The Detection Of Clinical Acute Leukemia Cell Membrane Related Antigen By A Cell Immunophenotyping Microarray

ObjectiveAt present,the leukemia immunophenotyping diagnosis mainly depends on the use of monoclonal antibody known(McAb) to identify leukemia cell surface antigens, or cell internal-CD antigens(cluster of differentiation),in accordance with the appearance and disappearance of different antigens in various stages,decide leukemia belongs to which series of decisions and which stage,make a immunological diagnosis. At present,doctors usually choose immunohistochemical technique or flow cytometry method.But both of them need large specimen volume,high price,complex procedures, and other restrictions such as analysis of 1～4 surface antigens,missing a lot of indicators to body immune system.Researchers have begun to use a biochip technological platform which is high-throughput,paralleled for the integration of leukemia immunophenotyping.The cell chip for leukemia immunophenotyping,a newly achievement of our laboratory,is based on specificity of antigen-antibody reactions and prepared by fixing monoclonal antibodies against CD antigens on the microarray supports,forming antibody array.Different monoclonal antibodies can automatically identify and capture the leukemic cells with CD antigens,thus leukemia immunophenotyping is achieved.On the basis of previous experiments,I have used the microarrays to immunophenotyped acute leukemia with peripheral blood samples.And detection the acute leukemia cell membrane related antigen CD6,LTK,CaSR by immunofluorescence technique.Get a quick,simple and complete immunization data for the clinical diagnosis and antibodies-target treatment. MethodsAccording to antibodies in our laboratory existing and internationally recognized acute leukemia immunophenotyping first-line,second-line antibodies,screen antibodies to adopt the best combination to build acute leukemia immunophenotyping chip.With acute leukemia immunophenotyping chip on 35 cases of acute leukemia peripheral blood to carry out Immunophenotyping:stain extracted leukocytes by acridine orange,then incubate the chip,scan the chip by fluorescence picture scanner to observe cells captured to determine antigen expression on the cell membrane.Wright -Giemsa staining,cell morphology was observed under the microscope after the antibody on the chip capture the cells,combining the results of immunology and morphology,the typing results were compared with clinical test results.Detection of 35 cases of acute leukemia cell membrane surface CD6,LTK,CaSR three membrane-associated antigens expression:leukemia cells captured by the chip, with indirect immunofluorescence technique to detect leukemia cell membrane associated antigen and observe under the fluorescence microscope for the results.ResultsAntibodies selected for Acute leukemia immunophenotyping chip:T-ALL:CD1,CD2,CD3,CD4,CD5,CD7,CD8-1,CD8-2,TCRαβ,TCRδγ.B-ALL:CD10,CD19,CD20,CD22,CD24,CD79b,k,λ.AML:CD13,CD14,CD15,CD33,CD41,CD61,CD64,CD103,CD235a.non-specific:TdT,HLA-DR,CD34.combination scanning results of cell membrane antigen expression with cell morphology under the microscope,35 cases of acute leukemia peripheral blood for immunophenotyping,11 cases of T-ALL,14 cases of B-ALL,10 cases of AML,typing results are consistent with clinical test results,proved the accuracy of diagnostic chips..peripheral blood of 35 cases of acute leukemia are tested,leukemia cells are captured by the cell chip.indirect immunofluorescence method are used to detect cell membrane associated antigen.the first antibody CD6,LTK,CaSR concentration of 1:300 37℃were incubated for 30 minutes,the second fluorescence antibody was incubated with concentration 1:200 37℃20 minutes.Under the Fluorescence microscope to observe.Leukemia cell membrane associated antigen express CD6 for T-ALL,LTK for B-ALL,CaSR for AML.ConclusionsIn this experiment typing results derived from acute leukemia imrnunophenotyping chips are the same with clinical detecting results,CD6,LTK,CaSR three acute leukemia cell membrane associated antigens with specific expression in T-ALL, B-ALL,AML.It can be used for clinical leukemia immune typing and antibody targeted therapy.The cell microarray can be easy to use,good repeatability and stability.For clinical leukemia immunophenotyping,it provides a fast, high-throughput,parallel,integrated technology platform.