**6. Pathophysiological coditions affecting RBC deformability**

Mechanical properties of RBCs is crucial for cell physiology of RBCs. This essential deformability is in turn affected by various physiological and pathological cues.

#### **6.1. Temperature**

Temperature plays important roles in RBC deformabilty. The elastic properties of RBC membrane were investigated as function of temperature using the micropipette aspiration

technique (Waugh and Evans,1979). Over the temperature range of 2-50C, both the shear modulus and the area expansion modulus decrease as temperature increased; the changes were -610-2 μN/mC and 6103 μN/mC, respectively. Due to the structual transitions of proteins occuring at certain critical tempertures, RBC deformabiltiy exhibits complex behaviors. At the transition temperature, RBCs undergo a sudden change from blocking to passing through a micropipette with a diameter of ~ 1 μm (Artmann, Kelemen et al.,1998). Body temperature or febril temperature are particularly important in various pathophysiology of RBCs. Membrane fluctuation measurements using DPM revealed that the shear modulus of *Pf-*RBCs significantly increases as temperature increases from body temperature to febrile temperature whereas healthy RBCs do not show noticible changes (Park, Diez-Silva et al., 2008). MTC study also reported that *Pf-*RBCs becomes significantly stiffened with temperature compared to the healthy RBCs (Marinkovic, Diez-Silva et al.,2009).

Measurement Techniques for Red Blood Cell Deformability: Recent Advances 183

Increased deformability of RBCs in abnormal shapes has been reported with various experimental methods. Ektacytometer measured increased DI values for SCs and ECs that were induced by 2,4-dinitrophenol treatment (Meiselman,1981). Recently, using DPM, the mechanical properteis of RBCs in different morphologies were quantified from dynamic membrane fluctuations (Park, Best et al.,2010). Bending modulus and area expansion modulus of ECs and SCs showed significantly high values compared to normal DCs. The shear moduli values show bimodal distributions (Fig. 11e), suggesting two independent conformations of the spectrin network: a soft configuration (*µ* ~ 7 *µ*N/m) and a stiff one (*µ* ~ 12 *µ*N/m). Aging of RBCs also cause significant morphological alterations: aged RBCs exhibit decreased surface area and volume (Waugh, Narla et al.,1992). The aged RBCs were found by ektacytometry to have decreases shear modulus mainly because of decreased

Different osmolalities of extracellular medium can bring significant changes in RBC shape and thus deformability. At normal physiological condition (295mOsm/kg), RBCs maintain their biconcave shapes. In hypotonic medium, RBCs are swollen due to water intake. At the osmotic pressure less than 100mOsm/kg, most of RBCs are lysed. In the hypertonic case, RBCs lose its volumes, which result in significant cell shrinkage. Although the total amount of Hb molecules in RBCs, or the mean corpuscular Hb (MCH), does not significantly change at different osmolality, Hb concentration can be considerably changed due to water influx and efflux. RBCs exhibit the maximum deformability at physiological condition; under either hypertonic or hypotonic condition, the deformability of RBCs decreases (Mohandas,

surface area and increased cytoplasmic viscosity.

**6.3. Osmotic pressure** 

Clark et al.,1980).

**DI (%)**

**0 100 200 300 400 500 <sup>0</sup>**

from (Park, Best et al.,2011).

**Osmolarity (mOsm/kg)**

**40**

**0 100 200 300 400 500 Osmolarity (mOsm/kg)**

A recent study, based on membrane fluctuation measurements, retrieved mechanical properties of RBC membrane under diffferent osmolarities (Park, Best et al.,2011). Although membrane fluctuation or deformability decreases either in hypotonic or hypertonic case; the

**Figure 12.** (a) DI of RBCs as a function osmolality, measured by ektacytometer. Modified, with permission, from (Mohandas, Clark et al.,1980). (b) Membrane fluctuations of RBCs as a function of osmotic pressure, measured by DPM. (c) Retrieved mechanical properties of RBCs from membrane fluctuations. 20 individual RBCs were measured at each osmotic pressure. Modified, with permission,

