MFI ratio estimation of ZAP-70 in B-CLL by flow cytometry can be improved by considering the isotype-matched antibody signal.

The IgV mutational status of B-cell chronic lymphocytic leukemia (B-CLL) is of prognostic value. Expression of ZAP-70 in B-CLL is a surrogate marker for IgV unmutated (UM). As determination of IgV mutational status involves a methodology currently unavailable for most clinical laboratories, it is important to have available a reliable technique for ZAP-70 estimation in B-CLL. Flow cytometry (FC) is a convenient technique for this purpose. However, there is still no adequate way for data analysis, which would prevent the assignment of false positive or negative expression. We have modified the currently most accepted technique, which uses the ratio of the mean fluorescent index (MFI) of B-CLL to T cells. The MFI for parallel antibody isotype staining is subtracted from the ZAP-70 MFI of both B-CLL and T cells. We validated this technique comparing the results obtained for ZAP-70 expression by FC with those obtained with quantitative PCR for the same patients. We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP-70 expression and IgV UM was obtained. Thus, the MFI ratio B-CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP-70 expression in B-CLL. [ABSTRACT FROM AUTHOR]