Advanced immunophenotyping: more is better!

Recent advances in multicolor flow cytometry have made possible the simultaneous analysis of up to 12 distinct cell surface molecules. This technical advance increases sensitivity and specificity for more accurate diagnosis and classification of various malignancies. In turn, this allows for a better assessment of disease prognosis and the establishment of a treatment regimen. In addition, multicolor flow cytometry provides a sensitive technique for investigating the occurrence of minimal residual disease. Studies of leukemias and lymphomas are particularly suitable for investigation by flow cytometry. Studies on solid tumors such as breast cancer, or on tumor infiltrating immune cells, are also possible if appropriate processing techniques are used to generate single-cell suspensions without altering cell surface receptor expression. In addition to analytical immunophenotyping, isolation of even very small (malignant) cell populations by multicolor flow cytometry provides a highly pure source for DNA and RNA analyses. Such combined sorting and molecular approaches provide a comprehensive basis for studying the molecular mechanisms underlying malignant transformation. Importantly, they also enable distinct diagnoses to be made when cell surface marker immunophenotyping alone is inconclusive. To exemplify the importance of combining various immunophenotyping tools, such as multicolor flow cytometry and gene expression analyses, with the classical tools of histopathology and differential blood count, we provide data on the analysis of a mouse model of human chronic B-cell leukemia (B-CLL): the New Zealand Black mouse. We relied for this study on gene expression databases generated by the National Cancer Institute for various human B-cell malignancies. B-CLL is one of the most common forms of leukemia in the Western world. No therapies currently exist against this usually slow-progressing but always fatal malignancy, making the development of a good animal model an important task. The data show that although the New Zealand Black mouse shows a spontaneous expansion of a CD5⁺ B-cell population (a hallmark of human B-CLL), it does not fit the classification of B-CLL by either phenotypic or genetic analysis criteria. In conclusion, increasing the number of simultaneous measurements for immunophenotyping and cell sorting by flow cytometry and combining it with comprehensive gene expression studies supports and expands the power of classical analysis tools.