YBCMD-01772; No of Pages 6 Blood Cells, Molecules and Diseases xxx (2013) xxx–xxx
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New insights provided by a comparison of impaired deformability with erythrocyte oxidative stress for sickle cell disease Viachaslau M. Barodka a,1, Enika Nagababu c,1, Joy G. Mohanty c, Daniel Nyhan a, Dan E. Berkowitz a, Joseph M. Rifkind c,⁎, John J. Strouse b a b c
Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, United States Molecular Dynamics Section, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States
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Article history: Submitted 30 July 2013 Revised 10 October 2013 Available online xxxx (Communicated by Narla) Keywords: Sickle cell disease Erythrocytes Hemoglobin Heme degradation Fluorescence Deformability
a b s t r a c t Sickle cell disease (SCD) is associated with increase in oxidative stress and irreversible membrane changes that originates from the instability and polymerization of deoxygenated hemoglobin S (HbS). The relationship between erythrocyte membrane changes as assessed by a decrease in deformability and oxidative stress as assessed by an increase in heme degradation was investigated. The erythrocyte deformability and heme degradation for 27 subjects with SCD and 7 with sickle trait were compared with normal healthy adults. Changes in both deformability and heme degradation increased in the order of control to trait to non-crisis SCD to crisis SCD resulting in a very significantly negative correlation between deformability and heme degradation. However, a quantitative analysis of the changes in deformability and heme degradation for these different groups of subjects indicated that sickle trait had a much smaller effect on deformability than on heme degradation, while crisis affects deformability to a greater extent than heme degradation. These findings provide insights into the relative contributions of erythrocyte oxidative stress and membrane damage during the progression of SCD providing a better understanding of the pathophysiology of SCD. Published by Elsevier Inc.
Introduction Erythrocytes are under continuous stress by oxygenation and deoxygenation cycles, strong shearing forces in narrow blood vessels and reactive oxygen species (ROS) generated both endogenously and exogenously during their 120 day life span. An increase in erythrocyte oxidative stress as assessed by measuring of membrane lipid peroxidation has been reported with erythrocyte disorders [15,26,38] and various other diseases including renal failure [10], Alzheimer's disease [3], and autoimmune thrombocytopenic purpura [33], as well as during cellular aging [32]. The endogenous reactive oxygen species (ROS) are generated by autoxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb) and superoxide, which is converted to hydrogen peroxide [14]. We have previously demonstrated that a fraction of the hydrogen peroxide that is not scavenged by the erythrocyte antioxidant defense system damages the heme moiety of hemoglobin producing fluorescent degradation products. These degradation products are relatively stable being retained in the membrane and only slowly released into the plasma. ⁎ Corresponding author at: Molecular Dynamics Section, National Institute on Aging, 251 Bayview Blvd., Baltimore, MD 21224, United States. Fax: +1 410 558 8397. E-mail address: [email protected] (J.M. Rifkind). 1 Equally contributing authors.
Therefore, a gradual increase in fluorescent degradation products occurs whenever the cell is exposed to oxidative stress and the determination of fluorescent heme degradation products in the erythrocyte provides an in vivo measure of erythrocyte oxidative stress [25–28]. In sickle cell disease (SCD), autoxidation and the associated production of endogenous ROS are augmented by the instability of sickle hemoglobin (HbS) [15]. We have recently confirmed the predicted increase in heme degradation for HbS using transgenic mice that produce Hb S [26]. In that study, erythrocytes from sickle cell transgenic mice had an average 5.6 fold increase in heme degradation relative to controls. The ability of erythrocytes to deform is critical to microvascular perfusion and oxygen delivery [21], with impaired deformability contributing to the severe vascular and end-organ pathology of SCD [23]. Membrane lipid and protein damage generated by oxidative stress contribute to impaired deformability. The resultant altered cytoskeletal network induces exposure of phosphatidylserine and altered cation homeostasis with increased intracellular calcium (Ca2+) levels due to both increased membrane permeability to Ca2+ and inhibition of CaATPase, which actively pumps Ca2+ out of the cell [11]. The increased concentration of intracellular Ca2+ activates the KCa3.1 channel, which permits potassium ions to escape followed by chloride (Cl−) and water (H2O) [8]. These processes produce dehydration and cell shrinkage, which result in impaired deformability [37].
1079-9796/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.bcmd.2013.10.004
Please cite this article as: V.M. Barodka, et al., Blood Cells Mol. Diseases (2013), http://dx.doi.org/10.1016/j.bcmd.2013.10.004
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In addition to increased oxidative stress associated with HbS, deformability is affected by a number of other factors. For sickle cell disease an important factor contributing to the impaired deformability is the oxygenation/deoxygenation cycles occurring in the circulation that result in cyclic aggregation/polymerization of deoxygenated HbS [16]. This process, which is exacerbated at higher concentrations of HbS produced by cell shrinkage [18], results in irreversible changes to the membrane and eventual sickling, which further impair erythrocyte deformability. This decrease in erythrocyte deformability becomes more profound 1 to 3 days before the development of painful vasoocclusive crisis and then reverses, gradually increasing to greater than steady-state levels by day 9. Oxidative stress in SCD also contributes to anemia both by producing rigid cells that are removed by the reticuloendothelial system (if still functional) [23], and the exposure of phosphatidylserine on the outer surface of the erythrocyte, which triggers the removal of cells by macrophages [13]. Anemia, which increases hypoxia, has also been shown to increase oxidative stress [16,27]. To evaluate the relative contributions of oxidative stress and hemoglobin polymerization to erythrocyte injury, we have measured both heme degradation, which is a direct measure of erythrocyte oxidative stress, and deformability, which is affected by both oxidative stress and hemoglobin polymerization.
as (L − W)/(L + W), where L and W are the major and minor axes of the ellipse. We determined EI at a range of shear stress values (0 to 20 Pa) and used the value at 3 Pa for comparative analysis of the deformability values of samples treated under different conditions, as suggested by Baskurt et. al.[7]. We also used values at 20 Pa to measure changes in the least deformable cells, which reflect the altered membrane structure of these cells. Measurement of heme degradation products
Design and methods
Heme degradation products were measured as previously described [26]. In brief, blood was collected into EDTA vacutainers and centrifuged at 3000 RPM for 10 min at 2–4 °C. The plasma was removed and 20 μl of unwashed erythrocytes were lysed in 3 ml deionized distilled water. The hemoglobin spectrum of the hemolysate was recorded from 490 to 640 nm using a PerkinElmer lambda 35 spectrophotometer. The concentrations of oxyHb and metHb were determined by a least square-fitting program using known concentrations of parent spectra of oxyHb and metHb. The total Hb concentration of the hemolysate was then adjusted to 50 μM by diluting with distilled water. The fluorescence emission spectrum was measured from 400 nm to 600 nm at an excitation wavelength of 321 nm using a PerkinElmer LS50B spectrofluorimeter. The fluorescence intensity of emission maximum at 485 nm was used as a measure of heme degradation. The excitation and emission slit widths were kept at 10 nm.
