Optimized whole-blood assay for measurement of ZAP-70 protein expression.
Chronic lymphocytic leukemia (CLL) is characterized by a clonal expansion of small lymphocytes commonly expressing cell surface markers (CD5 and CD19) that are consistent with a population of B lymphocytes. This unit describes a technique to measure ZAP-70 protein expression in whole-blood specimens from CLL samples. The protocols presented include an optimized fixation/permeabilization technique that allows labeling of cell surface markers and intracellular ZAP-70 protein with significantly improved signal-to-noise ratio, an optimized combination of antibodies-fluorophores to maximize ZAP-70 expression levels, standardized methodology for instrument setup, including compensation, to improve inter- and intra-laboratory reproducibility, and a method to index ZAP-70 protein expression levels to internal positive and negative cell populations. Residual normal T and B cells function as internal positive and negative controls. These are used to index ZAP-70 protein expression levels in the CLL population.
Shankey
T Vincent
TV
Beckman Coulter, Inc., Miami, Florida, USA.
Forman
Meryl
M
Scibelli
Paul
P
D015703:Antigens, CD / Q000097:blood; D001402:B-Lymphocytes / Q000276:immunology; D018956:CD5 Antigens / Q000097:blood; D002463:Cell Membrane Permeability; D005434:Flow Cytometry; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood* / Q000276:immunology; D016256:Lewis X Antigen / Q000097:blood; D012016:Reference Values; D013601:T-Lymphocytes / Q000276:immunology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000097:blood* / Q000235:genetics*
D015703:Antigens, CD; D018956:CD5 Antigens; D016256:Lewis X Antigen; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human