A Study Of The Immunocytochemistry Of Cells Captured By The Cell Microarray For Leukemia Immunophenotyping

ObjectiveBiochip was born in the early 1990s,is significant progress of characteristic of the times in recent years in the hi-tech field,is the high-tech integrated cross-formation of physics,microelectronics and molecular biology.It has been widely used in areas such as disease diagnosis.At present,the leukemia immunophenotyping diagnosis mainly depends on the use of monoclonal antibodies 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,decides leukemia to belong to which series of decisions and which stage,make a immunological diagnosis.The cell chip for leukemia immunophenotying,a newly achievement of our laboratory,is based on specificity of antigen-antibody reactions and prepared by fixing monoclonal antibodies against CD antigens(CD antibodies)on the microarray supports,forming antibody array.Different monoclonal antibodies can automatically identify and capture the leukemic cell with CD antigens(CD antibodies),thus leukemia immunophenotyping is achieved.Then compared with immunocytochemistry and flow cytometry method,this detection method of leukemia immunophenotyping is high-throughput,economical and practical;uses little sample,detects speedly and high sensitively.However,as a new type of diagnosis of leukemia immunophenotyping,it needs to testify using traditional immunocytochemical technique and flow cytometry method.In addition,the diagnosis of leukemia immunophenotyping,needs to detect both differentiation antigens on the surface and the ones within the cytoplasm,especially in the dual phenotypic and special phenotype testing,detection of intracellular antigen is more important.Australia Institute of Medicine of the University of Sydney Larssa Belov developed the cell chip to diagnosis the leukemia immunophenotyping,only to detect differentiation antigens on cellar membrane,and our laboratory has developed chips to take the glass with the transparency as the carrier,white blood cells with the corresponding CD antigens bind with the antibodies on the chip,if we carry on immunocytochemical method,we can detect the differentiation antigens within the cytoplasm,the results will be intuitive and stable,and will provide a more rapid, simple,complete and accurate diagnosis method of leukemia immunophenotyping for the clinical diagnosis.Methods1,Determination of the best fixation of cells on a chipThe cell chips were prepared,at room temperature,they were fixed with the following fixation:(1)methanol:acetone(1:1 v / v)for 90 seconds;(2)95%ethanol for three minutes;(3)95%ethanol:glacial acetic acid(AA liquid 95:5 v / v)for three minutes;(4)AF fluid(95%ethanol:formaldehyde:water=68.4:10:21.6 v / v)for one minute.After the cell chip fixed with AF was washed two times with distilled water,the other cell chips fixed with other fixations were naturally dried.The cell morphology, antigen expression and the color of dyed nuclear of cells captured by antibodies microarray were observed,and counting cells before and after fixing under the microscope.2,Determination of the best concentrations of monoclonal antibodies of the immunocytochemistry on cells captured by a cell microarrayUnder the same conditions,the cell chips were fixed with the best fixation,the streptavidin-peroxidase immunocytochemistry was carried on,concentrations of the first antibodies CD7,CD13,CD19,MPO were 4μg/ml,2μg/ml,1μg/ml,0.5 μg/ml,0.25μg/ml,negative control was dropped PBS,incubated at room temperature for one hour;all of them were colored with DAB,and observed under the microscope.3,Expression of the antigens on the membrane and within the cytoplasm of the cells captured by the cell chipThe cell chips,were fixed with the best methods and carried on immunocytochemistry using the first antibody immunocytochemical MPO,the best concentration of 1μg/ml,negative control used PBS buffer instead of the first antibody. Results were observed under the microscope.4,Detection of the specificity of the cell chips capturing cellsThe cell chips,were fixed with the best methods and carried on immunocytochemistry using the first antibodies CD7,CD13,CD19,the best concentration of 1μg/ml,negative control used PBS buffer instead of the first antibody,under the microscope three high-power microscope visions from each antibody points were picked,and counted the total number cells and the number of positive cells which had brown particles,at last the positive rates were calculated.5,Application of immunocytochemistry and the cell chip to diagnosis leukemia immunophenotypingThe cell chips to diagnosis leukemia immunophenotyping were prepared, leukemia WBC were isolated from the peripheral blood of patients,dyed with acridine orange,incubated on the chips,cleaned with PBS,scanned,signals were detected,then results of leukemia immunophenotypings were got.Immunocytochemistry was carried on to further confirm the results.Results1,The method olAF(95%ethanol:formaldehyde:water=68.4:10:21.6 v/v) fixing for one minute is the best fixation for the cells captured by cell chips.2,The best concentration of monoclonal antibodies in immunocytochemistry of cells captured by cell chips is 1μg/ml,incubated at room temperature for one hour.3,The first antibodies in immunocytochemistry of cells captured by cell chips are CD7,CD13 and CD19,brown particles are on the cell membrane;when MPO is the first antibody,the brown particles are in the cytoplasm.4,The positive rates of specificity of cell chips capturing are more than 96.7±0.4%,The positive rates of nonspecificity of cell chips capturing are less than 0.8±0.3%,it has obvious specificity.5,The positive rates of specificity of leukemia cell chips capturing are more than 97.4±0.2%,it has obvious specificity.Conclusion1,The method of AF(95%ethanol:formaldehyde:water=68.4:10:21.6 v / v) for one minute is the best fixation for the cell chip of diagnosising leukemia immunophenotyping.2,That the first antibodies 1μg/ml of immunocytochemistry incubated at room temperature for one hour are the best.3,Expression of intracellular and extracellular antigens is best.4,Immunocytochemistry tests that cell chip method is highly specific capture cells.5,30 cases of patients of leukemia were made the diagnosis of leukemia immunophenotyping with cell chips,carried out immunocytochemical analysis,the results of diagnosis are accurate and reliable.