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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.

Aguilar-Mahecha A, Kuzyk MA, Domanski D, Borchers CH, Basik M - PLoS ONE (2012)

Bottom Line: Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies.We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay.Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lady Davis Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.

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Cytokine levels at time 0.Concentration of cytokines in pg/ml from plasma collected in K2EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, + cytokines not detected in K2EDTA samples.
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pone-0038290-g001: Cytokine levels at time 0.Concentration of cytokines in pg/ml from plasma collected in K2EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, + cytokines not detected in K2EDTA samples.

Mentions: Our cytokine analysis was performed using plasma from blood collected in k2EDTA tubes and tubes containing k2EDTA and protease inhibitors (BD™ P100 Blood Collection Tubes). Each type of tube was processed at several time points after collection (T0, T2, and T6) using the processing protocol as shown in Table 1 (Protocol A or Protocol C). We observed that for at least 80% of the cytokines detected at time 0, the levels were higher in samples collected in k2EDTA tubes containing protease inhibitors (P100 tubes) and processed with the initial manufacturer guidelines (protocol A) than in samples collected in k2EDTA tubes processed with protocol C, a standard protocol for processing plasma samples (Figure 1). This observation was consistent for all time points analysed (Table S3). Paired t-tests comparing the levels of cytokines measured in both tube types at each of the time points showed significantly (p<0.05) higher mean concentrations in P100 plasma relative to k2EDTA plasma for 9 cytokines in samples analysed at T0 (mean percent change of 533% (82%–2480%)), for 10 cytokines in T2 samples (mean percent change of 134% (29%–324%)) and for 9 cytokines in T6 samples (mean percent change of 151% (35%–765%)). Interleukins 2, 8, 10 and 15 were not detected in k2EDTA samples in at least one of the time points analysed but were detectable at all time points in samples from P100 tubes (Figure 1 and Table S3). Only for IP-10 were higher levels detected in k2EDTA samples when compared to P100 at all time points.


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.

Aguilar-Mahecha A, Kuzyk MA, Domanski D, Borchers CH, Basik M - PLoS ONE (2012)

Cytokine levels at time 0.Concentration of cytokines in pg/ml from plasma collected in K2EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, + cytokines not detected in K2EDTA samples.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3368926&req=5

pone-0038290-g001: Cytokine levels at time 0.Concentration of cytokines in pg/ml from plasma collected in K2EDTA (white bars) and P100 (black bars) tubes processed immediately after collection (T0) using protocol A. The levels of 20 cytokines detected using the Bio-Plex® Assay are shown (RANTES levels not included in the figure, concentration is off scale). Paired t-test comparisons were performed for each cytokine between tube types. * p<0.05, + cytokines not detected in K2EDTA samples.
Mentions: Our cytokine analysis was performed using plasma from blood collected in k2EDTA tubes and tubes containing k2EDTA and protease inhibitors (BD™ P100 Blood Collection Tubes). Each type of tube was processed at several time points after collection (T0, T2, and T6) using the processing protocol as shown in Table 1 (Protocol A or Protocol C). We observed that for at least 80% of the cytokines detected at time 0, the levels were higher in samples collected in k2EDTA tubes containing protease inhibitors (P100 tubes) and processed with the initial manufacturer guidelines (protocol A) than in samples collected in k2EDTA tubes processed with protocol C, a standard protocol for processing plasma samples (Figure 1). This observation was consistent for all time points analysed (Table S3). Paired t-tests comparing the levels of cytokines measured in both tube types at each of the time points showed significantly (p<0.05) higher mean concentrations in P100 plasma relative to k2EDTA plasma for 9 cytokines in samples analysed at T0 (mean percent change of 533% (82%–2480%)), for 10 cytokines in T2 samples (mean percent change of 134% (29%–324%)) and for 9 cytokines in T6 samples (mean percent change of 151% (35%–765%)). Interleukins 2, 8, 10 and 15 were not detected in k2EDTA samples in at least one of the time points analysed but were detectable at all time points in samples from P100 tubes (Figure 1 and Table S3). Only for IP-10 were higher levels detected in k2EDTA samples when compared to P100 at all time points.

Bottom Line: Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies.We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay.Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Lady Davis Institute, McGill University, Montreal, Quebec, Canada.

ABSTRACT
Blood sample processing and handling can have a significant impact on the stability and levels of proteins measured in biomarker studies. Such pre-analytical variability needs to be well understood in the context of the different proteomics platforms available for biomarker discovery and validation. In the present study we evaluated different types of blood collection tubes including the BD P100 tube containing protease inhibitors as well as CTAD tubes, which prevent platelet activation. We studied the effect of different processing protocols as well as delays in tube processing on the levels of 55 mid and high abundance plasma proteins using novel multiple-reaction monitoring-mass spectrometry (MRM-MS) assays as well as 27 low abundance cytokines using a commercially available multiplexed bead-based immunoassay. The use of P100 tubes containing protease inhibitors only conferred proteolytic protection for 4 cytokines and only one MRM-MS-measured peptide. Mid and high abundance proteins measured by MRM are highly stable in plasma left unprocessed for up to six hours although platelet activation can also impact the levels of these proteins. The levels of cytokines were elevated when tubes were centrifuged at cold temperature, while low levels were detected when samples were collected in CTAD tubes. Delays in centrifugation also had an impact on the levels of cytokines measured depending on the type of collection tube used. Our findings can help in the development of guidelines for blood collection and processing for proteomic biomarker studies.

Show MeSH