Room:
Postergroup No. 1/1
Topic:
Solid organ transplantation
Form of presentation:
Poster Session
Duration:
60 Minutes
18:00
P1:
Helios expression and Foxp3 TSDR methylation of IFNy+ and IFNy- Treg from kidney transplant recipients with good long-term graft function
V. Daniel (Heidelberg, DE)
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Authors:
K. Trojan (Heidelberg, DE)
C. Unterrainer (Heidelberg, DE)
R. Weimer (Giessen, DE)
N. Bulut (Giessen, DE)
C. Morath (Heidelberg, DE)
M. Aly (Heidelberg, DE)
L. Zhu (Heidelberg, DE)
G. Opelz (Heidelberg, DE)
V. Daniel (Heidelberg, DE)
There is circumstantial evidence that IFNy+ Treg might have clinical relevance in transplantation. IFNy+ Treg express IFNy receptors and are induced by IFNy. In the present study we investigated kidney transplant recipients with good long-term stable graft function for the absolute cell counts of IFNy+ Treg subsets and whether their expression of Foxp3 is stable or transient. Helios expression determined by eight-color-fluorescence flow cytometry and methylation status of the Foxp3 Treg specific demethylation region (TSDR) served as indicators for stability of Foxp3 expression. Methylation status was investigated in enriched IFNy+ and IFNy- Treg preparations originating from peripheral blood using high resolution melt analysis. A total of 136 transplant recipients and 52 healthy controls were studied. Proportions of IFNy+ Treg were similar in patients and healthy controls (0.05% and 0.04% of all CD4+ lymphocytes; p=n.s.). Patients also had similar absolute counts of IFNy producing Helios+ and Helios- Treg (p=n.s.). Most of the IFNy+ and IFNy- Treg in transplant recipients had a methylated Foxp3 TSDR, however, there was a sizeable proportion of IFNy+ and IFNy- Treg with demethylated Foxp3 TSDR. Male and female patients showed more frequently methylated IFNy+ and IFNy- Treg than male and female controls (all p<0.05). Kidney transplant recipients with good long-term stable graft function have similar levels of IFNy+ Treg as healthy controls. IFNy+ and IFNy- Treg subsets in patients consist of cells with stable and cells with transient Foxp3 expression; however, patients showed more frequently methylated IFNy+ and IFNy- Treg than controls. The data show increased levels of Treg subsets with stable as well as transient Foxp3 expression in patients with stable allograft acceptance compared to healthy controls.
18:05
P2:
Angiotensin II type 1-receptor antibodies in highly sensitized renal transplant candidates
A. Vittoraki (Athens, GR)
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Authors:
A. Vittoraki (Athens, GR)
P. Markaki (Athens, GR)
N. Chouliari (Athens, GR)
D. Skoumi (Athens, GR)
S. Ioannou (Athens, GR)
M. Apostolaki (Athens, GR)
A. Iniotaki (Athens, GR)
Preformed before transplantation non-HLA antibodies (abs) are risk factors of acute rejection, appearance of de novo anti-HLA donor specific abs and poor graft survival with early graft loss. The aim of this study was to assess the incidence of angiotensin II type 1 receptor (AT1R) abs in highly sensitized renal transplant candidates (HSP, with PRAs>70%) compared to patients awaiting renal transplantation without anti-HLA-abs (PRAs=0%). Serum samples from 108 HSP (study group) and from 22 non-sensitized patients (control group-CG) were tested for AT1R-abs by ELISA (EIA-AT1R kit, One Lambda Inc). All sera were also tested for anti-HLA and anti-MICA antibody specificities by Single Antigen Bead Assay (Luminex, One Lambda Inc). Anti-AT1R-ab levels were classified as low <10 U/mL, at risk 10-17 U/mL and high>17 U/mL. For anti-HLA and anti-MICA abs an MFI>1000 was considered positive. Anti-AT1R-abs were detected in 41 HSP (37.9%) vs 2 patients (9.1%) from the CG (p=0.0057, Fisher exact p). The anti-AT1R-abs were detected either in intermediate (n=29, 26.8%) or high levels (n=12, 11%) in HSP with mean ±SD values of 12.4 ±1.9 and 25.4 ±10.5 U/ml respectively. The anti-AT1R-ab levels in the two positive patients from the CG were close to cut-off with mean ±SD values 11.2 ±1.5 U/ml. Sensitization in the study group was due to previous transplantation (n=78), pregnancies (n=25) or transfusions (n=78). The presence of anti-AT1R-abs was associated with pregnancies and/or graft loss. All HSP with transfusions history only had anti-AT1R <10 U/ml. Sixteen HSP had anti-MICA-abs (14.81%) vs none from the CG (p=0.04, Fisher exact p). The presence of anti-MICA-abs was significantly correlated with anti-AT1R-abs (p=0.01, Fisher exact p). Highly sensitized renal transplant candidates have additional donor HLA risk factors before renal transplantation. Detection of anti-AT1R-abs help to assess pre-transplant immunological status of these patients and force clinicians to establish therapeutic protocols in order to improve graft outcome.
