Random Healthy Donor Sera Show Varying Effectiveness in Hemolyzing ABO Incompatible Red Blood Cells.
Authors: Dumont Larry J. , AuBuchon James P. , Herschel Louise , Roger Jill , White Thayer , Stassinopoulos Adonis 
Published: April 2020

Abstract
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 (&gt;56 mg/dL; Elliot et al, Transfusion2003;43:297) or not. A total of 1462 plasma-RBC incubations were carried out, with each of the 110 test sera tested versus the test RBC. As expected, no significant hemolysis was detected for ABO-compatible combinations (p&gt;0.8). Complete hemolysis was not observed in any combination. Of the 89 O-sera, median hemolysis with A1-RBC was 3.9% (max 33.4%), but only a median of 0.9% (max of 17.5%) with A2-RBC. There was no significant effect of serum-donor gender on hemolysis (p=0.8). Group O-sera caused higher average hemolysis than B sera for both A1 and A2-RBC. A1-RBC had a larger hemolytic response to both sera types. Notably, not all sera of the same ABO-group caused equivalent hemolysis. We observed a larger variation in hemolysis between serum donors, with an intraclass correlation coefficient ρ=0.55, than between RBC units (ρ=0.028). Therefore, in vitro testing of hemolysis potential should be against a large panel of sera to capture the inherent person-to-person variability.
Hemolysis of ABO Incompatible Mixtures of RBC and Sera Phenotype Hemolysis (%) Hb (mg/dL) Visual Lysis RBC Serum N Mean±SE (min-max) p Mean±SE (min-max) &gt; 56 mg/dL (p&lt;0.0001) A1 O 686 6.2±0.2 (0–33.4) &lt;0.0001 264±10 (0–1276) 526/686 (77%) A1 B 79 3.3±0.5 (0–14.9) 0.0057 142±20 (0–570) 43/79 (54%) A2 O 178 2.5±0.3 (0–17.5) 0.0011 113±13 (0–790) 70/178 (39%) A2 B 9 1.3±0.6 (0–5.2 0.5160 60±28 (0–236) 3/9 (33%)