Postergroup No. 1/3
Solid organ transplantation
Form of presentation:
Virtual crossmatches impact on kidney discard rate
R. Prakash (New Providence, NJ, US)
A virtual crossmatch (VXM) is used to ascertain the presence of HLA antibodies in a transplant recipient’s serum and to determine, virtually, the flow cytometric crossmatch (FXM) results with an available donor. VXM testing is routinely used to aid in the selection of import donor kidneys for highly sensitized patients. A VXM can be a valuable tool in helping to reduce cold ischemia time and donor organ discard. This study evaluates the impact of the VXM on the discard rate of kidneys for a single Organ Procurement Organization (OPO).A calibration curve was developed for VXM by comparing the mean fluorescent intensity (MFI) values for the donor specific antibodies to the corresponding median channel shift (MCS) values. A linear regression was performed and the slope was calculated. The slope was entered into an algorithm for estimating the MCS for a given MFI value, therefore predicting a FXM result. A logistic regression model was utilized to compare the number of VXM tests performed between December 2014 and July 2016 to the monthly percent of discarded kidneys. A statistically significant relationship was observed between the possibility of a kidney being discarded and the number of virtual cross matches performed (p = 0.0347).
We see that for each unit increase in the number of virtual cross matches leads to a 0.04 decrease in the log-odds of a kidney being discarded.
The increase in the use of VXM is one of the factors responsible for a significant reduction in the discard rate of kidneys observed at our OPO.
Proper interpretation of DQB1 antibodies could minimize the risk to overlook relevant DSA
A. Dada (SA)
Detection and interpretation of DQB1 HLA antibodies has to be in the right manner especially in the presence of Donor Specific Antibody (DSA) and a mismatch in DQA1 between recipient and donor. A sensitive and specific method such as solid phase analysis can be useful with proper interpretation. In this review, due to presence of two or more DQB1 beads for the same antigen in all test kits offered in the market, interpretation of positive HLA antibody for DQB1 will be discussed considering strength and Mean Florescent Intensity (MFI). HLA typing was performed by One Lambda Sequence specific oligonucleotide probes and sequence specific primers (SSOP/SSP). DSA measurement was performed by a solid phase antibody assay using HLA class II single antigen beads with positive HLA Flow XM. HLA DSA for DQB1 was reviewed carefully and accurately for 5 pairs waiting for allogeneic renal transplant. More than one bead for the same DQB1 DSA has to be checked for the presence of combined antibody against donor DQA1 mismatches. It was found that DQB1 DSA has to be assigned according to their DQA1 mismatches and it shows good correlation with positivity in B cell flow cytometer XM in all 5 cases. This accurate assignment and procedure might be recommended in the interpretation of DQB1 DSA. Accurate pre- and post-transplantation workup, including immunological risk assessment for DQB1 DSA, could influence the outcome of organ transplantation significantly.
A normalization factor for an increased comparability within antibody results, tested by bead-based single antigen assays
M. Arnold (Erlangen, DE)
The Implementation of bead based techniques (Luminex) for anti-HLA antibody (Ab) detection has increased the complexity of Ab identification and specification. MFI values in Luminex Single Ag assays do not automatically mirror a predictable Ab concentration. The mean fluorescence intensity (MFI) of Abs is used for the definition of “unacceptable antigens”, even though there are considerable inter- and intra-HLA locus differences in the MFI readout of the Luminex assays. The aim of our study was to compare reaction patterns of Ab positive sera with respect to the various HLA class I and II Ab targets. MFI values of HLA specificites were measured and calculated in 278 anti HLA class I Ab positive and 281 anti HLA class II Ab positive sera from patients on the local waiting list in Erlangen, Germany using Luminex Single Ag assays. Anti HLA-C Abs reacted with a lower mean MFI level compared to anti HLA-A and anti HLA-B Abs. In contrast, anti HLA-DQ Abs showed higher MFI values than HLA-DR and HLA-DP Abs. In order to define a comparable MFI cutoff for all HLA Ab specificities, two normalization factors were calculated, based on a “positive” assumed cut-off of 3000 MFI: one for anti HLA-C Ab MFI (MFI x 2.11) and one for HLA-DQ Ab (MFI x 0.64) MFI. Due to the various Ag loading densities on the surface of the microbeads, MFI values of different HLA specificities are not comparable. Furthermore, HLA inter-specific differences in Ab binding cannot be ruled out. In the interest of treating all Ab MFI values equally, independent of the various HLA targets, a unique Ab MFI cutoff within a single patient’s serum should be calculated using a normalization factor for - at least - HLA-C and HLA-DQ Abs. In order to perform a “virtual crossmatch”, the definition of unacceptable antigens, based on MFI value without taking into account the inter-HLA locus variability should be discussed intensively. A comprehensive concept for the evaluation of this normalization factor would appear to be sensible.