18:10
P3:
Comparison of two Luminex based single antigen assays for the detection of anti-HLA antibodies and their complement variant (C3d and C1q)
A. Fylaktou (Athens, GR)
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Authors:
C. Zarras (GR)
T. Karampatakis (Thessaloniki, GR)
M. Papachristou (Thessaloniki, GR)
A. Boukla (Thessaloniki, GR)
G. Chatzika (Thessaloniki, GR)
G. Chatzianestis (Thessaloniki, GR)
F. Makrovasili (Thessaloniki, GR)
A. Fylaktou (Athens, GR)
Luminex based single antigen assays are widely used methods for defining anti-HLA specificities and their complement binding activity. The aim of the study was to compare two single antigen bead (SAB) assays in defining anti-HLA class I and II specificities and their complement binding capacity in association with Mean Fluorescence Intensity (MFI). We tested 30 pretransplant sera from 30 different patients (2016) with PRA>60%. All sera were analyzed by SAB for the presence of HLA class I and II antibodies with Immucor (IM) and One Lambda (OL). Positivity was defined according to manufacturer’s instructions. Analysis was followed by C3d and C1q assays. 769 class I and 612 class II specificities were totally assigned. The total concordance between IM and OL for class I and class II specificities was 65% and 48% respectively, with mean class I MFI values for IM and OL 9724 and 8946, respectively and mean class II MFI for IM and OL 8326 and 5524, respectively. 11% of class I specificities were detected only by IM with mean MFI 6317 and 23% only by OL with mean MFI 6409. 17% of class II specificities were detected only by IM with mean MFI 5524 and 35% only by OL with mean MFI 9009. Class I specificities were totally 44% C3d positive (mean MFI 16618) and 44.1% C1q positive (mean MFI 17134) and class II specificities were 66.7% C3d positive (mean MFI 19962) and 34.6% C1q positive (mean MFI 17938). From 489 class I specificities detected by both methods, 193 (39.5%) were both C3d and C1q positive, whereas 37 (7.6%) were only C3d positive and 43 (8.8%) only C1q positive. From 277 class II specificities detected by both methods, 104 (37.5%) were both C3d and C1q positive, whereas 82 (29.6%) were only C3d positive and 12 (4.3%) only C1q positive. OL detected more specificities in both class I and II than IM. Concordance was better in higher MFIs. Class II concordance was unexpectedly low compared to our previous results maybe due to lot differences. Higher rates of class II C3d positivity compared to C1q were observed totally and especially in concordant specificities.
18:15
P4:
Frequency and predictors of successful steroid withdrawal guided by surveillance biopsies
C. Wehmeier (Leiden, CH)
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Authors:
C. Wehmeier (Leiden, CH)
P. Hirt-Minkowski (Basel, CH)
P. Amico (Basel, CH)
A. Georgalis (Basel, CH)
H. Hopfer (Basel, CH)
J. Steiger (Basel, CH)
M. Dickenmann (Basel, CH)
S. Schaub (Basel, CH)
Steroid withdrawal following renal transplantation is controversial and has to balance the risk of rejection against the risk of steroid-related side effects. The aim of this retrospective study was to investigate the frequency and determinants of successful steroid withdrawal guided by surveillance biopsies. We investigated 156 ABO-compatible HLA-DSA negative renal transplants receiving basiliximab induction and maintenance immunosuppression with tacrolimus-mycophenolate-steroids. The absence of rejection in surveillance biopsies at 3 or 6 months post-transplant initiated steroid withdrawal, which was monitored by subsequent indication and/or surveillance biopsies. The primary outcome was the frequency of successful (i.e. rejection-free) steroid withdrawal at one year post-transplant. In the whole population, successful steroid withdrawal was achieved in 73/156 patients (47%). Steroid withdrawal was initiated in 98/156 patients (63%) having a rejection-free 3- or 6-months surveillance biopsy and was successful in 73/98 patients (74%). Subsequent clinical rejection occurred in only 1/98 patient (1%), whereas 24/98 patients (24%) experienced sub-clinical rejection. Steroid withdrawal was not initiated in 58/156 patients (37%) mainly due to current or prior severe (Banff equal or greater than IA) sub-clinical rejection (64%). Prediction of successful steroid withdrawal by pre-transplant or early post-transplant parameters was poor. By multivariable analysis, the only significant predictor was the absence of clinical rejection within the first 3 months post-transplant (odds ratio 2.84; 95% CI 1.07-8.52; p=0.04). In conclusion, (sub)clinical rejection-free steroid withdrawal can be expected in about half of pre-transplant HLA-DSA negative patients. As successful steroid withdrawal cannot be well predicted by pre- and early post-transplant parameters, guidance by surveillance biopsies is advisable.
