Advancing Plasma Protein Preservation

PRECISION™ T E C H N O L O G Y PRECISION™ T E C H N O L O G Y Powered by www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 The novel HEMAcollect™•PROTEIN blood collection tube designed for the extended preservation of plasma proteins Cameron Wood, Thanh Dang, Shaimaa Ahmed, Amit Arora, Kyle MacDonald, Graham Morse, Tara Crawford Parks DNA Genotek, Ottawa, Ontario, Canada Introduction Proteomics, the large-scale study of proteins and their functions, is a rapidly advancing field with broad applications in biomarker discovery, disease diagnostics, drug development and personalized medicine. These advancements have significantly deepened our understanding of complex disease areas such as neurology, oncology, metabolic disorders and immunology.1-3 Among the various biological matrices utilized in proteomic research, blood, particularly plasma, remains a critical sample type due to its accessibility and its ability to reflect systemic physiological and pathological states.4 Despite recent progress, large-scale proteomic interpretation remains in the early stages, with numerous technical and logistical barriers still to overcome.5 One of the most pressing challenges lies in the preanalytical stage of sample collection and handling, where delays in processing and non‑standardized workflows can result in significant alterations to the plasma proteome.6-7 Preanalytical variables such as blood collection techniques, centrifugation protocols, storage duration, temperature fluctuations and shipping delays can introduce variability that compromises analyte stability and reduces the reproducibility and interpretation of downstream proteomic analyses. Improper handling of blood samples can disrupt sample homeostasis, leading to hemolysis, platelet activation and proteolytic degradation ex vivo. Hemolysis and platelet activation can lead to the release of intracellular proteins into plasma, further confounding proteomic analyses by producing a sample that no longer accurately reflects the in vivo state.8-9 These disruptions are particularly problematic in studies aiming to detect low-abundance plasma proteins or subtle inter-cohort differences, where even minor preanalytical variations can mask or obscure biologically meaningful signals.10 To address the preanalytical issues that can arise during blood collection, we have developed a HEMAcollect™•PROTEIN blood collection tube (BCT), with novel ProteoPrecision™ technology, to support the generation of reliable proteomic data and the extended preservation of plasma proteins for up to 7 days. The HEMAcollect™•PROTEIN BCT is designed to stabilize proteins at the point of collection, minimizing proteomic changes and proteolytic degradation while preserving sample homeostasis. It also limits hemolysis and the release of intracellular and platelet-derived proteins by minimizing blood cells lysis and platelet activation. This paper demonstrates how the HEMAcollect™•PROTEIN BCT overcomes these key preanalytical variables to enable the generation of high-quality proteomic data across key platforms and technologies. Results The HEMAcollect™•PROTEIN BCT minimizes hemolysis and platelet activation after sample collection, storage and transport Hemolysis A visual assessment of hemolysis was performed for whole blood collected in a HEMAcollect™•PROTEIN BCT (n = 6) after storage at room temperature for a total duration of 7 days (Figure 1A). A quantitative assessment of hemolysis was conducted in HEMAcollect™•PROTEIN BCT-collected samples (n = 10) following exposure to simulated transportation conditions and subsequent storage for up to 7 days at room temperature as described in the methods (Figure 1B). All HEMAcollect™•PROTEIN BCT-collected samples remained at or below a 100 mg/dL concentration on the hemolytic index indicating minimal hemolysis11-12, while 50% of EDTA-collected samples had concentrations above 100 mg/dL at the 7-day timepoint. The plasma isolated from the www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 2 Hb