Summary of study ST001644

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001052. The data can be accessed directly via it's Project DOI: 10.21228/M8QD76 This work is supported by NIH grant, U2C- DK119886.

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Study IDST001644
Study TitleIn Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets
Study SummaryPlatelet concentrates are currently stored at room temperature (RPs) under constant agitation for up to 5-7 days depending on national regulations. However, platelet quality deteriorates during storage and room temperature storage also increases the risk of bacterial growth. Previous studies have shown that cold-stored platelets (CPs) have higher hemostatic function and can be stored for up to three weeks. While these studies have compared the metabolic phenotypes of CPs and RPs, they have not compared the impact of storage temperature and cold agitation (CPAs) on platelet function, nor have they identified metabolic correlates to such parameters. In vitro analysis showed CPAs and CPs had reduced count, faster CD62P expression and increased lactadherin binding. Furthermore, CPAs and CPs had higher maximal aggregation and a reduced aggregation lag phase compared to RPs. Metabolomic analysis revealed CPAs and CPs exhibited lower oxidative stress shown by preserved glutathione and pentose phosphate pools. CPAs and CPs also had reduced markers of beta-oxidation and amino acid catabolism demonstrating reduced needs for energy. Agitation did not significantly impact in vitro function or metabolomic parameters of cold-stored platelets. Correlation of in vitro and metabolomic results highlighted important metabolites that may contribute to stored platelet functions.
Institute
University of Colorado Anschutz Medical Campus
DepartmentBiochemistry and Molecular Genetics
LaboratoryAngelo D'Alessandro
Last NameD'Alessandro
First NameAngelo
Address12801 E 17th Ave L18-9403D
Emailangelo.dalessandro@cuanschutz.edu
Phone3037245798
Submit Date2021-01-08
Num Groups3
Total Subjects8
Raw Data AvailableYes
Raw Data File Type(s).raw
Analysis Type DetailLC-MS
Release Date2021-01-25
Release Version1
Angelo D'Alessandro Angelo D'Alessandro
https://dx.doi.org/10.21228/M8QD76
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001052
Project DOI:doi: 10.21228/M8QD76
Project Title:In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets
Project Summary:Platelet concentrates are currently stored at room temperature (RPs) under constant agitation for up to 5-7 days depending on national regulations. However, platelet quality deteriorates during storage and room temperature storage also increases the risk of bacterial growth. Previous studies have shown that cold-stored platelets (CPs) have higher hemostatic function and can be stored for up to three weeks. While these studies have compared the metabolic phenotypes of CPs and RPs, they have not compared the impact of storage temperature and cold agitation (CPAs) on platelet function, nor have they identified metabolic correlates to such parameters. In vitro analysis showed CPAs and CPs had reduced count, faster CD62P expression and increased lactadherin binding. Furthermore, CPAs and CPs had higher maximal aggregation and a reduced aggregation lag phase compared to RPs. Metabolomic analysis revealed CPAs and CPs exhibited lower oxidative stress shown by preserved glutathione and pentose phosphate pools. CPAs and CPs also had reduced markers of beta-oxidation and amino acid catabolism demonstrating reduced needs for energy. Agitation did not significantly impact in vitro function or metabolomic parameters of cold-stored platelets. Correlation of in vitro and metabolomic results highlighted important metabolites that may contribute to stored platelet functions.
Institute:University of Colorado Anschutz Medical Campus
Department:Biochemistry and Molecular Genetics
Laboratory:Angelo D'Alessandro
Last Name:D'Alessandro
First Name:Angelo
Address:12801 E 17th Ave L18-9403D, Aurora, Colorado, 80045, USA
Email:angelo.dalessandro@cuanschutz.edu
Phone:303-724-5798

Subject:

