Study On Key Markers Of Minimal Residual Disease In Children With B-ALL By Flow Cytometry And Establishment Of Ph~+ B-ALL Imatinib-Resistant Cell Line

Objective:(1)The antibodies commonly used in clinical laboratory for minimal residual disease(MRD)detection of B-lineage Acute Lymphoblastic Leukemia(B-ALL)were analyzed.According to the difference of detection rate of various antibodies,the combination of antibodies was optimized to provide reference for clinical flow detection of B-ALL MRD.(2)A stable Ph+ B-ALL cell line(SUP-B15/IM)resistant to imatinib(IM)was constructed,which provided a cell model for the study of drug resistance mechanism.Methods:(1)The subjects were B-ALL children who received formal chemotherapy in the first affiliated Hospital of Guizhou Medical University from September 2015 to July 2018.Five fixed of antibodies(B1:CD34/CD10/CD45/CD19/CD20/CD38,B2:cTdT/CD34/CD45/ CD19,B3:CD81/D10/CD45/CD19,B4:CD58/CD123/CD45/CD19,B5:CD34/CD13+CD33/CD45/CD19)were used to detect the MRD of children with B-ALL,and the leukemia related immunophenotypic(LAIP)was detected.(2)To establish a Ph+ B-ALL resistant cell line model(SUP-B15/IM),SUP-B15 cells were induced intermittently by increasing the concentration of imatinib in small doses.The cell resistance was detected by CCK-8 method,and the induction of apoptosis by IM was detected by flow cytometry(FCM).Results:(1)The combination of CD20 antibody at the late stage of B cell maturation with the CD10,CD34 and CD38 antibodies at early stage of B cells has a strong effect on the detection of MRD,and the detection rate is as high as 92.5%.The detection rates of CD10/CD81 and CD34/cTdT were 75.9% and60.2%,and the detection rate of CD123 alone was also high,accounting for 54.6%.However,the detection rates of CD58 and CD13+ CD33 accounted for only 38.1%and 41.6%,and other abnormal expressions were observed in cases where these threeantigens were abnormally expressed.(2)the drug resistance cell line of SUP-B15/IM was successfully established,and its drug resistance index to IM was 12.3.After stimulated with 100 ?mol/L IM for 24 h,the apoptosis rate in the control group SUP-B15/C cells was much higher than that in the drug resistant group SUP-B15/IM cells(P < 0.001).In addition,the expression levels of MDR1 and MRP5 gene in SUP-B15/IM cells treated with 100 ?mol/L IM for 24 h were significantly up-regulated(P < 0.001).Conclusion:(1)when the fluorescence channels of flow cytometry are limited,we can consider abandoning these three antibodies of CD58 and CD13+CD33 and combining CD34,CD10,CD45,CD19,CD20,CD38,CD81,CD123 and cTdT antibodies with high detection rate according to the actual number of fluorescence channels.(2)The IM-resistant Ph+ B-ALL cell line SUP-B15/IM was successfully constructed and can be used as an in vitro model for the study of Ph+B-ALL resistance to chemotherapeutic drugs.