concentration (mg/dL) 5 d 7 d T0 5 d 7 d HEMAcollect™•PROTEIN EDTA T0 0 50 100 150 200 B 250 HEMAcollect™•PROTEIN BCT has a slight brown or amber hue, which may darken after collection, unlike EDTA-derived plasma, which is straw-colored. This differentiation originates from the color of the stabilization liquid in the HEMAcollect™•PROTEIN BCT and is distinct from the pink-to-red discoloration that would be observed in hemolyzed samples collected in standard EDTA tubes. Together, these data demonstrate that the HEMAcollect™•PROTEIN BCT minimizes ex vivo hemolysis when exposed to simulated transport conditions and room temperature storage, while providing additional flexibility in sample handling compared with EDTA tubes. Platelet activation To evaluate platelet activation, ELISA-based assays were conducted for the surface protein marker P-selectin (CD62P), a classical platelet activation marker13, and epidermal growth factor (EGF), which is released upon platelet activation14. In this series of experiments, whole blood was collected into HEMAcollect™•PROTEIN and EDTA BCTs (n = 10 donors per BCT) and plasma was isolated within 2 hours to represent baseline (T0). Collected blood and isolated plasma samples were exposed to simulated transport conditions, followed by storage at room temperature for a total duration of 5 days (5 d) or 7 days (7 d). Broadly, the data demonstrate lower or equivalent concentrations of CD62P in all HEMAcollect™•PROTEIN BCT conditions compared with EDTA. Most notably, plasma isolated from whole blood collected in the HEMAcollect™•PROTEIN BCT (HCPP whole blood) in the 7 d condition had a lower average fold change (7 d/T0) compared with the matching 7 d condition in EDTA (EDTA whole blood) (1.14 in the HEMAcollect™•PROTEIN BCT compared with 2.10 in EDTA) (Figure 2A). Consistent with the CD62P findings, plasma EGF concentrations were lower or equivalent in HEMAcollect™•PROTEIN BCT-collected samples relative to EDTA across the same conditions (Figure 2B). Plasma isolated from whole blood collected in HEMAcollect™•PROTEIN BCTs (HCPP whole blood) in the 7 d condition had a lower average fold change (7 d/T0) compared with the matching 7 d condition in EDTA (EDTA whole blood) (12.24 in HCPP compared with 127.25 in EDTA). Collectively, these results indicate that the HEMAcollect™•PROTEIN BCT limits ex vivo platelet activation in whole blood samples, as represented by maintaining plasma concentrations of CD62P and EGF. A Figure 1A. Visual assessment of ex vivo hemolysis. Image shows whole blood collected in a HEMAcollect™•PROTEIN BCT after 7 days of storage at room temperature (20°C-26°C/68°F-79°F). Figure 1B. Quantitative measurement of ex vivo hemolysis after extended storage and simulated transport. Concentration of hemoglobin (mg/dL) in plasma derived from HEMAcollect™•PROTEIN BCT-collected and EDTA-collected samples from 10 healthy donors at (T0) baseline) or after simulated transport of whole blood subjected to temperature cycling (4°C-30°C/39°F-86°F for a minimum of 24 hours at each respective temperature), followed by storage at room temperature (20°C-26°C/68°F-79°F) for a total duration of (5 d) 5 days or (7 d) 7 days. www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 3 The HEMAcollect™•PROTEIN BCT maintains sample homeostasis, limiting preanalytical variability after sample collection, storage and transport To evaluate blood sample homeostasis, the expression of two biomarkers was evaluated: interleukin-8 (IL-8), an intracellular cytokine prone to release from leukocytes ex vivo15, and lectin-like oxidized LDL receptor-1 (LOX-1), a membrane-associated protein activated by proteolytic cleavage during sample degradation16. The measurement of these two proteins is indicative of the maintenance of blood sample homeostasis over time. Whole blood was collected into HEMAcollect™•PROTEIN and EDTA BCTs (n = 10 donors per BCT) and plasma was isolated within 2 hours to represent baseline (T0). Collected blood and isolated plasma samples were exposed to simulated transport conditions, followed