upper zone.

pneumoconioses (Ngatu et al, 2010; Jinkai et al, 2008).

then will progress towards the middle and lower zones.

*(Courtesy of Dr Hisao Shida)* 

fibrogenic dusts related diseases.

Fig. 7. Chest radiograph of asbestos-exposed worker showing the presence of small irregular opacities (of "t" type primarily and "s" type secondarily), predominantly on lower and middle lung zones. The right upper lobe is also involved. The profusion is 2/3. In addition, diffuse pleural thickening (DPT) is evident on the right and in-profile plaque on the left; also note the abnormal cardiac size. The film quality is of grade 2 (scapula overlap).  *(Courtesy of Prof. Dr Yukinori Kusaka, AIR PNEUMO)* 


Table 1. Summarized main radiographic findings related to silicosis and asbestos-related lung diseases

#### **3.3 Implementation of the two-hour training session 3.3.1 Participants and methods**

For the first reported training held in 2008, 102 Japanese physicians (72 males and 28 females; 2 to 44 years of experience) having different background (preventive medicine, internal medicine, psychiatrists, surgery, pediatrics) from different cities within the country. Participants were invited to the 2008 training session held in Kochi and Ehime prefecture through the staff of Japanese medical corporations, and the training session was conducted by a NIOSH-B reader from Kochi University Medical School, Japan.

We used three sets of chest radiographs were used, namely ILO/ICRP standard films, dustexposed workers' chest radiographs and a set of twelve test films. ILO standard films were used to describe pneumoconiotic lung abnormalities (shape, size, profusion of small opacities; large opacities, pleural plaques) during the lecture. Dust-exposed workers' chest radiographs, having different radiographic features of pneumoconioses, were provided by the Japan Pneumoconioses Study Group (JPSG). In order to read them, each film was put side by side with the ILO standard whose profusion, for example, seems to be close to that of the patient.

Our intervention was aimed at improving inexperienced physicians' skill in reading pneumo-coniotic chest radiographs. Participants had to listen to a pre-session talk to remind them the abnormalities to be checked on each of patients' radiographs (before the main lecture); then, they take a pre-test as described in the protocol (Fig. 8). Each participant received a reading form that had a list of 12 radiograph numbers (from 1 to 12) and they had to check for the presence (Yes) or the absence (No) of parenchymal or pleural abnormalities consistent with pneumoconiosis (opacities and pleural plaques) for each chest radiograph. Classifying the radiographs using categories of profusion of small opacities, location, width or extent of pleural plaques was not indicated. The main lecture was given after the pre-test for two-hours; afterwards a post-test was organized using the same test films that were displayed on viewboxes in the lecture room.

Fig. 8. Protocol of the intervention study (CXR: chest radiograph; JPSG: Japan pneumoconiosis study group)

## **3.3.2 Evaluation of physicians' reading skill and observed results**

The reading forms were collected and managed by s staff who no relationship with any of the participant and all participants were anonymously coded for the analysis of the tests

For the first reported training held in 2008, 102 Japanese physicians (72 males and 28 females; 2 to 44 years of experience) having different background (preventive medicine, internal medicine, psychiatrists, surgery, pediatrics) from different cities within the country. Participants were invited to the 2008 training session held in Kochi and Ehime prefecture through the staff of Japanese medical corporations, and the training session was conducted

We used three sets of chest radiographs were used, namely ILO/ICRP standard films, dustexposed workers' chest radiographs and a set of twelve test films. ILO standard films were used to describe pneumoconiotic lung abnormalities (shape, size, profusion of small opacities; large opacities, pleural plaques) during the lecture. Dust-exposed workers' chest radiographs, having different radiographic features of pneumoconioses, were provided by the Japan Pneumoconioses Study Group (JPSG). In order to read them, each film was put side by side with the ILO standard whose profusion, for example, seems to be close to that

Our intervention was aimed at improving inexperienced physicians' skill in reading pneumo-coniotic chest radiographs. Participants had to listen to a pre-session talk to remind them the abnormalities to be checked on each of patients' radiographs (before the main lecture); then, they take a pre-test as described in the protocol (Fig. 8). Each participant received a reading form that had a list of 12 radiograph numbers (from 1 to 12) and they had to check for the presence (Yes) or the absence (No) of parenchymal or pleural abnormalities consistent with pneumoconiosis (opacities and pleural plaques) for each chest radiograph. Classifying the radiographs using categories of profusion of small opacities, location, width or extent of pleural plaques was not indicated. The main lecture was given after the pre-test for two-hours; afterwards a post-test was organized using the same test films that were

> Training session Using ILO/ICRP

Post-intervention test (use of ILO standard Radiographs)

and

Comparison of CXR reading skill before and after intervention

**+**

JPSG films

The reading forms were collected and managed by s staff who no relationship with any of the participant and all participants were anonymously coded for the analysis of the tests

**3.3 Implementation of the two-hour training session** 

by a NIOSH-B reader from Kochi University Medical School, Japan.

**3.3.1 Participants and methods** 

displayed on viewboxes in the lecture room.

102 physicians enrolled

pneumoconiosis study group)

Brief explanation on lung abnormalities to check on CXR

1 2 <sup>3</sup> Enrollment

Fig. 8. Protocol of the intervention study (CXR: chest radiograph; JPSG: Japan

**3.3.2 Evaluation of physicians' reading skill and observed results** 

**+**

Pre-test

of the patient.

(pre-test and post-test) results. The difference in the physicians' reading skill before and after the intervention was evaluated using McNemar's chi-square test.

The general trend was towards improvement in terms of physicians' ability to detect pneumoconiotic parenchymal and pleural abnormalities on the test-radiographs. Regarding the dectection of the presence of pneumoconiotic small opacities, a significant increase in the specificity score was observed, 65% in the pre-test and 73% in post-test (p<0.0001); whereas the sensitivity score remained high, 84% and 81% in pre- and post-test (p>0.05), respectively (Table 2). It is noticeable that the high sensitivity score in the pre-test was due to a high proportion of physicians who considered the normal radiographs to have pneumoconiotic opacities (false positive or over reading) in the pre-intervention test. The over reading of test radiographs was noticeable for the pre-intervention test for most of the readers.


p: p-value by McNemar's chi-square test.

Table 2. Overall distribution of the sensitivity and specificity scores of the participants for detecting pneumoconiotic small opacities and pleural plaques on chest radiographs


p: p-value by McNemar's chi-square test.

Table 3. Distribution of sensitivity and specificity scores of readers for detecting pneumoconiotic abnormalities according to medical specialty.

For pleural plaques, a marked increase in the sensitivity score was noted, 46% in pre-test and 60% in post-test (p<0.0001), while an improvement was also observed in the specificity score but not significantly (77% and 79% in pre and post-test, respectively) (p>0.05) as shown in Table 2.

When compared according their medical specialties, a relatively similar reading skill improvement was observed between internists and physicians from working in other departments. Higher scores of sensitivity for the detection of small opacities and specificity for plaques were found in internists, and a similar trend was also noted in the group of physicians from other specialties in which a significant improvement of specificity score for pleural plaques was found (p<0.05) in the post-test (Table 3) (Ngatu et al, 2010).

The lack of training for medical doctors in the diagnosis of occupational diseases is the main factor leading to the misdiagnosis of pneumoconiosis as either chronic bronchitis or pulmonary tuberculosis (Murlidhar et al, 2005). One of the strong points of this training program is that, despite being carried out in relatively short time, it results in a noticeable improvement of physicians' ability to detect pneumoconiotic parenchymal and pleural abnormalities. Popularizing such program may contribute to the early diagnosis of pneumoconioses, improve their prognosis and give chances of survival for individuals with lung diseases related to dust exposure.

## **4. Conclusion**

This review article provides basic knowledge on pneumoconioses and a practical approach that may help physcians to diagnose occupational lung disorders related to exposure to mineral dust. Meanwhile, a training with demonstrations on the pneumoconiotic chest radiograph reading process is of utmost importance. Holding regular short period training courses for physicians, and even medical students who are about to graduate, focusing on main radiolographic features of lung parenchymal and pleural abnormalities related to occupational or environmental exposure to dust, with the use of typical ILO standard films, will contribute to improving their skill in the diagnosis of pneumoconioses.

## **5. Acknowledgments**

The authors thank Professor Yukinori Kusaka, Dr Hisao Shida and Dr Masanori Akira for providing materials used in this report.

## **6. References**


pulmonary tuberculosis (Murlidhar et al, 2005). One of the strong points of this training program is that, despite being carried out in relatively short time, it results in a noticeable improvement of physicians' ability to detect pneumoconiotic parenchymal and pleural abnormalities. Popularizing such program may contribute to the early diagnosis of pneumoconioses, improve their prognosis and give chances of survival for individuals with

This review article provides basic knowledge on pneumoconioses and a practical approach that may help physcians to diagnose occupational lung disorders related to exposure to mineral dust. Meanwhile, a training with demonstrations on the pneumoconiotic chest radiograph reading process is of utmost importance. Holding regular short period training courses for physicians, and even medical students who are about to graduate, focusing on main radiolographic features of lung parenchymal and pleural abnormalities related to occupational or environmental exposure to dust, with the use of typical ILO standard films,

The authors thank Professor Yukinori Kusaka, Dr Hisao Shida and Dr Masanori Akira for

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## **Lung Transplantation: Advances and Roadblocks in Treatment**

Matthew T. Hardison1 and J. Edwin Blalock2

*1Medical Genetics Laboratories, Department of Human and Molecular Genetics, Baylor College of Medicine 2Pulmonary, Allergy and Critical Care Medicine, Department of Medicine, University of Alabama at Birmingham United States of America* 

## **1. Introduction**

646 Lung Diseases – Selected State of the Art Reviews

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> The increasing population age, along with the prevalence of smoking and other environmental factors have contributed to a dramatic increase in the incidence of chronic pulmonary diseases with no known cures. Chronic obstructive pulmonary disease (COPD), lung cancer, and primary pulmonary hypertension are all conditions with well understood origins but few, if any therapeutic options. When taken in conjunction with the prevalence of genetic or poorly understood conditions such as cystic fibrosis (CF) and idiopathic pulmonary fibrosis (IPF) there is a large and growing cohort of patients that will eventually enter end-stage lung disease. The only currently available treatment option is lung transplantation which may potentially be extremely beneficial in terms of quality of life and life expectancy, although carries with it a vast array of considerations and complications. This chapter attempts to provide an account of the progress made in lung transplantation, complications associated with the procedure, current treatment for transplant associated conditions, and finally will discuss current research and possible future therapeutics.

## **2. History of lung transplantation**

Lung transplantation remains the final therapeutic option for treatment of patients with diverse diagnoses of end stage lung disease. (Arcasoy and Kotloff 1999) This however, has not always been the case. It was only within the last three decades that the medical community achieved reproducible outcomes that translated into clinical improvement in the condition of the patient. Vladimir Demikhov is considered by some to be the founder of the field due to his work in animal models in the mid 20th century, but it wasn't until 1963 that the first attempt at human lung transplantation occurred. (Cooper 1969; Hardy et al. 1963) This early trial was in a prisoner with both terminal lung cancer, and severe emphysema. Unfortunately, the patient died a mere 18 days post-transplantation. (Cooper 1969) It was almost another two decades before the first successful heart-lung transplantation was performed resulting in the multi-year survival of the recipient. This 1981 triumph was followed up by a group out of Toronto that proved once and for all that the one-time pipe dream of regular lung transplantation in humans was, in fact, a reality. (Cooper et al. 1987) It was in the early 1990s that a comparative "transplant boom" began and has since leveled off to the approximately 2,700/year lung transplants performed worldwide today. (Orens and Garrity 2009) The relatively limited number (compared to other whole organ transplant procedures) of lung transplantations is a factor of limited supply of acceptable organs and ability to procure donor tissue. (Dilling and Glanville 2011) Unfortunately, it is because of this dearth of patients that few multi-center trials have been performed to assist in the investigation of better pre-, peri-, and post-operative techniques. However, with the increase in surgeries seen in the 1990s, serious efforts at reducing the risk factors associated with lung transplantation were initiated.

## **3. Selection of candidates**

The majority of this chapter will deal with the complications and efforts to reduce them post-transplantation, but we will briefly touch on steps that have been implemented to reduce risk exposure prior to surgery. These preventative steps begin with a strict limitation on those who are eligible for transplant. Typically, individuals 55 and older are considered unsuitable for operation; although the mean age of recipients has been steadily increasing (it is currently over 50). (Dilling and Glanville 2011) Multiple organ failure, history of noncompliance, active/recent cigarette smoking, and active cancer are all contraindications for enrolling a patient in the United Network for Organ Sharing (UNOS). (Maurer et al. 1998) One of the most important factors that clinicians must consider when evaluating a patient for transplantation is the infection state of the possible recipient. While most bacterial and/or viral colonizations are permissible, there are many transplant centers that refuse to operate on patients with unremitting infection with pan-resistant *P. aeruginosa* and *B. cepacia*. Even so, there is at least one group out of Canada that is willing to consider transplantation of this patient population. (Nash et al. 2010; Flume et al. 1994) Prior to 2005, the allocation of lungs in the United States was dependant solely on waiting time; those who had been on the list longer were given priority over newly enrolled patients. Unfortunately, this obviously created a selection bias against the acutely ill. In 2005 the Lung Allocation System underwent an overhaul, utilizing a Lung Allocation Score (LAS) to determine those patients that were both most in need of transplantation, and most likely to survive the surgery and excel in recovery. (Egan et al. 2006) The new LAS system includes a number of factors (age, 6 minute walk test, forced vital capacity (FVC), body mass index (BMI), etc) to determine the patient's predicted waitlist survival time. The LAS is equal to (Calculated 1 year survival benefit) – (Calculated 1 year waiting list survival). (Morton and Glanville 2009) Obviously, there are limitations to this type of allocation system; certain subjective metrics like functional status and diagnosis can affect the patient's LAS, but the new method of lung allocation appears to have had a beneficial effect on median survival time posttransplantation.

