Establishment Of Point-of-Care Counting Method For CD4+ T Lymphocytes In Whole Blood Sample

AIDS(Acquired Immune Deficiency Syndrome)is a kind of infectious disease caused by HIV(Human Immunodeficiency Virus).Since HIV was discovered in 1981,nearly 40 million people have been infected with HIV in the worldwide,1.2 million people die each year from AIDS-related diseases.At present,the most effective means of AIDS treatment is highly active antiretroviral therapy(HAART).According to World Health Organization(WHO),while the number of CD4+ T lymphocytes is 200-350 per micro liter,recommended the best time for treatment of AIDS.The number of cells is also an important indicator to evaluate the therapeutic results.So developing rapid and accurate CD4+ T cell counting methods is urgently needed.CD4+ T lymphocytes counting gold standard is the flow cytometry(FCM).However because of its high cost,the operation is difficult,only developed cities and large hospital could have capability of cell counting by FCM.In China,the higher proportion of HIV infection is mainly concentrated in the areas of Yunnan,Henan,Guangxi,Xinjiang,where the economic conditions are not developed.In this study,we established a rapid CD4+ T cell detection method to monitor the number of CD4+T cells for backward areas.In the experiment,the whole blood CD4 fluorescent immune chromatographic test was established.The whole blood is pretreated by the capture column,and the influence caused by erythrocyte in the test is excluded.Then instrument detects results of the strip.The whole blood CD4+ T cells counting can be completed within 30 min.Compared with the result of flow cytometry,the fluorescent strip can detect the number of whole blood CD4+ T cells in the range of 50-1500 cells/μl,R2 is 0.84,have a good correlation.At the same time,we also cooperate with Beijing University of Chemical Technology,designing an automatic analyzer which integrates all experimental steps to finish the CD4+ T cells counting by instrument.Now we have completed the first prototype.However,the CD4 fluorescent immune chromatographic test method requires pre-treatment of whole blood sample,increasing the complexity of the operation,and the development of the corresponding automation equipment is more difficult.So we try to establish another method on the microfluidic chip platform,hoping to integrate the two parts of whole blood pretreatment and result detection to complete the whole blood CD4+ T cell quantitative detection.Quantitative detection chip of CD4+ T cells in the whole blood is on the basis of the microfluidic chip,with the centrifuge and detection equipment to complete the whole blood CD4+ T cells counting.The chip is made of PMMA and PC,with laser cutting and glue bonding.Chip separates erythrocytes from whole blood by density gradient centrifugation.Then the target cells transferred to the reaction chamber,and through the no-washing area to complete the immune detection.At present,the chip design has completed and the feasibility of the method has been verified.Compared with the CD4 ELISA kits,R2 is 0.54 when the number of CD4+ T cells in whole blood was 500-1200 cells/μl.We will continue to optimize the testing conditions and complete the integration of the instrument in the future.In this study,we established two CD4+ T cell counting methods.One of the whole blood CD4 fluorescent immune chromatographic test platform has been completed basically and entered into the stage of designing the automatic analyzer.The other method has been verified the feasibility of testing.In the next step,we will continue to optimize the testing conditions and complete the integration of the instrument.These two platforms are all suitable for remote areas,they have the merits of simple operation,time-consuming,without expensive equipment,on-site testing.It is of great significance for HIV testing and monitoring of antiretroviral therapy.