Use of a Monocyte Monolayer Assay to Evaluate Fcγ Receptor-mediated Phagocytosis

Abstract

Although originally developed for predicting transfusion outcomes of serologically incompatible blood, the monocyte monolayer assay (MMA) is a highly versatile in vitro assay that can be modified to examine different aspects of antibody and Fcγ receptor (FcγR)-mediated phagocytosis in both research and clinical settings. The assay utilizes adherent monocytes from peripheral blood mononuclear cells isolated from mammalian whole blood. MMA has been described for use in both human and murine investigations. These monocytes express FcγRs (e.g., FcγRI, FcγRIIA, FcγRIIB, and FcγRIIIA) that are involved in immune responses. The MMA exploits the mechanism of FcγR-mediated interactions, phagocytosis in particular, where antibody-sensitized red blood cells (RBCs) adhere to and/or activate FcγRs and are subsequently phagocytosed by the monocytes. In vivo, primarily tissue macrophages found in the spleen and liver carry out FcγR-mediated phagocytosis of antibody-opsonized RBCs, causing extravascular hemolysis. By evaluating the level of phagocytosis using the MMA, different aspects of the in vivo FcγR-mediated process can be investigated. Some applications of the MMA include predicting the clinical relevance of allo-or autoantibodies in a transfusion setting, assessing candidate drugs that promote or inhibit phagocytosis, and combining the assay with fluorescent microscopy or traditional Western immunoblotting to investigate the downstream signaling effects of FcγR-engaging drugs or antibodies. Some limitations include the laboriousness of this technique, which takes a full day from start to finish, and the requirement of research ethics approval in order to work with mammalian blood. However, with diligence and adequate training, the MMA results can be obtained within a 24-h turnover time.

Evaluation of the functional properties of cryopreserved buffy coat–derived monocytes for monocyte monolayer assay

“…Also, MMA showed very high levels of phagocytosis of anti‐AnWj–sensitized RBCs despite an IAT result showing that only a small amount (1+) of AnWj antibodies were bound to the RBCs. This phenomenon has been described in the past where studies have shown that the strength of opsonization does not necessarily correlate with significant monocyte interactions leading to phagocytosis …”

“…Additionally, we investigated the use of more readily available O+, D unphenotyped RBCs for MMA, which alleviated the need for finding an R 2 R 2 donor for each experiment or for the additional procedures of cryopreserving and preparing such control RBCs. Using the same PI acceptability criteria for R 2 R 2 ‐positive controls as in previous studies, we determined that all O D+ cells from the RBCs used in this study showed high rates of opsonization (IAT = 4+) and did not affect phagocytosis by the monocytes (PI > 60) and therefore could be deemed appropriate and valid positive controls. This further facilitates the implementation and interpretation of this assay, as we are able to set quantitative limits to control for the interdonor variability on phagocytosis in the MMA.…”

“…Anti‐D–sensitized RBCs consistently resulted in a strong (4+) IAT reactivity. Studies have generally indicated a requirement of R 2 R 2 ‐phenotyped cells for use as positive controls as this is the phenotype with the greatest expression of D antigen on the surface of the RBCs for sensitization . For all our experiments, group O D+ RBCs from donor units were used as positive controls.…”

“…As the number of RBCs phagocytosed by each monocyte solely determines PI, the presence of a few hyperactive monocytes could falsely increase the PI, thus misrepresenting the clinical importance of an alloantibody . We therefore examined the phagocytosis characteristics exhibited by MCBCs, MFBCs, and MFPBs.…”

“…Early reports by Hunt and colleagues, Schanfield and colleagues, and Nance and colleagues employed the MMA assay for the detection and determination of in vitro RBC alloantibody clinical significance. MMA utility has evolved over the years to include the examination of hemolysis associated with intravenous immunoglobulin administration and to evaluate receptor‐mediated phagocytosis …”