Flow Cytometric Detection Of Minimal Residual Disease In Patients With B Lineage Acute Lymphoblastic Leukemia

Objective:To establish and validate a triple-color flow cytometric assay for the detection of minimal residual disease(MRD) in B lineage acute lymphoblasticleukemia(B lineage-ALL).Methods: Using the five different monoclonal antibody(McAb) combinations (CD10/CD20/CD19, CD10/TdT/CD19, CD34/CD38/CD19, CD34/CD22/CD19, CD34/CD45/CD19) as the probes for the detection of MRD, a set of flow cytometric dot plots of immunophenotypic profiles of CD19+ cells in normal bone marrow(BM) and B lineage-ALL were obtained. Based on the characteristics of the profiles, a MRD detection assay with flow cytometry(MRD-DFCM), with a immunologic strategy for detecting MRD relying on combinations of leukocyte markers normally not seen in normal bone marrow ,was established. The sensitivity of the MRD-DFCM was tested with dilution experiments and thirteen bone marrow samples from patients with B iineage-ALL at diagnosis and twenty-two bone marrow samples in complete remission were analyzed. At the same time, the polymerase chain reaction(PCR) was applied to evalutate the efficacy of MRD-DFCM by amplifying the IgH-CDRlIIand TCR Y gene rearrangment for the detection of MRD in B Iineage-ALL.Results: 1 .The several characteristics of the flow cytometric dot plots of CD 19+ cells with five different McAb combinations were found out in this study as following: ㏕he dot plots of the normal BM with five McAb combinations had different patterns ,but the dot plots tested with the same McAb combination in different healthy persons had a similar profile (2)The dot plots reflected the different stages of development and the relative numbers of cells at each stage.(3)There were apparent differences in the characteristics of dot plot patterns between the B Iineage-ALL and the healthy persons, and the normal patterns reappeared in the patients who were in complete remission. 2.Dot plots of selected combinations of markers contained areasthat are consistently blank when normal bone marrow cells were analyed,and positive readings in these areas suggested residual leukemia. 3. At least one abnormal expression out of the five McAb combinations ocurred in twelve patients with B lineage ALL and the most informative combination was CD10/CD20/CD19 (61.5%). 4. When the flow cytometric system was optimized, there was a close correlation between the numbers of leukemic cells detected by the MRD-DFCM and the actual numbers of leukemic cells which were diluted into normal peripheral blood cells in concentration: 1:1 ~ 1:10000 (r=0.83, P=0.00) . 5. Twenty-two bone marrow samples in complete remission were analyzed by MRD-DFCM and PCR. The MRD-DFCM detected MRD among six samples, while leukemic cells were detected in nine by PCR. Leukemic cells were detected in six bone marrow samples by the two methods.Conclusion: The flow cytometric dot plots with five antibody combinations clearly indicate the different stages of the B cell development and the relative locations of CD19+ cells, and they form a reference for idetification of MRD.Based the normal dot plots,a MRD-DFCM has been established successfully in this study, when leukemic cells were serially diluted into normal peripheral blood cells,we found a sensitivity of detection of 1 leukemic cell in 104 normal marrow cells.