Effects of sample handling and storage on quantitative lipid analysis in human serum.

Author(s): Zivkovic AM, Wiest MM, Nguyen UT, Davis R, Watkins SM, German JB

Publication: Metabolomics, 2009, Vol. 5, Page 507-516

PubMed ID: 20046864 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of storage temperature, freeze-thaw cycling, and extraction of lipids from still frozen rather than thawed or refrigerated specimens on the lipid profile of serum. Serum from 3 healthy patients was shipped on cold packs from the supplier on the day after collection and analyzed first within 48 h or stored at 4, -20 or -80 degrees C.

   Summary of Findings:

   Overall <1% of the 262 metabolites assayed were significantly affected by freeze-thaw cycling (p<0.05), with decreases in levels of diacylglycerol (DG)20:3n9, free fatty acid (FFA)22:5n3, phosphatidylethanol-amine (PE)18:0, and PE20:4n3 noted after 1 freeze-thaw cycle, decreases in levels of DG20:3n9, DG20:2n6, cholesteryl ester (CE)20:2n6 and lysophosphatidylcholine (LY)20:5n3 noted after two freeze-thaw cycles, decreases in levels of CE20:2n6 noted after three freeze-thaw cycles and increases in levels of LY16:0 and triacylglycerol (TG)22:0 noted after 2 freeze-thaw cycles compared to refrigerated specimens. Thawing specimens prior to extraction rather than extracting lipids from still frozen serum led to significant changes in 9 of the 262 metabolites, with levels of DG18:3n6, LY20:4n6, LY22:5n3, PE16:0, LY polyunsaturated fatty acid (LYPUFA) and DG higher and phosphatidylcholine dimethyl (PCdm)18:0, PCdm18:1n9 and PE lower in specimens that were not thawed before extraction than those that were thawed. After 1 week of additional storage at 4 degrees C, levels of DGdm16:0, DG16:1n7, DG20:3n9, DGn7, DGdm and CE20:2n6 decreased compared to initial levels in refrigerated specimens (<48 h) (p<0.05). A week of storage at -20 degrees C led to decreased levels of CE20:2n6 and DG20:3n9 and increased levels of TG22:0 compared to levels in the refrigerated specimens prior to freezing (p<0.05, all). A week of storage at -80 degrees C led to decreased levels of DGdm and PCn7 and increased levels of TG22:0 compared to levels in the refrigerated specimens prior to freezing (p<0.05, all).

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Lipid	HPLC
   Lipid	GC- flame ionization
   Steroid	HPLC
   Steroid	GC- flame ionization

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Preservation	Type of fixation/preservation	Frozen Refrigeration
   Storage	Storage temperature	4 degrees C -20 degrees C -80 degrees C
   Storage	Freeze/thaw cycling	0 cycles 1 cycle 2 cycles 3 cycles
   Analyte Extraction and Purification	Analyte isolation method	Lipids extracted from still frozen serum Lipids extracted from thawed serum

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2. Study Purpose

   The purpose of this study was to determine the effects of freezing serum before fractionation on levels of lipids and lipid metabolites. Serum obtained from healthy volunteers was aliquoted within 60 min of collection and placed on ice or frozen at -80 degrees C for 2 h and then thawed. Fractionation was accomplished by ultracentrifugation, and all of the fractions were stored at -80 degrees C until lipid extraction.

   Summary of Findings:

   When specimens were frozen before fractionation, levels of 10 metabolites in the HDL fraction, 13 metabolites in the LDL fractionsand 15 metabolites in the VLDL fraction were significantly different than in specimens that were placed on ice until fractionation. The authors conclude that fractionation of serum should be conducted before freezing to minimize variability.

   Biospecimens

   • Bodily Fluid - Serum

   Preservative Types

   • Frozen
   • Other Preservative

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Lipid	HPLC
   Lipid	GC- flame ionization
   Steroid	HPLC
   Steroid	GC- flame ionization

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Analyte Extraction and Purification	Analyte isolation method	Serum fractionated before freezing Serum fractionated after freezing
   Biospecimen Preservation	Type of fixation/preservation	Frozen Refrigeration
   Storage	Storage temperature	-80 degrees C On ice

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