Improved flow cytometric detection of ZAP-70 in chronic lymphocytic leukemia using experimentally optimized isotypic control antibodies.
BACKGROUND
Expression of ZAP-70 by chronic lymphocytic leukemia (CLL) is associated with more aggressive disease and can help differentiate CLL using mutated immunoglobulin heavy chain variable genes (VH) from cases expressing unmutated VH genes. However, flow cytometric detection of ZAP-70 in CLL shows considerable variability and may be of questionable significance because most laboratories cannot correlate their results to clinical outcome or VH mutational data.


METHODS
Seventy cases of CLL were evaluated for ZAP-70 using a previously optimized staining procedure and two different methods to eliminate nonspecific background staining. One method, not previously reported, used isotypic control antibodies, where the concentrations were adjusted/optimized so that normal B-cells stained negatively for ZAP-70. The other used ZAP-70 stained peripheral blood B-cells from normal donors. The percentages of ZAP-70 stained CLL cells above the two thresholds were compared.


RESULTS
Concentrations of isotypic control antibodies had to be increased from manufacture's recommendations to insure normal B-cells were ZAP-70 negative. ZAP-70 levels among the CLL cases formed a bimodal distribution using the optimized isotypic control threshold, with 30 having low values (0-32% positive) and 40 high values (60-99% positive). In contrast, a continuous distribution was obtained with the ZAP-70 stained B-cell threshold. VH mutational status strongly correlated with the optimized control values as 29/30 low ZAP-70 cases had mutated VH genes and 37/40 high ZAP-70 cases used unmutated VH genes.


CONCLUSIONS
Use of an optimized isotypic control threshold could increase the reliability of flow based ZAP-70 detection and correlates well with VH mutational status.
ARUP (Associated Regional and University Pathologists) Institute of Clinical and Experimental Pathology, University of Utah, Salt Lake City, Utah, USA.
Sergey N
SN
Preobrazhensky
Philippe
P
Szankasi
David W
DW
Bahler
D000911:Antibodies, Monoclonal; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology; D014408:Biomarkers, Tumor / Q000235:genetics / Q000276:immunology; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D015203:Reproducibility of Results; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000097:blood* / Q000276:immunology
D000911:Antibodies, Monoclonal; D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human