Red Blood Cell Deformability Is Expressed by a Set of Interrelated Membrane Proteins.

Red blood cell (RBC) deformability, expressing their ability to change their shape, allows them to minimize their resistance to flow and optimize oxygen delivery to the tissues. RBC with reduced deformability may lead to increased vascular resistance, capillary occlusion, and impaired perfusion and oxygen delivery. A reduction in deformability, as occurs during RBC physiological aging and under blood storage, is implicated in the pathophysiology of diverse conditions with circulatory disorders and anemias. The change in RBC deformability is associated with metabolic and structural alterations, mostly uncharacterized. To bridge this gap, we analyzed the membrane protein levels, using mass spectroscopy, of RBC with varying deformability determined by image analysis. In total, 752 membrane proteins were identified. However, deformability was positively correlated with the level of only fourteen proteins, with a highly significant inter-correlation between them. These proteins are involved in membrane rafting and/or the membrane-cytoskeleton linkage. These findings suggest that the reduction of deformability is a programmed (not arbitrary) process of remodeling and shedding of membrane fragments, possibly mirroring the formation of extracellular vesicles. The highly significant inter-correlation between the deformability-expressing proteins infers that the cell deformability can be assessed by determining the level of a few, possibly one, of them.

Hemolytic Activity of Nanoparticles as a Marker of Their Hemocompatibility.

The potential use of nanomaterials in medicine offers opportunities for novel therapeutic approaches to treating complex disorders. For that reason, a new branch of science, named nanotoxicology, which aims to study the dangerous effects of nanomaterials on human health and on the environment, has recently emerged. However, the toxicity and risk associated with nanomaterials are unclear or not completely understood. The development of an adequate experimental strategy for assessing the toxicity of nanomaterials may include a rapid/express method that will reliably, quickly, and cheaply make an initial assessment. One possibility is the characterization of the hemocompatibility of nanomaterials, which includes their hemolytic activity as a marker. In this review, we consider various factors affecting the hemolytic activity of nanomaterials and draw the reader's attention to the fact that the formation of a protein corona around a nanoparticle can significantly change its interaction with the red cell. This leads us to suggest that the nanomaterial hemolytic activity in the buffer does not reflect the situation in the blood plasma. As a recommendation, we propose studying the hemocompatibility of nanomaterials under more physiologically relevant conditions, in the presence of plasma proteins in the medium and under mechanical stress.

Deformability of cord blood vs. newborns' red blood cells: implication for blood transfusion.

AIM: About 50% of premature neonates (PN) are treated with transfusion of packed red blood cells (PRBC) collected from adult donors, which has been suggested to potentially provoke PN pathologies, characterized as blood circulation disorders. RBC have properties that are key determinants of blood circulation, primarily the cell deformability. In previous studies we have shown that transfusion of RBC with reduced deformability impaired the transfusion outcome. Although RBC of PN (PN-RBC) are larger, and their microvessels are narrower than those of adults, their blood circulation is very efficient, pointing to the possibility that the deformability of adults' PRBC is inferior to that of PN-RBC, and that treating PN with PRBC transfusion might, therefore, introduce a risk to the recipients. This would infer that PN should be given RBC with high deformability. However, since using PN-RBC is not feasible, the use of cord blood RBC (CB-RBC) is a sound alternative, assuming that the deformability of CB-RBC is comparable to that of PN-RBC.The present study is aimed at testing this hypothesis. METHODS: We compared the deformability of (1) RBC of PN vs. the PRBC they received, and (2) PN-RBC vs. their autologous CB-RBC. RESULTS: 1. The deformability of the transfused PRBC is indeed inferior to that of PN-RBC. 2. The deformability of CB-RBC is equivalent to that of PN-RBC. CONCLUSION: This study supports the notion that treating PN with transfusion of adults' PRBC has the potential to introduce a circulatory risk to the recipients, while CB-RBC, with their superior deformability, provides a safer and more effective PN-specific transfusion therapy.

Determination of red blood cell adhesion to vascular endothelial cells: A critical role for blood plasma.

