Islet transplantation outcomes in type 1 diabetes and transplantation of HLA-DQ8/DR4: results of a single-centre retrospective cohort in Canada.

Background: In solid organ transplantation, HLA matching between donor and recipient is associated with superior outcomes. In islet transplantation, an intervention for Type 1 diabetes, HLA matching between donor and recipient is not performed as part of allocation. Susceptibility to Type 1 diabetes is associated with the presence of certain HLA types. This study was conducted to determine the impact of these susceptibility antigens on islet allograft survival. Methods: This is a single-centre retrospective cohort study. This cohort of transplant recipients (n = 268) received islets from 661 donor pancreases between March 11th, 1999 and August 29th, 2018 at the University of Alberta Hospital (Edmonton, AB, Canada). The frequency of the Type 1 diabetes susceptibility HLA antigens (HLA-A24, -B39, -DQ8, -DQ2 and-DQ2-DQA1∗05) in recipients and donors were determined. Recipient and donor HLA antigens were examined in relation to time to first C-peptide negative status/graft failure or last observation point. Taking into account multiple transplants per patient, we fitted a Gaussian frailty survival analysis model with baseline hazard function stratified by transplant number, adjusted for cumulative islet dose and other confounders. Findings: Across all transplants recipients of donors positive for HLA-DQ8 had significantly better graft survival (adjusted HRs 0.33 95% CI 0.17-0.66; p = 0.002). At first transplant only, donors positive for HLA-DQ2-DQA1∗05 had inferior graft survival (adjusted HR 1.96 95% CI 1.10-3.46); p = 0.02), although this was not significant in the frailty analysis taking multiple transplants into account (adjusted HR 1.46 95% CI 0.77-2.78; p = 0.25). Other HLA antigens were not associated with graft survival after adjustment for confounders. Interpretation: Our findings suggest islet transplantation from HLA-DQ8 donors is associated with superior graft outcomes. A donor positive for HLA-DQ2-DQA1∗05 at first transplant was associated with inferior graft survival but not when taking into account multiple transplants per recipient. The relevance of HLA-antigens on organ allocation needs further evaluation and inclusion in islet transplant registries and additional observational and interventional studies to evaluate the role of HLA-DQ8 in islet graft survival are required. Funding: None.

Updates in Kidney Transplantation From the 2022 Banff-Canadian Society of Transplantation Joint Meeting: Conference Report.

Purpose of the Conference: The 2022 Banff-Canadian Society of Transplantation Meeting in Banff, Alberta, brought together transplant professionals to review new developments across various aspects of solid organ transplantation (SOT) in Canada. Sources of Information: Presentations included consensus recommendations from expert-led forums; experiences with new procedures and legislation; reports from public health data repositories; original clinical and laboratory research; and industry updates regarding novel technologies. Speakers referenced articles and reports published in peer-reviewed journals and online, and unpublished data and preliminary findings. Methods: All authors attended presentations in-person or virtually. Recordings of select presentations were available for later review. Summaries emphasize concepts indicated by speakers as new and clinically relevant. Key Findings: The COVID-19 pandemic disproportionately affected solid organ transplant recipients (SOTRs), who experience worse outcomes of COVID-19 infection than the general population. Vaccinations demonstrate an attenuated immunological response in SOTRs yet provide meaningful protection. Monoclonal antibodies are effective for both passive immunization and treatment of COVID-19 in SOTRs. Infection control protocols have driven the development of virtual methods for clinical research, such as using home blood draws and virtual follow-up to evaluate vaccine efficacy in SOTRs; and patient care delivery, such as employing telerehabilitation post transplant. Access to living kidney donation is limited by various disincentives experienced by potential donors, which may be overcome by more efficient evaluations including a One-Day Living Kidney Donor Assessment Clinic. The International Donation and Transplantation Legislative and Policy Forum provided a means of establishing consensus guidance for organ donation and transplantation (ODT) program policy to standardize delivery across jurisdictions. The implementation of a deemed consent model for organ and tissue donation in Nova Scotia may provide insight as to whether this model indeed improves access to organs. Canada's Indigenous population experiences unique barriers to transplantation, prompting efforts for more inclusive ODT policy-making. The Pan-Canadian ODT Data and Performance Reporting System Project has defined performance quality indicators, of which iTransplant and other point-of-care software solutions may facilitate collection; however, these endeavors ultimately require information technology infrastructure that exceeds the capabilities of the existing Canadian Organ Replacement Register and Canadian Transplant Registry. Pig-to-human xenotransplantation requires genetic modification of pigs and xenoantibody testing in recipients but may yet prove viable. Serum cell-free DNA, urine biomarkers, and genetic markers offer an alternative to routine biopsy for identifying subclinical rejection. Modified perfusion temperatures and perfusion solutions with hydrogen sulfide donor compounds may improve organ preservation. Molecular compatibility tools provide another means of improving SOTR outcomes, and the Genome Canada Transplant Consortium has been examining important considerations of their implementation. Limitations: We were unable to capture all presentations and topics at the meeting due to the sizable quantity and variety. Topics ultimately excluded from this summary include those in pathology including Banff Classification updates; those unique to extra-renal SOT; as well as numerous abstract and poster presentations, allied health provider forums, and business meetings. A portion of the material was presented by speakers prior to peer-review or publication. Implications: The various conference presentations summarized in this report identify methods by which individual clinicians and provincial ODT programs may improve access, delivery, and quality of SOT care in Canada, while additionally identifying gaps in the literature that investigators are encouraged to pursue.

