ZAP‐70 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B‐cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP‐70 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.

A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4–17) to measure intracellular phospho‐epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP‐70 expression in positive T‐ or NK‐cells as compared to negative B‐cells in peripheral blood samples. A number of commercially available anti‐ZAP‐70 antibody‐conjugates were screened using this methodology, and the antibody‐conjugate showing the best performance was chosen to develop a four‐color, five antibody assays to measure ZAP‐70 levels in whole blood specimens.

Using the optimized fixation and permeabilization method, improvement in assay performance (signal‐to‐noise, S/N) was seen in most of the antibodies tested. The custom SBZAP conjugate gave the best S/N when used in conjunction with this optimized fixation /permeabilization method. In conjunction with carefully standardized instrument set‐up protocols, we obtained both intra‐ and interlaboratory reproducibility in the analysis of ZAP‐70 expression in whole blood samples from normal and CLL patients.

The development of a sensitive, specific and highly reproducible ZAP‐70 assay represents only the first essential step for any clinical assay. The universal implementation of a validated data analysis method and the establishment of methodology‐based cutoff points for clinical outcomes must next be established before ZAP‐70 protein analysis can be routinely implemented in the clinical laboratory. © 2006 International Society for Analytical Cytology