CCR4highCD4+ cell populations in kidney graft blood after steroid withdrawal: a prospective, randomized, controlled, parallel group study. Preliminary results
A. Caballero (Malaga, ES)
Steroids represent a mainstay of immunosuppression after kidney transplant. The infiltration into the graft of active T cells following KT depends on the expression of chemokines and their interaction with their T-cell receptors. However, the natural history of the expression of these molecules in patients who undergo steroid withdrawal after transplant is unknown.
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 (Group A) versus steroid withdrawal at the 3 post-KT month (Group B). We compared the evolution of CCR4highCD4+ and CXCR3highCD4+ lymphocyte sub-populations in graft blood (GB) extracted by fine needle aspiration puncture determined by flow cytometry in patients after steroid withdrawal at the 3 month post-KT versus patients who continue to receive conventional triple immunosuppression. Measurements were made at 3 (baseline) and 6 months post-KT in GB and in peripheral blood (PB). So far, 68 patients have been randomized (34 in each group). There were no significant differences in the clinical and demographic characteristics between the groups at baseline. The first analysis (at 3 months) in those patients who had completed 6 months of follow-up (Group A: n=13; Group B: n=15) showed a significant increase in the CCR4highCD4 subpopulations in GB versus PB in both groups. However, at six months a significant increase in GB versus PB was only seen in Group A. There were no significant differences in the CXCR3highCD4+ lymphocyte subpopulation at the third or sixth month between GB and PB in either group
These preliminary results could suggest a possible effect of prednisone that would favor the recruitment of CCR4highCD4+ cells into the renal graft.
A rapid method to isolate highly purified T or B cells from blood, lymph node or spleen samples for use in donor-recipient crossmatch analysis
K. McQueen (Vancouver, CA)
The crossmatch assay is used as part of a pre-transplant immunologic risk assessment to determine the compatibility between donor-recipient pairs. Isolated T or B cells from the donor are mixed with recipient serum and the presence of donor-specific antibodies is detected through a complement-dependent killing assay (CDC-crossmatch) or by flow cytometry (flow crossmatch). Isolation of specific cell types can be time consuming, and multiple methods must often be validated in laboratories that receive a variety of sample types. We have developed methods (EasySep) to isolate T or B cells directly from whole blood (WB) in 25 minutes, or lymph node (LN) and spleen samples in 11 minutes, without RBC lysis, sedimentation or density gradient centrifugation. Unwanted cells, platelets and RBCs were immunomagnetically labelled and then placed into a magnet. Labelled unwanted cells were retained, while untouched T or B cells were poured or pipetted off. Isolation of T or B cells using this method was tested on WB, peripheral blood mononuclear cells (PBMC) (model system for LN, which typically have few RBC) as well as on a suspension of PBMC/WB and a B cell line (model system for spleen, which has a high B cell content). Purities following T cell isolation were 97% +/-4 (n=10) from WB, 94% +/-6 (n=11) from PBMC (mock LN), and 94% +/-6 (n=12) from mock spleen (mean+/-SD). B cell purities were 97% +/-4 (n=10) from WB, 94% +/-7 (n=18) from PBMC, and 95% +/-9 (n=6) from mock spleen. On average, 650,000 T cells and 70,000 B cells were recovered per mL of WB. Starting with 5 x10e7 cells, 10 million T cells and 2.8 million B cells were recovered from PBMCs, while 10 million T cells and 1.8 million B cells were recovered from mock spleen samples. Isolations can be automated using RoboSep. This new method enables the isolation of highly purified T or B cells from multiple sample sources using the same reagents, thus simplifying validation for a busy HLA laboratory.
Long-term outcomes and discard rate of kidneys by decade of extended criteria donor age
A. Amoroso (Torino, IT)
Extended criteria donors represent nowadays a main resource for kidney transplantation, and recovery criteria are becoming increasingly inclusive. However, the limits of this approach are not clear as the effects of extreme donor ages on long-term kidney transplantation outcomes is not known. To address these issues, we performed a retrospective study on extended criteria donor kidney transplantation. In total, 647 consecutive extended criteria donor kidney transplantations performed over 11 years (2003–2013) were included. Donor, recipient, and procedural variables were classified according to donor age decades (group A, 50–59 years old [n=91]; group B, 60–69 years old [n=264]; group C, 70–79 years old [n=265]; and groupD, 80 years old [n=27]). Organs were allocated in single- or dual-kidney transplantation after a multi-step evaluation including clinical and histologic criteria. Long-term outcomes and main adverse events were analyzed among age groups and in either single- or dual-kidney transplantation. Kidney discard rate incidence and causes were evaluated. Median follow-up was 4.9 years (25th; 75th percentiles: 2.7; 7.6 years); patient and graft survival were comparable among age groups (5-year patient survival: group A, 87.8%; group B, 88.1%; group C, 88.0%; and group D, 90.1%; P=0.77; graft survival: group A, 74.0%; group B, 74.2%; group C, 75.2%; and group D, 65.9%; p=0.62) and between dual-kidney transplantation and single-kidney transplantation except for group D, with a better survival for dual-kidney transplantation (P=0.04). No difference was found analyzing complication incidence or graft function over time. Kidney discard rate was similar in groups A, B, and C (15.4%, 17.7%, and 20.1%, respectively) and increased in group D (48.2%; odds ratio, 5.1 with A as the reference group; 95% confidence interval, 2.96 to 8.79). Discard rate and long-term outcomes are similar among extended criteria donor kidney transplantation from donors ages 50–79 years old. Conversely, discard rate was strikingly higher among kidneys from octogenarian donors, but appropriate selection provides comparable long-term outcomes, with better graft survival for dual-kidney transplantation.
