| BackgroundCurrently, clinical organ transplants were carried out on the vast majority of the donors and recipients between which got no blood relationship. Because of the highly polymorphic of human HLA system, it is hard to find the dono r whose HLA is identical with the receptor in the crowd, there is a potention of rejection in every allogeneic transplantation. In order to reduce rejection, im prove the successful rate of transplantation, and prolong graft survival time wit h function, it is particularly important for strict immunological matching betwe en the donors and recipients before transplantation. Immunological factors of d onors and recipients including:①ABO blood type compatible:①HLA typing③Crossmatch④Panel Reactive Antibody.Human leukocyte antigen (HLA) is known as transplantation gene, HLA matching including:①HLA antigen typing;②HLA antibody analysis, that is i dentification of panel reactive antibody and analysis of the anti-donor-specific a ntibodies. Detection of the specificity of the HLA antibodies of the sera to ide ntify unacceptable HLA antigens(PRA), detection of the specific anti-donor HL A-antibodies to identify the acceptable HLA antigens(CDC). CDC is one of th e major histocompatibility experiment before or after kidney transplantation, it is the most important immunological experiment in selecting the donors and re cipients before transplantation, which is important for the prevention of hypera cute rejection and accelerated rejection, monitoring of the immune status of rec ipients, reducing side effects of immunosuppressive drugs and improving graft /recipients functional survival rate by the short and long term, CDC positive is a contraindications for the transplantion. If there are resistance of donor-specifi c anti-HLA antibodies (DSA) before transplantation in recipients, antibodies-me diated rejection (AMR) is likely to lead to early graft dysfunction. Circulating antibody levels of patients may fluctuate with hemodialysis frequency and effec ts, and (or) the patients received blood transfusion or other forms of sensitizati on (re-transplantation, pregnancy), although the cross-matching before transplant ation using the current sera of patients was negative, however, memory immun e response still exist, which may lead to hyperacute rejection, and (or) early g raft dysfunction, therefore it is necessary to subject to continuous monitoring a ntibodies of recipients after transplantation. CDC currently used mainly include complement dependent lymphocytotoxicity crossmatch (NIH-CDC) and flow cyt ometry crossmatch(FCXM), although the CDC result is positive, it is also poss ible to obtain good graft/recipient functional survival rate in short and long ter m. On the contrary, the CDC results is negative, but early graft dysfunction m ay also happen. The results of NIH-CDC and FCXM do not match with the e ffect of transplantation frequently, the reliability is poor, ELISA-PRA may be more accurately reflecting the situation of sensitization of recipients. Although the PRA strength is related to the survival rate of the graft, the antibodies det ected by ELISA-PRA are not necessarily specific against the donor. The presen t study show that ELISA has a stronger sensitivity and specificity in detection of HLA antibodies than the CDC method, the test results of ELISA is objectiv e and reproducible, detecting specific anti-HLA-IgG antibodies only, without th e interference of other antibodies; not only detecting complement binding HLA antibodies, but also non-complement binding HLA antibodies; no need for live lymphocytes, greatly facilitating the clinical applications. The method based o n ELISA is considered as the "gold standard " in HLA antibody screening. In order to improve the ability for detecting and prevent the occurrence of sever e rejection, many transplantation centers at home and abroad tend to use two kinds of cross-matching methods for detecting before transplantation currently, common combinations used including NIH-CDC + AHG-CDC or NIH-CDC + FCXM, etc, so establishing of a new cross-matching method can also be a ch oice for detecting. Based on the above, establishing of a new CDC method ba sed on ELISA principle for better predicting rejection can be considered.ObjectiveTo improve the sensitivity and specificity of DSA identification, provide a new method for monitoring DSA before and after transplantation and effectivel y predict rejection, based on the histocompatibility matching experimental condi tions of the domestic organ transplantation laboratory, innovatively establishing ELISA-CDC experimental techniques without adding special laboratory equipme nt. ELISA-CDC were performed using the PRA positive sera with the known specific antibodies of HLA, to compare the matching rate in recognition of the same specific HLA antibodies between ELISA-CDC and ELISA-PRA, and ex plore the value of ELISA-CDC before transplantation or in monitoring of DSA after transplantation. NIH-CDC-negative was the inclusion criteria for renal tra nsplant recipients receiving transplantation between January 2010～December 2010, patients were excluded from this transplantation if the ELISA-CDC was positive while the NIH-CDC was negative, close observation of the situation o f the transplant recipients within 5d after transplantation, pay attention to obser ve whether the hyperacute rejection or accelerated rejection happened.Methods1. subject1.1 ELISA-CDC establishment①Extraction of donor's lymphocytes and lymphocyte lysate preparation;②Establishment of quality control system:reagent control, lysate