Metabolic Influences Modulating Erythrocyte Deformability and Eryptosis

Many factors in the surrounding environment have been reported to influence erythrocyte deformability. It is likely that some influences represent reversible changes in erythrocyte rigidity that may be involved in physiological regulation, while others represent the early stages of eryptosis, i.e., the red cell self-programmed death. For example, erythrocyte rigidification during exercise is probably a reversible physiological mechanism, while the alterations of red blood cells (RBCs) observed in pathological conditions (inflammation, type 2 diabetes, and sickle-cell disease) are more likely to lead to eryptosis. The splenic clearance of rigid erythrocytes is the major regulator of RBC deformability. The physicochemical characteristics of the surrounding environment (thermal injury, pH, osmolality, oxidative stress, and plasma protein profile) also play a major role. However, there are many other factors that influence RBC deformability and eryptosis. In this comprehensive review, we discuss the various elements and circulating molecules that might influence RBCs and modify their deformability: purinergic signaling, gasotransmitters such as nitric oxide (NO), divalent cations (magnesium, zinc, and Fe++), lactate, ketone bodies, blood lipids, and several circulating hormones. Meal composition (caloric and carbohydrate intake) also modifies RBC deformability. Therefore, RBC deformability appears to be under the influence of many factors. This suggests that several homeostatic regulatory loops adapt the red cell rigidity to the physiological conditions in order to cope with the need for oxygen or fuel delivery to tissues. Furthermore, many conditions appear to irreversibly damage red cells, resulting in their destruction and removal from the blood. These two categories of modifications to erythrocyte deformability should thus be differentiated.

Changes in blood cell deformability in Chorea-Acanthocytosis and effects of treatment with dasatinib or lithium

Misshaped red blood cells (RBCs), characterized by thorn-like protrusions known as acanthocytes, are a key diagnostic feature in Chorea-Acanthocytosis (ChAc), a rare neurodegenerative disorder. The altered RBC morphology likely influences their biomechanical properties which are crucial for the cells to pass the microvasculature. Here, we investigated blood cell deformability of 5 ChAc patients compared to healthy controls during up to one-year individual off-label treatment with the tyrosine kinases inhibitor dasatinib or several weeks with lithium. Measurements with two microfluidic techniques allowed us to assess RBC deformability under different shear stresses. Furthermore, we characterized leukocyte stiffness at high shear stresses. The results show that blood cell deformability - including both RBCs and leukocytes - in general is altered in ChAc patients compared to healthy donors. Therefore, this study shows for the first time an impairment of leukocyte properties in ChAc. During treatment with dasatinib or lithium, we observe alterations in RBC deformability and a stiffness increase for leukocytes. The hematological phenotype of ChAc patients hints at a reorganization of the cytoskeleton in blood cells which partly explains the altered mechanical properties observed here. These findings highlight the need for a systematic assessment of the contribution of impaired blood cell mechanics to the clinical manifestation of ChAc.

Alterations of Selected Hemorheological and Metabolic Parameters Induced by Physical Activity in Untrained Men and Sportsmen

Optimal tissue oxygen supply is essential for proper athletic performance and endurance. It also depends on perfusion, so on hemorheological properties and microcirculation. Regular exercise is beneficial to the rheological status, depending on its type, intensity, and duration. We aimed to investigate macro and microrheological changes due to short, high-intensity exercise in professional athletes (soccer and ice hockey players) and untrained individuals. The exercise was performed on a treadmill ergometer during a spiroergometry examination. Blood samples were taken before and after exercise to analyze lactate concentration, hematological parameters, blood and plasma viscosity, and red blood cell (RBC) deformability and aggregation. Leukocyte, RBC and platelet counts, and blood viscosity increased with exercise, by the largest magnitude in the untrained group. RBC deformability slightly impaired after exercise, but showed better values in ice hockey versus soccer players. RBC aggregation increased with exercise, dominantly in ice hockey players. Lactate increased mostly in soccer players, and the respiratory exchange rate was the lowest in ice hockey players. Overall, short, high-intensity exercise altered macro and microrheological parameters, mostly in the untrained group. Significant differences were found between the two sports. The data can be useful in training status monitoring, selection, and in revealing the causes of physical loading symptoms.

