Tunisian experience in HLA antibody monitoring
A. Charfi (Sfax, TN)
During the period of 2012-2015, we analyzed 903 sera belonging to 513 patients awaiting or had already received a kidney transplantation. Anti-HLA antibodies were screened by complement dependent cytotoxicity (CDC) and Luminex assays (Labscreen Mixed 12 (LSM12). Luminex single antigen assays (LSA1 et/ou LSA2) were performed if anti-HLA screening was positive (NBG ratio>5). Anti-HLA antibody screening was positive by CDC in 10 patients and by Luminex assays (LSM12) in 79 patients distributed on 55 non-grafted patients and 24 transplanted patients. Sixty one patients were positive for HLA class I of which 17 were positive for HLA-C; forty six patients were positive for class II. Anti-DP antibodies were never isolated. They have been associated with anti-DR or anti-DQ antibodies. Among our 79 patients, 24 were positive for at least one of the anti HLA-C or DP. Among the 24 transplanted patients, donor-specific antibodies (DSA) were detected in 11 patients after transplantation, while non donor-specific antibodies (DSA) were detected in 13 patients. Seven of the 11 patients with DSA lost their graft.
An HLA-B7 specific antibody in an HLA-B*07 positive patient explained by a non-expressed allele (HLA-B*07:181N)
I. Faé (Vienna, AT)
HLA specific antibodies are a major cause for acute and chronic rejection after solid organ transplantations. Accurate definition of HLA class I and class II antibodies and their consideration for organ transplantations result in better outcome and longer graft survival. Antibody identification by a bead array assay in a kidney patient revealed several HLA-specific antibodies including one directed against HLA-B*07. Low resolution typing of the patient indicated the presence of an HLA-B*07 allele. To rule out an HLA-specific autoantibody the HLA-typing of the patient was further refined by nucleotide sequencing on a next-generation sequencing platform and eventually showed an HLA-B*39:01:01:03 and HLA-B*07:181N genotype. Thereby an allospecific nature of the antibody was proven. The anti-HLA-B7 could be explained by an immunization during the first kidney transplantation in 1996 with an HLA-B*07 positive donor. For plausibility of antibodies, the presence of non-expressed alleles that usually are not ruled out during typing should be taken into consideration.
Interferon-gama +874 polymorphism is not associated with chronic graft disease: evidence from a meta-analysis
B. Lima (Vilar Formoso, PT)
The success of renal transplantation and its clinical outcomes depends on many characteristics of both donors and recipients including ages, ethnicities, pre-formed antibodies, HLA mismatchs and other immune system agents such as cytokines and their receptors. Interferon-gamma (IFNg) is a pro-inflammatory cytokine that promotes the development of the TH1 response, regulates the presentation of HLA antigens, and induces apoptosis. Several studies have indicated that IFNg levels have significant effects on susceptibility to various autoimmune diseases, infections, and allergies. It may also contribute to the severity of the rejection episode by stimulating neopterin by monocytes derived macrophages. Chronic Graft Disease (CGD) is a multifactorial process which likely includes a combination of immunological, apoptotic and inflammatory factors. The application of individualized immunosuppressive therapies will also depend on the identification of risk factors that can influence chronic disease. The purpose of this study was to clarify the involvement of IFN-g +874 cytokine gene polymorphism and it possible association with CGD. Relevant published data were retrieved through Medline pertaining to kidney transplant outcome and IFN-g polymorphisms. Odds ratios (OR) with 95 % confidence intervals (CI) were used to assess the strength of the association. Z test was used to determine the significance of the pooled OR. Statistical heterogeneity was measured using the Q statistic. A total of 6 studies, including 394 CGD transplanted cases and 324 transplanted controls with stable graft function, were collected. For TT vs. TA or AA IFN-g genotypes data were combined using the fixed-effects model (Q=5.78; p=0.33). For the total population, we did not find a statistically significant association of the TT genotype and CGD, when compared with the SGF group: effect summary OR = 1.02; 95% CI = 0.67-1.54; p = 0.92. In this meta-analysis we didn’t find statistical evidence for the association of IFN-g genotypes and CGD. Further functional studies of IFN-g gene polymorphism will help to understand the underlying mechanisms of this cytokine.