**0 200 400 600 800 <sup>0</sup>**

**Osmolarity (mOsm/kg)**

**KA**

**0**

**20**

**[mPa.s],** 

**KA [mNm-1]**

**40**

**60**

**5**

**[mN/m],** 

 **[xkBT]**

**10**

**15**

**50**

**Membrane fluctuation (nm)**

**(a) (b) (c)**

**60**

**70**

#### **6.2. Morphology**

RBCs exhibit diverse morphological features depending on pathophysiological conditions (Diez-Silva, Dao et al.,2010). A healthy human RBC shows a smooth and biconcave disc shape (discocyte). However, atypical shapes of RBCs can be found under abnormal pathophysiological conditions, including acanthocyte, stomatocyte, schizocyte, and tear drop cells (Kenneth,2010). Our understanding of what determines RBC morphology and how RBC morphologies are related to the mechanics of RBCs still remains incomplete. One of the hypotheses describing RBC morphology is the bilayer-couple hypothesis (Sheetz and Singer,1974); small changes in the relaxed area difference between two layers of phospholipids. Later, this model can be used for explaning stomatocyte–discocyte– echinocyte morphological transitions (Lim HW, Wortis et al.,2002).

**Figure 11.** (a-c) Topographies of (a) discocyte, (b) echinocyte, and (c) spherocyte. (d-f) Retrieved mechanical properties: (d) bending modulus , (e) shear modulus *µ*, and (f) area modulus *KA* of discocytes (DCs), ATP-depleted discocytes [DCs (-ATP)], echinocytes (ECs), and spherocytes (SCs). Reproduced, with permission, from (Park, Best et al.,2010)

Increased deformability of RBCs in abnormal shapes has been reported with various experimental methods. Ektacytometer measured increased DI values for SCs and ECs that were induced by 2,4-dinitrophenol treatment (Meiselman,1981). Recently, using DPM, the mechanical properteis of RBCs in different morphologies were quantified from dynamic membrane fluctuations (Park, Best et al.,2010). Bending modulus and area expansion modulus of ECs and SCs showed significantly high values compared to normal DCs. The shear moduli values show bimodal distributions (Fig. 11e), suggesting two independent conformations of the spectrin network: a soft configuration (*µ* ~ 7 *µ*N/m) and a stiff one (*µ* ~ 12 *µ*N/m). Aging of RBCs also cause significant morphological alterations: aged RBCs exhibit decreased surface area and volume (Waugh, Narla et al.,1992). The aged RBCs were found by ektacytometry to have decreases shear modulus mainly because of decreased surface area and increased cytoplasmic viscosity.

#### **6.3. Osmotic pressure**

182 Blood Cell – An Overview of Studies in Hematology

**6.2. Morphology** 

technique (Waugh and Evans,1979). Over the temperature range of 2-50C, both the shear modulus and the area expansion modulus decrease as temperature increased; the changes were -610-2 μN/mC and 6103 μN/mC, respectively. Due to the structual transitions of proteins occuring at certain critical tempertures, RBC deformabiltiy exhibits complex behaviors. At the transition temperature, RBCs undergo a sudden change from blocking to passing through a micropipette with a diameter of ~ 1 μm (Artmann, Kelemen et al.,1998). Body temperature or febril temperature are particularly important in various pathophysiology of RBCs. Membrane fluctuation measurements using DPM revealed that the shear modulus of *Pf-*RBCs significantly increases as temperature increases from body temperature to febrile temperature whereas healthy RBCs do not show noticible changes (Park, Diez-Silva et al., 2008). MTC study also reported that *Pf-*RBCs becomes significantly stiffened with temperature

RBCs exhibit diverse morphological features depending on pathophysiological conditions (Diez-Silva, Dao et al.,2010). A healthy human RBC shows a smooth and biconcave disc shape (discocyte). However, atypical shapes of RBCs can be found under abnormal pathophysiological conditions, including acanthocyte, stomatocyte, schizocyte, and tear drop cells (Kenneth,2010). Our understanding of what determines RBC morphology and how RBC morphologies are related to the mechanics of RBCs still remains incomplete. One of the hypotheses describing RBC morphology is the bilayer-couple hypothesis (Sheetz and Singer,1974); small changes in the relaxed area difference between two layers of phospholipids. Later, this model can be used for explaning stomatocyte–discocyte–

compared to the healthy RBCs (Marinkovic, Diez-Silva et al.,2009).

echinocyte morphological transitions (Lim HW, Wortis et al.,2002).

**(a) (c)**

**(b)**

**(d) (e) (f)**

mechanical properties: (d) bending modulus

Reproduced, with permission, from (Park, Best et al.,2010)

**Figure 11.** (a-c) Topographies of (a) discocyte, (b) echinocyte, and (c) spherocyte. (d-f) Retrieved

discocytes (DCs), ATP-depleted discocytes [DCs (-ATP)], echinocytes (ECs), and spherocytes (SCs).

