Podium Presentation in Room 2 on Wednesday at 9:40 (Chair: Melanie Odenkirk)
Authors: Jessica Lukowski (1), Annapurna Pamreddy (2), Guanshi Zhang (2,3), Dusan Velickovic (1), Ljiljana Pasa-Tolic (1), Theodore Alexandrov (4,5), Kumar Sharma (2,3), Christopher Anderton (1,2) for the KPMP Consortium
Introduction: The Kidney Precision Medicine Project (KPMP) consortium in part, aims to create a human kidney tissue atlas from evaluating healthy and diseased biopsies. Our team’s role is to develop and optimize untargeted and targeted mass spectrometry imaging (MSI)-based spatial metabolomics analyses for these tissues, as well as to generate methods to link this data to other omics-based analyses being performed within the consortium. As biopsies are shared between tissue integration sites (TIS) within the consortium, tissue handling, and storage methods need to be considered and optimized to preserve this precious biopsy specimens.
Objective: To determine the optimal storage conditions for human kidney biopsy sections for spatial metabolomics, where our benchmark is in obtaining the maximum number of molecular annotations.
Methods: We evaluated human kidney biopsy sections along with those from a mimetic tissue on several different days throughout the span of a week using our established protocol for elucidating lipids in positive ion mode with 2,5 dihydroxybenzoic acid (DHB) matrix. Slides containing three kidney biopsy sections were stored in five different environments: at room temperature in a desiccator, open stored at -80°C, vacuumed sealed and stored at -80°C, under a nitrogen atmosphere and stored at -80°C, and with matrix pre-applied before storage at -80°C. Matrix application (HTX TM-Sprayer) and high mass resolution matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS Bruker 15T-FTICR-MS) analysis was performed on these tissue sections, and data was converted to the imzML format and uploaded to METASPACE. Putative molecular annotations were determined using the SwissLipids Database within METASPACE at a 20% FDR.
Results: Using MALDI-MSI we were able to putatively annotate 181±16 molecules in freshly cut kidney biopsy sections (n=12) from a pilot sample generously donated by the University of Michigan using the SwissLipids Database in METASPACE. However, storing sections overnight, regardless of storage method resulted in a decrease in molecular annotations. Slides openly stored at -80°C and under nitrogen showed the least amount of degradation overnight, indicating a 18.8% and 25.5% decrease in the number of molecular annotations, respectively. After a week of storage, the biopsies stored under nitrogen showed the largest number of retained molecular annotations at 70.7%. This was followed closely by slides stored at -80°C under a vacuum seal and slides sprayed with matrix at 60.2% each. Of note, our previously identified 14 QC positive ion mode-detectable lipids were detected in every analysis, showing their robustness overtime. Additionally, a reference mimetic tissue was evaluated alongside the biopsies. The reference mimetic tissue provides a robust approach to address instrument drift, QC of protocols, tissue integrity, and allows us to analyze biopsy sections the next day or even a week after sectioning as 89 ±8 molecules were consistently annotated in freshly cut mimetic tissue (n=6).
Conclusions: Our TIS anticipates receiving numerous fresh human kidney biopsies a weekly as part of the KPMP, so it is crucial to investigate the effects of analyzing the biopsy sections temporally. This data helps determine best practices for tissue handling and storage, by providing information about what to expect if samples are mishandled or about the ideal way to store and ship sections between TISs. Additionally, we found that having a mimetic sample is a key step to ensuring analysis reproducibility and in identifying unwanted batch effects, analysis drift, and instrumental bias.
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