Associations between obesity-related FTO rs9939609, PPARs gene variants and kidney transplantation
D. Piancatelli (L'Aquila, IT)
Obesity and lipid abnormalities have implications in various post-transplant pathological conditions, including infections, cardiovascular and metabolic diseases and chronic rejection. Some gene polymorphisms involved in metabolic processes, that could increase the medication-related post transplant risk, are poorly investigated in organ transplantation. FTO (fat mass and obesity-associated) gene variants affect obesity susceptibility in the general population; PPARs (peroxisome proliferator-activated receptors) are nuclear receptors both involved in lipid metabolism, insulin sensitization, inflammation and immune regulation. To evaluate if they could be additional risk factors for post transplant outcome, their associations with pre-post transplant clinical parameters were analyzed. Polymorphisms of FTO rs9939609, PPARA rs1800206 (Leu162Val), PPARG rs1801282 (Pro12Ala) and PPARD rs2016520 were detected using Taqman allelic discrimination methods. Variations of BMI, blood lipids, fasting plasma glucose and creatinine levels were examined in association with genotypes in 173 kidney transplant recipients. Results show that pre-transplant and post-transplant total cholesterol was increased in patients carrying the C allele of the PPARD rs2016520 polymorphism (pre-transplant: C/C+C/T, 190.18 ±43.23 mg/dl, T/T, 164.62 ±42.51 mg/dl, p=0.001; 1 year after transplantation: C/C+C/T, 206.73 ±47.42 mg/dl, T/T, 192.16 ±35.10 mg/dl, p=0.01). No other significant associations were found, although a trend to a slight increase of FTO A/A genotype frequency in obese patients in the first/second years after transplantation was present (1 year, obese, A/A=34.8%, non obese 19.3%, p=ns). In conclusion, analyzing a possible impact of the examined gene variants on some of the main post transplant risk factors for cardiovascular and metabolic diseases, only slight effects of FTO rs9939609 on the increase of body mass/obesity were detected; an association of PPARD rs2016520 with hypercholesterolemia in the early post-transplant period gives the indication for deeper investigations in the view of possible personalized interventions.
Frequency of anti-HLA antibodies in pre- and post- kidney transplant patients in the Republic of Macedonia
A. Petlichkovski (Skopje, MK)
Anti-HLA antibodies play crucial role in kidney graft survival, and should be routinely analyzed prior to kidney transplantation, but also monitored after the transplantation in order to predict and prevent graft failure. The anti-HLA antibodies could develop prior to transplantation due to different sensitization events, such as blood transfusions or pregnancies. We have analyzed in this study a total of 493 sera from kidney transplant patients at the Institute for Immunobiology and Human Genetics, at the Medical Faculty in Skopje, Republic of Macedonia. The samples were analyzed in a period of 4 years (2013 to 2016). One hundred and forty three samples were collected from patients before kidney transplantation, while the rest of 350 samples were post kidney transplantation. The presence of anti-HLA antibodies was determined using the LabScreen Kit with Luminex technology. We have found anti-HLA class I antibodies in 31.47% of the sera prior to transplantation, anti-HLA class 2 antibodies in 22.38% and anti-MICA antibodies in 10.49%. From the sera analyzed after kidney transplantation, 25.14% were found to be positive for anti-HLA class 1 antibodies, 19.14% for anti-HLA class 2 antibodies and 10% for anti-MICA antibodies. To assess the frequency of donor specific antibodies (DSA), we have analyzed total of 38 pairs for kidney transplantation. Only in 2 patients of the pairs analyzed were DSA detected. In 26 of the patients, DSA were not detected (in 4 of them non-DSA anti-HLA antibodies were detected). Sera from 10 patients were not analyzed for anti-HLA antibodies. In conclusion, we report relatively high percentage of sera positive for anti-HLA antibodies, while we have found a relatively low presence of DSA. This points out the importance of the anti-HLA screen and this fact should be taken into consideration when making policies for determination of anti-HLA antibodies and planning kidney transplantation.
HLA-DQB1*03:19: should we really consider DQ7 as its serological equivalent? A case report
V. Kheav (PARIS, FR)
The HLA-DQB1*03:19 allele was differentiated from HLA-DQB1*03:01 in 2007 by DNA Sequence-Based Typing with a single T185I polymorphism in exon 3. However, high resolution typing does not resolve this ambiguity, as EFI standards require “the identification of HLA alleles that encode the same protein sequence within the antigen recognition site”, i.e. the same exon 2 sequence for class II molecules. Hence, despite the structural differences between these two molecules, both alleles have been suggested to have the same DQ7 serological equivalent. A case of an HLA-DQB1*03:19 in a male patient who has developed anti-DQ7 antibodies questions this assumption.
