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  • Title: An improved flow cytometric method using FACS Lysing Solution for measurement of ZAP-70 expression in B-cell chronic lymphocytic leukemia.
    Author: Bekkema R, Tadema A, Daenen SM, Kluin-Nelemans HC, Mulder AB.
    Journal: Cytometry B Clin Cytom; 2008 Jan; 74(1):40-4. PubMed ID: 17703441.
    Abstract:
    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.
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