The effect of pre-analytical variability on the measurement of MRM-MS-based mid- to high-abundance plasma protein biomarkers and a panel of cytokines.
Author(s): Aguilar-Mahecha A, Kuzyk MA, Domanski D, Borchers CH, Basik M
Publication: PLoS One, 2012, Vol. 7, Page e38290
PubMed ID: 22701622 PubMed Review Paper? No
Purpose of Paper
This paper investigated whether a room temperature processing delay, and use of protease inhibitors (P100 tubes) or tube additives that prevent platelet activation (CTAD tubes), affect the levels of 56 medium to high abundance plasma proteins and 20 cytokines. Potential effects of centrifugation protocols on cytokine levels were also explored.
Conclusion of Paper
Peptide and cytokine levels were affected by the type of tube used for blood collection. Plasma from CTAD tubes displayed lower peptide levels in immediately processed specimens, and more stable levels in those subjected to a processing delay than plasma from either EDTA or P100 tubes. In fact, peptide levels were not significantly affected by a room temperature processing delay of up to 6 h when blood was collected in CTAD tubes, while plasma collected in other tube types exhibited significant changes over time. While 20 of the 27 cytokines targeted by the immunoassay were detectable in plasma collected in EDTA or P100 tubes, only 11 cytokines were detectable in plasma from CTAD tubes, which suggests elevated levels were due to platelet activation. Plasma from P100 tubes (the only tube investigated) displayed altered levels of up to 80% of the cytokines analyzed when centrifugation conditions were compared, specifically levels were elevated when specimens underwent centrifugation at 8°C compared to those centrifuged at room temperature.
Studies
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Study Purpose
This study investigated whether a room temperature processing delay and the use of protease inhibitors or tube additives that prevent platelet activation affect the detection or levels of 56 medium to high abundance plasma proteins. Blood was collected from 14 healthy but fasting individuals into K2EDTA tubes, BD P100 tubes (K2EDTA and a protease inhibitor cocktail), and CTAD tubes (citrate and additives that prevent platelet activation) and processed after a 0, 2, or 6 h room temperature delay. Specimens were processed similarly, and plasma was stored at -80°C prior to analysis.
Summary of Findings:
A processing delay of 2-6 h did not significantly alter plasma peptide levels when blood was collected in CTAD or EDTA tubes without a protease inhibitor. However, when blood was collected in P100 tubes 7 of the 56 peptides analyzed declined significantly after a 2 h delay (p<0.05), and 5 declined significantly after a 6 h delay (p<0.05) compared to immediately processed specimens. Differences between EDTA plasma collected with and without protease inhibitors over processing delays of up to 6 h were limited to a single peptide, as levels of plasminogen were significantly, but modestly higher (by 4%) in plasma collected in EDTA tubes compared to P100 tubes. Conversely, plasma collected in CTAD tubes displayed lower peptide levels than plasma collected in other tube types both in immediately processed specimens and those subjected to a processing delay, suggesting that release of these peptides may be influenced by platelet activation. Of the 56 peptides analyzed, 21, 28 and 41 peptides displayed 10-48% lower levels in plasma collected in CTAD tubes compared to plasma collected in one or both of the other tube types (K2EDTA and/or P100 tubes) after a 0 h, 2 h and 6 h processing delay, respectively. Fibronection was the only peptide with higher levels in CTAD tubes over the 6 h time course (64-128%) compared to EDTA tubes with and without a protease inhibitor.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Peptide MRM-MS Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Protease inhibitor Cocktail
No protease inhibitor added
Biospecimen Acquisition Type of collection container/solution BD P100 tube
CTAD tube
K2EDTA tube
Storage Time at room temperature 0 h
2 h
6 h
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Study Purpose
This study investigated whether a room temperature processing delay, use of protease inhibitors or tube additives that prevent platelet activation, and centrifugation conditions affect levels of 20 targeted cytokines. Blood was collected from 14 healthy but fasting individuals into K2EDTA tubes, BD P100 tubes (K2EDTA and a protease inhibitor cocktail), and CTAD tubes (citrate and additives that prevent platelet activation) and processed after a 0, 2, or 6 h room temperature delay. Blood collected into P100 tubes was subjected to three different centrifugation protocols: (1) 2000 x g for 5 min at 8°C; (2) 2500 x g for 20 min at room temperature; or (3) 1300 x g for 10 min followed by 2500 x g for 15 min at room temperature. Blood collected into EDTA tubes were centrifuged for 1300 x g for 10 min then 2500 x g for 15 min at room temperature. Blood collected into CTAD tubes was centrifuged at 1300 x g for 10 min at room temperature. Plasma was stored at -80°C prior to analysis.
Summary of Findings:
Levels of two cytokines differed after a processing delay of 2 h in plasma from P100 tubes compared to immediately processed controls, while cytokine levels remained stable in EDTA tubes. A 6 h processing delay increased levels of 6 cytokines in plasma from EDTA tubes (by 13-230%), but decreased levels of 8 cytokines in plasma from P100 tubes (by 9-43%). Approximately 80% of cytokines displayed higher levels when P100 tubes were processed by a single centrifugation at 8°C (2000 x g for 5 min) than two centrifugations at room temperature (1300 x g for 10 min, 2500 x g for 15 min). This was true for all specimens, regardless of the processing delay. Further, 50-970% higher levels of 15 cytokines were observed in specimens from P100 tubes centrifuged once at 8°C (2000 x g for 5 min ) than when centrifuged once at room temperature (2500 x g for 20 min). Cytokine levels were also significantly higher in plasma from P100 tubes than EDTA tubes (134-533% mean percent change) for 9 cytokines in immediately processed specimens, 10 cytokines in specimens subjected to a 2 h delay, and 9 cytokines subjected to a 6 h delay (all p<0.05) when processed under the same conditions (1300 x g for 10 min, 2500 x g for 15 min at room temperature). IP-10 alone displayed modestly but significantly higher levels in plasma from EDTA tubes compared to 100 tubes (p<0.05). Plasma collected in EDTA and P100 tubes and processed using identical centrifugation protocols without delay differed significantly for 4 cytokines, all of which were higher in plasma from P100 tubes (by 13-32%; p<0.05). However, only 11 of the 20 cytokines targeted by the assay were detectable in plasma from CTAD tubes, and 8 of these displayed significantly lower levels than observed in plasma from EDTA or P100 tubes.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Protein Immunoassay Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Multiple temperatures compared
Different number of centrifugation steps compared
Storage Time at room temperature 0 h
2 h
6 h
Analyte Extraction and Purification Protease inhibitor Cocktail
No protease inhibitor added
Biospecimen Acquisition Type of collection container/solution K2EDTA tube
BD P100 tube
CTAD tube