Evaluating the monocyte monolayer assay: assay conditions, inflammatory profile, and international review of best practice

In certain clinical scenarios, such as multiparity or repeated whole blood transfusions, patients develop antibodies that are directed against foreign red blood cell (RBC) antigens (alloantibodies). For patients with multiple alloantibodies, or rare blood groups, it may be difficult to source antigennegative RBC to transfuse. However, not all alloantibodies will initiate damage to transfused RBC following exposure to their concordant antigens. Antibodies may be clinically significant, not clinically significant, or have variable clinical significance. The monocyte monolayer assay (MMA) is used to determine whether an alloantibody will induce donor RBC damage in vivo. However, the mechanisms underpinning the MMA are poorly understood and there is a lack of standardisation between laboratories that use it. This project aimed to investigate aspects of the MMA, including global procedures and practices, underlying mechanisms, and alternative methods for a higher throughput and more objective results. Published international MMA procedures were assessed by review and a survey, which was distributed to blood providers internationally. Two commonly used readout indexes were then compared – the phagocytic index (PI) and the monocytic index (MI). The THP-1 cell line was investigated as an alternative monocyte source. To determine the suitability of THP-1 cells in the MMA, the MI and PI readouts of peripheral blood mononuclear cells (PBMC) and THP-1 were compared. Finally, the inflammatory profiles of MMA supernatants from assays that