A comparison of results from antihuman globulin-graded reactions with the monocyte monolayer assay

Blood transfusions are a common medical treatment. Risks arise when compatible blood is not available. This study assesses the correlation between antibody reaction strength at the antihuman globulin (AHG) phase of testing and the antibody clinical significance as predicted using the monocyte monolayer assay (MMA). Multiple examples of anti-K donor plasma samples were selected to sensitize K+k+ red blood cells (RBCs). Reactivity was confirmed by testing the sensitized K+k+ RBCs at saline-AHG. Antibody titers were determined by serial dilution using neat plasma. Sixteen samples were selected for the study based on comparable graded reactions with neat plasma (1+, 2+, 3+, and 4+) and similar titration endpoints. Each sample was used to sensitize the same Kk donor and then tested by monocytes to evaluate the clinical significance using the MMA, an in vitro procedure that mimics in vivo extravascular hemolysis to predict the survivability of incompatible transfused RBCs. The monocyte index (MI), i.e., the percentage of RBCs adhered, ingested, or both versus free monocytes, was calculated for each sample. Regardless of the reaction strength, all examples of anti-K were predicted to be clinically significant. While anti-K is known to be clinically significant, the immunogenicity rate of K ensures ample supply of antibody samples for inclusion in this project. This study demonstrates that in vitro antibody strength is highly subjective and variable. These results show no correlation between graded reaction strength at AHG and the predicted clinical significance of an antibody as assessed using the MMA.