ZAP-70 Detection Using Flow Cytometry

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Cytometry B Clin Cytom. 2006 Oct 18; [Epub ahead of print]

Comparative analysis of ZAP-70 expression and Ig VH mutational status

in B-cell chronic lymphocytic leukemia.

Munoz L, Lasa A, Carricondo MT, C, Ubeda J, Nomdedeu JF.

Laboratory UDIAT, Corporacio Sanitaria Parc Tauli, Sabadell, Spain.

BACKGROUND:: 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.

METHODS:: 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.

RESULTS:: 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.

CONCLUSIONS:: 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.

© 2006 International Society for Analytical Cytology.

PMID: 17051526 [PubMed - as supplied by publisher]