{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,17]],"date-time":"2026-09-17T22:13:06Z","timestamp":1789683186941,"version":"4.0.1"},"reference-count":31,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2006,8,11]],"date-time":"2006-08-11T00:00:00Z","timestamp":1155254400000},"content-version":"vor","delay-in-days":27,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Part B Clinical"],"published-print":{"date-parts":[[2006,7,15]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background:<\/jats:title><jats:p>ZAP\u201070 protein expression has been proposed as a marker for immunoglobulin heavy chain mutational status, which some studies have correlated with disease course in B\u2010cell chronic lymphocytic leukemia (CLL). Studies published to date measuring levels of expression of ZAP\u201070 intracellular protein using flow cytometry have demonstrated poor performance, as defined by the difference in signal in known positive and negative lymphocyte populations.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods:<\/jats:title><jats:p>A recently published method (Chow S, Hedley DW, Grom P, Magari R, Jacobberger JW, Shankey TV, Cytometry A 2005;67:4\u201317) to measure intracellular phospho\u2010epitopes was optimized using a design of experiments (DOE) approach to provide the best separation of ZAP\u201070 expression in positive T\u2010 or NK\u2010cells as compared to negative B\u2010cells in peripheral blood samples. A number of commercially available anti\u2010ZAP\u201070 antibody\u2010conjugates were screened using this methodology, and the antibody\u2010conjugate showing the best performance was chosen to develop a four\u2010color, five antibody assays to measure ZAP\u201070 levels in whole blood specimens.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results:<\/jats:title><jats:p>Using the optimized fixation and permeabilization method, improvement in assay performance (signal\u2010to\u2010noise, 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\u2010up protocols, we obtained both intra\u2010 and interlaboratory reproducibility in the analysis of ZAP\u201070 expression in whole blood samples from normal and CLL patients.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions:<\/jats:title><jats:p>The development of a sensitive, specific and highly reproducible ZAP\u201070 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\u2010based cutoff points for clinical outcomes must next be established before ZAP\u201070 protein analysis can be routinely implemented in the clinical laboratory. \u00a9 2006 International Society for Analytical Cytology<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.b.20135","type":"journal-article","created":{"date-parts":[[2006,8,12]],"date-time":"2006-08-12T00:17:04Z","timestamp":1155341824000},"page":"259-269","source":"Crossref","is-referenced-by-count":43,"title":["An optimized whole blood method for flow cytometric measurement of ZAP\u201070 protein expression in chronic lymphocytic leukemia"],"prefix":"10.1002","volume":"70B","author":[{"given":"T. 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