Comparison of flow cytometric methods for the measurement of ZAP‐70 expression in a routine diagnostic laboratory

Chronic lymphocytic leukaemia (CLL) follows a variable clinical course with patient survival ranging from only a few years despite treatment, to several decades in patients who may never require clinical intervention. Determination of the mutational status of a patient's immunoglobulin heavy chain variable region (Ig V_H) gene has been used to provide prognostic information, but this assay is not available in most laboratories. The discovery of the expression of the protein tyrosine kinase zeta‐associated protein (ZAP)‐70 in V_H‐unmutated CLL cases led to its proposal as a surrogate marker for V_H status. This study investigated the measurement of ZAP‐70 expression in CLL using different flow cytometric protocols. Two different antibodies and two different staining methods were compared. The Caltag ZAP‐70 antibody and Fix & Perm kit were the easiest to use and were the most sensitive and specific combination, with 91% concordance between ZAP‐70 expression and V_H status. Three patients (9%) were discordant (two V_H mutated/ZAP‐70 positive, and one V_H unmutated/ZAP‐70 negative). No correlation existed between CD38 and either ZAP‐70 expression or V_H status. Measurement of ZAP‐70 expression using the Caltag antibody/kit combination provides a standardized flow cytometric method that could be introduced into a routine CLL immunophenotyping panel in a clinical diagnostic laboratory.