#### **4. Selection of donor organs**

#### **4.1 Blood group matching and CMV status**

All of the evaluations discussed above are recipient-focused mechanisms designed to facilitate better outcomes in lung transplantation, but perhaps some of the most important protocols in place are related to obtaining the most viable donor organ available. There are several factors that contribute to the difficulty of procuring optimal lungs. Due to the

It was in the early 1990s that a comparative "transplant boom" began and has since leveled off to the approximately 2,700/year lung transplants performed worldwide today. (Orens and Garrity 2009) The relatively limited number (compared to other whole organ transplant procedures) of lung transplantations is a factor of limited supply of acceptable organs and ability to procure donor tissue. (Dilling and Glanville 2011) Unfortunately, it is because of this dearth of patients that few multi-center trials have been performed to assist in the investigation of better pre-, peri-, and post-operative techniques. However, with the increase in surgeries seen in the 1990s, serious efforts at reducing the risk factors associated with

The majority of this chapter will deal with the complications and efforts to reduce them post-transplantation, but we will briefly touch on steps that have been implemented to reduce risk exposure prior to surgery. These preventative steps begin with a strict limitation on those who are eligible for transplant. Typically, individuals 55 and older are considered unsuitable for operation; although the mean age of recipients has been steadily increasing (it is currently over 50). (Dilling and Glanville 2011) Multiple organ failure, history of noncompliance, active/recent cigarette smoking, and active cancer are all contraindications for enrolling a patient in the United Network for Organ Sharing (UNOS). (Maurer et al. 1998) One of the most important factors that clinicians must consider when evaluating a patient for transplantation is the infection state of the possible recipient. While most bacterial and/or viral colonizations are permissible, there are many transplant centers that refuse to operate on patients with unremitting infection with pan-resistant *P. aeruginosa* and *B. cepacia*. Even so, there is at least one group out of Canada that is willing to consider transplantation of this patient population. (Nash et al. 2010; Flume et al. 1994) Prior to 2005, the allocation of lungs in the United States was dependant solely on waiting time; those who had been on the list longer were given priority over newly enrolled patients. Unfortunately, this obviously created a selection bias against the acutely ill. In 2005 the Lung Allocation System underwent an overhaul, utilizing a Lung Allocation Score (LAS) to determine those patients that were both most in need of transplantation, and most likely to survive the surgery and excel in recovery. (Egan et al. 2006) The new LAS system includes a number of factors (age, 6 minute walk test, forced vital capacity (FVC), body mass index (BMI), etc) to determine the patient's predicted waitlist survival time. The LAS is equal to (Calculated 1 year survival benefit) – (Calculated 1 year waiting list survival). (Morton and Glanville 2009) Obviously, there are limitations to this type of allocation system; certain subjective metrics like functional status and diagnosis can affect the patient's LAS, but the new method of lung allocation appears to have had a beneficial effect on median survival time post-

All of the evaluations discussed above are recipient-focused mechanisms designed to facilitate better outcomes in lung transplantation, but perhaps some of the most important protocols in place are related to obtaining the most viable donor organ available. There are several factors that contribute to the difficulty of procuring optimal lungs. Due to the

lung transplantation were initiated.

**3. Selection of candidates** 

transplantation.

**4. Selection of donor organs** 

**4.1 Blood group matching and CMV status** 

extremely limited time that lungs are able to endure ischemia (less than 6 hours), there is little opportunity to perform traditional human leukocyte antigen (HLA) matching. Physicians face an unfortunate catch-22; better HLA matching reduces incidence of chronic rejection, but increased ischemic time in turn leads to a higher propensity for rejection. (Brugiere et al. 2008) Blood groups, rather, are the primary method of histological matching, with consideration being given to size and cytomegalovirus (CMV) sero-status. However, this is somewhat disputed in a review of the heart-lung transplant patient population at Stanford. (Deuse et al. 2010) CMV sero-negative recipients are at a higher risk of developing CMV infection when miss-matched with sero-positive donors than those recipients that are already CMV sero-positive. CMV infection has shown to be immunomodulative; creating an increased risk of acute and chronic rejection, and infections. (Freeman 2009) Like other opportunistic infections, there is an increased risk of CMV disease with the use of immunosuppressive treatment, although CMV in turn increases the risk of rejection, defeating the intent of the immunosuppressive therapies. (Fishman et al. 2007) CMV infection is not only limited to lung transplant recipients and thus there have been significant efforts from all corners to establish a gold-standard of prevention and treatment for CMV disease in all transplant patients. In studies performed in kidney and liver recipients, investigators demonstrated a significant reduction in incidence of CMV disease and viremia, along with significant reduction in the number of opportunistic infections in patients treated with prophylactic valganciclovir. (Humar et al. 2010) Valganciclovir is currently considered the gold-standard in CMV prevention, but other DNA polymerase inhibitors, including ganciclovir and valacyclovir have been in use for several years with a smaller impact. (Zamora et al. 2004) Unfortunately, lung transplant recipients face a higher incidence of CMV infection than any other transplant patient population. This is for two primary reasons; first, CMV dormancy and recurrence have been shown to be higher in the lungs than anywhere else, and, unlike other whole organ transplant surgeries, there is a preponderance of lymphatic tissue transplanted with the lungs that contains further amounts of dormant virus. (Zamora, Davis, and Leonard 2005)

#### **4.2 Other donor factors**

Although blood group matching and CMV sero-status are two of the most important criteria when evaluating potential donor lungs, there are a multiple other factors surgeons must consider when assessing a possible donor organ. Traditionally it has been protocol to decline any lungs from donors positive for hepatitis B, even so, recent work has cracked open the door to this pool of donors, potentially increasing the donor pool and number of available organs. A 1-year longitudinal study showed no significant difference in survival of hepatitis B positive donor lung recipients compared to the hepatitis B sero-negative group. (Dhillon et al. 2009) The physicians must also take into account something as simple as time and distance when determining the possibility of performing the potentially life-altering operation. Lungs, like any other tissue, are susceptible to ischemia-reperfusion (IR) injury and the flood of damaging cytokines that go along with it. Primary graft dysfunction, which can occur in up to 20% of patients, has been directly attributed to IR injury. (Christie, Sager et al. 2005) In addition to this, the lungs are more readily affected by the abundance of fluids that are typically given in end of life situations. (Arcasoy and Kotloff 1999) Current thinking is that non-heart beating donors (NHBD) are ill suited for the harvesting of lungs due to the perceived damage that warm extended periods of ischemia would incur. This has been challenged, however by a group in Canada demonstrating no ill-adverse effects from the use of this here-to-for off-limits pool of potential donor candidates. They state that there is an ameliorative effect on the levels of inflammatory cytokines present in these lungs which off sets the perceived ischemic damage. (Cypel et al. 2009)

Donor age is another consideration that is factored in when considering possible transplantation. Not surprisingly, elevated donor age and increased ischemic times correlate with worse outcomes in transplant recipients. (Meyers and Patterson 2000) While not optimal, there is such a shortage of available donors, NHBDs over the age of 60 now represent over 10% of the NHBD donor population. (Ojo et al. 1999) There are several techniques available to try and optimize the donor lung, but most common are the use of vasopressin, and thyroid hormone. (Botha et al. 2008) Finally, brain dead (BD) donors represent a potential donor pool that presents its own unique set of possible complications. In animal models of BD, investigators observed a three fold increase in the blood volume in the pulmonary circulation within minutes after onset of BD. (Avlonitis et al. 2005) This, in concert with loss of sympathetic tone, contributes heavily to a rapid onset of pulmonary edema which in and of itself is enough to dissuade many surgeons from considering using the tissue. Furthermore, several groups have reported a significant increase in the concentrations of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-2, IL-6, and tumor necrosis factor-alpha (TNFα). (Skrabal et al. 2005; Pratschke et al. 2000; Shohami et al. 1994) Additionally, the neutrophil chemokine, IL-8, has also been demonstrated to be elevated in the lungs of BD lung donors. (Fisher et al. 1999) Increased neutrophil burden has been strongly associated with increased matrix-metalloprotease activity and possible exposure of the autoantigen collagen V (Coll V). (Fisher et al. 2001; Hardison 2009) The exposure of Coll V directly correlates with the incidence of primary graft dysfunction. (Iwata et al. 2008)

All of the considerations discussed contribute to the limited number of lung transplants that are performed each year, but the lack of family consent is by far the largest obstacle to available donor organs. (Spital 2005) Since it is unlikely that an automatic-enrollment system similar to other countries is likely, it is vital that the best efforts are made to elucidate the mechanisms behind the numerous and varied complications inherent in lung transplantation by both basic scientists and clinicians alike. The litany of assessments and interventions discussed thus far are all employed to attempt to prevent the plethora of posttransplantation complications that can arise. The unfortunate reality is that while surgical techniques and medical therapeutics are far more advanced and elegant than when the initial forays in lung transplantation occurred, there is still ample room for improving the outcomes observed in lung transplant recipients. The remainder of this chapter will discuss post-transplantation complications, current therapies, recent research in the field, and the exciting possibilities for novel therapeutics that are on the horizon.

#### **5. Post transplantation complications**

Due to the myriad of risk factors associated with lung transplantation, it has one of the highest morbidity and mortality rates of any whole organ transplant procedure. (Trulock et al. 2007) Only recently has the mean survival time for transplant recipients risen to 5.7 years. (Ahmad, Shlobin, and Nathan 2011) There are four major causes of morbidity and mortality in all transplant recipients; primary allograft dysfunction, infection, acute rejection, and chronic rejection. These four primary causes are closely related; rather they exist on a spectrum with the incidence of chronic rejection (the most serious) rising with increasing occurrences of the PGD, infection and acute rejection.

#### **5.1 Primary graft dysfunction**

650 Lung Diseases – Selected State of the Art Reviews

of this here-to-for off-limits pool of potential donor candidates. They state that there is an ameliorative effect on the levels of inflammatory cytokines present in these lungs which off

Donor age is another consideration that is factored in when considering possible transplantation. Not surprisingly, elevated donor age and increased ischemic times correlate with worse outcomes in transplant recipients. (Meyers and Patterson 2000) While not optimal, there is such a shortage of available donors, NHBDs over the age of 60 now represent over 10% of the NHBD donor population. (Ojo et al. 1999) There are several techniques available to try and optimize the donor lung, but most common are the use of vasopressin, and thyroid hormone. (Botha et al. 2008) Finally, brain dead (BD) donors represent a potential donor pool that presents its own unique set of possible complications. In animal models of BD, investigators observed a three fold increase in the blood volume in the pulmonary circulation within minutes after onset of BD. (Avlonitis et al. 2005) This, in concert with loss of sympathetic tone, contributes heavily to a rapid onset of pulmonary edema which in and of itself is enough to dissuade many surgeons from considering using the tissue. Furthermore, several groups have reported a significant increase in the concentrations of pro-inflammatory cytokines such as interleukin-1 (IL-1), IL-2, IL-6, and tumor necrosis factor-alpha (TNFα). (Skrabal et al. 2005; Pratschke et al. 2000; Shohami et al. 1994) Additionally, the neutrophil chemokine, IL-8, has also been demonstrated to be elevated in the lungs of BD lung donors. (Fisher et al. 1999) Increased neutrophil burden has been strongly associated with increased matrix-metalloprotease activity and possible exposure of the autoantigen collagen V (Coll V). (Fisher et al. 2001; Hardison 2009) The exposure of Coll V directly

correlates with the incidence of primary graft dysfunction. (Iwata et al. 2008)

exciting possibilities for novel therapeutics that are on the horizon.

**5. Post transplantation complications** 

occurrences of the PGD, infection and acute rejection.

All of the considerations discussed contribute to the limited number of lung transplants that are performed each year, but the lack of family consent is by far the largest obstacle to available donor organs. (Spital 2005) Since it is unlikely that an automatic-enrollment system similar to other countries is likely, it is vital that the best efforts are made to elucidate the mechanisms behind the numerous and varied complications inherent in lung transplantation by both basic scientists and clinicians alike. The litany of assessments and interventions discussed thus far are all employed to attempt to prevent the plethora of posttransplantation complications that can arise. The unfortunate reality is that while surgical techniques and medical therapeutics are far more advanced and elegant than when the initial forays in lung transplantation occurred, there is still ample room for improving the outcomes observed in lung transplant recipients. The remainder of this chapter will discuss post-transplantation complications, current therapies, recent research in the field, and the

Due to the myriad of risk factors associated with lung transplantation, it has one of the highest morbidity and mortality rates of any whole organ transplant procedure. (Trulock et al. 2007) Only recently has the mean survival time for transplant recipients risen to 5.7 years. (Ahmad, Shlobin, and Nathan 2011) There are four major causes of morbidity and mortality in all transplant recipients; primary allograft dysfunction, infection, acute rejection, and chronic rejection. These four primary causes are closely related; rather they exist on a spectrum with the incidence of chronic rejection (the most serious) rising with increasing

sets the perceived ischemic damage. (Cypel et al. 2009)

#### **5.1.1 Characterization and etiology**

Primary graft dysfunction (PGD) is defined as having a **Pa**O2/**Fi** O2 of ≤ ~300 with radiographic infiltrates consistent with pulmonary edema. (Christie, Carby et al. 2005) PGD is further categorized into 3 gradations with Grades 1, 2 and 3 corresponding to **Pa**O2/**Fi** O2 greater than 300, between 200 and 300, and less than 200, respectively. Clinically, PGD is simply a unique form of acute lung injury (ALI), presenting within 72 hours post-surgery with replete pulmonary infiltrates/edema and impaired compliance. These guidelines for evaluating PGD were initially put forth in 2005 by the International Society for Heart and Lung Transplantation (ISHLT) Working Group on PGD and later confirmed by a retrospective study of approximately 400 lung transplant recipients. This study demonstrated that PGD grade 3 did indeed correlate with the worst mortality. (Prekker et al. 2006) PGD can affect up to 25% of patients and correlates with much higher levels of mortality in the first 30 days. (Christie et al. 2005) The precise cause(s) of PGD are still unknown, but it is widely accepted that ischemia-reperfusion (IR) injury plays a major role in initiating the destructive process. It is in the ischemic period that reactive oxygen species (ROS) are produced in large quantities and directly damage the pulmonary epithelium and endothelium. (Tasoulis et al. 2009; de Perrot et al. 2003) This influx of ROS incites a proinflammatory cascade that activates both the innate and complement immune systems. (Carter, Gelman, and Kreisel 2008)

#### **5.1.2 Risk factors for PGD**

Advanced age of organ donors is strongly associated with increased incidence of PGD in lung transplant recipients. What is interesting is that no definitive studies have been performed that indicate that recipients' age play any role in the development of PGD. (Barr et al. 2005; Christie et al. 2003) Additionally, race (African-American), sex (female), and smoking history are all donor-dependent risk factors in the development of PGD. (Lee and Christie 2009) No conclusive studies of transplant recipients have explicitly delineated whether indication for transplantation correlates with development but it appears that idiopathic pulmonary fibrosis confers at least some increased risk of PGD. (Barr et al. 2005) Pulmonary arterial hypertension (PAH) has emerged as the only clinically measureable metric that correlates with the development of primary graft dysfunction. While the precise pathway of PAH to PGD is as yet unknown, multiple studies have demonstrated the relationship. (Whitson et al. 2006)