, (e) shear modulus *µ*, and (f) area modulus *KA* of

Different osmolalities of extracellular medium can bring significant changes in RBC shape and thus deformability. At normal physiological condition (295mOsm/kg), RBCs maintain their biconcave shapes. In hypotonic medium, RBCs are swollen due to water intake. At the osmotic pressure less than 100mOsm/kg, most of RBCs are lysed. In the hypertonic case, RBCs lose its volumes, which result in significant cell shrinkage. Although the total amount of Hb molecules in RBCs, or the mean corpuscular Hb (MCH), does not significantly change at different osmolality, Hb concentration can be considerably changed due to water influx and efflux. RBCs exhibit the maximum deformability at physiological condition; under either hypertonic or hypotonic condition, the deformability of RBCs decreases (Mohandas, Clark et al.,1980).

**Figure 12.** (a) DI of RBCs as a function osmolality, measured by ektacytometer. Modified, with permission, from (Mohandas, Clark et al.,1980). (b) Membrane fluctuations of RBCs as a function of osmotic pressure, measured by DPM. (c) Retrieved mechanical properties of RBCs from membrane fluctuations. 20 individual RBCs were measured at each osmotic pressure. Modified, with permission, from (Park, Best et al.,2011).

A recent study, based on membrane fluctuation measurements, retrieved mechanical properties of RBC membrane under diffferent osmolarities (Park, Best et al.,2011). Although membrane fluctuation or deformability decreases either in hypotonic or hypertonic case; the

reasons for the decreased deformability are different. Under hypotonic cases, both shear moduli and area expansion moduli increase, suggesting nonlinear stiffening in streached membrane structure. Under hypertonics cases, other mechanical parameters are not significantly changed except that cytoplasmic viscosity increases.

Measurement Techniques for Red Blood Cell Deformability: Recent Advances 185

Healthy RBC

*Pf-*RBC (Schizont stage)

**(b)**

**sporozites**

**Schizont**

**Liver**

**Ring**

**gamatocyte**

**6.6. Genetic diseases: sickle cell disease** 

**sexual cycle**

**gamates Ookinete Oocyst**

**Inside the mosquito**

**Salivary gland**

**Hemozoin**

**Trophozoite**

**Parasitophorus vacuoles** 

tweezers. Reproduced, with permission, from (Suresh, Spatz et al.,2005).

conditions and the deformabiltiy of sickle RBCs significantly decreases.

**Inside the liver**

**merozoite**

**Inside Red Blood Cells**

**Figure 13.** Malaria parasite life cycle in human body. Reproduced, with permission, from (Cho, Kim et al.,2011). (b) Optical images of a healthy RBC and a *Pf*-RBC (schizont stage) stretched by optical

Sickle cell disease, characterized by abnormal rheological properties and a sickle-shape of RBCs, is an autosomal recessive inherited blood disorder. A point mutation in β-globin gene encoding Hb results in the production of sickle Hb (HbS) instead of normal Hb (HbA) (Barabino, Platt et al.,2010). Under deoxygenated conditions, HbS molecules becomes selfassembled and grows to fibers inside RBCs up to a few micrometer lengths. Due to these highly stiff HbS fibers, sickle RBCs have elongated- and crescent-shape at deoxygenated

Sickle RBCs have different morphologies depending on its density (Kaul, Fabry et al.,1983; Evans, Mohandas et al.,1984). After repeated sicklings, a fraction of RBCs becomes irreversibly sickled cells and they exhibit the most significant loss in deformability. While Hb concentrations does not affect static rigidity of normal RBCs, static rigidity of sickle RBCs depends on Hb concentration (Evans, Mohandas et al.,1984). Earlier studies using ektacytometry and filteration techniques reported decreased deformability of sickle RBCs

even under oxygenated conditions (Chien, Usami et al.,1970; Klug, Lessin et al.,1974).

deformability compared to healthy RBCs (Maciaszek and Lykotrafitis,2011).

