Table 1.

Suggested recommendations for ensuring lipid stability

Sample collection	Pre-collection considerations	While fresh samples allow for the best representation of an unaltered lipidome, frozen samples are acceptable as long as procedures are employed to ensure lipid stability. Plasma preparation from whole blood collected in tubes containing an anticoagulant is preferred, as anti-clotting agents such as EDTA can capture heavy metals, which are pro-oxidants. Because studies have shown the type of anticoagulant used to impact lipid extraction, create interferences, and/or result in the degradation of certain lipid classes, the same anticoagulant should be utilized for all samples within a study and among studies that will be compared.
Sample handling	Post-collection considerations	After collection, all samples should be kept cold until sample preparation can be performed. Samples should ideally be immediately stored frozen until sample analysis at −20 °C or lower. If samples cannot be stored frozen, the time between collection and sample analysis should be minimal.
Sample preparation	Internal standards	If available, structurally similar internal standards should be spiked into samples before sample preparation to assess sample variability. The internal standards selected should be absent in the sample matrix and should closely represent the lipid class of interest. Lipid markers such as sphingadienine 1-phosphate, spingosine-1-phosphate, and lysophosphatidylcholine 18:2 have been proposed for use in assessing sample quality and pre-analytical variation. However, caution should be employed as these markers have not been assessed in larger cohort studies, as a panel of independent markers, or in other matrices apart from human serum and plasma.
	Flash-freezeing	If applicable, samples can be flash frozen in liquid nitrogen to quench enzymatic activity. Sample preparation should continue to be performed in a cold environment.
	Additivies	The use of additives such as phenylmethanesulfonyl fluroride (PMSF) have been shown to reduce the incidences of oxidization and enzymatic degradation of lipid species.
	Antioxidants	The use of antioxidants is encouraged during sample preparation to prevent the degradation of lipid species by (per)oxidation.
	Heat-treatment	For sample matrices where additives and antioxidant addition is challenging such as with tissue samples, heat treatment, which can be directly applied to the sample or solvent, has been shown to improve the stability of phospholipids, triacylglycerols, and sphingolipids during sample preparation through the inhibition of lipases. However, it should be noted that the non-enzymatic physical and chemical degradation of lipids are not accounted for with this technique.
Storage conditions	Short-term	The short-term storage of lipid extracts at room temperature and at 4 °C should be avoided as enzymatic activity has been shown to still be present. Lipids should be stored in an environment free of water, oxygen, and/or light to avoid the chemical transformation of certain lipid species.
	Long-term	If applicable, samples can be flash frozen in liquid nitrogen and stored at −20°C or lower. Lipid extracts should be stored absent of oxygen (under nitrogen), light, metal ions, and peroxides in an organic solvent to avoid sublimation. Lipid extracts should be stored at −20 °C or lower.
	Freeze–thaw cycles	Laboratories should assess the effect of multiple freeze thaw cycles on the lipid class(es) of interest in a similar matrix as this is sample-dependent. If the lipid class of interest is affected by multiple freeze thaw cycles, biofluids should be aliquoted and tissues should be sliced appropriately to allow for multiple analyses.
Standardization efforts	Targeted	Depending on the analyte, laboratories can participate in the following QA/QC programs to ensure accuracy in lipid measurements: • [CDC] Lipid Standardization Program (LSP) for total cholesterol, triacylglycerols, high-density lipoprotein cholesterol, apolipoprotein A-I, and apolipoprotein B • [NIST and NIH ODS] Health Assessment Measurements Quality Assurance Program (HAMQAP) for free fatty acids
	Untargeted	While no formal lipid measurement standardization/harmonization programs are currently in place for untargeted lipidomics studies, the following efforts exist: • LipidQC is a method validation tool established by NIST for robust untargeted lipidomics platforms, which can be used to compare experimental results from NIST SRM 1950 to consensus mean concentrations derived from the 2017 NIST Lipidomics Interlaboratory Comparison Exercise. • The results from various lipidomics international ring trials and interlaboratory studies have been reported in literature and can be used as a point of reference.