#### **5.1.3 Treatment of PGD**

Although over-used to the point of being a cliché, the maxim "an ounce of prevention is worth a pound of cure" is distinctly applicable to the treatment of primary graft dysfunction. Increasing emphasis is being placed on improved preservation of the donor organ. The main weapon in the clinician's arsenal of preservative techniques is induced hypothermia of the tissue. It has been demonstrated that by maintaining the organ at 4oC, the metabolic rate is drastically reduced compared to that of control temperature tissue. (de Perrot et al. 2005) Two methods of organ cooling are typically employed; core cooling and pulmonary arterial flush (PAF). (Okada and Kondo 2009) The core cooling method occurs prior to organ explantation and is initiated with cardio-pulmonary bypass to reduce the body temperature to ~14oC. Upon harvesting, the donor lungs are submerged in 4oC saline. PAF is accomplished by instilling 60ml/kg into the pulmonary artery, there have been some recent studies investigating the effects of a dual flush with the addition of flushing the pulmonary veins as well. The hypothesis being that this would ensure the complete removal of micro-thrombi in the capillaries and result in a more equitable distribution of the preservative solution. (Struber et al. 2002) There are several different "recipes" of preservative solution but the first in wide use (Euro-Collins solution), with high a high K+ and low Na+ concentrations to mimic intracellular fluid, was originally developed for use in liver preservation. (Okada and Kondo 2009) Since then, more sophisticated preservation solutions have been developed and are in the process of being tested. The Okada group developed a solution termed extra-cellular phosphate buffered saline type 4 (Ep4) that included dextran 40. This solution was capable of preserving canine lungs for up to 96 hrs post-explantation. Dextran 40 appeared to exert an anti-coagulative effect, ostensibly by forming a protective coat on the endothelial surface. (Handa et al. 1989; Okada et al. 1997; Colombat et al. 2004)

Sadly, if efforts to prevent the development of primary graft dysfunction fail, the similarity to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) results in little that the clinician can do other than provide supportive care. Due to the increased concentration of pro-inflammatory cytokines in the pulmonary circulation it is of vital importance that fluid administration is closely monitored to decrease the risk of fulminant edema. (Shargall et al. 2005) In addition to the vigilance given to the administration of fluids in PGD patients, ventilator settings can play a major role in outcomes of those individuals. A multicenter, randomized, controlled trial demonstrated that lower tidal volumes, combined with elevated positive end expiratory pressure (PEEP) were significantly protective in individuals with ARDS. The authors of the study hypothesize that this was due to the decreased alveolar damage in the hyper-compliant lungs. (Petrucci and Iacovelli 2003)

#### **5.1.4 Current research and future directions**

Much of the current research in this area is focused on the development of better therapeutics and the identification of biomarkers in PGD to provide a deeper knowledge of the genetic and biochemical forces that are integral in the genesis of the condition. With the recent advances made in high through-put proteomics and DNA micro-array technology there are ample platforms to rapidly pursue numerous avenues of investigation in great detail.

#### **5.1.4.1 Potential Biomarkers of PGD**

A 2006 study by Kaneda et al, utilizing quantitative real time polymerase chain reaction (qPCR) revealed that the IL-6/IL-10 ratio in the donor lung was predictive of 30 day mortality in the transplant recipient. (Kaneda et al. 2006) Luminex analysis of blood samples from 25 Grade 3 PGD patients and 25 Grade 0 control patients by Hoffman, et al revealed a vastly different chemokine profile in the two population's plasma. Severe PGD patients had significantly elevated levels of monocyte chemotactic protein-1 (MCP1) and CXC motif ligand 10 (CXCL10) compared to controls. (Hoffman et al. 2009) The detection of novel biomarkers is not limited to new sophisticated techniques, more traditional approaches also have also yielded results. Indeed, a recent study by Diamond, et al, using a conventional "sandwich" enzyme linked immunosorbent assay (ELISA) provided evidence that increased Clara cell secretory protein 16 (CC16) was significantly associated with the development of PGD. (Diamond et al. 2011) There are numerous other studies that have produced a litany of possible biomarkers (soluble receptor for advanced glycosylation end-products (sRAGE), soluble P-selectin, Protein C, etc.) for primary graft dysfunction and that may one day provide the key to its early detection and prevention. (Kawut et al. 2009; Christie et al. 2007; Christie et al. 2009)

#### **5.1.4.2 Novel/Future Therapeutics for PGD**

The goal of those developing novel therapeutics for PGD is the prevention of the condition itself. A multi-center, randomized, placebo controlled trial by Keshavjee, et al showed a protective effect in the use of soluble complement receptor-1 (sCR1) resulting in decreased time to extubation and a trend toward abbreviated intensive care unit (ICU) stays. (Keshavjee et al. 2005) In an intriguing study by Eriksson, and colleagues, induced hypothermia appeared to be beneficial in case studies of PGD patients and was recapitulated in an animal model. (Eriksson and Steen 1998; Eriksson et al. 1999) Significant forays into the use of inhaled Nitric Oxide (iNO) as a therapeutic agent have also been made in recent years. There have been discordant results, however as to the efficacy, ranging from no change in outcomes to a decreased incidence of severe PGD and diminished concentrations of IL-6 and IL-8 in plasma compared to controls. (Meade et al. 2003; Ardehali et al. 2001) Due to the prevalence of PGD in lung transplant recipients, and the comparative lack of knowledge regarding the pathogenesis of this condition, it is reasonable to expect considerable resources to continue to be directed toward the investigation and prevention of this syndrome.

#### **5.2 Infection**

652 Lung Diseases – Selected State of the Art Reviews

PAF is accomplished by instilling 60ml/kg into the pulmonary artery, there have been some recent studies investigating the effects of a dual flush with the addition of flushing the pulmonary veins as well. The hypothesis being that this would ensure the complete removal of micro-thrombi in the capillaries and result in a more equitable distribution of the preservative solution. (Struber et al. 2002) There are several different "recipes" of preservative solution but the first in wide use (Euro-Collins solution), with high a high K+ and low Na+ concentrations to mimic intracellular fluid, was originally developed for use in liver preservation. (Okada and Kondo 2009) Since then, more sophisticated preservation solutions have been developed and are in the process of being tested. The Okada group developed a solution termed extra-cellular phosphate buffered saline type 4 (Ep4) that included dextran 40. This solution was capable of preserving canine lungs for up to 96 hrs post-explantation. Dextran 40 appeared to exert an anti-coagulative effect, ostensibly by forming a protective coat on the endothelial surface. (Handa et al. 1989; Okada et al. 1997;

Sadly, if efforts to prevent the development of primary graft dysfunction fail, the similarity to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) results in little that the clinician can do other than provide supportive care. Due to the increased concentration of pro-inflammatory cytokines in the pulmonary circulation it is of vital importance that fluid administration is closely monitored to decrease the risk of fulminant edema. (Shargall et al. 2005) In addition to the vigilance given to the administration of fluids in PGD patients, ventilator settings can play a major role in outcomes of those individuals. A multicenter, randomized, controlled trial demonstrated that lower tidal volumes, combined with elevated positive end expiratory pressure (PEEP) were significantly protective in individuals with ARDS. The authors of the study hypothesize that this was due to the decreased alveolar damage in the hyper-compliant lungs. (Petrucci and Iacovelli 2003)

Much of the current research in this area is focused on the development of better therapeutics and the identification of biomarkers in PGD to provide a deeper knowledge of the genetic and biochemical forces that are integral in the genesis of the condition. With the recent advances made in high through-put proteomics and DNA micro-array technology there are ample platforms to rapidly pursue numerous avenues of investigation in great

A 2006 study by Kaneda et al, utilizing quantitative real time polymerase chain reaction (qPCR) revealed that the IL-6/IL-10 ratio in the donor lung was predictive of 30 day mortality in the transplant recipient. (Kaneda et al. 2006) Luminex analysis of blood samples from 25 Grade 3 PGD patients and 25 Grade 0 control patients by Hoffman, et al revealed a vastly different chemokine profile in the two population's plasma. Severe PGD patients had significantly elevated levels of monocyte chemotactic protein-1 (MCP1) and CXC motif ligand 10 (CXCL10) compared to controls. (Hoffman et al. 2009) The detection of novel biomarkers is not limited to new sophisticated techniques, more traditional approaches also have also yielded results. Indeed, a recent study by Diamond, et al, using a conventional "sandwich" enzyme linked immunosorbent assay (ELISA) provided evidence that increased Clara cell secretory protein 16 (CC16) was significantly associated with the development of PGD. (Diamond et al. 2011) There are numerous other studies that have produced a litany of

Colombat et al. 2004)

detail.

**5.1.4 Current research and future directions** 

**5.1.4.1 Potential Biomarkers of PGD** 

#### **5.2.1 Characterization and etiology**

Infection in post lung transplantation begins as any other infection, however with the host being in the unenviable position of possessing immune systems under active and permanent suppression, in addition to the mechanical and physiologic stress that is inherent to any major surgery. Infection is, in fact, the primary source of mortality in the first year posttransplant and continues to be a significant source of morbidity and mortality for the remainder of the recipient's life. (Corris and Christie 2008) Unlike all other solid organ transplants, the lungs are open to the external environment and all of the pathogens, both virulent and opportunistic, that entails. Furthermore, the denervation that is a necessary component of organ harvesting results in at least a temporary impairment in the cough reflex and thus diminished clearance. (Ahmad, Shlobin, and Nathan 2011) It is important to note that any infection, particularly recurrent infection, is a risk factor for the occurrence of rejection, both acute and chronic. Rapid identification of the pathogen and appropriate treatment is optimal to decrease morbidity and mortality in this patient population.

#### **5.2.2 Bacterial infection**

Bacterial infections are the most common type of infection in lung transplant recipients and occur in a bi-modal, temporal-dependant manner. Pulmonary bacterial infections are common both early, due to the previously mentioned impaired cough reflex and damaged lymphatic system, and late, as an element of chronic rejection. (Kramer et al. 1993) Due to the nature of the pre-transplant disease, those patients with cystic fibrosis (CF) are the most difficult to maintain in an infection free state. This is especially true in the individual with a single lung transplant due to the potential for colonization from the native lung. Interestingly, the CF patient population also has the best long-term results despite the proclivity towards infection. (Lease and Zaas 2010)

*Pseudomonas aeruginosa* is the most common bacterial organism isolated from the posttransplant lung. (Kramer et al. 1993) This opportunistic infection has a higher incidence in CF patients and has been estimated to be responsible for up to 33% of pneumonias in all transplant recipients. (Aguilar-Guisado et al. 2007; Campos et al. 2008) *P. aeruginosa* airway infection prior to transplant does not appear to demonstrate a predictive relationship for chronic rejection. However, this is not the case for colonization in the post-operative period. (Botha et al. 2008; Vos et al. 2008) *P. aeruginosa* infection is not limited to pneumonias; the pathogen has been reported to be responsible for nearly half of all post-transplant infections. (Valentine et al. 2008)

*Mycobacterium* is a designation indicating acid-fast bacteria that are capable of causing a wide range of diseases. Perhaps the most well known of these *M. tuberculosis*, has the robust cell wall that is a calling card of the classification, and helps to confer resistance to broad spectrum antibiotics. Although, not nearly as common as *P. aeruginosa* infection, *M. tuberculosis* colonization presents a unique set of treatment hurdles, especially in the presence of multi-drug resistant (MDR) strains. (Lee et al. 2003)

Opportunistic infections, hospital-acquired infections, and resistant strains of bacteria are all significantly more common in lung transplant recipients than the general population. *Staphylococci* strains have demonstrated a propensity for antibiotic resistance and this is only magnified in the already immunosuppressed transplant patient. Specifically, *S. aureus* is the second most common organism isolated from the airway, with multi-drug resistant *S. aureus* (MRSA) being extremely difficult to treat due to its almost pan-resistant nature. (Kotloff and Thabut 2011)

#### **5.2.3 Treatment of bacterial infection**

The treatment of infection in lung transplant recipients is a challenging task due to the patients' significant cocktail of immunosuppressive therapies. Many of these drugs, including cyclosporine and tacrolimus can be highly cross-reactive. There is unfortunately no "magic bullet" specific for transplant patients, the traditional regimen of broad spectrum antibiotics, with specific coverage for gram negative, acid-fast, etc. being added upon differential diagnosis.

### **5.2.4 Fungal infection**

While colonization by widespread fungi such as *Aspergillus* and *Candida* is common, a much smaller percentage of patients will develop a clinically relevant fungal infection. (Singh 2003) *Aspergillus* and *Candida* are by far the most common, with *Cryptococcus* species and other molds playing a much smaller role. (Neofytos et al. 2010) The presence of foreign material (sutures) in the airway anastomosis, in addition to the inherent risk with ventilation, increases the risk of fungal infection in post-operative transplant patients.

#### **5.2.5 Prevention/treatment of fungal infection**

Similar to the treatment of bacterial infections, the therapeutic approach to fungal infection in lung transplant patients is primarily unchanged from the normal treatment options in other patients. Treatment regimens will typically consist of aerosolized Amphotericin B in the acute post-operative period, with voriconazole and itraconazole prophylactic treatment extending for up to a year post-transplantation. (Ahmad, Shlobin, and Nathan 2011)

#### **5.2.6 Viral infection**

654 Lung Diseases – Selected State of the Art Reviews

*Pseudomonas aeruginosa* is the most common bacterial organism isolated from the posttransplant lung. (Kramer et al. 1993) This opportunistic infection has a higher incidence in CF patients and has been estimated to be responsible for up to 33% of pneumonias in all transplant recipients. (Aguilar-Guisado et al. 2007; Campos et al. 2008) *P. aeruginosa* airway infection prior to transplant does not appear to demonstrate a predictive relationship for chronic rejection. However, this is not the case for colonization in the post-operative period. (Botha et al. 2008; Vos et al. 2008) *P. aeruginosa* infection is not limited to pneumonias; the pathogen has been reported to be responsible for nearly half of all post-transplant infections.

*Mycobacterium* is a designation indicating acid-fast bacteria that are capable of causing a wide range of diseases. Perhaps the most well known of these *M. tuberculosis*, has the robust cell wall that is a calling card of the classification, and helps to confer resistance to broad spectrum antibiotics. Although, not nearly as common as *P. aeruginosa* infection, *M. tuberculosis* colonization presents a unique set of treatment hurdles, especially in the

Opportunistic infections, hospital-acquired infections, and resistant strains of bacteria are all significantly more common in lung transplant recipients than the general population. *Staphylococci* strains have demonstrated a propensity for antibiotic resistance and this is only magnified in the already immunosuppressed transplant patient. Specifically, *S. aureus* is the second most common organism isolated from the airway, with multi-drug resistant *S. aureus* (MRSA) being extremely difficult to treat due to its almost pan-resistant nature. (Kotloff and

The treatment of infection in lung transplant recipients is a challenging task due to the patients' significant cocktail of immunosuppressive therapies. Many of these drugs, including cyclosporine and tacrolimus can be highly cross-reactive. There is unfortunately no "magic bullet" specific for transplant patients, the traditional regimen of broad spectrum antibiotics, with specific coverage for gram negative, acid-fast, etc. being added upon

While colonization by widespread fungi such as *Aspergillus* and *Candida* is common, a much smaller percentage of patients will develop a clinically relevant fungal infection. (Singh 2003) *Aspergillus* and *Candida* are by far the most common, with *Cryptococcus* species and other molds playing a much smaller role. (Neofytos et al. 2010) The presence of foreign material (sutures) in the airway anastomosis, in addition to the inherent risk with ventilation, increases the risk of fungal infection in post-operative transplant patients.

Similar to the treatment of bacterial infections, the therapeutic approach to fungal infection in lung transplant patients is primarily unchanged from the normal treatment options in other patients. Treatment regimens will typically consist of aerosolized Amphotericin B in the acute post-operative period, with voriconazole and itraconazole prophylactic treatment

extending for up to a year post-transplantation. (Ahmad, Shlobin, and Nathan 2011)

presence of multi-drug resistant (MDR) strains. (Lee et al. 2003)

(Valentine et al. 2008)

Thabut 2011)

differential diagnosis.

**5.2.4 Fungal infection** 

**5.2.3 Treatment of bacterial infection** 

**5.2.5 Prevention/treatment of fungal infection** 

As has previously been mentioned, the sero-status of cytomegalovirus (CMV) is an important factor in opting to transplant or harvest donor organs. CMV is by far the most common viral pathogen observed after lung transplant procedures, affecting at least a third of all patients. CMV usually presents with fever, exhaustion, and flu-like symptoms with associated leukopenia. (Snydman et al. 2011) Diagnosis of CMV syndrome is made with PCR evaluation of viral titers in peripheral blood. If CMV pneumonia is suspected, the definitive test must be performed on a biopsy specimen of the in tissue. (Kotton et al. 2010) CMV infection has been shown to predispose the patient to infection with other community acquired respiratory viruses (CARVs). (Sims and Blumberg 2011)

In a recent three year longitudinal study, Kumar et al showed the presence of virulent strains of H1N1 influenza, adenovirus, flu A, flu B, and rhinovirus in over half of transplant recipients. (Kumar et al. 2010) Diagnosis of these viral infections has become much more straightforward with the advent of qPCR to provide rapid evaluation of viral titer loads in bronchoalveolar lavage (BAL) fluid of patients. Antibody based assays are also available to determine the infection state of an individual, although these may vary from center to center.

#### **5.2.7 Prevention/treatment of viral infection**

Intravenous (IV) ganciclovir and the oral pro-drug valganciclovir are given prophylactically for anywhere from 6 to 12 months post-transplant. (Jaksch et al. 2009) CMV prophylaxis is standard protocol for all patients regardless of center and has been shown to be effective in reducing the CMV syndrome. (Palmer et al. 2010) Treatment for CARVs is dependant upon specific diagnosis of pathogen and strain and can include zanamivir, amantidine, oseltamivir, etc. (Ison and Michaels 2009) Unlike CMV therapy, prolonged treatment with these anti-virals is contraindicated for fear of contributing to the development of resistance.

#### **5.3 Acute rejection**

#### **5.3.1 Characterization/etiology of acute rejection**

Acute rejection (AR) presents clinically with non-specific symptoms including dyspnea, mild fever, malaise, cough, and leukocytosis. Although not present in all AR exacerbations, radiographic imaging may show mild pleural effusion, and interstitial opacities with a concurrent decline in oxygenation and spirometric measurements. (Millet et al. 1989; Otulana et al. 1990) AR is definitively diagnosed by BAL and TBB followed by histological analysis showing perivascular infiltrates. (Stewart et al. 2007) TBB may not always be possible due to patient status and thus AR is tentatively determined based upon clinical symptoms and radiologic evidence. Many transplant centers have post-transplant protocols that include routine monitoring by BAL which is also capable of confirming AR. (Chakinala et al. 2004) Most importantly, as indicated by the name, acute rejection is just that, a brief intermittent condition that is most often easily resolved with or without hospitalization.

Acute rejection is an extremely common complication of lung transplant patients, occurring in as many as 90% of lung recipients over their lifespan. (Arcasoy 2004) The incidence of AR is highest within the first year post-transplant, afflicting approximately one third of all patients. (Christie et al.) It is difficult to determine precise results in these retrospective studies due to the fact that AR can be clinically silent, only detectable upon transbronchiole biopsy (TBB). (Hopkins et al. 2002) The precise etiology of AR is still unknown but stratified risk appears to be heavily weighted toward donor-dependent factors in the immediate postoperative period, with recipient-dependent factors playing the predominant role after the first year. HLA-B mismatching, increasing donor age, non-O donor blood type, and increasing body-surface area (most likely corresponding to elevated BMI) all correspond with increased AR incidence. With a recipient history of diabetes, and recent transplant accounting for recipient driven risk in the intermediate and late phase AR. (Mangi et al. 2011)

The International Society for Heart-Lung Transplantation (ISHLT) has delineated the standard nomenclature to describe the various gradations of AR based solely on histological findings. Grade A0 is the absence of AR, no cellular infiltrates and healthy lung parenchyma. Grade A1 is characterized by patchy mononuclear cell infiltrates in alveolar parenchyma. Grade A2 is described as having more prevalent lymphocytic infiltrates centered in the perivascular area with the presence of some activated macrophages and eosinophils. Grade A3 features dense perivascular infiltrates, significant eosinophil presence, with the first observation of neutrophils in the intra-alveolar space. Grade A4 describes the finding of diffuse mononuclear infiltrates, pneumocyte damage, macrophage and neutrophil presence. (Stewart et al. 2007)

#### **5.3.2 Treatment of acute rejection**

Similar to many aspects of lung transplantation, treatment for AR varies dependant upon hospital setting with some centers opting to not treat AR considered to be <Grade A2. (Orens and Garrity 2009) In more severe cases however, conventional treatment consists of a hospital admission with a three day course of solumedrol followed by an oral prednisone taper. (Aboyoun et al. 2001; Yousem et al. 1994) Additional treatment with methotrexate, lymphoid irradiation, and antithymocyte globulin, among others, has been reported to be effective in alleviating the transient AR condition. (Hachem 2009)

#### **5.3.3 Biomarkers of acute rejection**

Although ISHLT has defined acute rejection based solely upon histological findings, there is significant effort to describe biomarkers specific to patients undergoing AR to better classify and determine treatment. A recent study by a group out of Copenhagen described elevated mRNA of the regulatory T cell (Treg) cytokine FoxP3, along with cytotoxic T cell-derived granzyme B in BAL fluid of patients in AR. (Madsen et al. 2010) A 2007 study by Lande et al describes the use of microarray analysis to observe relative gene expression levels of cytokines thought in be involved in acute rejection in the BAL fluid of AR patients. (Lande et al. 2007)

#### **5.3.4 Novel/future therapeutics of acute rejection**

Due to the incredible amounts of data generated by high through methods such as massspectrometry-assisted proteomics and gene microarray analysis there have never been more targets identified for the design of targeted therapeutics in acute rejection. The use of animal models of acute rejection have also significantly improved the ability to design and test novel therapeutics for acute rejection allograft rejection in lung transplantation.

Jung et al (2006) have developed a spontaneous model of acute rejection in rats by performing lung transplantation in a manner similar to that used in humans. By utilizing a small molecule, irreversible inhibitor of dipeptidylpeptidase IV (DPPIV/CD26) the investigators reduced the incidence of acute rejection, preserved lung function, and

operative period, with recipient-dependent factors playing the predominant role after the first year. HLA-B mismatching, increasing donor age, non-O donor blood type, and increasing body-surface area (most likely corresponding to elevated BMI) all correspond with increased AR incidence. With a recipient history of diabetes, and recent transplant accounting for recipient driven risk in the intermediate and late phase AR. (Mangi et al.

The International Society for Heart-Lung Transplantation (ISHLT) has delineated the standard nomenclature to describe the various gradations of AR based solely on histological findings. Grade A0 is the absence of AR, no cellular infiltrates and healthy lung parenchyma. Grade A1 is characterized by patchy mononuclear cell infiltrates in alveolar parenchyma. Grade A2 is described as having more prevalent lymphocytic infiltrates centered in the perivascular area with the presence of some activated macrophages and eosinophils. Grade A3 features dense perivascular infiltrates, significant eosinophil presence, with the first observation of neutrophils in the intra-alveolar space. Grade A4 describes the finding of diffuse mononuclear infiltrates, pneumocyte damage, macrophage

Similar to many aspects of lung transplantation, treatment for AR varies dependant upon hospital setting with some centers opting to not treat AR considered to be <Grade A2. (Orens and Garrity 2009) In more severe cases however, conventional treatment consists of a hospital admission with a three day course of solumedrol followed by an oral prednisone taper. (Aboyoun et al. 2001; Yousem et al. 1994) Additional treatment with methotrexate, lymphoid irradiation, and antithymocyte globulin, among others, has been reported to be

Although ISHLT has defined acute rejection based solely upon histological findings, there is significant effort to describe biomarkers specific to patients undergoing AR to better classify and determine treatment. A recent study by a group out of Copenhagen described elevated mRNA of the regulatory T cell (Treg) cytokine FoxP3, along with cytotoxic T cell-derived granzyme B in BAL fluid of patients in AR. (Madsen et al. 2010) A 2007 study by Lande et al describes the use of microarray analysis to observe relative gene expression levels of cytokines thought in be involved in acute rejection in the BAL fluid of AR patients. (Lande

Due to the incredible amounts of data generated by high through methods such as massspectrometry-assisted proteomics and gene microarray analysis there have never been more targets identified for the design of targeted therapeutics in acute rejection. The use of animal models of acute rejection have also significantly improved the ability to design and test

Jung et al (2006) have developed a spontaneous model of acute rejection in rats by performing lung transplantation in a manner similar to that used in humans. By utilizing a small molecule, irreversible inhibitor of dipeptidylpeptidase IV (DPPIV/CD26) the investigators reduced the incidence of acute rejection, preserved lung function, and

novel therapeutics for acute rejection allograft rejection in lung transplantation.

2011)

and neutrophil presence. (Stewart et al. 2007)

effective in alleviating the transient AR condition. (Hachem 2009)

**5.3.2 Treatment of acute rejection** 

**5.3.3 Biomarkers of acute rejection** 

**5.3.4 Novel/future therapeutics of acute rejection** 

et al. 2007)

maintained normal histological structure in rat lung transplantees. (Jung et al. 2006) They previously published that DPPIV/CD26 was elevated in plasma of rats undergoing AR after cardiac transplant and hypothesize that the pulmonary protective effect is due to reducing the co-stimulatory effect of DPPIV/CD26 on T cells. (Korom et al. 1997) In a similar model, a group out of Fukouka, Japan employed a Janus kinase 3 (Jak3) inhibitor to prevent the development of AR. Jak3 is located at a biochemical bottleneck in the pathway of T cell clonal expansion. Higuchi, et al demonstrated a dose-dependant inhibition of the development of acute rejection by the AG490 in the experimental population. (Higuchi et al. 2005)

Animal models are not the only avenue available to scientists to pursue better methods of treating and preventing acute rejection. Investigators at the University Clinics of Leipzig reported a reduction in the incidence of acute rejection with preemptive administration of the traditional therapeutic methylprednisone. By simply giving bolus doses 2 hours prior to incision and immediately prior to completion of the transplant procedure they observed a significant improvement in outcomes. (Bittner et al. 2010)

Clearly this is not meant to be an exhaustive review of the potential novel therapeutics currently under development. We are merely presenting a representative sample indicating the myriad of pathways that are being studied to yield targeted countermeasures to acute rejection.

## **5.4 Chronic rejection (bronchiolitis obliterans syndrome)**

### **5.4.1 Characterization/etiology of bronchiolitis obliterans syndrome**

Chronic rejection, clinically termed bronchiolitis obliterans syndrome (BOS), is the primary source of morbidity and mortality seen in the lung transplant patient population. (Bando et al. 1995; Stewart et al. 2007) Due to the nature of BOS, primarily occurring irregularly in the small airways, diagnosis by transbronchial biopsy is ineffective. (Chamberlain et al. 1994) Diagnosis of BOS is difficult due to its similarities with other post-transplant complications. There are no tests to specifically determine BOS, rather, it is a diagnosis of exclusion. A persistent, unexplainable drop in forced expiratory volume in one second (FEV1) (~80% of baseline) with accompanying decline in FEV25-75 (less than or equal to 75% of baseline) is defined as BOS stage 0. (Belperio et al. 2009; Estenne et al. 2002) BOS is staged 0-3 based upon progressive declines in percent of expected FEV1.

BOS is unfortunately observed in over half of lung transplant patients who survive five years or more post-transplantation. (Bando et al. 1995) Chronic rejection initially presents clinically in a manner similar to that of other complications with dyspnea, cough, and progressive airway obstruction. (Estenne and Hertz 2002) X-ray analysis is often unremarkable, but computer assisted tomography (CT) may reveal air trapping and bronchiectasis. (Morrish et al. 1991)

BOS is characterized pathologically by a prominent neutrophilic component with a definite increase in pulmonary fibrosis and extra-cellular remodeling. (Billings et al. 2002; Boehler and Estenne 2003) The BAL fluid of the majority of BOS patients reveals chronic pulmonary neutrophilia. Indeed, when >20% of cells are neutrophils in BAL fluid patients fail to survive past 7 years post-transplant. (Neurohr et al. 2009) Unlike acute rejection, where the cellular infiltrates are centered around the vasculature, in BOS, the cells are located primarily in and around the airways with a striking increase in cell number and activation level of leukocytes. (Vanaudenaerde et al. 2008) The causes of BOS are only partially understood with much unknown about the exact causative events that lead to disease.

