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

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BlankAn optimized whole blood method for flow cytometric measurement of ZAP-70

protein expression in chronic lymphocytic leukemia.

T Shankey, Meryl Forman, Scibelli, Cobb, Cecilia M ,

Rhonda Mills, Holdaway, Bernal-Hoyos, Mafalda Van Der Heiden,

Jan Popma, and Mike Keeney

Cytometry B Clin Cytom, August 11, 2006; 70B(4): 259-269.

Beckman Coulter Incorporated, Miami, Florida.

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,

berger 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. © 2006 International Society for Analytical Cytology.

PMID: 16906581