control and negative control, reading absorbance A value with a microplate reader, dec iding the positive and negative samples by comparing the OD value of the ne gative control samples;③Based on the ELISA principle, specifically detection of HLA-IgG antib odies.1.2 Validation of ELISA-CDC methods1.2.1 Specimen collectionAll specimens were selected from the 5 cases of lymphocytes with the kn own HLA-A, B, DR genes and the 24 Cases of ELISA-PRA positive sera wit h the known specificity of HLA antibodies:groupⅠ, ELISA-CDC were perfor med between sera selected from 24 cases of ELISA-PRA positive ones and ly mphocytes with the corresponding HLA-A, B, DR genes, the sera targeted wit h the corresponding HLA-A, B genes were 51 copies, and targeted with HLA-DR genes were 44 Copies; groupⅡ, ELISA-CDC were performed between 24 PRA positive sera and lymphocytes without the corresponding HLA-A, B, DR genes, the sera targeted with no corresponding HLA-A, B genes were 13 co pies, and targeted with no corresponding HLA-DR genes were 11 copies. PRA-negative sera were for the negative control group.1.2.2 Results treatmentAll data were recorded and analyzed by using statistical software packag es SPSS13.0, paired;χ2test (McNemar) was used between the two methods. Ka ppa test was used for the goodness of fit between the two methods. Determini ng statistical significance for the difference if P≤0.05.1.3 Clinical transplantation study and Statistics1.3.1 Clinical transplantation studyNIH-CDC-negative was the inclusion criteria for the renal transplant recipi ents receiving transplantation between January 2010-December 2010, patients w ere excluded from this transplantation if the ELISA-CDC was positive while th e NIH-CDC was negative, close observation of the situation of the transplant r ecipients after transplantation, pay attention to observe whether the hyperacute rejection or accelerated rejection happened.1.3.2 Results treatmentAll data were recorded and analyzed by using statistical software packages SPSS13.0, comparison of two independent sample rateχ2test was used betwee n the PRA positive and negative group. Determining statistical significance for the difference if P≤0.05.2,Test methods2.1 Capture antigens lymphocytes were isolated from the donor blood with lymphocyte separatio n medium, and then cracked with cell lysate, packaged and used immediately or placed preservation in -80℃.2.2 ELISA testAll the reagents were removed from the 4℃refrigerator, balanced to roo m temperature, removing the strip, marking the location of negative control, ly sate control, positive control and blank wells on the record paper. The Lysate for measuring ClassⅠantibodies was 8-fold dilution by lysate and coupled dil ution (LCD), and 4-fold dilution by LCD for ClassⅡantibodies. Stem cell co ntrol lysates were 4-fold dilution by LCD. Except the wells specified as the p ositive control and blank control, diluted lysates of donor's lymphocyte 15ul w ere added in the rest of the wells, "control cell lysates" were added into the p ositive control wells, the blank were without any reagent. incubated at 37℃fo r 30min, removing and drying, washing strips for 4 times. The sera of recipie nts, negative and positive control were all 1:3 diluted by specimen diluent (SD) , adding the diluted sera 15ul at corresponding wells, LCD 15ul were added i nto lysate control wells, incubated 30min at 37℃, removing and drying, washi ng strips 4 times. Anti-IgG antibodies were 1:100 diluted by SD, lysis control reagents LCR1 and LCR2 were 1:100 diluted by LCD, each well added 15ul of the diluted anti-IgG antibodies except the blank and lysate control, diluted LCR1 and LCR2 15ul were added into ClassⅠand ClassⅡlysate control w ells respectively, Incubator at 37℃for 30 minutes, removing the drying, washi ng strip 4 times. Nitrophenyl phosphate (PNPP) powder was dissolved with 0. 5ml deionized water, each strip added 0.5ml enzyme solution and 5ul PNPP so lution, each well added diluted enzyme solution 50ul except the blank, dark re action in 22-25℃for 30min, each well added stop solution 50ul, the control a dded stop solution 100ul. reading the value of absorbance A at the microplate reader in the wavelength of 405nm within 30min.2.3 Results decision and analysisBy comparing the OD value of the negative control to identify the positiv e and negative samples. Read the value of absorbance A at the microplate rea der in a wavelength of 405nm, sample testing values≥2 times of the negative values, then the sentence is positive; sample testing values<2 times of the n egative values, the sentence is negative.Results1. ELISA-CDC establishment:(1) Lymphocyte lysate can be placed in -80℃cryopreservation for using within 2 years, which can be used for retrospectively lymphocytotoxicity cross-matching and immunology monitoring after transplantation;.(2) Experimental time:The average time required 2.5-3h;(3) Experimental detection system includes 3 sets of quality control:reage nt control, lysate control and negative control, read absorbance A value with a microplate reader, the results are stable and objective;(4) Determination results:ELISA tests showed that the A value of lysate control well> 0.900, indicating that full amount of HLA glycoproteins have be en captured; A value of reagent control well> 1.000, indicating reagent system is working properly; A value of negative control well<0.300;(5) HLA-ⅠandⅡantigens were extracted from the donor lymphocytes lys ate, and curing in the ELISA plate by the specific monoclonal antibodies, res ponsing with the recipients sera by ELISA, specifically detection of HLA-IgG antibodies, IgM, IgA antibodies and non-anti-donor specific HLA-IgG antibodie s were washed off because they did not combine, excluding the influence of n on-HLA antibodies, non-IgG antibodies and non-donor-specific antibodies;.