Immune Risk Assessment in Pediatric Heart Transplant Patients
Creators: Halpin, Anne M
Publisher: name: University of Alberta Library
Year: 2022

<p>Background: Although pediatric heart transplantation is a life-saving and effective therapy for children with cardiac disease, barriers remain due to a lack of available organs as well as transplant failure due to rejection. This dissertation aimed to increase knowledge related to immune risk assessment in pediatric heart transplant patients. There are recognised differences in immune responses across the age spectrum. Pediatric transplant patients require studies that evaluate these differences as they may alter pre- and post-transplant care. Chapters 2 and 3 evaluated HLA and AT1R immune responses in pediatric heart transplant populations. Chapter 4 involved the creation of a novel assay for use in ABO-incompatible pediatric heart transplantation for more accurate assessment of antibodies to ABO glycans specific to the heart; this assay also has applications beyond this population. </p>

<p>Methods: Three studies comprise this thesis. For the project described in Chapter 2, retrospective HLA antibody data were collected from pre- and post-VAD therapy patients. HLA antibody testing was performed using solid phase antibody testing methodologies. Transfusion data were also collected. Changes in HLA antibodies following VAD implantation were examined and association to blood transfusion was evaluated. AT1R antibody levels were measured in pediatric heart transplant patients and non-transplant controls (described in Chapter 3). A commercially available ELISA assay used in all recent literature in this field was employed (Cell Trend GmbH). Non-specific, non-AT1R reactivity in this assay was explored using another commercially available reagent, Adsorb Out™. Chapter 4 describes a methods development project in which a novel ABO antibody assay was created and tested. This assay was developed by optimising coupling of ABH subtype carbohydrate structures to Luminex beads. The beads were tested using healthy control sera to demonstrate proof of concept. Detection of ABO antibodies using this assay was compared to the antibody titre detected by standard red cell agglutination.</p>

<p>Results: HLA antibodies in VAD therapy: While both pediatric and adult patients were at risk of developing HLA antibodies following VAD therapy, adult patients demonstrated a statistically significant increase in only class I PRA levels, whereas children did not; neither group showed a significant increase in class II PRA. The proportion of adult vs pediatric patients who developed new HLA antibodies was similar despite differences in overall PRA level. Transfusion was not associated with the development of HLA antibodies. AT1R antibodies in pediatric heart transplant patients: AT1R antibodies were detected in a high proportion of pediatric heart transplant recipient and control samples, 55% and 56%, respectively. Positive samples were very likely to be converted to a negative value following adsorption treatment. AT1R antibody status changes were not consistent from pre- to post-transplant for either non-adsorbed or adsorbed patient samples. Neu5Gc glycans were detected on the Adsorb Out™ microparticles, suggesting this may be a source of non-specific reactivity removed in this procedure. ABO antibody detection: A Luminex bead-based ABO antibody detection assay was developed, optimised, and tested. It was shown to be reproducible between laboratories. Each ABO anti-A and anti-B titre was shown to include a wide range of ABO IgG and IgM antibodies. Additionally, differences between A and B glycan subtype-specific antibody patterns were observed that will be the subject of future study. </p>

<p>Summary and Conclusions: The projects described in this dissertation generated new data that will assist in immune risk assessment for pediatric heart transplant patients. While larger studies are more easily conducted in adult transplant populations due to higher transplant volumes in these cohorts, findings may vary across the age spectrum. In these projects, there are measurable differences in HLA antibody development following VAD therapy as well as AT1R antibody status between children and other published studies in adults. Test method interferences may also vary between adults and children; this should be a consideration in the design (including controls), development, and validation of laboratory assays such as the AT1R assay used in Chapter 3. Differences in tissue and cell distribution of ABH glycans are, in part, a driver for the necessity for new assays such as the Luminex ABO assay presented in this thesis. Pediatric heart transplant patients will be better served by accurate determination of their immune risk status in pre- and post-transplant phases to enable improved donor selection and more precise post-transplant monitoring for rejection.</p>