Research On Cytochip Applying To Immunophenotyping For Leukemia

ObjectiveLeukemia is one of the common malignant neoplasms. Before thel980s diagnosis of Leukemia mainly based on morphology, Monoclonal antibody application for the typing of leukemia has opened a new way. Immunophenotyping for Leukemia overcomes the shortcoming of FAB classification ( morphology) , accuracy for distinguishing leukemia of myeloid and lymphoid origin. FAB classify provides more than 60% and Immunophenotyping provides 90% accuracy.Now clinical leukemia Immunophenotyping mainly in FAB classification no clear, conductde 1 ~ 2 antibody immunohistochemistry testing to bone marrow slides. It is difficult in its clinical application by the specimen volume;price restrictions. Flow cytometric analysis of leukemia with panels of monoclonal antibodies now provides 90% accuracy for immunophenotyping. But their equipment is expensive and labor - intension, requiring fluorescently labeled and allowing concurrent analysis for a limited number of CD antigens, usually one to four. It is difficult for widespread application.This experiment constructs a cytochip applying to immunophenotyping of leukemia. We choose an arm element that has a strong ability to immobilize protein , through antigen - antibody reaction to capture cells, and selection of a group of suitable antibodies to form microarray. Using a cytochip to immunophe-notype leukemia patients'vein blood specimens provides a simple clinical Immunophenotyping of leukemia diagnostic tools.Methods1. Slide decorationThe effects of different chemical reagent on slide were compared and slide immobilization efficiency was detected by fluorescence labled protein. We chose a chemical reagent that has a strong ability to immobilize protein.2. Cytochip constructionMonoclonal antibodies that are applied to immunophenotyping of leukemia were fixed slide to form cytochip.3. Immunophenotyping of leukemia by cytochipA suspension of mononuclear cells is applied to the cell array and cells only bind to antibody dots for which they express the corresponding CD antigen. The cytochip that had captured cells were dyed with Wrights - Giemsa.Results1. The glasses slides modified with aldehyde have the highest efficiencies of protein immobilization and activities of the proteins.2. Each point of the glasses modified with glutaraldehyde has same efficiency of protein immobilization.3. The cells dots were easy to be observed by the scanner or the common optics microscope. The bound cells were special according to corresponding antibody detected by fluorescently labeled antibodies and morphology.4. 7 cases were diagnosed ALL. 2 cases T - ALL expressed CD2, CD5, CD7, HLA -DR;5 cases B -ALL expressed CD19, CD1O,CD11 ,CD37.5. 23 cases were diagnosed AML. The incidence of HLA - DRNCD33N CD14NCD34 expression was respective 69. 6% ,69. 6% ,30. 4% ,21. 7%. M3 didn' t express HLA -DR, CD14 was only expressed in M4,M5.ConclusionThe arm molecules chosen have higher efficiencies of protein immobilization and maintain activities of the proteins. Monoclonal antibodies were spotted on the slide modified with aldehyde to make cytochip. The results of Immunopheno-typing of leukemia by cytochip are consistent with relevant paper. The results can be used as further support and complementarity to clinical FAB classifies. The cell array in our lab has a relatively simple technique route, easy to operate and repeat. It provides a rapid and high - throughput diagnostic tool for clinical Immunophenotyping of leukemia.