Red blood cell (RBC) adhesion to vascular endothelial cells (EC) is considered a potent effector of circulatory disorders, and its enhancement is implicated in the pathophysiology of numerous conditions, mainly hemoglobinopathies. The actual RBC/EC interaction is determined by both cellular and plasmatic factors, and the differentiation between them is essential for understanding its physiological implications. Yet, RBC/EC adhesion has been studied predominantly in protein-free media. To explore the plasma contribution to RBC/EC adhesion, we examined the adhesion of human RBC to human vascular endothelial cells in the presence of fresh frozen plasma (FFP) and compared it to that in a protein-free phosphate-buffered saline (PBS). RBC from blood samples freshly-collected from five healthy donors and from fifteen units of packed RBC units were used. The same FFP sample was used in all measurements. In FFP, the RBC form strongly adherent aggregates, which are dispersed as the shear stress (τ) increases to 3.0 Pa, and even at 5.0 Pa a large portion of the RBC are still adherent. In PBS, the RBC are singly dispersed and their adhesion becomes insignificant already at τ = 0.5 Pa. No cross-correlation was found between the adhesion in PBS vs. that in FFP at the same τ. However, in both media, under conditions that form singly dispersed adherent RBC, an inverse correlation between RBC/EC adhesion in PBS vs. that in FFP was observed. This study clearly implies that for understanding the physiological relevance of RBC/EC adhesion it should be determined in plasma.

Inter-donor variability in deformability of red blood cells in blood units.

OBJECTIVE: This study aimed to examine the donor-to-donor variability in the deformability of red blood cells (RBCs) from freshly collected blood donations (F-RBC) and packed RBCs. BACKGROUND: Packed RBCs are supplied for transfusion by the first-in-first-out (FIFO) criterion, assuming that their quality is the same for packed RBCs with equal storage duration. To challenge this notion, we determined the deformability of F-RBC and packed RBCs stored for different durations. METHODS: Three RBC groups were employed: A. 79 samples of F-RBC; B. 76 samples of packed RBC units, randomly used for transfusion at different storage durations; and C. 65 samples of outdated packed RBCs stored for 35 to 37 days. All packed RBC units were non-leukofiltrated and stored in Citrate-phosphate-dextrose solution with adenine (CPDA-1). RBC deformability was determined using a computerised cell-flow properties analyser, which monitors the shape change of cells directly visualised in a narrow-gap flow chamber and provides the cells' deformability distribution in a large RBC population. RESULTS: The F-RBC deformability exhibited a wide-range inter-donor variability. The cold storage of packed RBCs exerted a mild reduction of deformability, which became significant, compared to the initial inter-donor variability, only after 3 weeks of storage. CONCLUSION: Packed RBCs are generally supplied for transfusion by the FIFO criterion based on the assumption that the storage duration is a key factor of RBC quality. This study demonstrates that the deformability of red blood cells is significantly different in donors, and substantial variability persists throughout the entire process of their storage. Therefore, the FIFO criterion is not sufficient for assessing the RBC deformability, which should, therefore, be specifically characterised for each unit.

Unit-to-unit variability in the deformability of red blood cells.

BACKGROUND: In blood banking practice, the storage duration is used as the primary criterion for inventory management, and usually, the packed red blood cells (PRBC) units are supplied primarily according to first-in-first-out (FIFO) principle. However, the actual functionality of individual PRBC units is mostly ignored. One of the main features of the RBCs not accounted for under this approach is the deformability of the red cells, i.e., their ability to affect the recipients' blood flow. The objective of the study was to analyze unit-to-unit variability in the deformability of PRBCs during their cold storage. METHODS: RBC samples were obtained from twenty leukoreduced PRBC units, stored in SAGM. The deformability of cells was monitored from the day of donation throughout 42 days. RBC deformability was determined using the computerized cell flow-properties analyzer (CFA) based on cell elongation under a shear stress of 3.0 Pa, expressed by the elongation-ratio (ER). The image analysis determines the ER for each cell and provides the ER distribution in the population of 3000-6000 cells. RESULTS: The deformability of freshly-collected RBCs exhibited marked variability already on the day of donation. We also found that the aging curve of PRBC deformability varies significantly among donors. SIGNIFICANCE: The present study has demonstrated that storage duration is only one of the factors, and seemingly not even the major one, affecting the PRBCs functionality. Therefore, the FIFO approach is not sufficient for assessing the potential transfusion outcome, and the PRBC functionality should be determined explicitly for each unit.

Non-oxidative band-3 clustering agents cause the externalization of phosphatidylserine on erythrocyte surfaces by a calcium-independent mechanism.

