Immunophenotyping is key to the diagnostics of hematologic neoplasms. It is used to complete diagnosis, classify diseases, assess prognoses and to quantify malignant cells over the course of treatment (minimal/measurable 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 peripheral blood or bone marrow aspirate with heparin as anticoagulant 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 infiltrated peripheral blood. Therefore, bone marrow aspirate should be tested if peripheral blood is not infiltrated.
Characterization involves multi-parametric flow cytometry (MFC) of the analyzed cell populations based on 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.