After a kidney transplantation from a HLA-A1, 9, B8, 18, DR5, 6 serologically-typed donor, anti-HLA antibody screenings by Luminex were still negative just before the transplant failed. Numerous de novo antibodies were then identified by Luminex Single Antigen from the very first serum after graft failure. Among them, anti-DQ7 antibodies (not directed at the DQ alpha chain) were identified with a peak MFI at 19687. We retrospectively verified this observation by crossmatching this serum and two others by flow cytometry to a donor against which we identified no other HLA-A, -B, -C, -DRB1 or -DQB1 specific antibodies than the anti-DQ7 antibody by Luminex Single Antigen. All crossmatches were strongly positive (ratio 4.3-10.6 depending on the serum; cut-off= 1.7), confirming the presence of the anti-DQ7 antibodies. To solve the issue of the serological equivalent of HLA-DQB1*03:19, we propose to distinguish the donor from the recipient. For the donor, as long as no HLA-DQB1*03:19 bead will be available in either one of the two commercially available Luminex Single Antigen identification kits, we propose to keep considering DQ7 as the serological equivalent. By contrast, DQB1*03:19 recipients should be assigned a phenotypic blank to allow the selection of all potential DQ3 unacceptable antigens for organ allocation.
Endothelial precursor cell crossmatch using Tie-2-enriched spleen cells
V. Daniel (Heidelberg, DE)
Non-HLA-antibodies against human endothelial progenitor cells (EPC) in pre-transplant recipient serum can have a deleterious influence on the graft. EPC enriched from peripheral blood have been commonly used for EPC crossmatching. In the present study, we describe crossmatches using EPC enriched from fresh or frozen-thawed spleen cell preparations, thereby widening the sample source for deceased-donor crossmatching and retrospective studies. EPC crossmatches were performed retrospectively using spleen cells and the flow cytometric XM-ONE crossmatch test kit. One of 29 (3%) sera of healthy controls contained IgG and IgM EPC antibodies, and three (10%) only IgM EPC antibodies. When sera of 11 random dialysis patients were studied, two patients (18%) showed IgG EPC antibodies, one (9%) additional IgM EPC and one (9%) only IgM EPC antibodies. When pre-transplant sera of 20 graft recipients with good long-term graft outcome (serum creatinine 1.0 ±0.2 mg/dl measured 2463 ±324 days post-transplant) were investigated using frozen-thawed and then separated Tie-2-enriched spleen cells of the original transplant donor, 2 patients (10%) had pre-transplant IgG EPC antibodies, one (5%) additional IgM and one (5%) only IgM EPC antibodies. When pre-transplant sera of five patients with intra-operative graft loss were studied employing the original donor spleen cells, four (80%) patients had IgG EPC antibodies and two had, in addition, IgM EPC antibodies. Three of the four (75%) patients with graft loss and IgG EPC antibodies had, in addition, antibodies against angiotensin II type I receptor (AT1R), and one patient had, in addition, antibodies against endothelin ETA receptor (ETAR). Crossmatches with spleen cell-derived EPC using the XM-ONE assay are technically possible. Our very preliminary experience suggests clinical relevance.
Detection of anti-HLA antibodies in pre-kidney transplantation candidates in the Kurdistan Region of Iraq
R. Al-Qadi (Duhok, Duhok Governorate, IQ)
The presence of anti-HLA antibodies in the sera of patients waiting for kidney transplantation is a well-known risk factor for development of antibody-mediated rejection (AMR), which eventually might lead to graft loss. The Luminex based bead detection of anti-HLA antibodies has facilitated the task of determining the sensitization status of these patients. In this study, we aim to determine the presence or absence of anti-HLA antibodies in candidates of kidney transplantation in the Kurdistan region of Iraq. Also, to determine the correlation between the Luminex data and the CDC crossmatches that we routinely perform for such patients. From the period between September 2014 and December 2016 we tested 462 sera for the presence of anti-HLA antibodies using Immucor’s Deluxe LifeScreen, Class I and Class II ID (PRA), and LIFECODES LSA class I and II Single Antigens. Out of 462 sera, 170 (37%) were either sensitized for class I or class II anti-HLA antibodies or both. Of the sensitized sera, 30/170 (18%) had only class I anti-HLA antibodies, 61/170 (36%) had only class II anti-HLA antibodies, and 79/170 (47%) had both class I and class II anti-HLA antibodies. In the same period of time there were 16 positive CDC crossmatches between potential recipients and donors, of which 3 of them (19%) had only class I anti-HLA antibodies, 13 (81%) had both class I and class II anti-HLA antibodies and none had class II anti-HLA antibodies alone. The mean fluorescence intensity (MFI) values for the positive CDC crossmatch were all greater than 8000. This study is the first study to be done in the Kurdistan region of Iraq for the determination of the anti-HLA antibodies by using Luminex bead technology. Further studies in the region are required for better understanding the immunological patterns of the patients of the region.
