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

Objective To definate leukemia-associated immunophenotypes(LAIPs)and establish the method of detecting minimal residual disease(MRD)in acute myeloid leukemia(AML)by multiparameter flow cytometry(MFC).Methods 158 bone marrow specimens were selected from the Second Hospital of Shanxi Medical University,the People Hospital of Shanxi and the Children Hospital of Shanxi from April,2005 to March,2006.130 new AML bone marrow samples and 28 bone marrow samples after chemtherapy were included.At first,immunophenotypes were assayed using a sets of commercial 3-color antibodies combination(IgGFITC/IgGPE/CD45ECD,CD34/CD33/ CD45,CD19/CD10/CD45,CD14/CD13/CD45,HLA-DR/CD7/CD45,cyMPO/cyCD79 a /cyCD3(added CD45CY5).The patients of AML diagnosised by immunology,following-up patients and normal bone marrow were detected using 7 sets of 4-color antibody panels (IgG1FITC/IgG1PE/CD45Cy5/IgG1ECD,CD34/CD13/CD45/CD19,HLA-DR/CD33/CD45/CD 34,CD15/CD11b/CD45/CD34,CD15(orD11b)/CD117/CD45/CD34,CD34/CD2/CD33/CD45, CD34/CD56/CD33/CD45)。According to the results from normal marrow specimens,myeloid precursors in normal and leukemic bone marrow was compared by relatively quantitative, leukemia- associated immunophenotypes(LAIPs)was definated.28 bone marrow samples after chemtherapy were monitored accoeding to the LAIPs after initial chemtherapy。Results 81 patients in 130 new AML(62.31%),in 90 non-M₃ AML(90%)had more than one LAIPs.25 LAIPs were identified by multiparameter flow cytometry.The panal of highest frenquence was the CD11b/CD117/CD45/CD34 combination,72.22%.CD15/CD117/CD45/ CD34(56.00%),HLA-DR/CD33/CD45/CD34(43.21%),CD34/CD13/CD45/CD19(37.03%), HLA-DR/CD7/CD45(33.33%),CD34/CD56/CD33/CD45(7.50%),CD15/CD11b/CD45/CD34 (5.66%)and CD34/CD2/CD33/CD45(2.50%))nect to it.Conclusion 1,25 LAIPs were definated.They were adapted in 62.31%AML,90% non-M₃ AML.2,SSC/CD45 contribute to identify LAIPs.3,LAIPs with some regularity in different AML showed obvious peculiarity of stage and lineage.4,Immunophenotype chaging was the result of leukemia cells differentiation.5,Immunophenotype chaging is advantage in detecting AML MRD by MFC.6,New LAIPs can be identified in relapse AML. Objective To monitor the effect aftert initial induction therapy and minimal residual disease in acute promyelocytic leukemia(APL or M₃)by a panel of four-color combinations of monoclonal antibodies(CD15/CD11b/CD33/CD45).Methods Using a panel of four-color combinations of monoclonal antibodies (CD15/CD11b/CD33/CD45),detect 40 borrow ample of patients with M₃ diagnosised by FAB and immunophenotype before threapy and 12 follow-up borrow specimens after initial induction therapy,at the same time,8 borrow specimens from normal controls were detected.Result Leukemia cells expressed CD15-CD11b-CD33+ in M₃ before therapy at CD45/SSC gate.The immunophenotype is leukemia-associated immunophenotype.CD15-CD11b-CD33+ leukemia cells obviously decreasad after initial induction therapy,the cells began to express CD15+ and/or CD11b+,and CD15++CD11b++ mature neutrocyte.The frenquent of cells with CD15-CD11b-CD33+ was 0,0.002%,0.03%,0.08%,0.14%,0.18%, 0.25%,0.66%and 1.73%respectively in granulocytes with high SSC in remission M3,and was 12.24%,14.76%and 45.07%respectively in leukemia cells of M3 without remission.Conclusion Four-color combinations of monoclonal antibodies(CD15/CD11b/CD33/ CD45),can monitor the effect of induction therapy in M₃ and MRD in some M₃ with high SSC. Objective To establish the method of detecting minimal residual disease(MRD)in B lineage acute lymphoblastic leukemia(B-ALL)by multiparameter flow cytometry(MFC),and definate leukemia-associated immunophenotypes(LAIPs).Methods 105 bone marrow specimens(60 from new B-ALL,17 after initial induction therapy from the new patients and 28 regenerating bone marrow samples after chemotherapy without initial data)from B-ALL and 8 normal marrow specimens was detected by using four sets of 4-color antibody panels(TdT/CD10/CD19/CD45,CD20/CD10/CD19/CD45,CD34/CD38/CD19/CD45,CD34/CD22/CD19/CD45).CD34/CD13/CD19/CD45,HLA-DR/ CD33/CD19/CD45 or CD10/CD14/CD19/CD45 was detected when CD13,CD33 or CD14 was expressed at diagnosis.According to the results from normal marrow specimens and regenerating bone marrow samples,B-cell precursors in normal and leukemic bone marrow was compared by relatively quantitative,leukemia-associated immunophenotypes(LAIPs)was definated.These LAIPs were not expressed or very low at diagnosis of B-ALL.The bone marrow specimens after initial induction therapy from the new patients were monitored by the LAIPs.Results 21 LAIPs were identified by multiparameter flow cytometry.The most informative panal was 60%for CD20/CD10/CD19/CD45.Valid LAIPs were found in 91.67% of 60 new B-ALL。In 17 patients after initial induction therapy,the frequencies of LAIPs in 12 patients which reached phenotype remission were 0.25%,1.2%,0.003%,0.08%,0.06%,0.66%,0.3%,0.08%,0.22%,0.09%,3.6%and 0.07%,respectively.The other 5 patients which not reached phenotype remission were 12.25%,38.2%,7.02%,5.08%å’Œ13.06%,respectively. The sensitivity of this method was 10,much higher than microscopic inspection.Conclusion 21 LAIPs were identified.Detection of MRD by multiparameter flow cytometry is adapted 90%B-ALL.B-lymphocyte precursors in regenerating bone marrow samples after chemotherapy were increased compared with that in normal marrow specimens.These were important in identifying LAIPs.