### **5.4.2 Risk Factors for bronchiolitis obliterans syndrome**

What facts are known about the pathogenesis of BOS are the multiple risk factors that have been detailed via epidemiologic investigations. Both alloimmune and alloimmuneindependent factors appear to play principle roles in the development of BOS. (Knoop and Estenne 2006) Primary graft dysfunction, along with gastroesophageal reflux (GER), and infection are all nonalloimmune factors that are associated with occurrence of BOS. (Estenne and Hertz 2002) CMV infection resulting in disease has been reported to enhance the relative risk of developing BOS, although there are conflicting reports regarding this. (Belperio et al. 2009) Alloimmune factors closely associated with BOS are recurrent, or persistent AR, and HLA mismatching. (Palmer et al. 2002)

### **5.4.3 Treatment of bronchiolitis obliterans syndrome**

Currently there are no demonstrated effective treatments for BOS. Most present strategies rely on augmenting immunosuppression with the use of corticosteroids, cyclosporine, methotrexate, etc. (Date et al. 1998; Dusmet et al. 1996; Iacono et al. 1996) These, however have been met with little success. The majority of existing treatment methods rely on unproven, anecdotal evidence with few multi-center, randomized, controlled trials.

#### **5.4.4 Current research and future directions in bronchiolitis obliterans syndrome**

Our previously published hypothesis regarding the onset of BOS is that there is some initial insult (infection by CMV or other pathogens, inflammatory damage due to recurrent AR, etc.) that awakens the adaptive immune system to over-ride the potent cocktail of immunosuppressive drugs present. The adaptive immune (Type 1, and Type 2 T cells) further damages the transplanted organ by responding to the foreign epitopes innately present in the donor lung. This repeated damage induces a persistent release of proinflammatory cytokines that recruit neutrophils into the interstitium and airways where they remain and participate in a fibroproliferative and proteolytically destructive process. (Hardison et al. 2009)

#### **5.4.4.1 Potential Biomarkers in Bronchiolitis Obliterans Syndrome**

We, along with others, have reported on the activation of neutrophils by glutamate-leucinearginine positive (ELR+) CXC chemokines such as IL-8. (Xu et al. 2011; Chakrabarti and Patel 2005) Upon activation, neutrophils degranulate which releases potent proteases such as matrix metalloproteinase-8, -9, and prolyl endopeptidase. (Xu 2011, O'Reilly 2010) It has been well established that collagen fragments are chemotactic to neutrophils, and in 1995, Pfister et al. were able to determine the sequence, proline-glycine-proline (Pro-Gly-Pro, PGP) confers chemotactic potential to collagen breakdown products. (Pfister et al. 1995) In a 2006 paper, our group demonstrated the mechanism by which PGP is able to attract neutrophils into tissue. PGP shares significant sequence and structural homology with almost all ELR+CXC chemokines, which act via CXC receptors 1 and 2 in human (CXCR1, CXCR2). We reported that PGP competes with the CXCR ligand, IL-8, for binding, causes chemotaxis in CXCR transfected cells and elicits a similar oxidative burst to IL-8 stimulation. (Weathington et al. 2006) Recently we have also published the step-wise manner in which it is produced. (Gaggar et al. 2008)

MMP-8 and -9 are capable of initially digesting collagen but are incapable of performing the final cleavage to PGP and a second step is necessary for the matrikine's production. Our lab demonstrated that the serine protease prolyl endopeptidase (PE) performs the final

What facts are known about the pathogenesis of BOS are the multiple risk factors that have been detailed via epidemiologic investigations. Both alloimmune and alloimmuneindependent factors appear to play principle roles in the development of BOS. (Knoop and Estenne 2006) Primary graft dysfunction, along with gastroesophageal reflux (GER), and infection are all nonalloimmune factors that are associated with occurrence of BOS. (Estenne and Hertz 2002) CMV infection resulting in disease has been reported to enhance the relative risk of developing BOS, although there are conflicting reports regarding this. (Belperio et al. 2009) Alloimmune factors closely associated with BOS are recurrent, or

Currently there are no demonstrated effective treatments for BOS. Most present strategies rely on augmenting immunosuppression with the use of corticosteroids, cyclosporine, methotrexate, etc. (Date et al. 1998; Dusmet et al. 1996; Iacono et al. 1996) These, however have been met with little success. The majority of existing treatment methods rely on

unproven, anecdotal evidence with few multi-center, randomized, controlled trials.

**5.4.4.1 Potential Biomarkers in Bronchiolitis Obliterans Syndrome** 

**5.4.4 Current research and future directions in bronchiolitis obliterans syndrome**  Our previously published hypothesis regarding the onset of BOS is that there is some initial insult (infection by CMV or other pathogens, inflammatory damage due to recurrent AR, etc.) that awakens the adaptive immune system to over-ride the potent cocktail of immunosuppressive drugs present. The adaptive immune (Type 1, and Type 2 T cells) further damages the transplanted organ by responding to the foreign epitopes innately present in the donor lung. This repeated damage induces a persistent release of proinflammatory cytokines that recruit neutrophils into the interstitium and airways where they remain and participate in a fibroproliferative and proteolytically destructive process.

We, along with others, have reported on the activation of neutrophils by glutamate-leucinearginine positive (ELR+) CXC chemokines such as IL-8. (Xu et al. 2011; Chakrabarti and Patel 2005) Upon activation, neutrophils degranulate which releases potent proteases such as matrix metalloproteinase-8, -9, and prolyl endopeptidase. (Xu 2011, O'Reilly 2010) It has been well established that collagen fragments are chemotactic to neutrophils, and in 1995, Pfister et al. were able to determine the sequence, proline-glycine-proline (Pro-Gly-Pro, PGP) confers chemotactic potential to collagen breakdown products. (Pfister et al. 1995) In a 2006 paper, our group demonstrated the mechanism by which PGP is able to attract neutrophils into tissue. PGP shares significant sequence and structural homology with almost all ELR+CXC chemokines, which act via CXC receptors 1 and 2 in human (CXCR1, CXCR2). We reported that PGP competes with the CXCR ligand, IL-8, for binding, causes chemotaxis in CXCR transfected cells and elicits a similar oxidative burst to IL-8 stimulation. (Weathington et al. 2006) Recently we have also published the step-wise manner in which it

MMP-8 and -9 are capable of initially digesting collagen but are incapable of performing the final cleavage to PGP and a second step is necessary for the matrikine's production. Our lab demonstrated that the serine protease prolyl endopeptidase (PE) performs the final

**5.4.2 Risk Factors for bronchiolitis obliterans syndrome** 

persistent AR, and HLA mismatching. (Palmer et al. 2002)

**5.4.3 Treatment of bronchiolitis obliterans syndrome** 

(Hardison et al. 2009)

is produced. (Gaggar et al. 2008)

proteolysis. (Gaggar et al. 2008) PE cleaves after a proline in a peptide of ~100 amino acids or less.

We have reported the potential for PGP as a biomarker of disease in multiple chronic inflammatory lung diseases such as cystic fibrosis (CF), chronic obstructive pulmonary disease (COPD), and most relevant to this manuscript, BOS. (Gaggar et al. 2007; O'Reilly et al. 2009; Hardison et al. 2009) In matched BAL samples of patients obtained three months prior to, and concurrent with diagnosis of BOS, we described a temporal shift in the chemokine/matrikine profile. MMP-8, -9 and PE activities were increased in the samples collected at diagnosis of BOS compared to a transplant control population, and matched samples collected prior to confirmation of disease. By employing a previously published mass spectrometry technique, we detected measureable amounts of PGP in both pre- and post-diagnosis of BOS, however, there was a dramatic and significant increase in the levels observed in BAL fluid collected at the time of diagnosis. Through the use of neutralizing antibodies to IL-8 and PGP, first individually and then in concert, we demonstrated a shift from the relative importance of the classical PMN chemoattractant, IL-8, to the more novel molecule, PGP, upon diagnosis of BOS.

Clearly PGP is not the only molecule with the potential to serve as a novel biomarker for BOS diagnosis. There is a litany of research underway on a wide variety of proteins and cell types that may potentially one day play a role in the more precise classification of this condition. Endothelin-1, mesenchymal stromal cells, and serum KL-6 (a glycoprotein) have all been published as possible markers of disease progression. (Salama et al. 2011; Badri et al. 2011; Haberman et al. 2010)

#### **5.4.4.2 Current Research in Bronchiolitis Obliterans Syndrome**

Perhaps the most exciting development in the quest for reliable preventative therapy for BOS has been the recent establishment of reproducible rodent models of BOS which closely mimic human disease. The most recent model described by Jungraithmayr, et al., utilizing single lung transplantation, is a vast improvement over the traditional tracheal transplant which had the obvious limitation of leaving the native lung in the rodent. (Jungraithmayr et al. 2010) The Swiss group reports that a T cell response and cytokine presence, similar to that of humans with BOS, was observed. Another group, from the University of Pittsburgh, has developed a human-mouse chimeric model of BOS in which the allograft and immune effector cells are of human origin. (Xue et al. 2011) They state that the formation of chimeric allogenic T cells, and the resulting infiltration into small human airways is definitive in delineating the role T cells play in the development of BOS.

#### **5.4.4.3 Future Therapeutics for Bronchiolitis Obliterans Syndrome**

This is, in part, a continuation of the previous section due to the experimental nature of some of the procedures/drugs described herein. As was previously discussed, there is an association of GER and BOS incidence. A study out of Duke University describes improved pulmonary function in patients who underwent proactive treatment for GER. Surgical fundoplication was performed to reduce the possibility of bile aspiration and potential damage to the airway. (Davis et al. 2003) Additionally, a retrospective study of transplant recipients with BOS identified a possible advantage of treatment with Azithromycin (Az), though the mechanism of action remains unclear. (Jain et al. 2010) Patients with a higher initial neutrophil burden responded better to the treatment and had better outcomes. What is most interesting is the fact that azithromycin is a macrolide antibiotic, a class known to be inhibitors of matrix metalloproteinases. It is possible that Az is preventing the generation of PGP and thus relieving some of the neutrophil burden associated with BOS.

A recent publication by our group in *Science* detailed a bi-functional enzyme, Leukotriene A4 Hydrolase (LTA4H), which serves to degrade PGP endogenously. This enzyme is present in both neutrophils and airway epithelium and is elevated in a mouse model of pulmonary infection and inflammation. (Snelgrove et al. 2010) Modulation of the activity of this important enzyme may provide a welcome opportunity to utilize patients' own biology to help resolve some of the destructive chronic neutrophilic inflammation seen in BOS. We have also described two specific inhibitors of PGP, arginine-threonine-arginine (RTR), and an all "D" isomer of PGP (DD-PGP) that are potent in preventing the mechanism of action of PGP *in vitro* and *in vivo*. (Jackson et al. 2011; van Houwelingen et al. 2008)

## **6. Conclusion**

Even though long-term survival of lung transplant recipients has improved over time, the overall mortality rate in lung transplantation remains significantly higher than any other whole organ transplant population. The relatively recent advent of a more appropriate organ allocation system, along with improved ability to preserve donor organs has increased the pool of available organs to all-time highs. Even so, the number of actual lungs harvested each year compared to the estimated viable donor lungs is a small fraction of what is possible. With continued advancement in animal models, and description of biomarkers of the various complications associated with transplantation, there has been marked, if gradual, improvements in the therapeutic armament clinicians have at their disposal. It may yet be that lung transplantation one day be viewed as an early intervention in progressive irreversible pulmonary conditions such as idiopathic pulmonary fibrosis and confer a permanent, rejuvenative improvement in the lifespan and quality of life of such patients.

#### **7. References**


inhibitors of matrix metalloproteinases. It is possible that Az is preventing the generation of

A recent publication by our group in *Science* detailed a bi-functional enzyme, Leukotriene A4 Hydrolase (LTA4H), which serves to degrade PGP endogenously. This enzyme is present in both neutrophils and airway epithelium and is elevated in a mouse model of pulmonary infection and inflammation. (Snelgrove et al. 2010) Modulation of the activity of this important enzyme may provide a welcome opportunity to utilize patients' own biology to help resolve some of the destructive chronic neutrophilic inflammation seen in BOS. We have also described two specific inhibitors of PGP, arginine-threonine-arginine (RTR), and an all "D" isomer of PGP (DD-PGP) that are potent in preventing the mechanism of action of

Even though long-term survival of lung transplant recipients has improved over time, the overall mortality rate in lung transplantation remains significantly higher than any other whole organ transplant population. The relatively recent advent of a more appropriate organ allocation system, along with improved ability to preserve donor organs has increased the pool of available organs to all-time highs. Even so, the number of actual lungs harvested each year compared to the estimated viable donor lungs is a small fraction of what is possible. With continued advancement in animal models, and description of biomarkers of the various complications associated with transplantation, there has been marked, if gradual, improvements in the therapeutic armament clinicians have at their disposal. It may yet be that lung transplantation one day be viewed as an early intervention in progressive irreversible pulmonary conditions such as idiopathic pulmonary fibrosis and confer a permanent, rejuvenative improvement in the lifespan and quality of life of such

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**6. Conclusion** 

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## **Bronchial Atresia**

Lirios Sacristán Bou and Francisco Peña Blas *Hospital General de Tomelloso & Centro de Salud de Monforte del Cid España* 

## **1. Introduction**

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Gaggar. 2011. A self-propagating matrix metalloprotease-9 (MMP-9) dependent

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infection in lung transplant recipients: evidence-based recommendations.

2004. Following universal prophylaxis with intravenous ganciclovir and cytomegalovirus immune globulin, valganciclovir is safe and effective for prevention of CMV infection following lung transplantation. *Am J Transplant* 4 Bronchial atresia is an interesting congenital abnormality because of its variable appearance and its semblance to certain acquired diseases. It is characterized by a branching mass formed by mucus that dilates the proximal bronchi to the atretic segment. The distal lung to atresia can develop normally but it shows a paucity of blood vessels and is hyperinflated due to unidirectional collateral air drift through intraalveolar pores of Kohn, bronchoalveolar channels of Lambert and interbronchiolar pores of Martin from the adjacent normal lung. These collateral communications act as a check-valve mechanism allowing the air to enter but not to leave the distal lung.

More than 150 cases of bronchial atresia have been reported since 1953, when it was first described by Ramsay & Byron. The exact mechanism that ends in bronchial atresia is still unknown, but there are two hypotheses about the pathogenesis which have in common that they should occur before birth because the bronchial pattern to the site of stenosis is entirely normal. Many of the most relevant case reports and published series of cases are reviewed in this chapter to update our knowledge of bronchial atresia. They have been obtained as the result of a bibliographical research at Pubmed; ninety five articles were found using the Medical Subject Headings (MeSH) thesaurus descriptors congenital and bronchial atresia.

## **2. Pathogenesis**

Bronchial buds appear in the fifth week of gestation and then complete branching takes place in the sixteenth week. Although bronchial atresia is associated with a decreased amount of alveoli, the number of airways is not reduced.