Aging of red blood cells (RBCs) is associated with alteration in a wide range of RBC features, occurring each on its own timescale. A number of these changes are interrelated and initiate a cascade of biochemical and structural transformations, including band-3 clustering and phosphatidylserine (PS) externalization. Using specific band-3 clustering agents (acridine orange (AO) and ZnCl2), we examined whether treatment of RBCs with these agents may affects PS externalization and whether this process is Ca2+-dependent. RBCs were isolated from the blood of eight healthy donors upon obtaining their informed consent. The suspension was supplemented with increasing concentrations of AO or ZnCl2 (from 0.5 to 2.0 mM) and incubated at 25 °C for 60 min. To detect PS at the RBC surface, we used allophycocyanin-conjugated recombinant human Annexin V. We demonstrated, that treatment of RBCs with both clustering agents caused an elevation in the percent of cells positively labeled by Annexin-V (RBCPS), and that this value was not dependent on the presence of calcium in the buffer: RBCs treated with AO in the presence of either EDTA, EGTA or calcium exhibited similar percentage of RBCPS. Moreover, the active influx of Zn2+ into RBCs induced by their co-incubation with both ZnCl2 and A23187 did not increase the percent of RBCPS as compared to RBCs incubated with ZnCl2 alone. Taken together, these results demonstrate that the band-3 clustering agents (AO or ZnCl2) induce PS externalization in a Ca2+ independent manner, and we hereby suggest a possible scenario for this phenomenon.

Preparation of packed red blood cell units in the blood bank: Alteration in red blood cell deformability.

BACKGROUND: Donated blood is stored in the blood bank as packed red blood cell units. In the process of packed cells preparation, the red blood cells (RBCs) are subjectedto high level of shear stress, which can induce alterations in their properties. In the present study, we examined the effect of packed RBCs preparation (which included leuko-filtration) on red cell deformability. METHODS: Blood samples were collected from 25 healthy donors and from corresponding units of packed RBCs. The portion of undeformable cells (%UDFC) was determined for each sample. RESULTS: The median value of %UDFC was equal to 6.75 ± 0.70 %, for freshly-donated RBCs, and to 6.36 ± 0.51 %, for packed cells. Wherein, %UDFC may increase or decrease following packed cells preparation, depending upon the initial portion of undeformable cells. CONCLUSION: Likely, exposure of RBCs to high shear stress, during packed cells preparation, induces opposing effects: (a) removal/destruction of rigid (undeformable) cells, thereby reducing their total amount (i.e., decreasing the %UDFC) on the one hand, and (b) mechanical damage to the cell membrane and subsequent reduction of the cell deformability (thereby increasing the %UDFC) on the other. As a consequence, the final impact of packed cells preparation is primarily determined by the initial state of erythrocytes in the blood of the donor.

Phototherapy decreases red blood cell deformability in patients with psoriasis.

BACKGROUND: Alternations in erythrocyte deformability (ED), namley, the ability of erythrocytes to change shape under flow in the microcirculation, can contribute to cardiovascular diseases. Psoriasis, a systemic inflammatory skin disorder, is associated with an increased cardiovascular risk. The effect of psoriasis and psoriasis treatment on ED was only scarcely evaluated. OBJECTIVE: To evaluate ED changes in psoriasis patients following narrow band-ultraviolet B (NB-UVB) treatment. METHODS: Erythrocyte deformability was determined using a computerized cell flow properties analyzer in 9 patients with psoriasis before and after a course of Goeckerman regimen. ED was quantified using two parameters: average elongation ratio (AER) in the cell population, and the fraction of low deformable cells (% LDFC). RESULTS: All 9 patients showed decreased ED (i.e. impaired deformability) following NB-UVB treatment. There was a significant (p = 0.003) decrease in AER after treatment (AER±SD; 1.58±0.06) compared to the starting values (1.69±0.1). Additionally, there was a significant (p = 0.002) increase in the fraction of low deformable cells (% LDFC±SD; 60.00±9.05) compared to their fraction before treatment (34.86±11.44). CONCLUSIONS: The decreased ED observed following phototherapy could have clinical influences on psoriasis patients, and may partially explain why phototherapy does not decrease the cardiovascular risk in psoriasis compared to other treatments.

Hemodynamic Functionality of Transfused Red Blood Cells in the Microcirculation of Blood Recipients.

