Quantitative flow cytometry of ZAP‐70 levels in chronic lymphocytic leukemia using molecules of equivalent soluble fluorochrome

Abstract

Background: ZAP-70 has emerged as a potential pivotal prognostic marker for patients with chronic lymphocytic leukemia (CLL), which could replace immunoglobulin heavy chain mutation status. Although several flow cytometry assays have been described for assessing ZAP-70 in CLL, certain technical and scientific issues remain unsolved, which have prevented results of this crucial test from being reported, even in the best routine flow cytometry laboratories. In this report, we aimed to solve some of these issues by providing a computerized quantitative flow cytometric assay for ZAP-70 within the entire CLL population, which would be easy to perform and enable standardization between laboratories.Methods

Correlation between ZAP‐70, phospho‐ZAP‐70, and phospho‐Syk expression in leukemic cells from patients with CLL

“…demonstrated that phospho-ZAP-70, phospho-Syk, and p21 cip1 are stable in cells maintained at room temperature ex vivo for 24 hours (Table 3). It should be noted that others have shown that ZAP-70 levels decline over this period of time (15)(16)(17)(18)(19), but we have found they are relatively stable for 5 hours at room temperature, which is the time limit for processing in our procedure. Thus, the data we have obtained is reproducible, quantitative, stable upon freezing/thawing, and stable under the parameters of sample collection used in our procedure.…”

“…For the most part, the median fluorescence ratios determined by EAS correlated well with the percent positive cells obtained by standard methodology; however, two samples were significantly discrepant. Although we cannot be certain of the explanation for these two discrepancies, the detection of ZAP-70 is known to be significantly affected by cellular fixative, reporting methods, and deterioration of the signal (15)(16)(17)(18)(19)32). Additionally, the sample size for the analytical method comparison was relatively small.…”

Flow cytometry in clinical pathology

“…23 Although 'broad-brush' quantitative leukaemia panels are yet to find a role, quantitation has been shown to be of value in specific prognostic situations such as CD38 and ZAP-70 expression in CLL. 24,25 Internal quality control, external quality assessment Provision of a clinical flow cytometry service requires rigorous internal quality control (IQC) and participation in external quality assessment (EQA). Specific details vary according to the nature of the assay.…”

A standardized ZAP‐70 assay—Lessons learned in the trenches

“…There remains the need for additional studies to examine the relative performance of our assay and other quantitative approaches, such as the ratio metric method described by Bakke et al (21) or the work described by Kay et al, who added a level of quantification to the flow cytometric analysis of ZAP-70 expression by calibrating the fluorescence intensity of the ZAP-70 immunofluorescence utilizing calibrated fluorescence bead standards (6). We hope the laboratory community can cooperatively agree on a standardized method for ZAP-70 reporting or this assay will follow the fate of DNA content assays and we will again be forced back into the trenches.…”

“…And, it was the conclusion of this CLL cooperative study group that ZAP-70 expression, defined by greater than 20% ZAP-70 positive CLL cells above the highest B cell ZAP-level, was a strong predictor of the need for earlier treatment. Recently, Kay et al (6) added a level of quantification to the flow cytometric analysis of ZAP-70 expression by calibrating the fluorescence intensity of the ZAP-70 immunofluorescence, utilizing calibrated fluorescence bead standards, and demonstrated that normal T and B cells exhibited characteristic levels of ZAP-70 expression, and that tumor cells demonstrated variable levels of ZAP-70 expression, with higher ZAP-70 levels linked to more aggressive disease.…”