Donor variability affects the outcome for the monocyte monolayer assay (MMA)

The monocyte monolayer assay (MMA) is used to predict transfusion outcomes when antibodies to red blood cell (RBC) antigens are present and compatible antigen-negative blood isn’t available. Typically, the monocytes used are peripheral blood mononuclear cells (PBMC) isolated from healthy volunteers. This study aimed to determine the between-volunteer variability in monocyte reactivity in the MMA. PBMCs were isolated (Ficoll density gradient) from freshly collected whole blood from 28 individuals and incubated with RBCs sensitised with anti-D (1 hour, 37oC, 5% CO2; positive control). Monocytic index (MI%) was calculated after microscopic analysis of the proportion of monocytes with phagocytosed or adherent RBCs. Mean MI was 23.6% (SD 10.35); however, between-volunteer variation was wide (MI range: 1.6–45.9%). Based on a 20% MI as a cut off, 11 volunteers (39.3%) did not reach the positive threshold for the anti-D positive control, which would result in assay failure and inconclusive results that could not be used to inform transfusion decision making. These results indicate MMA outcomes are impacted by individual volunteer monocytes. Therefore, a curated pool of volunteers with demonstrated monocyte reactivity is required for a reliable MMA. Further investigation of monocyte characteristics that contribute to the variability of MMA results between individuals is warranted.