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Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: a methodological approach

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Abstract

Zeta-associated protein 70 (ZAP-70), determined by flow cytometry, has been advocated a surrogate marker of immunoglobulin (Ig)VH unmutated status in B chronic lymphocytic leukemia (CLL). The aim of the current study was to test the applicability of an easy flow cytometry protocol for ZAP-70 measurement in CLL samples. Samples from 61 CLL patients and 44 normal subjects were analyzed using a commercial ZAP-70 monoclonal antibody (1E7.2 clone) conjugated with phycoerythrin (PE) and Alexa 488 fluorochromes. Modifications of the published methods led to the structure of a simplified in-house method of ZAP-70 measurement. A three-color approach was used with CD19, CD3 gating comparing with the isotype control provided by the same manufacturer. The cutoff levels for ZAP-70 positivity were defined from a receiver operator characteristic curve in relation to the IgVH mutational status and from the ln normalized mean value +2 SD of normal controls. Using the 20% cutoff value for ZAP-70 positivity in CLL patients defined by the literature, ZAP-PE had a sensitivity of 55% and a specificity of 98% in predicting the IgVH mutational status, whereas the corresponding values for ZAP-Alexa were 55% and 84%, respectively. Using the 7% cutoff value for CD38 positivity, the sensitivity was 55%, whereas the specificity was 76%. ZAP-70-positive patients showed a shorter time to disease progression in comparison with ZAP-70-negative patients (p<0.001). In conclusion, the 100% specific prediction of mutational status is accompanied by reduced sensitivity, thus limiting ZAP-70’s applicability either as a single marker or combined with CD38 for the assessment of the mutational status of CLL.

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Authors and Affiliations

  1. Immunology Department and National Histocompatibility Centre, “G Gennimatas” General Hospital, Athens, Greece

    Freda Passam, Dimitra Skoumi, Aikaterini Stavropoulos-Giokas & Georgios Paterakis

  2. Hematology Department and Hematopoietic Cell Transplantation Unit, “G. Papanicolaou” Hospital, Thessaloniki, Greece

    Varvara Tachynopoulou, Aliki Tsompanakou, Chrysanthi Vadikolia & Achilles Anagnostopoulos

  3. 21 Leonidou Str, 15341, Athens, Greece

    Georgios Paterakis

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  1. Freda Passam
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Correspondence to Georgios Paterakis.

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Passam, F., Tachynopoulou, V., Skoumi, D. et al. Feasibility of an easily applicable method of ZAP-70 measurement in chronic lymphocytic leukemia in the routine flow cytometry setting: a methodological approach. Ann Hematol 85, 795–805 (2006). https://doi.org/10.1007/s00277-006-0159-4

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