by storage at room temperature for a total duration of 5 days (5 d) or 7 days (7 d). Plasma isolated from whole blood collected in HEMAcollect™•PROTEIN BCTs (HCPP whole blood) in the 7 d condition had a lower average fold change for IL-8 levels (7 d/T0) compared with the matching 7 d condition in EDTA (EDTA whole blood) (0.90 in HCPP compared with 111.7 in EDTA) (Figure 3A). In addition, the HEMAcollect™•PROTEIN BCT preserved LOX-1 levels near baseline (2.40-fold increase at 7 d), indicating the suppression of protease activity to support the maintenance of the proteomic profile.16 In contrast, LOX-1 levels increased markedly in EDTA-collected samples (11.26‑fold increase at 7 d) reflecting elevated protease activity.16 These data indicate the effective preservation of cellular integrity and reduced ex vivo cytokine release in HEMAcollect™•PROTEIN BCT-collected samples. T0 5 d 7 d 7 d T0 5 d 7 d 7 d 0 50 100 150 200 CD62P (ng/mL) HEMAcollect™•PROTEIN EDTA HCPP whole blood HCPP plasma EDTA plasma EDTA whole blood A Storage condition Figure 2A. ELISA-based evaluation of ex vivo platelet activation via the measurement of CD62P concentrations in HEMAcollect™•PROTEIN BCT-collected and EDTA-collected samples. Samples collected from 10 healthy donors at (T0) baseline or after simulated transport of whole blood subjected to temperature cycling (4°C-30°C/39°F-86°F for a minimum of 24 hours at each respective temperature) or plasma isolated at T0 (-10°C to 30°C/14°F to 86°F for a minimum of 24 hours at each respective temperature), followed by room temperature storage (20°C-26°C/68°F-79°F) for a total duration of (5 d) 5 days or (7 d) 7 days. Figure 2B. ELISA-based evaluation of ex vivo platelet activation via the measurement of EGF concentrations in HEMAcollect™•PROTEIN BCT-collected and EDTA-collected samples. Samples collected from 10 healthy donors at (T0) baseline or after simulated transport of whole blood subjected to temperature cycling (4°C-30°C/39°F-86°F for a minimum of 24 hours at each respective temperature) or plasma isolated at T0 (-10°C to 30°C/14°F to 86°F for a minimum of 24 hours at each respective temperature), followed by storage at room temperature (20°C-26°C/68°F-79°F) for a total duration of (5 d) 5 days or (7 d) 7 days. 0 200 400 600 800 1000 EGF (pg/mL) HCPP whole blood HCPP plasma EDTA plasma EDTA whole blood Storage condition T0 5 d 7 d 7 d T0 5 d 7 d 7 d HEMAcollect™•PROTEIN EDTA B www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 4 The HEMAcollect™•PROTEIN BCT shows superior performance and direct compatibility with downstream proteomic technologies The compatibility and robustness of the HEMAcollect™•PROTEIN BCT was evaluated across a variety of downstream proteomic technologies, including untargeted mass spectrometry and multi‑target, high-throughput affinity-based proteomic platforms. Mass spectrometry To evaluate the compatibility of the HEMAcollect™•PROTEIN BCT with mass spectrometry workflows, whole blood was collected into HEMAcollect™•PROTEIN and EDTA BCTs (n = 4 donors per BCT ) and plasma was isolated within 2 hours to represent baseline (T0). First, compatibility with an LC-MS/MS workflow was investigated, which included 2 upfront abundant protein depletion kits. Following depletion, HEMAcollect™•PROTEIN BCT-derived samples showed a reduction in the most abundant proteins, similar to that observed in EDTAderived samples, as measured using a Bradford assay (93% in HCPP vs. 85% in EDTA for Thermo Fisher™; 76% in HCPP vs. 78% in EDTA for Norgen™, data not shown). This reduction demonstrates the HEMAcollect™•PROTEIN BCT compatibility with 2 common upfront depletion kits. Next, following depletion and analysis using an LC-MS/ MS workflow, the total number of proteins detected was found to be comparable between EDTA-collected and HEMAcollect™•PROTEIN BCT-collected samples with some notable differences (increased or decreased abundance) in the EDTA samples (Figure 4A, 4B). For example, some proteins known to be associated with blood cell lysis and platelet activation