Objectifs de la conférence: En 2022, le congrès annuel de la Société canadienne de transplantation qui s'est tenu à Banff (Alberta) a réuni des professionnels de la transplantation qui se sont penchés sur les nouveaux développements dans divers aspects de la transplantation d'organes solides (TOS) au Canada. Sources: Les présentations portaient notamment sur : les recommandations consensuelles issues de forums dirigés par des experts; l'expérience avec les nouvelles procédures et lois; des rapports provenant de dépôts de données de santé publique; des recherches cliniques et des recherches de laboratoire originales; et les mises à jour du secteur sur les nouvelles technologies. Les intervenants ont fait référence à des articles et rapports publiés en ligne et dans des revues évaluées par les pairs, ainsi qu'à des données non publiées et des conclusions préliminaires. Méthodologie: Tous les auteurs ont assisté aux présentations en personne ou virtuellement. Les enregistrements de certaines présentations étaient disponibles pour visionnement ultérieur. Les résumés mettent l'accent sur les concepts jugés comme nouveaux et cliniquement pertinents par les intervenants. Principaux résultats: La pandémie de COVID-19 a affecté les receveurs d'une transplantation d'organe solide (RTOS) de manière disproportionnée; ces derniers ayant suivi une évolution plus défavorable que la population générale à la suite d'une infection à la COVID-19. La vaccination, bien qu'elle offre une protection significative, montre une réponse immunologique plus faible chez les RTOS. Les anticorps monoclonaux sont efficaces à la fois pour l'immunization passive et le traitement de la COVID-19 chez les RTOS. Les protocoles de contrôle des infections ont mené au développement de méthodes virtuelles pour la recherche clinique (p. ex. prélèvements sanguins à domicile, suivi virtuel pour évaluer l'efficacité du vaccin chez les RTOS) et la prestation de soins aux patients (p. ex. rééducation à distance) après la transplantation. L'accès au don de rein vivant est limité par divers facteurs dissuasifs pour les donneurs potentiels, mais ces obstacles peuvent être surmontés par des évaluations plus efficaces, notamment par une clinique d'un jour pour évaluer la candidature des donneurs vivants de rein. Le Forum législatif et politique international sur le don et la transplantation a fourni un moyen d'établir des lignes directrices consensuelles pour la politique du program de dons d'organes et de transplantation (DOT), dans l'objectif de normaliser la prestation d'une juridiction à l'autre. La mise en œuvre en Nouvelle-Écosse du consentement présumé pour le don d'organes et de tissus pourrait aider à déterminer si un tel modèle améliore effectivement l'accès aux organes. Les populations autochtones du Canada sont confrontées à des obstacles uniques en matière de transplantation, ce qui encourage les efforts visant l'élaboration de politiques plus inclusives en matière de DOT. Le Projet de système pancanadien de données et de mesure de la performance pour les DOT a défini des indicateurs de performance, dont iTransplant et d'autres solutions logicielles pour les points de soins, qui peuvent faciliter la collecte des données; ces derniers nécessitent toutefois une infrastructure informatique qui dépasse les capacités du Registre canadien des insuffisances et des transplantations d'organes et du Registre canadien de transplantation. La xénogreffe de porc à humain requiert une modification génétique des porcs et le dépistage de xénoanticorps chez les receveurs, mais elle peut encore s'avérer viable. L'ADN acellulaire sérique, les biomarqueurs urinaires et les marqueurs génétiques offrent une alternative à la biopsie de routine pour identifier les rejets subcliniques. Des températures de perfusion modifiées et des solutions de perfusion contenant des composés générateurs de sulfure d'hydrogène peuvent améliorer la conservation des organes. Les outils de compatibilité moléculaire offrent un autre moyen d'améliorer les résultats des RTOS, et le Genome Canada Transplant Consortium a examiné les aspects importants à prendre en considération pour leur mise en œuvre. Limites: Nous n'avons pas été en mesure d'assister à toutes les présentations en raison du grand nombre et de la grande diversité des sujets abordés. Les sujets exclus de ce résumé incluent la pathologie, notamment les mises à jour de la classification Banff; les sujets propres à la TOS extrarénale; ainsi que de nombreux résumés et présentations d'affiches, forums de prestataires de soins de santé alliés et réunions d'affaires. Une partie du matériel présenté l'a été avant l'examen par les pairs ou la publication. Conclusion: Les présentations du congrès qui sont résumées dans le présent rapport identifient les méthodes que les programs provinciaux de DOT et les cliniciens pourraient employer pour améliorer l'accès, la prestation et la qualité des soins en TOS au Canada, et soulignent des lacunes dans la littérature que les chercheurs sont encouragés à creuser.

