Discordant results of flow cytometric ZAP-70 expression status in B-CLL samples if different gating strategies are applied.

BACKGROUND

Recent studies have identified ZAP-70 expression status as an excellent prognostic parameter in chronic lymphocytic leukemia (CLL). ZAP-70 expression can be determined by direct antibody staining followed by flow cytometric analysis. However, there are currently several different gating strategies for analysis. We compared those strategies for ZAP-70 expression analysis.

METHODS

One hundred and one patients with B-CLL were analyzed employing a directly labeled alexa-fluor-488-ZAP-70-antibody. In 27 cases, we additionally measured and analyzed ZAP-70 expression, together with healthy controls as described previously.

RESULTS

Applying either T-/NK-cell isotype or healthy control analysis strategies on patient samples that were processed in parallel revealed discrepant results in 48% (12/25) of all cases. Taken together with the 74 B-CLL patients, who were analyzed with regard to average reference values, disconcordant results were obtained in 58% of the samples. We demonstrate that high variances in ZAP-70 T-/NK-cell staining within B-CLL patients, paired with a close proximity of ZAP-70 B-cell values to the suggested cut-off levels, may lead to interpretation difficulties of ZAP-70 status.

CONCLUSIONS

We conclude that different gating strategies for determining flow cytometric ZAP-70 expression status produce highly discordant results. Further standardization is required before ZAP-70 can be used as a reliable prognostic parameter in immunophenotyping of B-CLL.