were increased in EDTA samples compared with HEMAcollect™•PROTEIN BCT samples, depending on the depletion kit used (HBB, PRDX2, ACTB, PBP and PF-4).17-20 Affinity-based proteomic platforms The direct compatibility and performance of HEMAcollect™•PROTEIN BCTs across a broader subset of proteins was evaluated on the Olink™ proximity extension assay (PEA) and SomaLogic™ SomaScan™ assay. For this set of experiments, whole 0 10 20 30 40 500 1000 1500 2000 IL-8 (pg/mL) HCPP whole blood HCPP plasma EDTA plasma EDTA whole blood Storage condition T0 5 d 7 d 7 d HEMAcollect™•PROTEIN T0 5 d 7 d 7 d EDTA A Figure 3A. ELISA-based evaluation of blood cell release via the measurement of IL-8 concentrations in HEMAcollect™•PROTEIN BCT-collected and EDTA-collected samples. Samples collected from 10 healthy donors at (T0) baseline or after simulated transport of whole blood subjected to temperature cycling (4°C-30°C/39°F to 86°F for a minimum of 24 hours at each respective temperature) or plasma isolated at T0 (-10°C to 30°C/14°F to 86°F for a minimum of 24 hours at each respective temperature), followed by storage at room temperature (20°C-26°C/68°F-79°F) for a total duration of (5 d) 5 days or (7 d) 7 days. Figure 3B. ELISA-based evaluation of changes in LOX-1 concentrations in HEMAcollect™•PROTEIN BCT-collected and EDTA-collected samples. Samples collected from 10 healthy donors at (T0) baseline or after simulated transport of whole blood subjected to temperature cycling (4°C-30°C/39°F-86°F for a minimum of 24 hours at each respective temperature) or plasma isolated at T0 (-10°C to 30°C/14°F to 86°F for a minimum of 24 hours at each respective temperature), followed by storage at room temperature (20°C-26°C/68°F-79°F) for a total duration of (5 d) 5 days or (7 d) 7 days. 0 20 40 60 80 100 500 1000 1500 2000 3000 4000 5000 6000 7000 8000 9000 10000 LOX-1 (pg/mL) HCPP whole blood HCPP plasma EDTA plasma EDTA whole blood Storage condition T0 5 d 7 d 7 d HEMAcollect™•PROTEIN T0 5 d 7 d 7 d EDTA B www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 5 blood was collected into HEMAcollect™•PROTEIN BCTs and EDTA BCTs and plasma was isolated within 2 hours to represent baseline (T0). Collected blood samples were either stored at room temperature for 5 days (5 d) and 7 days (7 d) or exposed to simulated transport conditions as described in the methods for a total duration of 7 days (7 d+). HEMAcollect™•PROTEIN BCT-derived samples evaluated on the Olink™ Target 48 Cytokine Panel revealed similar proteomic profiles relative to baseline (T0) samples across all conditions tested (5 d, 7 d, 7 d+) unlike EDTA-collected samples tested under the same conditions (Figure 5A, 5B). After 7 days of storage, 85% (34/40) of proteins remained stable within an average log2 fold change of ±1 (7 d/T0) in HEMAcollect™•PROTEIN BCT-collected samples across donors (n = 4), contrasted with EDTA-collected samples, which revealed that only 47.5% (19/40) of proteins remained within the same threshold. Similarly, 80% (32/40) of proteins remained stable within an average log2 fold change (7 d+/T0) of ±1 in the HEMAcollect™•PROTEIN BCT within the 7 d+ condition, whereas only 32.5% (13/40) of proteins remained stable within EDTA (Figure 5A, 5B). Consistent with the Olink™ panel data, HEMAcollect™•PROTEIN BCT-derived samples evaluated on a custom 100-protein SomaScan™ panel demonstrated proteomic profiles similar to T0 samples across all conditions tested (5 d, 7 d, 7 d+), comparable to EDTA-collected samples under matched conditions (Figure 5C, 5D). After 7 days of storage, 93% (93/100) of proteins remained stable within an average log2 fold change (7 d/T0) of ±1 in HEMAcollect™•PROTEIN BCT-collected samples across donors (n = 5) in contrast with EDTA-collected samples which revealed only 76% (76/100) of proteins remained within the same threshold. Similarly, 93% (93/100) of proteins remained stable within an average log2 fold change (7 d+/T0) of ±1 in the HEMAcollect™•PROTEIN BCT in the 7 d+ condition, whereas only 69% (69/100) of proteins remained