Postmortem Donors Research Articles

Chapter 1 - The Netherlands Brain Bank for Psychiatry

The Netherlands Brain Bank (NBB) performs rapid autopsies of donors who gave written informed consent during life for the use of their brain tissue and medical files for research. The NBB initiated the Netherlands Brain Bank for Psychiatry (NBB-Psy), a prospective donor program for psychiatric diseases. NBB-Psy wants to expand the tissue collections in order to provide a strong incentive to increase research in psychiatry. The ultimate goal of NBB-Psy is to reduce the burden of psychiatric disorders for patients, their families, and for society as a whole. NBB-Psy consists of an antemortem and postmortem donor program. This chapter focuses on the design of NBB-Psy and the antemortem donor program, where patients and relatives are actively informed on the possibility to become a brain donor. Since the initiation of NBB-Psy, the number of registered donors with a psychiatric diagnosis has increased from 149 in 2010 to 1018 in May 2016.

Acceptance of post-mortem organ donation in Germany : Representative cross-sectional study

The German post-mortem organ donation rate has dropped by one third since 2010. Furthermore, 958 patients died in 2015 in Germany while waiting for an organ. To decrease organ shortage, an amendment of the transplantation law was established in 2012. An information package including an organ donor card is sent to all German citizens via the postal service. Avoluntary national transplantation register was introduced in 2016 to improve transparency in the organ donation process. The influence of several transplantation scandals starting in 2012 on organ donation rates is in question. Therefore, the objective of this article is to discuss approval and objections to post-mortem organ donation among the next of kin of potential donors and the general public in Germany. Binary logistic regression of data from the 2014 survey by the Federal Centre for Health Education on attitudes towards organ and tissue donation in Germany was conducted, aiming to identify influencing factors on the likelihood of organ donor card possession. Additionally, data of the German Organ Transplantation Foundation on post-mortem organ donations in Germany in 2014 were studied to highlight reasons for approval and objections by next of kin of potential and explanted post-mortem organ donors. Methods of documentation of the deceased's will according to data of the German Organ Transplantation Foundation were analyzed. Male gender and lack of knowledge about organ donation decrease the likelihood of having an organ donor card. Of the respondents in the survey of the Federal Centre for Health Education 71.0% would donate their own organs, whereas only one third possess an organ donor card. Health insurances and physicians are the most important providers of organ donor cards in Germany. An increase in the percentage of organ donor card possession following the amendment of the transplantation law could not be observed by 2016. Fear of organ trade and unjust organ allocation are the main reasons for rejecting organ donation among the general public. Previous transplantation scandals are aprimary reason for anegative change in attitudes. Main reasons for objection among the next of kin of potential organ donors are known objections of the deceased and the lack of knowledge about the will of the deceased. In addition, only 58.1% of all explanted organ donors documented their will in written or verbal form. Education on organ donation can be ameans to increase organ donation rates. The effects of the change in legislation and the establishment of the transplant register need to be evaluated. Further research regarding the influence of religion, especially among religious minorities, on organ donation rates in Germany needs to be conducted to identify possible obstacles. Moreover, the use of social networks to address potential organ donors has proven to increase registration numbers and could easily be implemented in Germany.

Discovering the 3' UTR-mediated regulation of alpha-synuclein.

Recent evidence indicates a link between Parkinson's Disease (PD) and the expression of a-synuclein (SNCA) isoforms with different 3′ untranslated regions (3′UTRs). Yet, the post-transcriptional mechanisms regulating SNCA expression are unknown. Using a large-scale in vitro /in silico screening we identified RNA-binding proteins (RBPs) that interact with SNCA 3′ UTRs. We identified two RBPs, ELAVL1 and TIAR, that bind with high affinity to the most abundant and translationally active 3′ UTR isoform (575 nt). Knockdown and overexpression experiments indicate that both ELAVL1 and TIAR positively regulate endogenous SNCA in vivo. The mechanism of regulation implies mRNA stabilization as well as enhancement of translation in the case of TIAR. We observed significant alteration of both TIAR and ELAVL1 expression in motor cortex of post-mortem brain donors and primary cultured fibroblast from patients affected by PD and Multiple System Atrophy (MSA). Moreover, trans expression quantitative trait loci (trans-eQTLs) analysis revealed that a group of single nucleotide polymorphisms (SNPs) in TIAR genomic locus influences SNCA expression in two different brain areas, nucleus accumbens and hippocampus. Our study sheds light on the 3′ UTR-mediated regulation of SNCA and its link with PD pathogenesis, thus opening up new avenues for investigation of post-transcriptional mechanisms in neurodegeneration.

Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS

ALS is a devastating and debilitating human disease characterized by the progressive death of upper and lower motor neurons. Although much effort has been made to elucidate molecular determinants underlying the onset and progression of the disorder, the causes of ALS remain largely unknown. In the present work, we have deeply sequenced whole transcriptome from spinal cord ventral horns of post-mortem ALS human donors affected by the sporadic form of the disease (which comprises ~90% of the cases but which is less investigated than the inherited form of the disease). We observe 1160 deregulated genes including 18 miRNAs and show that down regulated genes are mainly of neuronal derivation while up regulated genes have glial origin and tend to be involved in neuroinflammation or cell death. Remarkably, we find strong deregulation of SNAP25 and STX1B at both mRNA and protein levels suggesting impaired synaptic function through SNAP25 reduction as a possible cause of calcium elevation and glutamate excitotoxicity. We also note aberrant alternative splicing but not disrupted RNA editing.

The development of a narrative describing the bereavement of families of potential organ donors: A systematic review.

Families of potential post-mortem organ donors face various challenges in the unfamiliar hospital context and after returning home. This review of sources published between 1968 and 2017 seeks to understand their journey as a bereavement experience with a number of unique features. Grief theory was used to identify ways that staff can assist family members to tolerate ambiguities and vulnerabilities while contributing to an environment characterised by compassion and social inclusion. Staff can guide families and create opportunities for meaningful participation, building resilience and developing bereavement-related skills that could assist them in the months that follow.

Strong Humoral Anti-HLA Immune Response Upon Arbitrarily Chosen Allogeneic Arterial Vessel Grafts

Forty-three patients were grafted with forty-four fresh or cryopreserved allogeneic arterial vessels in order to treat infections of synthetic vascular implants as these often lead to sepsis, amputation and death. All the patients were HLA-typed whereas typing results of the post-mortem donors were inquired or genotyped from residuary vessels’ segments. 84% of the patients were cured from the underlying infections with a re-infection rate of only 9% attesting this therapeutic procedure a high success rate for recovering from the bacterial infections. Since the allografts were chosen without considering HLA-histocompatibility between donors and recipients 95% of the patients developed a humoral anti-HLA immune response with 89% of them giving rise to virtually definable donor-specific antibodies. Regarding the clinical outcome 16% of the patients exhibited thromboses as the most frequent complication. In addition to clinical follow up analyses of all patients, tissue excisions of 11 patients’ allografts due to various complications but not resulting from persisting infections were analyzed in order to compare these vessels’ pre-and post-transplant histological appearance. In contrast to three early excised homografts (after 14 to 45 days) all of the later on explanted vessels (after 8 to 96 months) clearly showed chronic degeneration processes induced by the alloreactive immune response. Although not leading to acute graft dysfunctions comparable to those of solid organs, arbitrarily selected vessels exhibit a high degree of alloimmunization with consequent chronic degeneration processes such as the loss of smooth muscle cells. Additionally, clear signs of fibrosis of the different arterial vessel layers lead to obliterative arteriopathy and thromboses. Thus, apparently the overall practicability of the unmatched allocation of arterial homografts is clearly limited. Further approaches of arterial vessel allo-grafting are required to investigate the hypothesis that the clinical outcome of well HLA-matched homografts may lead to a significantly decreased number of thromboses.

