Abstract
Abstract 2325
Red blood cell (RBC) hemolysis during hypothermic storage is a principal criterion for assessing the quality of RBC units. The presence of free hemoglobin in RBC units can promote post transfusion injuries, such as vascular dysfunction, via accelerated nitric oxide scavenging and reactive oxygen species. It has been noted in clinical practice that specific donor red cell units are more likely to hemolyze during storage, and that female gender is associated with less hemolysis following 42 days of hypothermic storage, and during experimental mechanical stress. These findings lead us to our overarching hypothesis that genetic variations, including gender, modulate the propensity of RBCs to hemolyze under diverse stressors that may be maximized during red blood cell storage. This study further examines the relationship between gender and hemolysis in human and mouse RBCs.
Washed RBCs were obtained from RBC units stored in ADSOL for short (3–10 days old) or long (42–47 days old, expired units) time periods. The donors' age averaged 43.9±14.8 and 39.3±14.6 years, women versus men, respectively. RBCs were also obtained from 10-week-old C57BL/6J male and female mice and stored for 3 days in phosphate buffered saline. Hemolytic propensity was assessed by various stressors validated in our laboratory for 96-well plate high throughput assays. Osmotic stress was induced by incubating RBCs in hypotonic buffer containing glycerol. Oxidative hemolysis was induced by 2,2'-azobis (2-methylpropionamidine) dihydrochloride (AAPH). Mechanical stress was achieved by shaking RBC suspensions in the presence of a 3/32” stainless steel bead over 90 minutes. Percent hemolysis was measured by supernatant cell-free hemoglobin using Drabkin's assay.
Hemolysis levels (%) in response to osmotic stress were significantly (p=0.0108, Mann-Whitney test) lower in RBCs donated by women and stored for 8±2 days (16±7.1 % versus 24±7.3 % in men, n=14). This difference was also observed in expired units (31±6.0 % versus 40±6.1 % in men, n=12-16, p=0.0017). The mechanical fragility index (MFI) of premenopausal women RBCs stored for 10 days was significantly (p=0.0286) lower than that of men from a matched age group (MFI= 7.3±0.9 versus 9.4±1, respectively; n=4). Conversely, women RBC response to AAPH-induced oxidative hemolysis treatment was similar to that of men (49±4.8 % versus 48±4.7 %, respectively, n=14). Similar assays done in C57BL/6J mice revealed that female RBCs hemolyze less in response to osmotic stress (60±5.6 % versus 69±8.4 % in males, n=10, p=0.0435) or AAPH treatments (58±6 % versus 65±3.3 % in males, n=10, p=0.0106). These gender differences became noticeable after 2–3 days of storage in PBS.
Our study correlates gender with the propensity of RBCs to hemolyse under various stresses in human and mouse. It implies that under the tested conditions, RBCs collected by women are more resilient to mechanical and osmotic stresses, whereas female mouse RBCs exhibit higher resistance to osmotic and AAPH-induced oxidative stress compared with males. The mechanisms of this phenomenon are yet to be resolved and may be related to estrogen protective effects in reproductive females. Our experimental model suggests that gender differences are intrinsic to the RBC, as cells were washed and plasma or additive solution was removed. Understanding the molecular mechanisms of hemolysis can contribute to the process of RBC donor screening, reduce storage hemolysis, and possibly post-transfusion complications.
Disclosures:
Gladwin: Patents filed related to treating hemolysis.: Patents & Royalties.
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