Study population
Measurement of total hemoglobin, HbS and fetal hemoglobin
We enrolled participants with sickle cell disease and sickle cell trait (HbAS) N4 years of age from outpatient clinics at Johns Hopkins Hospital. Exclusion criteria included pregnancy, weight b10 kg, transfusion in the last 2 months, and, for patients on hydroxyurea, a change in dose in the last 2 months or known poor adherence to treatment. Participants in steady state had not reported pain consistent with sickle cell crisis for at least 2 weeks. Participants during the beginning of acute painful episodes receiving parenteral opiates for acute sickle cell pain were classified as having vaso-occlusive crisis. The study was approved by the Johns Hopkins Medicine Institutional Review Board. Blood samples from normal healthy subjects without SCD were obtained at the National Institute of Aging (NIA), National Institutes of Health (NIH) through IRB approved protocol (2003-071) for tissue procurement for biomedical research.
Complete blood and reticulocyte counts were performed using an automated analyzer (Sysmex XE-2100, Roche, Indianapolis, IN) and proportion of hemoglobins by high performance liquid chromatography (Biorad Variant Hemoglobin Testing System, Biorad Laboratories, Hercules, CA) respectively. The proportion of erythrocytes and reticulocytes containing Hemoglobin F (HbF) was measured by flow cytometry [24] (Table 1).
Sample preparation Up to 6 ml of venous blood anticoagulated with K2EDTA was placed on ice and then kept at 4 °C till measurements were performed. The tests for deformability and heme degradation were done in parallel, blinded to the diagnosis of the patient and within 24 h of blood collection. Measurement of erythrocyte deformability Erythrocyte deformability was measured using a microfluidic RheoScan-D slit-flow ektacytometer (Rheo Meditech, Seoul, South Korea) [36]. Briefly, this instrument consists of a diode laser, a CCD camera, a pressure sensor, a vacuum generating mechanism and a sample holder containing a disposable microfluidic chip. For erythrocyte deformability measurement, cells were suspended (final hct ~ 0.5%) by slowly mixing them in the highly-viscous PVP360 solution (viscosity ~ 30 cP) and loaded onto the sample reservoir of the chip (Rheo Meditech). The erythrocytes are pulled through the microchannel at a range of shear stresses, while the elliptical diffraction patterns are projected on a screen by a laser, captured by a CCD-video camera, and analyzed by ellipse-fitting-software. Deformability of erythrocytes is expressed as Elongation Index (EI), which is defined
Results Comparison of levels of total Hb, HbS and HbF for SCD We enrolled 27 participants with sickle cell disease (21 participants with sickle cell anemia (HbSS), 3 with Hemoglobin SC disease (HbSC), 2 with HbS β-thalassemia plus and 1 with HbS Osler) and 7 with sickle cell trait (Table 1). To evaluate the anemia associated with SCD, we measured the total hemoglobin for each group. Anemia for SCD is indicated by the reduced level of total hemoglobin relative to that of sickle trait (Fig. 1A), which is similar to that of controls (data not shown). Samples obtained toward the beginning of acute painful episodes (in crisis) do not demonstrate a further reduction in total hemoglobin. Heterozygotes with sickle cell trait have an average of 38% HbS, less than those with SCD. Crisis, however, does not result in a significant increase in HbS (Fig. 1B). The subjects with SCD that did not have HbSS were intermediate between those with sickle trait and HbSS, both with respect to the HbS level and the total Hb level (Table 1). Table 1 Clinical characteristics of participants. Variable
HbSS on HU
HbSS no HU
Other SCD
Sickle cell trait
Number Age (years) Male sex Total Hb (g/dl) Reticulocytes (%) Hb F (%) Hb S (*5) Pain crisis
13 27 ± 11 46% 8.4 ± 1.2 8.4 ± 5.4 16 ± 7.1 77 ± 8 31%
8 18 ± 11 50% 8.4 ± 1.0 12.6 ± 1.9 9.2 ± 5.1 83 ± 5 37%
6 28 ± 13 50% 10.7 3.4 ± 1.4 2.8 50% 67%
7 39 ± 5 14% 12.7 1.1 0.4 0.8 38% 0%
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Fig. 1. (A) Total hemoglobin in erythrocytes of participants with sickle cell crisis, steady-state, and trait. (B) Percent HbS for participants with sickle cell crisis, steady-state, and trait. (C) Difference in the percent HbF for participants with sickle cell disease undergoing crisis.