Quality Assessment of Postmortem Tissue Donors in a University Hospital: Detection and Generation Tissue Indicators

BackgroundDefining quality assessment and measurement tools in the area of tissue donation should be considered to be one of the most important strategies for developing health centers. The aim of this project was to identify, define, and analyze a set of indicators to assess the most important steps in the tissue donor detection and generation processes. MethodsA prospective, descriptive, and comparative study of all potential tissue donors (TDs) detected and generated in a university hospital was performed. All deceased patients after cardiocirculatory death were evaluated in 2015 by the transplant coordinators (TCs). We defined as detection indicators: total deaths, percentage of detection and evaluation, percentage of clinical contraindications, tissue donor potentiality (TDP; corneal or multitissue potentiality), and the functional detection time (FDT); and as generation indicators: generation rate (corneal or multitissue generation), family request time, number of interviewed relatives, and TC experience (y). ResultsThe detection and evaluation rate was 100% (n = 1,235); tissue clinical contraindications were 57%, and TDP was 43% (n = 528; corneal, 80%; multitissue, 20%). The FDT was 24 ± 30 minutes. The generation rate was 53.4% (n = 282): corneal, 57% (n = 241); and multitissue, 40% (n = 41). Family request time was 10 ± 17 minutes, average number of interviewed relatives was 2.2 ± 1.6, and 35% of TCs had experience in the field for >5 years. ConclusionsObtaining indicators for quality assessment in the area of tissue donation is useful in predicting the outcome of the TD process as well as promoting the approach of continuous improvement.

TGFβ induces BIGH3 expression and human retinal pericyte apoptosis: a novel pathway of diabetic retinopathy.

PurposeOne of the earliest hallmarks of diabetic retinopathy is the loss of retinal pericytes. However, the mechanisms that promote pericyte dropout are unknown. In the present study, we propose a novel pathway in which pericyte apoptosis is mediated by macrophages, TGFβ and pro-apoptotic BIGH3 (TGFβ-induced Gene Human Clone 3) protein.Patients and methodsTo elucidate this pathway, we assayed human retinal pericyte (HRP) apoptosis by TUNEL assay, BIGH3 mRNA expression by qPCR, and BIGH3 protein expression by western blot analysis. HRP were treated with BIGH3 protein, TGFβ1 and TGFβ2 and inhibition assays were carried out by blocking with antibodies against BIGH3. The distribution of BIGH3 and CD68+ macrophages were compared in a post-mortem donor eye with 7-year history of Type II diabetes and histopathogically confirmed non-proliferative diabetic retinopathy (NPDR).ResultsTGFβ induced a significant increase in BIGH3 mRNA and protein expression, and HRP apoptosis. BIGH3 treatment showed HRP undergo apoptosis in a dose-dependent manner. At 5 μg/ml, BIGH3 induced 3.5-times more apoptosis in HRP than in retinal endothelial cells. TGFβ induced apoptosis was inhibited by blocking with antibodies against BIGH3. In an example of NPDR, BIGH3 accumulated within the walls of the inner retina arterioles. Macrophage infiltrates were frequently associated with these vessels and the inner nuclear layer.ConclusionTogether with our previously published results on macrophage-induced retinal endothelial cell apoptosis, the present study supports a novel inflammatory pathway mediated by macrophages and the BIGH3 protein leading to HRP apoptosis. As shown in human post-mortem globes, these observations are clinically relevant, suggesting a new mechanism underlying pericyte dropout during NPDR.

Aging Shapes the Population-Mean and -Dispersion of Gene Expression in Human Brains.

Human aging is associated with cognitive decline and an increased risk of neurodegenerative disease. Our objective for this study was to evaluate potential relationships between age and variation in gene expression across different regions of the brain. We analyzed the Genotype-Tissue Expression (GTEx) data from 54 to 101 tissue samples across 13 brain regions in post-mortem donors of European descent aged between 20 and 70 years at death. After accounting for the effects of covariates and hidden confounding factors, we identified 1446 protein-coding genes whose expression in one or more brain regions is correlated with chronological age at a false discovery rate of 5%. These genes are involved in various biological processes including apoptosis, mRNA splicing, amino acid biosynthesis, and neurotransmitter transport. The distribution of these genes among brain regions is uneven, suggesting variable regional responses to aging. We also found that the aging response of many genes, e.g., TP37 and C1QA, depends on individuals' genotypic backgrounds. Finally, using dispersion-specific analysis, we identified genes such as IL7R, MS4A4E, and TERF1/TERF2 whose expressions are differentially dispersed by aging, i.e., variances differ between age groups. Our results demonstrate that age-related gene expression is brain region-specific, genotype-dependent, and associated with both mean and dispersion changes. Our findings provide a foundation for more sophisticated gene expression modeling in the studies of age-related neurodegenerative diseases.

