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Cell-Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer.

ABSTRACT: Optimized blood collection tubes (BCT) have been developed to expand the utility of plasma cell-free DNA (cfDNA) and are in clinical use. The appropriateness of plasma collected and stored in these tubes for proteomic analysis is unknown.Paired blood samples were collected in BCT and traditional K3EDTA (EDTA) tubes from healthy controls and from colorectal cancer (CRC) patients before and after surgery, and stored for between 45 min and 48 h at room temperature. Plasma proteins were analyzed following high-abundant plasma protein depletion in quantitative discovery and targeted proteomics by liquid chromatography tandem-mass spectrometry (LC-MS/MS).BCT reduced cellular protein contamination in healthy controls over time, and increased the number of high confident low-abundant protein identifications in CRC blood samples compared to matched samples collected in EDTA tubes. The known CRC plasma protein biomarker, carcinoembryonic antigen (CEA), showed elevated levels across patients pre-operatively when collected and stored in BCT compared to EDTA tubes. Emerging CRC biomarkers, Dickkopf-3 (DKK3) and Gelsolin (GSN), showed elevated levels pre-operatively when collected in BCT.Optimized BCT are appropriate for low-abundant plasma protein analysis and can be used with confidence for clinical proteomics.

SUBMITTER: Almazi JG

PROVIDER: S-EPMC5947838 | biostudies-other | 2018 May

REPOSITORIES: biostudies-other

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Cell-Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer.

Almazi Juhura G JG Pockney Peter P Gedye Craig C Smith Nathan D ND Hondermarck Hubert H Verrills Nicole M NM Dun Matthew D MD

Proteomics. Clinical applications 20180330 3

<h4>Background</h4>Optimized blood collection tubes (BCT) have been developed to expand the utility of plasma cell-free DNA (cfDNA) and are in clinical use. The appropriateness of plasma collected and stored in these tubes for proteomic analysis is unknown.<h4>Methods</h4>Paired blood samples were collected in BCT and traditional K3EDTA (EDTA) tubes from healthy controls and from colorectal cancer (CRC) patients before and after surgery, and stored for between 45 min and 48 h at room temperature ...[more]

PMID: 29476593

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Project description:ImportanceBlood collection for laboratory testing in intensive care unit (ICU) patients is a modifiable contributor to anemia and red blood cell (RBC) transfusion. Most blood withdrawn is not required for analysis and is discarded.ObjectiveTo determine whether transitioning from standard-volume to small-volume vacuum tubes for blood collection in ICUs reduces RBC transfusion without compromising laboratory testing procedures.Design, setting, and participantsStepped-wedge cluster randomized trial in 25 adult medical-surgical ICUs in Canada (February 5, 2019 to January 21, 2021).InterventionsICUs were randomized to transition from standard-volume (n = 10 940) to small-volume tubes (n = 10 261) for laboratory testing.Main outcomes and measuresThe primary outcome was RBC transfusion (units per patient per ICU stay). Secondary outcomes were patients receiving at least 1 RBC transfusion, hemoglobin decrease during ICU stay (adjusted for RBC transfusion), specimens with insufficient volume for testing, length of stay in the ICU and hospital, and mortality in the ICU and hospital. The primary analysis included patients admitted for 48 hours or more, excluding those admitted during a 5.5-month COVID-19-related trial hiatus.ResultsIn the primary analysis of 21 201 patients (mean age, 63.5 years; 39.9% female), which excluded 6210 patients admitted during the early COVID-19 pandemic, there was no significant difference in RBC units per patient per ICU stay (relative risk [RR], 0.91 [95% CI, 0.79 to 1.05]; P = .19; absolute reduction of 7.24 RBC units/100 patients per ICU stay [95% CI, -3.28 to 19.44]). In a prespecified secondary analysis (n = 27 411 patients), RBC units per patient per ICU stay decreased after transition from standard-volume to small-volume tubes (RR, 0.88 [95% CI, 0.77 to 1.00]; P = .04; absolute reduction of 9.84 RBC units/100 patients per ICU stay [95% CI, 0.24 to 20.76]). Median decrease in transfusion-adjusted hemoglobin was not statistically different in the primary population (mean difference, 0.10 g/dL [95% CI, -0.04 to 0.23]) and lower in the secondary population (mean difference, 0.17 g/dL [95% CI, 0.05 to 0.29]). Specimens with insufficient quantity for analysis were rare (≤0.03%) before and after transition.Conclusions and relevanceUse of small-volume blood collection tubes in the ICU may decrease RBC transfusions without affecting laboratory analysis.Trial registrationClinicalTrials.gov Identifier: NCT03578419.