Pre-transplant HLA antibody screening by solid phase assays: incidence of anti-HLA IgM antibodies in kidney transplant candidates
E. Poggi (Rome, IT)
A positive lymphocytotoxic crossmatch represents an absolute contraindication to kidney transplantation. This paradigm is widely accepted for IgG antibodies but few data on the clinical relevance of IgM antibodies are available and so their role is still controversial. Some studies report that they can be protective for transplant, while other suggest that IgM antibodies are harmful for the graft. Pre-transplant antibody screening by CDC technique does not allow us to discriminate IgG and IgM isotypes. Instead, serum treatment with DTT can indirectly highlight this because it is able to inactivate the IgM pentamer. Moreover, in both cases it is not possible to know if these antibodies are specific for HLA molecules. The new solid-phase techniques permit us to investigate anti-HLA antibodies and to discriminate their isotype. Since September 2016, 189 kidney transplant candidates on the waiting list at the Lazio Regional Transplant Center, were analyzed to evaluate the incidence of IgM antibodies. The antibody characterization was performed using FlowPRA Screening Test to detected either anti-HLA IgM alone, otherwise unknown, or in combination with IgG antibodies and by Luminex Single Antigen Beads to identify antibody specificity. The incidence of anti-HLA IgM antibodies was 10% (19/189). Only IgM antibodies were detected in ten (53%) patients, both IgM/IgG antibodies were presented in nine (47%) patients. IgM positive-group showed in 6 cases only anti-HLA class I antibodies (2000≥MFI≤7000) and 4 cases only class II antibodies (4000≥MFI≤12000). The antibody characterization of IgM/IgG positive-group evidenced in 3 patients the same IgM and IgG specificity, while 6 patients showed additional IgM specificity respect to evidenced IgG antibody specificity. In conclusion, our study suggests widening the antibody screening to anti-HLA IgM antibodies in transplant candidates. The strength and specificity of detected IgM antibodies highlighted the importance of their accurate characterization to understand the clinical significance and to improve graft survival.
Feasibility of eplet-based matching for allocation of deceased donor renal transplants
S. Fidler (Perth, AU)
Retrospective studies have shown a poor correlation between the number of broad and eplet mismatches at the same HLA locus. If eplet mismatches are to be incorporated in the deceased donor allocation pathway, using high resolution HLA typing to calculate the number of eplet mismatches is not practical in the short time-frame available. We aimed to determine whether low / intermediate resolution HLA typing could be used to accurately calculate eplet mismatches. 264 patients who underwent renal transplant between 2003 and 2007 were included. Prospective serological HLA typing and retrospective 4-digit Sanger sequencing was performed for donor / recipient pairs. Two-digit molecular typing was derived from the 4-digit sequencing results. The number of eplet mismatches was calculated using HLAMatchmaker for 2-digit typing and compared with the 4-digit typing derived eplet mismatches. Correlation and agreement of HLA-A, -B, -C, -DR and –DQ mismatches between the 2- and 4-digit results were analysed. There was close correlation between the number of eplet mismatches calculated using serological and four-digit molecular typing methods at HLA-A, -B and -DR loci with coefficients above 0.95 (Spearman correlation). In contrast, there was less correlation at HLA-C and -DQ loci with coefficients of 0.87 and 0.80, respectively. The correlation coefficients between the number of eplet mismatches calculated using 2-digit and 4-digit molecular typing at all loci were above 0.98 (p <0.001). Consistency and absolute agreement in the number of eplet mismatches was similar using 2-digit and 4-digit molecular typing across class I and II loci. In contrast, consistency and absolute agreement was generally lower for serological typing, particularly at HLA-C (consistency: 0.875 and absolute agreement: 0.875) and HLA-DQ loci (consistency: 0.801 and absolute agreement: 0.792). There is good correlation and agreement between 2- and 4-digit typing for total eplet mismatches at all loci. These results suggest that 2-digit molecular HLA typing may be sufficient for the allocation of donor kidneys by eplet-based matching in deceased donor allocation pathways. Further studies evaluating the correlation and agreement in a broader ethnically diverse population group are required.
