Effect of red blood cell lysing agent in leukemia/lymphoma immunophenotyping by flow cytometry

RBC lysing agent’s effect on flow cytometry

Abstract

Background: Getting rid of the red blood cells (RBC) is an integral part of flow cytometry (FCM)-based leukemia/lymphoma immunophenotyping. It is routinely done using an RBC lysing agent. RBC lysing has numerous consequences for the physical and immunophenotypic characteristics of the cell. We aimed to analyse the effect of different RBC lysing agents (with or without paraformaldehyde) on the antibody staining pattern. Methods: Three different RBC lysis preparations were used, namely BD FACSLyse (FL) and BD PharmLyse (PL), and a laboratory-prepared (home-brewed, HB) ammonium chloride-based reagent. A lyse-stain-wash protocol was used to prepare the cells, which were stained using a single tube of five-color antibodies (CD20-BV605, CD8-BV510, CD3-FITC, CD4-CD19-PeCy7, and CD45-APCR700). The effects of different lysings on different cellular characteristics, including the median fluorescence intensity (MFI) of CD45 in lymphocytes, CD3 in T lymphocytes, CD19 in B lymphocytes, CD20 in B lymphocytes, CD4 in T helper cells, CD8 in cytotoxic T cells, and CD4 in monocytes, were noted. Results: The duration needed for the RBC lysis using PL and HB was longer than that with FL (15–20 minutes vs. 10 minutes). The morphology of leukocytes was better preserved with the FL as compared to the HB and PL. The MFI of most of the antigens was similar for the HB and PL, but it was significantly lower for the FL. Conclusion: We should be aware of the changes that these agents may bring to the physical or immunophenotypic properties so as to choose the appropriate agent.