{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,6]],"date-time":"2026-10-06T22:35:29Z","timestamp":1791326129394,"version":"4.3.1"},"reference-count":17,"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:p>The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold\u2010standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta\u2010associated protein (ZAP\u201070) expression was associated with unmutated IgVH genes and ZAP\u201070 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP\u201070 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP\u201070 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti\u2010ZAP\u201070 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti\u2010ZAP\u201070 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP\u201070 levels were expressed as percentage positive (POS) relative to isotype control or normal B\u2010cells or normal T\u2010cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T\u2010cells. The reported level of ZAP\u201070 expression varied greatly depending on the antibody and the method used to express the results. The CLL\/T\u2010cell ZAP\u201070 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP\u201070 expression. We assessed the variation of ZAP\u201070 expression levels in fresh cells according to storage time, which demonstrated that ZAP\u201070 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures. \u00a9 2006 International Society for Analytical Cytology<\/jats:p>","DOI":"10.1002\/cyto.b.20132","type":"journal-article","created":{"date-parts":[[2006,8,12]],"date-time":"2006-08-12T00:17:54Z","timestamp":1155341874000},"page":"309-314","source":"Crossref","is-referenced-by-count":61,"title":["Evaluation of ZAP\u201070 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization 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