Background: Unfortunately, many children suffer kidney failure for a variety of reasons. Kidney transplantation provides the best outcomes for these children. However, kidney transplants are a finite resource and are unlikely to last beyond 25 years, meaning most children will need re-transplanting in the future. We aim to identify biomarkers that may help diagnose transplant rejection earlier and with greater sensitivity than monitoring serum creatinine.
Methods: First, we de-paraffinised and then stained five kidney transplant biopsy samples with allograft dysfunction with haematoxylin and eosin (H&E). We then used a proteinase digestion protocol to remove barriers that will impede RNA collection. The degraded samples were then stained using immunofluorescence techniques with a variety of antigen markers. The samples were then imaged using confocal microscopy.
Results: We confirmed a diagnosis of acute T-cell-mediated rejection (aTMR) with H&E and identified areas of borderline rejection (BR) in the same sample. We optimised our immunofluorescence staining protocol and decided on using CD45, Pan-CK, and Syto-13 stains for identifying leucocytes, tubular epithelial cells, and nuclei, respectively. We are now able to segment areas around leucocyte involvement in BR and aTMR.
Conclusions: We now plan on proceeding with spatial transcriptomics and measuring RNA transcripts in areas of BR and comparing it with areas of aTMR using Bruker GeoMx technology.