Random Healthy Donor Sera Show Varying Effectiveness in Hemolyzing ABO Incompatible Red Blood Cells.

Mixing of allogeneic serum with RBC that are ABO-incompatible is well known to cause lysis of the RBC. However, the severity and extent of such hemolysis may not be the same for every mismatched pairing and is not documented in the literature. We were interested in quantitating the lytic potential of a large panel of normal sera when exposed to ABO-incompatible RBC. We collected 80mL of whole blood in red-top tubes from 110 random healthy subjects (84 female 26 male) with blood Groups A (n=6), AB (n=5), B (n=10) and O (n=89). Sera were prepared by clotting at room temp, frozen in aliquots, stored at −80°C. CPD whole blood (N=14) from healthy subjects were obtained commercially, shipped and stored per standard methods (A₁: 8, A₂: 2, O: 4). Forward ABO typing on all serum subjects and whole blood was with Immucor-Gamma murine monoclonal antibody reagents. Within 48 h of donation, packed RBC were prepared by centrifugation of an aliquot at 900xg for 5 min. RBC were resuspended in AS-3 to a final hematocrit of 10%. Total Hb was measured on a BayerAdvia120 analyzer, and hematocrit was determined by capillary tube centrifugation. Aliquots of 125μL of this RBC suspension were incubated with 250μL aliquots of the serum panel for 30 min at 37°C followed by double centrifugation at 900xg for 5 min. Supernatant (sup) was assayed for Hb by the cyanmethemoglobin method reading on a spectrophotometer at 540nm with a turbidity correction for 680nm absorbance. Results were corrected for test serum and RBC plasma absorbances. Percent hemolysis was calculated as 100xHb(sup, mg/dL)x(1-hct)/Hb(total, mg/dL). A repeated measures analysis of variance mixed-effects model was used to test the effect of serum-donor sex, serum-donor ABO group and RBC ABO group on hemolysis. Sup Hb was categorized into visually detectable hemolysis (>56 mg/dL;