Elevated levels of HbF are frequently found in patients with SCD and are increased by treatment with hydroxyurea. The presence of HbF has also been shown to inhibit the polymerization of deoxygenated HbS. Consistent with the protective effects of HbF, we find reduced levels of HbF in samples obtained during crisis in the beginning of acute painful episodes (Fig. 1C). This likely reflects the higher frequency of crisis in patients with SCD and low HbF. Erythrocytes deformability and sickle cell disease Fig. 2A shows the erythrocyte deformability for normal healthy controls and those with sickle trait and SCD during steady state and crisis at 3 Pa, which reflects changes in the average deformability. The sensitivity of the deformability measurement is indicated by a significant (p b 0.001) 11% decrease in deformability in those with sickle trait. For SCD, an additional significant (p b 0.0001) decrease in deformability was found, which depended on the severity of disease resulting in about a 50% decrease in the EI for those in crisis relative to control subjects. Fig. 2B shows the deformability for the same subjects at a 20 Pa. The decrease in deformability at 20 Pa indicates structural changes in the membrane of sickle cell subjects. The beneficial effects of fetal hemoglobin have been attributed to an inhibition of the aggregation of sickle hemoglobin that triggers sickling [17]. The significant correlation between deformability and HbF (Fig. 2B) is consistent with a contribution of hemoglobin aggregation, and its effect on the membrane, to deformability. Thirteen of the 21 participants with HbSS were treated with hydroxyurea. On average they had a higher percentage of HbF (16 ± 7.1 vs. 9.2 ± 5.1) than
those with HbSS not treated with hydroxyurea. As indicated in Fig. 2B treatment with hydroxyurea does not have a significant effect on the observed relationship between HbF and deformability. Heme degradation and sickle cell disease Fluorescent heme degradation products are measures of oxidative stress originating in the erythrocyte [28,31]. Fig. 3 shows that the fluorescent heme degradation products significantly (p b 0.0001) increased relative to the control by a factor of 1.7, 2.1 and 2.5 fold for sickle trait, SCD in steady state and SCD in crisis, respectively. When we evaluated the correlation between HbF and heme degradation we found a decrease in heme degradation with increasing HbF consistent with a protective effect of HbF. However unlike the very significant effect found for deformability this correlation was not significant (data not shown). Comparison of deformability and heme degradation The contribution of oxidative stress to deformability is indicated by the significant negative correlation (R = 0.68; p b 0.001) between heme degradation and deformability (Fig. 4A). Although this plot indicates that these 2 parameters are highly correlated, the results with HbF (highly correlated with deformability but not heme degradation) indicate that the aggregation process affects deformability to a greater extent than heme degradation. At the same time a comparison of Figs. 2A and 3 indicate a proportionally greater increase in heme degradation than decrease in deformability when comparing sickle cell trait
Fig. 2. (A&B) Deformability of erythrocytes of control, sickle trait, and sickle cell disease participants. Elongation index (E.I.) as a measure of deformability was measured as described in the methods section. Values are mean ± standard deviation, N control = 50, Trait = 7, steady state = 16, crisis = 11. Significance relative to control; ⁎p b 0.001; ⁎⁎p b 0.0001. (C) Correlation of deformability at 3 Pa with fetal hemoglobin percentage for SCD subjects. (▲), participants on hydroxyurea (R = 0.509). (■), participants not receiving hydroxyurea (R = 0.541).
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Fig. 3. Heme degradation in control and erythrocytes from participants with SCD and trait. Heme degradation was determined as mentioned in methods section. Values are given as arbitrary fluorescent units (F.A.U.) mean ± standard error. N, crisis 11, steady state = 16, trait = 7 and control = 50. Significance relative to control; ⁎p b 0.0001.
with controls. The dominant effect of sickle cell trait on heme degradation can be attributed to the increase in autoxidation of HbS, which causes oxidative stress. While this process determines heme degradation, deformability is affected by both oxidative stress and hemoglobin aggregation, which is most pronounced during acute vaso-occlusive episodes (crisis). The different contribution of oxidative stress and hemoglobin aggregation during SCD is demonstrated in Fig. 4B where we plotted the percent changes in heme degradation and deformability for the four different groups (control, trait, non-crisis and crisis). Heme degradation, which is a direct measure of the ROS generated by autoxidation of hemoglobin is increased for trait because of the significant proportion (typically ~40%) of the less stable HbS. Deformability, which is affected by hemoglobin aggregation/polymerization in addition to any effects of oxidative stress will be modified to a greater extent by the changes that take place during acute vaso-occlusive episodes. Discussion SCD is caused by a substitution of valine for glutamic acid at the β-6 position in the hemoglobin β-chain [9]. The valine to glutamic acid modification of HbS facilitates aggregation/polymerization of hemoglobin in
the deoxygenated state and can cause vascular occlusion that dramatically affects oxygen delivery [9]. Erythrocyte deformability is essential to pass through narrow capillaries required for oxygen delivery, and is expected to contribute to the impaired microcirculatory flow of SCD [2,4–6]. Before, during, and after crisis show a decrease in deformability 1–3 days before the onset of pain. This deformability further decreases in the beginning of crisis, but increases later during crisis as the pain subsides. The improved deformability has been attributed to the removal of the poorly dense deformable cells and replacement by young deformable cells. Consistent with these studies, we have observed a significant decrease in deformability when comparing steady state subjects who have not experienced any painful episodes for at least 2 weeks with patients in the initial stage of painful crisis. Erythrocyte deformability is affected by various factors including cytoplasmic viscosity, cell shrinkage and membrane properties. The decreased deformability of erythrocytes in SCD is in part associated with aggregation/polymerization of HbS, which eventually leads to morphological sickling that affects the ability of the membrane to deform [18,19,22,23]. An additional factor that can affect deformability in SCD is the increased oxidative stress associated with the instability of HbS. This instability results in a higher rate of autoxidation for HbS than normal hemoglobin A [35]. Autoxidation of oxyHb produces metHb and superoxide [20]. Superoxide is converted to hydrogen peroxide by superoxide dismutase [14]. Most of the hydrogen peroxide is neutralized by antioxidant enzymes including catalase, glutathione peroxidase, and peroxiredoxin. However, a fraction of the hydrogen peroxide has been shown to escape from these antioxidant enzymes and reacts with the heme of hemoglobin forming fluorescent heme degradation products [28,31]. Because of the instability of HbS, elevated levels of these heme degradation products are detected. The observed increase in heme degradation products for SCD is further elevated because of the reported higher affinity of HbS for band 3 of the erythrocyte membrane than HbA [34]. Hydrogen peroxide generated by this membrane associated Hb is less accessible to cytosolic antioxidants [28], resulting in an increase in ROS that is not neutralized by the erythrocyte antioxidant system. These degradation products are more stable than the ROS produced during autoxidation and, therefore, reflect the cumulative exposure of erythrocytes to reactive oxygen species [29,30]. The observed level of heme degradation products has been identified as an in vivo measure of erythrocyte induced oxidative stress [26,29,31]. This in vivo measure of oxidative stress was previously used to show that transgenic mice expressing human HbS, human hemoglobin C and
Fig. 4. (A) Negative correlation between deformability and heme degradation for all participants with SCD or sickle trait. The experimental details are the same as for Figs. 2A and 3. E.I. = elongation index. F.A.U = Arbitrary fluorescence units. (B) Percent changes relative to SCD with crisis set at 100% for the average values of deformability and heme degradation for sickle trait and SCD without crisis.