Degradation of Red Blood Cell Deformability during Cold Storage in Blood Bags

RBC transfusions are a life-saving procedure, aiding both chronic and acute patients in restoring tissue oxygenation. The ability to store collected RBC units for prolonged periods has been one of the most transformative advances in medicine, significantly improving the reliability and the speed of access to blood. However, RBCs undergo a number of metabolic, structural, and biochemical changes during storage, collectively known as the storage lesion, that is detrimental to the quality of the RBC. A major challenge is the ability to evaluate the extent of the storage lesion, and thus the quality of the stored RBC unit directly prior to transfusion. The storage lesion can directly or indirectly reduce the ability of the RBC to deform through the small openings in the microvasculature. Rigid RBCs pose a risk of sequestration in capillaries, impeding blood flow and reducing tissue oxygenation, and are more likely to be cleared out by endothelial macrophages. Studies have shown that there is a loss in RBC deformability during storage and that the rate of RBC deformability loss is donor-dependent. Thus, RBC deformability can be a valuable and reliable biophysical marker of RBC unit quality. Currently, there is a need for a reliable measurement technique that is repeatable and sensitive enough to observe individual differences in RBC deformability in healthy donors, to enable quality control testing of RBC units. We have developed the microfluidic ratchet device, which sorts RBCs based on their deformability, allowing the measurement of both rigid and deformable sup-populations of RBCs within the sample, and generating a unique deformability curve. Here, we use this assay to predict the quality of stored RBC units. We assessed the deformability of 14 healthy donor RBC units through 8 weeks of cold storage at 4°C, which is 2 weeks beyond the Canadian Blood Services approved 6-week standard in Canada. We measured RBC deformability, standard hematological parameters (MCV, MCHC, MCH, and RDW), and hemolysis levels at the time of RBC unit manufacture (week 0), followed by weeks 2, 4, 6, and 8. The microfluidic ratchet device operates by forcing RBCs to deform and travel through rows of tapered constrictions. Constriction size changes from 7.5 to 1.5 µm and is reflective of the microvasculature and vessel opening sizes encountered by RBCs in circulation. RBCs are sorted into 12 distinct outlets based on their deformability. Distribution of RBCs in outlets 1-12 can be quantified and used to calculate the cumulative distribution curve. The cumulative distribution curve provides a distinct deformability signature of each individual RBC sample, which can be defined as rigidity score (RS). RS provides an easy metric to compare the changes in RBC deformability throughout storage (ΔRS) in a single donor as well as across multiple donors. We show that there are both donor- and sex-specific differences in the RBC deformability signatures of stored RBC units. We observed significant inter-donor variability in RBC deformability measured on the day of the RBC unit manufacture, where male donors showed a more stable RBC deformability range (n=8, RS=3.00±0.18) compared to female donors (n=6, RS= 3.29±0.48). The average RS scores were stable between weeks 0-2 (ΔRS 0.07) and showed a reduction in deformability between weeks 1-6 (ΔRS 0.35), with the greatest loss seen between weeks 6-8 (ΔRS 0.42) of cold storage. Interestingly, the response to cold storage is variable, with ΔRS 0.22 to 0.90, suggesting that some donors are more susceptible to storage related changes in RBC deformability than others. Notably, the change in RS over time was donor-specific and did not correlate with RBC deformability at week 0. The majority of RBCs from male donors (ΔRS 0.485, p<0.05), but none of the female donors (ΔRS 0.172) showed changes in deformability during cold storage, suggesting that RBCs from female donors degrade at a slower rate compared to RBCs from male donors. The ability to profile RBC deformability at the individual blood bag level may help identify more stable RBCs for use in chronic and sensitive patients, or RBC units that can be safely stored beyond the 6-week storage window. Disclosures No relevant conflicts of interest to declare.

