Detection Of Minimal Residual Disease By Flow Cytometry In Acute Myeloid Leukemia Patients

Objective: To detect the AML-MRD with leukemia associated immunophenotypes(LAIP) by multiparameter flow cytometry. The present analysis aimed at improving the applicability of this approach to more patients with AML and more abroad clinical use.Methods: Bone marrow samples from unselected patients with AML at diagnosis,complete remission , relapse and from healthy volunteers were immnunophenotyped applying triple-stainings of 6 panals of antigens. A live-gate strategy was used according to the antibody combinations. Leukemia-associated immunophenotypes were defined by gating on populations displaying an abberrant or infrequent immunophenotypes. The discrepancy of immunophenotypes between AML cases and normal controls was calculated by the logarithm difference(LD), i.e. LD=log(frequency of AML immunophenotype in leukemic bone marrow / frequency of AML immunophenotype in normal bone marrow). Immunophenotypes with ld>2.0 were defined as LAIPs. The expression patterns of these LAIPs were also described in AML patients and in normal controls. Based on the LAIP and LD values, MRD levels were determined in 11 AML patients.Results: 21 of 48 immunophenotypes were were identified as LAIP according to the LD value>2.0. The antigen asynchronous expression is the most common abberant type in present study. At least one aberrant/infrequent immunophenotypes was identified(median:4;range 0 to 5) in 92.8% (53/57) analyzed AML patients. Most of these LAIPs distribute regularly and much lowercompared to AML in normal controls. An analysis rule was outlined for detection of MRD in unselected AML patients. With the analysis rule, the MRD were identified in all of the 11 AML patients. One patient's relapse, who had entered complete remission three years before, was sucessfully predicted two monthes before his obvious relapse in morphology because his very small amount of bone marrow cells, 0.45% of LAIP with CD117+/CD15+/CD34+ was detected by our fow cytometry analysis.Conclusions: 1. 21 of LAIP were obtained according to the LD >2.0, and 95.2%(20/21) of these LAIP were CD34+,CD13+ or CD33+; 2. LD value can be used as the standard for LAIP; 3. At least one LAIP can be detected in 92.8%(53/57) of patients; 4.Phenotype switch was not found in the present study; 5.Most LAIPs express regularly but minimally in normal bone marrows. The express patterns could be used as controls in MRD analysis. 5. The rule used to analyze MRD in our study emphasize the importance of FCM data at diagnosis. Comparing with other normal cells in the same data also provide useful information for recognizing abberant cell populations. 6.The application of multiparameter fiwo cytometry to identify LAIP and MRD is feasible in unselected patients with AML.