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mouse models of beta-thalassemia [26] are exposed to increased oxidative stress. In that study, we reported an in vivo increase in oxidative stress ranging between 1.5 and 6.9 fold. In the present study, we for the first time in human subjects demonstrate a 2 fold increase in fluorescent heme degradation products in SCD, which depends on the severity of the disease (Fig. 3). Oxidative stress can directly damage the membrane inducing exposure of phosphatidylserine, increasing membrane stiffness, and impairing cation homeostasis. Membrane damage by oxidative stress results in increased intracellular calcium levels that activate the KCa3.1 channel, permitting potassium to escape the cell followed by Cl− and H2O [8,11]. This causes dehydration and cell shrinkage with increased cytoplasmic viscosity and a stiffened cytoskeleton that is less deformable [37]. Consistent with the expected effect of oxidative stress on the deformability we found a very significant correlation between deformability and heme degradation (Fig. 4A). To differentiate between the deformability changes induced by hemoglobin polymerization and oxidative stress we have compared the deformability with increased levels of HbF, for which the primary beneficial effect involves inhibition of HbS aggregation and polymerization, and the levels of fluorescent heme degradation products, which measure oxidative stress. Both HbF and heme degradation are highly correlated with deformability (Figs. 2B and 4A). However the lack of a correlation between heme degradation and HbF implies that these two factors contribute independently to erythrocyte deformability. By measuring both deformability and heme degradation, we were able to distinguish between the deformability changes that occur during crisis and to further delineate the changes occurring among those with SCD and trait. Even though sickle cell trait has moderate levels of HbS (Table 1, Fig. 1B), hemoglobin aggregation, sickling and other aspects of sickle cell pathology are rarely seen. Because of the negligible contribution of aggregation to sickle trait deformability, the relative decrease in deformability for trait is only 20% as much as seen in SCD with crisis (Figs. 2A and 4B). This increase in oxidative stress for sickle trait is consistent with the deleterious changes reported during exercise in people with sickle trait [12]. The mechanism of these changes is likely an additive effect of the increased oxidative stress from sickle trait and from exercise [1]. Our analysis shown in Fig. 4B provides insight into the differences induced by crisis. For stable SCD the increased proportion of HbS (Fig. 1B), as well as the reduced total hemoglobin (Fig. 1A; Table 1), increase oxidative stress [27]. The aggregation of Hb during deoxygenation of the cells also contributes to a change in deformability. However, the decreased deformability due to increased aggregation, polymerization and sickling for the group with acute vaso-occlusion (crisis) is not present in steady state SCD. These changes during crisis explain the proportionally greater decrease in deformability (39.4% of the total decrease in deformability) than increase in heme degradation (only 19% of the total increase in heme degradation) that takes place when steady state SCD goes into crisis. Limitations of our study include the cross-sectional design and potential confounding by unmeasured variables that may affect the severity of SCD. In addition, the participants with sickle trait were significantly older than those with SCD. The relatively small size of our study did not permit adjustment for age or other possible confounders. Conclusions In this paper we compare levels of heme degradation, which provides a direct measure of erythrocyte oxidative stress, with erythrocyte deformability. We find that, with increasing the severity of sickle disease, there is an increase in heme degradation and a decrease in deformability resulting in a significant negative correlation between heme degradation and deformability. These results indicate that oxidative stress, which is determined by heme degradation, plays a role in
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Blood Cells, Molecules and Diseases journal homepage: www.elsevier.com/locate/bcmd
New insights provided by a comparison of impaired deformability with erythrocyte oxidative stress for sickle cell disease Viachaslau M. Barodka a,1, Enika Nagababu c,1, Joy G. Mohanty c, Daniel Nyhan a, Dan E. Berkowitz a, Joseph M. Rifkind c,⁎, John J. Strouse b a b c
Department of Anesthesiology/Critical Care Medicine, Johns Hopkins University School of Medicine, Baltimore, MD, United States Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, MD, United States Molecular Dynamics Section, National Institute on Aging, National Institutes of Health, Baltimore, MD, United States
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Article history: Submitted 30 July 2013 Revised 10 October 2013 Available online xxxx (Communicated by Narla) Keywords: Sickle cell disease Erythrocytes Hemoglobin Heme degradation Fluorescence Deformability
a b s t r a c t Sickle cell disease (SCD) is associated with increase in oxidative stress and irreversible membrane changes that originates from the instability and polymerization of deoxygenated hemoglobin S (HbS). The relationship between erythrocyte membrane changes as assessed by a decrease in deformability and oxidative stress as assessed by an increase in heme degradation was investigated. The erythrocyte deformability and heme degradation for 27 subjects with SCD and 7 with sickle trait were compared with normal healthy adults. Changes in both deformability and heme degradation increased in the order of control to trait to non-crisis SCD to crisis SCD resulting in a very significantly negative correlation between deformability and heme degradation. However, a quantitative analysis of the changes in deformability and heme degradation for these different groups of subjects indicated that sickle trait had a much smaller effect on deformability than on heme degradation, while crisis affects deformability to a greater extent than heme degradation. These findings provide insights into the relative contributions of erythrocyte oxidative stress and membrane damage during the progression of SCD providing a better understanding of the pathophysiology of SCD. Published by Elsevier Inc.
Introduction Erythrocytes are under continuous stress by oxygenation and deoxygenation cycles, strong shearing forces in narrow blood vessels and reactive oxygen species (ROS) generated both endogenously and exogenously during their 120 day life span. An increase in erythrocyte oxidative stress as assessed by measuring of membrane lipid peroxidation has been reported with erythrocyte disorders [15,26,38] and various other diseases including renal failure [10], Alzheimer's disease [3], and autoimmune thrombocytopenic purpura [33], as well as during cellular aging [32]. The endogenous reactive oxygen species (ROS) are generated by autoxidation of oxyhemoglobin (oxyHb) to methemoglobin (metHb) and superoxide, which is converted to hydrogen peroxide [14]. We have previously demonstrated that a fraction of the hydrogen peroxide that is not scavenged by the erythrocyte antioxidant defense system damages the heme moiety of hemoglobin producing fluorescent degradation products. These degradation products are relatively stable being retained in the membrane and only slowly released into the plasma. ⁎ Corresponding author at: Molecular Dynamics Section, National Institute on Aging, 251 Bayview Blvd., Baltimore, MD 21224, United States. Fax: +1 410 558 8397. E-mail address: [email protected] (J.M. Rifkind). 1 Equally contributing authors.