Abstract 4500: Normal tissue and data resources for cancer research from the GTEx program

Abstract The NIH Common Fund's Genotype-Tissue Expression (GTEx) project aims to study gene expression and regulation across multiple human tissues from approximately 1000 healthy normal postmortem donors. GTEx will provide valuable insights into gene regulation and its tissue specificity, to identify correlations between genetic variations and variations in gene expression levels as expression quantitative trait loci (eQTLs), and to help understand inherited susceptibility to disease. Initiated in 2010, the GTEx program has generated a large volume of data associated with each donor, including clinical and histopathological, as well as genotyping and gene expression data from whole genome sequencing, whole exome sequencing, expression array, and RNAseq data. The program has published research results in multiple scientific journals over the past year. The following public resources are available from the GTEx program: 1. GTEx Portal: an open access database of GTEx expression data and analysis results: http://www.gtexportal.org 2. Database of Genotypes and Phenotypes (dbGaP): controlled access of comprehensive GTEx clinical data and raw sequencing data: http://www.ncbi.nlm.nih.gov/gap 3. Access to residual GTEx biospecimens for research: http://www.gtexportal.org/home/samplesPage 4. SOPs and best practices from GTEx biospecimen collections: http://biospecimens.cancer.gov/resources/sops/library.asp 5. GTEx histological image viewer: http://biospecimens.cancer.gov/resources/tissue_image_library.asp 6. GTEx donors’ families website: a lay description of the GTEx project tailored to the GTEx donors’ families: http://www.genome.gov/gtex GTEx data can be used in combination with other data sets such as The Cancer Genome Atlas (TCGA) and genome-wide association studies (GWAS) to further explore the genetic causes and biology of cancer. The eQTL data from multiple tissues will help prioritize candidate genes within GWAS-associated loci; allow evaluation of tissue specificity of associated loci, and pinpoint target tissues for disease studies. Researchers are using GTEx resources to: study cancer heterogeneity by subtracting normal tissue expression; identify mutations in protein-truncating variants which may cause cancer; and explore gene activity from multiple donors across multiple tissue types to better understand the age/gender bias in cancer and other diseases. Citation Format: Ping Guan, Abhi Rao, Simona Volpi, Susan Koester, Helen M. Moore, GTEx consortium. Normal tissue and data resources for cancer research from the GTEx program. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4500.

Cornea donors who have had prior refractive surgery: data from the Eye Bank Association of America.

Millions of Americans have undergone refractive surgeries, including radial keratotomy, photorefractive keratectomy, and laser-assisted in situ keratomileusis. Eye Bank Association of America medical standards do not permit corneas from patients who have undergone refractive procedures to be used in penetrating keratoplasty, anterior lamellar keratoplasty, or tectonic grafting procedures. Such corneas, can, however, be used for endothelial corneal transplantation. The objective of this article is to provide an update on current trends for the screening and usage of corneas that have undergone refractive surgery. Several case reports have highlighted the difficulty in using postrefractive surgery corneas in penetrating keratoplasty. However, tissue with anterior stromal flaws, including a history of refractive surgery, has been used in endothelial keratoplasty with equivalent outcomes in topography, endothelial cell count, and visual acuity. Many modalities for proper identification of postmortem donor corneas that have undergone refractive surgery have been studied. Corneas with a history of refractive surgery have found use in endothelial keratoplasty. Multiple objective methods of tissue identification have been investigated to avoid the use of these corneas in penetrating or anterior keratoplasty surgeries.

An atlas of gene expression and gene co-regulation in the human retina.

The human retina is a specialized tissue involved in light stimulus transduction. Despite its unique biology, an accurate reference transcriptome is still missing. Here, we performed gene expression analysis (RNA-seq) of 50 retinal samples from non-visually impaired post-mortem donors. We identified novel transcripts with high confidence (Observed Transcriptome (ObsT)) and quantified the expression level of known transcripts (Reference Transcriptome (RefT)). The ObsT included 77 623 transcripts (23 960 genes) covering 137 Mb (35 Mb new transcribed genome). Most of the transcripts (92%) were multi-exonic: 81% with known isoforms, 16% with new isoforms and 3% belonging to new genes. The RefT included 13 792 genes across 94 521 known transcripts. Mitochondrial genes were among the most highly expressed, accounting for about 10% of the reads. Of all the protein-coding genes in Gencode, 65% are expressed in the retina. We exploited inter-individual variability in gene expression to infer a gene co-expression network and to identify genes specifically expressed in photoreceptor cells. We experimentally validated the photoreceptors localization of three genes in human retina that had not been previously reported. RNA-seq data and the gene co-expression network are available online (http://retina.tigem.it).