Prospective examination of HLA sensitization after VAD implantation in children and adults.

BACKGROUND: Ventricular assist devices (VADs) have improved survival to heart transplantation (HTx). However, VADs have been associated with development of antibodies against human leukocyte antigen (HLA-Ab) which may limit the donor pool and decrease survival post-HTx. Since HLA-Ab development after VAD insertion is poorly understood, the purpose of this prospective single-center study was to quantify the incidence of and evaluate risk factors for HLA-Ab development across the age spectrum following VAD implantation. METHODS: Adult and pediatric patients undergoing VAD placement as bridge to transplant or transplant candidacy between 5/2016 and 7/2020 were enrolled. HLA-Ab were assessed pre-VAD and at 1-, 3-, and 12-months post-implant. Factors associated with HLA-Ab development post-VAD implant were explored using univariate and multivariate logistic regression. RESULTS: 15/41 (37%) adults and 7/17 (41%) children developed new HLA-Ab post-VAD. The majority of patients (19/22) developed HLA-Ab within two months of implant. New class I HLA-Ab were more common (87% adult, 86% pediatric). Prior pregnancy was strongly associated with HLA-Ab development in adults post-VAD (HR 16.7, 95% CI 1.8-158, p = 0.01). Of the patients who developed new HLA-Ab post-VAD, in 45% (10/22) the HLA-Ab resolved while in 55% (12/22) the HLA-Ab persisted. CONCLUSION: More than one-third of adult and pediatric VAD patients developed new HLA-Ab early after VAD implant with the majority having class I antibodies. Prior pregnancy was strongly associated with post-VAD HLA-Ab development. Further studies are needed to predict regression or persistence of HLA-Ab developed post-VAD, to understand modulation of individuals' immune responses to sensitizing events, and to determine whether transiently detected HLA-Ab post-VAD recur and have long-term clinical impact post-heart transplantation.

Successful ABO incompatible heart transplantation after desensitization therapy in an older child.

BACKGROUND: ABO-incompatible heart transplantation (HTx) has become a standard procedure for children below 2 years of age due to an immunologically immature immune system and associated low isohemagglutinin titers. METHODS: We report a case of an ABO-incompatible HTx (recipient blood group O, donor blood group A) at the age of 5 years and 11 months with a fully matured immune system and previously high isohemagglutinin titers that diminished as a result of human leucocyte antigen (HLA) desensitization therapy with rituximab and immunoglobulins. RESULTS: The anti-A titer at the time of HTx was 1:16 with post-transplant isoagglutinin titers never exceeding 1:4 without any signs of rejection with now 3 years of post-HTx follow-up. CONCLUSIONS: ABO isohemagglutinin titers should be routinely assessed in children undergoing desensitization therapy since ABOi transplantation can be considered in selected cases to expand the donor pool with the option of crossing the ABO barrier to find a better-matched allograft.

Pancreas and Islet Transplantation: Comparative Outcome Analysis of a Single-centre Cohort Over 20-years.