18:20
P5:
Donor-specificity but not broadness of sensitization is associated with antibody-mediated rejection and graft loss in renal allograft recipients
C. Wehmeier (Leiden, CH)
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Authors:
C. Wehmeier (Leiden, CH)
G. Hoenger (Basel, CH)
H. Cun (Basel, CH)
P. Amico (Basel, CH)
P. Hirt-Minkowski (Basel, CH)
A. Georgalis (Basel, CH)
H. Hopfer (Basel, CH)
M. Dickenmann (Basel, CH)
J. Steiger (Basel, CH)
S. Schaub (Basel, CH)
Panel-reactive antibodies are widely regarded as an important immunological risk factor for rejection and graft loss. The broadness of sensitization against HLA is most appropriately measured by the ‘calculated population-reactive antibodies’ (cPRA) value. In this study, we investigated whether cPRA represent an immunological risk in times of sensitive and accurate determination of pre-transplant DSA. 527 consecutive transplantations were divided into four groups: cPRA 0% (n=250), cPRA 1-50% (n=129), cPRA 51-100% (n=43), and DSA (n=105). Patients without DSA were considered as normal risk and received standard immunosuppression without T-cell depleting induction. Patients with DSA received an enhanced induction therapy and maintenance immunosuppression. Surveillance biopsies were performed at 3 and 6 months. Median follow-up was 5.7 years. Among the three cPRA groups there were no differences regarding the one-year incidence of ABMR (p=0.16) and TCMR(p=0.75). 5-year allograft survival was similar and around 87% (p=0.28). eGFR at last follow-up was 50-53 ml/min (p=0.45). By multivariable Cox proportional hazard analysis, the strongest independent predictor for ABMR and (death-censored) graft survival were pre-transplant DSA. cPRA were not predictive for ABMR, TCMR, and (death-censored) graft survival. We conclude that with current DSA assignment the broadness of sensitization measured by cPRA does not imply an immunological risk.
18:25
P6:
Expression of CXCR3 monocytes increases significantly in the graft blood compared to peripheral blood in patients with stable renal function
A. Caballero (Malaga, ES)
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Authors:
A. Caballero (Malaga, ES)
P. Ruiz-Esteban (Malaga, ES)
E. Palma (Malaga, ES)
A. Torio (Albacete, ES)
M. Cabello (Malaga, ES)
V. Lopez (Malaga, ES)
A. Duarte (Malaga, ES)
T. Vazquez (Malaga, ES)
J. Alonso-Titos (Malaga, ES)
D. Hernandez (Malaga, ES)
We have recently reported that some lymphocyte populations do not maintain the same proportion in graft blood as in peripheral blood, despite a stable function of the transplanted kidney. These results suggest that the comparative study between leukocyte cells from the graft blood and those obtained in peripheral blood could provide information about the inflammatory state of the transplanted organ. In this work we selected the population of monocytes expressing CXCR3 to test this hypothesis. The study was performed by flow cytometry during the third, sixth and twelfth months after transplantation in 69 patients who received an isolated kidney transplant and the same immunosuppressive regimen. The peripheral blood sample was obtained by venipuncture and the graft blood by fine needle aspiration. We found a significant decrease in CXCR3+ monocytes throughout the first year of transplantation in peripheral blood (15.8 ±20.7 vs. 12.6 ±12.4 vs. 6.3 ±9.0, p=0.001), whereas the percentage of CXCR3+ monocytes in the graft blood did not change over this period. This situation resulted in a significant percentage difference between the CXCR3+ monocytes of the graft blood and those of the peripheral blood during the sixth (16.19 ±9.54 vs. 12.6 ±12.4, p=0.027) and twelfth months (14.10 ±8.94 vs. 6.3 ±9.0, p<0.001). We can conclude, therefore, that the significant percentage increase of CXCR3+ monocytes in the graft blood with respect to the peripheral blood suggests the presence of inflammatory activity despite having stable renal function during the second half of the first year after transplant.
