Quantitative In-Vitro Diagnostic NMR Spectroscopy for Lipoprotein and Metabolite Measurements in Plasma and Serum: Recommendations for Analytical Artefact Minimization with Special Reference to COVID-19/SARS-CoV-2 Samples.

Quantitative nuclear magnetic resonance (NMR) spectroscopy of blood plasma is widely used to investigate perturbed metabolic processes in human diseases. The reliability of biochemical data derived from these measurements is dependent on the quality of the sample collection and exact preparation and analysis protocols. Here, we describe systematically, the impact of variations in sample collection and preparation on information recovery from quantitative proton (1H) NMR spectroscopy of human blood plasma and serum. The effects of variation of blood collection tube sizes and preservatives, successive freeze-thaw cycles, sample storage at -80°C, and short term storage at 4°C and 20°C on the quantitative lipoprotein and metabolite patterns were investigated. Storage of plasma samples at 4oC for up to 48 hr, freezing at -80°C and blood sample collection tube choice have few and minor effects on quantitative lipoprotein profiles, and even storage at 4°C for up to 168 hr caused little information loss. In contrast, the impact of heat-treatment (56°C for 30 minutes), which has been used for inactivation of SARS CoV-2 and other viruses, that may be required prior to analytical measurements in low level biosecurity facilities induced marked changes. It was conclusively demonstrated that this heat inactivation procedure degrades lipoproteins and changes metabolic information in complex ways. Plasma from control individuals and SARS CoV-2 infected patients are differentially altered resulting in the creation of artefactual pseudo-biomarkers and destruction of real biomarkers to the extent that data from heat-treated samples are largely uninterpretable. We also present several simple blood sample handling recommendations for optimal NMR-based biomarker discovery investigations in SARS CoV-2 studies and general clinical biomarker research.