Feasible extended HLA typing of deceased donors in solid organ transplantation
G. Ozzella (Rome, IT)
In solid organ transplantation the HLA-A,-B,-DR,-DQ donor-recipient matching results are insufficient for highly sensitized patients. In fact, solid phase single antigen assays, used to define HLA antibodies in transplant candidates, show the presence of antibodies specific for all HLA molecules. Thus, an extended HLA typing of deceased donor is necessary to improve selection of the most suitable transplant candidate. Until July 2016, in our laboratory all donors were typed for HLA-A,-B,-C,-DR,-DQ loci by a PCR-SSP technique; when a sensitized patient was selected, the donor typing was prospectively enlarged to pertinent HLA molecules. To simplify this procedure and improve organ allocation we introduced the new RT PCR-SSP technique, based on an innovative chemistry (Linkage Bioscience Inc.), it enables us to provide intermediate resolution typing of 11 HLA loci, in less than 90 minutes. Allele-specific amplification combined with SYBR Green and real-time PCR instruments are used to detect amplification products and to collect dissociation data for automatic interpretation by SureTyper software. Since August 2016, 76 potential deceased donors were typed by this technique. No allelic ambiguities were evidenced but rather high resolution typing were obtained in several cases: 11 HLA-A alleles, 55 HLA-B alleles, 48 HLA-C alleles, 41 HLA-DRB1 alleles, 37 HLA-DRB345 alleles, 20 HLA-DQA1 alleles, 30 HLA-DQB1 alleles, one HLA-DPA1 allele, and 50 HLA-DPB1 alleles. Moreover, the extended HLA typing obtained by RT PCR-SSP avoided additional HLA-C, -DRB1, -DQA1 and -DPB1 typing in 12 cases (15.8%) and allowed us to define donor molecules against which 10 patients (13.2%) showed preformed high fluorescence intensity antibodies (MFI>5000). In conclusion, RT PCR-SSP is less hands-on and, considering the number of typed HLA loci, cheaper than traditional PCR-SSP techniques. The extended donor HLA typing gives useful information for a more precise pre-transplant virtual crossmatch and for a better donor-recipient selection to improve clinical transplant outcome.
Prediction of flow cytometric crossmatch outcome from bead array data
S. Wenda (Wien, AT)
In order to improve the virtual crossmatch for our centre we studied the association of single antigen specific bead array results with the outcome of the flow cytometric crossmatch. Sera from 168 consecutive patients undergoing solid organ transplantation (kidney, liver, heart and lung) between May 2016 and November 2016 were drawn on the day of transplantation and analysed in a solid phase screening assay and in a lymphocytotoxic test (CDC) for the presence of HLA antibodies. HLA antibody containing sera were further analysed in a single antigen bead array test for the presence of donor specific antibodies (DSA). In addition, all sera were used for flow cytometric T and B cell crossmatches and CDC crossmatches. Regarding the bead array results, DSA could be detected in 12 out of the 168 patients, with MFI values between 1000 and 20000. The flow cytometric crossmatch was positive in eight patients when analysing T cells and positive in 14 patients when analyzing B cells. Overall an MFI value of 6000 in the bead array test seems for our centre to be the best cut-off value for the prediction of a positive flow cytometric crossmatch. We observed one discrepant false negative flow cytometric crossmatch and four false positive ones. These could be due to non HLA-specific alloantibodies and medication related artefacts. We conclude that there is a fairly good association between bead array tests and flow cytometric crossmatch. A larger number of parallel testing is however needed, before either test can be abandoned.
Anti-HLA antibodies in liver transplantation: is there any importance? A single center experience.
A. Fylaktou (Athens, GR)
Donor specific human leukocyte antigen (HLA) alloantibodies (DSA) are important among solid organ transplantations. The aim of the present study is to investigate if there is any importance of anti-HLA antibodies in liver transplantation and patient-graft survival. A total of 72 primary liver only transplant patients (2010-2016) were tested for anti-HLA antibodies, with single antigen bead technology, before and 1, 6, 12 months after transplantation and thereafter annually. HLA typing was performed for all donor-recipient pairs. Pre-formed anti-HLA antibodies (PA) were detected in 32 patients (44.4%) who had 47.7% one-year survival rate vs 77.8% in patients negative for PA (p=0.030). Only 10% of these patients had pre-existing DSA class I or II with 60% one-year survival rate vs 65.1% in negative patients (difference not statistically significant). 28 out of 72 patients (39.5%) developed de novo anti-HLA antibodies after transplantation and 15 of them (20.9%) had de novo DSA (five HLA class I and 13 class II). The difference in graft survival rate between de novo DSA positive (77.8%) and negative (86.2%) patients was not statistically important may be due to the small number of transplanted patients with de novo DSA. Patient and graft survival rate seemed to be affected by preformed anti-HLA antibodies but not by de novo DSA, although independent validation is needed. Long term outcome in patients with post-transplant DSA needs further study.