De novo donor-specific HLA antibodies after steroid withdrawal in kidney transplant recipients: a prospective, randomized, controlled, parallel group study. Preliminary results
A. Caballero (Malaga, ES)
Steroids represent one of the mainstays of immunosuppression after kidney transplant (KT). Steroid withdrawal reduces metabolic and cardiovascular complications, but whether it increases the risk of acute rejection and the generation of donor-specific anti-HLA antibodies (DSA) is currently undetermined. In a controlled clinical trial (NCT02284464), a total of 176 KT patients with low immunological risk were recruited to randomly receive either conventional triple immunosuppression: steroids, TAC and MMF versus steroid withdrawal at the third post-KT month. We compared the incidence of de novo DSA, determined by Luminex Mixed and Luminex Single Antigen (One Lambda®), and its impact on graft histology in patients with steroid withdrawal at the 3 post-KT month (after a protocol biopsy) versus patients who continue to receive conventional triple immunosuppression. So far, 68 patients have been randomized (34 per group), with no significant differences in the clinical and demographic characteristics between the groups. The intermediate analysis in those patients who had completed one year of follow-up (n=28) showed no significant differences in the formation of DSA (0% vs. 0%), nor was there rejection in those patients in whom prednisone was withdrawn after randomization. Patients with triple therapy showed a trend toward better renal function compared to those without steroids at the first post-KT year (1.29 ±0.25 vs. 1.56 ±0.42 mg/dL, P=0.088). HbA1c levels were similar between both group at the first post-KT year (5.79 ±0.59 vs. 5.68 ±0.81%, P=0.734). The preliminary results show that steroid withdrawal at the 3 month post-KT seems safe when assessing the appearance of rejection and formation of DSA compared to the patients who continued to receive conventional triple immunosuppression.
Inflammatory and regulatory cytokine changes in HIV-Positive-to-HIV-Positive renal transplant recipients
S. Rautenbach (Cape Town, ZA)
Since 2008, 42 renal transplants have been performed from HIV+ deceased donors to HIV+ recipients with renal failure due to HIV associated nephropathy. Despite transplantation across HLA mismatches, these transplants have been relatively rejection-free, safe and successful in a 3-5 year follow-up. Anti-thymocyte globulin (ATG), as a conditioning regimen given immediately post transplant for 5 days, is known to deplete the majority of T-cells, B-cells, macrophages, monocytes, and dendritic cells. Recipients also receive maintenance immunosuppression of prednisone, mycophenolate mofetil and tacrolimus. The impact of ATG and immunosuppression along with transplantation on host inflammatory and regulatory cytokines was assessed to gain insight into immune homeostatic mechanisms after ATG treatment. Despite the HLA mismatches, there were no rejection events, losses of graft function or deaths at 1 year PT. CD4 counts at transplant had a wide range (132 – 973 cells/ml) but did not change significantly over the first year PT. All recipients are adherent to their combined anti-retroviral therapy (cART) and all viral loads were undetectable throughout follow-up, indicating effective anti-retroviral treatment. In a sub group of 10 participants, the Luminex 200 system was used to measure the concentrations of 37 cytokines in plasma immediately pre-transplant and then at 1, 3, 6 and 12 weeks post-transplant. In all these recipients, when compared to baseline, IL-35 significantly decreased at 1, 3, 6 and 12-weeks post-transplant, whilst IL-10 increased significantly at 1-week post-transplant in 5 recipients. Hierarchical clustering showed a decrease over time in IL-35, IFN-g, IL-20, IL-28A, and IL-11 for all assayed participants. This analysis showed that both inflammatory and regulatory cytokines decline over the 12 weeks of follow-up, although IL-10 is transiently increased after ATG treatment, suggestive of induction of an immune regulatory environment. In this environment, the organ recipients tolerate their grafts and have stable HIV suppression in the presence of cART.