Congenital bronchial atresia pathogenesis is not completely understood, but there are two pathogenic hypotheses. The first one proposes that proliferating cells at the distal tip of the developing bud are disconnected from the normal branch at any time from the fifth to the sixteenth weeks by a still unknown agent (Bucher et al., 1961; Kuhn et al., 1992).

The other one postulates that focal ischemic insults at the fifth week or later result in necrosis and obliteration of the already completed bronchus (Reid, 1977 or Waddell et al., 1965). The experiment of Louw & Barnard (1955), in which they tied off a mesenteric arterial branch in puppies two weeks before birth, showed that atresia may result from a vascular occlusion and develop late in intra-uterine life. Their findings favour vascular occlusion rather than a failure of growth as the cause.

The pulmonary arterial development is closely associated with the developing lung bud. The primitive pulmonary artery is already present as a major branch and grows toward the lung bud. Subsequently, it gives off a branch to accompany each airway branch in the preacinar region. Pulmonary vascular abnormalities have been documented in bronchial atresia (Ko et al., 1998; Lacquet et al., 1971).

## **3. Respiratory symptoms and epidemiology**

Bronchial atresia has a male predominance, with an estimated prevalence of 1.2 cases per 100,000 males and 0.6 cases per 100,000 females - a male: female ratio of 2:1.

About two thirds of the reported patients were asymptomatic before diagnosis and occurred as an incidental radiological discovery. It is usually diagnosed in the second or third decade of life, and the diagnosis is infrequent during childhood. One third of patients may present with cough, shortness of breath and recurrent infections and less frequently with refractory wheezing, haemoptysis, chest pain, or pneumothorax (Agarwal et al., 2005; Kameyama et al., 2006; Morikawa et al., 2005 (Table 1).


Table 1. Frequency of symptoms from more to less frequent (Jederlinic et al., 1987).

The bronchi involved in this congenital anomaly are the apico-posterior segment of left upper lobe, right upper lobe, right middle lobe and right lower lobe, in decreasing order of frequency (Acosta Gordillo et al., 2005; Psathakis et al., 2004).

## **4. Imaging techniques for the diagnosis**

Currently clinicians have lot of many imaging techniques at their disposal for the investigation of patients with this congenital anomaly.

## **4.1 Plain Chest Radiography**

The radiographic findings mirror the pathologic changes.

On chest radiography the typical finding of a mucocele is that of a nodule or a shadow-like mass close to the hilum, with well-defined margins, presenting as a tubular, round, ovoid or branching structure (Sacristán Bou et al., 2010) (Fig 1). A mucocele with an air-fluid level is considered by some authors as a variance of congenital bronchial atresia (Matsushima et al., 2003).

The branching bronchocele mimics a glove-finger shadow, but is not pathognomonic of bronchial atresia.

It has been proposed that the impacted mucous can also liquefy producing the radiological sign of an air-fluid level-mimicking a cavitatory lesion. When the hyperinsuflated area gets

The pulmonary arterial development is closely associated with the developing lung bud. The primitive pulmonary artery is already present as a major branch and grows toward the lung bud. Subsequently, it gives off a branch to accompany each airway branch in the preacinar region. Pulmonary vascular abnormalities have been documented in bronchial

Bronchial atresia has a male predominance, with an estimated prevalence of 1.2 cases per

About two thirds of the reported patients were asymptomatic before diagnosis and occurred as an incidental radiological discovery. It is usually diagnosed in the second or third decade of life, and the diagnosis is infrequent during childhood. One third of patients may present with cough, shortness of breath and recurrent infections and less frequently with refractory wheezing, haemoptysis, chest pain, or pneumothorax (Agarwal et al., 2005; Kameyama et

100,000 males and 0.6 cases per 100,000 females - a male: female ratio of 2:1.

Table 1. Frequency of symptoms from more to less frequent (Jederlinic et al., 1987).

The bronchi involved in this congenital anomaly are the apico-posterior segment of left upper lobe, right upper lobe, right middle lobe and right lower lobe, in decreasing order of

Currently clinicians have lot of many imaging techniques at their disposal for the

On chest radiography the typical finding of a mucocele is that of a nodule or a shadow-like mass close to the hilum, with well-defined margins, presenting as a tubular, round, ovoid or branching structure (Sacristán Bou et al., 2010) (Fig 1). A mucocele with an air-fluid level is considered by some authors as a variance of congenital bronchial atresia (Matsushima et al.,

The branching bronchocele mimics a glove-finger shadow, but is not pathognomonic of

It has been proposed that the impacted mucous can also liquefy producing the radiological sign of an air-fluid level-mimicking a cavitatory lesion. When the hyperinsuflated area gets

atresia (Ko et al., 1998; Lacquet et al., 1971).

al., 2006; Morikawa et al., 2005 (Table 1).

**3. Respiratory symptoms and epidemiology** 

Asymptomatic 58.00% Recurrent Infection 21.00% Dysnea 14.00% Coughing 6.00%

frequency (Acosta Gordillo et al., 2005; Psathakis et al., 2004).

**4. Imaging techniques for the diagnosis** 

**4.1 Plain Chest Radiography** 

2003).

bronchial atresia.

investigation of patients with this congenital anomaly.

The radiographic findings mirror the pathologic changes.

Fig. 1. Polylobulated round mass near to the hilum in the right lower lobe (Sacristán Bou et al., 2010).

infected and presents as a pneumonia, the walls of bronchoceles develop tiny breaks that enables the air to enter in its lumen (Psathakis et al., 2009; Rahalkar et al., 2005).

Occasionally, the dilated bronchi may appear as purely air-filled, lucent bands of odd shapes (Nussbaumer-Ochsner & Kohler, 2011; Van Klaveren et al., 1992).

The distal lung is always distended, forming the apex of a roughly triangular zone of hyperlucency of the lung parenchyma (due to oligoemia and hyperinflation). On radiography obtained at expiration, obstructive emphysema is obvious, however, the synchronous appearance of both the mucocele and lung hyperlucency is only seen in 69% of cases (Jederlinic et al., 1987).

Sometimes bronchial atresia is associated with other congenital anomalies like pectum excavatum (Van Klaveren et al., 1992).

## **4.2 Computed Tomography (CT) and High-Resolution Computed Tomography**

Computed tomography is the procedure of choice for the diagnosis and study of congenital bronchial atresia.

The bronchocele presents as a round/ovoid/branching structure near the hilum, with or without a fluid level, without contrast enhancement and it exhibits a density between 10 to 25 Hounsfield Units due to mucoid material. Computed tomography allows characterization of the lack of communication between the mucocele and hilum (Fig. 2).

High resolution techniques can display the characteristic features of the mucocele, being more sensitive than the plain chest radiography to show oligaemia, reduced caliber of the pulmonary vessels, hyperinflation of the lung parenchyma (Fig. 2), small mucoceles invisible to conventional radiography, associated mass effect and even calcification (Kinsella et al., 1992 and Primetis et al., 2011).

Fig 2. Polilobulated round mass located in the posterobasilar right subsegmental bronchi with a distal segmental area of radiolucency and lack of communication between the mucocele and hilum on high-resolution computed tomography (Sacristán Bou et al., 2010).

### **4.3 Magnetic Resonance Imaging (MRI)**

MRI shows a very high signal intensity within the bronchocele on T1W and T2W modes due to mucoid contents; however, it cannot depict regional air-trapping (Matsushima et al., 2003) and it does not have the same sensivity as computed tomography to evaluate the lung parenchyma (Naidich et al., 1988). So this technique has a limited role in the diagnosis of congenital brochial atresia, although a small number of case reports have dealt specifically with differential diagnosis and have established its value.

Magnetic resonance imaging seem to be useful for the evaluation of either anomalous vessels or fluid collections that are usually associated with pulmonary abnormalities (Cohen et al., 1987).

### **5. Bronchoscopy**

Before the advent of computed tomography, the diagnosis of bronchial atresia was made by bronchography, which allowed confirmation of the atretic segmental bronchus showing non-filling of the involved bronchus.

Flexible-bronchoscopy identifies blind-ending bronchi. In clinical practice however, any absence of a segmental or sub-segmental bronchus that is found by chance during bronchoscopy in the absence of the characteristic radiographic features may be considered as a normal anatomic variance of the bronchial tree rather than a bronchial atresia.

In the majority of the cases therefore, congenital bronchial atresia remains a radiological diagnosis.

Some authors suggest that similar findings could be found in other disorders as well, such as lung cancer or bronchial adenoma (Jeung et al., 2002; Woodring, 1990). The role of bronchoscopy is to exclude these disorders and demonstrate the patency of the central bronchi, especially in doubtful cases (Daoud et al., 2001; Ward &, Morcos, 1999).

Fig 2. Polilobulated round mass located in the posterobasilar right subsegmental bronchi with a distal segmental area of radiolucency and lack of communication between the mucocele and hilum on high-resolution computed tomography (Sacristán Bou et al., 2010).

MRI shows a very high signal intensity within the bronchocele on T1W and T2W modes due to mucoid contents; however, it cannot depict regional air-trapping (Matsushima et al., 2003) and it does not have the same sensivity as computed tomography to evaluate the lung parenchyma (Naidich et al., 1988). So this technique has a limited role in the diagnosis of congenital brochial atresia, although a small number of case reports have dealt specifically

Magnetic resonance imaging seem to be useful for the evaluation of either anomalous vessels or fluid collections that are usually associated with pulmonary abnormalities (Cohen

Before the advent of computed tomography, the diagnosis of bronchial atresia was made by bronchography, which allowed confirmation of the atretic segmental bronchus showing

Flexible-bronchoscopy identifies blind-ending bronchi. In clinical practice however, any absence of a segmental or sub-segmental bronchus that is found by chance during bronchoscopy in the absence of the characteristic radiographic features may be considered

In the majority of the cases therefore, congenital bronchial atresia remains a radiological

Some authors suggest that similar findings could be found in other disorders as well, such as lung cancer or bronchial adenoma (Jeung et al., 2002; Woodring, 1990). The role of bronchoscopy is to exclude these disorders and demonstrate the patency of the central

as a normal anatomic variance of the bronchial tree rather than a bronchial atresia.

bronchi, especially in doubtful cases (Daoud et al., 2001; Ward &, Morcos, 1999).

**4.3 Magnetic Resonance Imaging (MRI)** 

et al., 1987).

diagnosis.

**5. Bronchoscopy** 

non-filling of the involved bronchus.

with differential diagnosis and have established its value.

## **6. Lung function tests**

Pulmonary function tests do not aid in diagnosis. They are normal in comparison with the magnitude of the radiological abnormality. The most interesting point is the normal physiological dead space that implies air trapping in the emphysematous area as a result of collateral ventilation that causes the inflation.

## **7. Differential diagnosis**

The singular finding of a dilated bronchus due to mucoid impaction (bronchocele) can be seen in a variety of conditions apart from bronchia atresia. It is important to differentiate between congenital and acquired causes of obstruction (Table 2).

## **7.1 Congenital obstructive illnesses**

## **7.1.1 Lung Aplasia**

Pulmonary agenesis refers to undeveloped pulmonary vessels, bronchi, and parenchyma. It may be unilateral or bilateral. In unilateral aplasia, the remaining lung contains twice as much alveoli as normal, but has normal bronchi. Although aplasia does not have the same structures, it has a rudimentary bronchus.

Pulmonary aplasia (agenesis) is thought to result from the negative effects that occur in the 4th week of fetal life. Although its etiology is not fully understood, Vitamin A or folic acid deficiency or the use of salicylates may be responsible. The incidence in males and females and the occurrence of the anomaly in the right or left lung are about the same. Hypoplasia and aplasia are often observed together with other malformations (diaphragm defects, kidney anomalies, extrapulmonary sequestration, muscular or skeletal system defects). Nearly one-third of the patients have congenital heart diseases. Although the most common is the atrial septal defect, ventricular septal defect, patent ductus arteriosus, or aorta coarctation can also be observed. Clinical findings change with the presence of comorbid anomalies and their severity. Recurrent infections can increase respiratory dysfunction. Although patients with unilateral lung aplasia (agenesis) are believed to die usually in the neonatal period, there are patients who live up to adulthood, some of whom are completely asymptomatic. In the diagnosis of this condition, methods such as contrast-enhanced CT, bronchography, bronchoscopy, pulmonary angiography, and magnetic resonance imaging are also employed.

#### **7.1.2 Congenital Lobar Emphysema**

A congenital lobar emphysema (CLE) refers to an over inflation of one or more lung lobes presumably due to various factors including a possible obstructive check valve mechanism at the bronchial level. It is more common in males and often detected in neonates or identified during in utero ultrasound. Anomalies are rather infrequent. Patients will typically have respiratory distress within the first 6 months of life.

CLE almost always involves one lobe, with rates of occurrence as follows: 41% left upper lobe, 34% right middle lobe, 21% right upper lobe. Congenital lobar emphysema has two forms of presentation: hypoalveolar: fewer than the expected number of alveoli, and polyalveolar: greater than the expected number of alveoli.

There are many presumed mechanisms for progressive overdistension of a lobe including obstruction, cartilage deficiency, dysplasia, immaturity and idiopathic. It can be associated with cardiac anomalies such as: a ventricular septal defect, patent ductus arteriosus and tetralogy of Fallot. Congenital lobar emphysema appears in the immediate postpartum period.

Radiography of the chest in anteroposterior and lateral projections identifies the involved lobe, the degree of involvement, and the effect on surrounding structures. If a decubitus position radiograph is obtained, the involved lung does not collapse. Computed tomography scanning can provide details about the involved lobe and its vascularity, as well as information about the remaining lung. MRI can be used as an adjunctive modality to evaluate vascular supply and distribution to the involved lobe but is not routinely employed. In congenital lobar emphysema, the abnormal lobe usually has a normal vascular supply.

## **7.1.3 Congenital Cystic Adenomatoid Malformation (CAM)**

The first cystic adenomatoid malformation (CAM) was described as a distinct entity by Ch'in and Tang in 1949. CAM is a developmental hamartomatous abnormality of the lung, with adenomatoid proliferation of cysts resembling bronchioles. CAM represents approximately 25% of all congenital lung lesions (Colin et al., 2006).

CAM is subdivided into three major types:


CAM receives its blood supply from pulmonary circulation and is not sequestered from the tracheobronchial tree. However, type II and III lesions can occasionally coexist with extralobar sequestration, and in such cases, they may receive a systemic arterial supply. CAM may also occur in combination with a polyalveolar lobe. This is a form of congenital emphysema with an increased number of alveoli with normal bronchi and pulmonary vasculature. CAM usually occurs early in fetal life, whereas the polyalveolar lobe occurs later.