Subject ID:SU001721
Subject Type:Human
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Treatment Day
SA15130060_B6_D14_4NS+4NS 14
SA15130170_B7_D14_4NS+4NS 14
SA15130280_B8_D14_4NS+4NS 14
SA15130350_B5_D14_4NS+4NS 14
SA15130440_B4_D14_4NS+4NS 14
SA15130520_B2_D14_4NS+4NS 14
SA15130610_B1_D14_4NS+4NS 14
SA15130710_B1_D14_4NS-4NS 14
SA15130820_B2_D14_4NS-4NS 14
SA15130960_B6_D14_4NS-4NS 14
SA15131070_B7_D14_4NS-4NS 14
SA15131180_B8_D14_4NS-4NS 14
SA15131250_B5_D14_4NS-4NS 14
SA15131340_B4_D14_4NS-4NS 14
SA15131430_B3_D14_4NS+4NS 14
SA15131530_B3_D14_4NS-4NS 14
SA1513164_B1_D2_4NS+4NS 2
SA15131744_B5_D2_4NS+4NS 2
SA15131834_B4_D2_4NS+4NS 2
SA15131924_B3_D2_4NS+4NS 2
SA1513204_B1_D2_4NS-4NS 2
SA15132154_B6_D2_4NS+4NS 2
SA15132264_B7_D2_4NS+4NS 2
SA15132374_B8_D2_4NS+4NS 2
SA15132414_B2_D2_4NS-4NS 2
SA15132554_B6_D2_4NS-4NS 2
SA15132664_B7_D2_4NS-4NS 2
SA15132744_B5_D2_4NS-4NS 2
SA15132874_B8_D2_4NS-4NS 2
SA15132914_B2_D2_4NS+4NS 2
SA15133024_B3_D2_4NS-4NS 2
SA15133134_B4_D2_4NS-4NS 2
SA15133237_B4_D7_4NS-4NS 7
SA1513337_B1_D7_4NS+4NS 7
SA15133447_B5_D7_4NS-4NS 7
SA15133527_B3_D7_4NS-4NS 7
SA15133667_B7_D7_4NS-4NS 7
SA15133717_B2_D7_4NS+4NS 7
SA15133877_B8_D7_4NS-4NS 7
SA15133957_B6_D7_4NS-4NS 7
SA15134067_B7_D7_4NS+4NS 7
SA15134117_B2_D7_4NS-4NS 7
SA1513427_B1_D7_4NS-4NS 7
SA15134377_B8_D7_4NS+4NS 7
SA15134457_B6_D7_4NS+4NS 7
SA15134537_B4_D7_4NS+4NS 7
SA15134647_B5_D7_4NS+4NS 7
SA15134727_B3_D7_4NS+4NS 7
SA15134879_B8_D14_4S+4S 14
SA1513499_B1_D14_4S-4S 14
SA15135069_B7_D14_4S+4S 14
SA15135119_B2_D14_4S-4S 14
SA1513529_B1_D14_4S+4S 14
SA15135339_B4_D14_4S-4S 14
SA15135449_B5_D14_4S-4S 14
SA15135559_B6_D14_4S-4S 14
SA15135669_B7_D14_4S-4S 14
SA15135729_B3_D14_4S-4S 14
SA15135859_B6_D14_4S+4S 14
SA15135949_B5_D14_4S+4S 14
SA15136039_B4_D14_4S+4S 14
SA15136129_B3_D14_4S+4S 14
SA15136219_B2_D14_4S+4S 14
SA15136379_B8_D14_4S-4S 14
SA15136453_B6_D2_4S-4S 2
SA15136523_B3_D2_4S-4S 2
SA15136613_B2_D2_4S-4S 2
SA15136733_B4_D2_4S-4S 2
SA15136843_B5_D2_4S-4S 2
SA15136963_B7_D2_4S-4S 2
SA15137013_B2_D2_4S+4S 2
SA15137173_B8_D2_4S-4S 2
SA1513723_B1_D2_4S+4S 2
SA15137373_B8_D2_4S+4S 2
SA15137423_B3_D2_4S+4S 2
SA15137563_B7_D2_4S+4S 2
SA1513763_B1_D2_4S-4S 2
SA15137733_B4_D2_4S+4S 2
SA15137843_B5_D2_4S+4S 2
SA15137953_B6_D2_4S+4S 2
SA15138026_B3_D7_4S+4S 7
SA15138146_B5_D7_4S+4S 7
SA15138236_B4_D7_4S+4S 7
SA15138366_B7_D7_4S+4S 7
SA1513846_B1_D7_4S-4S 7
SA15138576_B8_D7_4S+4S 7
SA15138656_B6_D7_4S+4S 7
SA15138746_B5_D7_4S-4S 7
SA15138876_B8_D7_4S-4S 7
SA15138916_B2_D7_4S+4S 7
SA1513906_B1_D7_4S+4S 7
SA15139166_B7_D7_4S-4S 7
SA15139256_B6_D7_4S-4S 7
SA15139326_B3_D7_4S-4S 7
SA15139436_B4_D7_4S-4S 7
SA15139516_B2_D7_4S-4S 7
SA1513961_B1_D1_RT-RT 1
SA15139721_B3_D1_RT-RT 1
SA15139811_B2_D1_RT-RT 1
SA15139931_B4_D1_RT-RT 1
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Collection:

Collection ID:CO001714
Collection Summary:Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003).
Sample Type:Platelets

Treatment:

Treatment ID:TR001734
Treatment Summary:Whole blood was collected by Canadian Blood Services from donors providing signed informed consent. Buffy coat platelet concentrates suspended in plasma were produced by the Canadian Blood Services Blood4Research Facility in Vancouver, Canada. For each biological replicate, three buffy coat platelet units were pooled and split into three identical platelet units to minimize the effect of donor variability. One unit (RP) was stored at 22°C under constant agitation (Helmer Scientific, PC1200-Pro Platelet Incubator, Noblesville, IN). One unit (CPA) was stored in a walk-in fridge (4°C) in a platelet agitator (Forma platelet agitator, Thermo Electron Corporation. Waltham, MA). The last unit (CP) was stored in walk-in fridge (4°C) without agitation. Platelet units were sampled through a coupling port (Fresenius Kabi, Bad Homburg, Germany) using a sterile syringe in a biosafety cabinet. Platelet units were sampled on days 1, 2, 4, 7, 9, 11, and 14 of storage. This study was approved by both the University of British Columbia Ethics Committee (H17-02708) and the Canadian Blood Services Ethics Board (2018-003).

Sample Preparation:

Sampleprep ID:SP001727
Sampleprep Summary:A volume of 50μl of platelet concentrates were extracted at a 1:10 dilution in methanol:acetonitrile:water (5:3:2, v/v/v). After vortexing at 4°C for 30 min, extracts were separated from the protein pellet by centrifugation for 10 min at 10,000g at 4°C and stored at −80°C until analysis. For polar metabolites, half of the extracts were dried down via SpeedVac prior to resuspension in ddH2O + 0.1% formic acid, to facilitate ionization and chromatographic resolution of certain polar metabolites (e.g., betaine, kyurenine, etc.).

Combined analysis:

Analysis ID AN002689 AN002690
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um) Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive Orbitrap Thermo Q Exactive Orbitrap
Ion Mode NEGATIVE POSITIVE
Units Relative Abundance Relative Abundance

Chromatography:

Chromatography ID:CH001983
Chromatography Summary:UHPLC-MS metabolomics analyses were performed as described,(1, 3) using a Vanquish UHPLC system coupled online to a high-resolution Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7 µm; Phenomenex, Torrance, CA, USA) at 45°C. A volume of 10 μl of sample extracts was injected into the UHPLC-MS. Each sample was injected and run with two different chromatographic and MS conditions using a 5 minute gradient at 450 µL/minute from 5-95% B (A: 5% acetonitrile, 95%water/1 mM ammonium acetate; B:95%acetonitrile/5% water, 1 mM ammonium acetate) and the MS was operated in negative ion mode.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
Chromatography Type:Reversed phase
  
Chromatography ID:CH001984
Chromatography Summary:UHPLC-MS metabolomics analyses were performed as described,(1, 3) using a Vanquish UHPLC system coupled online to a high-resolution Q Exactive mass spectrometer (Thermo Fisher, Bremen, Germany). Samples were resolved over a Kinetex C18 column (2.1x150 mm, 1.7 µm; Phenomenex, Torrance, CA, USA) at 45°C. A volume of 10 μl of sample extracts was injected into the UHPLC-MS. Each sample was injected and run with two different chromatographic and MS conditions, using a 5 minute gradient at 450 µL/minute from 5-95% B (A: water/0.1% formic acid; B:acetonitrile/0.1% formic acid) and the MS was operated in positive mode.
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Kinetex C18 (150 x 2.1mm, 2.6 um)
Chromatography Type:Reversed phase

MS:

MS ID:MS002488
Analysis ID:AN002689
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode at 70,000 resolution in the 60-900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative or positive ion mode (separate runs).
Ion Mode:NEGATIVE
  
MS ID:MS002489
Analysis ID:AN002690
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:The UHPLC system was coupled online with a Q Exactive (Thermo, San Jose, CA, USA) scanning in Full MS mode at 70,000 resolution in the 60-900 m/z range, 4 kV spray voltage, 15 sheath gas and 5 auxiliary gas, operated in negative or positive ion mode (separate runs).
Ion Mode:POSITIVE
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