The relevance of tumor necrosis factor-alpha levels in kidney transplant recipients
H. Senturk Ciftci (Istanbul, TR)
Allograft rejection and Cytomegalovirus (CMV) viremia are major predictors of long-term graft survival in kidney transplant recipients. The role of tumor necrosis factor-alpha (TNF-alpha) has not been well defined in this complex entities before. The aim of this study was to determine the diagnostic value of serum and urine TNF-alpha levels in allograft rejection and CMV viremia. A total of 65 patients (61.5% male; mean age 36 ±12 years) who underwent living kidney transplantation between 2013 and 2015 were included. Serum and urinary TNF-alpha levels were measured at post-transplant 1st, 7th day; 1st, 3rd and 6th month. Additionally serum creatinine, proteinuria, serum CMV DNA levels are monitored during post-transplant follow-up. The mean follow-up time was 26 ±9 months. Standard enzyme-linked immuno-absorbant assay (ELISA) was used for detection of TNF-alpha levels. Nine patients (9/65 (13.8%)) had biopsy proven rejection during the follow-up period. Serum TNF-alpha levels were significantly higher in the allograft rejection group in 7th day and 1st month (11.5 ±4.7 vs 15.4 ±5.8 p=0.029, 11.1 ±4.8 vs 17.8 ±10.9 p=0.003, respectively). Elevated urine TNF-alpha levels were found in the allograft rejection group compared to non-rejection group at 1st and 7th day, 1st, 3rd and 6th month (10.2 ±2.5 vs 14.1 ±6.8 p=0.002, 9.8 ±2.2 vs 14.5 ±2.7 p<0.001, 8.0 ±1.7 vs 11.8±2.4 p<0.001, 7.7±1.6 vs 9.6±1.7 p=0.002, 7.4±1.6 vs 8.9±0.9 p=0.005, respectively). Both serum and urine TNF-alpha levels were significantly higher at 1st day (11.6 ±4.7 vs 19.6 ±4.4 p=0.002, 10.4 ±2.7 vs 15.6 ±9.9 p=0.004, respectively). Serum TNF-alpha levels were elevated at 7th day, and 3rd month in CMV viremia group (11.7 ±4.9 vs 16.9 ±5.3 p=0.042, 10.9 ±4.3 vs 23.3 ±10.3 p<0.001, respectively). Serum and urine TNF-alpha levels may be a possible predictor for allograft rejection. Besides that serum and urine TNF-alpha levels may be related to CMV viremia.
Association of effluent parameters, BMI, liver quality and liver transplant outcomes
M. Sadeghi (Heidelberg, DE)
Predictors of postoperative complications are considerable parameters to save patients and organs after liver transplantation. The aim of our study is to evaluate whether effluent parameters prior to re-perfusion show any correlation with post-transplant outcomes such as mortality, early graft dysfunction (EAD), acute rejection and viral and bacterial infections in clinical liver transplant recipients. Pre-transplant concentrations of HMGB1, M65, M30, ALT, AST, GGT, and ALP were measured in available effluent samples of 15 adult recipients who died during the first year post-transplant and 38 age and gender matched liver transplant recipients who survived the first year post-transplant. Effluent concentration of ALP (p=0.006), AST (p=0.050), and Ca++ (p=0.003) were higher in patients with one year post-transplant bacteremia than those without. ALP (p=0.015) was higher in patients with EAD than those without. Multivariate analysis of effluent parameters showed that Ca++>0.30 mmol/l (p=0.012: OR=7.12, CI 1.56-32.58) and ALP ≥27 IU/l (p=0.033: OR=5.31, CI 1.14-27.74) are significant associated factors of 1 year post-transplant bacteremia. They also showed that ALP ≥27 IU/l (p=0.020: OR=5.56, CI 1.32-23.46) is a significant associated factor of EAD. Donors with fatty liver >20% had significantly higher HMGB1 (140.87 vs 42.38 pg/ml: p=0.0001). HMGB1 > 54 pg/ml (p=0.008: OR=6.05, CI 1.59-23.00) is a significant associated factors of BMI and fatty liver (p=0.005: OR=11.68, CI 2.10-64.01). Effluent parameters are indicators of liver quality and predict outcome of liver transplantation. High effluent Ca++ and ALP are risk factors of post-transplant bacteremia, high ALP is a risk factor of EAD and HMGB1 an indicator of donor liver quality.