Prenatal ultrasonography is accurate in diagnosing CAM. Prenatally diagnosed lesions may be asymptomatic at birth (71%), and they have normal radiographic findings (57%). A concurrent sequestration may not be identified. Usually, radiographic findings are apparent in a symptomatic individual, but they may not be as apparent in an asymptomatic child.

Most often, the diagnosis can be made by using plain radiographs. CT scans may be used for confirmation and when planning surgery. Overlapping CT features exist among cases of CAM, pulmonary sequestration, bronchogenic cyst, and other foregut malformations.

There are many presumed mechanisms for progressive overdistension of a lobe including obstruction, cartilage deficiency, dysplasia, immaturity and idiopathic. It can be associated with cardiac anomalies such as: a ventricular septal defect, patent ductus arteriosus and tetralogy of Fallot. Congenital lobar emphysema appears in the immediate postpartum

Radiography of the chest in anteroposterior and lateral projections identifies the involved lobe, the degree of involvement, and the effect on surrounding structures. If a decubitus position radiograph is obtained, the involved lung does not collapse. Computed tomography scanning can provide details about the involved lobe and its vascularity, as well as information about the remaining lung. MRI can be used as an adjunctive modality to evaluate vascular supply and distribution to the involved lobe but is not routinely employed. In congenital lobar emphysema, the abnormal lobe usually has a normal vascular

The first cystic adenomatoid malformation (CAM) was described as a distinct entity by Ch'in and Tang in 1949. CAM is a developmental hamartomatous abnormality of the lung, with adenomatoid proliferation of cysts resembling bronchioles. CAM represents

 Type I lesions, the most common, are composed of 1 or more cysts measuring 2-10 cm in diameter. Larger cysts are often accompanied by smaller cysts, and their walls contain muscle, elastic, or fibrous tissue. Cysts are frequently lined by pseudostratified columnar epithelial cells, which occasionally produce mucin. Mucinogenic

 Type II lesions are characterized by small, relatively uniform cysts resembling bronchioles. These cysts are lined by cuboid-to-columnar epithelium and have a thin

Type III lesions have the appearance of solid masses without obvious cyst formation

CAM receives its blood supply from pulmonary circulation and is not sequestered from the tracheobronchial tree. However, type II and III lesions can occasionally coexist with extralobar sequestration, and in such cases, they may receive a systemic arterial supply. CAM may also occur in combination with a polyalveolar lobe. This is a form of congenital emphysema with an increased number of alveoli with normal bronchi and pulmonary vasculature. CAM

Prenatal ultrasonography is accurate in diagnosing CAM. Prenatally diagnosed lesions may be asymptomatic at birth (71%), and they have normal radiographic findings (57%). A concurrent sequestration may not be identified. Usually, radiographic findings are apparent in a symptomatic individual, but they may not be as apparent in an asymptomatic child.

Most often, the diagnosis can be made by using plain radiographs. CT scans may be used for confirmation and when planning surgery. Overlapping CT features exist among cases of CAM, pulmonary sequestration, bronchogenic cyst, and other foregut malformations.

fibromuscular wall. The cysts generally measure 0.5-2 cm in diameter.

although adenomatoid cysts can be detected microscopically.

usually occurs early in fetal life, whereas the polyalveolar lobe occurs later.

**7.1.3 Congenital Cystic Adenomatoid Malformation (CAM)** 

differentiation is unique to this subtype of CAM.

CAM is subdivided into three major types:

approximately 25% of all congenital lung lesions (Colin et al., 2006).

period.

supply.

#### **7.1.4 Bronchogenic cysts**

Bronchogenic cysts are part of a spectrum of congenital abnormalities of the lung including pulmonary sequestration, congenital cystic adenomatoid malformation and congenital lobar hyperinflation (emphysema). There exists a predilection in all of them for the left upper lobe that could be due to the embryologic instability of this area (Sadler, 1990).

Although relatively rare, cysts represent the most common lesion of the mediastinum. In infants and small children, these cysts can be life threatening when they compress vital structures. In particular, subcarinal cysts can pose life-threatening airway compromise. In infants, the initial presentation may be respiratory distress. More than one half of patients are asymptomatic.

These are usually found using antenatal ultrasonography or routine chest radiography and during evaluations for gastro-intestinal or cardiac symptomatology. Bronchogenic cysts are the result of anomalous development of the ventral foregut; they are usually single but may be multiple and can be filled with fluid or mucus. They have been found all along the tracheoesophageal course, in perihilar or intraparenchymal sites, with a predilection for the area around the carina. Those in the mediastinum frequently attach to, but do not communicate with, the tracheobronchial tree. Bronchogenic cysts have also been described in more remote locations, including the interatrial septum, neck, abdomen, and retroperitoneal space. Chest pain and dysphagia are the most common symptoms in adults with bronchogenic cysts; in infants, symptoms are most often produced as a result of airway or esophageal compression.

Bronchogenic cysts are usually an incidental finding, and differentiating them from other pathologic conditions is important. On conventional radiographs, the appearances of mediastinal or lung masses are nonspecific and should be evaluated further using computed tomography (CT) scanning or magnetic resonance imaging (MRI). Intrapulmonary cysts are difficult to diagnose and must usually be aspirated to confirm the diagnosis.

#### **7.1.5 Anomalous pulmonary venous return**

Abnormal development of the pulmonary veins may result in either partial or complete anomalous drainage back into the systemic venous circulation. Three major clinical patterns of total anomalous pulmonary venous return (TAPVR) are seen: severe pulmonary venous obstruction; early heart failure; mildly symptomatic or asymptomatic.

## **7.1.6 Pulmonary sequestration**

Pulmonary sequestration is a cystic or solid mass composed of nonfunctioning primitive tissue that does not communicate with the tracheobronchial tree and has an anomalous systemic blood supply rather than the pulmonary circulation. In 15-20% of cases multiple feeding vessels may be present. The two forms of pulmonary sequestration are intrapulmonary, which is surrounded by normal lung tissue, and extrapulmonary, which has its own pleural investment. Demonstration of a dominant feeding vessel, usually from the aorta or its major vessels, and venous drainage to the pulmonary veins suggests the diagnosis. Other congenital malformations may be present.

Chest radiographs can provide a reasonable diagnostic clue to pulmonary sequestration. A mass in the posterobasal segment of the lung in young patients with recurrent, localized pulmonary infections is suggestive of pulmonary sequestration. Computed tomography scanning, angiography, magnetic resonance imaging or bronchography may be helpful in excluding other diagnoses. CT scans have 90% accuracy in the diagnosis of pulmonary sequestration. Arteriography is helpful in differentiating the lesion from other abnormalities of the lung, such as pulmonary arteriovenous fistulae. Magnetic resonance angiography can provide information similar to that on CT scans.

## **7.1.7 Cystic Fibrosis (CF)**

The name cystic fibrosis refers to the characteristic scarring (fibrosis) and cyst formation within the pancreas, first recognized in the 1930s. Difficulty breathing is the most serious symptom and results from frequent lung infections that are treated with, though not cured by, antibiotics and other medications. A multitude of other symptoms, including sinus infections, poor growth, diarrhea, and infertility result from the effects of CF in other parts of the body.

CF is caused by a mutation in the gene for the protein: the cystic fibrosis transmembrane conductance regulator (CFTR). This gene is required to regulate the components of sweat, digestive juices, and mucus. Although most people without CF have two working copies of the CFTR gene, only one is needed to prevent cystic fibrosis. CF develops when neither gene works normally and therefore has autosomal recessive inheritance.

Individuals with cystic fibrosis can be diagnosed before birth by genetic testing, or by a sweat test in early childhood. Ultimately, lung transplantation is often necessary as CF worsens. CF mainly affects the lungs, pancreas, liver, intestines, sinuses, and sex organs.

## **7.2 Acquired obstructive illnesses**

## **7.2.1 Allergic bronchopulmonary aspergillosis (ABPA)**

Allergic bronchopulmonary aspergillosis (ABPA) can be classified as an eosinophilic hypersensitivity disease. It appears concomitantly in patients with long standing asthma, and occasionally in patients with cystic fibrosis. Rarely, it can appear in patients with no other identifiable pulmonary illness. In general, patients are diagnosed before the age of 40 years.

Clinically, patients have atopic symptoms and they present with recurrent chest infection. A clinical staging system has been developed: Stage I Acute, Stage II Remission, Stage III Recurrent Exacerbation, Stage IV Steroid-Dependent Asthma and Stage V Pulmonary Fibrosis.

Laboratory findings include elevated Aspergillus specific IgE, elevated precipitating IgG against Aspergillus, peripheral eosinophilia and positive skin test.

In patients with ABPA radiological findings will be reversible after appropriate treatment or may show progression from multi-focal and non-segmental consolidations to pulmonary fibrosis and central varicose type of bronchiectasis (stage V).

Chest radiographs can provide a reasonable diagnostic clue to pulmonary sequestration. A mass in the posterobasal segment of the lung in young patients with recurrent, localized pulmonary infections is suggestive of pulmonary sequestration. Computed tomography scanning, angiography, magnetic resonance imaging or bronchography may be helpful in excluding other diagnoses. CT scans have 90% accuracy in the diagnosis of pulmonary sequestration. Arteriography is helpful in differentiating the lesion from other abnormalities of the lung, such as pulmonary arteriovenous fistulae. Magnetic resonance angiography can

The name cystic fibrosis refers to the characteristic scarring (fibrosis) and cyst formation within the pancreas, first recognized in the 1930s. Difficulty breathing is the most serious symptom and results from frequent lung infections that are treated with, though not cured by, antibiotics and other medications. A multitude of other symptoms, including sinus infections, poor growth, diarrhea, and infertility result from the effects of CF in other parts

CF is caused by a mutation in the gene for the protein: the cystic fibrosis transmembrane conductance regulator (CFTR). This gene is required to regulate the components of sweat, digestive juices, and mucus. Although most people without CF have two working copies of the CFTR gene, only one is needed to prevent cystic fibrosis. CF develops when neither gene

Individuals with cystic fibrosis can be diagnosed before birth by genetic testing, or by a sweat test in early childhood. Ultimately, lung transplantation is often necessary as CF worsens. CF mainly affects the lungs, pancreas, liver, intestines, sinuses, and sex organs.

Allergic bronchopulmonary aspergillosis (ABPA) can be classified as an eosinophilic hypersensitivity disease. It appears concomitantly in patients with long standing asthma, and occasionally in patients with cystic fibrosis. Rarely, it can appear in patients with no other identifiable pulmonary illness. In general, patients are diagnosed before the age of 40

Clinically, patients have atopic symptoms and they present with recurrent chest infection. A clinical staging system has been developed: Stage I Acute, Stage II Remission, Stage III Recurrent Exacerbation, Stage IV Steroid-Dependent Asthma and Stage V Pulmonary

Laboratory findings include elevated Aspergillus specific IgE, elevated precipitating IgG

In patients with ABPA radiological findings will be reversible after appropriate treatment or may show progression from multi-focal and non-segmental consolidations to pulmonary

works normally and therefore has autosomal recessive inheritance.

**7.2.1 Allergic bronchopulmonary aspergillosis (ABPA)** 

against Aspergillus, peripheral eosinophilia and positive skin test.

fibrosis and central varicose type of bronchiectasis (stage V).

provide information similar to that on CT scans.

**7.1.7 Cystic Fibrosis (CF)** 

**7.2 Acquired obstructive illnesses** 

of the body.

years.

Fibrosis.

## **7.2.2 Other causes**

Bronchial obstruction may be due to many acquired conditions including inflammatory diseases (broncholithiasis and foreign body aspiration), benign neoplastic processes (bronchial hamartoma, lipoma, and papillomatosis) and malignancies (bronchogenic carcinoma, carcinoid tumor, and metastases) (Gipson et al., 2009; Wilson et al., 2009). They can even take place after a segmentectomy as a surgical complication (Okuda et al., 2006).

All these situations can produce an appearance of a round, oval or branching (glove-finger shadow) type of bronchocele. Most of these conditions can be differentiated by appropriate history (as all will be symptomatic), the progressive nature of the disease, imaging techniques, bronchoscopy and biopsy.


Table 2. The differential diagnosis of bronchial atresia.

## **8. Treatment of bronchial atresia**

Treatment of bronchial atresia is controversial. The majority of patients are asymptomatic and therefore no treatment is necessary. It is currently felt that surgical excision should be reserved for patients with secondary complications to the atretic bronchus, such as infection or significant compromise of adjacent lung parenchyma. However, some physicians advocate performing surgery on all patients because a definitive diagnosis could only be made by exeresis of the lung (Cohen et al., 1987). Lobar resection and segmentectomy have been used to preserve as much normal lung parenchyma as possible to maintain pulmonary function (Miyahara et al., 1999; Pamer et al., 2008).

## **9. Prenatal bronchial atresia**

Antenatal ultrasonography can raise the suspicion of bronchial atresia in the prenatal period by the presence of a hyperechogenic mass at the hilum of the lung which corresponds to a mucocele (Kamata et al., 2003; McAlister et al., 1987). Those hyperechogenic lesions are more likely to regress compared with cystic or mixed lesions (Hadchouel et als. 2011a, 2011b).

The routine use of prenatal steroids for microcystic congenital cystic adenomatoid malformation seemed to enhance regression (Curran et al., 2010). There are no documented cases on regression of bronchial or lobar bronchial atresia. Despite the lack of evidence, it would be advisable to follow an expectant management (Bonnefoy et al., 2011).

## **10. Diagnostic algorithm**

Fig. 3. Diagnostic algorithm in suspected bronchial atresia.

#### **11. State of art during the last three years**

Due to a wave of new interest in bronchial atresia among clinicians, there have been many more articles and case descriptions published in the last three years than previously. The aim of this section is to summarize the main aspects of these reports.

been used to preserve as much normal lung parenchyma as possible to maintain pulmonary

Antenatal ultrasonography can raise the suspicion of bronchial atresia in the prenatal period by the presence of a hyperechogenic mass at the hilum of the lung which corresponds to a mucocele (Kamata et al., 2003; McAlister et al., 1987). Those hyperechogenic lesions are more likely to regress compared with cystic or mixed lesions (Hadchouel et als. 2011a,

The routine use of prenatal steroids for microcystic congenital cystic adenomatoid malformation seemed to enhance regression (Curran et al., 2010). There are no documented cases on regression of bronchial or lobar bronchial atresia. Despite the lack of evidence, it

Due to a wave of new interest in bronchial atresia among clinicians, there have been many more articles and case descriptions published in the last three years than previously. The

would be advisable to follow an expectant management (Bonnefoy et al., 2011).

