Comprehensive lipidomic profiling by plasma separation cards

Abstract

Large-scale lipidomic analyses have been limited by the cost and accessibility of traditional venipuncture sampling. Microsampling techniques offer a less-invasive and more accessible alternative. From a single drop of blood, plasma separation cards (PSC) deliver two volumetric dried plasma samples which are studied here for profiling endogenous blood lipids. Six lots of EDTA-treated human whole blood were used to compare PSC, dried blood spot analyses (DBS), and classic wet plasma extractions. Six replicate extractions were performed for each lot. Nontargeted lipidomics was performed by liquid chromatography-high resolution tandem mass spectrometry. Lipids were annotated by accurate mass/retention time matching and MS/MS spectral library matching using peak intensities for quantitation. Four hundred ninety-eight compounds covering 24 lipid subclasses were annotated. Inter-lot repeatability was evaluated by the percent relative standard deviation (%RSD) for each lot, giving median %RSD values across the lots at 14.6% for PSC, 9.3% for DBS, and 8.6% for wet plasma. Strong correlations of lipid peak intensities between wet plasma and PSCs were observed, but less for DBS. Lipid recovery and stability were comparable between the PSC and DBS samples, with roughly 60% of annotated lipids stable at room temperature after 28 days. Overall, PSCs provide a better alternative for quantitative blood lipidomic analyses compared to dried blood spots. However, problems with lipid stability for samples handled and shipped at room temperature are currently unavoidable outside of a clinical setting. Data transferability and comparability to standard plasma is lipid and lipid class dependent.

Keywords: Lipidomics; Mass spectrometry; Method validation; Microsampling.

Fig. 1

Illustration of a commercial plasma separation card

Fig. 2

Classification of lipids found in ≥95% of samples (n=315). Abbreviations of lipid classes are given in the supplementary data.

Fig. 3

a Median percent relative standard deviation (%RSD) values across all annotated lipids for EDTA-treated samples. Median %RSD for plasma, PSC, and DBS samples is 8.6%, 14.6%, and 9.3%, respectively. b Median %RSD of individual lipids for citrate-treated samples. Overall %RSD is 10.5% and 13.6% for plasma and PSC samples, respectively

Fig. 4

a Correlation between average peak intensities in EDTA-treated PSC and EDTA plasma samples. b Correlation between average peak intensities in EDTA-treated DBS and EDTA plasma samples. c Correlation between average peak intensities in citrate-treated PSC and citrate plasma samples. The median percent changes of lipid intensities for the three sample types in comparison to plasma are −34%, +118%, and −26%, respectively

Fig. 5

a Differences in peak intensities of regularly extracted lipid internal standards versus internal standards directly spotted onto the sample. b Intensity fold changes for each lipid class represented by the internal standards. c The fold changes applied as correction factors to the individual lipid intensities, improving the median percent change of PSCs to plasma to −6%

Fig. 6

a Proportion of lipids at each temperature assigned to each stability category (n=344). Green indicates stability across days 1–28, blue indicates stability after 7 days of storage, and red indicates limited or no stability over the 28-day storage period. b Frequency of stability category per lipid class for PSC and DBS samples stored at room temperature