Validation of a flow-cytometry-based red blood cell antigen phenotyping method

Background and Objectives: Current manual and automated phenotyping methods are based on visual detection of the antigen–antibody interaction. This approach has several limitations including the use of large volumes of patient and reagent red blood cells (RBCs) and antisera to produce a visually detectable reaction. We sought to determine whether the flow cytometry could be developed and validated to perform RBC phenotyping to enable a high-throughput method of phenotyping using comparatively miniscule reagent volumes via fluorescence-based detection of antibody binding. Materials and Methods: RBC phenotyping by flow cytometry was performed using monoclonal direct typing antisera (human IgM): anti-C, -E, -c, -e, -K, -Jk^a, -Jk^b and indirect typing antisera (human IgG): anti-k, -Fy^a, -Fy^b, -S, -s that are commercially available and currently utilized in our blood transfusion services (BTS) for agglutination-based phenotyping assays. Results: Seventy samples were tested using both flow-cytometry-based-phenotyping and a manual tube standard agglutination assay. For all the antigens tested, 100% concordance was achieved. The flow-cytometry-based method used minimal reagent volume (0.5–1 μl per antigen) compared with the volumes required for manual tube standard agglutination (50 μl per antigen). Conclusion: This study demonstrates the successful validation of flow-cytometry-based RBC phenotyping. Flow cytometry offers many benefits compared to common conventional RBC phenotyping methods including high degrees of automation, quantitative assessment with automated interpretation of results and extremely low volumes of reagents. This method could be used for high-throughput, low-cost phenotyping for both blood suppliers and hospital BTS.