stable within EDTA (Figure 5C, 5D). Several markers (IL-8, LOX-1, PF-4, CTAP-III, NAP-2, BTG, PDGF-AA and BDNF PBEF) associated with cellular leakage, platelet activation, protease activity and leukocyte activation are more controlled in the HEMAcollect™•PROTEIN BCT compared with the EDTA-collected samples.21-24 These findings collectively support that the HEMAcollect™•PROTEIN Figure 4. Detection of proteins using an LC-MS/MS workflow with common upfront depletion columns. Pooled HEMAcollect™•PROTEIN BCT-derived samples were compared with pooled EDTA-derived samples (n = 4 donors per BCT) using a (A) Norgen™ depletion column or (B) Thermo Fisher™ depletion column. Orange dots show proteins that are detected in greater abundance in EDTA-derived samples. Blue dots show proteins that are detected in lower abundance in EDTA-derived samples. Grey dots show proteins that are not differentially detected in EDTA-derived samples and are comparable to HEMAcollect™•PROTEIN BCT-derived samples. www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 6 14 12 10 8 6 4 2 0 -2 -4 Average log2 fold change relative to T0 HEMAcollect™•PROTEIN EDTA 5 d 7 d 7 d+ 5 d 7 d 7 d+ A Figure 5A. Protein stability over time in the HEMAcollect™•PROTEIN BCT and EDTA BCT using the Olink™ platform. Average fold change of protein targets for the Olink™ Target 48 Cytokine Panel in HEMAcollect™•PROTEIN BCT-collected and EDTA whole blood-collected samples (n = 4 donors per BCT) after storage at (5 d) 5 days and (7 d) 7 days) at room temperature, or (7 d+) up to 7 days subjected to temperature cycling from 4°C to 30°C for 24 hours at each temperature. Figure 5B. Heatmap of protein stability over time in the HEMAcollect™•PROTEIN BCT and EDTA BCT using the Olink™ platform. Heatmap of the average fold change of individual protein targets for the Olink™ Target 48 Cytokine Panel in HEMAcollect™•PROTEIN BCT-collected and EDTA whole blood-collected samples (n = 4 donors per BCT) after storage at (5 d) 5 days and (7 d) 7 days at room temperature, or (7 d+) up to 7 days subjected to temperature cycling from 4°C to 30°C for 24 hours at each temperature. Figure 5C. Protein stability over time in the HEMAcollect™•PROTEIN BCT and EDTA BCT using SomaLogic™ platform. Average fold change of protein targets for the custom 100-protein SomaScan™ panel in HEMAcollect™•PROTEIN BCT-collected and EDTA whole blood-collected samples (n = 5 donors per BCT) after storage at (5 d) 5 days and (7 d) 7 days at room temperature, or (7 d+) up to 7 days subjected to temperature cycling from 4°C to 30°C for 24 hours at each temperature. Figure 5D. Heatmap of protein stability over time in the HEMAcollect™•PROTEIN BCT and EDTA BCT using SomaLogic™ platform. Heatmap of the average fold change of individual protein targets for the custom 100-protein SomaScan™ panel in HEMAcollect™•PROTEIN BCT-collected and EDTA whole blood-collected samples (n = 5 donors per BCT) after storage at (5 d) 5 days and (7 d) 7 days at room temperature, or (7 d+) up to 7 days subjected to temperature cycling from 4°C to 30°C for 24 hours at each temperature. 5 d 7 d 7 d+ 5 d 7 d 7 d+ Average log2 fold change relative to T0 B Avg. fold change log2 HEMAcollect™•PROTEIN EDTA 8 Average log fold change relative to T0 HEMAcollect™•PROTEIN EDTA 5 d 7 d 7 d+ 5 d 7 d 7 d+ 6 4 2 0 -2 -4 C www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 7 BCT effectively minimizes preanalytical sample changes by maintaining sample homeostasis, which enables high-quality proteomic data generation. Discussion During the preanalytical phase, blood samples collected for proteomics are vulnerable to stressors that can lead to hemolysis, platelet activation, leukocyte activation and protein degradation.7 These factors can significantly compromise sample quality, potentially bias downstream analyses and obscure the discovery of relevant biomarkers.25 Our findings demonstrate that the HEMAcollect™•PROTEIN BCT significantly mitigates these issues across a range of storage and transport conditions, providing a more robust BCT for