CD Markers for Flow Cytometry

The immune system is composed of a variety of cell types each with unique roles and functions. These different cell types can be identified by a unique signature of cell-surface proteins called CD (cluster of differentiation) markers. Well known examples of CD markers are CD4 and CD8, which are used to identify T lymphocytes involved in regulating the adaptive immune response. CD markers for flow cytometry are used in many applications, including immunophenotyping (see below).

The CD nomenclature has been developed and maintained by the HLDA (Human Leukocyte Differentiation Antigens) workshop. This initiative, starting in 1982, originally aimed to classify the monoclonal antibodies (mAbs) raised against cell surface molecules of leukocytes being generated by laboratories worldwide. The proposed marker is assigned a CD number once two specific mAbs have been shown to bind to it. Markers that are not sufficiently characterized are labeled with a provisional “w” (as in “CDw145”).

There are now over 370 CD unique clusters and subclusters described in humans, including cell types other than leukocytes. CD antibodies are used widely for studying cell structure, function and distribution in research, differential diagnosis, and monitoring and treatment of disease (Table 1).

Applications for CD markers

Immunophenotyping 

Immunophenotyping is a powerful tool that uses flow cytometry to gain insight into the composition and dynamics of cell populations in an immune response. The technique involves identifying cell types in heterogeneous cell populations by using different fluorescently-tagged antibodies that target various CD markers. Different combinations of antibodies can be used to identify different groups and sub-groups of cells. For example, CD3 can be used as a general marker for T cells followed by other CD markers to identify regulatory T cells (CD4 and CD25), T helper cells (CD4), and cytotoxic T cells (CD8). Immunophenotyping can be used for discovery and can also be applied in clinical settings, for example to help diagnose and classify a leukemia or lymphoma, or refine the analysis of cancer cell lines by determining the presence or absence of cancer cell markers that correlate with different degrees of severity. 

Figure 1. Human peripheral blood leukocytes, stained with CD3-FITC (Clone UCHT-1) and CD5-PE (Clone UCHT-2). Double positive cells in the upper right quadrant represent 27.6% of the cell population, while 67.6% were negative for both CD5 and CD3 (lower left quadrant).

Immunohistochemistry

Many of the antigens used in immunohistochemistry (IHC) are CD markers. For example, CD15 and CD30 can be used to identify Hodgkin’s disease, while CD20 and CD3 can be used to differentiate between B-cell lymphomas and T-cell lymphomas, respectively.

Spatial Phenotyping

Spatially resolved, highly multiplexed biomarker analysis (spatial phenotyping) is being used to analyze the interactions between neighboring cells or groups of cells that are key to important biological processes. This analysis of context frequently involves the use of CD markers — for example, determining the difference between a CD4 T cell in breast cancer tissue surrounded by CD8 T cells to a CD4 T cell surrounded by luminal epithelial cells.

Figure 3. Sample image for spatial phenotyping of renal cell carcinoma tissue stained for 7 different markers including Ki67, CD8, CD4, Podo, SMA, CD20, and CD31.