OBJECTIVE: To provide the largest single-center analysis of islet (ITx) and pancreas (PTx) transplantation. SUMMARY BACKGROUND DATA: Studies describing long-term outcomes with ITx and PTx are scarce. METHODS: We included adults undergoing ITx (n=266) and PTx (n=146) at the University of Alberta from January 1999 to October 2019. Outcomes include patient and graft survival, insulin independence, glycemic control, procedure-related complications, and hospital readmissions. Data are presented as medians (interquartile ranges, IQR) and absolute numbers (percentages, %) and compared using Mann-Whitney and χ2 tests. Kaplan-Meier estimates, Cox proportional hazard models and mixed main effects models were implemented. RESULTS: Crude mortality was 9.4% and 14.4% after ITx and PTx, respectively ( P= 0.141). Sex-adjusted and age-adjusted hazard-ratio for mortality was 2.08 (95% CI, 1.04-4.17, P= 0.038) for PTx versus ITx. Insulin independence occurred in 78.6% and 92.5% in ITx and PTx recipients, respectively ( P= 0.0003), while the total duration of insulin independence was 2.1 (IQR 0.8-4.6) and 6.7 (IQR 2.9-12.4) year for ITx and PTx, respectively ( P= 2.2×10 -22 ). Graft failure ensued in 34.2% and 19.9% after ITx and PTx, respectively ( P =0.002). Glycemic control improved for up to 20-years post-transplant, particularly for PTx recipients (group, P= 7.4×10 -7 , time, P =4.8×10 -6 , group*time, P= 1.2×10 -7 ). Procedure-related complications and hospital readmissions were higher after PTx ( P =2.5×10 -32 and P= 6.4×10 -112 , respectively). CONCLUSIONS: PTx shows higher sex-adjusted and age-adjusted mortality, procedure-related complications and readmissions compared with ITx. Conversely, insulin independence, graft survival and glycemic control are better with PTx. This study provides data to balance risks and benefits with ITx and PTx, which could improve shared decision-making.

Evaluating the Potential for ABO-incompatible Islet Transplantation: Expression of ABH Antigens on Human Pancreata, Isolated Islets, and Embryonic Stem Cell-derived Islets.

BACKGROUND: ABO-incompatible transplantation has improved accessibility of kidney, heart, and liver transplantation. Pancreatic islet transplantation continues to be ABO-matched, yet ABH antigen expression within isolated human islets or novel human embryonic stem cell (hESC)-derived islets remain uncharacterized. METHODS: We evaluated ABH glycans within human pancreata, isolated islets, hESC-derived pancreatic progenitors, and the ensuing in vivo mature islets following kidney subcapsular transplantation in rats. Analyses include fluorescence immunohistochemistry and single-cell analysis using flow cytometry. RESULTS: Within the pancreas, endocrine and ductal cells do not express ABH antigens. Conversely, pancreatic acinar tissues strongly express these antigens. Acinar tissues are present in a substantial portion of cells within islet preparations obtained for clinical transplantation. The hESC-derived pancreatic progenitors and their ensuing in vivo-matured islet-like clusters do not express ABH antigens. CONCLUSIONS: Clinical pancreatic islet transplantation should remain ABO-matched because of contaminant acinar tissue within islet preparations that express ABH glycans. Alternatively, hESC-derived pancreatic progenitors and the resulting in vivo-matured hESC-derived islets do not express ABH antigens. These findings introduce the potential for ABO-incompatible cell replacement treatment and offer evidence to support scalability of hESC-derived cell therapies in type 1 diabetes.

ABO blood group is involved in the quality of the specific immune response anti-SARS-CoV-2.

Since December 2019, the coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has spread throughout the world. To eradicate it, it is crucial to acquire a strong and long-lasting anti-SARS-CoV-2 immunity, by either natural infection or vaccination. We collected blood samples 12-305 days after positive polymerase chain reactions (PCRs) from 35 recovered individuals infected by SARS-CoV-2. Peripheral blood mononuclear cells were stimulated with SARS-CoV-2-derived peptide pools, such as the spike (S), nucleocapsid (N) and membrane (M) proteins, and we quantified anti-S immunoglobulins in plasma. After 10 months post-infection, we observed a sustained SARS-CoV-2-specific CD4+ T-cell response directed against M-protein, but responses against S- or N-proteins were lost over time. Besides, we demonstrated that O-group individuals presented significantly lower frequencies of specific CD4+ T-cell responses against Pep-M than non O-group individuals. The non O-group subjects also needed longer to clear the virus, and they lost cellular immune responses over time, compared to the O-group individuals, who showed a persistent specific immune response against SARS-CoV-2. Therefore, the S-specific immune response was lost over time, and individual factors might determine the sustainability of the body's defenses, which must be considered in the future design of vaccines to achieve continuous anti-SARS-CoV-2 immunity.

