Correlation and agreement between eplet mismatches calculated using serological, low-intermediate and high resolution molecular human leukocyte antigen typing methods.

Structural human leukocyte antigen (HLA) matching at the eplet level can be identified by HLAMatchmaker, which requires the entry of four-digit alleles. The aim of this study was to evaluate the agreement between eplet mismatches calculated by serological and two-digit typing methods compared to high-resolution four-digit typing. In a cohort of 264 donor/recipient pairs, the evaluation of measurement error was assessed using intra-class correlation to confirm the absolute agreement between the number of eplet mismatches at class I (HLA-A, -B, C) and II loci (HLA-DQ and -DR) calculated using serological or two-digit molecular typing compared to four-digit molecular typing methods. The proportion of donor/recipient pairs with a difference of >5 eplet mismatches between the HLA typing methods was also determined. Intra-class correlation coefficients between serological and four-digit molecular typing methods were 0.969 (95% confidence intervals [95% CI] 0.960-0.975) and 0.926 (95% CI 0.899-0.944), respectively; and 0.995 (95% CI 0.994-0.996) and 0.993 (95% CI 0.991-0.995), respectively between two-digit and four-digit molecular typing methods. The proportion of donor/recipient pairs with a difference of >5 eplet mismatches at class I and II loci was 4% and 16% for serological versus four-digit molecular typing methods, and 0% and 2% for two-digit versus four-digit molecular typing methods, respectively. In this small predominantly Caucasian population, compared with serology, there is a high level of agreement in the number of eplet mismatches calculated using two-compared to four-digit molecular HLA-typing methods, suggesting that two-digit typing may be sufficient in determining eplet mismatch load in kidney transplantation.

Modeling the benefits and costs of integrating an acceptable HLA mismatch allocation model for highly sensitized patients.

BACKGROUND: The Eurotransplant acceptable mismatch program has improved transplantation access for highly sensitized recipients. However, the benefits and costs of implementing such a program remain unknown. METHODS: Using decision analytical modeling, we compared the average waiting time for transplantation, overall survival gains (in life-years and quality-adjusted life-years gained), and costs of integrating an acceptable mismatch allocation model compared with the current deceased-donor kidney allocation model in Australia. RESULTS: Acceptable mismatches were identified in 12 of 28 (43%) highly sensitized recipients using HLAMatchmaker. Inclusion of acceptable mismatches in the current allocation model improved the transplantation access for four (14%) highly sensitized recipients, with an average reduction in waiting time of 34 months (from 86 to 52 months). Compared with the current allocation model, incorporating an acceptable mismatch allocation model achieved an overall lifetime gain of 0.034 quality-adjusted life-years and savings of over $4,000 per highly sensitized patient, with a small consequential loss of 0.005 quality-adjusted life-years and extra costs of $800 for every reallocated patient. CONCLUSIONS: Despite modest overall health gains, application of an acceptable mismatch allocation model is an equitable approach to improve transplantation access for highly sensitized transplant candidates without compromising the overall health benefits among the other patients on the deceased-donor waitlist in Australia.

Peri-operative third party red blood cell transfusion in renal transplantation and the risk of antibody-mediated rejection and graft loss.

Historic red blood cell transfusion (RBCT) may induce anti-HLA antibody which, if donor specific (DSA), is associated with increased antibody-mediated rejection (AMR). Whether post-operative RBCT influences this risk is unknown. We examined the RBCT history in 258 renal transplant recipients stratified according to prevalent recipient HLA antibody (DSA, Non-DSA or No Antibody). AMR occurred more frequently in patients who received RBCT both pre and post transplant compared with all other groups (Pre+Post-RBCT 21%, Pre-RBCT 4%, Post-RBCT 6%, No-RBCT 6%, HR 4.1 p=0.004). In the 63 patients who received Pre+Post-RBCT, 65% (13/20) with DSA developed AMR compared with 0/6 in the Non-DSA group and 2/37 (5%) in the No-Antibody group (HR 13.9 p<0.001). In patients who received No-RBCT, Pre-RBCT or Post-RBCT there was no difference in AMR between patients with DSA, Non-DSA or No-Antibody. Graft loss was independently associated with Pre+Post-RBCT (HR 6.5, p=0.001) AMR (HR 23.9 p<0.001) and Non-AMR (6.0 p=0.003) after adjusting for DSA and delayed graft function. Re-exposure to RBCT at the time of transplant is associated with increased AMR only in patients with preformed DSA, suggesting that RBCT provides additional allostimulation. Patients receiving Pre+Post-RBCT also had an increased risk of graft loss independently of AMR or DSA. Both pre and post procedural RBCT in renal transplantation is associated with modification of immunological risk and warrants additional study.