Body mass index before kidney transplantation — principal risk factor for NODAT

Purpose. Pretransplant obesity is a well-known risk factor for post-transplant outcomes such as patient and graft survival, delayed graft function, rejection, and wound complications. According to the recommendations of the European Renal Best Practice, patients who have body mass index (BMI) value of more than 30 kg/m2 before kidney transplantation should reduce their weight. Materials and methods. In the group of 297 patients who had undergone primary kidney transplantation from post-mortem donors, we found that assessed the impact of BMI on the development of new onset diabetes after transplantation (NODAT). Additionally, relationships between immunosuppression, weight gain and BMI in patients after kidney transplantation were also analysed. We measured the value of the patients’ BMI and weight before kidney transplantation, 12 months after kidney transplantation, and 5 years after kidney transplantation. The group contained only those patients who, at the time of the kidney transplantation, did not suffer from diabetes mellitus. According to the development of NODAT in the monitored period, the group of patients was divided into the control group and NODAT group. We detected analysed the data on the type of immunosuppression (tacrolimus, cyclosporine A, mTOR inhibitor) and the average levels in the monitored period and identified whether BMI or increased weight 12 months and 5 years after kidney transplantation is related to the level (or dose) of the used immunosuppression. Results. In our group, the patients who developed NODAT in the post-transplant period were significantly older in the 12-month analysis (p < 0.0001) and also in the 5-year analysis (p = 0.0001); had higher BMI at the time of transplantation (p = 0.0003) and higher BMI 12 months after kidney transplantation (p = 0.0004) and a significantly higher weight gain 12 months after kidney transplantation (p = 0.0469). We discovered that neither the level of immunosuppression nor the dose of prednisone had any effect on the increase in BMI or weight gain during the monitored period. Conclusion. The patients in the waiting list, who have any risk factors for the development of diabetes mellitus, should be informed how to eliminate these risk factors (weight control, diet, physical exercises, etc.). In addition to the above, all candidates for kidney transplantation are recommended to stop smoking, to control blood pressure, and perform a lipidogram.

Imputing Gene Expression in Uncollected Tissues Within and Beyond GTEx

Gene expression and its regulation can vary substantially across tissue types. In order to generate knowledge about gene expression in human tissues, the Genotype-Tissue Expression (GTEx) program has collected transcriptome data in a wide variety of tissue types from post-mortem donors. However, many tissue types are difficult to access and are not collected in every GTEx individual. Furthermore, in non-GTEx studies, the accessibility of certain tissue types greatly limits the feasibility and scale of studies of multi-tissue expression. In this work, we developed multi-tissue imputation methods to impute gene expression in uncollected or inaccessible tissues. Via simulation studies, we showed that the proposed methods outperform existing imputation methods in multi-tissue expression imputation and that incorporating imputed expression data can improve power to detect phenotype-expression correlations. By analyzing data from nine selected tissue types in the GTEx pilot project, we demonstrated that harnessing expression quantitative trait loci (eQTLs) and tissue-tissue expression-level correlations can aid imputation of transcriptome data from uncollected GTEx tissues. More importantly, we showed that by using GTEx data as a reference, one can impute expression levels in inaccessible tissues in non-GTEx expression studies.

Long Intergenic Noncoding RNAs Mediate the Human Chondrocyte Inflammatory Response and Are Differentially Expressed in Osteoarthritis Cartilage.

