title: Kolmogorov-Smirnov statistical test for analysis of ZAP-70 expression in B-CLL, compared with quantitative PCR and IgV(H) mutation status.
abstract: 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.
authors.affiliation: Department of Clinical Chemistry, Microbiology and Immunology, Ghent University, Ghent, Belgium.
authors.forename: Femke
authors.initials: F
authors.lastname: Van Bockstaele
authors.forename: Ann
authors.initials: A
authors.lastname: Janssens
authors.forename: Anne
authors.initials: A
authors.lastname: Piette
authors.forename: Filip
authors.initials: F
authors.lastname: Callewaert
authors.forename: Valerie
authors.initials: V
authors.lastname: Pede
authors.forename: Fritz
authors.initials: F
authors.lastname: Offner
authors.forename: Bruno
authors.initials: B
authors.lastname: Verhasselt
authors.forename: Jan
authors.initials: J
authors.lastname: Philippé
mesh_terms: 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
chemical_list: D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human