Standardization & harmonization of cytometric analysis of ZAP70 and CD38

Background

Biomarkers need to fulfil 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 in the framework of the ERIC.

Goals

  1. To review the techniques used in the different participating center
  2. To propose a multicentric evaluation of selected procedures
  3. To reach a minimal consensus on good practices
  4. To reach a minimal consensus on the future techncal approach

Participants

The study included the following ERIC members :
Nancy BOECKX Leuven Belgium, Sebastien BOETTCHER Kiel Germany, Anne Mette BUHL Copenhagen Denmark, Jan DUERIG Essen Germany, Paolo GHIA Milano Italy, Rachel IBBOTSON Bournemouth United Kingdom, Alexander KROEBER Ulm Germany, Anton LANGERAK Rotterdam The Netherlands, Magali LE GARFF-TAVERNIER Paris France, Ian MOCKRIDGE Southampton United Kingdom, Alison MORILLA London United Kingdom, Ruth PADMORE Ottawa Canada, Laura RASSENTI San Diego USA, Andy RAWSTRON Leeds United Kingdom, Matthias RITGEN Kiel Germany, Medhat. SHEHATA Vienna Austria, Piotr SMOLEWSKI Lodz Poland, Peter STAIB Koln Germany, Michel TICCHIONI Nice France, Neus VILLAMOR Barcelona Spain, Clare WALKER Sheffield United Kingdom, Florence CYMBALISTA and Rémi LETESTU Bobigny France

Approach

  1. First stage: a consensus meeting
    Briefly, a one-day meeting of participants from 20 laboratories from 12 countries led to a consensus on several steps including fixation and permeabilization procedures and combination of surface markers.
  2. Second stage: A multicentric evaluation was subsequently designed.
    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. It was decided to address these issues through an exchange of raw data for reanalysis.
  3. Third stage: the electronic-trial
    This e-trial has allowed pointing out 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.
  4. Fourth stage: design of a new technique
    Conclusions from the e-trial were taken into account in the new technique designed and tested at Avicenne hospital. The procedure is described in details and available on the ERIC website since January 2010:
    PDF ERIC ZAP-70 staining technique
  5. Fifth stage: evaluation of the new technique
    This new technique 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 even if the MFI ratio appears more adequate for harmonization, in particular for non-experienced labs.

Time-line

  • First stage: completed march 2005
  • Second stage: completed july 2006
    Publication: Cytometry B Clin Cytom. 2006 Jul 15;70(4):309-14
  • Third stage: completed 2007
    presented at ERIC meeting (EHA Vienna)
  • Fourth stage: partly completed in 2010
    manuscript in preparation: submission late 2011
  • Fifth stage: mid 2012 (see ongoing project)

Coordination

Pr. Florence Cymbalista, MD, PhD
Bobigny France
E-mail:

Dr Rémi Letestu, MD, PhD
Bobigny France
E-mail: