Standardization & harmonization of cytometric analysis of ZAP70 and CD38

(contact person: Prof. F. Cymbalista, Bobigny/France)

Biomarkers need to fulfill the preliminary steps of technical validation and standardization before proceeding to clinical validation for widespread use.

Standardization of technical determination of ZAP-70 expression has proven very challenging. Several factors were evidenced as introducing variability in the results: antibody clone, conjugate, staining strategy, expression of results.

The need for standardization led to the organization of the first international effort on ZAP-70 harmonization (Cytometry B Clin Cytom. 2006 Jul 15;70(4):309-14 ). Briefly, a one day meeting of participants from 20 laboratories from 12 countries led to a consensus on several steps including fixation and permeabilization procedure and combination of surface markers.

The first trial was run on stabilized blood samples that proved unsuitable. Although this study provided valuable information, an international consensus was not reached on several technical issues such as the most appropriate technique and the expression of the results. Moreover, as new reagents were also becoming available, exchange of raw data was therefore considered as mandatory to address the issues of gating strategy and interpretation of results. This e-trial was allowed to point out to the difficulty in discriminating between normal residual B cells and CLL cells and the need for a reference population. Different modes of expression of the results for ZAP-70 were evaluated and Fluorescence ratio proved better than % on either isotype or T cells. But, when using MFI ratio, threshold of positivity has to be set up for each procedure. Conclusions from this electronic trial (e-trial) helped design a new technique that was further tested among 5 labs on an exchange of fresh samples. A standardization of the settings resulted in similar compensation matrix. Results were homogeneous and consistent with the previous trial. The homogeneity of the results expressed as percentages improved of even if the MFI ratio appears more adequate for harmonization, in particular for non experienced labs. This final consensual technique is described here in details:

PDF ERIC ZAP-70 staining technique