**2. Exhaled breath condensate**

Compared to the currently widespread invasive and semi-invasive diagnostic methods, the analysis of exhaled breath condensate (EBC) is relatively new and has the first-rate potential to become a preferred and completely noninvasive alternative. EBC is a biological matrix reflecting the composition of the bronchoalveolar extra-cellular lung fluid. The main advantage of EBC as of a matrix is its specificity for the respiratory tract (the liquid is not influenced by process occurring in other parts of human organism). Many important biomolecules are present in exhaled breath in the form of an aerosol [4, 5] (**Figure 1**) which is condensed by cooling during the collection, forming the EBC matrix.

assessing the type and severity of ongoing pathological process or even the efficiency of a

Molecular Diagnostics of Pulmonary Diseases Based on Analysis of Exhaled Breath Condensate

rienes (cys-LTs), lipoxins (LXs), malverines, resolvins, isoprostanes, prostaglandines, glutathione, adenosine, thiobarbituric acid, aldehydes, nitrotyrosine, cytokines represent a specific group of biomarkers and their concentration levels are elevated (eventually lowered) in airways and lungs as a result of an ongoing allergic reaction, inflammation, oxidative stress, and

The most significant advantage of EBC compared to other biological matrices (as are, for example, urine and blood) is the fact that EBC is a highly specific fluid for the respiratory

During the collection of EBC, the exhaled air is led through the condenser, where some components are condensed. The patients should breath calmly and regularly during the whole

system, so any other biochemical processes in human organism do not influence it.

O2

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143

, cysteinyl leukot-

therapeutic procedure, etc. In case of numerous pulmonary diseases, H2

other processes [9–12].

**Figure 1.** Formation of exhaled breath condensate.

**3. The collection of EBC**

The collection of EBC is performed while using the condenser, which is currently available at a specialized clinical facility. During the collection, the exhaled air is led through the condenser into the cooling box that is pre-cooled to the temperature −20°C. In the cooling box, the aerosol particles are obtained and the gaseous phase is liquidized.

In the obtained liquid, typically known as EBC, more than 2000 compounds [6] have been identified so far and many of them are considered to represent sensitive biomarkers of lung diseases [7, 8]. The determination of the concentration of these molecules in EBC allows

Molecular Diagnostics of Pulmonary Diseases Based on Analysis of Exhaled Breath Condensate http://dx.doi.org/10.5772/intechopen.74402 143

**Figure 1.** Formation of exhaled breath condensate.

physiological states. Via different odors, sounds, and breath dynamics often attributed to supernatural powers and superstitious believes, various lung diseases could be relatively well diagnosed and further progression could be predicted. For instance, the odors in EB as, for example, fruity traces of acetone aided to identify diabetes; a rather pungent characteristic odor was associated with a lung inflammation, while volatile vapors from urine revealed a kidney disease [1]. Modern investigations enlisted approx. 250 frequently detected volatile organic compounds (VOC's) in EB [2]. Early analyses did not incorporate sample pre-treatments as sample concentration and exclusively depended on relatively modest gas chromatography (GC) methods. The progress of technology, however, over the decades has permitted much more precise and sophisticated analyses of EB, some of which have been implemented to the clinical practice, as, for example, ethanol levels in blood or typical inflammations caused by common pathogens as *Helicobacter pylori* using 13/14C-urea [3]. As mentioned above, the prime advantage of EB analysis is the patient's comfort, especially eliminating the stressful intrusions to human organisms, yet there are challenges ahead. For instance, a breakthrough task is to find common internal standard reliably standardizing diagnoses for each pathological status. Furthermore, an opposite selection of multi-marker panel is to be conspicuously correlated to different health phenomena, providing the knowledge of characteristic concentrations. Moreover, it is often unclear which metabolic pathways in relation to different measured biomarkers are involved and some are probably yet to be discovered or decoded. Last but not the least, technological and procedural challenges include also the standardization in terms of the sample collection and treatment, and conceivably, endeavors to automatization

Compared to the currently widespread invasive and semi-invasive diagnostic methods, the analysis of exhaled breath condensate (EBC) is relatively new and has the first-rate potential to become a preferred and completely noninvasive alternative. EBC is a biological matrix reflecting the composition of the bronchoalveolar extra-cellular lung fluid. The main advantage of EBC as of a matrix is its specificity for the respiratory tract (the liquid is not influenced by process occurring in other parts of human organism). Many important biomolecules are present in exhaled breath in the form of an aerosol [4, 5] (**Figure 1**) which is condensed by

The collection of EBC is performed while using the condenser, which is currently available at a specialized clinical facility. During the collection, the exhaled air is led through the condenser into the cooling box that is pre-cooled to the temperature −20°C. In the cooling box, the

In the obtained liquid, typically known as EBC, more than 2000 compounds [6] have been identified so far and many of them are considered to represent sensitive biomarkers of lung diseases [7, 8]. The determination of the concentration of these molecules in EBC allows

of the complete process in the clinical practice.

cooling during the collection, forming the EBC matrix.

aerosol particles are obtained and the gaseous phase is liquidized.

