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

Abstract

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 ( 1 H) 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 and 20 °C on the quantitative lipoprotein and metabolite patterns were investigated. Storage of plasma samples at 4 °C for up to 48 h, 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 h caused little information loss. In contrast, the impact of heat-treatment (56 °C for 30 min), 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 in both lipoprotein and low molecular weight metabolite profiles. 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 artifactual 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.

Altered high-density lipoprotein composition and functions during severe COVID-19

“…In viral sepsis, the situation is less clear-cut even if lipid profile disorders have been reported in Dengue, HIV or Hepatitis infections 27 – 29 . A recent study in COVID-19 patients reports reduced plasma ApoA-I, ApoA-II and Apo-B levels as well as decreased LDL particle number and HDL subfractions assessed by nuclear magnetic resonance 30 . They also report increased LDL triglycerides and of VLDL parameters (particle number, free cholesterol and triglycerides) in these patients.…”

Low Volume in Vitro Diagnostic Proton NMR Spectroscopy of Human Blood Plasma for Lipoprotein and Metabolite Analysis: Application to SARS-CoV-2 Biomarkers

“…Plasma samples were centrifuged and processed according to a standard protocol. 10 , 11 Prior to NMR analysis, frozen plasma samples were thawed at 20 °C for 30 min and centrifuged for 10 min at 13 000 g at 4 °C. Plasma samples were prepared using both 5 mm SampleJet NMR tubes, following the recommended procedures for in vitro analytical and diagnostics procedures, 12 and 3 mm SampleJet NMR tubes.…”

Changes to the sebum lipidome upon COVID-19 infection observed via rapid sampling from the skin

“…Samples were transferred from the hospital to the University of Surrey by courier within 4 h of collection, whereupon the samples were then quarantined at room temperature for seven days to allow for virus inactivation, avoiding heat treatment given potential for lipid degradation [15] . Finally, the vials were transferred to −80 °C storage until required.…”