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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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Related in: MedlinePlus

Comparison of cytokine levels between tube types and processing protocols.Concentration of 20 cytokines in pg/ml from plasma collected in K2EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.
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pone-0038290-g002: Comparison of cytokine levels between tube types and processing protocols.Concentration of 20 cytokines in pg/ml from plasma collected in K2EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.

Mentions: The higher levels of cytokines observed in tubes containing protease inhibitors may be related to the fact that different protocols were used to process each type of tube. Therefore, in order to further evaluate P100 tubes, we analysed these tubes again, this time comparing different processing protocols recommended by the company (Protocol A and B), in addition to the protocol used regularly in our laboratory for plasma processing (Protocol C) (Table 1). All samples were processed immediately after blood collection (T0). As mentioned above, 20 cytokines out of the 27 could be measured by the assay. Of the three different protocols used to process P100 tubes, the most significant impact on cytokine levels was observed when we compared the BD initial protocol (protocol A) to the other 2 protocols, i.e., BD updated protocol (protocol B) and protocol C (in-house protocol). As shown in Figure 2, the mean levels of 16 cytokines were higher when samples were processed with protocol A compared to protocol B. In other words, centrifugation under refrigerated conditions for a shorter period of time resulted in statistically significant higher levels for 15 of these analytes (nine of which were interleukins), with percent differences ranging between 50% and 970% (Figure 2A). Protocols B and C are quite similar as the differences between them were mainly differences in centrifugation speed and the addition of a second centrifugation step, without changing centrifugation temperature. These differences did not significantly affect plasma cytokine levels since we found comparable levels for all analytes measured with both methods. No statistically significant differences were observed, except for FGF-b, the levels of which were slightly higher (15%) in samples processed with Protocol C (pā€Š=ā€Š0.03) (Figure 2B). Thus it appears that results with the P100 tubes are largely dependent on the temperature at which centrifugation of the plasma samples is performed. Interestingly, we identified three proteins whose levels were very stable irrespective of the P100 tube processing protocol: these include Eotaxin, MCP-1 and MIP-1b (Figure 2).


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)

Comparison of cytokine levels between tube types and processing protocols.Concentration of 20 cytokines in pg/ml from plasma collected in K2EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0038290-g002: Comparison of cytokine levels between tube types and processing protocols.Concentration of 20 cytokines in pg/ml from plasma collected in K2EDTA tubes, P100 tubes processed with different protocols and CTAD tubes. All tubes were processed immediately after collection (T0). Brackets depict groups compared using paired t-tests and for which statistical significance * p<0.05 was found.
Mentions: The higher levels of cytokines observed in tubes containing protease inhibitors may be related to the fact that different protocols were used to process each type of tube. Therefore, in order to further evaluate P100 tubes, we analysed these tubes again, this time comparing different processing protocols recommended by the company (Protocol A and B), in addition to the protocol used regularly in our laboratory for plasma processing (Protocol C) (Table 1). All samples were processed immediately after blood collection (T0). As mentioned above, 20 cytokines out of the 27 could be measured by the assay. Of the three different protocols used to process P100 tubes, the most significant impact on cytokine levels was observed when we compared the BD initial protocol (protocol A) to the other 2 protocols, i.e., BD updated protocol (protocol B) and protocol C (in-house protocol). As shown in Figure 2, the mean levels of 16 cytokines were higher when samples were processed with protocol A compared to protocol B. In other words, centrifugation under refrigerated conditions for a shorter period of time resulted in statistically significant higher levels for 15 of these analytes (nine of which were interleukins), with percent differences ranging between 50% and 970% (Figure 2A). Protocols B and C are quite similar as the differences between them were mainly differences in centrifugation speed and the addition of a second centrifugation step, without changing centrifugation temperature. These differences did not significantly affect plasma cytokine levels since we found comparable levels for all analytes measured with both methods. No statistically significant differences were observed, except for FGF-b, the levels of which were slightly higher (15%) in samples processed with Protocol C (pā€Š=ā€Š0.03) (Figure 2B). Thus it appears that results with the P100 tubes are largely dependent on the temperature at which centrifugation of the plasma samples is performed. Interestingly, we identified three proteins whose levels were very stable irrespective of the P100 tube processing protocol: these include Eotaxin, MCP-1 and MIP-1b (Figure 2).

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
Related in: MedlinePlus