**2. Exhaled breath condensate**

142 Biomarker - Indicator of Abnormal Physiological Process

assessing the type and severity of ongoing pathological process or even the efficiency of a therapeutic procedure, etc. In case of numerous pulmonary diseases, H2 O2 , cysteinyl leukotrienes (cys-LTs), lipoxins (LXs), malverines, resolvins, isoprostanes, prostaglandines, glutathione, adenosine, thiobarbituric acid, aldehydes, nitrotyrosine, cytokines represent a specific group of biomarkers and their concentration levels are elevated (eventually lowered) in airways and lungs as a result of an ongoing allergic reaction, inflammation, oxidative stress, and other processes [9–12].

The most significant advantage of EBC compared to other biological matrices (as are, for example, urine and blood) is the fact that EBC is a highly specific fluid for the respiratory system, so any other biochemical processes in human organism do not influence it.

#### **3. The collection of EBC**

During the collection of EBC, the exhaled air is led through the condenser, where some components are condensed. The patients should breath calmly and regularly during the whole process. The exhaled air flows through the mouthpiece and the one-way valve into the cooling cuff that is pre-cooled at the temperature of −20°C. In the cooling cuff, the aerosol particles and the obtained gaseous phase are condensed. This liquid is then gathered in the sample collection vial (the temperature remains the same) [13]. The whole process lasts approximately 7–12 min. It is necessary to obtain 120 l of EB in total, which corresponds to 1–2 ml of the condensate. The obtained condensate is then conserved in a micro-test-tube. In order to monitor the degrading process, the samples were labeled by deuterium-labeled internal standards. The prepared samples are then subsequently frozen and stored for a period not exceeding 6 months (−80°C).

As the collection of EBC is a noninvasive diagnostic method that does not burden the patient, it can be used in several different clinical studies. A regular collection of EBC enables, for example, monitoring of the impact of climate conditions on the patients. Globally, collection of EBC is a method that is suitable for clinical studies that are trying to understand the process in the organism which corresponds to some external impulses (physical activity, air quality, allergens, etc.)

> products, eosinophils and neutrophils, cause mucus hypersecretion, edema, and constriction of smooth muscles. This is usually associated with the late asthma phase and it causes the

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There are several options for the diagnostics of asthma; however, only an early and correct diagnosis of this life-threatening disease permits the physician to timely initiate an effective therapy and minimize the harm to the patient [18]. Several noninvasive methods are already in clinical use (e.g., spirometry, bronchomotoric tests, etc.). In some cases, invasive and *semi*invasive methods appear to be an inevitable option to gain the correct diagnosis (e.g., openlung biopsy and bronchoalveolar lavage) [21], yet it is to an unambiguous expense of the

Currently, a significant part of the relevant research centers focuses on methods of the socalled personalized diagnostics (or methods of personalized medicine), with the aim to stratify patients to characteristic groups (e.g., phenotypes) and thus achieve a more efficient therapy reflecting an individual phenotypic disposition (inclusive of genomic, proteomic and metabolomic profiles) [22, 23]. One of the examples of these endeavors (particularly for diagnostics of pulmonary diseases) is the measurement of a fractional exhaled nitric oxide (FeNO) [24–26] in EBC, helping to distinguish asthma from other pathogenetic processes diagnosed as chronic cough, gastroesophageal reflux disease (GERD), vocal cord dysfunction, bronchi-

As asthma is a disease affecting millions of people of all ages worldwide, many criteria can be used for its classification. Nevertheless, the predominantly used criterion is the severity of the disease, as is presented in **Figure 3**, followed by the age of the first exacerbation [9, 26].

patient and often the health cost as well as a demanding laboratory examination.

tis, chronic obstructive pulmonary disease (COPD), etc.

second drop in FEV1.