Fig. 3. Diagnostic algorithm in suspected bronchial atresia.

aim of this section is to summarize the main aspects of these reports.

**11. State of art during the last three years** 

function (Miyahara et al., 1999; Pamer et al., 2008).

**9. Prenatal bronchial atresia** 

**10. Diagnostic algorithm** 

2011b).


## **12. Conclusions**

Bronchial atresia is a congenital abnormality with characteristic radiological features: a nodule or a mass like a shadow close to the hilum, with well-defined margins, presenting as a tubular, round, ovoid or branching structure and distal oligaemia and hyperinflation.

When it is required to do differential diagnosis over bronchial obstruction, bronchial atresia should be kept in mind.

Knowledge of this condition in patients with suspected bronchial obstruction would avoid unnecessary surgery. Currently, surgical excision is reserved only for patients with secondary complications to the atretic bronchus. Most surgeons try to preserve as much normal lung parenchyma as possible to maintain pulmonary function, whilst others prefer to resect the atretic segment.

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## **A Case of Adult Congenital Cystic Adenomatoid Malformation of the Lung with Atypical Adenomatous Hyperplasia**

Ho Sung Lee1, Jae Sung Choi1, Ki Hyun Seo1, Ju Ock Na1, Yong Hoon Kim1, Mi Hye Oh2 and Sung Shick Jou3 *1Department of Internal Medicine Diagnostic 2Department of Pathology and 3Department of Radiology Soonchunhyang University College of Medicine, Cheonan Korea* 

## **1. Introduction**

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Congenital cystic adenomatoid malformation of the lung is a rare disease that shows multiple cystic lesions in pulmonary tissues in the development process. It was first described by Chin et al.1 in 1949 and its incidence is known to be 1:25,000 to 1:35,0002. With the development of prenatal diagnosis, this disease can be diagnosed in 60% and detected within 2 years because of such symptoms as respiratory distress by compression of surrounding lung tissues immediately after birth and repeated respiratory infections in infancy. Among adults, it is detected accidentally on X-ray or by such symptoms as pneumonia, pneumothorax, and hemoptysis. In Korea, the first case in a 28 year-old woman was reported by Geun-Heung Ki et al.3 in 1989. Since then, about 25 adult cases were reported until 2006.

Atypical adenomatous hyperplasia is pathologically defined as the proliferation of atypical cuboidal or columnar cepithelial cells which are typically 5 mm or smaller in size along the alveolus or respiratory bronchiole4,5. In 1999, the WHO classified this disease as a precancerous lesion together with squamous dysplasia and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia5. It is known to be found in about 12% of lung cancers and highly correlated with glandular cancer6.

## **2. Case**

Patient: O-Back Kim, Female, 37

Current case history: The patient had been treated in a private clinic due to purulent sputum and cough and hemoptysis which had started two weeks before transfer to our hospital.

Past case history: The patient had been treated for hyperthyroidism from 1999 to 2003.

Examination findings: Vital signs were stable. Chest examination detected reduced bilateral breathing sounds only.

Laboratory findings: The peripheral blood examination found 13.2 g/dL of hemoglobin, 39.5 % of hematocrit, 5210 /mm3 of white blood cells (neutrophils 43%, lymphocytes 44.5%, monocytes 9.2%), and 255,000 /mm3 of platelets. The results of serum biochemical examination, hepatitis marker test, urinalysis, mycoplasma antigen test, and pulmonary function test were normal. The thyroid function test found minor hypothyroidism with 8.72 nIU/ml of thyroid stimulating hormone (TSH), 0.894 ng/dl and 1.19ng/ml of free T4 and T3 respectively. The results of the sputum culture and bronchial washing cytodiagnosis were also negative. The results of arterial blood gas analysis which was conducted in atmosphere were 7.407 of pH, 41.2 mmHg of carbon dioxide tension, 82.6 mmHg of oxygen tension, 25.4 mEq/L of bicarbonates, and 96.2% of oxygen saturation. The tumor marker test result was also normal with 1.49 ng/mL of CEA and 1.57 ng/mL of cyfra 21-1.

Chest radiology: The chest X-ray showed a 2.5cm cavitating lesion in the left upper zone. (Figure 1).

Fig. 1. Chest PA shows cavitatory lesion in the left upper zone

On the second day after hospitalization, the chest CT showed a diffuse ground glass appearance and cystic lesions that spread in a branching pattern were found in the left lung (Figure 2).

Clinical progress: The patient received symptomatic therapy as there were no features of infection. The bronchial endoscopy did not reveal any lesions. The bronchoalveolar lavage fluid test found 82% of macrophages, 8% of neutrophils, 0.5% of eosinophils, and 9.5% of lymphocytes. Endobronchial lung biopsy was performed at the left lower lobe and only chronic inflammatory changes were noted. On the 9th day after hospitalization, a wedge resection of the top section of the left upper lobe was performed. On the 20th day after hospitalization, the patient was discharged with no complications.

Biopsy: The microscopic findings of the resected lung showed cystic lesions of various sizes. The cells covering them were diverse, including ciliated pseudostratified columnar cells, monolayer columnar cell, and cuboid cells, which corresponded to type 2 of the congenital cystic adenomatoid malformation(Figure 3).

Laboratory findings: The peripheral blood examination found 13.2 g/dL of hemoglobin, 39.5 % of hematocrit, 5210 /mm3 of white blood cells (neutrophils 43%, lymphocytes 44.5%, monocytes 9.2%), and 255,000 /mm3 of platelets. The results of serum biochemical examination, hepatitis marker test, urinalysis, mycoplasma antigen test, and pulmonary function test were normal. The thyroid function test found minor hypothyroidism with 8.72 nIU/ml of thyroid stimulating hormone (TSH), 0.894 ng/dl and 1.19ng/ml of free T4 and T3 respectively. The results of the sputum culture and bronchial washing cytodiagnosis were also negative. The results of arterial blood gas analysis which was conducted in atmosphere were 7.407 of pH, 41.2 mmHg of carbon dioxide tension, 82.6 mmHg of oxygen tension, 25.4 mEq/L of bicarbonates, and 96.2% of oxygen saturation. The tumor marker test result was

Chest radiology: The chest X-ray showed a 2.5cm cavitating lesion in the left upper zone.

On the second day after hospitalization, the chest CT showed a diffuse ground glass appearance and cystic lesions that spread in a branching pattern were found in the left lung

Clinical progress: The patient received symptomatic therapy as there were no features of infection. The bronchial endoscopy did not reveal any lesions. The bronchoalveolar lavage fluid test found 82% of macrophages, 8% of neutrophils, 0.5% of eosinophils, and 9.5% of lymphocytes. Endobronchial lung biopsy was performed at the left lower lobe and only chronic inflammatory changes were noted. On the 9th day after hospitalization, a wedge resection of the top section of the left upper lobe was performed. On the 20th day after

Biopsy: The microscopic findings of the resected lung showed cystic lesions of various sizes. The cells covering them were diverse, including ciliated pseudostratified columnar cells, monolayer columnar cell, and cuboid cells, which corresponded to type 2 of the congenital

also normal with 1.49 ng/mL of CEA and 1.57 ng/mL of cyfra 21-1.

Fig. 1. Chest PA shows cavitatory lesion in the left upper zone

hospitalization, the patient was discharged with no complications.

cystic adenomatoid malformation(Figure 3).

(Figure 1).

(Figure 2).

Fig. 2. Chest CT shows diffuse ground glass opacity and multiple branching cystic lesion in left lung

Fig. 3. The lining cells are pseudostratified ciliated columnar cells to columnar and cuboidal cells like bronchiolar epithelial cells (H&E, x100).

As there were minor atypical nuclei locally and linear structures filled with mucus in the cytoplasm, it looked similar to the mucoid bronchioloalveolar carcinoma, but because their sizes were all smaller than 5mm, they were judged to be atypical adenomatous proliferation (Figure 4).

Fig. 4. Focal mucinous epithelial lesion with mild atypism is revealed in the intervening parenchyma (H&E, x200).

Treatment and progress: After discharge, the patient was transferred to another hospital at her request; As it was checked, she was being followed up with chest CT every 3 months with no specific treatment.

## **3. Discussion**

The congenital cystic adenomatoid malformation of the lung is very rare and its incidence is known to be 1:25,000 - 1:35,0002. The cause is not known, but two hypotheses have been suggested: cessation in the development of the lung tissues and no development of aveoli during the development process of the respiratory system7,8. The time when the malformation occurs is estimated to be between 5 and 6 weeks before the lobe divides and prechondrial tissues are formed in the bronchus. It has been reported that it causes stillborn and premature infants. Immediately after birth, respiratory distress is frequent in most cases, and as the baby grows, repeated infections and pneumothorax are more frequent than respiratory distress. Accompanying malformations include kidney growth failure, diaphragmatic hernia, jejunul atresia, and colon growth failure1. A histological characteristic is the arrangement of polyp-shaped cysts of various sizes in the bronchial epithelium or simple columnar epithelium with no cartilaginous tissues or inflammatory reaction. Stoker et al. classified them based on size and pathological findings into type I (only a few large thick walled cyst), type II (numerous, evenly spaced cyst, less than 1cm), and type III (less numerous, firm and bulky masses) in 1977. In 1994, they re-classified them into 5 types based on bronchial invasion: type 0 (bronchial), type I (bronchial/bronchiolar), type II

Fig. 4. Focal mucinous epithelial lesion with mild atypism is revealed in the intervening

Treatment and progress: After discharge, the patient was transferred to another hospital at her request; As it was checked, she was being followed up with chest CT every 3 months

The congenital cystic adenomatoid malformation of the lung is very rare and its incidence is known to be 1:25,000 - 1:35,0002. The cause is not known, but two hypotheses have been suggested: cessation in the development of the lung tissues and no development of aveoli during the development process of the respiratory system7,8. The time when the malformation occurs is estimated to be between 5 and 6 weeks before the lobe divides and prechondrial tissues are formed in the bronchus. It has been reported that it causes stillborn and premature infants. Immediately after birth, respiratory distress is frequent in most cases, and as the baby grows, repeated infections and pneumothorax are more frequent than respiratory distress. Accompanying malformations include kidney growth failure, diaphragmatic hernia, jejunul atresia, and colon growth failure1. A histological characteristic is the arrangement of polyp-shaped cysts of various sizes in the bronchial epithelium or simple columnar epithelium with no cartilaginous tissues or inflammatory reaction. Stoker et al. classified them based on size and pathological findings into type I (only a few large thick walled cyst), type II (numerous, evenly spaced cyst, less than 1cm), and type III (less numerous, firm and bulky masses) in 1977. In 1994, they re-classified them into 5 types based on bronchial invasion: type 0 (bronchial), type I (bronchial/bronchiolar), type II

parenchyma (H&E, x200).

with no specific treatment.

**3. Discussion** 

(bronchiolar), type III (bronchiolar/alveolar), and type IV (peripheral). In 1994, they reported that type I was the most frequent at 50 - 70% and type III showed the worst prognosis. Radiographic diagnoses include chest X-ray test, CT, and prenatal ultrasonography. Among them, chest CT can observe lesions that contain cysts with multiple large and small thin walls. It must be differentiated from pneumonia accompanied by pneumatosis, pulmonary sequestration, congenital lobar emphysema, and bronchiectasis. Definite diagnosis is only possible by pathological tests. Some claim that it is associated with malignant tumors of the lung. There was a report of pulmonary rhabdomyosarcoma in 22-month old boy, and several papers reported the accompaniment of mucoid bronchioloalveolar carcinoma in adults and children3,9. Hence, Ioachimescu et al.9 recommended surgical removal even if there were no symptoms, because it may become malignant.

 Pathological findings in this patient were accompanied by atypical adenomatous hyperplasia. Atypical adenomatous hyperplasia is pathologically defined as the proliferation of atypical cuboidal epithelial cells or columnar epithelial cells along the alveolus or respiratory bronchiole4. Their sizes are mostly 5 mm or smaller, although a size of 19mm has been reported. As it is difficult to differentiate from bronchioloalveolar carcinoma, one researcher suggested 5 mm as the reference size for differentiation. It occurs in up to 5% of normal people and is usually asymptomatic. It is known to develop in 2.9% of total population and increases to 10 - 23.2% in lung cancer 11,12,13,14,15. The recent diagnostic rate is increasing due to low dose chest CT as a lung cancer screening test. Although there are no specific CT findings, the most frequent finding are nodules with a good boundary accompanied by a ground glass apperance10. In 1999, the WHO classified this disease as a precancerous lesion together with squamous dysplasia and diffuse idiopathic pulmonary neuroendocrine cell hyperplasia5. Chapman et al.6 analyzed the pathological findings of 554 patients with primary lung cancer and found that atypical adenomatous hyperplasia was accompanied in 67 cases (12.1%), and that the percentage of glandular cancer was the highest (glandular cancer 23.2%, giant cell undifferentiated cancer 12.5%, epithelial carcinoma 2.2%). There are many different opinions among pathologists and no established views on the differentiation level of atypical adenomatous hyperplasia, classification based on this, and its relationship with adenocarcinoma. However, there are some reports related to morphological changes of the nuclei, expression of Ki-67 and p53, and K-ras mutation, which are expected to be helpful for better investigation of the characteristics of atypical adenomatous hyperplasia as a precancerous lesion5,10. As there are no principles in therapy yet, careful follow-up is needed.

Although there is controversy about the treatment of this patient, the authors believe that pneumonectomy of the left lung will be necessary because the patient has both congenital cystic adenomatoid malformation which can be accompanied by bronchioloalveolar carcinoma and atypical adenomatous hyperplasia which is a precancerous lesion.

After a literature review, it is believed that this is the first case report of congenital cystic adenomatoid malformation accompanied by atypical adenomatous hyperplasia.

## **4. References**

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## *Edited by Elvis Malcolm Irusen*

The developments in molecular medicine are transforming respiratory medicine. Leading clinicians and scientists in the world have brought their knowledge and experience in their contributions to this book. Clinicians and researchers will learn about the most recent advances in a variety of lung diseases that will better enable them to understand respiratory disorders. This treatise presents state of the art essays on airways disease, neoplastic diseases, and pediatric respiratory conditions. Additionally, aspects of immune regulation, respiratory infections, acute lung injury/ ARDS, pulmonary edema, functional evaluation in respiratory disorders, and a variety of other conditions are also discussed. The book will be invaluable to clinicians who keep up with the current concepts, improve their diagnostic skills, and understand potential new therapeutic applications in lung diseases, while scientists can contemplate a plethora of new research avenues for exploration.

Lung Diseases - Selected State of the Art Reviews

Lung Diseases

Selected State of the Art Reviews

*Edited by Elvis Malcolm Irusen*