Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.
BACKGROUND
ZAP-70 has been proposed as a surrogate marker for immunoglobulin heavy-chain variable region (IgV(H)) mutation status, which is known as a prognostic marker in B-cell chronic lymphocytic leukemia (CLL). The flow cytometric analysis of ZAP-70 suffers from difficulties in standardization and interpretation. We applied the Kolmogorov-Smirnov (KS) statistical test to make analysis more straightforward.


METHODS
We examined ZAP-70 expression by flow cytometry in 53 patients with CLL. Analysis was performed as initially described by Crespo et al. (New England J Med 2003; 348:1764-1775) and alternatively by application of the KS statistical test comparing T cells with B cells. Receiver-operating-characteristics (ROC)-curve analyses were performed to determine the optimal cut-off values for ZAP-70 measured by the two approaches. ZAP-70 protein expression was compared with ZAP-70 mRNA expression measured by a quantitative PCR (qPCR) and with the IgV(H) mutation status.


RESULTS
Both flow cytometric analyses correlated well with the molecular technique and proved to be of equal value in predicting the IgV(H) mutation status. Applying the KS test is reproducible, simple, straightforward, and overcomes a number of difficulties encountered in the Crespo-method.


CONCLUSIONS
The KS statistical test is an essential part of the software delivered with modern routine analytical flow cytometers and is well suited for analysis of ZAP-70 expression in CLL.
Van Bockstaele
Femke
F
Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium.
Janssens
Ann
A
Piette
Anne
A
Callewaert
Filip
F
Pede
Valerie
V
Offner
Fritz
F
Verhasselt
Bruno
B
Philippé
Jan
J
D000328:Adult; D000368:Aged; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis; D005260:Female; D005434:Flow Cytometry / Q000379:methods / Q000706:statistics & numerical data; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics*; D007135:Immunoglobulin Variable Region / Q000235:genetics*; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000235:genetics / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D009154:Mutation; D011379:Prognosis; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D013194:Staining and Labeling; D018709:Statistics, Nonparametric; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000096:biosynthesis
D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human