Experimental Research Of Six-color Flow Cytometry Detect Leukemia Associated Immunophenotype In Childhood B-lineage Acute Lymphocytic Lleukemia

Background:B-linage acute lymphocytic leukemia was the malignantly clonal disease which expressed early leukocyte differentiation antigens and its incidence among children malignant cancer was the first. With the development of flow cytometry and the application of monoclonal antibodies, as the necessary complement of the traditional morphological and cytochemical examination, immunophenotype has became a basic mean for acute lymphoblastic leukemia research. Because of the leukemia cells were neoplasm cells, which could express the different immunophynotype from normal cells, as Leukemia associated immunophenotype. LAIP could't only as the base for the diagnosis of leukemia, but LAIP also provided the basis for monitoring MRD. By immunophenotyping, flow cytometry could determine the cell type and differentiation stage of B-linage acute lymphocytic leukemia's, and also became an important mean for minimal residual disease detection based on leukemia associated immunophenotyp e as firstly diagnosed.Formerly, Leukemia associated immunophenotype of B-linage lymphocytic leukemia detection was based on three-color or four-color flow cytometry. In recent years, with new fluorescent antibody technology appearing, multi-color fluorescence labeling antibodies developing, and flow cytometry equipment improving, six-color flow cytometry for Leukemia associated immunophenotype detection was possible. The aim of this study was to discuss applying six-color flow cytometry to detect leukemia associated immunophenotype of B-linage acute lymphocytic leukemia on the base of previous three-color flow cytometric immunophenotype of our hospital.Objective:1,To discuss leukemia associated immune phenotype of one hundred and five children with B-lineage acute lymphocytic leukemia in Guangzhou, provide the basis and reference for selecting antibody combination of six-color flow cytometry to detect leukemia associated immune phenotype.2,To evaluate the stability of antibody expression in B-lineage acute lymphobla stic leukemia detected by six-color flow cytometry, discuss superiority of six-color flow cytometry in detecting leukemia associated immune phenotype of acute B-lineage lymphoblastic leukemia, provide the basis and reference for using six-color flow cytometry to detect minimal residual disease.Methods:1,The immunophenotypes of 105 children with B-linage acute lymphocytic leukemia firstly diagnosed by three-color flow cytometry from 2008.8 to 2010.2 in Guangzhou Women and Children's Medical Center were analyzed. Meanwhile, 10 non-leukemic children's bone marrows were detected as control.2,Two groups of six-color antibody combinations CD20/CD34/CD10/CD22/CD 45/CD19, CD7/CD5/CD13/CD33/CD45/CD19 were used to detect bone marrow of 25 patients diagnosed as B-ALL by three-color flow cytometry. Meanwhile,10 non-leukemic children's bone marrows were detected as control.Results:1,98 of 105 patients were detected LAIP. The frequencies of LAIP were : CD19,CD10,CD34,CD22,HLA-DR abnormal over-expression was 25%,47%,33%,11%,9% respectively; CD45, CD38, HLA-DR abnormal weakly positive or loss expression was 66%, 16%, 5% respectively; CD34, CD20 cross-phase abnormal positive expression was 25%; Myeloid lineage associated antigens CD13, CD33, CD15, CD64 cross-lineage expression was 23%,18%, 16%,1% respectively; T- lineage associated antigens CD7 cross-linage expression was 1%. 2,In three-color flow cytometric immunophenotyping, the positive expression rate of CD19, CD22, CD10, CD20, CD5, CD13, CD33, CD34, CD45 was 96%, 100%, 76%, 40%, 4%, 36 %, 36%, 84%, 36% respectively. In Six-color flow cytometry, the positive expression rate of CD19, CD22, CD10, CD20, CD5, CD13, CD33, CD34, CD45 was 100%, 96%, 80%, 40%, 4%, 44%, 32 %, 84%, 40% respectivel. Three-color flow cytometric immunophenotype using 23 kinds of antibod y detected 10 LAIPs of CD19^strong,CD10^strong,CD34^strong,CD22^strong,CD45^-/dim,CD34⁺/CD38-,CD13⁺,CD15⁺,CD33⁺,CD34⁺/CD20⁺. 23 of 25 patients can detect LAIP. 1 case with 1 LAIP, 4 cases with 2 LAIPs, 7 cases with 3 LAIPs, 5 cases with 4 LAIPs, 5 cases with 5 LAIPs,1 case with 6 LAIPs. Six-color flow cytometry using 10 kinds of antibody detected 13 LAIPs of CD10^strong,CD34^strong,CD22^strong,CD45^-/dim,CD19⁺/CD13⁺,CD19⁺/CD33⁺,CD34⁺/CD20⁺,CD34⁺/CD22⁺⁺,CD10⁺⁺/CD20⁺,19⁺/CD10⁺⁺/CD22⁺⁺,19⁺/CD34⁺/CD10dim⁺,19⁺/CD45^-/dim+/CD34^-/dim+,19⁺/CD10-/ CD20-/CD34-. 23 of 25 patients can detect LAIP, 1 case with 1 LAIP, 2 cases with 2 LAIPs, 4 cases with 3 LAIPs, 4 cases with 4 LAIPs, 2 cases with 5 LAIPs, 5 cases with 6 LAIPs,4 cases with 7 LAIPs, 1 cases with 8 LAIPs.Conclusion:1,Among the leukemia associated immunophenotype of B-linage acute lymphocytic leukemia in Guangzhou, the mainly antibody over expression were CD10,CD34,CD19,the rate was 47%,33%,25% respectively; The mainly antibody weakly positive expression or absence was CD45,the rate is 66%; The mainly antigen cross-linage abnormal expression were CD13,CD33,the rate was 23%,18% respectively.2,In Six-color flow cytometric, the positive rate of antibody detected was stable. The positive rate of antibody detected was no significant differences compared with three-color flow cytometric immunophenotype. Although the total number of antibody used in Six-color flow cytometry was few, through FSC/SSC,SSC/CD45,SSC/CD19 gating, analyzing na?ve cell populations,CD19⁺ cell populations on multiparametric two dimensional point maps, we can detect more Leukemia associated immunopheno- type than the three-color Flow cytometry. Moreover, Six-color flow cytometry can detect six antibodies in one tube. It also can flexibly adjust antibody combinations according to Leukemia associated immunophenotype as firstly diagnosed, which was more conducive to detect Minimal residual disease after chemotherapy complete remission.