T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B-cells due to ZAP70 overexpression in patient T-cells

Authors

  • David Rizzo,

    1. Department of Biology, Laboratory of Haematology, University Hospital Dupuytren, Limoges, France
    2. Centre National de la Recherche Scientifique, UMR CNRS 7276 CRIBL, Faculté de Médecine de Limoges, France
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  • Gaëlle Bouvier,

    1. Beckman-Coulter Immunotech, Marseille, France
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  • Ibtissam Youlyouz-Marfak,

    1. Centre National de la Recherche Scientifique, UMR CNRS 7276 CRIBL, Faculté de Médecine de Limoges, France
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  • Estelle Guerin,

    1. Department of Biology, Laboratory of Haematology, University Hospital Dupuytren, Limoges, France
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  • Franck Trimoreau,

    1. Department of Biology, Laboratory of Haematology, University Hospital Dupuytren, Limoges, France
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  • Dominique Bordessoule,

    1. Centre National de la Recherche Scientifique, UMR CNRS 7276 CRIBL, Faculté de Médecine de Limoges, France
    2. Department of Oncology and Haematology, Haematology and Cellular Therapy Unit, University Hospital Dupuytren, Limoges, France
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  • Arnaud Jaccard,

    1. Centre National de la Recherche Scientifique, UMR CNRS 7276 CRIBL, Faculté de Médecine de Limoges, France
    2. Department of Oncology and Haematology, Haematology and Cellular Therapy Unit, University Hospital Dupuytren, Limoges, France
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  • Nathalie Gachard,

    1. Department of Biology, Laboratory of Haematology, University Hospital Dupuytren, Limoges, France
    2. Centre National de la Recherche Scientifique, UMR CNRS 7276 CRIBL, Faculté de Médecine de Limoges, France
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  • Jean Feuillard

    Corresponding author
    1. Department of Biology, Laboratory of Haematology, University Hospital Dupuytren, Limoges, France
    2. Centre National de la Recherche Scientifique, UMR CNRS 7276 CRIBL, Faculté de Médecine de Limoges, France
    • Laboratoire d'Hématologie and UMR CNRS 7276, 2 Avenue Martin Luther King, 87042 Limoges Cedex, France
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  • How to cite this article: Rizzo D, Bouvier G, Youlyouz-Marfak I, Guerin E, Trimoreau F, Bordessoule D, Jaccard A, Gachard N, Feuillard J. T/B ratio does not reflect levels of ZAP70 expression in clonal CLL B-cells due to ZAP70 overexpression in patient T-cells. Cytometry Part B 2013; 84B: 125–132.

Abstract

Flow cytometry is the reference technique for assessing ZAP70 expression, a marker of poor prognosis in CLL. One of the most common methods is to assess ZAP70 levels in CLL cells by calculating the ratio between ZAP70 mean fluorescence intensities (MFIs) in residual T-cells and CLL B-cells (ZAP70 T/B ratio). In this study, we developed a new method for ZAP70 labeling. Cells were labeled with a combination of anti ZAP70 phycoerythrin-conjugated SBZAP monoclonal antibody (mAb) and mAbs against CD45, CD19, and CD5. The latter three were used to specifically gate on different lymphocyte subsets. Staining was performed in absence (test) or in presence of excess unconjugated SBZAP mAb (isoclonic control). A so-called ZAP70 isoclonic ratio between SBZAP MFIs in the test and isoclonic control was calculated. A series of 32 patients with CLL and 10 normal controls were studied. Prediction of IGHV mutation status by ZAP70 isoclonic and T/B ratios was similar. By using the ZAP70 isoclonic ratio, we showed that ZAP70 expression was increased in T-cells from CLL patients. Nearly all cases with increased ZAP70 expression in CLL cells were associated with high ZAP70 expression in cognate T-cells. Therefore, the ZAP70 isoclonic ratio was more likely to closely reflect the biology of ZAP70 dysregulation rather than the T/B ratio. These results also explained why ZAP70 T/B ratios were artefactually close to normal in cells from CLL patients with high levels of ZAP70. © 2012 International Clinical Cytometry Society

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