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The effect of blood sampling and preanalytical processing on human N-glycome.

Author(s): Dědová T, Grunow D, Kappert K, Flach D, Tauber R, Blanchard V

Publication: PLoS One, 2018, Vol. 13, Page e0200507

PubMed ID: 29995966 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   This study investigated the effects of clot time, tube type, anticoagulant, post-centrifugation storage duration and temperature, and hemolysis on the glycomic profile as determined by CE-LIF and MALDI-TOF MS. ­­­Blood was collected from 10 healthy blood donors into S-Monovette tubes containing K₃EDTA (1 tube), trisodium citrate (2 tubes), and heparin (1 tube) and into serum tubes containing clot-activating silica-covered beads without separation gel (4 tubes) and with separation gel (one tube) by free flow and using vacuum into a single evacuated trisodium citrate tube. To investigate the effects of delayed centrifugation, specimens in serum tubes were stored for 2 h and 6 h at room temperature and for 2 h at 4˚C. Specimens used to compare serum tube types were stored at room temperature for 6 h after centrifugation. To investigate the effects of post-centrifugation storage, serum was stored for 0, 24, and 48 h at 4˚C and for >2 months at -80˚C and -20˚C. The effect of hemolysis was investigated in citrated plasma from five donors by shaking the citrated blood at 1400 rpm overnight and through the addition of deionized water to citrated blood of all 10 donors. Plasma and serum were collected by centrifugation at 1500 x g for 15 min and stored at -80˚C until analysis, unless otherwise specified. Specimens were diluted in phosphate buffer with dithioerythritol, alkylated with iodoacetamide, and glycan release was performed by overnight incubation at 37˚C using peptide-N-glycosidase. Glycans were purified using C18 cartridges and desalted with graphitized carbon columns. Specimens were analyzed by MALDI-TOF and CE-LIF.

   Summary of Findings:

   Of the 46 glycans investigated by MALDI-TOF, 19 were affected by preanalytical variables such as clot time, tube type, anticoagulant, or post-centrifugation storage. The 19 affected glycans included high-mannose structures (4), asialylated complex-type structures (4), monosialylated structures (5), disialylated structures (2), one tetrasialylated structure, fucosylated structures (2) and fucosylated sialylated structures (1). Only four of the 19 glycans affected by preanalytical factors had relative intensities >1%. Similar to MALDI-TOF results, CE-LIF analysis showed 19 of 23 peaks were significantly affected by preanalytical factors but ten of these had relative intensities >1%.

   Precentrifugation storage of specimens in clot-activating tubes for 2 or 6 h at room temperature or for 2 h at 4˚C had no effect on the glycan profile. Frozen storage of serum for 2 months at -20˚C affected one glycan and storage at -80 ˚C affected another glycan. Use of a serum gel tube rather than the standard tube increased one glycan peak as determined by CE-LIF. In contrast, no effects of frozen storage or serum tube type were observed on the MALDI-TOF glycan peaks. Two MALDI-TOF peaks were significantly smaller in EDTA and heparin plasma than citrated plasma and three CE-LIF peaks were higher and three lower in heparin plasma than citrate plasma. Inducing hemolysis by the addition of deionized water increased one CE-LIF peak and decreased another but no effects were found by MALDI-TOF. In contrast, hemolysis induced by overnight shaking at 1400 rpm did not affect the CE-LIF or MALDI-TOF glycan peak intensities. Very strong negative correlations were observed with the degree of hemolysis for one glycan (r=-0.958) and with the author’s glycan-based ovarian cancer biomarker (r=-0.958). The degree of hemolysis due to the addition of deionized water was correlated with the relative intensities of four asialo complex N-glycans and two oligomannose structures (r=0.634-0.852) and was negatively correlated with two N-glycans (r=-0.667 and r=-0.695) but was not correlated with the author’s glycan-based ovarian cancer biomarker. However, there was no correlation between the area of the glycan peaks and the degree of hemolysis when analyzed by CE-LIF.

   Biospecimens

   • Bodily Fluid - Blood
   • Bodily Fluid - Serum
   • Bodily Fluid - Plasma

   Preservative Types

   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Carbohydrate	LC-MS or LC-MS/MS

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Biospecimen Acquisition	Anticoagulant	Lithium heparin Citrate Potassium EDTA
   Biospecimen Acquisition	Type of collection container/solution	Serum tube without separation gel Serum tube with separation gel
   Storage	Storage temperature	4˚C -20˚C -80˚C
   Biospecimen Aliquots and Components	Hemolysis	Distilled water-induced Shaking-induced Not induced
   Storage	Storage duration	2 h 6 h 24 h 48 h
   Biospecimen Aliquots and Components	Centrifugation	Centrifugation delays investigated

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