proteomic applications. Specifically, the performance evaluation of the HEMAcollect™•PROTEIN BCT highlights superior flexibility in sample handling and processing enabled by the ProteoPrecision™ technology, which minimizes hemolysis, controls platelet activation and maintains sample homeostasis in whole blood across a broad temperature range. In addition, the reduced variability observed across the data collected for HEMAcollect™•PROTEIN BCT samples compared with EDTA samples highlights the potential of HEMAcollect™•PROTEIN BCTs to offer standardization within current workflows for venous blood collection and processing for proteomic applications. Importantly, the performance data demonstrates direct compatibility of the HEMAcollect™•PROTEIN BCT on both traditional targeted immunoassays (ELISA), untargeted mass spectroscopy (LC-MS/MS) and 2 leading next generation, high-throughput affinity-based proteomic platforms (Olink™ proximity extension assay (PEA) and SomaLogic™ SomaScan™ assay). In addition to having direct compatibility with HEMAcollect™•PROTEIN BCTs, these multi-target protein panels emphasize the robust performance of HEMAcollect™•PROTEIN BCTs in stabilizing the plasma protein profile during storage and transport conditions, showcasing the unique innovation of ProteoPrecision™ technology. Through the effective preservation of the whole blood sample, the HEMAcollect™•PROTEIN BCT provides a sample management solution that can facilitate discovery using downstream proteomic technologies. Conclusions The HEMAcollect™•PROTEIN BCT sets a new standard for venous blood sample collection in the rapidly growing proteomics field. With a purpose-built design to meet the unique demands of proteomic discovery research, the HEMAcollect™•PROTEIN BCT streamlines sample handling, enables extended sample storage and transport at ambient temperature and eliminates the constraints associated with standard blood collection tubes. The HEMAcollect™•PROTEIN BCT offers a scalable, highly assay-compatible, fieldready solution that is positioned as a foundational tool for the next generation of precision biomarker discovery and translational research. Materials and methods Sample collection and processing For all studies, paired venous blood samples were collected from 6-10 healthy donors into a HEMAcollect™•PROTEIN BCT and BD Vacutainer® K2 EDTA (K2E) Plus BCT (Thermo Fisher Scientific, Cat. No. 02-657-32). Baseline plasma (T0, control) was isolated within 2 hours of collection and across subsequent timepoints. At all timepoints, plasma was isolated from the whole blood via centrifugation (1,900 × g for 15 minutes at 4°C) or as recommended by the downstream assay manufacturer. All plasma samples were stored at -80°C until they were tested. Storage and transport conditions The samples collected in the HEMAcollect™•PROTEIN BCT and BD Vacutainer® K2 EDTA (K2E) Plus BCT were exposed to 2 temperature conditions, which are defined as room temperature storage (20°C–26°C/68°F– 79°F) and simulated transport (4°C-30°C/39°F-86°F for whole blood; -10°C to 30°C/14°F to 86°F for isolated plasma). The samples were stored for at least 24 hours at each simulated transport temperature before being kept at room temperature for up to 7 days. www.dnagenotek.com • support@dnagenotek.com © 2025 DNA Genotek Inc., a subsidiary of OraSure Technologies, Inc., all rights reserved. PD-WP-80 v1 8 Performance evaluation The centrifuged BCTs were visually evaluated for hemolysis following sample processing via a quantitative assay for hemolysis (Abcam, Cat. No. ab234046). To assess the maintenance of blood sample homeostasis, ELISA-based immunoassays were carried out for representative markers as a proxy for ex vivo platelet activation (CD62P: Abcam, Cat. No. ab292202; EGF: Abcam, Cat. No. ab217772), blood cell protein release (IL-8: Abcam, Cat. No. ab214030) and broad protease activity (LOX-1: Abcam, Cat. No. ab212161) using plasma isolated from HEMAcollect™•PROTEIN and EDTA BCTs. Multi-target protein analysis on plasma isolates was evaluated using