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 ID | ST001644 |
Study Title | In Vitro Characterization and Metabolomic Analysis of Cold-Stored Platelets |
Study 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 |
angelo.dalessandro@cuanschutz.edu | |
Phone | 3037245798 |
Submit Date | 2021-01-08 |
Num Groups | 3 |
Total Subjects | 8 |
Raw Data Available | Yes |
Raw Data File Type(s) | raw(Thermo) |
Analysis Type Detail | LC-MS |
Release Date | 2021-01-25 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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 |
Species Group: | Mammals |
Factors:
Subject type: Human; Subject species: Homo sapiens (Factor headings shown in green)
mb_sample_id | local_sample_id | Treatment | Day |
---|---|---|---|
SA151300 | 60_B6_D14_4NS+ | 4NS | 14 |
SA151301 | 70_B7_D14_4NS+ | 4NS | 14 |
SA151302 | 80_B8_D14_4NS+ | 4NS | 14 |
SA151303 | 50_B5_D14_4NS+ | 4NS | 14 |
SA151304 | 40_B4_D14_4NS+ | 4NS | 14 |
SA151305 | 20_B2_D14_4NS+ | 4NS | 14 |
SA151306 | 10_B1_D14_4NS+ | 4NS | 14 |
SA151307 | 10_B1_D14_4NS- | 4NS | 14 |
SA151308 | 20_B2_D14_4NS- | 4NS | 14 |
SA151309 | 60_B6_D14_4NS- | 4NS | 14 |
SA151310 | 70_B7_D14_4NS- | 4NS | 14 |
SA151311 | 80_B8_D14_4NS- | 4NS | 14 |
SA151312 | 50_B5_D14_4NS- | 4NS | 14 |
SA151313 | 40_B4_D14_4NS- | 4NS | 14 |
SA151314 | 30_B3_D14_4NS+ | 4NS | 14 |
SA151315 | 30_B3_D14_4NS- | 4NS | 14 |
SA151316 | 4_B1_D2_4NS+ | 4NS | 2 |
SA151317 | 44_B5_D2_4NS+ | 4NS | 2 |
SA151318 | 34_B4_D2_4NS+ | 4NS | 2 |
SA151319 | 24_B3_D2_4NS+ | 4NS | 2 |
SA151320 | 4_B1_D2_4NS- | 4NS | 2 |
SA151321 | 54_B6_D2_4NS+ | 4NS | 2 |
SA151322 | 64_B7_D2_4NS+ | 4NS | 2 |
SA151323 | 74_B8_D2_4NS+ | 4NS | 2 |
SA151324 | 14_B2_D2_4NS- | 4NS | 2 |
SA151325 | 54_B6_D2_4NS- | 4NS | 2 |
SA151326 | 64_B7_D2_4NS- | 4NS | 2 |
SA151327 | 44_B5_D2_4NS- | 4NS | 2 |
SA151328 | 74_B8_D2_4NS- | 4NS | 2 |
SA151329 | 14_B2_D2_4NS+ | 4NS | 2 |
SA151330 | 24_B3_D2_4NS- | 4NS | 2 |
SA151331 | 34_B4_D2_4NS- | 4NS | 2 |
SA151332 | 37_B4_D7_4NS- | 4NS | 7 |
SA151333 | 7_B1_D7_4NS+ | 4NS | 7 |
SA151334 | 47_B5_D7_4NS- | 4NS | 7 |
SA151335 | 27_B3_D7_4NS- | 4NS | 7 |
SA151336 | 67_B7_D7_4NS- | 4NS | 7 |
SA151337 | 17_B2_D7_4NS+ | 4NS | 7 |
SA151338 | 77_B8_D7_4NS- | 4NS | 7 |
SA151339 | 57_B6_D7_4NS- | 4NS | 7 |
SA151340 | 67_B7_D7_4NS+ | 4NS | 7 |
SA151341 | 17_B2_D7_4NS- | 4NS | 7 |
SA151342 | 7_B1_D7_4NS- | 4NS | 7 |
SA151343 | 77_B8_D7_4NS+ | 4NS | 7 |
SA151344 | 57_B6_D7_4NS+ | 4NS | 7 |
SA151345 | 37_B4_D7_4NS+ | 4NS | 7 |
SA151346 | 47_B5_D7_4NS+ | 4NS | 7 |
SA151347 | 27_B3_D7_4NS+ | 4NS | 7 |