Quantitative phase microscopy measured decreased membrane fluctuations for sickle RBCs (Shaked, Satterwhite et al.,2011). FTLS showed significantly altered elastic and viscous membrane properties in sickle RBCs (Kim, Higgins et al.,2012). Recently, four important mechanical properties of sickle RBCs were retrieved with memebrane fluctuations measurements (Byun, Higgins et al.,under review). Using AFM technique, decresed deformability was measured in sickle RBCs (Maciaszek, Andemariam et al.,2011). RBCs in sickle cell trait, having only one abnormal allele of the Hb beta gene, also exhibit decreased

**(a)**

#### **6.4. ATP effect**

The presence of adenosine 5'-triphosphate (ATP) is important in maintaining the biconcave shape of RBCs and also significantly affects the RBC deformability. In the absence of ATP, RBCs loss its biconcave shapes and become flattened echinocytes (Sheetz and Singer,1977). The metabolic state of RBCs, determined by the level of ATP, is crucial for maintaining cellular deformability. When celullar ATP level decreases, the stored RBCs significantly lose the deformability (Weed, LaCelle et al.,1969). Micropipette aspiration technqiue measured mechanical properties of RBCs upon ATP depletion; shear modulus and elastic area compression modulus increase by 17% and 14%, respectively (Meiselman, Evans et al.,1978). Decreased membrane fluctuation in the absence of ATP was first observed by using darkfield microscopy (Tuvia, Levin et al.,1998). Membrane fluctuation measurements studied the effects of ATP to the mechanical properties of RBCs (Betz, Lenz et al.,2009; Park, Best et al.,2010). Analysis on dynamic membrane fluctutions further showed non-Gaussian dynamics in the presence of ATP, suggesting the metabolic remodelling in the lipid membrane and spectrin network structure (Park, Best et al.,2010). ATP-dependent RBC deformability has been also studied using theoretical models (Gov and Safran,2005; Ben-Isaac, Park et al.,2011).

## **6.5. Malaria: Parasite invasion**

Pathogenesis of malaria causes structural and mechanical modifications to the host RBCs. During intra-erythrocytic development, the malaria-inducing parasite exports proteins that interact with the host cell membrane and spectrin cytoskeletal network (Simmons, Woollett et al.,1987). Parasite-exported proteins modify material properties of host RBCs, resulting in altered cell circulation. Despite the genetic and biochemical approaches identified, proteins exported by parasites have remained elusive as well as the mechanism and effect of these proteins on the host cells.

*Pf*-RBCs exhibits significantly decreased deformability. Microfluidic technique demonstrated the occlusion of small channels by infected RBCs (Shelby, White et al.,2003). Optical tweezers technique measured that membrane shear modulus continuously increases as the disease progesses during the intraerythrocytic cycle (Suresh, Spatz et al.,2005). Employing genetic knock-out assay, the effects of RESA protein to the host RBC deformabiltiy has been studied (Mills, Diez-Silva et al.,2007). Membrane fluctuation measurement also showed increased shear modulus of malaria-invaded RBCs (Park, Diez-Silva et al.,2008). Recently, the loss of deformability in the malaria-invaded RBCs has been simulated using multiscale numerical models (Fedosov, Lei et al.,2011).

**Figure 13.** Malaria parasite life cycle in human body. Reproduced, with permission, from (Cho, Kim et al.,2011). (b) Optical images of a healthy RBC and a *Pf*-RBC (schizont stage) stretched by optical tweezers. Reproduced, with permission, from (Suresh, Spatz et al.,2005).

#### **6.6. Genetic diseases: sickle cell disease**

184 Blood Cell – An Overview of Studies in Hematology

**6.4. ATP effect** 

Isaac, Park et al.,2011).

proteins on the host cells.

numerical models (Fedosov, Lei et al.,2011).

**6.5. Malaria: Parasite invasion** 

reasons for the decreased deformability are different. Under hypotonic cases, both shear moduli and area expansion moduli increase, suggesting nonlinear stiffening in streached membrane structure. Under hypertonics cases, other mechanical parameters are not

The presence of adenosine 5'-triphosphate (ATP) is important in maintaining the biconcave shape of RBCs and also significantly affects the RBC deformability. In the absence of ATP, RBCs loss its biconcave shapes and become flattened echinocytes (Sheetz and Singer,1977). The metabolic state of RBCs, determined by the level of ATP, is crucial for maintaining cellular deformability. When celullar ATP level decreases, the stored RBCs significantly lose the deformability (Weed, LaCelle et al.,1969). Micropipette aspiration technqiue measured mechanical properties of RBCs upon ATP depletion; shear modulus and elastic area compression modulus increase by 17% and 14%, respectively (Meiselman, Evans et al.,1978). Decreased membrane fluctuation in the absence of ATP was first observed by using darkfield microscopy (Tuvia, Levin et al.,1998). Membrane fluctuation measurements studied the effects of ATP to the mechanical properties of RBCs (Betz, Lenz et al.,2009; Park, Best et al.,2010). Analysis on dynamic membrane fluctutions further showed non-Gaussian dynamics in the presence of ATP, suggesting the metabolic remodelling in the lipid membrane and spectrin network structure (Park, Best et al.,2010). ATP-dependent RBC deformability has been also studied using theoretical models (Gov and Safran,2005; Ben-

