Comparative analysis of flow cytometric techniques in assessment of ZAP-70 expression in relation to IgVH mutational status in chronic lymphocytic leukemia.

We compared 1 subjective and 5 objective flow cytometric methods to evaluate zeta-associated protein (ZAP-70) expression in relation to immunoglobulin heavy-chain variable-region (IgVH) gene mutational status in 154 samples from 125 patients with chronic lymphocytic leukemia (CLL). ZAP-70 expression determined by all methods used correlated with IgVH gene mutational status, but none of them demonstrated high concordance rates. Of the objective methods, ZAP-70 staining determined as a ratio of molecules of equivalent soluble fluorochrome intensity in CLL cells to that in normal B cells (ZAP-70+ staining in IgVH germline cases, 59%; ZAP-70- in IgVH mutated cases, 75%) or T cells (ZAP-70+ in IgVH germline cases, 66%; ZAP-70- in IgVH mutated cases, 57%) provides the best combination for assigning ZAP-70+ status to IgVH germline and ZAP-70- status to IgVH mutated cases. The subjective method based on ZAP-70 expression in natural killer/T cells gave a similar result, but reproducibility between laboratories may be difficult. Further studies on ZAP-70 expression in relation to clinical parameters may address whether ZAP-70 is an independent prognostic marker for CLL.

Optimized whole-blood assay for measurement of ZAP-70 protein expression.

Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP-70 protein expression in whole-blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly improved signal-to-noise ratio, an optimized combination of antibodies-fluorophores to maximize ZAP-70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter- and intra-laboratory reproducibility, and a method to index ZAP-70 protein expression levels to internal positive and negative cell populations. Residual normal T and B cells function as internal positive and negative controls. These are used to index ZAP-70 protein expression levels in the CLL population.

An optimized whole blood method for flow cytometric measurement of ZAP-70 protein expression in chronic lymphocytic leukemia.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.