(6) Using the pyrolysis products of donor lymphocytes, not affected by ly mphocyte activity;(7) Specifical HLA-Ⅰ,Ⅱantibodies could be detected respectively by a cle avage product, HLA-Ⅱantibodies can be detected in the strong presence of cla ss I antibodies;(8) Both complement binding antibodies and non-complement binding anti bodies can be detected.2. Validation of ELISA-CDC methodsCrossmatchings were divided into two groups:groupⅠ, ELISA-CDC wer e performed between sera selected from 24 Cases of ELISA-PRA positive ones and lymphocytes with the corresponding HLA-A, B, DR genes, the sera targe ted with the corresponding HLA-A, B genes were 51 copies, and targeted HL A-DR genes were 44 Copies, the results were all positive; groupⅡ, ELISA-CDC were performed between 24 ELISA-PRA positive sera and lymphocytes without the corresponding HLA-A, B, DR genes, the sera targeted with no cor responding HLA-A, B genes were 13 copies, and targeted with no correspondi ng HLA-DR genes were 11 copies, the results were all negative. PRA-negative sera were for the negative control group, the results were also negative. Paire d;χ2test (McNemar) was used in recognition of the same specific HLA antibo dies between ELISA-CDC and ELISA-PRA. P=1.000, show that there was not statistically significant in recognition of the same specific HLA antibodies bet ween ELISA-CDC and ELISA-PRA. Kappa test was used for the goodness of fit between the two methods, Kappa= 1.000. P< 0.001, show that the coincidenc e of the two methods was statistically significant, and the goodness of fit was strong.3. Clinical transplantation study groupNIH-CDC-negative was the inclusion criteria for the renal transplant recip ients receiving transplantation, patients were excluded from this transplantation if the ELISA-CDC was positive while the NIH-CDC was negative. A total of 101 cases of recipients received transplantation,74 cases were male, female 27 cases; 85 patients with negative PRA, PRA positive in 16 cases. Close obser vation of the condition of the transplanted recipients, the hyperacute rejection a nd accelerated rejection did not occur in all recipients including PRA- positive and PRA-negative ones. Comparison of two independent sample rate;χ2test w as used between the PRA positive and negative group, P= 1.000, show that the re was not statistically significant in rejection rate between the PRA positive a nd negative group.ConclusionThe result of CDC currently used do not match with the effect of transp lantation frequently, the reliability is poor, ELISA-PRA may be more accuratel y reflecting the sensitization status of recipients. ELISA-CDC and ELISA-PRA are all based on ELISA principle, ruled out the impact of non-HLA antibodie s and non-IgG antibodies. ELISA-CDC can express the consistent specificity of HLA antibodies with ELISA-PRA. Although the survival rate of graft is relat ed to the PRA strength, but the antibodies detected by ELISA-PRA are not ne cessarily specific against the donor, ELISA-CDC extract HLA antigens from th e lymphocytes lysate of the donor, excluding the impact of non-donor specific antibodies.ELISA-CDC has several advantages over the traditional CDC tests, such as no need for viable cells; ELISA-CDC is stable and objective, it is possible to achieve the automation of the detection process; HLA-Ⅰ,Ⅱantibodies can be detected respectively using one kind of pyrolysis products, classⅡHLA an tibodies can be detected when there is presence of strong classⅠantibodies; I mmunosuppressive therapy has no impact on the results; Purified donor's HLA antigens can be long-term cryopreserved, it is not only helpful for the crossm atchings between several recipients and a single donor before transplantation, b ut also a simple, objective and effective method to monitor the DSA using HL A genes of the same donor after transplantation.ELISA-CDC may be more accurately reflects the sensitization status of t he patients, patients were excluded from this transplantation if the ELISA-CDC was positive while the NIH-CDC was negative, which can effectively reduce the incidence of the hyperacute rejection and acceleration rejection, providing a more reliable cross-matching method for the recipients, especially for the PR A-positive ones to find a suitable donor. In addition, the operation of ELISA-CDC is simple, time-consuming is not long, when the HLA specificity is unce rtain or the positive results of the current CDC needs to be confirmed, the res ults of ELISA-CDC also can be used as important reference. ELISA-CDC has certain advantages in crossmatch of the donor and recipient before transplantat ion and monitoring of DSA after transplantation, there is promotional value in clinical.In this study, ELISA-CDC results were fully consistent with the ELISA-PRA, which may be related to insufficient sample size, so further research cou Id consider increasing the sample size. This study only had a comparison of match rate between ELISA-CDC and ELISA-PRA, and observed whether hyper acute rejection or accelerated rejection occurred in the recipients with ELISA-C DC-negative before operation, but not applied to monitor after operation for ob serving the relationship between the test results and graft function and prognos is, next research could conduct in this area. |
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