Project description:IntroductionPrecision medicine has revolutionized the understanding and treatment of cancer by identifying subsets of patients who are amenable to specific treatments according to their molecular characteristics, as exemplified by epidermal growth factor receptor (EGFR) mutations in non-small cell lung cancer (NSCLC). Although tissue biopsy is the gold standard for determining molecular alterations in tumors, its limitations have prompted the development of new techniques for studying tumor biomarkers in liquid biopsies, such as mutation analysis in cell-free DNA (cfDNA). cfDNA analysis can accurately determine tumor progression and prognosis and more effectively identify appropriate targeted therapies. However, cfDNA is vulnerable, particularly during plasma sample shipping.ObjectiveWe compared the cell- and DNA-stabilizing properties of cell-free DNA blood collection tubes (BCTs) with those of the traditional shipping method (frozen plasma) for EGFR mutation testing using the cobas® EGFR Mutation Test v2 in a prospective cohort of 49 patients from three different Spanish hospitals.MethodsIn total, 98 NSCLC samples, two from each patient, were studied; five of the 49 cases were considered invalid by cobas® with one of the two shipping methods analyzed. After excluding these samples, we analyzed 88 samples from 44 patients. Considering the current methodology (frozen plasma) for sending samples as the gold standard, we evaluated the sensitivity and specificity of cfDNA BCT shipment.ResultsThe global agreement between the two methods was 95.4%, with 100% sensitivity and 94.6% specificity for the cfDNA BCTs. cfDNA BCTs had a positive predictive value of 81.8% and negative predictive value of 100%.ConclusioncfDNA BCTs have the same sensitivity for EGFR mutation analysis in liquid biopsy as the current methodology and very high specificity. They also have some additional advantages in terms of collection and further shipment. Therefore, cfDNA BCTs can be perfectly incorporated into the routine practice for EGFR mutation determination.FundingRoche Farma S.A., Spain.

Project description:Background:One of the most significant challenges in colorectal cancer (CRC) management is the use of compliant early stage population-based diagnostic tests as adjuncts to confirmatory colonoscopy. Despite the near curative nature of early clinical stage surgical resection, mortality remains unacceptably high-as the majority of patients diagnosed by faecal haemoglobin followed by colonoscopy occur at latter stages. Additionally, current population-based screens reliant on fecal occult blood test (FOBT) have low compliance (~ 40%) and tests suffer low sensitivities. Therefore, blood-based diagnostic tests offer survival benefits from their higher compliance (≥ 97%), if they can at least match the sensitivity and specificity of FOBTs. However, discovery of low abundance plasma biomarkers is difficult due to occupancy of a high percentage of proteomic discovery space by many high abundance plasma proteins (e.g., human serum albumin). Methods:A combination of high abundance protein ultradepletion (e.g., MARS-14 and an in-house IgY depletion columns) strategies, extensive peptide fractionation methods (SCX, SAX, High pH and SEC) and SWATH-MS were utilized to uncover protein biomarkers from a cohort of 100 plasma samples (i.e., pools of 20 healthy and 20 stages I-IV CRC plasmas). The differentially expressed proteins were analyzed using ANOVA and pairwise t-tests (p < 0.05; fold-change > 1.5), and further examined with a neural network classification method using in silico augmented 5000 patient datasets. Results:Ultradepletion combined with peptide fractionation allowed for the identification of a total of 513 plasma proteins, 8 of which had not been previously reported in human plasma (based on PeptideAtlas database). SWATH-MS analysis revealed 37 protein biomarker candidates that exhibited differential expression across CRC stages compared to healthy controls. Of those, 7 candidates (CST3, GPX3, CFD, MRC1, COMP, PON1 and ADAMDEC1) were validated using Western blotting and/or ELISA. The neural network classification narrowed down candidate biomarkers to 5 proteins (SAA2, APCS, APOA4, F2 and AMBP) that had maintained accuracy which could discern early (I/II) from late (III/IV) stage CRC. Conclusion:MS-based proteomics in combination with ultradepletion strategies have an immense potential of identifying diagnostic protein biosignature.

Dataset Information

Cell-Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer.

Publications

Cell-Free DNA Blood Collection Tubes Are Appropriate for Clinical Proteomics: A Demonstration in Colorectal Cancer.

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