title: Changes to serum sample tube and processing methodology does not cause Intra-Individual [corrected] variation in automated whole serum N-glycan profiling in health and disease.
abstract: BACKGROUND
  Serum N-glycans have been identified as putative biomarkers for numerous diseases. The impact of different serum sample tubes and processing methods on N-glycan analysis has received relatively little attention. This study aimed to determine the effect of different sample tubes and processing methods on the whole serum N-glycan profile in both health and disease. A secondary objective was to describe a robot automated N-glycan release, labeling and cleanup process for use in a biomarker discovery system.
  
  
  METHODS
  25 patients with active and quiescent inflammatory bowel disease and controls had three different serum sample tubes taken at the same draw. Two different processing methods were used for three types of tube (with and without gel-separation medium). Samples were randomised and processed in a blinded fashion. Whole serum N-glycan release, 2-aminobenzamide labeling and cleanup was automated using a Hamilton Microlab STARlet Liquid Handling robot. Samples were analysed using a hydrophilic interaction liquid chromatography/ethylene bridged hybrid(BEH) column on an ultra-high performance liquid chromatography instrument. Data were analysed quantitatively by pairwise correlation and hierarchical clustering using the area under each chromatogram peak. Qualitatively, a blinded assessor attempted to match chromatograms to each individual.
  
  
  RESULTS
  There was small intra-individual variation in serum N-glycan profiles from samples collected using different sample processing methods. Intra-individual correlation coefficients were between 0.99 and 1. Unsupervised hierarchical clustering and principal coordinate analyses accurately matched samples from the same individual. Qualitative analysis demonstrated good chromatogram overlay and a blinded assessor was able to accurately match individuals based on chromatogram profile, regardless of disease status.
  
  
  CONCLUSIONS
  The three different serum sample tubes processed using the described methods cause minimal inter-individual variation in serum whole N-glycan profile when processed using an automated workstream. This has important implications for N-glycan biomarker discovery studies using different serum processing standard operating procedures.
authors.affiliation: Gastrointestinal Unit, Centre for Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
authors.forename: Nicholas T
authors.initials: NT
authors.lastname: Ventham
authors.affiliation: Ludger Ltd, Culham Science Centre, Oxford, Oxfordshire, OX14 3EB, United Kingdom.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gardner
authors.affiliation: Gastrointestinal Unit, Centre for Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
authors.forename: Nicholas A
authors.initials: NA
authors.lastname: Kennedy
authors.affiliation: Ludger Ltd, Culham Science Centre, Oxford, Oxfordshire, OX14 3EB, United Kingdom.
authors.forename: Archana
authors.initials: A
authors.lastname: Shubhakar
authors.affiliation: Gastrointestinal Unit, Centre for Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
authors.forename: Rahul
authors.initials: R
authors.lastname: Kalla
authors.affiliation: Gastrointestinal Unit, Centre for Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
authors.forename: Elaine R
authors.initials: ER
authors.lastname: Nimmo
authors.affiliation: Ludger Ltd, Culham Science Centre, Oxford, Oxfordshire, OX14 3EB, United Kingdom.
authors.forename: Daryl L
authors.initials: DL
authors.lastname: Fernandes
authors.affiliation: Gastrointestinal Unit, Centre for Genetics and Molecular Medicine, University of Edinburgh, Western General Hospital, Edinburgh, EH4 2XU, United Kingdom.
authors.forename: Jack
authors.initials: J
authors.lastname: Satsangi
authors.affiliation: Ludger Ltd, Culham Science Centre, Oxford, Oxfordshire, OX14 3EB, United Kingdom.
authors.forename: Daniel I R
authors.initials: DI
authors.lastname: Spencer
mesh_terms: D000328:Adult; D015415:Biomarkers / Q000097:blood; D001774:Blood Chemical Analysis; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism; D001800:Blood Specimen Collection / Q000295:instrumentation*; D002851:Chromatography, High Pressure Liquid; D005260:Female; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006801:Humans; D015212:Inflammatory Bowel Diseases / Q000097:blood; D008297:Male; D008875:Middle Aged; D011134:Polysaccharides / Q000097:blood* / Q000302:isolation & purification
chemical_list: D015415:Biomarkers; D001798:Blood Proteins; D006023:Glycoproteins; D011134:Polysaccharides
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