Combined normal donor and CLL: Single tube ZAP-70 analysis.
BACKGROUND
Zeta-chain-associated protein kinase 70 (ZAP-70) has been identified as an independent prognostic marker in chronic lymphocytic leukemia (CLL). Based on our previous studies, we have developed a combined one-tube technology with multiple internal controls to optimize ZAP-70 assessment.


METHODS
Forty-eight untreated CLL cases were examined for ZAP-70 expression using a modified 7-color one-tube assay. Normal donor (ND) whole blood is stained with CD3 APC-Cy7 and CD19 APC. In a second tube, patient whole blood is stained with CD5 PE-Cy7, CD19 PerCP-Cy5.5, and CD20 eFluor450. After surface staining and fixation, these two tubes are combined. After saponin permeabilization, the cells were stained with two anti-ZAP-70 clones (1E7.2/AF488 and SBZAP/PE). The results obtained from this modified tube were compared with those obtained concurrently using the non-mixed single sample tubes. Five different methods of ZAP-70 expression analysis were evaluated: percentage positive cells using ND T-cells as a reference; the internal patient T-cell/clone ratio; ND T-cell/clone ratio; clone/ND B-cell ratio; and modified Z-index.


RESULTS
Overall, the combined patient and ND mix tube performed better than the non-mixed single sample tube. The strongest correlations between ZAP-70 expression and immunoglobulin heavy chain variable (IGHV) mutational status were seen with percentage positive ND T-cell, ND T-cell/clone ratio, and clone/ND B-cell ratio for both 1E7.2 and SBZAP clone (P < 0.0001).


CONCLUSIONS
The modified one tube method combining the ND and patient sample provides highly reliable results that correlate with the IGHV mutational status. This method should be considered as part of the next step in standardization of the ZAP-70 assay in CLL.
Center for Biologics Evaluation and Research, FDA, Bethesda, Maryland, USA.
Heba A
HA
Degheidy
David J
DJ
Venzon
Mohammed Z H
MZ
Farooqui
Fatima
F
Abbasi
Diane C
DC
Arthur
Wyndham H
WH
Wilson
Adrian
A
Wiestner
M A
MA
Stetler-Stevenson
Gerald E
GE
Marti
D000368:Aged; D000369:Aged, 80 and over; D000911:Antibodies, Monoclonal; D018941:Antigens, CD19 / Q000276:immunology; D018951:Antigens, CD20 / Q000276:immunology; D001402:B-Lymphocytes / Q000166:cytology; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis; D017252:CD3 Complex / Q000276:immunology; D018956:CD5 Antigens / Q000276:immunology; D005260:Female; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000032:analysis / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis*; D008297:Male; D008875:Middle Aged; D013194:Staining and Labeling / Q000379:methods; D013601:T-Lymphocytes / Q000166:cytology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000097:blood*
D000911:Antibodies, Monoclonal; D018941:Antigens, CD19; D018951:Antigens, CD20; D014408:Biomarkers, Tumor; D017252:CD3 Complex; D018956:CD5 Antigens; D007143:Immunoglobulin Heavy Chains; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human