Donor exchange programs were designed to allocate organs for highly sensitized (HS) patients. The allocation algorithm differs slightly among countries and includes different strategies to improve access to transplants in HS patients. However, many HS patients with a calculated panel reactive of antibodies (cPRA) of 100 % remain on the waiting list for a long time. Some allocation algorithms assume immunological risk, including Imlifidase treatment, to increase the chance of transplantation in very HS patients. Here, we describe our unicenter experience of low-risk delisting strategy in 15 HS patients included in the Spanish donor exchange program without donor offers. After delisting, 7 out of 15 HS patients reduced the cPRA below 99.95 % and impacted the reduction of time on the waiting list (p = 0.01), where 5 out of 7 achieved transplantation. Within those HS that remained above 99.95 %, 1 out of 8 was transplanted. All the HS were transplanted with delisted DSA, and only one with DSA level rebounded early after transplantation. All HS transplanted after delisting maintain graft function. The transplant immunology laboratories are challenged to search intermediate risk assessment methods for delisting high HS patients.

Imlifidase recently received early access authorization for highly sensitized adult kidney transplant candidates with a positive crossmatch against an ABO-compatible deceased donor. These French consensus guidelines have been generated by an expert working group, in order to homogenize patient selection, associated treatments and follow-up. This initiative is part of an international effort to analyze properly the benefits and tolerance of this new costly treatment in real-life. Eligible patients must meet the following screening criteria: cPRA ≥ 98%, ≤ 65-year of age, ≥ 3 years on the waiting list, and a low risk of biopsy-related complications. The final decision to use Imlifidase will be based on the two following criteria. First, the results of a virtual crossmatch on recent serum, which shall show a MFI for the immunodominant donor-specific antibodies (DSA) > 6,000 but the value of which does not exceed 5,000 after 1:10 dilution. Second, the post-Imlifidase complement-dependent cytotoxicity crossmatch must be negative. Patients treated with Imlifidase will receive an immunosuppressive regimen based on steroids, rATG, high dose IVIg, rituximab, tacrolimus and mycophenolic acid. Frequent post-transplant testing for DSA and systematic surveillance kidney biopsies are highly recommended to monitor post-transplant DSA rebound and subclinical rejection.

The aim of this study was to investigate if human IgM is a cleavable substrate for imlifidase and to explain an observed effect in anti-HLA IgM single antigen bead (SAB) assays in sensitized patients.
Serum samples collected pre- and 24 h post-imlifidase administration from sensitized patients enrolled in a phase II trial were investigated for anti-HLA IgG and IgM using SAB assays, with and without in vitro IgG depletion using a CaptureSelect™ affinity matrix. In addition, pre-dose samples and purified human IgM samples were treated with imlifidase in vitro and evaluated by SDS-PAGE, Western blot (PE-conjugated anti-human IgM) and SAB (IgG, IgM) assays.
By comparing the mean fluorescence intensity (MFI) of HLA-beads, pre- and post-imlifidase administration, three IgM-related patterns were observed; IgM-specific HLA-SABs with an increased MFI post-imlifidase, IgM-specific HLA-SABs with a decreased MFI post-imlifidase, and IgM-specific HLA-SABs with a marginal MFI difference between the pre- and post-imlifidase administration. These IgM signal patterns were observed despite neither purified IgM nor serum IgM could be cleaved by imlifidase. After removing IgG, the effects observed on anti-HLA IgM was largely eliminated with the biggest differences seen in patients with very high anti-HLA IgG in pre-dose samples.
We demonstrate that imlifidase does not cleave human IgM, including HLA-specific IgM antibodies from highly sensitized subjects. Observed decreases of SAB-HLA IgM signals after imlifidase treatment may result from the cleavage of IgG-IgM complexes which are bound to SAB-HLA. Serum analysis of patients with high levels of anti-HLA IgG will result in a more accurate SAB-HLA IgM reading after IgG depletion.

Antibody-mediated rejection (AMR) of cardiac allografts mediated by anti-HLA Donor Specific Antibodies (DSA) is one of the major barriers to successful transplantation for the treatment of end-stage heart failure. Therapeutic plasma exchange (TPE) is a first-line treatment for pre-transplant desensitization. However, indications for treatment regimens and treatment end-points have not been well established. In this study, we investigated how sera dilutions could guide TPE regimens for effective peri-operative desensitization and early AMR treatment. Our data show that 1:16 dilutions of EDTA-treated sera and 1.5 volume TPE reduce anti-HLA class I and class II antibody levels in the same manner and, therefore, allows to predict which antibodies would respond to peri-operative TPE. We successfully applied this approach to transplanting three highly sensitized cardiac recipients (CPRA 85-93%) with peri-operative desensitization based on a virtual crossmatch performed on 1:16 diluted serum. Furthermore, we have used sera dilutions to guide DSA treatment post-transplant. Although these findings have to be confirmed in a larger prospective study, our data suggest that serum dilutions can serve as a predictive biomarker to guide peri-operative desensitization and post-transplant immunologic management.