SA151348 | 79_B8_D14_4S+ | 4S | 14 |
SA151349 | 9_B1_D14_4S- | 4S | 14 |
SA151350 | 69_B7_D14_4S+ | 4S | 14 |
SA151351 | 19_B2_D14_4S- | 4S | 14 |
SA151352 | 9_B1_D14_4S+ | 4S | 14 |
SA151353 | 39_B4_D14_4S- | 4S | 14 |
SA151354 | 49_B5_D14_4S- | 4S | 14 |
SA151355 | 59_B6_D14_4S- | 4S | 14 |
SA151356 | 69_B7_D14_4S- | 4S | 14 |
SA151357 | 29_B3_D14_4S- | 4S | 14 |
SA151358 | 59_B6_D14_4S+ | 4S | 14 |
SA151359 | 49_B5_D14_4S+ | 4S | 14 |
SA151360 | 39_B4_D14_4S+ | 4S | 14 |
SA151361 | 29_B3_D14_4S+ | 4S | 14 |
SA151362 | 19_B2_D14_4S+ | 4S | 14 |
SA151363 | 79_B8_D14_4S- | 4S | 14 |
SA151364 | 53_B6_D2_4S- | 4S | 2 |
SA151365 | 23_B3_D2_4S- | 4S | 2 |
SA151366 | 13_B2_D2_4S- | 4S | 2 |
SA151367 | 33_B4_D2_4S- | 4S | 2 |
SA151368 | 43_B5_D2_4S- | 4S | 2 |
SA151369 | 63_B7_D2_4S- | 4S | 2 |
SA151370 | 13_B2_D2_4S+ | 4S | 2 |
SA151371 | 73_B8_D2_4S- | 4S | 2 |
SA151372 | 3_B1_D2_4S+ | 4S | 2 |
SA151373 | 73_B8_D2_4S+ | 4S | 2 |
SA151374 | 23_B3_D2_4S+ | 4S | 2 |
SA151375 | 63_B7_D2_4S+ | 4S | 2 |
SA151376 | 3_B1_D2_4S- | 4S | 2 |
SA151377 | 33_B4_D2_4S+ | 4S | 2 |
SA151378 | 43_B5_D2_4S+ | 4S | 2 |
SA151379 | 53_B6_D2_4S+ | 4S | 2 |
SA151380 | 26_B3_D7_4S+ | 4S | 7 |
SA151381 | 46_B5_D7_4S+ | 4S | 7 |
SA151382 | 36_B4_D7_4S+ | 4S | 7 |
SA151383 | 66_B7_D7_4S+ | 4S | 7 |
SA151384 | 6_B1_D7_4S- | 4S | 7 |
SA151385 | 76_B8_D7_4S+ | 4S | 7 |
SA151386 | 56_B6_D7_4S+ | 4S | 7 |
SA151387 | 46_B5_D7_4S- | 4S | 7 |
SA151388 | 76_B8_D7_4S- | 4S | 7 |
SA151389 | 16_B2_D7_4S+ | 4S | 7 |
SA151390 | 6_B1_D7_4S+ | 4S | 7 |
SA151391 | 66_B7_D7_4S- | 4S | 7 |
SA151392 | 56_B6_D7_4S- | 4S | 7 |
SA151393 | 26_B3_D7_4S- | 4S | 7 |
SA151394 | 36_B4_D7_4S- | 4S | 7 |
SA151395 | 16_B2_D7_4S- | 4S | 7 |
SA151396 | 1_B1_D1_RT- | RT | 1 |
SA151397 | 21_B3_D1_RT- | RT | 1 |
SA151398 | 11_B2_D1_RT- | RT | 1 |
SA151399 | 31_B4_D1_RT- | RT | 1 |
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,2.6um) | Phenomenex Kinetex C18 (150 x 2.1mm,2.6um) |
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,2.6um) |
Column Temperature: | 45 |
Flow Gradient: | 5 minute gradient from 5-95% B |
Flow Rate: | 450 µL/min |
Solvent A: | 5% acetonitrile/95% water; 1 mM ammonium acetate |
Solvent B: | 95% acetonitrile/5% water; 1 mM ammonium acetate |
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,2.6um) |
Column Temperature: | 45 |
Flow Gradient: | 5 minute gradient from 5-95% B |
Flow Rate: | 450 µL/min |
Solvent A: | 5% acetonitrile/95% water; 1 mM ammonium acetate |
Solvent B: | 95% acetonitrile/5% water; 1 mM ammonium acetate |
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 |