An improved flow cytometric method using FACS Lysing Solution for measurement of ZAP-70 expression in B-cell chronic lymphocytic leukemia.
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.
Bekkema
Roelof
R
Department of Clinical Chemistry, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands. r.bekkema@lc.umcg.nl
Tadema
Afke
A
Daenen
Simon M G J
SM
Kluin-Nelemans
Hanneke C
HC
Mulder
André B
AB
D000937:Antigen-Antibody Reactions; D001402:B-Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000473:pathology; D014408:Biomarkers, Tumor / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002469:Cell Separation; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis / Q000378:metabolism*; D011933:Reagent Kits, Diagnostic; D012996:Solutions / Q000737:chemistry / Q000494:pharmacology*; D013601:T-Lymphocytes / Q000737:chemistry / Q000187:drug effects / Q000473:pathology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000737:chemistry / Q000276:immunology / Q000378:metabolism*
D014408:Biomarkers, Tumor; D011933:Reagent Kits, Diagnostic; D012996:Solutions; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human