Quality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profiling.

Author(s): Kamlage B, Maldonado SG, Bethan B, Peter E, Schmitz O, Liebenberg V, Schatz P

Publication: Clin Chem, 2014, Vol. 60, Page 399-412

PubMed ID: 24305685 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 delayed centrifugation of blood, microclotting and induced hemolysis on the levels of 267 metabolites in EDTA plasma. Blood from 13 females and 7 males was collected into K3EDTA tubes after an overnight fast. Blood was collected first into 3 9 mL S-Monovette tubes, then 1 mL was collected into a neutral S-Monovette and discarded before collection into a 9 mL neutral S-Monovette and 3 9 mL EDTA S-Monovette tubes. Individual tubes of blood were pooled for experimentation. Hemolysis was induced in 2 6 mL pools of blood by passage through a 25 gauge needle (grade 1) or a 27 gauge needle (grade 2). To determine the effects of microclotting blood, a 9 mL S-Monovette was decanted into a 9 mL EDTA S-Monovette, and plasma was prepared after 5 min. After centrifugation, EDTA plasma was stored at -80 degrees C until analysis.

   Summary of Findings:

   Storage of blood at room temperature for 2 h or on ice for 2 or 6 h led to significant increases in 10%, 16% and 17% of the 267 metabolites tested, respectively and decreases in 12%, 12%, and 11% of the tested metabolites, respectively in EDTA plasma. Similarly, when blood was subjected to hemolysis by passage through a 25 or 27 gauge needle, 6% and 19% of tested metabolites, respectively, were increased, and 11% and 12% of the 267 metabolites, respectively, were decreased in EDTA plasma. Microclotting of the blood led to increases in 1% and decreases in 10% of the tested metabolites in EDTA plasma. Interestingly, storing blood on ice had effects more like those seen after hemolysis than after storage of blood at room temperature. Metabolites affected by preanalytical handling of blood included some signaling metabolites, carbohydrates, energy metabolites, amino acids and lipids, but the magnitude of the effects depended on the metabolite in question and preanalytical processing.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Carbohydrate	LC-MS or LC-MS/MS
   Protein	LC-MS or LC-MS/MS
   Peptide	GC-MS
   Peptide	LC-MS or LC-MS/MS
   Protein	GC-MS
   Small molecule	GC-MS
   Small molecule	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage duration	2 h 6 h
   Storage	Storage temperature	19-22 degrees C (room temperature) 0 degrees C (on ice)
   Biospecimen Aliquots and Components	Hemolysis	Fine needle aspiration-induced Not induced
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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2. Study Purpose

   The purpose of this study was to determine the effects of storage of plasma and contamination with buffy coat on the levels of 262 metabolites in EDTA plasma. Blood from 13 females and 7 males was collected into K3EDTA tubes after an overnight fast. Blood was collected first into 3 9 mL S-Monovette tubes, then 1 mL was collected into a neutral S-Monovette and discarded before collection into a 9 mL neutral S-Monovette and 3 9 mL EDTA S-Monovette tubes. Individual tubes of blood were pooled for experimentation. After centrifugation, EDTA plasma or EDTA plasma contaminated with buffy coat was stored at -80 degrees C until experimental storage.

   Summary of Findings:

   When plasma was contaminated with the buffy coat during separation, levels of only 3/267 metabolites increased significantly, and none of the metabolites were significantly decreased. With increasing storage of EDTA plasma at 4 or 12 degrees C or room temperature, an increasing number of metabolites were significantly affected. The number of metabolites affected by storage of EDTA plasma for 16 h depended on the storage temperature with 11%, 14%, and 23% of of the 267 tested metabolites affected by storage at 4 degrees C, 12 degrees C, and room temperature, respectively. Increasing storage duration and increasing storage temperature led to similar changes in metabolite levels. For instance, effects seen after 5 h at room temperature were similar to those stored for 16 h at 12 degrees C. Metabolites affected by storage of plasma or contamination of plasma included some signaling metabolites, amino acids and lipids, but the magnitude of the effects depended on the metabolite in question and preanalytical processing.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Peptide	LC-MS or LC-MS/MS
   Peptide	GC-MS
   Protein	GC-MS
   Protein	LC-MS or LC-MS/MS
   Small molecule	GC-MS
   Small molecule	LC-MS or LC-MS/MS
   Carbohydrate	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Storage temperature	4 degrees C 12 degrees C Room temperature
   Storage	Storage duration	0 h 0.5 h 2 h 5 h 16 h
   Biospecimen Aliquots and Components	Blood and blood products	Plasma Plasma with peripheral blood mononuclear cells

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