The primary goal of red blood cell (RBC) transfusion is to supply oxygen to tissues and organs. However, due to a growing number of studies that have reported negative transfusion outcomes, including reduced blood perfusion, there is rising concern about the risks in blood transfusion. RBC are characterized by unique flow-affecting properties, specifically adherence to blood vessel wall endothelium, cell deformability, and self-aggregability, which define their hemodynamic functionality (HF), namely their potential to affect blood circulation. The role of the HF of RBC in blood circulation, particularly the microcirculation, has been documented in numerous studies with animal models. These studies indicate that the HF of transfused RBC (TRBC) plays an important role in the transfusion outcome. However, studies with animal models must be interpreted with reservations, as animal physiology may not reflect human physiology. To test this concept in humans, we have directly examined the effect of the HF of TRBC, as expressed by their deformability and adherence to vascular endothelium, on the transfusion-induced effect on the skin blood flow and hemoglobin increment in ß-thalassemia major patients. The results demonstrated, for the first time in humans, that the TRBC HF is a potent effector of the transfusion outcome, expressed by the transfusion-induced increase in the recipients' hemoglobin level, and the change in the skin blood flow, indicating a link between the microcirculation and the survival of TRBC in the recipients' vascular system. The implication of these findings for blood transfusion practice and to vascular function in blood recipients is discussed.

Biophysical and Biochemical Markers of Red Blood Cell Fragility.

BACKGROUND: Red blood cells (RBCs) undergo a natural aging process occurring in the blood circulation throughout the RBC lifespan or during routine cold storage in the blood bank. The aging of RBCs is associated with the elevation of mechanical fragility (MF) or osmotic fragility (OF) of RBCs, which can lead to cell lysis. The present study was undertaken to identify RBC properties that characterize their susceptibility to destruction under osmotic/mechanical stress. METHODS: RBCs were isolated from freshly donated blood or units of packed RBCs (PRBCs) and suspended in albumin-supplemented phosphate-buffered saline (PBS). In addition, PRBCs were separated by filtration through a microsphere column into two fractions: enriched with rigid (R-fraction) and deformable (D-fraction) cells. The RBCs were subjected to determination of deformability, MF and OF, moreover, the level of cell surface phosphatidylserine (PS) and the stomatin level in isolated RBC membranes were measured. RESULTS: In the RBC population, the cells that were susceptible to mechanical and osmotic stress were characterized by low deformability and increased level of surface PS. The OF/MF was higher in the R-fraction than in the D-fraction. Stomatin was depleted in destroyed cells and in the R-fraction. CONCLUSION: RBC deformability, the levels of surface PS, and membrane stomatin can be used as markers of RBC fragility.

The dielectric spectroscopy of human red blood cells: the differentiation of old from fresh cells.

OBJECTIVE: The objective of the study was to gauge the effect of storage lesions on the dielectric response of red blood cells (RBC), in particular those processes linked to deformations of the cellular membrane known as the ß-dispersion. APPROACH: The dielectric response of RBC suspensions, exposed to blood-bank cold storage, was studied using time-domain dielectric spectroscopy (TDDS) in the frequency range of 500 kHz up to 1 GHz. The measured dielectric processes are characterized by their dielectric strength (Δε) and relaxation time (τ). Changes in the dielectric properties of the RBC suspensions due to storage-related lesions were evaluated. For a quantitative characterization of RBC lesions, we measured the deformability of fresh and stored RBC as expressed by their elongation ratio (ER), which was achieved under a shear stress of 3.0 Pa. MAIN RESULT: The results show that the storage of RBC induced a statistically significant decrease of dielectric relaxation times. In addition, a sound correlation between the mean values of ER and the relaxation times was observed (Spearman's correlation coefficient ρ = 0.847). We draw the conclusion that those alterations in the relaxation time are induced by changes in the shape of the RBC that happen during cold-storage. SIGNIFICANCE: The evolution of the ß-dispersion of RBC opens new possibilities in the blood bank inventory management.

Dielectric Response of Cytoplasmic Water and Its Connection to the Vitality of Human Red Blood Cells. II. The Influence of Storage.

