Page 253 - 2021_10-Haematologica-web

Letters to the Editor
Figure 3. Schematic summary of the sex-specific, time-dependent changes in packed red blood cells derived from men (n=12) and women (n=12). Packed red blood cells (pRBC) were cold-stored for 8 weeks (weeks 7 and 8 being additional time points exceeding expiration date). Physiological parameters were assessed with use of biochemical analysis, morphological analysis, flow cytometry, ektacytometry and atomic force microscopy. fRBC: female RBC; mRBC: male RBC; LDH: lactate dehydrogenase; RMP: red blood cell-derived mircoparticle.
clearance17 and proove that basal composition of RBC membrane is sex specific.
Expression of phosphatidylserine (PS, a marker of senescence or damage) was found to increase during fRBC and mRBC storage18 but our results suggest that mRBC are more prone to undergo membranopathies that result in higher PS exposure, at least at some point of their lifespan (Figure 1H; Online Supplementary Figure S2). In both sexes the values of maximum deformability were decreasing age-independently at the same speed during storage, whereas fRBC retained higher deformability compared to mRBC at every time point of storage (Figure 1I; Online Supplementary Figure S3). These data agree with previous research,19 which showed lower RBC deforma- bility in males. RBC, hemoglobin (HGB), hematocrit (HCT) and mean corpuscular hemoglobin concentration (MCHC) were significantly higher in mRBC compared to fRBC throughout entire storage time (Figure 1). No differ- ences in mean corpuscular hemoglobin (MCH) values between mRBC and fRBCs were detected. Mean corpus- cular volume (MCV) (Figure 1J) was relatively stable up to the fifth week of storage, at which point we saw a signif- icant increase in MCV only in mRBC. This is indicative of greater swelling of mRBC during long-term storage, which is due to higher alterations in their membranes, as reflected by higher leakage of membrane lipid and their lower deformability. Apart from the MCV increase, a slight decrease in MCHC (Figure 1L) levels during storage of mRBC confirms cell membrane loss.20
In order to study the size of RMP appearing on the RBC surface in mRBC and fRBC during storage, we applied our recently published AFM methodology7 (Figure 2). Different changes in the RMP size were observed in two clear-cut time intervals. During the first interval lasting from the first to fourth week, fluctuation of the RMP mean size, oscillating around 150 nm, was observed in mRBC. In case of fRBC a different tendency was observed: the values representing RMP sizes significantly increased from around 150 nm in the first week, up to around 200 nm in the fourth week. During the second
time interval, observed phenomena reversed in both sexes. In mRBC, a significant increase in the values from around 150 nm up to 200 nm was observed, while in fRBC the RMP sizes stabilized on the level of approxi- mately 160 nm until the end of the storage.
Our findings shed a new light on pRBC sex-derived dif- ferences in the performance during storage related to RBC membranopathy, which are summed up in Figure 3. To the best of our knowledge, for the first time we have presented significant sex-related differences between the kinetics of both cholesterol and triglyceride level changes observed in mRBC and fRBC in this work. Our results show that increased kinetics of RBC membrane lipid leakage is related with shedding of RMP of smaller size (approximately 150 nm) until the fourth week of storage and bigger RMP (approximately 200 nm) starting from the fifth week. On the other hand, our results reveal that shedding of large RMP until the fourth week of storage, seems to be an adaptation of fRBC to adverse conditions and demonstrates their higher resistance in comparison to mRBC. This is reflected by a slower membrane lipid leakage and higher deformability of fRBC during the whole time of storage. A relation observed between membrane lipids leakage and RMP formation suggests that the mechanism of RMP shedding, RMP composition and RBC response to storage conditions are all sex-relat- ed. These changes are accompanied by a higher level of hemolysis and a decreased deformability at each time point of pRBC storage observed for male donors. A sta- tistically significant increase in MCV and CD47 expres- sion of mRBC in comparison to fRBC during prolonged storage confirmed stronger swelling of mRBC, which originates from greater alterations of their membranes including rearrangements of the phospholipid bilayer. fRBC were shown to be more resilient to an unfavorable mileu which was reflected by their higher deformability, no changes in MCV, slower membrane lipid leakage and lower hemolysis. Cold storage seems to affect mRBC in a different, yet more severe way, while fRBC cope with adverse conditions more efficienty, a fact that should be
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