Accurate identification of HLA antibodies using the single antigen bead (SAB) assay is critical for assessment of pre/post-transplant immunological risk and successful virtual crossmatching. Unfortunately, high titer HLA antibodies can be missed or underestimated in the SAB assay as a result of interference with the detection of IgG. This so called prozone effect has been attributed to both complement- and IgM-dependent mechanisms and can be minimized with serum dilution or treatment with heat, EDTA, or DTT. In this study we describe the frequency, nature, and degree of prozone in a cohort of highly sensitized patients (cPRA ≥ 95%), in whom accurate detection of HLA antibodies and virtual crossmatching is of paramount importance. Sera were tested by the SAB assay ± EDTA treatment, ±1:10 dilution to identify the prozone effect. The relative contribution of complement vs IgM to prozone was assessed using anti-C3d and anti-IgM reporter antibodies, respectively. We found that prozone was very frequent in highly sensitized patients (80%), especially those with a history of previous transplantation (87%). Class I HLA specificities were more commonly affected than class II and the susceptibility to prozone was locus dependent with HLA-A(31%), -B(29%) and -DQ(26%) being affected more frequently than HLA-DP(17%), -C(16%) and -DR(5%) antigens. Interestingly, the presence of prozone could be predicted by C3d positivity (MFI ≥ 4000; sensitivity = 95.2%, specificity = 97.2%) and the degree of prozone correlated directly with the extent of C3d deposition. The role of IgM was less clear. However, serum dilution studies suggested that IgM may contribute to interference in a small subset of prozone positive specificities. Our study underscores the importance of serum treatment to inhibit complement activation and minimize prozone in the SAB assay, especially in highly sensitized patients.

Calculated panel reactive antibodies (CPRA) is a sensitization measure used to classify and prioritize transplant candidates in different kidney transplant allocation systems. CPRA is based on identification of HLA unacceptable on potential organ donors making a transplant candidate ineligible for transplantation. Here, we present a CPRA online estimator based on HLA allelic and haplotypic frequencies from Portuguese donors. We also compare the values we obtained from our CPRA estimator (Portuguese [PT]-CPRA) against CPRA values obtained from: Eurotransplant virtual PRA calculator (ET-CPRA); Canadian CPRA calculator (Canadian [CN]-CPRA) and Organ Procurement and Transplantation Network CPRA calculator (United States [US]-CPRA). When we analyzed correlations between CPRA values obtained from pairs of calculators, we observed that they are significantly and highly correlated. Bland-Altman plots for the comparison between PT-CPRA calculator against the other calculators, show higher differences between PT and CN than between PT and ET and PT and US. Also, the lowest value for Lin\'s concordance coefficient was obtained for the comparison between PT and CN calculators. CPRA values reliability depend on donors\' pool from which it is calculated and it is crucial for classify correctly highly sensitized patients. A CPRA calculator must use donors\' HLA frequencies similar from those who would be actual organ donors.

Liver-kidney transplantation is a rare procedure in children, with just ten to 30 cases performed annually worldwide. The main indications are autosomal recessive polycystic liver-kidney disease and primary hyperoxaluria. This study aimed to report outcomes of liver-kidney transplantation in a cohort of pediatric patients.
We retrospectively analyzed all pediatric liver-kidney transplantations performed in our center between September 2000 and August 2015. Patient data were obtained by reviewing inpatient and outpatient medical records and our transplant database.
A total of 14 liver-kidney transplants were performed during the study period, with a median patient age and weight at transplant of 144.4 months (131.0-147.7) and 27.3 kg (12.0-45.1), respectively. The indications for liver-kidney transplants were autosomal recessive polycystic liver-kidney disease (8/14), primary hyperoxaluria -1 (5/14), and idiopathic portal hypertension with end-stage renal disease (1/14). Median time on waiting list was 8.5 months (5.7-17.3). All but two liver-kidney transplants were performed simultaneously. Patients with primary hyperoxaluria-1 tended to present a delayed recovery of renal function compared with patients transplanted for other indications (62.5 vs 6.5 days, respectively, P 0.076). Patients with liver-kidney transplants tended to present a lower risk of acute kidney rejection than patients transplanted with an isolated kidney transplant (7.2% vs 32.7%, respectively; P < 0.07). Patient and graft survival at 1, 3, and 5 years were 100%, 91.7%, 91.7%, and 91.7%, 83.3%, 83.3%, respectively. No other grafts were lost.
Long-term results of liver-kidney transplants in children are encouraging, being comparable with those obtained in isolated liver transplantation.

OBJECTIVE: The calculated panel reactive of antibodies (cPRAs) necessary for kidney donor-pair exchange and highly sensitized programs are estimated using different panel reactive antibody (PRA) calculators based on big enough samples in Eurotransplant (EUTR), United Network for Organ Sharing (UNOS), and Canadian Transplant Registry (CTR) websites. However, those calculators can vary depending on the ethnic they are applied. Here, we develop a PRA calculator used in the Spanish Program of Transplant Access for Highly Sensitized patients (PATHI) and validate it with EUTR, UNOS, and CTR calculators.
METHODS: The anti-human leukocyte antigen (HLA) antibody profile of 42 sensitized patients on waiting list was defined, and cPRA was calculated with different PRA calculators.
RESULTS: Despite different allelic frequencies derived from population differences in donor panel from each calculator, no differences in cPRA between the four calculators were observed. The PATHI calculator includes anti-DQA1 antibody profiles in cPRA calculation; however, no improvement in total cPRA calculation of highly sensitized patients was demonstrated.
CONCLUSIONS: The PATHI calculator provides cPRA results comparable with those from EUTR, UNOS, and CTR calculators and serves as a tool to develop valid calculators in geographical and ethnic areas different from Europe, USA, and Canada.