Maintaining an appropriate inventory of packaged blood products is a critical part of modern medicine. Consequently, the assessment of red blood cell (RBC) functionality is instrumental for the monitoring of the quality of stored RBC (sRBC) in the blood bank. We present a comprehensive study of sRBC lesion kinetics in SAGM (saline, adenine, glucose, mannitol) solution, using microwave dielectric spectroscopy (0.5-50 GHz) and cell deformability. As part of the research, we have isolated the microwave dielectric response of cytoplasmic water in sRBC. The extracted dielectric parameters are sensitive to the age of the cells and, in particular, to the critical moment of transition from discocyte to echinocyte. From the analysis of the dielectric relaxation as a function of storage-duration, we postulate that the behavior is rooted in the delicate interplay between bound and bulk water in the cellular interior. In particular, the microwave dielectric response reflects the moment when the continuous diffusion of oxygen to the cell and the oxygenation of hemoglobin affects the role played by water in the maintenance of cell integrity. These results open a possible new avenue for the noninvasive inspection of stored red blood cells, permitting a true inventory system for the modern blood bank.

Phospholipase D messenger RNA expression and clinical role in high-grade serous carcinoma.

The objective of this study was to analyze the expression and clinical role of phospholipase D (PLD) in high-grade serous carcinoma (HGSC). PLD1 and PLD2 isoform expression was studied in 125 HGSC specimens (73 effusions, 28 ovarian tumors, 24 solid metastases) using quantitative real-time reverse-transcription polymerase chain reaction. Expression levels were analyzed for association with clinicopathological parameters, including chemoresponse, and survival. PLD1 and PLD2 isoforms were found in most specimens at all anatomic sites, and their levels were strongly positively related (P<.001 for effusions and solid lesions). PLD2 messenger RNA (mRNA) expression was significantly higher in effusions compared with both carcinomas in the ovary and solid metastases (P<.001). Higher levels of both isoforms were associated with higher CA 125 levels at diagnosis (P<.001), and higher PLD2 mRNA levels in effusions were associated with unfavorable response to chemotherapy (P=.021). Expression levels of the studied isoforms were unrelated to the levels of previously studied mRNAs that form part of the phospholipase A2 pathway or to survival. The present study provides the first evidence of PLD expression in HGSC and suggests a role in mediating progression to effusions and chemoresistance in this cancer.

Erythrocyte survival is controlled by microRNA-142.

Hematopoietic-specific microRNA-142 is a critical regulator of various blood cell lineages, but its role in erythrocytes is unexplored. Herein, we characterize the impact of microRNA-142 on erythrocyte physiology and molecular cell biology, using a mouse loss-of-function allele. We report that microRNA-142 is required for maintaining the typical erythrocyte biconcave shape and structural resilience, for the normal metabolism of reactive oxygen species, and for overall lifespan. microRNA-142 further controls ACTIN filament homeostasis and membrane skeleton organization. The analyses presented reveal previously unappreciated functions of microRNA-142 and contribute to an emerging view of small RNAs as key players in erythropoiesis. Finally, the work herein demonstrates how a housekeeping network of cytoskeletal regulators can be reshaped by a single micro-RNA denominator in a cell type specific manner.

Deformability of transfused red blood cells is a potent determinant of transfusion-induced change in recipient's blood flow.

OBJECTIVE: There is a growing concern regarding the risks in the transfusion of PRBC, as numerous studies have reported negative transfusion outcomes, including reduced blood perfusion. In search of this phenomenon's mechanism, the effect of PRBC deformability, a major determinant of blood flow, on transfusion outcome was explored. METHODS: The effect of PRBC deformability was examined by the transfusion-induced change in recipients' ∆SBF, in ß-TM patients, who are routinely treated with lifelong frequent transfusions. SBF was determined using a laser Doppler imager. RESULTS: ∆SBF was examined vs PRBC deformability, the transfusion-induced increase in ∆Hct and the recipients' SBF before transfusion (SBFB ). ∆SBF elevated with increasing PRBC deformability, with a highly significant dependence, while its elevation with ∆Hct was much less significant. ∆SBF was inversely proportional to the SBFB . CONCLUSIONS: This study provides, for the first time in humans, direct evidence that the deformability of transfused PRBC is a potent effector of transfusion outcome. Currently, PRBC are supplied primarily by the first-in-first-out criteria, while their functionality is ignored. The testing of PRBC hemodynamic quality would introduce a new paradigm into blood banking, which would contribute substantially to improving transfusion therapy.