Hypoxia Impact on Red Blood Cell-Mediated Microvascular Occlusion and Adhesion in Sickle Cell Disease and Sickle Cell Trait

Introduction: Sickle cell disease (SCD) is a recessively inherited anemia caused by a single gene mutation leading to sickle hemoglobin production. Sickle cell trait (SCT) is the carrier state. Abnormal hemoglobin polymerization and resultant red blood cell (RBC) sickling, decreased deformability and increased adhesion, are well-known features of homozygous SCD. However, the overall pathophysiological impact of SCT on the RBC remains incompletely characterized. Here we use microfluidic techniques designed by us, the OcclusionChip and SCD Biochip (previously published), and commercially available ektacytometry to investigate hypoxia impact on RBC biophysical properties in SCT. Methods: Venous blood samples were collected in EDTA from subjects with homozygous HbSS, SCT (HbAS), and non-anemic controls (HbAA) under an IRB-approved protocol. OcclusionChip devices were fabricated using standard soft lithography protocols [1]. RBCs were isolated from whole blood, re-suspended in PBS at 20% hematocrit, and passed through the OcclusionChip device with a constant inlet pressure. Following a wash step, the OcclusionChip microchannel was imaged, and Occlusion Index (OI), a standardized generalizable parameter we developed, representing the overall microcapillary network occlusion, was quantified. SCD Biochip microchannels were fabricated by lamination and were functionalized with human laminin (LN-511) [2]. Undiluted whole blood was injected into the microchannel at 1 dyne/cm 2, a shear stress value typically observed in the post-capillary venules. Following a wash step, the SCD Biochip microchannel was imaged, and the number of adherent RBCs in a 32-mm 2 window was quantified. For hypoxia experiments, a hypoxic setup was fabricated for blood deoxygenation (pO 2 ~45 mmHg) [3, 4]. Ektacytometry measurements were performed according to the manufacturers' specifications (Lorrca Maxsis). Data are reported as mean ± standard deviation (SD). Results: We initially analyzed RBC-mediated microvascular occlusion under normoxia or hypoxia using the OcclusionChip (Figure 1A). Under normoxia, HbSS-containing RBCs had relatively greater OI values compared to HbAA- and HbAS-containing RBCs (Figure 1B, P = 0.057 for HbSS vs HbAA and P = 0.060 for HbSS vs HbAS). However, exposure to hypoxia led to significantly elevated OI values in the HbAS- and HbSS-containing RBCs (Figure 1B, 0.05 ± 0.02% vs 33.62 ± 18.31%, P = 0.015 for HbAS, and 0.27 ± 0.24% vs 49.37 ± 24.47%, P = 0.001 for HbSS, normoxia vs hypoxia). Negligible occlusion was observed in HbAA-containing RBCs (Figure 1B). We then analyzed RBC adhesion to LN under normoxia or hypoxia using the SCD Biochip (Figure 1C). Hypoxia led to greater number of adherent RBCs on LN in the HbSS-containing RBCs (Figure 1D, 141 ± 91 vs 497 ± 392, P = 0.089, normoxia vs hypoxia), but this effect was not present in HbAA- or HbAS-containing RBCs (Figure 1B, 2 ± 1 vs 3 ± 1, P > 0.05 for HbAA, and 10 ± 7 vs 12 ± 3, P > 0.05 for HbAS, normoxia vs hypoxia). Further, under normoxia, we found that the HbAS-containing RBCs had slightly greater number of adherent RBCs on LN compared to the HbAA-containing RBCs (Figure 1D, P = 0.057 for HbAA vs HbAS). As previously reported, HbSS-containing RBCs showed greatest adhesion to LN under normoxia compared to the HbAA- and HbAS-containing RBCs (Figure 1D, P = 0.027 for HbSS vs HbAA and P = 0.033 for HbSS vs HbAS)., Finally, we preformed Lorrca oxyscan and found that ektacytometry is less sensitive to RBC deformability change under hypoxia in SCT (Figure 1E). Conclusions: Findings in this study suggest that although RBCs from subjects with SCT are deformable under normoxia and are able to clear narrow capillaries similar to normal RBCs, hypoxia changes deformability, presumably due to hypoxic polymer formation, and could contribute to microvascular occlusion in SCT. The OcclusionChip is a single cell-based technology, and may be more sensitive to single RBC deformability. Future studies will prospectively focus on analyzing RBC adhesion on activated microvascular endothelial cells in physiologic flow to further interrogate the impact of hypoxia on pathophysiology in SCT. References: [1] Man et al., LabChip, 2020, 20, 2086-2099. [2] Kim et al., Microcirculation, 2017, 24, e12374. Figure 1 Figure 1. Disclosures An: Hemex Health, Inc.: Patents & Royalties. Kucukal: BioChip Labs: Current Employment, Patents & Royalties. Nayak: BioChip Labs: Patents & Royalties. Little: Biochip Labs: Patents & Royalties; Hemex Health, Inc.: Patents & Royalties. Gurkan: Dx Now Inc.: Patents & Royalties; Hemex Health, Inc.: Current Employment, Patents & Royalties; Biochip Labs: Patents & Royalties; Xatek Inc.: Patents & Royalties.