**4.2. Asthma phenotypes**

**4.1. The diagnostics of asthma**

**Figure 2.** Immunopathogenesis of asthma [20].

#### **4. Bronchial asthma**

Bronchial asthma is a relatively common pulmonary disease, which is usually characterized by dyspnoea combined with intervals of a normal breathing [14–19]. Typical symptoms of asthma include constricted bronchial tubes and an increased secretion of sputum, which is abnormally dense and viscous [16]. Various sources agree that on the global scale, the asthma incidence accounts for around 300 million people, while the prognoses that are negative in the sense of the future number will keep rising. On the other hand, wide ranges of relatively efficient anti-asthmatic therapies are available (e.g., glucocorticoid therapy, β<sup>2</sup> -receptors agonists, etc.) [17] enabling the majority of patients to live normal lives. However, there is still a small group of patients, who do not respond to any kind of current therapy. These patients are usually diagnosed as sever refractory asthmatics (SRA) [6], whose common feature is a lack of any response to any contemporarily available pharmacotherapy. SRA accounts for approximately 5% of all asthmatics, which represents 10 million of people [6].

**Figure 2** describes the immunopathogenesis of asthma [20]. The asthma attack starts by exposure to an allergen, which causes synthesis of immunoglobulin E (IgE). IgE then binds to the surface of mast cells. As there occurs a re-exposure to the same allergen, the interaction between allergen and antibody triggers the release of mediators as are prostaglandins (PGDs), cys-LTs, LTB4 and platelet-activating factor (PAF). These mediators cause bronchoconstriction that is connected to an immediate drop in FEV1 (= forced expiratory volume in 1 s; the FEV1 is the volume exhaled during the first second of a forced expiratory maneuver started from the level of total lung capacity). The allergen-antibody interaction also causes production of a wide range of cytokines (e.g., interleukin 4 and 5 (IL-4 and IL-5), tumor necrosis factor (TNF) and tissue growth factor (TGF)). These cytokines then activate neutrophils and eosinophils. Neutrophils produce proteases and PAF, and at the same time, eosinophils produce eosinophil cationic protein (ECP) and major basic protein (MBP). These

Molecular Diagnostics of Pulmonary Diseases Based on Analysis of Exhaled Breath Condensate http://dx.doi.org/10.5772/intechopen.74402 145

**Figure 2.** Immunopathogenesis of asthma [20].

process. The exhaled air flows through the mouthpiece and the one-way valve into the cooling cuff that is pre-cooled at the temperature of −20°C. In the cooling cuff, the aerosol particles and the obtained gaseous phase are condensed. This liquid is then gathered in the sample collection vial (the temperature remains the same) [13]. The whole process lasts approximately 7–12 min. It is necessary to obtain 120 l of EB in total, which corresponds to 1–2 ml of the condensate. The obtained condensate is then conserved in a micro-test-tube. In order to monitor the degrading process, the samples were labeled by deuterium-labeled internal standards. The prepared samples are then subsequently frozen and stored for a period not exceeding

As the collection of EBC is a noninvasive diagnostic method that does not burden the patient, it can be used in several different clinical studies. A regular collection of EBC enables, for example, monitoring of the impact of climate conditions on the patients. Globally, collection of EBC is a method that is suitable for clinical studies that are trying to understand the process in the organism which corresponds to some external impulses (physical activity, air quality,

Bronchial asthma is a relatively common pulmonary disease, which is usually characterized by dyspnoea combined with intervals of a normal breathing [14–19]. Typical symptoms of asthma include constricted bronchial tubes and an increased secretion of sputum, which is abnormally dense and viscous [16]. Various sources agree that on the global scale, the asthma incidence accounts for around 300 million people, while the prognoses that are negative in the sense of the future number will keep rising. On the other hand, wide ranges of relatively

nists, etc.) [17] enabling the majority of patients to live normal lives. However, there is still a small group of patients, who do not respond to any kind of current therapy. These patients are usually diagnosed as sever refractory asthmatics (SRA) [6], whose common feature is a lack of any response to any contemporarily available pharmacotherapy. SRA accounts for

**Figure 2** describes the immunopathogenesis of asthma [20]. The asthma attack starts by exposure to an allergen, which causes synthesis of immunoglobulin E (IgE). IgE then binds to the surface of mast cells. As there occurs a re-exposure to the same allergen, the interaction between allergen and antibody triggers the release of mediators as are prostaglandins

constriction that is connected to an immediate drop in FEV1 (= forced expiratory volume in 1 s; the FEV1 is the volume exhaled during the first second of a forced expiratory maneuver started from the level of total lung capacity). The allergen-antibody interaction also causes production of a wide range of cytokines (e.g., interleukin 4 and 5 (IL-4 and IL-5), tumor necrosis factor (TNF) and tissue growth factor (TGF)). These cytokines then activate neutrophils and eosinophils. Neutrophils produce proteases and PAF, and at the same time, eosinophils produce eosinophil cationic protein (ECP) and major basic protein (MBP). These

and platelet-activating factor (PAF). These mediators cause broncho-


efficient anti-asthmatic therapies are available (e.g., glucocorticoid therapy, β<sup>2</sup>

approximately 5% of all asthmatics, which represents 10 million of people [6].