Pathogenesis of malaria causes structural and mechanical modifications to the host RBCs. During intra-erythrocytic development, the malaria-inducing parasite exports proteins that interact with the host cell membrane and spectrin cytoskeletal network (Simmons, Woollett et al.,1987). Parasite-exported proteins modify material properties of host RBCs, resulting in altered cell circulation. Despite the genetic and biochemical approaches identified, proteins exported by parasites have remained elusive as well as the mechanism and effect of these

*Pf*-RBCs exhibits significantly decreased deformability. Microfluidic technique demonstrated the occlusion of small channels by infected RBCs (Shelby, White et al.,2003). Optical tweezers technique measured that membrane shear modulus continuously increases as the disease progesses during the intraerythrocytic cycle (Suresh, Spatz et al.,2005). Employing genetic knock-out assay, the effects of RESA protein to the host RBC deformabiltiy has been studied (Mills, Diez-Silva et al.,2007). Membrane fluctuation measurement also showed increased shear modulus of malaria-invaded RBCs (Park, Diez-Silva et al.,2008). Recently, the loss of deformability in the malaria-invaded RBCs has been simulated using multiscale

significantly changed except that cytoplasmic viscosity increases.

Sickle cell disease, characterized by abnormal rheological properties and a sickle-shape of RBCs, is an autosomal recessive inherited blood disorder. A point mutation in β-globin gene encoding Hb results in the production of sickle Hb (HbS) instead of normal Hb (HbA) (Barabino, Platt et al.,2010). Under deoxygenated conditions, HbS molecules becomes selfassembled and grows to fibers inside RBCs up to a few micrometer lengths. Due to these highly stiff HbS fibers, sickle RBCs have elongated- and crescent-shape at deoxygenated conditions and the deformabiltiy of sickle RBCs significantly decreases.

Sickle RBCs have different morphologies depending on its density (Kaul, Fabry et al.,1983; Evans, Mohandas et al.,1984). After repeated sicklings, a fraction of RBCs becomes irreversibly sickled cells and they exhibit the most significant loss in deformability. While Hb concentrations does not affect static rigidity of normal RBCs, static rigidity of sickle RBCs depends on Hb concentration (Evans, Mohandas et al.,1984). Earlier studies using ektacytometry and filteration techniques reported decreased deformability of sickle RBCs even under oxygenated conditions (Chien, Usami et al.,1970; Klug, Lessin et al.,1974).

Quantitative phase microscopy measured decreased membrane fluctuations for sickle RBCs (Shaked, Satterwhite et al.,2011). FTLS showed significantly altered elastic and viscous membrane properties in sickle RBCs (Kim, Higgins et al.,2012). Recently, four important mechanical properties of sickle RBCs were retrieved with memebrane fluctuations measurements (Byun, Higgins et al.,under review). Using AFM technique, decresed deformability was measured in sickle RBCs (Maciaszek, Andemariam et al.,2011). RBCs in sickle cell trait, having only one abnormal allele of the Hb beta gene, also exhibit decreased deformability compared to healthy RBCs (Maciaszek and Lykotrafitis,2011).

Measurement Techniques for Red Blood Cell Deformability: Recent Advances 187

membrane shape and deformability. In addition, it will be possible to study RBC deformability in vivo in the near future, by directly imaging and manipulating RBCs through highly scattering skin tissues. Recent works have demonstrated that it is indeed possible to control and suppress multiple light scattering (Vellekoop, Lagendijk et al.,2010; Vellekoop and Aegerter,2010; Mosk, Lagendijk et al.,2012; Park, Park et al.,2012; Park, Park et al.,2012). In diabetes mellitus, RBCs exhibit reduced deformability (McMillan, Utterback et al.,1978), which has been attributed to the changes in lipid composition of the membranes. This impaired RBC deformability in diabetes occurs before significant histological vascular changes (Diamantopoulos, Kittas et al.,2004). RBCs from the patients with diabetes mellitus undergoes substantial alterations in the lipid composition, membrane proteins, and Hb molecules. Saturated fatty acid levels in diabetes mellitus were significantly elevated compared to normal RBCs while the amount of polyunsaturated fatty acids were decreased

