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Implementation of lipidomics in clinical routine: Can fluoride/citrate blood sampling tubes improve preanalytical stability?

Author(s): Hahnefeld L, Gurke R, Thomas D, Schreiber Y, Schäfer SMG, Trautmann S, Snodgrass IF, Kratz D, Geisslinger G, Ferreirós N

Publication: Talanta, 2020, Vol. 209, Page 120593

PubMed ID: 31892006 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of collection tube type, delayed centrifugation (at room temperature or in ice water), and storage of plasma in ice water versus at room temperature on levels of lipids in plasma. Blood was collected from six healthy men and four healthy women into K₃EDTA (9 mL and 2.7 mL) and sodium fluoride/citrate-containing GlucoExact (3.1 mL) tubes. To test the effects of delayed centrifugation, blood in 2.7 mL K₃EDTA and GlucoExact tubes was stored in ice water or at room temperature for 1 min (reference), 20 min, 1 h, 4 h, or 24 h before plasma was obtained by centrifugation at 2000 x g for 10 min at 4°C, frozen at -20°C, and then transferred to -80°C. To examine the stability of analytes in plasma, 9 mL K₃EDTA and GlucoExact tubes were immediately centrifuged at 2000 x g for 10 min at 4°C and plasma was stored for 0 min, 20 min, 1 h, 2 h, 4 h, or 24 h at room temperature or in ice water before freezing and -20°C and storage at -80°C. Lysophosphatidic acid (LPA), sphingolipids, and endocannabinoids were quantified by LC-MS. For non-targeted analysis, lipids were extracted from plasma using a methyl-tert-butyl ether (MTBE) extraction protocol, dried under a nitrogen stream, and stored frozen at -80°C. Lipids were reconstituted in methanol separated using a Zorbax RRHD Eclipse Plus C8 column with a SecurityGuard Ultra C8 pre-column using a Nexera X2 system and analyzed using a TripleTOF 6600 equipped with a DuoSpray ion source. Protein content was analyzed using a Bradford assay and pH was determined using a pH meter.

   Summary of Findings:

   Levels of 206 of the 248 analytes investigated differed significantly between K₃EDTA and GlucoExact tubes and total protein levels as measured by Bradford assay were 11% lower in plasma from GlucoExact tubes. Levels of sphingosine-1-phosphate increased significantly when K₃EDTA or GlucoExact blood was stored at room temperature (approximately 2-fold by 2 h, P=Not specified), but were not affected by storage of blood in ice water or in plasma stored under either condition. Levels of lysophosphatidic acid (LPA) 16:0, 18:1, 18:2, and 20:4 increased rapidly with storage of K₃EDTA whole blood or plasma at room temperature (1.6 to 2.5-fold after 1 h). A smaller increase in LPA 16:0, 18:1, 18:2, and 20:4 was found after storage of K₃EDTA whole blood or plasma in ice water for 24 h, but levels remained stable in GlucoExact blood or plasma stored at either temperature. Storage of K₃EDTA blood in ice water increased levels of 1-AG, 2-AG, and AEA by 60%, 95%, and 30%; respectively, after 20 min and levels of palmitoyl ethanolamide (PEA) increased by 30% after 1 h, but levels of 1-AG declined in the first 20 min when K₃EDTA blood was stored at room temperature and then increased, levels of 2-AG declined in the first 20 min and then plateaued, and levels of AEA and PEA increased. The effects of storage on levels of AEA was partially attenuated by the use of GlucoExact tubes instead of K₃EDTA and while the window of stability in ice water of 1-AG and 2-AG was extended by storage of blood in GlucoExact tubes, 1-AG and 2-AG increased more rapidly at room temperature when stored in GlucoExact tubes. In K₃EDTA plasma, levels of 1-AG increased within 20 min when stored at either temperature but levels of 2-AG, AEA, PEA, and oleoyl ethanolamide (OEA)/vaccenic acid ethanolamide (VEA) were stable when plasma was stored for up to 4 h at room temperature or in ice water. Use of GlucoExact rather than K₃EDTA plasma extended the window of stability for 1-AG, 2-AG, and AEA in plasma stored at either temperature. Levels of sphingolipids including ceramides, glucosylceramides, and lactosylceramides were stable when K₃EDTA whole blood or plasma was stored at either temperature.

   Semi-targeted analysis showed a time-dependent increase in free fatty acids (FA), diglycerides (DG), lysophosphatidylcholines (LPC), and lysophosphatidylethanolamines (LPE) with storage of K₃EDTA whole blood or plasma at room temperature but decreased phosphatidylcholines (PC), phosphatidylethanolamines (PE), and triglycerides (TG) when stored at room temperature for 24 h. Storage of the K₃EDTA blood or plasma in ice water or use of GlucoExact tubes attenuated the effects of storage. Of the 3198 aligned features, 360 were affected by storage of K₃EDTA whole blood at room temperature for 24 h but only linoleyl carnitine was affected by storage of K₃EDTA whole blood in ice water. Of the 360 features affected by room temperature storage of blood, 73 of these were identifiable and included an increase in acylcarnitines which were not affected by storage of plasma at either temperature. Of the 3084 aligned features, 208 were found to be significantly affected by storage of whole blood for 24 h in GlucoExact tubes but changes were smaller than those observed for K₃EDTA whole blood. The affected features included increases in DG, LPC, and LPE and decreases in steryl esters and TG.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Protein	Colorimetric assay
   Lipid	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Type of collection container/solution	K3EDTA tube GlucoExact tube
   Biospecimen Acquisition	Anticoagulant	Potassium EDTA Sodium fluoride/citrate
   Storage	Storage conditions	As plasma As whole blood
   Storage	Storage duration	1 min 20 min 1 h 2 h 4 h 24 h
   Storage	Storage temperature	Room temperature In ice water
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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