high-throughput biomarker platforms, including Olink™ Target 48 Cytokine Panel (Thermo Fisher Scientific, Cat. No. CRLB-GMEL) and the custom 100-protein SomaScan™ panel assay (SomaLogic™). For the Olink™ panel, targets for which at least 50% of donor values were below the limit of detection (LoD) or were undetectable under all tested conditions, were excluded from analysis, as their inclusion would preclude a valid comparison. For the SomaScan™ assay, plasma isolated from HEMAcollect™•PROTEIN BCTs did not require upfront buffer exchange processing. Plasma isolated from four individual venous whole blood donor samples were pooled and 5 replicates were evaluated in HEMAcollect™•PROTEIN BCTs for compatibility with 2 commercially available protein depletion kits for preanalytical LC-MS sample processing workflows, including the High-Select™ Top 14 Abundant Depletion Columns (Thermo Fisher Scientific, Cat. No. A36371) and the ProteoSpin Abundant Serum Depletion Kit (Norgen, Cat. No. 17300). Protein depletion efficiency was evaluated by quantifying protein concentration using the Quick Start Bradford Protein Assay Kit 1 (Bio-Rad, Cat. No. 5000201). Protein-depleted plasma samples collected in a HEMAcollect™•PROTEIN BCT were compared with BD Vacutainer® K2 EDTA (K2E) Plus BCT (Thermo Fisher Scientific) and their respective undepleted samples using Nano LC-MS/MS analysis performed on an Ultimate3000 nano RLSC coupled to an Orbitrap Fusion™ Lumos™ (Proteomics Core Facility, University of Ottawa, April 2025) and analyzed using MaxQuant. References 1 Kline RA, Lößlein L, Kurian D, et al. (2022). An optimized comparative proteomic approach as a tool in neurodegenerative disease research. Cells. 11(17):2653. doi: 10.3390/cells11172653 2 Park Y, Kim MJ, Choi Y, et al. (2022). Role of mass spectrometry-based serum proteomics signatures in predicting clinical outcomes and toxicity in patients with cancer treated with immunotherapy. J Immunother Cancer. 10(3):e003566. doi: 10.1136/jitc-2021-003566 3 Costanzo M, Zacchia M, Bruno G, et al. (2017). Integration of proteomics and metabolomics in exploring genetic and rare metabolic diseases. 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J Proteome Res. 9(10):4982-91. doi: 10.1021/pr100646w 9 Kamlage B, Maldonado SG, Bethan B, et al. (2014). Quality markers addressing preanalytical variations of blood and plasma processing identified by broad and targeted metabolite profiling. Clin Chem. 60(2):399-412. doi: 10.1373/clinchem.2013.211979 10 Chantada-Vázquez MDP, Bravo SB, Barbosa-Gouveia S, et al. (2022). Proteomics in inherited metabolic disorders. Int J Mol Sci. 23(23):14744. doi: 10.3390/ijms232314744 11 Candia J, Fantoni G, Moaddel R, et al. (2025). Effects of in vitro hemolysis and repeated freeze–thaw cycles in protein abundance quantification using the SomaScan and Olink assays. Journal of Proteome Research. 24(5): 2517-2528. doi: 10.1021/acs.jproteome.5c00069 12 Anwar M, Renu K, Singh A, et al. (2025). Impact of hemolysis on the levels of proteins associated with aging and age-related neurodegenerative diseases in a multicentric clinical research. Practical Laboratory Medicine. 44; e00455. doi: 10.1016/j.plabm.2025.e00455 The Olink and SomaScan trademarks are properties of their respective owners. Some DNA Genotek Inc. products are not available in all geographic regions. HEMAcollect and ProteoPrecision are trademarks of DNA Genotek Inc., registered in various jurisdictions. All other brands and names contained herein are the property of their respective owners. HEMAcollect™•PROTEIN BCT is for Research Use Only, not for use in diagnostic procedures. All DNA Genotek™ protocols, white papers and application notes are available in the support section of our website at www.dnagenotek.com. Customers must review the labels of sample collection devices and ensure compliance with their intended requirements. For more information or to request samples or a quote, visit www.dnagenotek.com or email info@dnagenotek.com. 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