We have highlighted techniques for studying RBC deformabilty. Due to various deformability test techniques developed in the last years, our understandings on pathophysiology of RBCs have been significantly improved. Recent advances have enabled the precise measurements of various biomechanical properties of RBCs under systemically controlled conditions that mimic complex *in vivo* physiological environments. However, three major technical issues should be resolved in order to bring a much significant impact. First, the molecular mechanisms on RBC deformability should be directly accessed and studied. Employing biochemical assays such as molecular imaging and genetic knock-out methods, the relation between molecule-level changes and cellular-level deformability alterations can be studied. Second, such measurements should be performed at individual cell levels. Profiling mechanical, chemical, and biological properties at the cellular levels and their correlations may allow accessing to unexplored regimes of diseases mechanisms. Third, interactions between cell-to-protein, cell-to-cell, and cell-to-vessel should be considered, since these interactions can be affected and *in turn* modify RBC deformability. As more knowledge is gained about the pathophysiology of RBCs and their circulation through biomechanical studies, the potential for the development of novel diagnostic and treatment strategies for various RBC-related disease will become real and answer to

*Department of Physics, Korea Advanced Institute of Science and Technology, Daejeon, South Korea* 

The authors wish to acknowledge supports from KAIST, KAIST Institute for Optical Science and Technology, Korean Ministry of Education, Science and Technology (MEST) grant No.

in diabetes (Prisco, Paniccia et al.,1989).

**7. Conclusion and outlook** 

important questions in hematology.

Youngchan Kim, Kyoohyun Kim and YongKeun Park

**Author details** 

**Acknowledgement** 

**Figure 14.** (a) Illustration showing structural modifications inside a sickle RBC. Modified, with permission, from (Barabino, Platt et al.,2010). (b-d) Typical morphologies of sickle RBCs measured by DPM; (b) echinocyte, (c) discocyte, and (d) crescent-shaped irreversibly sickled cell. Reproduced, with permission, from (Kim, Higgins et al.,2012).

#### **6.7. Other conditions altering RBC deformability**

There are still many pathophysiological conditions that affect the deformabiltiy of RBCs, which are not covered in the above sections. Several hereditary disorders associated with formation of RBC membrane structures and Hb protein can result into altered RBC deformability. Thalassemias, causing the formation of abnormal Hb molecules due to the limited synthesis of the globin chain, results into loss of RBC deformability. Thalassemia is thus often accompanied by the destruction of a large number of RBCs in spleen, accompanying with the enlargement of spleen. In addition, abnormal Hb molecules in thalassemia often caues the formation of Heinz bodies, inclusions within RBCs composed of denatured Hb, and it causes the local rigidification of RBC membrane (Reinhart, Sung et al., 1986). Ektacytometer study measured that RBCs in hereditary spherocytosis showed markedly diminished deformability while their surface/volume ratio was normal (Nakashima and Beutler,1979). RBCs from the patients with homozygous hereditary elliptocytosis exhibits marked abnormalities in deformability and membrane fragility; these changes are closely related to the reduced levels of band 4.1 proteins (Tchernia, Mohandas et al.,1981). Since band 4.1 plays an important role in the modulation of spectrin-actin interaction, it has been suggested to be closely related to the maintenance of normal membrane shape and deformability. In addition, it will be possible to study RBC deformability in vivo in the near future, by directly imaging and manipulating RBCs through highly scattering skin tissues. Recent works have demonstrated that it is indeed possible to control and suppress multiple light scattering (Vellekoop, Lagendijk et al.,2010; Vellekoop and Aegerter,2010; Mosk, Lagendijk et al.,2012; Park, Park et al.,2012; Park, Park et al.,2012). In diabetes mellitus, RBCs exhibit reduced deformability (McMillan, Utterback et al.,1978), which has been attributed to the changes in lipid composition of the membranes. This impaired RBC deformability in diabetes occurs before significant histological vascular changes (Diamantopoulos, Kittas et al.,2004). RBCs from the patients with diabetes mellitus undergoes substantial alterations in the lipid composition, membrane proteins, and Hb molecules. Saturated fatty acid levels in diabetes mellitus were significantly elevated compared to normal RBCs while the amount of polyunsaturated fatty acids were decreased in diabetes (Prisco, Paniccia et al.,1989).
