Standardization of the Monocyte Monolayer Assay: Lessons Learned From Laboratory Optimization and Literature Review

ABSTRACT Objectives Several variables influence the outcome of cellular assays. We conducted a comprehensive literature review to identify the factors affecting the Monocyte Monolayer Assay (MMA) and subsequently standardized the assay through systematic in‐house laboratory trials. Methods The study was conducted in two phases. Phase 1 involved a literature review using PubMed and Scopus databases to identify factors influencing the assay. Phase 2 included experimental trials to evaluate the effects of these factors. Monocytes were isolated from buffy coat bags, and assay parameters, such as centrifugation speed, monocyte adherence conditions, RBC sensitization, incubation time, and staining, were systematically standardized. A blinded evaluation was employed for slide reading to assess the influence of procedural factors on assay reliability. Results A literature review using the PubMed and Scopus databases identified key variables influencing MMA performance. Twelve sequential trials were conducted to identify the effects of these variables on assay outcome. Optimal centrifugation speed and time was 600 × g for 15 min, and the use of four buffy coat bags improved monocyte yield to 8.3 × 10 6 /mL. Dilution with Phosphate buffer saline (PBS, pH 7.2) and use of Histopaque provided consistent cell recovery. A 1‐h incubation yielded optimal monocyte adherence with minimal lysis. The final protocol demonstrated high reproducibility and improved visualization of phagocytosis. Conclusion This study emphasizes the importance of standardizing the MMA procedure before implementation. Optimizing the parameters to ensure enhanced monocyte yield, reduced cell lysis, and reliable interpretation for assessing the clinical significance of red cell alloantibodies is essential.