Human Leukocyte Antigen Antibody Sampling in Ventricular Assist Device Recipients: Are We Talking?

BACKGROUND: Ventricular assist devices (VADs) are commonly used as a bridge to transplantation but may yield HLA sensitization. We evaluated the prevalence of HLA antibody (Ab) sampling pre- and post-VAD placement in pediatric and adult patients and notification of VAD status to the HLA laboratory. METHODS: All pediatric and adult patients who received a first-time VAD between 2005 and 2013 were included in this single-center retrospective review. Data were collected from the University of Alberta Hospital histocompatibility laboratory's information system and a local VAD database. RESULTS: In total, 106 patients were included (40 pediatric, median 3.0 years [interquartile range, 0.3-10.7]; 66 adult, 55.0 years [46.8-61.2]). HLA Ab sampling within 1-month pre-VAD occurred in 70% of pediatric and 79% of adult recipients (P = .215). Testing within 1 month of VAD placement occurred in 89% of pediatric and 67% of adult recipients (P = .012). For those with HLA Ab sampling within 30 days postimplant, notification to the HLA laboratory of VAD status occurred in 19 of 27 (70%) pediatric and 24 of 33 (73%) adult patients (P = .533). Of patients transplanted post VAD with HLA Ab samples collected, 12 of 28 (43%) and 13 of 38 (34%) adult recipients did not have notification of VAD status to the HLA laboratory (P = .322). CONCLUSIONS: There were inconsistencies in HLA Ab sampling and communication to the HLA laboratory surrounding VAD placement. Standardization of both HLA Ab assessment frequency after VAD implantation and communication regarding changes in clinical status and the occurrence of key sensitizing events such as VAD placement are imperative as patients await transplantation.

Characterization of ABH-subtype donor-specific antibodies in ABO-A-incompatible kidney transplantation.

ABO-incompatible (ABOi) transplantation requires preemptive antibody reduction; however, the relationship between antibody-mediated rejection (AMR) and ABO-antibodies, quantified by hemagglutination (HA), is inconsistent, possibly reflecting variable graft resistance to AMR or HA assay limitations. Using an ABH-glycan microarray, we quantified ABO-A antigen-subtype (A-subtype)-specific IgM and IgG in 53 ABO-O recipients of ABO-A kidneys, before and after antibody removal (therapeutic plasma exchange [TPE] or ABO-A-trisaccharide immunoadsorption [IA]) and 1-year posttransplant. IgM binding to all A-subtypes correlated highly (R2  ≥ .90) and A-subtype antibody specificities was reduced equally by IA versus TPE. IgG binding to the A-subtypes (II-IV) expressed in kidney correlated poorly (.27 ≤ R2  ≤ .69). Reduction of IgG specific to A-subtype-II was equivalent for IA and TPE, whereas IgG specific to A-subtypes-III/IV was not as greatly reduced by IA (p < .005). One-year posttransplant, IgG specific to A-II remained the most reduced antibody. Immunostaining revealed only A-II on vascular endothelium but A-subtypes II-III/IV on tubular epithelium. These results show that ABO-A-trisaccharide is sufficient for IgM binding to all A-subtypes; this is true for IgG binding to A-II, but not subtypes-III/IV, which exhibits varying degrees of specificity. We identify A-II as the major, but importantly not the sole, antigen relevant to treatment and immune modulation in adult ABO-A-incompatible kidney transplantation.

New hepatitis C virus infection, re-infection and associated risk behaviour in male Irish prisoners: a cohort study, 2019.