Therefore, a gradual increase in fluorescent degradation products occurs whenever the cell is exposed to oxidative stress and the determination of fluorescent heme degradation products in the erythrocyte provides an in vivo measure of erythrocyte oxidative stress [25–28]. In sickle cell disease (SCD), autoxidation and the associated production of endogenous ROS are augmented by the instability of sickle hemoglobin (HbS) [15]. We have recently confirmed the predicted increase in heme degradation for HbS using transgenic mice that produce Hb S [26]. In that study, erythrocytes from sickle cell transgenic mice had an average 5.6 fold increase in heme degradation relative to controls. The ability of erythrocytes to deform is critical to microvascular perfusion and oxygen delivery [21], with impaired deformability contributing to the severe vascular and end-organ pathology of SCD [23]. Membrane lipid and protein damage generated by oxidative stress contribute to impaired deformability. The resultant altered cytoskeletal network induces exposure of phosphatidylserine and altered cation homeostasis with increased intracellular calcium (Ca2+) levels due to both increased membrane permeability to Ca2+ and inhibition of CaATPase, which actively pumps Ca2+ out of the cell [11]. The increased concentration of intracellular Ca2+ activates the KCa3.1 channel, which permits potassium ions to escape followed by chloride (Cl−) and water (H2O) [8]. These processes produce dehydration and cell shrinkage, which result in impaired deformability [37].
1079-9796/$ – see front matter. Published by Elsevier Inc. http://dx.doi.org/10.1016/j.bcmd.2013.10.004
Please cite this article as: V.M. Barodka, et al., Blood Cells Mol. Diseases (2013), http://dx.doi.org/10.1016/j.bcmd.2013.10.004
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In addition to increased oxidative stress associated with HbS, deformability is affected by a number of other factors. For sickle cell disease an important factor contributing to the impaired deformability is the oxygenation/deoxygenation cycles occurring in the circulation that result in cyclic aggregation/polymerization of deoxygenated HbS [16]. This process, which is exacerbated at higher concentrations of HbS produced by cell shrinkage [18], results in irreversible changes to the membrane and eventual sickling, which further impair erythrocyte deformability. This decrease in erythrocyte deformability becomes more profound 1 to 3 days before the development of painful vasoocclusive crisis and then reverses, gradually increasing to greater than steady-state levels by day 9. Oxidative stress in SCD also contributes to anemia both by producing rigid cells that are removed by the reticuloendothelial system (if still functional) [23], and the exposure of phosphatidylserine on the outer surface of the erythrocyte, which triggers the removal of cells by macrophages [13]. Anemia, which increases hypoxia, has also been shown to increase oxidative stress [16,27]. To evaluate the relative contributions of oxidative stress and hemoglobin polymerization to erythrocyte injury, we have measured both heme degradation, which is a direct measure of erythrocyte oxidative stress, and deformability, which is affected by both oxidative stress and hemoglobin polymerization.
as (L − W)/(L + W), where L and W are the major and minor axes of the ellipse. We determined EI at a range of shear stress values (0 to 20 Pa) and used the value at 3 Pa for comparative analysis of the deformability values of samples treated under different conditions, as suggested by Baskurt et. al.[7]. We also used values at 20 Pa to measure changes in the least deformable cells, which reflect the altered membrane structure of these cells. Measurement of heme degradation products
Design and methods
Heme degradation products were measured as previously described [26]. In brief, blood was collected into EDTA vacutainers and centrifuged at 3000 RPM for 10 min at 2–4 °C. The plasma was removed and 20 μl of unwashed erythrocytes were lysed in 3 ml deionized distilled water. The hemoglobin spectrum of the hemolysate was recorded from 490 to 640 nm using a PerkinElmer lambda 35 spectrophotometer. The concentrations of oxyHb and metHb were determined by a least square-fitting program using known concentrations of parent spectra of oxyHb and metHb. The total Hb concentration of the hemolysate was then adjusted to 50 μM by diluting with distilled water. The fluorescence emission spectrum was measured from 400 nm to 600 nm at an excitation wavelength of 321 nm using a PerkinElmer LS50B spectrofluorimeter. The fluorescence intensity of emission maximum at 485 nm was used as a measure of heme degradation. The excitation and emission slit widths were kept at 10 nm.
Study population
Measurement of total hemoglobin, HbS and fetal hemoglobin
We enrolled participants with sickle cell disease and sickle cell trait (HbAS) N4 years of age from outpatient clinics at Johns Hopkins Hospital. Exclusion criteria included pregnancy, weight b10 kg, transfusion in the last 2 months, and, for patients on hydroxyurea, a change in dose in the last 2 months or known poor adherence to treatment. Participants in steady state had not reported pain consistent with sickle cell crisis for at least 2 weeks. Participants during the beginning of acute painful episodes receiving parenteral opiates for acute sickle cell pain were classified as having vaso-occlusive crisis. The study was approved by the Johns Hopkins Medicine Institutional Review Board. Blood samples from normal healthy subjects without SCD were obtained at the National Institute of Aging (NIA), National Institutes of Health (NIH) through IRB approved protocol (2003-071) for tissue procurement for biomedical research.
Complete blood and reticulocyte counts were performed using an automated analyzer (Sysmex XE-2100, Roche, Indianapolis, IN) and proportion of hemoglobins by high performance liquid chromatography (Biorad Variant Hemoglobin Testing System, Biorad Laboratories, Hercules, CA) respectively. The proportion of erythrocytes and reticulocytes containing Hemoglobin F (HbF) was measured by flow cytometry [24] (Table 1).
