Clinical immunology
Standard immunophenotyping of leukemia cells in acute myeloid leukemia (AML)

Immunophenotyping of leukemia cells plays crucial role in identification of leukemia cell line, definition of maturation stage and finding possible aberrant antigens what in turn serves for individual treatment monitoring and detection of residual disease. The purpose of this study was to develop standard laboratory procedure for immunophenotyping of leukemia cells in acute myeloid leukemia (AML), to establish the kit of necessary antibodies and to work out strategy of typing in acute proliferations.
116 patients with AML type M1-M5 (according to French-American-British classification, FAB) were tested. Immunophenotype determination was carried out by direct antibody staining and analysis with FACSCanto Becton Dickinson flow cytometer.
Flow cytometry analysis revealed that for proper determination of leukemia cells’ phenotype the first step is to separate leukemia cells with pan-leukocyte CD45 antigen. The next step is to determine
line-specific antigens: CD13, CD14, CD15, CD33, CD64, CD117, CD36, MPO as well as antigens associated with maturation of hematopoietic cells: CD34, CD38, TdT and myeloid cells: CD16, CD66.
We established useful screening kits for three- and four-color flow cytometry. Proposed three color kit is: surface: CD45/CD14, CD19/CD13/CD45, CD7/CD33/CD45, CD64/CD16/CD45 and cytoplasm: TdT/CD79a, MPO/cCD3. Proposed four color kit is: surface: CD14/CD64/CD45, CD19/CD13/CD45/CD3, CD7/CD33/CD45/CD117, CD66/CD16CD45 and cytoplasm: CD79a/MPO/CD3/TdT.
Auxiliary but important role plays determination of non-line specific antigens that are characteristic for myeloid and monocytic cells like CD11c, CD11b. Also, it is purposeful to look for lymphoid antigens like CD7, CD19, CD56 which, if co-expressed, are extremely useful to monitor residual disease.