Blood collection device components: issues, innovations, and recommendations for clinical laboratories and manufacturers—a narrative review

Introduction

Background

Laboratory testing informs approximately 60–70% of clinical decisions; however, its accuracy is highly susceptible to preanalytical errors that occur during sample collection, transport, and storage (1). Components of blood collection devices (BCDs), such as blood collection tubes (BCTs), catheters, syringes, and needles, can substantially affect test accuracy. These errors can delay diagnosis and treatment, increase healthcare costs (estimated at approximately $1.2 million annually for a typical 650-bed U.S. hospital), and erode trust in laboratory data quality (2). Despite advancements in identifying BCD-related sources of error and developing technologies to mitigate their effects, the influence of BCD components on clinical laboratory testing has remained a persistent issue over the past decade, as demonstrated in this narrative review.

Rationale and knowledge gap

Laboratory testing is central to clinical decision-making, yet BCDs remain an often-overlooked source of variability that can compromise test accuracy. Components of BCTs, including tube walls, surfactants (SFs), rubber stoppers, lubricants, separators, clot activators, anticoagulants, and glycolysis inhibitors, can alter analyte composition and interfere with assay performance, as illustrated in Figure 1 (3-7). This issue is particularly concerning in high-complexity testing areas, such as metabolomics and proteomics, where even minor inaccuracies can undermine the clinical utility of results.

Figure 1 This figure illustrates various blood collection devices and their components that may influence specimen integrity and/or analyte measurements. (A) Schematic diagram of a standard blood collection tube showing major components, including the tube wall, stopper, additive layer, and separator gel. Adapted with permission from Bowen RAR, Hortin GL, Csako G, Otanez O, Remaley AT. Impact of blood collection devices on clinical chemistry assays. Clin Biochem 2010;43:4-5, with permission from Elsevier. (B) Peripheral catheter illustration. Sources: https://unsplash.com/@ads and Wikimedia Commons (https://commons.wikimedia.org/wiki/File:Urinary_catheterization_01.JPG). (C) Syringe with components illustration. Sources: https://commons.wikimedia.org/wiki/File:Insulin_syringe_in_disassembled_form.jpg and https://commons.wikimedia.org/wiki/File:Syringe_with_insulin_for_a_cat.jpg). (D) Venipuncture needle commonly used in routine blood collection illustration. Sources: https://unsplash.com/photos/a-close-up-of-a-pen-sRErS-oguq0. (E) Butterfly needle illustration. Source: https://en.m.wikipedia.org/wiki/File:Butterfly_needle.png. All figures in this manuscript were vector-based illustrations developed using Adobe Animate CC (version 24.0.7, Adobe Creative Cloud Suite) and digitally rendered on a Wacom Cintiq interactive pen display to ensure precision in graphical representation.

Given the wide range of affected assays and the complex array of interference mechanisms, the authors argue that a narrative review is the most suitable approach to synthesize interdisciplinary findings, identify recurrent interference patterns, and critically evaluate the clinical and operational implications of BCD-related variability. Despite growing awareness, the impact of BCT materials, manufacturing inconsistencies, and improper handling on test results remains underappreciated. Variations in polymer composition, SFs, and proprietary formulations contribute to inconsistencies in laboratory results. Major manufacturers, including Becton Dickinson (BD; Franklin Lakes, NJ, USA), Terumo (Tokyo, Japan), Greiner Bio-One (Kremsmünster, Austria), and Sarstedt (Nümbrecht, Germany), utilize unique formulations that complicate interlaboratory reproducibility. Laboratories should proactively request detailed technical specifications from manufacturers and prioritize BCTs with validated, low-interference profiles for clinical assays.

In response to these risks, regulatory agencies have strengthened their oversight frameworks. In the U.S., the Food and Drug Administration (FDA) regulates BCTs through the Center for Devices and Radiological Health. In May 2024, the FDA finalized a rule placing LDTs under formal medical device oversight, requiring registration, adverse event reporting, and premarket review for high-risk tests (8). While this rule represents progress, exemptions for legacy and in-house LDTs may limit its effectiveness. However, on March 31, 2025, the U.S. District Court for the Eastern District of Texas ruled that the FDA lacks statutory authority to regulate LDTs and vacated the May 2024 rule (8). Despite this decision, the FDA continues to regulate specimen collection devices and may pursue enforcement against clinical laboratories involved in both the manufacturing and distribution of collection kits intended for uses not consistent with FDA-cleared indications (8). The FDA may consider such kits to be unauthorized and inaccurately labeled medical devices (8). Given this evolving regulatory environment, clinical laboratories should proactively verify the performance of BCDs, particularly for high-risk assays, rather than wait for regulatory mandates.