Stroke mechanisms associated with coronavirus disease (COVID-19)

The purpose is to identify the pathophysiological mechanisms of stroke development in patients with COVID-19. Methods A total of 92 patients (46.72+1.72 years) with impairments of cerebral circulation due to COVID-19 (confirmed by positive PCR test results) had been examined. Among them, 67 patients had an ischemic stroke, 14–a transient ischaemic attack, 11–an intracerebral hemorrhage, 3– a subarachnoid hemorrhage. The parameters of hemostasis were measured by standard methods, electrical and viscoelastic parameters of red blood cells (RBC) by the dielectrophoresis method was carried out. Results Most of the surveyed persons (60 patients) showed signs of an intravascular coagulation and thrombosis: accelerated platelet-leukocyte aggregation, increased levels of coagulation products, reduced fibrinolysis activity (p=0.002–0.04). The levels of D-dimer, fibrinogen, ESR, platelet count were significantly higher in this group compared to patients in the second one (p<0.01). A moderate increase in summarized rigidity, viscosity of RBC was noted. The level of RBC hemolysis was associated with platelet count (r=0.727, p<0.05), D-dimer level (r=0.422, p=0.04), and fibrinogen level (r=0.318, p<0.05). In the second group of patients (32 persons with a predominance of men - 25 male subjects), the markers of thrombosis had moderate deviations. A sharply reduced RBC deformability with increased summarized rigidity and viscosity was dominant coupled with the background of high electrical conductivity of cell membranes compared to the indicators in the first group (p<0.01). There was a decrease in membrane capacity, surface charge, electrical dipole moment of the cells and polarizability at all frequencies of the electric field than those in the first group (p=0.0001–0.05). A sharp decrease in RBC deformability creates obstacles to overcoming small-diameter capillaries, leading to violations of microcirculatory blood flow. The RBC deformability was associated with levels of ferritin (r=0.407, p=0.024), HbA1c (r=0.419, p=0.033), uric acid (r=−0.303, p<0.05), and LDL cholesterol (r=0.426, p=0.029). Incubation of blood samples in vitro for 10 min with riboflavin, nicotinamide, inosine, which ensures RBC energy metabolism (anaerobic glycolysis and pentose phosphate pathway) restored the reduced the RBC deformability (p<0.01), and decreased the RBC aggregation (p<0.001). Similar changes were obtained when coenzymes were administered to patients in vivo. Conclusion Two independent mechanisms of cerebral circulatory disturbances have been identified in COVID-19 stroke patients: both thrombotic and hemorheologic. The thrombotic variant is associated with a procoagulant state and with an activity of inflammation. The hemorheologic variant is caused by a decrease in the activity of the RBC energy metabolism enzymes; it is associated with the presence of metabolic disorders and is to be eliminated by an administration of coenzymes and metabolites. FUNDunding Acknowledgement Type of funding sources: Other. Main funding source(s): This research was done within the framework of the topic “Epidemiological monitoring of the population state of health and studies on molecular genetic and molecular biological mechanisms of the development of common internal diseases in Siberia for improvement of the relevant diagnostic, preventive, and therapeutic methods” in State Assignment No. 0324-2018-0001, registration No. AAAA-A17-117112850280-2

Nocturnal Hypoxemia Rather Than Obstructive Sleep Apnea Is Associated With Decreased Red Blood Cell Deformability and Enhanced Hemolysis in Patients With Sickle Cell Disease

