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An improved flow cytometric method using FACS Lysing Solution for measurement of ZAP‐70 expression in B‐cell chronic lymphocytic leukemia
Author(s) -
Bekkema Roelof,
Tadema Afke,
Daenen Simon M. G. J.,
KluinNelemans Hanneke C.,
Mulder André B.
Publication year - 2008
Publication title -
cytometry part b: clinical cytometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.646
H-Index - 61
eISSN - 1552-4957
pISSN - 1552-4949
DOI - 10.1002/cyto.b.20367
Subject(s) - flow cytometry , microbiology and biotechnology , lysis , staining , chronic lymphocytic leukemia , biology , leukemia , immunology , medicine , pathology
Background: B‐cell expression of ZAP‐70, normally expressed in T and NK cells, correlates with poor prognosis in B‐CLL. Poor discrimination between ZAP‐70 positive and negative cells hampers routine application of flow cytometry. We examined the usefulness of FACS Lysing Solution. Methods: ZAP‐70 expression in 65 healthy volunteers was measured by four‐color flow cytometry, comparing FACS Lysing Solution for fixation and permeabilization with the Fix & Perm kit. Separation between ZAP‐70 positive T cells and negative B cells was based on a ratio of median ZAP‐70 staining of T cells to B cells. In 25 B‐CLL patients, ZAP‐70 expression was estimated using the lower limit of the fluorescence range corresponding with 98% of ZAP‐70 positive T cells as threshold marker as well as a ratio of B‐CLL cell to internal T‐cell median ZAP‐70 staining. Results: Use of FACS Lysing Solution resulted in approximately fourfold increased separation between ZAP‐70 positive T cells and negative B cells, when compared with the Fix & Perm kit. In B‐CLL samples, ZAP‐70 negative and positive B‐cell expression could be clearly discerned. Conclusions: FACS Lysing Solution is a simple procedure that markedly improves discrimination between ZAP‐70 positive and negative cells. © 2007 Clinical Cytometry Society.