National standardization of ZAP-70 determination by flow cytometry: the French experience.
BACKGROUND
ZAP-70, after being considered as a potential surrogate for VH mutational status, has seen its own prognostic value emerge. We aimed at standardizing a simple, fast, and reproducible flow cytometry method.


METHODS
AntiZAP-70 antibody 2F3.2 was used with indirect labeling and secondary anti-IgG2a antibody. The reference values for the expression of the results were determined on 45 normal blood samples. ZAP-70 protein expression was investigated in 192 CLL samples. The indirect technique was compared with FITC-conjugated 2F3.2 clone, and with clone 1E7.2-FITC, -PE or -AlexaFluor 488.


RESULTS
Using FITC or PE-conjugated antibodies, 2F3.2 and 1E7.2 clones allowed a much less adequate discrimination between positive and negative cells and discordant cases were most likely true negative cases. Using the AlexaFluor 488 conjugated 1E7.2 clone, the discordant cases were mostly negative with the conjugated antibody and positive with the 2F3.2 clone but Western blotting or RNA microarray confirmed discordant cases were false negative with the conjugated antibody. Subsequently, recommendations were used by 13 centers participating in an interlaboratory quality control protocol. The use of MFI ratio appeared to be more reliable.


CONCLUSIONS
Results suggested that slight differences in the procedure had little impact on the interpretation in characteristic cases; however, careful interpretation was required for values close to threshold.
Le Garff-Tavernier
Magali
M
Hematology Biology Department, CHU Pitié Salpêtrière, Paris, France.
Ticchioni
Michel
M
Brissard
Martine
M
Salmon
Céline
C
Raynaud
Sophie
S
Davi
Frédéric
F
Bernard
Alain
A
Merle-Béral
Hélène
H
Ajchenbaum-Cymbalista
Florence
F
Letestu
Rémi
R
D000906:Antibodies / Q000276:immunology; D001402:B-Lymphocytes / Q000378:metabolism; D016022:Case-Control Studies; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D005602:France; D006801:Humans; D015451:Leukemia, Lymphocytic, Chronic, B-Cell / Q000097:blood / Q000175:diagnosis*; D012015:Reference Standards; D014407:Tumor Cells, Cultured; D051746:ZAP-70 Protein-Tyrosine Kinase / Q000097:blood* / Q000276:immunology
D000906:Antibodies; D051746:ZAP-70 Protein-Tyrosine Kinase