ABO-incompatible matching significantly enhances transplant rates in kidney paired donation.

BACKGROUND: Although preformed donor-specific anti-human leukocyte antigen antibodies (DSA) can be overcome by plasmapheresis-based strategies with some success in renal transplantation, kidney paired donation (KPD) is a more effective strategy to avoid DSA. In contrast, ABO incompatibility can be crossed with outcomes equivalent to ABO-compatible transplantation. Here, we report the ability of accepting human leukocyte antigen-compatible but ABO-incompatible donors to increase the number of exchanges in a KPD program. METHODS: In the Australian KPD program, virtual crossmatch is used to allocate suitable donors to recipients. Acceptance of ABO-incompatible donors is allowed in cases where anti-blood group antibody titres are deemed amenable to removal by apheresis or immunoabsorption. The number of matched recipients, identified chains, and transplants performed with and without acceptance of ABO incompatibility was analyzed. RESULTS: In 2 years, 115 pairs were included in nine quarterly match runs. Incompatibility due to DSA accounted for 86% of the listed pairs and 52% were also blood group incompatible to their coregistered donor. Median calculated panel-reactive antibody in registered recipients was 83% (mean, 67%±37%). ABO-incompatible donors were accepted for 36 patients. Two waitlist recipients and 48 KPD candidates were matched and transplanted. Ten recipients (20%) of an ABO-incompatible donor kidney were distributed across 8 chains that resulted in 21 recipients being transplanted. Thus, without ABO-incompatible matching, only 27 recipients in 12 chains would have been transplanted. CONCLUSION: Acceptance of blood group-incompatible donors for patients with low to moderate anti-blood group antibody significantly increases transplant rates for highly sensitized recipients.

Pre-transplant donor specific anti-HLA antibody is associated with antibody-mediated rejection, progressive graft dysfunction and patient death.

BACKGROUND: The long term effect of donor specific antibodies (DSA) detected by Luminex Single Antigen Bead (SAB) assay in the absence of a positive complement-dependant cytotoxicity (CDC) crossmatch is unclear. DSA at the time of transplant were determined retrospectively in 258 renal transplant recipients from 2003 to 2007 and their relationship with rejection and graft function prospectively evaluated. After a median of 5.6 years follow-up 9% of patients had antibody mediated rejection (AMR) (DSA 11/37 (30%), DSA-Neg 13/221 (6%), HR 6.6, p<0.001). Patients with anti-HLA class II (HR 6.1) or both class I+II (HR 10.1) DSA had the greatest risk for AMR. The Mean Fluorescent Intensity (MFI) of the DSA was significantly higher in patients with AMR than those with no rejection (p=0.006). Moreover, the strength of the antibody was shown to be important, with the risk of AMR significantly greater in those with DSA >8000 MFI than those with DSA <8000 MFI (HR 23, p<0.001). eGFR progressively declined in patients with DSA but was stable in those without DSA (35.7 ± 20.4 mls/min vs 48.5 ± 22.7) and composite patient and graft survival was significantly worse in those with class II (HR 2.9) or both class I+II (HR 3.7) but not class I DSA. Class II DSA alone, or in combination with class I DSA had the strongest association with graft loss and patient death. Patients with DSA not only have increased rates of acute AMR, but also chronic graft dysfunction, graft loss and death. Antibody burden quantified by SAB assay may identify patients at highest immunological risk and therefore influence patient management and improve long-term patient outcome.

Transnational validation of the Australian algorithm for virtual crossmatch allocation in kidney paired donation.

An independent pool of 16 incompatible live donor-recipient pairs registered at the Vienna transplant unit was applied to test whether virtual crossmatch allocation used in the Australian kidney paired donation (KPD) program reliably predicts negative crossmatches. High resolution HLA data were entered into the computer-matching algorithm and allocation was performed excluding any DSA>2000MFI. CDC and flow crossmatch data of recipients against any of the donors were available for 112 crossmatch combinations. The computer program identified 19 possible pairings in 2-way or 3-way chains in multiple combinations. The top ranked combination included one 3-way and two 2-way ABO-compatible chains. Where crossmatches were available all recipients were CDC crossmatch negative with the computer-matched donor. Excluding allocation of KPD donors in the presence of DSA>2000MFI had a negative predictive of 99.9% for CDC and 96.4% for flow crossmatch. In the 12 pairings with ⩾1 DSA against crossmatched donors there was a negative CDC and flow crossmatch. These results show excellent correlation between matching using virtual crossmatch and actual crossmatch results. Using the 2000MFI cut-off the number of potentially unacceptable CDC and flow crossmatch positive pairings identified by virtual crossmatching is low, but some potential crossmatch negative pairings are missed.

Comparison of time on the deceased donor kidney waitlist versus time on the kidney paired donation registry in the Australian program.

