Immunophenotyping detects the presence or absence of white blood cell (WBC) . These antigens are structures found on the surface or interior of WBCs. Typical groupings of antigens are present on normal WBCs. Atypical but characteristic groupings are seen with specific leukaemias and lymphomas. This allows immunophenotyping to be useful in helping to diagnose and classify these blood cell cancers dependent on the particular pattern of antigens present or absent.
Leukaemias are caused by abnormal types of WBC termed or myeloid (granular) WBC that begin to clone themselves. This leads to monoclonal lymphocytic or myeloid leukaemias. Lymphomas are caused by abnormal lymphocytes that become monoclonal and lead to cancer of the lymph system. The monoclonal cells produced do not fight infections like normal WBCs, and they do not die at a normal rate. They accumulate in the or in the lymph node where they originated. As the number of WBC clones increases, they may crowd out and inhibit the production of normal red and white blood cells platelets and the leukaemia or lymphoma cells may be released into the blood stream.
Full blood count (FBC) and differential tests performed on a sample of blood from someone with leukaemia or lymphoma will usually reveal an increased number of white blood cells with a predominance of one type of WBC. These tests may suggest lymphoma or leukaemia, but more information is generally needed to confirm a diagnosis. FBC and differential testing often cannot confirm monoclonal WBCs or detect the subtle differences that may exist between different types of blood cell cancers and they cannot distinguish between the different types of lymphocytes or myeloid WBCs.
With immunophenotyping, a blood, bone marrow, or other sample can be tested to gather this information – information that is then used to identify a specific type of leukaemia or lymphoma and, where possible, used to predict its likely aggressiveness and/or responsiveness to certain treatment. The identifications and predictions made are based upon a "library" of antigen associations and patterns that have been established over time.
Most of the antigens that immunophenotyping detects are identified by a CD (clusters of differentiation) number, such as: CD1a, CD2, CD3, CD4, CD8, CD13, CD19, CD20, CD33, CD61, or CD235. CD numbers represent a naming convention that is based upon international consensus. Several hundred antigens have been identified and received CD designations, but only a small number of these are routinely tested for clinical use. The pattern of expression of multiple CD markers can be unique to particular subtypes of leukaemia or lymphoma.
Flow cytometry is the technique most used for immunophenotyping. It is performed by processing a blood, bone marrow, tissue, or fluid sample to remove red cells, and then adding specific antibodies that have been tagged with fluorescent markers. These antibodies attach to corresponding antigens on the white blood cells when the antigens are present. The WBCs are then drawn up into a single-cell fluid stream under pressure past multiple lasers and detectors and each cell is analysed individually.
The flow cytometer rapidly measures characteristics about each cell, such as its size and granular complexity, and evaluates the type and quantity of fluorescent antigen-antibody complexes that are present. Thousands of cells are evaluated during the test. Results are then graphed and compared to "normal" results and to patterns that are known to be associated with different leukaemias and lymphomas. This process allows the person interpreting the test results to determine the types of WBCs present, their maturity, and to determine the types and quantities of antigens on or in these cells.