Globally, similar regulatory frameworks exist. In the European Union, BCTs are classified as Class IIa medical devices under the Medical Device Regulation, requiring clinical evaluation and post-market surveillance. Health Canada applies comparable standards. However, regulatory requirements vary among nations, and guidance specific to interference from BCT components is lacking. We advocate international harmonization of regulatory standards, emphasizing the need for manufacturers to disclose chemical compositions, validate matrix compatibility, and conduct interference testing across assay platforms.

While current oversight mechanisms continue to evolve, clinical laboratories should adopt a proactive approach by requiring transparent BCT specifications, conducting local validation studies, and integrating BCT-related risks into their quality management systems.

Objectives

In this review, we aim to inform clinical laboratory professionals, diagnostic manufacturers, and regulatory stakeholders about how BCTs and their components impact the accuracy and reproducibility of laboratory test results. By synthesizing current evidence, we identify key sources of preanalytical variability that arise from interactions between BCD constituents and analytes across various assays, including routine chemistry, immunoassays, mass spectrometry (MS), and specialized areas such as metabolomics and proteomics.

This review places particular emphasis on material-related interferences, additive-related assay bias, analyte adsorption, and variability introduced by inconsistent manufacturing practices. To support diagnostic reliability, it advocates the implementation of standardized validation and verification protocols tailored to local laboratory conditions and specific assay requirements. Furthermore, it recommends incorporating BCD-related considerations into quality control (QC) and proficiency testing (PT) programs to enhance the detection of tube-related interferences.

We emphasize the need for local validation studies before adopting new or alternative BCTs, even when these products are marketed as functionally equivalent. By promoting awareness, encouraging collaboration with manufacturers, and aligning laboratory practices with evidence-based validation strategies, we aim to reduce BCD-related diagnostic errors and enhance the overall quality of laboratory testing, without attempting to replace existing clinical guidelines. We present this article in accordance with the Narrative Review reporting checklist (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-5/rc).

Methods

To support the objectives of this review, we conducted a literature search across multiple biomedical databases, including PubMed, ScienceDirect, Google Scholar, Scopus, Embase, and Web of Science. The search included original research articles, review papers, regulatory publications, and clinical guidelines published between January 1, 2014, and January 31, 2025. Seminal studies published prior to this date range were also included if they provided foundational insights into the impact of BCD components on laboratory assay performance.

The PubMed search was conducted from January 15, 2024 to January 31, 2025, and was limited to studies involving human subjects. Search terms were structured using Boolean operators (“AND” and “OR”) and relevant medical subject headings (MeSH), emphasizing BCT constituents (e.g., tube walls, SFs, rubber stoppers, lubricants, separators, clot activators, anticoagulants, glycolysis inhibitors) and BCDs such as catheters, syringes, and needles. Search strings were developed from the following three thematic categories: (I) blood specimen collection methods and instrumentation; (II) BCD-related components; and (III) downstream effects on assay performance, including diagnostic errors, specimen handling, test variability, and blood preservation.

Additional topics of interest included analyte instability, trace metal contamination, drug adsorption, hemolysis, and gas diffusion. Validation strategies, such as BCT lot-to-lot variability and regulatory quality requirements, were also considered.

The PubMed search utilized combinations of MeSH terms, including “Blood Specimen Collection/methods”, “Surface-Active Agents”, “Equipment Contamination”, and “Immunoassay/methods”, along with free-text keywords such as “blood collection device”, “glycolysis inhibitor”, “test accuracy”, “routine chemistry”, and “rubber stopper”. Studies were included if they reported quantitative discrepancies, assay-specific interferences, or clinically significant measurement bias associated with BCD components.

Table 1 and the Appendix 1 summarize the search strategy, whereas key findings from the included studies are outlined in Table S1 . Studies on microcollection devices or capillary blood self-sampling systems were excluded owing to unique preanalytical factors. The following sections analyze the impact of BCD and additives on test accuracy, focusing on BCT components (walls, SFs, rubber stoppers and lubricants, separators (gel and mechanical), clot activators, anticoagulants, and glycolysis inhibitors), as well as catheters, syringes, and needles. This analysis highlights vulnerabilities across routine assays and advanced diagnostics. The review concludes with recommendations to reduce errors associated with BCD components and their use.