In the Australian kidney paired donation (KPD) program matching is based on acceptable mismatches, whereas deceased donor waitlist (DDWL) patients are allocated kidneys based on HLA antigen matching rules. Herein, we compared waiting time for a KPD match to the waiting time on the DDWL and the occurrence of matching in the DDWL for patients who were registered in both programs. Data on first dialysis, matches on the DDWL, KPD program entry, matches and transplant dates were assessed in 26 KPD recipients of the Australian program. There were 22 recipients who were listed in the DDWL and received kidney transplants by KPD. Time on dialysis until KPD transplantation was 808 ± 646 days. Eleven patients had never been matched with a deceased donor (waiting time 345 ± 237 days) and 11 had been matched on average 3 ± 5 times (waiting time 1227 ± 615 days, P < 0.0001 vs. never matched), but did not progress to transplantation because of positive crossmatch or class II donor-specific antibody. Mean time from registration in the KPD program until kidney transplantation was 153 ± 92 days (P < 0.0001 vs. DDWL). KPD allocation using the acceptable mismatch approach is effective in identifying suitable live donors for some recipients within a relatively short time-frame.

High transplant rates of highly sensitized recipients with virtual crossmatching in kidney paired donation.

BACKGROUND: In kidney paired donation (KPD), flexibility in the allocation of incompatible pairs is required if a critical mass of pairs to efficiently find matches cannot be reached. METHODS: In the Australian KPD program, virtual crossmatch is used for the allocation of suitable donors to registered recipients. Matching is based on acceptable mismatches, and donors are excluded from matching to recipients with donor-specific antibodies (DSAs) greater than 2000 mean fluorescence intensity (MFI). Match and transplant rates in the first year of the program were reviewed with respect to recipient and donor characteristics, including blood group distribution, level of recipient's sensitization, and postallocation crossmatches. RESULTS: Four quarterly match runs were performed, which included 53 pairs and 2 altruistic donors. Human leukocyte antigen incompatibility accounted for 90% of the listed pairs. In the second run, the DSA threshold was increased to greater than 8000 MFI, because no matches were found with standard allocation. Optional ABO-incompatible matching was introduced from run 3. Matches were identified in 37 (70%) patients, of whom 92% had a negative crossmatch with their matched donor. Crossmatch positive results were found only in recipients with DSAs greater than 2000 MFI in the second run. In 4 cases immunological reasons and in 4 cases other reasons resulted in breakdown of chains and 17 patients not progressing to transplantation. Eventually, 20 (38%) patients received a KPD transplant, and 35% of these had a calculated panel-reactive antibody greater than 90%. CONCLUSIONS: KPD using virtual crossmatch is a valid and effective solution for patients with immunologically incompatible donors even in the context of highly sensitized recipients.

HLA typing using bead-based methods.

The LABType(®) SSO (One Lambda, Inc) and Gen-Probe LIFECODES HLA-SSO HLA Typing tests are rapid and efficient assays for determining human leukocyte antigens (HLAs). The principle of these assays involves the hybridization of reverse sequence-specific oligonucleotide probes each attached to a unique colour coded microsphere to identify HLA class I and class II alleles. Target DNA is polymerase chain reaction (PCR) amplified using group-specific primers and then biotinylated which allows it to be detected using R-Phycoerythrin-conjugated Streptavidin. The PCR product is then denatured and allowed to hybridise to complementary DNA probes conjugated to fluorescently code microsperes. The Luminex(®) Flow Analyser achieves detection by determining the fluorescent intensity of PE on each microsphere. The assignment of HLA alleles is based on the reaction pattern of the various beads compared to patterns with known HLA alleles.

Separation and cryopreservation of lymphocytes from spleen and lymph node.

Spleen and lymph node retrieved post-mortem from deceased organ donors are a rich source of lymphocytes. Storage of lymphocytes separated from these sources can be valuable where post-transplant testing (crossmatching) is required. DNA extraction from stored lymphocytes also allows further genetic testing where required, for example additional HLA typing not performed at the time of transplant for donor-specific antibody monitoring. Methods for the isolation and freezing of such cells is described.

Crossmatching by complement-dependent lymphocytotoxicity.

The presence of preformed donor-specific HLA antibodies detected by Complement-dependent cyto-toxicity (CDC) crossmatch assay is associated with a high incidence of hyperacute or accelerated rejection and remains one of the gold standard tests pre-transplant. The standard CDC crossmatch detects IgG1, IgG3, and IgM antibody, i.e. complement fixing, bound to the native viable cell surface of lymphocytes. The crossmatch can be enhanced with the addition of anti-human-globulin to detect non-complement fixing antibodies (IgG2 and IgG4), and sensitivity can be improved with prolonged incubation times.