Comparison of anti-HLA antibody detection methods in cadaveric transplant candidates in Turkey: a single centre experience
M. Soyöz (Izmir, TR)
Chronic kidney failure can result from diabetes, obesity and hypertension which are the most abundant diseases worldwide. The definitive treatment of chronic kidney failure is transplantation. However organ rejections can occur due to the presence of anti-HLA antibodies after transplantation. Therefore detection of anti-HLA antibodies is important to prevent hyperacute or acute rejections. In this study crossmatch tests were performed before transplantation to 416 cadaveric transplant candidates who were admitted to Tepecik Training and Education Hospital Tissue Typing Laboratory between 2014 and 2016. The lymphocyte cells were obtained from 113 cadaveric donors. 31.7% of DSA and 35.3% of Flow XM results of CI antibodies and 33.3% of DSA and 47.6 Flow XM results of CII antibodies were positive while determined as negative by CDCXM. 68.3% of CI antibodies were detected as positive by both CDC and DSA and 64.7% were positive by both CDCXM and FCXM. 66.7% and 52.4 % of CII antibodies were detected as positive by DSA and FCXM, respectively while determined as positive by CDCXM as well. It has been known that CDCXM is a gold standard to detect the complement dependent anti-HLA antibodies. However the sensitivity of CDCXM is lower than bead based methods. Thus, at least two different crossmatch tests should be performed before cadaveric transplantation.
The importance of flow cytometric crossmatch, panel reactive antibody and single antigen bead tests in detecting donor specific antibody in renal transplantation
Ö. SENOL (izmir, TR)
Pre-existing donor-specific antibodies (DSA) to donor HLA antigens cause antibody-mediated rejection. In this study, we aimed to determine the most appropriate method for detecting DSA before renal transplantation. 100 patients who were living donors who applied to Ege University Organ Transplant Application and Research Center were included in the study. PRA levels and SAB levels of these patients were determined by a Luminex FC-XM test and CDC-XM test were applied to each patient. MFI values for FCXM-T were 20; the MFI value for FCXM-B was determined to be 40. The results of FCXM-T and FCXM-B of the patients studied were evaluated according to the determined reference values. When the HLA class I and class I values of the patients were compared with FCXM and CDCXM results, the results of FCXM-T and B and CDCXM-T and B of 25 patients with HLA class I PRA level positive were negative. The FCXM-T result were negative for 29 patients with HLA-class II PRA levels positive; FCXM-B value positive and CDCXM-T result negative. The CDCXM-B value of five patients were found to be positive. All of the patients studied were negative for FCXM-T; and an FCXM-B value of 32 was positive. Of the 25 patients with HLA CL1 PRA level positive, ten were positive for SAB. In addition, DSA was not detectable in the Luminex PRA test. Of the 29 patients with HLA CL2 PRA levels positive, nine were negative for SAB and 20 were positive for SAB. Antibodies that were not detectable in the PRA test in class II antibodies were also detected in the SAB test as in the HLA CL1 SAB test. Patients with PRA CL1 positive were found to be statistically insignificant (p> 0.05). Results of PRA CL1 and SAB CL1 of these patients were statistically significant (p <0.05). FCXM-B results of patients with PRA CL2 positive were statistically significant (p <0.05). Statistically significant results were obtained when the results of PRA CL2 and SAB CL2 of these patients were compared (p <0.05). Anti-HLA antibodies that can not be detected by serologic methods can be detected by flow cytometric methods and SAB test is more sensitive test for identification of these antibodies than PRA test. We found that the SAB test was a more reliable method for detecting DSA in renal transplant recipients than other antibody detection tests, according to the statistical values of our results.
