A six-color flow cytometry assay for immunophenotyping classical Hodgkin lymphoma in lymph nodes.

OBJECTIVES We have recently demonstrated that classical Hodgkin lymphoma (CHL) can be immunophenotyped by flow cytometry (FC), thus obviating the need for immunohistochemistry in many cases. The previously described nine-color assay, however, cannot be used by laboratories that do not have access to a nine- or ten-color flow cytometer. Therefore, a six-color FC tube was designed employing the following combination: CD64-FITC/CD30-PE/CD40-PeCy5.5/CD20-PECy7/CD95-APC/CD3-APC-H7. METHODS To validate this assay, we analyzed 408 tissue specimens (including 55 CHL cases, 26 of which had been previously cryopreserved). Specimen inclusion criteria included the identification of an abnormal population by FC or (if no abnormal population was identified) greater than 50,000 viable events and specimen age less than 4 days. All FC studies were examined blinded to any clinical, laboratory, or histologic information. RESULTS The diagnostic sensitivity and specificity of the six-color FC assay was 85.4% and 99.7%, respectively. CONCLUSIONS Taken together, these results suggest that the six-color FC assay has acceptable sensitivity and specificity for clinical use, allowing more FC laboratories to immunophenotype CHL by this method.