6 months (−80°C).

144 Biomarker - Indicator of Abnormal Physiological Process

allergens, etc.)

**4. Bronchial asthma**

(PGDs), cys-LTs, LTB4

products, eosinophils and neutrophils, cause mucus hypersecretion, edema, and constriction of smooth muscles. This is usually associated with the late asthma phase and it causes the second drop in FEV1.

#### **4.1. The diagnostics of asthma**

There are several options for the diagnostics of asthma; however, only an early and correct diagnosis of this life-threatening disease permits the physician to timely initiate an effective therapy and minimize the harm to the patient [18]. Several noninvasive methods are already in clinical use (e.g., spirometry, bronchomotoric tests, etc.). In some cases, invasive and *semi*invasive methods appear to be an inevitable option to gain the correct diagnosis (e.g., openlung biopsy and bronchoalveolar lavage) [21], yet it is to an unambiguous expense of the patient and often the health cost as well as a demanding laboratory examination.

Currently, a significant part of the relevant research centers focuses on methods of the socalled personalized diagnostics (or methods of personalized medicine), with the aim to stratify patients to characteristic groups (e.g., phenotypes) and thus achieve a more efficient therapy reflecting an individual phenotypic disposition (inclusive of genomic, proteomic and metabolomic profiles) [22, 23]. One of the examples of these endeavors (particularly for diagnostics of pulmonary diseases) is the measurement of a fractional exhaled nitric oxide (FeNO) [24–26] in EBC, helping to distinguish asthma from other pathogenetic processes diagnosed as chronic cough, gastroesophageal reflux disease (GERD), vocal cord dysfunction, bronchitis, chronic obstructive pulmonary disease (COPD), etc.

#### **4.2. Asthma phenotypes**

As asthma is a disease affecting millions of people of all ages worldwide, many criteria can be used for its classification. Nevertheless, the predominantly used criterion is the severity of the disease, as is presented in **Figure 3**, followed by the age of the first exacerbation [9, 26].

**5.1. Chronic bronchitis**

**5.2. Emphysema**

ing to the characteristic cough.

damage the pulmonary tissue [29].

**Table 1.** Asthma and COPD comparison.

**5.3. Asthma and COPD**

In chronic bronchitis [28], a typical symptom is a permanent constriction of bronchial tubes. Furthermore, an inhalation of harmful substances cause impairment of the respiratory mucous membrane, while a repeated damage to the membrane makes it thicker and lowers the tissue transparency. As a result, the affected cells increase the production of mucus, lead-

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Emphysema is characterized by a loss of the pulmonary tissue, while the respiratory ways are

The main cause of emphysema is smoking. The substances that are inhaled during smoking are led through the respiratory ways to bronchioles. In bronchioles, the substances provoke a local immune reaction, which is linked with the production of aggressive compounds via leucocytes (mainly free radicals responsible for oxidative stress). This reaction thus initiates a degradation of bronchioles. The afflicted bronchioles merge into huge lung sacs. These sacs have a smaller surface of the pulmonary tissue and thus the gas exchange between lungs and blood is limited. The second cause of this disease can be disequilibrium between proteases and their inhibitors—anti-proteases. Some COPD patients suffer from the lack of alfa-1-tripsin (an anti-protease), which is the reason for a higher number of proteases in the respiratory ways, which

Similar to asthma, COPD is a pulmonary disease and shares many similar symptoms (e.g., pulmonary obstruction, over-production of mucus, attacks of cough and dyspnoea, etc.).

Dyspnoea Rather abrupt, variable Often, rather permanent Pulmonary obstruction Mainly reversible Often irreversible Smoking Not very common 80% of cases Allergy Often (or parents) Rarely

Inflammation (can differ) Rather eosinophil Rather neutrophil Bronchial hyperreactivity Distinct Less common Glucocorticoid therapy Mainly efficient Rather inefficient Mortality (inhabitants per year (world)) 300 million (decreases) 600 million (increases)

**Parameter Asthma COPD** Age (origin of the disease) Childhood, anytime 40+ Development of the disease Abrupt attack Slower

abnormally widened distantly from terminal bronchioles [28].

**Figure 3.** Asthma phenotypes.
