Flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia: correlation with immunocytochemistry and Western blot analysis.

Abstract

Expression of ZAP-70 in chronic lymphocytic leukemia (CLL) predicts worse clinical outcome in patients with early-stage disease. It has become important to include ZAP-70 in the immunophenotyping panel used to diagnose CLL, commonly performed by flow cytometry (FC). Nevertheless, the methodology used to detect ZAP-70 by FC has not been extensively evaluated. To describe our FC method for detecting ZAP-70 in CLL and assess whether this assay is useful in estimating the ZAP-70 protein level in CLL cells. ZAP-70 expression was assessed by FC in 45 consecutive newly diagnosed CLL patients, and the results were correlated with those of immunocytochemistry and Western blot analysis. With >25% ZAP-70-positive B cells as the cutoff, the FC results had a perfect concordance with those of immunocytochemistry (39/39, 100%) and Western blot analysis (7/7, 100%). The use of autofluorescence controls was found to be superior to other alternatives. Overall, 19 (42%) of 45 cases were ZAP-70 positive in our series. Since only 7 cases (16%) had >20% to 30% ZAP-70-positive B cells, the cutoff of >25% readily separated CLL into positive and negative groups in most cases. ZAP-70 positivity was significantly associated with atypical morphology but not other laboratory parameters evaluated. With proper specimen processing and the use of directly fluorescence-conjugated anti-ZAP-70 antibody, one can readily incorporate ZAP-70 into the routine FC study panel for CLL. Our data suggest that FC is a rapid and useful method to estimate the ZAP-70 protein expression level in CLL.