CD34+ cell subpopulations detected by 8-color flow cytometry in bone marrow and in peripheral blood stem cell collections: application for MRD detection in leukemia patients.

Fast development in polychromatic flow cytometry (PFC) makes it possible to study CD34+ cells with two scatter and eight fluorescence parameters. Minimal residual disease (MRD) is determined as persistence of leukemic cells at submicroscopic levels in bone marrow (BM) of patients in complete remission. MRD can be present in collections of hematopoietic stem cell from blood (HSC-B). Using PFC, we have defined patterns of antigen expression in CD34+ cell subpopulations in BM and applied them as templates in MRD analysis. Twelve BM samples from hospital control (HC) patients with no signs of hematological malignancy were studied using five 8-color monoclonal antibody combinations detecting subsets of CD34+ cells. These patterns have been used as templates to determine levels of MRD in HSC-B collections from six AML patients. Several subsets of CD34+ precursor cells were found to be present at very low frequencies (<10(-4)) in BM and/or HSC-B collections. All six HSC-B collections from AML patients showed MRD by 8-color technique and only three by previously applied 3-color method. The 8-color technique showed promising results in efficient detection of different CD34+ subpopulations of HSC-B and in MRD quantification. Monitoring of MRD should become a part of quality control of HSC-B collections.