Pathologists use flow cytometry to analyze the physical and chemical properties of cells moving in single file in a fluid stream (Fig 3-2A, a). The most common use of flow cytometry in clinical practice is for immunophenotyping hematopoietic proliferations. This procedure may be performed on ocular adnexal tissue, aqueous humor, or vitreous. One example of flow cytometry is leukocyte immunophenotyping. For this procedure, the cells need to be fresh (unfixed, unfrozen). Fluorochrome-labeled antibodies targeted against various leukocyte antigens bind to the surface of lymphoid cells, and a suspension of labeled cells is sequentially illuminated by a light source (usually an argon laser) for approximately 10−6 second (Fig 3-2A, b). As the excited fluorochrome returns to its resting energy level, a specific wavelength of light is emitted (Fig 3-2A, c), which is sorted by wavelength stream (Fig 3-2A, d) and received by a photodetector (Fig 3-2A, e). The flow cytometer then converts this signal to electronic impulses, which are analyzed by computer software. The results may be represented by a multicolored dot-plot histogram (Fig 3-2B, C).
An advantage of flow cytometry is that it shows the proportion of particular cells in a specimen in histogram format. In addition, multiple antibodies and cellular size can be analyzed simultaneously. For example, the proportion of CD4 (helper T cells), CD8 (suppressor T cells), both CD4+ and CD8+, or either CD4+ or CD8+ may be displayed for a given lymphocytic infiltrate. The disadvantages of flow cytometry are its failure to show the location and distribution of these cells in tissue and the possibility of sampling error. Depending on the number of cells in the sample and on clinical information, the flow cytometrist chooses the panel of antibodies to be tested. Flow cytometric data should therefore be used as an adjunct to routine light microscopy and immunohistochemistry.
The ability of a laboratory to test small and viscous specimens such as vitreous with flow cytometry varies on an individual basis. Consultation with the pathologist is recommended before tissue is submitted for this type of testing.
Excerpted from BCSC 2020-2021 series: Section 4 - Ophthalmic Pathology and Intraocular Tumors. For more information and to purchase the entire series, please visit https://www.aao.org/bcsc.