Microsampling-Based Metabolic Phenotyping: Innovations and Enhanced Techniques for Comprehensive Lipid and Lipoprotein Profiling

Jayden Lee Roberts

Microsamples are miniaturised biofluid collections (< 50 μL), such as dried blood spots (DBS), which offer an alternative to venous phlebotomy. DBS samples address challenges such as the need for trained personnel, cold-chain logistics, and invasiveness; enabling self-sampling for at-home or remote collection. Although widely used in neonatal screening, DBS remain underutilised in metabolic phenotyping, despite their potential to support predictive, preventive, personalised, and participatory (P4) medicine. Metabolic phenotyping can profile metabolites to understand disease mechanisms using analytical technologies such as liquid chromatography-mass spectrometry and nuclear magnetic resonance (NMR) spectroscopy. These techniques have previously highlighted the role of lipids in inflammation, energy metabolism, and cellular signalling. However, challenges remain for microsamples, including equivalence to venous samples, the need for workflows specific to metabolic profiling of DBS, and the assessment of storage stability and self-collection feasibility (discussed in Chapter 2). This thesis addressed these gaps. Chapter 3 uses proton NMR spectroscopy to assess and confirm capillary blood microsampling (500 μL MiniCollect tubes) as a viable alternative to venous blood (10 mL vacutainers) for NMR IVDr methodologies that measure lipoprotein subfractions, and inflammatory supramolecular phospholipid composites (SPC) and α-1-acid glycoprotein (Glyc) parameters. Strong agreement (Adj. R² ≥ 0.8, p < 0.001) was observed between matched capillary and venous samples for 86% of plasma and 88% of serum parameters (n = 120 total IVDr parameters). Further, we demonstrated the ability for decentralised and point-of-care assessment of capillary plasma using a low-field benchtop measurement of SPC and Glyc parameters. Chapter 4 presents an optimised DBS-based untargeted lipidomics workflow using ultrahigh-performance liquid chromatography – high-resolution mass spectrometry (UHPLC–HRMS) to enable the comprehensive analysis of 432 unique lipid features in 10 μL self-collected DBS samples. Optimised lipid extraction and stability assessments were performed on commonly employed substrates (Ahlstrom 222, Perkin-Elmer 226, Whatman 903) for advanced microsampling devices, revealing that most lipid subclasses remain stable under storage at conventional fridge and freezer temperatures, but prolonged exposure above room temperature should be avoided. The incorporation of isotopically labelled internal standards allowed for semi-quantitation and improved reproducibility (PQC replicates < 25% variability). The DBS lipidomics workflow was translated into a preliminary cohort of diabetic participants (n = 17) in Chapter 5, and a lipid signature comparable to that of traditional plasma was identified. These findings support the potential of microsamples for scalable and minimally invasive metabolic phenotyping in remote and resource-limited settings.

Microsampling-Based Metabolic Phenotyping: Innovations and Enhanced Techniques for Comprehensive Lipid and Lipoprotein Profiling

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