Long-term de novo anti-HLA antibody development in kidney transplant recipients from a Spanish multicenter study
E. Asensio (Santander, ES)
Our aim was to analyze the frequency and rate of production of anti-HLA antibodies (both donor and non-donor specific) in a multi-centric prospective cohort from 9 renal transplant units in Spain. The cohort included 742 patients. Recruitment was conducted from 2009 to 2012 and all patients gave informed consent. Serum samples were collected at transplantation and at 3, 12 and 24 months after transplantation. Presence of anti-HLA antibodies was screened by Luminex LabScreen (One Lambda Inc). If positive, specificities were detected with Luminex Single Antigen bead arrays and monitored from first positive serum. Positivity was defined for those sera that fulfilled the following conditions: MFI (mean fluorescence intensity) >25% of positive control of the assay and MFI >1500. Donor HLA typing was performed by PCR-SSP (One Lambda Inc). 600 of the 742 patients (80.8%) included in the study did not present anti-HLA antibodies pre-transplantation. 39 of the 742 patients (5.26%) had non-donor specific anti-HLA antibodies, whereas the 103 remaining (13.88%) had donor-specific anti-HLA antibodies (DSA) (19 class I, 63 class II and 21 class I and II). We studied the evolution of the de novo DSA at the different time points. 23 of 730 patients (3.15%) analyzed prior transplantation developed de novo DSA at 3 months, 17 of 629 (2.7%) at 12 months and 2 of 428 (0.47%) at 24 months. This is a descriptive study, but analysis of relationship with clinical data and graft and patient outcome is being performed at present.
The assessment Of HLA compatibility effect on 10 year graft function and survival in renal transplantation. North Greece immunogenetic laboratory experience
A. Fylaktou (Athens, GR)
HLA incompatibility is associated with post-transplant adverse consequences in renal transplantation. In this study, the effects of HLA incompatibility on 10 year graft function and survival in renal transplantation were assessed. Outcome analysis was performed in 109 diseased (67) or living (42) donor renal transplantations during 2005-2007. Patients were grouped according to the level of HLA-A, -B and -DR mismatching into two groups, group A with 0-3 incompatibilities (n=74) and group B with 4-6 incompatibilities (n=35). Serum creatinine levels (mg/dl), 24 hour urine protein levels (mg/24h) in 1st, 5th and 10th year post-transplantation were measured along with graft survival, time on waiting list, cold ischemia time, donor and recipient age, donor type (diseased or living) and acute rejection episodes. HLA antibody reaction frequency (% PRA) was also measured. Groups A and B were compared in association with the previous parameters. Statistical analysis was performed through SPSS using t-test, chi-square and Fisher’s exact test at a level of p<0.05. The total graft survival was 85.3%, 81.7% and 80.7% in 1st, 5th, 10th year, respectively. Graft survival was higher in group A in 1st, 5th and 10th year post-transplantation (98.1%, 92.6%, 88.9%, respectively) versus group B (66.7%, 61.9%, 61.9%) (p<0.001, 0.001, 0.007 respectively). 24 hour urine protein in 5th and 10th year was 266.52 and 197.11 in group A, versus 413.29 and 401.6 in group B (p>0.05). 93.1%, 4.2% and 2.8% of patients had PRA 0%, 5-69% and >70%, respectively. No statistically significant differences were observed between the groups in serum creatinine levels, waiting list time, cold ischemia time, donor and recipient age, donor type, % PRA and acute rejection episodes. Although only 2.8% of patients had PRA>70% with no differences in pre-sensitization between the groups, graft survival was significantly superior in group A. This demonstrates that HLA incompatibility is an independent risk factor affecting the 10 year graft survival.
Contribution of Luminex–based virtual crossmatch during renal transplantation
A. Charfi (Sfax, TN)
The purpose of our study was to evaluate the contribution of the use of the Luminex Single Antigen kit (LSA) for the detection of anti-HLA antibodies as compared to the serological cross-match (XM). We aimed to study the sensitivity and specificity of this technology and evaluate the possibility of predicting the evolution of the graft in post-transplantation. During the period from January 2011 to December 2015 we achieved 182 XM by complement dependent cytotoxicity (CDC). These XM were performed as part of the preparation for a renal transplant (RT) or post-transplant follow-up.
Each serum was studied by the Labscreen Mixed 12 (LSM12) screening kit, and then, in case of positivity, the specificity and the raw of the anti-HLA antibodies were defined by the LSA1 and / or LSA2 kits. We realized a virtual XM by the EpViX software for each positive serum in LSA1 and / or 2. The cut-off of the virtual cross-match was 2000. A negative virtual auto-XM was a necessary condition for the validation of the analysis. We studied the sensitivity and specificity of the virtual XM with short-term graft success.
For 182 donor / recipient pairs, the sensitivity and specificity of the virtual XM compared to the XM results by CDC were 100% and 92.1%, respectively. With T lymphocytes, the sensitivity and specificity of the virtual XM were 100% and 93.2%, respectively. As for B lymphocytes, the sensitivity and specificity of the virtual XM were 66.7% and 96.6%, respectively. One hundred fifteen RT were performed during this period. Five acute rejections were diagnosed. The sensitivity and specificity of the virtual XM compared to the short-term graft evolution, were 40% and 99.1% respectively. Indeed, two cases of humoral rejection (positive with virtual XM) could be prevented. Our results confirm those of the literature. The use of the LSA kits and the realization of a virtual XM by the EpViX software makes it possible to reduce the number of acute rejections.