CD20 is a very useful biomarker for B-cell chronic lymphocytic leukemia (CLL). The quantification of CD20 is based on known CD4 expression on T helper cells from Cyto-Trol control cells, both stained in APC. The whole blood sample was stained with CD45 FITC, CD19 PE-Cy7 and CD20 APC, and Cyto-Trol was stained with CD45 FITC, CD3 V450 and CD4 APC in two separate sample tubes. After staining and washing, the two samples were combined in a single tube and run on a calibrated flow cytometer. (A) two individual lymphocyte gates (CD45+ and Low SSC) were drawn as ‘Cyt’ for Cyto-Trol cells and ‘Lymph’ for unknown whole blood sample in CD45 FITC vs. SSC-A; (B) gated on ‘Cyt’, T cells were identified in a dot plot of CD45 FITC vs. CD3 V450; (C) under T-cell gate, CD4 histogram shows the positive CD4+ gate, which was used to obtain respective MFI value of CD4; (D) gated on ‘Lymph’, B cells were identified in a dot plot of CD45 FITC vs. CD19 PE-Cy7; (E) gated on B cells, CD20 histogram shows the positive CD20+ gate that was used to obtain the MFI value of CD20. With measured MFI values of CD20 and CD4, CD20 expression in ABC can be obtained. The use of CD4 expression as the reference control drastically reduces the variability of CD20 expression measurements and enables quantitative measure of CD20 expression that is independent of cytometer platforms used.