Sample preparation Up to 6 ml of venous blood anticoagulated with K2EDTA was placed on ice and then kept at 4 °C till measurements were performed. The tests for deformability and heme degradation were done in parallel, blinded to the diagnosis of the patient and within 24 h of blood collection. Measurement of erythrocyte deformability Erythrocyte deformability was measured using a microfluidic RheoScan-D slit-flow ektacytometer (Rheo Meditech, Seoul, South Korea) [36]. Briefly, this instrument consists of a diode laser, a CCD camera, a pressure sensor, a vacuum generating mechanism and a sample holder containing a disposable microfluidic chip. For erythrocyte deformability measurement, cells were suspended (final hct ~ 0.5%) by slowly mixing them in the highly-viscous PVP360 solution (viscosity ~ 30 cP) and loaded onto the sample reservoir of the chip (Rheo Meditech). The erythrocytes are pulled through the microchannel at a range of shear stresses, while the elliptical diffraction patterns are projected on a screen by a laser, captured by a CCD-video camera, and analyzed by ellipse-fitting-software. Deformability of erythrocytes is expressed as Elongation Index (EI), which is defined
Results Comparison of levels of total Hb, HbS and HbF for SCD We enrolled 27 participants with sickle cell disease (21 participants with sickle cell anemia (HbSS), 3 with Hemoglobin SC disease (HbSC), 2 with HbS β-thalassemia plus and 1 with HbS Osler) and 7 with sickle cell trait (Table 1). To evaluate the anemia associated with SCD, we measured the total hemoglobin for each group. Anemia for SCD is indicated by the reduced level of total hemoglobin relative to that of sickle trait (Fig. 1A), which is similar to that of controls (data not shown). Samples obtained toward the beginning of acute painful episodes (in crisis) do not demonstrate a further reduction in total hemoglobin. Heterozygotes with sickle cell trait have an average of 38% HbS, less than those with SCD. Crisis, however, does not result in a significant increase in HbS (Fig. 1B). The subjects with SCD that did not have HbSS were intermediate between those with sickle trait and HbSS, both with respect to the HbS level and the total Hb level (Table 1). Table 1 Clinical characteristics of participants. Variable
HbSS on HU
HbSS no HU
Other SCD
Sickle cell trait
Number Age (years) Male sex Total Hb (g/dl) Reticulocytes (%) Hb F (%) Hb S (*5) Pain crisis
13 27 ± 11 46% 8.4 ± 1.2 8.4 ± 5.4 16 ± 7.1 77 ± 8 31%
8 18 ± 11 50% 8.4 ± 1.0 12.6 ± 1.9 9.2 ± 5.1 83 ± 5 37%
6 28 ± 13 50% 10.7 3.4 ± 1.4 2.8 50% 67%
7 39 ± 5 14% 12.7 1.1 0.4 0.8 38% 0%
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Fig. 1. (A) Total hemoglobin in erythrocytes of participants with sickle cell crisis, steady-state, and trait. (B) Percent HbS for participants with sickle cell crisis, steady-state, and trait. (C) Difference in the percent HbF for participants with sickle cell disease undergoing crisis.
Elevated levels of HbF are frequently found in patients with SCD and are increased by treatment with hydroxyurea. The presence of HbF has also been shown to inhibit the polymerization of deoxygenated HbS. Consistent with the protective effects of HbF, we find reduced levels of HbF in samples obtained during crisis in the beginning of acute painful episodes (Fig. 1C). This likely reflects the higher frequency of crisis in patients with SCD and low HbF. Erythrocytes deformability and sickle cell disease Fig. 2A shows the erythrocyte deformability for normal healthy controls and those with sickle trait and SCD during steady state and crisis at 3 Pa, which reflects changes in the average deformability. The sensitivity of the deformability measurement is indicated by a significant (p b 0.001) 11% decrease in deformability in those with sickle trait. For SCD, an additional significant (p b 0.0001) decrease in deformability was found, which depended on the severity of disease resulting in about a 50% decrease in the EI for those in crisis relative to control subjects. Fig. 2B shows the deformability for the same subjects at a 20 Pa. The decrease in deformability at 20 Pa indicates structural changes in the membrane of sickle cell subjects. The beneficial effects of fetal hemoglobin have been attributed to an inhibition of the aggregation of sickle hemoglobin that triggers sickling [17]. The significant correlation between deformability and HbF (Fig. 2B) is consistent with a contribution of hemoglobin aggregation, and its effect on the membrane, to deformability. Thirteen of the 21 participants with HbSS were treated with hydroxyurea. On average they had a higher percentage of HbF (16 ± 7.1 vs. 9.2 ± 5.1) than
those with HbSS not treated with hydroxyurea. As indicated in Fig. 2B treatment with hydroxyurea does not have a significant effect on the observed relationship between HbF and deformability. Heme degradation and sickle cell disease Fluorescent heme degradation products are measures of oxidative stress originating in the erythrocyte [28,31]. Fig. 3 shows that the fluorescent heme degradation products significantly (p b 0.0001) increased relative to the control by a factor of 1.7, 2.1 and 2.5 fold for sickle trait, SCD in steady state and SCD in crisis, respectively. When we evaluated the correlation between HbF and heme degradation we found a decrease in heme degradation with increasing HbF consistent with a protective effect of HbF. However unlike the very significant effect found for deformability this correlation was not significant (data not shown). Comparison of deformability and heme degradation The contribution of oxidative stress to deformability is indicated by the significant negative correlation (R = 0.68; p b 0.001) between heme degradation and deformability (Fig. 4A). Although this plot indicates that these 2 parameters are highly correlated, the results with HbF (highly correlated with deformability but not heme degradation) indicate that the aggregation process affects deformability to a greater extent than heme degradation. At the same time a comparison of Figs. 2A and 3 indicate a proportionally greater increase in heme degradation than decrease in deformability when comparing sickle cell trait
Fig. 2. (A&B) Deformability of erythrocytes of control, sickle trait, and sickle cell disease participants. Elongation index (E.I.) as a measure of deformability was measured as described in the methods section. Values are mean ± standard deviation, N control = 50, Trait = 7, steady state = 16, crisis = 11. Significance relative to control; ⁎p b 0.001; ⁎⁎p b 0.0001. (C) Correlation of deformability at 3 Pa with fetal hemoglobin percentage for SCD subjects. (▲), participants on hydroxyurea (R = 0.509). (■), participants not receiving hydroxyurea (R = 0.541).
