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.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

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
Email	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

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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
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

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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).

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.).

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