BACKGROUND: Prisoners are recognised as a high-risk population and prisons as high-risk locations for the transmission of hepatitis c virus (HCV) infection. Injecting drug use (IDU) is the main driver of HCV infection in prisoners and harm reduction services are often suboptimal in prison settings. HCV prevalence and incident data in prisoners is incomplete which impacts the public health opportunity that incarceration provides in identifying, treating and preventing HCV infection. The aim of this study is to identify new HCV infection and associated risk factors in an Irish male prison. METHODS: We conducted a follow up (18-month) cohort study on prisoners who had previously tested negative, self-cleared or had been successfully treated for HCV infection. We conducted the study in a male medium security prison located in Dublin Ireland (Mountjoy Prison) using HCV serology, a review of medical records and a researcher-administered questionnaire. RESULTS: 99 prisoners with a mean age of 33.2 yrs. participated in the study and 82(82.8%) completed a research-administered questionnaire. Over half (51%) had a history of drug use from a young age (14.8 yrs.), 49.9% a history of heroin use and 39% a history of IDU. The prevalence of HIV and hepatitis B virus core antibody was 3% and HCV antibody was 22.2%. No new HCV infections were identified in those who had never been infected (n = 77), had self-cleared (n = 9) or achieved sustained virological response (n = 12). Small numbers of prisoners continued to engage in risk-behaviour including, IDU both in the prison (n = 2) and the community (n = 3), sharing syringes (n = 1) and drug taking paraphernalia (n = 6) and receiving non-sterile tattoos (n = 3). CONCLUSION: Despite the high numbers of Irish prisoners with a history of IDU and HCV infection, new HCV infection is low or non-existent in this population. Small numbers of prisoners continue to engage in risk behaviour and larger studies are required to further understand HCV transmission in this cohort in an Irish and international context.

A single nucleotide polymorphism genetic risk score to aid diagnosis of coeliac disease: a pilot study in clinical care.

BACKGROUND: Single nucleotide polymorphism-based genetic risk scores (GRS) model genetic risk as a continuum and can discriminate coeliac disease but have not been validated in clinic. Human leukocyte antigen (HLA) DQ gene testing is available in clinic but does not include non-HLA attributed risk and is limited by discrete risk stratification. AIMS: To accurately characterise both HLA and non-HLA coeliac disease genetic risk as a single nucleotide polymorphism-based GRS and evaluate diagnostic utility. METHODS: We developed a 42 single nucleotide polymorphism coeliac disease GRS from a European case-control study (12 041 cases vs 12 228 controls) using HLA-DQ imputation and published genome-wide association studies. We validated the GRS in UK Biobank (1237 cases) and developed direct genotyping assays. We tested the coeliac disease GRS in a pilot clinical cohort of 128 children presenting with suspected coeliac disease. RESULTS: The GRS was more discriminative of coeliac disease than HLA-DQ stratification in UK Biobank (receiver operating characteristic area under the curve [ROC-AUC] = 0.88 [95% CIs: 0.87-0.89] vs 0.82 [95% CIs: 0.80-0.83]). We demonstrated similar discrimination in the pilot clinical cohort (114 cases vs 40 controls, ROC-AUC = 0.84 [95% CIs: 0.76-0.91]). As a rule-out test, no children with coeliac disease in the clinical cohort had a GRS below 38th population centile. CONCLUSIONS: A single nucleotide polymorphism-based GRS may offer more effective and cost-efficient testing of coeliac disease genetic risk in comparison to HLA-DQ stratification. As a comparatively inexpensive test it could facilitate non-invasive coeliac disease diagnosis but needs detailed assessment in the context of other diagnostic tests and against current diagnostic algorithms.

HLA Alloimmunization Following Ventricular Assist Device Support Across the Age Spectrum.

BACKGROUND: Ventricular assist device (VAD) therapy has become an important tool for end-stage heart failure. VAD therapy has increased survival but is associated with complications including the development of human leukocyte antigen (HLA) antibodies. We sought to determine the incidence of HLA antibody development post-VAD insertion, across the age spectrum, in patients receiving leukocyte-reduced blood products, with standardized HLA antibody detection methods and to investigate factors associated with antibody development. METHODS: This was a retrospective analysis of all patients who underwent durable VAD placement between 2005 and 2014. Inclusion criteria included availability of pre- and post-VAD HLA antibody results. Associations between HLA antibody development in the first-year postimplant and patient factors were explored. RESULTS: Thirty-nine adult and 25 pediatric patients made up the study cohort. Following implant, 31% and 8% of patients developed new class I and class II antibodies. The proportion of newly sensitized patients was similar in adult and pediatric patients. The class I HLA panel reactive antibody only significantly increased in adults. Pre-VAD sensitization, age, sex (pediatrics), and transfusion were not associated with the development of HLA antibodies. CONCLUSIONS: In a cohort of VAD patients receiving leukocyte-reduced blood products and standardized HLA antibody testing, roughly one-third developed new class I antibodies in the first-year postimplant. Adults showed significantly increased class I panel reactive antibody following VAD support. No patient-related factors were associated with HLA antibody development. Larger prospective studies are required to validate these findings and determine the clinical impact of these antibodies following VAD insertion.

