title: Evaluation of ZAP-70 expression by flow cytometry in chronic lymphocytic leukemia: A multicentric international harmonization process.
abstract: The clinical course of patients with chronic lymphocytic leukemia (CLL) is heterogeneous with some patients requiring early therapy whereas others will not be treated for years. The evaluation of an individual CLL patient's prognosis remains a problematic issue. The presence or absence of somatic mutations in the IgVH genes is currently the gold-standard prognostic factor, but this technique is labor intensive and costly. Genomic studies uncovered that 70 kDa zeta-associated protein (ZAP-70) expression was associated with unmutated IgVH genes and ZAP-70 protein expression was proposed as a surrogate for somatic mutational status. Among the available techniques for ZAP-70 detection, flow cytometry is most preferable as it allows the simultaneous quantification of ZAP-70 protein expression levels in CLL cells and residual normal lymphocyte subsets. However, several factors introduce variability in the results reported from different laboratories; these factors include the anti-ZAP-70 antibody clone and conjugate, the staining procedure, the gating strategy, and the method of reporting the results. The need for standardization of the approach led to the organization of an international working group focused on harmonizing all aspects of the technique. During this workshop, a technical consensus was reached on the methods for cell permeabilization and immunophenotyping procedures. An assay was then designed that allowed comparison of two clones of anti-ZAP-70 antibody and the identification of the expression of this molecule in B, T, and NK cells identified in a four multicolor analysis. This procedure was applied to three stabilized blood samples, provided by the UK NEQAS group to all participating members of this study, in order to minimize variability caused by sample storage and shipment. Analysis was performed in 20 laboratories providing interpretable data from 14 centers. Various gating strategies were used and the ZAP-70 levels were expressed as percentage positive (POS) relative to isotype control or normal B-cells or normal T-cells; in addition the levels were reported as a ratio of expression in CLL cells relative to T-cells. The reported level of ZAP-70 expression varied greatly depending on the antibody and the method used to express the results. The CLL/T-cell ZAP-70 expression ratio showed a much lower interlaboratory variation than other reporting strategies and is recommended for multicenter studies. Stabilization results in decreased expression of CD19 making gating more difficult and therefore stabilized samples are not optimal for multicentric analysis of ZAP-70 expression. We assessed the variation of ZAP-70 expression levels in fresh cells according to storage time, which demonstrated that ZAP-70 is labile but sufficiently stable to allow comparison using fresh samples distributed between labs in Europe. These studies have demonstrated progress toward a consensus reporting procedure, and further work is underway to harmonize the preparation and analysis procedures.
authors.affiliation: Service d'Hématologie Biologique, Hôpital Avicenne, Bobigny, France.
authors.forename: Remi
authors.initials: R
authors.lastname: Letestu
authors.forename: Andy
authors.initials: A
authors.lastname: Rawstron
authors.forename: Paolo
authors.initials: P
authors.lastname: Ghia
authors.forename: Neus
authors.initials: N
authors.lastname: Villamor
authors.forename: Nancy
authors.initials: N
authors.lastname: Boeckx
authors.forename: Nancy Boeckx
authors.initials: NB
authors.lastname: Leuven
authors.forename: Sebastian
authors.initials: S
authors.lastname: Boettcher
authors.forename: Anne Mette
authors.initials: AM
authors.lastname: Buhl
authors.forename: Jan
authors.initials: J
authors.lastname: Duerig
authors.forename: Rachel
authors.initials: R
authors.lastname: Ibbotson
authors.forename: Alexander
authors.initials: A
authors.lastname: Kroeber
authors.forename: Anton
authors.initials: A
authors.lastname: Langerak
authors.forename: Magali
authors.initials: M
authors.lastname: Le Garff-Tavernier
authors.forename: Ian
authors.initials: I
authors.lastname: Mockridge
authors.forename: Alison
authors.initials: A
authors.lastname: Morilla
authors.forename: Ruth
authors.initials: R
authors.lastname: Padmore
authors.forename: Laura
authors.initials: L
authors.lastname: Rassenti
authors.forename: Matthias
authors.initials: M
authors.lastname: Ritgen
authors.forename: Medhat
authors.initials: M
authors.lastname: Shehata
authors.forename: Piotr
authors.initials: P
authors.lastname: Smolewski
authors.forename: Peter
authors.initials: P
authors.lastname: Staib
authors.forename: Michel
authors.initials: M
authors.lastname: Ticchioni
authors.forename: Clare
authors.initials: C
authors.lastname: Walker
authors.forename: Florence
authors.initials: F
authors.lastname: Ajchenbaum-Cymbalista
mesh_terms: D000911:Antibodies, Monoclonal / Q000737:chemistry; D000918:Antibody Specificity; D000925:Anticoagulants / Q000494:pharmacology; D000937:Antigen-Antibody Reactions; D014408:Biomarkers, Tumor / Q000032:analysis / Q000096:biosynthesis / Q000276:immunology; D032921:Consensus; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D007143:Immunoglobulin Heavy Chains / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000235:genetics; D007391:International Cooperation; D007694:Killer Cells, Natural / Q000187:drug effects / Q000276:immunology; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology; D009154:Mutation; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000187:drug effects / Q000276:immunology; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000032:analysis* / Q000096:biosynthesis / Q000276:immunology
chemical_list: D000911:Antibodies, Monoclonal; D000925:Anticoagulants; D014408:Biomarkers, Tumor; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D051746:ZAP-70 Protein-Tyrosine Kinase; C497261:ZAP70 protein, human