Forbidden HLA antigens in kidney re-transplantation – a single center study
A. Slavcev (Prague, CZ)
One of the allocation strategies intended to reduce the incidence of antibody-mediated rejection (AMR) is the definition of unacceptable, or “forbidden” HLA antigens before transplantation, however, the approaches for definition of these antigens applied by different centers (even in the same country) considerably vary. In our center, forbidden HLA antigens were defined as mismatched antigens from previous transplantation(s) against which patients have HLA antibodies detectable by Luminex. 144 patients were included in our study and were divided into two cohorts: historical cohort including 90 patients who were transplanted without taking into consideration forbidden antigens and 54 patients who were transplanted after applying the forbidden HLA antigens approach (test cohort). After the implementation of this allocation policy, there was a significant decrease in the proportion of re-transplanted patients compared with the total number of organ transplants (21% vs. 13%; p = 0.0008). As far as the incidence of AMR during the first year after transplantation, 30 patients in the historical cohort experienced AMR and 22 in the test cohort (33% vs. 41% p = 0.3772). The incidence of T cell mediated rejection was also similar in the two patient groups 20 (22%) vs. 6 (11%) p = 0.1185. On the contrary, in case AMR was diagnosed in the first month after transplantation, graft failure was lower in the test cohort in comparison with the historical cohort p = 0.048. The introduction of forbidden HLA antigens in re-transplanted patients in our center did not positively influence the incidence of AMR, however resulted into a significant decrease in the number of re-transplantations and into accumulation of these high-risk patients on the waiting list.
Case Report: the importance of C3d assays in kidney transplant
e. pacquola (Camposampiero, IT)
Antibody-mediated rejection (AMR) is a major cause of kidney graft loss. Here we tried to test whether the capacity of anti-HLA antibodies to bind complement components allows accurate risk stratification at the time of AMR diagnosis. We report a case of a young female (35 years old), who was admitted to Padua Hospital for a cadaveric kidney transplant. The Complement Dependent Cytotoxicity (CDC) Crossmatch was repeatedly negative. Anti-HLA antibodies pre-transplant were tested by using Luminex Technologies and no DSA were identified. The serum creatinine level was 4.1 mg/dL at 1 day after transplantation. Open biopsy was performed immediately. Histology revealed moderate peritubular capillaritis and mild glomerulitis without C4d immunoreactivity. Sera banked at the time of the renal biopsy were screened for the presence of donor-specific anti-HLA antibodies (DSAs) and our laboratory examination showed that the patient had donor-specific antibodies (DSAs) to DR52 and to DRB1*11. In parallel serum was tested for the presence of C3d-binding anti-HLA using a novel single-antigen flow bead assay in order to asses the ability of these antibodies (DSAs) to bind complement components. We found a positive correlation between the absence of C4d deposition and non C3d-fixing DSAs, and a better outcome of patient kidney transplant. The young patient was not treated with plasmapheresis, but we continued to monitoring her antibody status as per protocol. The presence of Donor-Specific anti-HLA Antibodies (DSA) was determined by testing Lifecodes Single Antigen and Lifecodes C3d (Immucor Transplant Diagnostic), detected by X-MAP Luminex Technology. This result was supported by recent studies showing that the presence of C4d deposition into the graft was not associated with higher risk of graft. In contrast, the presence of circulating C3d-binding DSA at the time of rejection was strongly associated with higher risk for kidney-graft failure.