A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies.

The analysis and validation of flow cytometry-based biomarkers in clinical studies are limited by the lack of standardized protocols that are reproducible across multiple centers and suitable for use with either unfractionated blood or cryopreserved PBMCs. Here we report the development of a platform that standardizes a set of flow cytometry panels across multiple centers, with high reproducibility in blood or PBMCs from either healthy subjects or patients 100 days after hematopoietic stem cell transplantation. Inter-center comparisons of replicate samples showed low variation, with interindividual variation exceeding inter-center variation for most populations (coefficients of variability <20% and interclass correlation coefficients >0.75). Exceptions included low-abundance populations defined by markers with indistinct expression boundaries (e.g., plasmablasts, monocyte subsets) or populations defined by markers sensitive to cryopreservation, such as CD62L and CD45RA. Automated gating pipelines were developed and validated on an independent data set, revealing high Spearman's correlations (rs >0.9) with manual analyses. This workflow, which includes pre-formatted antibody cocktails, standardized protocols for acquisition, and validated automated analysis pipelines, can be readily implemented in multicenter clinical trials. This approach facilitates the collection of robust immune phenotyping data and comparison of data from independent studies.

Concordance and discordance in anti-HLA antibody testing.

BACKGROUND: Correct identification of the specificity of antibodies directed against HLA using single antigen Luminex beads (SALB) is essential in current HLA laboratory practice for transplantation. The aim of this study was to investigate the magnitude of concordance and discordance among laboratories in testing for anti-HLA antibodies using SALB. METHOD: 35 sera were distributed by the ASHI Proficiency Testing Program to HLA laboratories worldwide. We analyzed 4335 test results submitted between April 2010 and April 2013 by participating laboratories. RESULTS: SALB was used by approximately 94% of the participating laboratories, yet concordant assignment of antibody specificity was imperfect. For each serum, the assignment of an average of 10 antibody specificities was discordant. Disagreement was observed for antibodies directed against common as well as uncommon antigens. The assignment of an average of 15 antibody specificities in each "positive" serum appeared to be influenced by vendor-dependent causes. Inter-vendor concordance was lower than intra-vendor concordance, indicating that vendor dependent factors may be a central cause for disagreement. CONCLUSIONS: Our study illustrates the prevalence of concordance and discordance, also affected by unpremeditated causes, in reporting SALB antibody results. Insufficient concordance and standardization in antibody testing may have practical implications for organ allocation and organ sharing programs.

Glutaraldehyde treatment of allograft tissue decreases allosensitization after the Norwood procedure.

OBJECTIVE: Cryopreserved allograft tissue used in the Norwood procedure for infants with hypoplastic left heart syndrome causes a marked immunologic sensitization that may complicate future heart transplantation. Treatment of the allograft tissue before implantation may prevent this sensitization. The purpose of this study was to assess the anti-human leukocyte antigen antibody response to glutaraldehyde-treated allograft tissue used in the repair of hypoplastic left heart syndrome. METHODS: Since June 2005, the University of Alberta has subjected allograft vascular tissue used in the Norwood procedure to glutaraldehyde treatment. An observational study was designed to assess whether glutaraldehyde treatment of the allograft tissue affected subsequent panel reactive antibody after patch implantation. Panel reactive antibodies for class I (human leukocyte antigen-A, B, C) and class II (human leukocyte antigen-DR, DQ) antibodies were measured 4 months postoperatively using flow cytometry. RESULTS: Fourteen patients underwent a Norwood procedure using glutaraldehyde-treated allograft tissue. Historical controls consisted of 12 patients who underwent a Norwood procedure using untreated allograft tissue. At 4 months, infants who had received glutaraldehyde-treated allograft tissue had lower class I panel reactive antibody (7.3% +/- 17.4% [median, 0%] vs 61.9% [median, 73%] +/- 39.9%; P = .0005) and class II panel reactive antibody (6.1% [median, 0%] +/- 22.7% vs 49.3% [median, 63%] +/- 41.9%, P = .001) compared with the historical controls. CONCLUSION: Intraoperative glutaraldehyde treatment of allograft tissue used in hypoplastic left heart syndrome repair prevents the profound immunologic sensitization that occurs in the majority of infants undergoing surgical palliation. In patients requiring subsequent heart transplantation, this decreases the risk of antibody-mediated rejection and increases the likelihood of finding a suitable donor, thus improving access to transplantation.