Background: Although obstructive sleep apnea (OSA) could act as a modulator of clinical severity in sickle cell disease (SCD), few studies focused on the associations between the two diseases.Research Question: The aims of this study were: (1) to explore the associations between OSA, nocturnal oxyhemoglobin saturation (SpO2) and the history of several acute/chronic complications, (2) to investigate the impact of OSA and nocturnal SpO2 on several biomarkers (hematological, blood rheological, and coagulation) in patients with SCD.Study Design and Methods: Forty-three homozygous SCD patients underwent a complete polysomnography recording followed by blood sampling.Results: The proportion of patients suffering from nocturnal hypoxemia did not differ between those with and those without OSA. No association between OSA and clinical severity was found. Nocturnal hypoxemia was associated with a higher proportion of patients with hemolytic complications (glomerulopathy, leg ulcer, priapism, or pulmonary hypertension). In addition, nocturnal hypoxemia was accompanied by a decrease in RBC deformability, enhanced hemolysis and more severe anemia.Interpretation: Nocturnal hypoxemia in SCD patients could be responsible for changes in RBC deformability resulting in enhanced hemolysis leading to the development of complications such as leg ulcers, priapism, pulmonary hypertension or glomerulopathy.Clinical Trial Registration:www.ClinicalTrials.gov, identifier: NCT03753854.

A Novel Fragmentation Sensitivity Index Determines the Susceptibility of Red Blood Cells to Mechanical Trauma

Supraphysiological shear stresses (SSs) induce irreversible impairments of red blood cell (RBC) deformability, overstretching of RBC membrane, or fragmentation of RBCs that causes free hemoglobin to be released into plasma, which may lead to anemia. The magnitude and exposure tisme of the SSs are two critical parameters that determine the hemolytic threshold of a healthy RBC. However, impairments in the membrane stability of damaged cells reduce the hemolytic threshold and increase the susceptibility of the cell membrane to supraphysiological SSs, leading to cell fragmentation. The severity of the RBC fragmentation as a response to the mechanical damage and the critical SS levels causing fragmentation are not previously defined. In this study, we investigated the RBC mechanical damage in oxidative stress (OS) and metabolic depletion (MD) models by applying supraphysiological SSs up to 100 Pa by an ektacytometer (LORRCA MaxSis) and then assessed RBC deformability. Next, we examined hemolysis and measured RBC volume and count by Multisizer 3 Coulter Counter to evaluate RBC fragmentation. RBC deformability was significantly impaired in the range of 20–50 Pa in OS compared with healthy controls (p < 0.05). Hemolysis was detected at 90–100 Pa SS levels in MD and all applied SS levels in OS. Supraphysiological SSs increased RBC volume in both the damage models and the control group. The number of fragmented cells increased at 100 Pa SS in the control and MD and at all SS levels in OS, which was accompanied by hemolysis. Fragmentation sensitivity index increased at 50–100 Pa SS in the control, 100 Pa SS in MD, and at all SS levels in OS. Therefore, we propose RBC fragmentation as a novel sensitivity index for damaged RBCs experiencing a mechanical trauma before they undergo fragmentation. Our approach for the assessment of mechanical risk sensitivity by RBC fragmentation could facilitate the close monitoring of shear-mediated RBC response and provide an effective and accurate method for detecting RBC damage in mechanical circulatory assist devices used in routine clinical procedures.

Deep Learning Image Classification of Red Blood Cell Deformability

Red blood cells (RBCs) must be highly deformable to transit through the microvasculature to deliver oxygen to tissues. The loss of RBC deformability resulting from pathology, natural aging, or storage in blood bags can impede the proper function of these cells. A variety of methods have been developed to measure RBC deformability, but these methods require specialized equipment, long measurement time, and highly skilled personnel. To address this challenge, we investigated whether a machine learning approach could be applied to determine donor RBC deformability using single cell microscope images. We used the microfluidic ratchet device to sort RBCs based on deformability. Sorted cells are then imaged and used to train a deep learning model to classify RBCs based on deformability. This model correctly predicted deformability of individual RBCs with 84 ± 11% accuracy averaged across ten donors. Using this model to score the deformability of RBC samples were accurate to within 4.4 ± 2.5% of the value obtained using the microfluidic ratchet device. While machine learning methods are frequently developed to automate human image analysis, our study is remarkable in showing that deep learning of single cell microscopy images could be used to measure RBC deformability, a property not normally measurable by imaging. Measuring RBC deformability by imaging is also desirable because it can be performed rapidly using a standard microscopy system, potentially enabling RBC deformability studies to be performed as part of routine clinical assessments.