| Objectives:Human Leukocyte Antigen(HLA)is the main component that induces transplant rejection.After the receptor immune cells recognize the HLA of the surface of the transplant,they can produce anti-HLA antibodies against the graft.The effective detection of anti-HLA antibodies is the key to reduce the rejection.Lymphocyte crossmatch experiment and Panel Reactive Antibody(PRA)experiment can be used to judge the pre-existing anti-HLA antibodies of the receptors,which is of great significance to predict the risk of transplant rejection.At present,Complement Dependent Cytotoxicity(CDC)experiment and ELISA PRA experiment have low detection sensitivity due to the limitation of methodology.In practical work,false negative results may appear,which will affect the diagnosis and treatment of transplant patients.In order to improve the sensitivity of the detection method,this study will establish a new method of lymphocyte crossmatch and PRA experiment based on flow cytometry and apply it to kidney transplant patients.Methods:1.Research object and sample source(1)Lymphocyte cross-matching experiment donor specimens came from 30 donors conducted by our Organ Acquisition Organization(OPO)from March 2019 to January 2020.For 48 recipients,the negative control sera came from the mixed sera of 2 AB male health checkers in our hospital,and the positive control sera came from the mixed sera of 20 PRA positive patients in our hospital.(2)The PRA experimental specimens were from 2907 outpatients and inpatients from September 2016 to April 2019.2.Establishment of flow cytometry crossmatch(FCXM)experimental method and methodological comparisonIsolate donor lymphocytes by density gradient centrifugation or immunomagnetic bead method(negative selection method),compare the purity and recovery rate of lymphocytes separated by two different methods,and choose the method with better separation effect as the lymphocyte separation method.Incubate the cells with the receptor serum,add anti-CD3-PE,anti-CD 19-APC,anti-IgG-FITC and other fluorescent antibodies to identify B lymphocytes,T lymphocytes,and anti-HLA IgG antibodies,and use flow cytometry to identify The data is analyzed.14 donor lymphocytes and 48 recipient sera were collected,and FCXM and CDC experiments were performed simultaneously to compare the consistency of the two methods.In the same period,the recipient sera were grouped according to the results of flow fluorometry PRA,and the coincidence rates of the FCXM and CDC methods with the PRA results were compared.3.Establishment and experimental comparison of experimental methods for PRA detection by flow fluorescenceIncubate the receptor serum with fluorescent microspheres adsorbed with HLA antigen,so that the anti-HLA antibody in the receptor serum binds to the corresponding antigen,add R-phycoerythrin-labeled goat anti-human IgG antibody,and bind to the microsphere Recognize the anti-HLA antibody and analyze the data by Luminex 200.Sera of 2907 patients were collected,of which 1142 were detected by ELISA and 1765 were detected by flow fluorometry.During the same period,serum of 40 patients was drawn and tested by two methods.4.Statistical analysis of experimental dataStatistical analysis was performed using SPSS V22.0 statistical software.The measurement data results were statistically analyzed using t test,one-way ANOVA,chi-square test,Mann-Whitney U test and other methods according to the data type.Chi-square test was used to compare the count data rate between groups.The paired count data was tested by McNemar test and Kappa test.Results:1.Establishment of FCXM experimental method and methodological comparison(1)The purity and recovery of lymphocytes separated by magnetic activated cell sorting(Negative selection)are 95.1±1.9%and 84.1±7.8%respectively.The purity and recovery of separated by density gradient centrifugation are 77.0±9.2%and 14.5±7.7%respectively.The purity and recovery of magnetic activated cell sorting(Negative selection)are significantly higher than that of density gradient centrifugation(P<0.01).(2)In FCXM experiment,the cutoff value of anti-IgG-FITC of T-lymphocyte is 222.5,and that of anti-IgG-FITC of B-lymphocyte was 210.1.(3)In the PRA negative group,the consistent result between CDC and PRA is 100%(7/7),that between FCXM and PRA is 42.9%(3/7),that between FCXM and PRA is 48%(24/50),and that between CDC and PRA is 8%(4/50).(4)57 crossmatching experiments were carried out by FCXM and CDC at the same time.There were 4 cases of CDC positive,including 2 cases of FCXM positive and 2 cases of FCXM negative and 53 cases of CDC negative,including 27 cases of FCXM negative and 26 cases of FCXM positive.The consistency of the two methods is poor.(McNemar P<0.01;Kappa<0.4).2.Establishment of experimental method of PRA detection by flow fluorometry and methodological comparison(1)PRA of 40 patients was detected by flow cytometry and ELISA at the same time.There were 4 cases of PRA positive in ELISA,including 4 cases of PRA positive and 0 case of PRA negative in flow cytometry and 36 cases of PRA negative in ELISA,including 2 cases of PRA negative and 34 cases of PRA positive in flow cytometry.The consistency of the two methods is poor.(McNemar P<0.01;Kappa<0.4).(2)In ELISA,PAR ?,PAR ? and total positive rates are 9.0%(103/1142),8.1%(92/1142)and 13.7%(156/1142)respectively.In flow cytometry,they are 22.9%(404/1765),21.9%(387/1765)and 34.3%(606/1765)respectively.The positive rates in flow cytometry are significantly higher than those of ELISA(P<0.01).Conclusions:1.The purity and recovery of lymphocyte separated by magnetic activated cell sorting(Negative selection)is higher than that by density gradient centrifugation.2.The sensitivity of FCXM experiment is higher than that of CDC experiment,and the interpretation of experimental results is less affected by the subjective factors of experimental operators,so it is easy to standardize.3.In PRA experiment,the sensitivity of flow cytometry is higher than that of ELISA.The detection procedure of flow cytometry takes less time and is suitable for the detection of samples in bulk. |
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