18:30
P7:
A successful DCD strategy with minimal cold ischaemic time – a single centre review
M. Mishra (Lucknow, UP, GB)
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Authors:
M. Mishra (Lucknow, UP, GB)
A. Courtney (Belfast, GB)
M. Bernadette (Belfast, GB)
J. Martin (Belfast, GB)
Belfast is the sole renal transplant centre in Northern Ireland (population 1.8 million) and the local H&I laboratory supports approximately 120 transplants annually. Donation after Circulatory Death (DCD) donor organs have been used in this centre since 2013. We present the strategy employed to achieve this and the outcomes for the year 2015-16. Samples from 35 local potential DCD donors were received in the laboratory for HLA typing. Eighteen were subsequently ‘stood down’ and in two instances both kidneys were exported to mainland UK leaving 15 that were used locally. In 5 cases both kidneys were transplanted locally, culminating in 20 transplants. HLA typing was commenced on all 35 potential donors by Luminex rSSO. The CDC crossmatch procedure was amended in that peripheral blood, received at the time of sampling for typing, was used instead of waiting for retrieval of spleen cells. Crossmatching then commenced immediately as a recipient was chosen using cells extracted from donor peripheral blood and the latest serum sample available from the potential recipient; some donors were stood down during this process. Virtual crossmatch was considered suitable for three recipients and CDC crossmatch for the 17 remaining prospective recipients. Allele mismatches varied from 2-7 (mean 5.7) on considering mismatches at HLA–A, -B, -C, -DRB1,-DRB3,4,5,-DQB1 and –DPB1 loci. Mean age of recipients and donors was 58.6 and 48.8 years respectively. The mean CIT was 8.4 hours (range 4.4 – 18.1). This is substantially better than that achieved in any other UK transplant centres. The routine use of cells extracted from peripheral blood instead of waiting for retrieval of the spleen for crossmatching, facilitated a significant reduction in cold ischemia time in this sub optimal group of donors. Frequently crossmatch was reported before withdrawal of life support thus allowing timely transplantation. Other centres may proceed to surgery in a similar manner by restricting DCD transplants to recipients suitable for virtual crossmatching only. The Belfast method allows equity of access to DCD organs to all on the waiting list.
18:34
P8:
New HLA genotyping technologies offer the opportunity to better assess the donor-recipient HLA mismatch in solid organ transplantation
V. Van Sandt (Mechelen, BE)
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Authors:
V. Van Sandt (Mechelen, BE)
A. Senev (Mechelen, BE)
L. Daniëls (Mechelen, BE)
K. Johan (Mechelen, BE)
M. Hestand (Leuven, BE)
J. Vermeesch (Leuven, BE)
M. Naesens (Leuven, BE)
M. Emonds (Mechelen, BE)
HLA antigens are divided into 1st field groups based on shared serological patterns. Although this categorization was meant to allow matching algorithms that minimize allo-immunization after transplantation, it fails to do so. Low resolution typing and antigen categorization does not discriminate between alleles with different presentation and it excludes entire allele families based on cross-reactivity (CREG) and the assumption that they all share the same immunogenic risk. This matching strategy therefore does not correctly reflect the actual risk of allo-immunization and could benefit from fine-tuning. Epitope matching is currently suggested as an opportunity to improve donor-recipient matching. To do so we need in depth molecular characterization of the antigens. New techniques offer the opportunity to do this and even include new relevant loci such as DPB1 in a cost effective manner. Pacific Biosciences' Single-Molecule Real Time (SMRT) sequencing technology offers read lengths >15 kb which enable direct haplotyping of long range PCR products derived from individual antigens. Reducing the need for fragmentation this permits direct phasing of variants instead of sophisticated error prone imputation from short reads. We are currently integrating this technique into an HLA genotyping workflow for organ transplant recipients and donors, allowing us to correlate high resolution single bead antibody with allelic resolution genotyping data in a retrospective analysis. We are already able to multiplex up to 31 samples for HLA class I in a single SMRTcell, resulting in high-quality data without any mistypes compared to the consensus. Our setup provides sufficient coverage to correct for the high individual molecule error rates with costs that are comparable to that of commercial SSO methods, making this approach feasible for implementation in organ transplant recipient, related donor and post-transplant deceased donor allelic resolution genotyping. A similar approach for HLA class II molecules (HLA- DRB1,3,4,5, DQB1,and DPB1) is in development. To allow future epitope discrimination in prospective donor typing we are currently evaluating the potential of real-time PCR kits such as the single antigen bead resolution kit (SABR, Linkage Biosciences), which discriminates between the epitopes present in current antibody identification kits.