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Fig. 3. Heme degradation in control and erythrocytes from participants with SCD and trait. Heme degradation was determined as mentioned in methods section. Values are given as arbitrary fluorescent units (F.A.U.) mean ± standard error. N, crisis 11, steady state = 16, trait = 7 and control = 50. Significance relative to control; ⁎p b 0.0001.
with controls. The dominant effect of sickle cell trait on heme degradation can be attributed to the increase in autoxidation of HbS, which causes oxidative stress. While this process determines heme degradation, deformability is affected by both oxidative stress and hemoglobin aggregation, which is most pronounced during acute vaso-occlusive episodes (crisis). The different contribution of oxidative stress and hemoglobin aggregation during SCD is demonstrated in Fig. 4B where we plotted the percent changes in heme degradation and deformability for the four different groups (control, trait, non-crisis and crisis). Heme degradation, which is a direct measure of the ROS generated by autoxidation of hemoglobin is increased for trait because of the significant proportion (typically ~40%) of the less stable HbS. Deformability, which is affected by hemoglobin aggregation/polymerization in addition to any effects of oxidative stress will be modified to a greater extent by the changes that take place during acute vaso-occlusive episodes. Discussion SCD is caused by a substitution of valine for glutamic acid at the β-6 position in the hemoglobin β-chain [9]. The valine to glutamic acid modification of HbS facilitates aggregation/polymerization of hemoglobin in
the deoxygenated state and can cause vascular occlusion that dramatically affects oxygen delivery [9]. Erythrocyte deformability is essential to pass through narrow capillaries required for oxygen delivery, and is expected to contribute to the impaired microcirculatory flow of SCD [2,4–6]. Before, during, and after crisis show a decrease in deformability 1–3 days before the onset of pain. This deformability further decreases in the beginning of crisis, but increases later during crisis as the pain subsides. The improved deformability has been attributed to the removal of the poorly dense deformable cells and replacement by young deformable cells. Consistent with these studies, we have observed a significant decrease in deformability when comparing steady state subjects who have not experienced any painful episodes for at least 2 weeks with patients in the initial stage of painful crisis. Erythrocyte deformability is affected by various factors including cytoplasmic viscosity, cell shrinkage and membrane properties. The decreased deformability of erythrocytes in SCD is in part associated with aggregation/polymerization of HbS, which eventually leads to morphological sickling that affects the ability of the membrane to deform [18,19,22,23]. An additional factor that can affect deformability in SCD is the increased oxidative stress associated with the instability of HbS. This instability results in a higher rate of autoxidation for HbS than normal hemoglobin A [35]. Autoxidation of oxyHb produces metHb and superoxide [20]. Superoxide is converted to hydrogen peroxide by superoxide dismutase [14]. Most of the hydrogen peroxide is neutralized by antioxidant enzymes including catalase, glutathione peroxidase, and peroxiredoxin. However, a fraction of the hydrogen peroxide has been shown to escape from these antioxidant enzymes and reacts with the heme of hemoglobin forming fluorescent heme degradation products [28,31]. Because of the instability of HbS, elevated levels of these heme degradation products are detected. The observed increase in heme degradation products for SCD is further elevated because of the reported higher affinity of HbS for band 3 of the erythrocyte membrane than HbA [34]. Hydrogen peroxide generated by this membrane associated Hb is less accessible to cytosolic antioxidants [28], resulting in an increase in ROS that is not neutralized by the erythrocyte antioxidant system. These degradation products are more stable than the ROS produced during autoxidation and, therefore, reflect the cumulative exposure of erythrocytes to reactive oxygen species [29,30]. The observed level of heme degradation products has been identified as an in vivo measure of erythrocyte induced oxidative stress [26,29,31]. This in vivo measure of oxidative stress was previously used to show that transgenic mice expressing human HbS, human hemoglobin C and
Fig. 4. (A) Negative correlation between deformability and heme degradation for all participants with SCD or sickle trait. The experimental details are the same as for Figs. 2A and 3. E.I. = elongation index. F.A.U = Arbitrary fluorescence units. (B) Percent changes relative to SCD with crisis set at 100% for the average values of deformability and heme degradation for sickle trait and SCD without crisis.
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mouse models of beta-thalassemia [26] are exposed to increased oxidative stress. In that study, we reported an in vivo increase in oxidative stress ranging between 1.5 and 6.9 fold. In the present study, we for the first time in human subjects demonstrate a 2 fold increase in fluorescent heme degradation products in SCD, which depends on the severity of the disease (Fig. 3). Oxidative stress can directly damage the membrane inducing exposure of phosphatidylserine, increasing membrane stiffness, and impairing cation homeostasis. Membrane damage by oxidative stress results in increased intracellular calcium levels that activate the KCa3.1 channel, permitting potassium to escape the cell followed by Cl− and H2O [8,11]. This causes dehydration and cell shrinkage with increased cytoplasmic viscosity and a stiffened cytoskeleton that is less deformable [37]. Consistent with the expected effect of oxidative stress on the deformability we found a very significant correlation between deformability and heme degradation (Fig. 4A). To differentiate between the deformability changes induced by hemoglobin polymerization and oxidative stress we have compared the deformability with increased levels of HbF, for which the primary beneficial effect involves inhibition of HbS aggregation and polymerization, and the levels of fluorescent heme degradation products, which measure oxidative stress. Both HbF and heme degradation are highly correlated with deformability (Figs. 2B and 4A). However the lack of a correlation between heme degradation and HbF implies that these two factors contribute independently to erythrocyte deformability. By measuring both deformability and heme degradation, we were able to distinguish between the deformability changes that occur during crisis and to further delineate the changes occurring among those with SCD and trait. Even though sickle cell trait has moderate levels of HbS (Table 1, Fig. 1B), hemoglobin aggregation, sickling and other