Immunophenotyping is key to the diagnostics of hematologic neoplasia. It is used to complete diagnosis, classify diseases, assess prognoses and to quantify malignant cells over the course of treatment (minimal residual disease, MRD). Diagnostic application of the method is based on the characterization of antigen expression patterns among malignant cells and their discrimination from healthy cells.
Between 5 and 10 ml of bone marrow with heparin anticoagulant, i.e. peripheral blood, are needed for immunophenotyping. Alternatively, EDTA can also be used as anticoagulant, although it is associated with a faster loss of cell viability. Peripheral blood is sufficient as a test material, provided that malignant cells have passed into the blood. Therefore, bone marrow should be tested if passage has not occurred.
Characterization involves multi-parametric flow cytometry (MFC) of the analyzed cell populations at the base of their light-scattering properties. This procedure uses fluorescence-marked monoclonal antibodies that are targeted at diagnostically relevant antigens on the cell membrane and in the cytoplasm. Modern flow cytometers enable simultaneous detection of several different fluorochromes and hence allow a precise description of antigen expression patterns for approximately 1,000 cells per second. This means that even cell populations with a frequency of 1% or less can be characterized very quickly.