Technique / Immunology / Immunophenotyping

Immunophenotyping technique analyze population of heterogenous cells using immunophenotypical markers.

Flow cytometric immunophenotyping remains an indispensable tool for the diagnosis, classification, staging, and monitoring of hematologic neoplasms. The last 10 years have seen advances in flow cytometry instrumentation and availability of an expanded range of antibodies and fluorochromes that have improved our ability to identify different normal cell populations and recognize phenotypic aberrancies, even when present in a small proportion of the cells analyzed. Phenotypically abnormal populations have been documented in many hematologic neoplasms, including lymphoma, chronic lymphoid leukemias, plasma cell neoplasms, acute leukemia, paroxysmal nocturnal hemoglobinuria, mast cell disease, myelodysplastic syndromes, and myeloproliferative disorders. The past decade has also seen refinement of the criteria used to identify distinct disease entities with widespread adoption of the 2001 World Health Organization (WHO) classification. This classification endorses a multiparametric approach to diagnosis and outlines the morphologic, immunophenotypic, and genotypic features characteristic of each disease entity. When should flow cytometric immunophenotyping be applied? The recent Bethesda International Consensus Conference on flow cytometric immunophenotypic analysis of hematolymphoid neoplasms made recommendations on the medical indications for flow cytometric testing. This review discusses how flow cytometric testing is currently applied in these clinical situations and how the information obtained can be used to direct other testing.

Flow cytometry (FCM) immunophenotyping is frequently used as an ancillary technique for the diagnosis of hematological malignancies or for measurement of DNA content. In recent years, we applied FCM to the diagnosis of metastatic adenocarcinoma and malignant mesothelioma in effusions. We established a panel of antibodies that allows for rapid and effective differentiation between epithelial cells, mesothelial cells, and leukocytes. FCM was subsequently used for quantitative analysis of integrin subunits. Recently, we studied different parameters of the immune response, including HLA molecules and chemokine receptors, using this method. Our data suggest that FCM is an effective method for the characterization of cancer cells in clinical effusion specimens in both the diagnostic and research setting, and that this method is comparable to immunohistochemistry in terms of sensitivity and specificity, with the additional advantage of providing quantitative data. This review discusses previous work in this area and the future potential of this method in the characterization of tumor cells in serous effusions.

Flow cytometry-based immunophenotyping assays have become increasingly multiparametric, concomitantly analyzing multiple cellular parameters. To maximize the quality of the information obtained, antibody conjugate panels need to be developed with care, including requisite controls at every step. Such an optimization procedure for multicolor immunophenotyping assays is time consuming, but the value of having a reliable antibody conjugate panel that provides for sensitive detection of all molecules of interest justifies this time investment. This article outlines important considerations and procedures to undertake for the successful design and development of multicolor flow cytometry panels.