ObjectiveTo identify long noncoding RNAs (lncRNAs), including long intergenic noncoding RNAs (lincRNAs), antisense RNAs, and pseudogenes, associated with the inflammatory response in human primary osteoarthritis (OA) chondrocytes and to explore their expression and function in OA.MethodsOA cartilage was obtained from patients with hip or knee OA following joint replacement surgery. Non‐OA cartilage was obtained from postmortem donors and patients with fracture of the neck of the femur. Primary OA chondrocytes were isolated by collagenase digestion. LncRNA expression analysis was performed by RNA sequencing (RNAseq) and quantitative reverse transcriptase–polymerase chain reaction. Modulation of lncRNA chondrocyte expression was achieved using LNA longRNA GapmeRs (Exiqon). Cytokine production was measured with Luminex.ResultsRNAseq identified 983 lncRNAs in primary human hip OA chondrocytes, 183 of which had not previously been identified. Following interleukin‐1β (IL‐1β) stimulation, we identified 125 lincRNAs that were differentially expressed. The lincRNA p50‐associated cyclooxygenase 2–extragenic RNA (PACER) and 2 novel chondrocyte inflammation–associated lincRNAs (CILinc01 and CILinc02) were differentially expressed in both knee and hip OA cartilage compared to non‐OA cartilage. In primary OA chondrocytes, these lincRNAs were rapidly and transiently induced in response to multiple proinflammatory cytokines. Knockdown of CILinc01 and CILinc02 expression in human chondrocytes significantly enhanced the IL‐1–stimulated secretion of proinflammatory cytokines.ConclusionThe inflammatory response in human OA chondrocytes is associated with widespread changes in the profile of lncRNAs, including PACER, CILinc01, and CILinc02. Differential expression of CILinc01 and CIinc02 in hip and knee OA cartilage, and their role in modulating cytokine production during the chondrocyte inflammatory response, suggest that they may play an important role in mediating inflammation‐driven cartilage degeneration in OA.

Schizophrenia: From genetics to physiology at last.

The identification of a set of genetic variations that are strongly associated with the risk of developing schizophrenia provides insights into the neurobiology of this destructive disease. See Article p.177 The strongest genetic association found in schizophrenia is its association to genetic markers across the major histocompatibility complex (MHC) locus, first described in three Nature papers in 2009. The association signal at the MHC is extremely complex. Here Steven McCarroll and colleagues report a dissection of the MHC association to schizophrenia. They find a strong contribution from many structurally diverse alleles of the complement component 4 (C4) genes. The linkage was higher for C4 alleles that promoted greater expression of C4A, measured in the brain tissues of adult post-mortem donors with or without schizophrenia. The authors suggest that C4 may work with other components of the classical complement cascade to promote synaptic pruning, and demonstrate that C4 mediates synaptic refinement in a mouse model.

Understanding how non-coding genomic polymorphisms affect gene expression.

The NIH Common Fund GTEx project is designed to serve as a data and post-mortem tissue resource to the research community. The project is testing the role of genomic variation in altering gene expression across a wide array of tissues in a large number of human post-mortem donors. Both data and tissue samples are available to the research community for additional studies.

CFH Y402H polymorphism and the complement activation product C5a: effects on NF-κB activation and inflammasome gene regulation

Background/aimsThe Y402H polymorphism in the complement factor H (CFH) gene is an important risk factor for age-related macular degeneration (AMD). Complement activation products and proinflammatory cytokines are associated with this...

Living Kidney Donation in Germany – A Topical Overview

In 2014, a total of 2128 kidney transplants were performed in 40 centers. Of these, 620 were living kidney transplants, which corresponds to around 30 % of all kidney transplants. As the number of postmortem donors has been declining, living donation is becoming more and more important when discussing kidney transplants. In the following paper, both the obvious advantages for patients, as well as legal principles are to be discussed, so as to emphasize the importance of living kidney transplants against the background of current problems in transplantation medicine in Germany. The age of kidney transplants in humans began with a living kidney donation between identical twins by Murray et al. After introducing medicinal immunosuppression and constantly improving available substance groups, postmortem transplants became more common in the time following. Nevertheless, the importance of living donations is currently rising rapidly, as it is superior to postmortem transplantation and the number of organ donors has decreased dramatically since the allocation scandal in Germany in 2012.

Allocation strategies for elderly kidney transplant recipients.

In all modern societies, the percentage of elderly people is increasing. However, this does not necessary reflect the percentage of patients waiting for a new kidney and the availability of organs for this entity differs markedly between countries. Thus, allocation strategies for elderly kidney recipients should be based on the characteristics of the specific countries and take into account: access to the waiting list and availability of living as well as marginal and conventional post-mortem donors.