M-E Marquez1, P-A Deglesne, G Suarez, E Romano. 1. Laboratorio de Patología Celular y Molecular, Centro de Medicina Experimental, Instituto Venezolano de Investigaciones Científicas, Caracas, Venezuela. marmarqu@ivic.gob.ve
Abstract
INTRODUCTION: The IgV(H) 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(H) unmutated (UM). As determination of IgV(H) 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. METHODS: 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. RESULTS: We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP-70 expression and IgV(H) UM was obtained. CONCLUSIONS: Thus, the MFI ratio B-CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP-70 expression in B-CLL.
INTRODUCTION: The IgV(H) 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(H) unmutated (UM). As determination of IgV(H) 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. METHODS: 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. RESULTS: We applied the technique in a series of 53 patients. With this modification, a better correlation between ZAP-70 expression and IgV(H) UM was obtained. CONCLUSIONS: Thus, the MFI ratio B-CLL/T cell corrected by isotype is a reliable analysis technique to estimate ZAP-70 expression in B-CLL.