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Allosensitization prolongs waiting time and increases waitlist mortality in children awaiting heart transplantation. We present a case of 12 year-old female, sensitized by prior heart transplant against DQ7, 8, 9 and DR53, who required re-transplantation. Multiple offers were unacceptable because of high titer ( ⩾ 1:16) and C1q positive DSA. She underwent desensitization therapy consisting of rituximab and 9 doses of bortezomib with concomitant plasmapheresis. HLA-Abs were monitored by serial dilutions and C1q assay. Early into her third cycle of BTZ a DQ7/DQA1 ∗ 05-positive, DR53-negative donor was accepted based on predicted weak flow virtual CXM. The impact of desensitization on Class II HLA-Ab and CXM is summarized in Fig. 1. DQ7 DSA became C1q negative and dropped to 10,000 MFI at 1:16) at 1 month and >4000 MFI at 1:16 with and C1q positive thereafter. Flow and CDC CXM were performed retrospectively on pre-transplant sera. As expected the flow CXM was weak B positive but unexpectedly the CDC-CXM was strong B positive. The B cell reactivity seen by CDC did not correlate with the level of DSA ( Download high-res image (216KB) Download full-size image

This case report summarizes the spectrum of anti–human leukocyte antigen (HLA) antibody reactivity determined by single-allele Luminex immunoglobulin G and C1q binding assays before transplant, during an episode of antibody-mediated rejection (AMR), and following treatment in a sensitized pediatric heart transplant (Tx) recipient. We were able to discriminate between complement- and non-complement-binding epitope-specific antibodies present against a single donor antigen (HLA-A2) during the progression of AMR and its resolution. Our findings illustrate the usefulness of determining antibody specificities against epitopes using various Luminex-based assays.

Failure to develop antibodies to nonself A and B blood group antigens is well described after infant ABO-incompatible heart transplantation and suggests that exposure to incompatible ABO antigens early in life may lead to tolerance rather than immunogenicity. If this finding is also true following ABO-incompatible cryopreserved homograft implantation, then such patients who require transplantation may be able to accept certain ABO-incompatible organs. In this study, we measured anti-A and -B antibody titers (isohemagglutinins) and allosensitization to human leukocyte antigens (HLA) in 21 patients after homograft placement (12 of whom were

Aim Antibody mediated rejection (AMR) remains problematic in solid organ transplantation. New treatment modalities are continually being considered, especially those interfering with the complement cascade. However, assays predicting the efficacy of AMR treatments are poor at best, despite the sensitivity of single antigen bead (SAB) testing and new SAB functional assays. Recently, the therapeutic use of C1 esterase inhibitor (C1-INH) has been proposed to prevent/minimize complement-mediated allograft damage by donor specific HLA antibodies by blocking initial complement activation. Here, we test a novel modification of the Luminex C1q Screen® SAB assay using C1-INH, in vitro, to predict the susceptibility of HLA antibodies to C1-INH inhibition. Methods Highly sensitized heart (n=6) and small bowel (n=3) patient sera were tested for HLA antibody by complement dependent cytotoxicity (CDC) T and B cell crossmatch(XM), T cell cytotoxic PRA and by Luminex C1q Screen®. C1-INH (Berinert®, CSLBehring) was added to assays (0.025U/μl–0.4U/μl) and inhibition of cell death or MFI was measured. As a control, human α1-proteinase inhibitor (Zemaira®) was substituted for C1-INH to demonstrate specificity. Results C1-INH added to sera inhibited CDC-XM positivity in a dose-dependent manner (0.4U/μl to 0.025U/μl). Similarly, the MFI of C1qScreen positive HLA antibodies was reduced >80% when 0.4U/μl C1-INH was added to the assay. High-titer (>1:16) C1q positive HLA specificities were less affected by C1-INH compared to lower-titer ( Conclusions The in vitro efficacy of C1-INH to inhibit complement activating HLA antibodies in both CDC and C1q Screen® was dependent on the titer of C1q reactive IgG antibodies. These assays may potentially define HLA antibodies susceptible to C1-INH therapy. Lunz: CSL Behring: Grant Research. Vensak: CSL Behring: Employee. Berger: CSL Behring: Employee. Zeevi: CSL Behring: Grant Research.

Aim Sensitized heart transplant candidates are evaluated for donor specific HLA antibody (DSA) by Luminex single antigen bead (SAB) testing to assess donor suitability and predict a positive complement dependent cytotoxicity (CDC) crossmatch (XM) by virtual XM (VXM). However, SAB testing imperfectly correlates with VXM, and antibody thresholds for XM prediction are center-specific. A novel Luminex SAB-based assay detecting C1q-binding HLA antibodies adds functional data to SAB testing, but the relationship between SAB strength and C1q positivity is unclear. In this retrospective study, we examine the relationship between HLA antibody titer and complement-binding ability in cardiac allograft recipients and correlate C1q DSA to antibody-mediated rejection (AMR). Methods We identified 15 sensitized pediatric and adult heart recipients with a calculated panel reactive antibody >50%. Sera were tested by conventional SAB antibody testing using undiluted or 1:16 diluted sera and were compared to C1q results. Pre- and post-transplant DSA by both methods were correlated with XM and early AMR. Results In 9 samples, individual Class I antibodies measured by SAB testing in undiluted sera correlated poorly with C1q reactivity (R 2 = 0.07). In contrast, using sera diluted 1:16, conventional SAB results correlated better with C1q testing ( R 2 = 0.73). Receiver operator characteristic curve analysis also showed superior area under the curve (AUC) for predicting C1q positive results in sera diluted 1:16 than in undiluted sera (AUC = 0.99 vs 0.82). Low MFI by conventional SAB testing in alleles with high C1q MFI increased upon dilution or heat-treating sera and correlated with C1q results. Clinically, in 13 recipients C1q DSA predicted CDC-XM positivity and early AMR (positive predictive value (PV) 87.5%; negative PV 100%). Conclusions In conclusion, SAB titer correlated with and predicted C1q reactivity. The risk for positive crossmatch and early AMR in sensitized heart recipients strongly correlated with C1q binding DSA.