ABO titers: harmonization and identifying clinically relevant ABO antibodies

M inor ABO-incompatible transfusions and transplants are commonplace in medicine. Estimating the antibody concentration with a titer method is the standard by which clinical decisions are made for ABO-incompatible plasma-containing transfusions, solid organs, and stem cell transplants. Administeringminor ABO-incompatible products carries some risk to the patient: recipients of minor ABO-incompatible platelet transfusions can develop anemia due to overt vascular hemolysis (rare) or insidious extravascular hemolysis; the full impact on trauma patients of anti-A and anti-B in low-titer whole blood is unknown; some group B solid-organ recipients simply do not qualify for an A2or A2B-incompatible organ due to arbitrarily defined “high” titers; and delayed engraftment or failure to engraft is still a potential outcome for ABOincompatible stem cell transplantation. Our basic understanding of IgM ABO naturally occurring and immune antibodies can be traced to studies from the 1960s and 1970s. ABO antibodies develop within 3 to 6 months of life, likely a result of microbial exposure, and peak by the fifth to seventh year of life. Studies performed on blood donors revealed that these antibodies wane by midlife and can significantly decrease or may be absent in adults 65 years of age or older. Naturally occurring ABO antibodies typically react at “room temperature” or lower and can be a single class or a mixture of classes: IgM, IgG, and IgA. For unknown reasons, IgG2 antibodies, which poorly activate complement, are commonly formed against carbohydrate moieties like A and B antigens. Among group O individuals, anti-A titers are often higher than anti-B titers. Hemolysin activity of ABO antibodies has been observed for many years, with the ability of both IgM and IgG to hemolyze red blood cells (RBCs). “Immune” ABO antibodies, however, can be either of the IgM or IgG class and are deemed immune because they 1) readily hemolyze human RBCs and 2) agglutinate porcine A-like RBCs. Thus, the complement-activating property of a particular anti-A or anti-B may be more important than the titer, and hemolytic activity is not restricted to the IgM class alone. There is a real need to revisit what defines a hemolysin and begin to isolate the factors that make a particular ABO antibody a “hemolysin”. Sensitive methods can demonstrate that 98% of all ABO antibodies can activate complement C3, and therefore complement activation alone does not define “hemolysin. ” Group O individuals apparently possess a minor portion of “linked” anti-A,B that can be demonstrated only by adsorption/elution studies. However, group A and B RBCs used in these studies might be inadequate to determine the existence of anti-A,B. This situation occurs because the A and B glycosyltransferases are leaky, whereby the critical group A– and group B–defining amino acids in the globular (enzymatic) region infrequently add the incorrect terminal sugar residue to RBCs. Adsorption/elution studies using group A RBCs are “contaminated” with small amounts of group B antigen. Thus, the existence of anti-A,B is difficult to prove with human RBCs, and its clinical relevance is limited since the antigen structure has yet to be identified. On the other hand, we do know that anti-A,B cannot be obtained from group A blood. Suffice it to say that anti-A,B if it does exist is in low amounts and found only among group O individuals. Moieties expressing ABO blood group antigens represent a widely expressed biologically diverse set of molecules that participate in immune recognition. Six core substrate configurations are possible when expressing ABO sugars, but only Types II, III, and IV are found on RBCs. Differences in expression are commonly observed in blood banks, as A2 individuals have one-tenth the amount of A antigen on RBCs, and the A2-associated glycosyltransferase fails to add N-acetyl-galactosamine to Type III and IV glycolipid chains. Transfusion medicine has seen a transformation from the initial studies performed on ABO isoagglutinins to significant advancements in the semiquantitation of antibody levels. Studies to understand the relative difference between titration methods and interlaboratory variation has been the topic of College of American Pathologists proficiency challenge questions. When reporting proficiency sample results, clear distinction of key elements of the method of testing is necessary for comparative purposes. Yet, there is a problem since the interlaboratory differences can be three or more doublingdilution tubes. The bottom line is that harmonizing titer methods does not lower the interlaboratory variability. Attempts to standardize ABO titers with an automated instrument are steps in the right direction. In this issue of TRANSFUSION, Lally et al. provide data on the conversion of ABO titers from a manual gel method to an automated instrument (Galileo, Immucor). They compared two critical steps: the conversion from gel to solid phase and from manual dilutions to automated dilutions performed by the instrument. The investigators abandoned routine testing with dithiothreitol (DTT), and have some 10 to 12 yearsʼ experience with the manual gel method. Certainly, ABO titers performed with DTT-treated serum at room temperature do not reflect a doi:10.1111/trf.15726 © 2020 AABB TRANSFUSION 2020;60;441–443