Optimization and validation of a flow cytometric method for the measurement of ZAP-70 in B-cell Chronic Lymphocytic Leukemia

Abstract

B-cell Chronic Lymphocytic Leukemia (B-CLL) is a clonal lymphoproliferative disorder which exhibits clinical heterogeneity (Bosch, et al., 2006). A prognostic marker for B-CLL is Zeta-chain-associated protein kinase-70 (ZAP-70) which is an intracellular protein. When expressed in high levels in B-CLL cells, ZAP-70 implicates a poor prognostic outcome (Shults, Miller, Davis, Flye, Hobbs, & Stelzer, 2006). The aim of this study was to introduce ZAP-70 as part of the diagnostic and prognostic services in Malta by establishing a method for the determination of ZAP-70 in BCLL patients. A technically validated method of sample preparation used at the Royal Marsden Hospital, Sutton UK was optimized for use within the Haematology Laboratory, Pathology Department at Mater Dei Hospital. This method was then compared to two other methods of sample preparation to determine the best method to use at the Haematology laboratory for ZAP-70 testing. The Percentage Positivity (PP) and the Mean Fluorescent Intensity (MFI) were determined for each sample and the best approach was chosen to report ZAP-70 positivity. Statistical Analysis of the three methods showed that our optimized method, cytoplasmic to membrane staining (C&M) discriminates better between ZAP-70 positive and negative populations. Moreover, false positives were reported with the MFI approach and hence, the PP approach was chosen to identify ZAP-70 positivity. This study established the best method for ZAP-70 testing to be applied at the Haematology Laboratory, Mater Dei Hospital. This will improve the diagnostic protocol of B-CLL patients, which consequently will allow better patient management.