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Cytometry B Clin Cytom. 2007 Mar;72(2):96-102.

Comparative analysis of ZAP-70 expression and Ig VH mutational status in B-cell chronic lymphocytic leukemia.

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  • 1Laboratory UDIAT, Corporació Sanitaria Parc Taulí, Sabadell, Spain.



B-cell chronic lymphocytic leukemia (CLL) is a heterogeneous disorder with respect to its clinical course. Accurate identification of prognostic factors is becoming increasingly important in order to determine those patients requiring aggressive treatments. Two of the most predictive outcome markers are the Ig VH mutational gene status and ZAP-70 expression. In earlier reports, both parameters have shown a high degree of concordance. To assess the value of these determinations in clinical practice, we simultaneously analyzed Ig VH mutations and ZAP-70 expression in a consecutive series of B-CLL.


Fifty-three consecutive B-CLL cases were included in the study. ZAP-70 expression was investigated by flow cytometry. Positivity was established using two methods: comparing ZAP70 expression in B-cells with T-cells using cytoplasmic CD3 (ZAP-70/T) and with NK-cell reactivity (ZAP-70/NK). The complete immunophenotype was recorded in each case. Ig VH mutational gene status was determined employing purified RNA from peripheral blood samples. Retrotranscribed DNA was PCR-amplified, direct-sequenced, and compared with available public databases. VH3.21 family use was also recorded.


Using a T-cell marker, 58% of patients were ZAP-70+ and 42% were ZAP-70-. NK-cell comparisons gave only 6% of ZAP-70 positivity in B-CLL, and in six cases the absence of a clearly defined NK-cell population precluded the ZAP70 analysis. Twenty-four (45%) patients had mutated Ig VH genes and 29 (55%) had unmutated Ig VH genes. The results showed a statistical association between ZAP-70/T expression and VH mutational status. Despite this, in 30% of cases there was a discordant result. Immunophenotypic analysis showed no major differences in Matutes'score between mutated and nonmutated cases. Only FMC7 was more commonly expressed in the unmutated B-CLL cases. VH3.21 was present in 7.5% of cases, mostly having an unmutated pattern.


ZAP-70 reactivity using a T-cell marker as a control allows to identify the majority of patients with an unmutated Ig VH genotype. Parallel analysis revealed that discordances with Ig VH analysis are quite common with currently employed flow cytometry reagents and techniques.

(c) 2006 International Society for Analytical Cytology.

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