18:39
P9:
Detection of HLA-DQ compatibility of recipient-donor couples in renal transplants from deceased donor and investigation of de novo antibodies after transplantation
T. Kilicaslan Ayna (Izmir, TR)
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Authors:
I. Totur (Izmir, TR)
T. Kilicaslan Ayna (Izmir, TR)
A. Özkızılcık Koçyiğit (Izmir, TR)
M. Soyöz (Izmir, TR)
Kidney tranpslantation is used as treatment for end-stage chronic kidney failure. Blood group and HLA tissue type compatibility, DSA presence, and PRA positivity between recipient and donor are important for the success of kidney transplants. In this study, a total of 25 recipient-deceased donor couples who applied to our laboratory in January 2014-September 2016 were included. HLA tissue typing of the couples (HLA-A, -B, -C, -DRB1, -DQA1, and -DQB1) were performed by a Luminex-SSO method. Pre- and post-transplant anti-HLA antibodies were investigated by a Luminex-PRA method and the association of the results with graft survival were assessed. The patients were divided into two groups according to their alloimmunizations. The patients with previous transplantation, blood transfusion, and pregnancy were in group 1 (20.8% (n=5)), while the patients with blood transfusion and pregnancy were in group 2 (79.2% (n=19)). One of the patients had no alloimmunization. One patient of the first group (20%) and two patients of the second group (10.5%) were pre-transplant PRA positive. Post-transplant PRA of two patients in the first group (40%) and second group (10.5%) were positive. When the results were evaluated according to graft survival, it was found that one patient in the first group lost the allograft due to de novo donor specific antibodies. In the second group, de novo antibody did not develop and graft failure was not observed. In conclusion, it was determined that transplantation was an important determinant for anti-HLA antibody production and post-transplant donor specific antibody development was important for graft failure.
18:44
P10:
Early post-transplant monitoring of anti-HLA antibodies in heart transplant recipients fails to detect patients at risk of humoral rejection
J. Irure (Cantabria, ES)
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Authors:
J. Irure (Cantabria, ES)
L. Riesco (Santander, ES)
M. Llano-Cardenal (Santander, ES)
S. Rubio-Ruiz (Santander, ES)
I. Romón (Santander, ES)
M. López-Hoyos (Santander, ES)
M. Cobo (Santander, ES)
D. San Segundo Arribas (Santander, ES)
There is growing interest in the monitoring of anti-HLA antibodies after heart transplantation as their presence has been linked with coronary allograft vasculopathy (CAV) and implicated in allograft injury, but there is no consensus in their follow-up timing. 93 consecutive patients that underwent heart transplantation between 2011 and 2015 at the Marques de Valdecilla Universitary Hospital were included in the study. The presence of anti-HLA antibodies was assessed in pre-transplant sera and during the first year every three months after transplantation by using LABScreen® Mixed and Single Antigen assays (One Lambda, Canoga Park, CA, USA), according to the manufacturer's protocol. Positive specificities were considered when two out of three criteria were fulfilled: raw mean fluorescence intensity (MFI) above 1500, baseline MFI above 1000 and above 25% of maximum MFI. Anti-HLA antibodies were found prior to transplant in 6.5% of all recipients (two against HLA class I; two against class II and two against both class I and class II antigens). 8.1% of the patients analyzed prior transplantation had anti-HLA antibodies at three months and 8.6% at 12 months. Two out of six de novo reactions were donor-specific (all against class II antigens) and both with rejection episode confirmed. 37.6% of patients had transplant rejection, being humoral rejection in 20% of the cases. C4d staining was positive in 71.4% of the biopsies. 74.3% of the patients who lost the graft did not present anti-HLA antibodies at any time. Therefore, anti-HLA antibody monitoring in heart transplant patients did not allow predicting graft rejection. Multi-centre and prospective studies on heart transplant candidates should be addressed in order to assess the best plan of anti-HLA antibodies monitoring in heart transplant recipients.