Poster Presentation ESA-SRB-ANZBMS 2024 in conjunction with ENSA

Effect of fixation and decalcification on the lipidome of bone marrow trephine biopsies using mass spectrometry (#339)

Mackenzie Skinner 1 2 , Jacob Truong 2 3 , Laura Trainor 1 2 , Marten Snel 4 5 , Andrew Zannettino 1 2 , Kate Vandyke 1 2 , Melissa Cantley 1 2 , Paul Trim 4 5
  1. Myeloma Research Laboratory, School of Biomedicine, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, Australia
  2. Precision Cancer Medicine Theme, South Australian Health and Medical Research Institute, Adelaide, SA, Australia
  3. South Australian immunoGENomics Cancer Institute, Adelaide Medical School, Faculty of Health and Medical Sciences, The University of Adelaide, Adelaide, SA, Australia
  4. Metabolomics and MS-imaging Core Facility, South Australian Health and Medical Research Institute, Adelaide, SA, Australia
  5. The University of Adelaide, Adelaide, SA, Australia

The bone marrow microenvironment is a highly cellular and metabolically active site, where study of the spatial lipidome could provide insight into disease. However, application of mass spectrometry imaging (MSI) to bone marrow trephine (BMT) biopsies is limited by the difficulty obtaining high-quality undecalcified bone sections. Fresh-frozen samples are preferred for MSI, due to lipid signal suppression associated with fixation. Decalcification softens bone for sectioning; however, little is known of its effect on lipid signal. Consequently, we aimed to characterise the effect of both fixation and decalcification on the lipid signal obtained from BMTs.

BMTs were obtained from a sheep pelvis, and snap-frozen (undecalcified)(n=6), decalcified in 10% ethylenediaminetetraacetic acid (EDTA)(n=6), or fixed in 4% paraformaldehyde (PFA) and decalcified in 10% EDTA (n=6). For liquid chromatography (LC-MS) lipidomic analysis, samples were homogenised and analysed using the Xevo-G2XS-QTOF (Waters, USA). For MSI, BMTs were embedded in 8% gelatin and frozen (-80°C), then cryosections mounted on conductive slides, before matrix sublimation. MSI data was acquired on a timsTOF-fleX and processed with SCiLS Lab 2023a (Bruker, Germany). Statistical analysis was conducted using GraphPad Prism.

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The contribution of lipid class to total signal detected by LC-MS revealed significant loss of phosphatidylethanolamines (PE, PE O-, PE P-) in fixed and decalcified BMTs, compared to undecalcified controls (Figure 1). The relative abundance of 16 PE lipids significantly decreased upon fixation and decalcification, MSI confirmed. LC-MS suggested decalcification alone led to a significant loss of PE O- lipids, and significant increase in sphingomyelins (SM), compared to undecalcified BMTs (Figure 1). However, MSI did not reveal significant changes to lipid spatial distribution or intensity with decalcification.

Decalcification of BMTs may provide section integrity for MSI, while minimising chemical modification, presenting the opportunity to study differences in the spatial lipidome of BMTs, pertinent to the study of molecular disease processes.