aspects of sickle cell pathology are rarely seen. Because of the negligible contribution of aggregation to sickle trait deformability, the relative decrease in deformability for trait is only 20% as much as seen in SCD with crisis (Figs. 2A and 4B). This increase in oxidative stress for sickle trait is consistent with the deleterious changes reported during exercise in people with sickle trait [12]. The mechanism of these changes is likely an additive effect of the increased oxidative stress from sickle trait and from exercise [1]. Our analysis shown in Fig. 4B provides insight into the differences induced by crisis. For stable SCD the increased proportion of HbS (Fig. 1B), as well as the reduced total hemoglobin (Fig. 1A; Table 1), increase oxidative stress [27]. The aggregation of Hb during deoxygenation of the cells also contributes to a change in deformability. However, the decreased deformability due to increased aggregation, polymerization and sickling for the group with acute vaso-occlusion (crisis) is not present in steady state SCD. These changes during crisis explain the proportionally greater decrease in deformability (39.4% of the total decrease in deformability) than increase in heme degradation (only 19% of the total increase in heme degradation) that takes place when steady state SCD goes into crisis. Limitations of our study include the cross-sectional design and potential confounding by unmeasured variables that may affect the severity of SCD. In addition, the participants with sickle trait were significantly older than those with SCD. The relatively small size of our study did not permit adjustment for age or other possible confounders. Conclusions In this paper we compare levels of heme degradation, which provides a direct measure of erythrocyte oxidative stress, with erythrocyte deformability. We find that, with increasing the severity of sickle disease, there is an increase in heme degradation and a decrease in deformability resulting in a significant negative correlation between heme degradation and deformability. These results indicate that oxidative stress, which is determined by heme degradation, plays a role in
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decreasing deformability, which is also affected by hemoglobin polymerization. However, the enhanced effect of polymerization and sickling on deformability is indicated by a proportionally greater effect on deformability than heme degradation when comparing participants undergoing vaso-occlusive crisis with steady state SCD. These results indicate that polymerization of HbS is the principal factor producing reduced deformability during sickle cell crisis. Conflicts of interest The authors have no conflicts of interest to report. Source of funding Supported in part by grants from National Institutes of Health, NIH 5R01 HL105296 to (DEB) and the Doris Duke Charitable Foundation (JJS) and from the Intramural Research Program of National Institute of Aging. References [1] R.S. Ajmani, J.L. Fleg, A.A. Demehin, et al., Oxidative stress and hemorheological changes induced by acute treadmill exercise, Clin. Hemorheol. Microcirc. 28 (2003) 29–40. [2] N.O. Akinola, S.M. Stevens, I.M. Franklin, et al., Rheological changes in the prodromal and established phases of sickle cell vaso-occlusive crisis, Br. J. Haematol. 81 (1992) 598–602. [3] I. Baldeiras, I. Santana, M.T. Proenca, et al., Peripheral oxidative damage in mild cognitive impairment and mild Alzheimer's disease, J. Alzheimers Dis. 15 (2008) 117–128. [4] S.K. Ballas, Sickle cell anemia with few painful crises is characterized by decreased red cell deformability and increased number of dense cells, Am. J. Hematol. 36 (1991) 122–130. [5] S.K. Ballas, E.D. Smith, Red blood cell changes during the evolution of the sickle cell painful crisis, Blood 79 (1992) 2154–2163. [6] G.A. Barabino, M.O. Platt, D.K. Kaul, Sickle cell biomechanics, Annu. Rev. Biomed. Eng. 12 (2010) 345–367. [7] O.K. Baskurt, M. Boynard, G.C. Cokelet, et al., New guidelines for hemorheological laboratory techniques, Clin. Hemorheol. Microcirc. 42 (2009) 75–97. [8] C. Brugnara, Membrane transport of Na and K and cell dehydration in sickle erythrocytes, Experientia 49 (1993) 100–109. [9] H.F. Bunn, Pathogenesis and treatment of sickle cell disease, N. Engl. J. Med. 337 (1997) 762–769. [10] G. Caimi, Erythrocyte peroxide metabolism, plasma lipid pattern and hemorheological profile in chronic renal failure, J. Nephrol. 15 (2002) 104–108. [11] M. Canessa, Red cell volume-related ion transport systems in hemoglobinopathies, Hematol. Oncol. Clin. N. Am. 5 (1991) 495–516. [12] P. Connes, O. Hue, J. Tripette, M.D. Hardy-Dessources, Blood rheology abnormalities and vascular cell adhesion mechanisms in sickle cell trait carriers during exercise, Clin. Hemorheol. Microcirc. 39 (2008) 179–184. [13] J. Connor, C.C. Pak, A.J. Schroit, Exposure of phosphatidylserine in the outer leaflet of human red blood cells. Relationship to cell density, cell age, and clearance by mononuclear cells, J. Biol. Chem. 269 (1994) 2399–2404. [14] C. Giulivi, P. Hochstein, K.J. Davies, Hydrogen peroxide production by red blood cells, Free Radic. Biol. Med. 16 (1994) 123–129. [15] R.P. Hebbel, The sickle erythrocyte in double jeopardy: autoxidation and iron decompartmentalization, Semin. Hematol. 27 (1990) 51–69. [16] R.P. Hebbel, Beyond hemoglobin polymerization: the red blood cell membrane and sickle disease pathophysiology, Blood 77 (1991) 214–237. [17] D.K. Kaul, X.D. Liu, H.Y. Chang, et al., Effect of fetal hemoglobin on microvascular regulation in sickle transgenic-knockout mice, J. Clin. Invest. 114 (2004) 1136–1145. [18] V.L. Lew, R.M. Bookchin, Osmotic effects of protein polymerization: analysis of volume changes in sickle cell anemia red cells following deoxy-hemoglobin S polymerization, J. Membr. Biol. 122 (1991) 55–67. [19] V.L. Lew, R.M. Bookchin, Ion transport pathology in the mechanism of sickle cell dehydration, Physiol. Rev. 85 (2005) 179–200. [20] H.P. Misra, I. Fridovich, The generation of superoxide radical during the autoxidation of hemoglobin, J. Biol. Chem. 247 (1972) 6960–6962. [21] N. Mohandas, J.A. Chasis, S.B. Shohet, The influence of membrane skeleton on red cell deformability, membrane material properties, and shape, Semin. Hematol. 20 (1983) 225–242. [22] N. Mohandas, W.M. Phillips, M. Bessis, Red blood cell deformability and hemolytic anemias, Semin. Hematol. 16 (1979) 95–114. [23] N. Mohandas, R.P. Hebbel, Erythrocyte deformability, fragility and rheology, in: S.H. Embury, R.P. Hebbel, N. Mohandas, M.H. Steinberg (Eds.), Sickle Cell Disease, Raven Press, New York, 1994, pp. 205–216. [24] Y. Mundee, N.C. Bigelow, B.H. Davis, J.B. Porter, Flow cytometric method for simultaneous assay of foetal haemoglobin containing red cells, reticulocytes and foetal haemoglobin containing reticulocytes, Clin. Lab. Haematol. 23 (2001) 149–154.
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