Immunophenotypic analysis by flow cytometry
Flow cytometry is a clinical method is used routinely to monitor bone marrow and peripheral blood samples. For instance, the standard "CBCs" with which parents of children with cancer are so familiar are done on flow cytometer machines.
Flow cytometry was first introduced in the 1960s, and now includes several different types of machines. In general terms, flow cytometry refers to an automated procedure in which a suspension of cells flows past a detector. What the detector measures depends both on the design of the machine and the design of the experiment. Flow cytometry can detect and measure:
- the number, size, and cellular granularity of cells
- RNA/DNA content
- ploidy and DNA index
- cell antigens
The measurement of cell antigens relies on an antigen/antibody response. First, they acquire an antibody to a specific cell antigen and connect it to a fluorescent marker. Then, they incubate the cell sample that they want to study with this antibody/fluorescent marker. If the antibody attaches to a specific cell antigen in the sample being tested, the cell will fluoresce as it passes the flow cytometry detector.
Flow cytometry can give the medical team a lot of information about the patient's type of leukemia. Still, routine flow cytometry can only detect a leukemic cell population if it is more than 1% of the total cell population.
A few good web sites:
- Flow Cytometry on the University of Washington, Laboratory Medicine pages
Use of flow cytometry in the detection of MRD
Coustan-Smith et al (13) present a method for detection of MRD in ALL using multiparameter immunological detection by flow cytometry that they claim is capable of detecting one leukemic cell among 10,000 normal cells. The principle behind this assay is that leukemia cells display certain surface, cytoplasmic and nuclear leukocyte antigens, or proteins, which normal cells do not. Monoclonal antibodies to these antigens will attach to them when they see them. So, they tag a fluorescent dye to a monoclonal antibody, incubate it with a sample of bone marrow aspirate from a leukemia patient, and then monitor the cells by flow cytometry for the attachment of fluorescent dyes to the cells. If the cells fluoresce, they have leukemia antigens.
According to current concepts, all B-lymphocytes arise from pluripotent stem cells in the bone marrow. These cells are arrested at various stages in the normal differentiation scheme. (7) The diagram below shows the different antigens that are expressed during B-cell development. The different types of leukemias represent development arrested at different stages, as reflected in the antigens these leukemias display. The earliest antigens are terminal deoxynucleotidyl transferase (TdT) and HLA-Dr, neither of which is B lineage specific. Later, CD19, 20,10 are expressed; these are all B-cell specific.
Reference websites for leukemia antigens and immunochemistry:
- University of Washington, diagnosis of acute leukemia: immunophenotype chart
- A remedy for CDphobia, by Margaret Uthman, MD: Hematopathologic Phenotypes Made Mockingly Simple
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