ZAP-70 Can be Tested for by Flow Cytometry, Immunohistochemistry

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[4994] A Comparison of Zap-70 by Immunohistochemistry and Flow

Cytometry Using Two Different Flow Analysis Methods. Session Type:

Publication Only

Lawrence Hertzberg, Karina Baggianni, lba Tamayo, Browne,

Teri Oldaker Hematopathology, Genzyme Genetics, Los Angeles, CA, USA

Zap-70 expression by flow cytometry has been shown to be a surrogate

marker for un-mutated/germline IgH status in chronic lymphocytic

leukemia (CLL).1 Reproducibility and confidence in the accuracy of

results can make this assay problematic.

We compared our current single-parameter/one-color,

immunohistochemistry (IHC) Zap-70 assay with our flow cytometry Zap-

70 assay. Twenty-one (21) patient samples submitted to our lab for

Zap-70 testing were assayed by IHC and flow cytometry (FC). In

addition, two FC analysis methods were used. One analysis method uses

isotype controls to establish negative regions and a 20% cut-off for

positivity1, with BD CellQuestTM software (CQ). The other analysis

method uses internal negative and positive mean channel fluorescence

(MCF) differences using BD Paint-A-GateTM software (PAG). The IHC

assay resulted in nine positive, six negative and six equivocal

results for Zap-70. The equivocal results were due to very low

proportions of CLL cells (three cases) or other problems identified

on review of stained slides (three cases).

The FC assay resulted in six positive, thirteen negative and two

equivocal results for both analysis methods. When comparing IHC Zap-

70 and FC Zap-70, there was 29% (6/21) discordance using the CQ

analysis and 24% discordance (5/21) using the PAG analysis (equivocal

cases included in the denominator only). Comparison of the CQ and PAG

analysis demonstrated 16% (3/19) discordance (equivocal cases

excluded from the denominator). The FC Zap-70 PAG analysis method

correlated somewhat better with the IHC Zap-70 results than did the

FC Zap-70 CQ analysis method. This may be due to the variability

introduced by user-defined/isotype cursor settings.

We conclude that Zap-70 expression can be tested for using either IHC

or FC, and that IHC testing may have higher sensitivity. Correlation

between the results utilizing these different methods is not ideal,

and improvement in both assays, likely including flow analysis

methods, is needed. We plan additional, comparative evaluation of

more cases.

1. Crespo, M. et al., Zap-70 Expression as a Surrogate for

Immunoglobulin-Variable-Region Mutations in Chronic Lymphocytic

Leukemia. N Engl J Med 2003 348:18:1764-1775.