Failure of prophylactic intravenous immunoglobulin to prevent sensitization to cryopreserved allograft tissue used in congenital cardiac surgery.

OBJECTIVE: Cryopreserved allograft tissue used in the Norwood procedure for infants with hypoplastic left heart syndrome causes profound immunologic sensitization, which may complicate future transplantation. Intravenous immunoglobulin has been shown to reduce sensitization after it has developed, allowing successful transplantation. The purpose of this pilot trial was to determine whether intravenous immunoglobulin given before and after the procedure could prevent sensitization to cryopreserved allograft patches used in the initial repair of hypoplastic left heart syndrome. METHODS: Intravenous immunoglobulin (2 g/kg) was given preoperatively, 3 weeks postoperatively, and 4 months postoperatively to 7 infants undergoing the Norwood procedure. Panel-reactive antibodies were measured with flow cytometry preoperatively and at 1, 4, 6, and 12 months postoperatively and compared with values from a contemporary cohort of 12 infants undergoing the Norwood procedure who did not receive intravenous immunoglobulin. RESULTS: The groups were well matched for length and weight at time of surgery. Control infants were somewhat younger than the cohort receiving intravenous immunoglobulin (8 +/- 5 vs 17 +/- 14 days, P = .021). There were no differences in transfusion requirements. There was no difference in the degree of sensitization between control and intravenous immunoglobulin groups at 1 month (class I panel-reactive antibodies 20% +/- 30% vs 4% +/- 9%, P = .443, class II panel-reactive antibodies 17% +/- 27% vs 20% +/- 17%, P = .400), 4 months (class I panel-reactive antibodies 62% +/- 40% vs 73% +/- 41%, P = .813, class II panel-reactive antibodies 49% +/- 42% vs 54% +/- 41%, P = .706), and 12 months (class I panel-reactive antibodies 49% +/- 42% vs 58% +/- 39%, P = .686, class II panel-reactive antibodies 44% +/- 36% vs 49% +/- 42%, P = .651). CONCLUSION: Despite studies showing intravenous immunoglobulin to reduce sensitization, we were unable to demonstrate that intravenous immunoglobulin prevented sensitization after exposure to allograft tissue in neonates undergoing congenital cardiac surgery.

Use of an allograft patch in repair of hypoplastic left heart syndrome may complicate future transplantation.

OBJECTIVE: Cryopreserved allograft tissue used in the Norwood procedure for infants with hypoplastic left heart syndrome (HLHS) has the potential to cause marked immunologic sensitization which may complicate potential future heart transplantation, if required. The purpose of this study was to assess the anti-HLA antibody response to allograft patches used in the initial repair of HLHS. METHODS: A prospective cohort study was conducted comparing the panel-reactive antibody levels (PRA) in 12 infants undergoing repair of HLHS with cryopreserved allograft patch to 10 infants undergoing arterial switch for transposition of the great arteries (no allograft tissue used). PRA for Class I (HLA-A, B, C) and Class II (HLA-DR, DQ) antibodies were assessed preoperatively and postoperatively using flow cytometry. RESULTS: The two groups were well matched at the time of surgery (age, weight, gender). Infants in both groups received blood from multiple donors; however, the allograft group received significantly more (12+/-10 vs. 5+/-1 units; P<0.001). By 4 months, most infants receiving allograft tissue had become highly sensitized for both Class I PRA (62+/-40 vs. 0; P=0.002) and Class II PRA (49+/-42 vs. 2+/-3; P=0.022). This response continued to increase at 12 months: Class I PRA (79+/-21 vs. 0; P=0.008) and Class II PRA (66+/-27 vs. 5+/-6; P=0.008). Specificity analysis confirmed antibodies were specific for the donor allograft HLA type. In addition, infants who were coincidently HLA-matched with their allograft did not develop an elevated PRA. CONCLUSIONS: Allograft tissue used in the repair of HLHS is associated with profound donor specific immunologic sensitization in the majority of recipients and may complicate or jeopardize future transplantation. Methods to reduce the immunogenicity of cryopreserved allograft tissue used for arch reconstruction requires further investigation.