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Metabolomics

, 14:151 | Cite as

Impact of post-collection freezing delay on the reliability of serum metabolomics in samples reflecting the California mid-term pregnancy biobank

  • Michael R. La Frano
  • Suzan L. Carmichael
  • Chen Ma
  • Macy Hardley
  • Tong Shen
  • Ron Wong
  • Lorenzo Rosales
  • Kamil Borkowski
  • Theresa L. Pedersen
  • Gary M. Shaw
  • David K. Stevenson
  • Oliver Fiehn
  • John W. NewmanEmail author
Original Article

Abstract

Background

Population-based biorepositories are important resources, but sample handling can affect data quality.

Objective

Identify metabolites of value for clinical investigations despite extended postcollection freezing delays, using protocols representing a California mid-term pregnancy biobank.

Methods

Blood collected from non-pregnant healthy female volunteers (n = 20) underwent three handling protocols after 30 min clotting at room temperature: (1) ideal—samples frozen (− 80 °C) within 2 h of collection; (2) delayed freezing—samples held at room temperature for 3 days, then 4 °C for 9 days, the median times for biobank samples, and then frozen; (3) delayed freezing with freeze–thaw—the delayed freezing protocol with a freeze–thaw cycle simulating retrieved sample sub-aliquoting. Mass spectrometry-based untargeted metabolomic analyses of primary metabolism and complex lipids and targeted profiling of oxylipins, endocannabinoids, ceramides/sphingoid-bases, and bile acids were performed. Metabolite concentrations and intraclass correlation coefficients (ICC) were compared, with the ideal protocol as the reference.

Results

Sixty-two percent of 428 identified compounds had good to excellent ICCs, a metric of concordance between measurements of samples handled with the different protocols. Sphingomyelins, phosphatidylcholines, cholesteryl esters, triacylglycerols, bile acids and fatty acid diols were the least affected by non-ideal handling, while sugars, organic acids, amino acids, monoacylglycerols, lysophospholipids, N-acylethanolamides, polyunsaturated fatty acids, and numerous oxylipins were altered by delayed freezing. Freeze–thaw effects were assay-specific with lipids being most stable.

Conclusions

Despite extended post-collection freezing delays characteristic of some biobanks of opportunistically collected clinical samples, numerous metabolomic compounds had both stable levels and good concordance.

Keywords

Delayed freezing Data quality Metabolite stability Metabolomics Biorepositories 

Abbreviations

CBP

California Biobank Program

PM

Primary metabolism

CL

Complex lipids

TA

Targeted assays

IP

Ideal protocol

DFP

Delayed freezing protocol

DFFTP

Delayed freezing with freeze–thaw protocol

CE

Cholesteryl esters

PC

Phosphatidylcholines

PE

Phosphotidylethanolamines

LPC

Lysophosphotidylcholines

LPE

Lysophosphotidylethanolamines

Cers

Ceramides

SM

Sphingomyelins

TAG

Triacylglycerides

DAG

Diacylglycerols

MAG

Monoacylglycerols

ICC

Intraclass correlation coefficients

AA

Amino acids

NEFA

Non-esterified fatty acids

NAE

N-Acylethanolamides

PUFA

Polyunsaturated fatty acids

Notes

Acknowledgements

This research was supported by: the March of Dimes Foundation Prematurity Research Center at Stanford University School of Medicine (22-FY18-808; GMS, DKS); the Lucile Packard Foundation for Children’s Health; the Stanford Child Health Research Institute, the National Institutes of Health (UL1-TR001085, [SLC, CM, MH, GMS, DKS]; U24-DK097154, [OF, JWN]) and the USDA (2032-51530-022-00D, JWN). The USDA is an equal opportunity employer and provider.

Author contributions

Project design—SLC, TLP, GMS, DKS, JWN. Performed research—MRL, TS, MH, RW, TLP, OF, JWN. Analyzed data—MRL, CM, KB, JWN. Wrote paper—MRL, SLC, LR, JWN. Reviewed manuscript—all authors.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Ethical approval

This study was approved by Stanford University IRB and conducted in accordance with the ethical standards set forth by the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Informed consent

Written informed consent was obtained from all study participants.

Supplementary material

11306_2018_1450_MOESM1_ESM.xlsx (891 kb)
Supplementary material 1 (XLSX 891 KB)
11306_2018_1450_MOESM2_ESM.docx (1004 kb)
Supplementary material 2 (DOCX 1004 KB)

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Copyright information

© This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply  2018

Authors and Affiliations

  • Michael R. La Frano
    • 1
    • 2
    • 3
  • Suzan L. Carmichael
    • 4
  • Chen Ma
    • 4
  • Macy Hardley
    • 4
  • Tong Shen
    • 1
  • Ron Wong
    • 4
  • Lorenzo Rosales
    • 3
  • Kamil Borkowski
    • 1
    • 7
  • Theresa L. Pedersen
    • 6
  • Gary M. Shaw
    • 4
  • David K. Stevenson
    • 4
  • Oliver Fiehn
    • 1
    • 5
  • John W. Newman
    • 1
    • 2
    • 7
    • 8
    Email author
  1. 1.West Coast Metabolomics Center, Genome CenterUniversity of California DavisDavisUSA
  2. 2.Department of NutritionUniversity of California DavisDavisUSA
  3. 3.Department of Food Science and NutritionCalifornia Polytechnic State UniversitySan Luis ObispoUSA
  4. 4.Department of PediatricsStanford UniversityStanfordUSA
  5. 5.Department of Biochemistry, Faculty of SciencesKing Abdulaziz UniversityJeddahSaudi Arabia
  6. 6.Advanced AnalyticsWoodlandUSA
  7. 7.USDA-ARS Western Human Nutrition Research CenterDavisUSA
  8. 8.Obesity and Metabolism Research UnitUSDA-ARS-WHNRCDavisUSA

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