Use of a blocking antibody method for the flow cytometric measurement of ZAP-70 in B-CLL.
BACKGROUND
In this study we developed a method to measure the amount of ZAP-70 [zeta accessory protein] in B-CLL cells without relying on the ZAP-70 expression of patient B or T cells to normalize fluorescence intensity.


METHODS
B-CLL cells were fixed with formaldehyde before surface staining with gating antibodies CD19PC5 and CD5FITC. The cells were permeabilized with saponin, and the ZAP-70 antigen was blocked in one tube with unlabeled antibody to ZAP-70 [clone 1E7.2]. Zap-70-PE was then added to this tube. ZAP-70-PE was added to a second tube without unlabeled antibody to ZAP-70. The mean fluorescence intensity of the ZAP-70 in the tube without unlabeled antibody divided by the mean fluorescence intensity of the ZAP-70 in the tube with unlabeled antibody equals the RATIO of total fluorescence to non-specific ZAP-70 fluorescence in the B-CLL cells. In a second method of analysis, a region is created in the histogram showing ZAP-70 fluorescence intensity in the tube with unlabeled antibody to ZAP-70. This region is set to 0.9% positive cells. This same region is then used to measure the % positive [%POS] ZAP-70 cells in the tube without unlabeled antibody to ZAP-70. The brighter the ZAP-70 fluorescence above the non-specific background, the higher the %POS.


RESULTS
Due to the varying amount of non-specific staining between patient B-CLL cells and other cells, the blocking antibody method yielded a more quantitative and reproducible measure of ZAP-70 in B-CLL cells than other methods, which use the ratio of B-CLL fluorescence to normal B or T-cell fluorescence. Using this improved method, ZAP-70 was determined to be negative if the RATIO was less than 2:1 and positive if the RATIO was greater than 2:1. ZAP-70 was determined to be negative if the %POS was less than 5% and positive if the %POS was greater than 5%, a cut-off value lower than previous values published, due to exclusion of non-specific staining. Both cut-offs were based upon patient specimen distribution profiling.


CONCLUSIONS
Use of a blocking antibody resulted in a robust, reproducible clinical B-CLL assay that is not influenced by the need to measure the amount of ZAP-70 in other cells. ZAP-70 results segre gate patients into indolent and aggressive groups suggested by published clinical outcomes.
Shenkin
Mark
M
AmeriPath, Inc, Orlando, FL and Shelton, CT, USA. mshenkin@ameripath.com
Maiese
Russell
R
D051997:ADP-ribosyl Cyclase 1 / Q000032:analysis / Q000276:immunology; D019138:Antibodies, Blocking / Q000737:chemistry*; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D016130:Immunophenotyping; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000276:immunology; D015203:Reproducibility of Results; D013194:Staining and Labeling; D016707:Tissue Fixation / Q000379:methods; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000276:immunology
D019138:Antibodies, Blocking; D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human; D051997:ADP-ribosyl Cyclase 1