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.