BCTs and preanalytical variability

Preanalytical errors stemming from BCD components, particularly BCTs, are a considerable source of variability in clinical laboratory testing. Variations in tube composition and design can affect analyte stability, assay performance, and overall diagnostic reliability. This section discusses how BCT components contribute to this variability.

BCT components as sources of preanalytical errors

BCTs contain various components, including tube wall materials, SFs, rubber stoppers with lubricants, separator gels or mechanical barriers, clot activators, anticoagulants, and glycolysis inhibitors. These components may introduce preanalytical variability and interfere with clinical assay results, as shown in Figure 1A.

BCT wall material

BCTs, as illustrated in Figure 1A, are cylindrical containers typically measuring 50–150 mm in length and 10–20 mm in diameter, with standard tubes usually 75–100 mm long and 13 mm in diameter, capable of holding 2–10 mL of blood. These tubes are commonly used to collect blood from patients for diagnostic testing in clinical laboratories (3,7). Most modern BCTs are made from polyethylene terephthalate (PET) or polypropylene (PP), offering advantages such as reduced breakage, compatibility with automated processing, and resistance to high centrifugal forces. However, the material composition of BCT walls may negatively affect test accuracy, particularly in sensitive or specialized assays.

In the context of Alzheimer’s disease diagnostics, the material properties of collection tubes considerably influence the recovery and stability of amyloid-beta (Aβ) peptides. Strand et al. investigated the adsorption of Aβ1-42 and Aβ1-40 across different PP tubes (9). The study found that peptide recovery varied, with losses ranging from 6.5% to 32.2%, depending on the tube brand. However, Sarstedt tubes (Sar10CSF and Sar5CSF), designed for cerebrospinal fluid handling, exhibited minimal adsorption and more consistent Aβ recovery. Both the Sar10CSF tube, endorsed by the European BIOMARKAPD consortium, and the Sar5CSF tube, a reduced-volume variant, demonstrated similarly low adsorption, possibly due to shared proprietary polymer properties; however, the limited sample size (n=14) and narrow temperature range (room temperature and 37 °C) constrain the generalizability of these findings (9).

Building on Strand’s findings, Ladang et al. examined the effects of BCT material and preanalytical conditions on plasma concentrations of Aβ and total tau (tTau) in a cohort of seven healthy male volunteers (10). PET-based BD Vacutainer tubes yielded Aβ1-42 and Aβ1-40 concentrations that were 15% and 13% higher, respectively, than those obtained with PP microtainer tubes. Although tTau levels did not differ considerably across the tube types, Vacutainer tubes yielded a 16% higher median tTau recovery. These findings suggest that absolute peptide levels are more susceptible to tube material variability than peptide ratios are. Furthermore, Aβ1-40 concentrations decreased by 14–15% when PET tubes were stored horizontally for 1 h, underscoring the importance of tube orientation. While the small sample size (n=7) and the unexpectedly higher recovery in PET tubes—contrary to prior cerebrospinal fluid findings—limit generalizability, the authors proposed that protein and matrix interactions, particularly those involving albumin, may influence surface adsorption. Notably, the Aβ1-42 to Aβ1-40 ratio remained stable across tube types, possibly because both peptides were affected proportionally by adsorption and recovery conditions. This reinforces the utility of the Aβ1-42 to Aβ1-40 ratio as a more reliable and analytically robust metric for assessing plasma Aβ concentrations (10).

Beyond protein adsorption, trace element contamination has also emerged as a key source of preanalytical error from BCT walls. Yang et al. investigated antimony (Sb) contamination in PET-based BD vacutainer ethylenediaminetetraacetic acid (EDTA) tubes (11). In samples from healthy volunteers, median whole-blood Sb levels were substantially higher in blood collected in BD tubes (3.45 µg/L) than in blood collected in Greiner sodium-heparin tubes (1.11 µg/L; P<0.0001), with 98% of BD samples exceeding the clinical reference interval of <3 µg/L. Contamination was traced to residual antimony trioxide, a catalyst used in PET manufacturing. Acid rinse experiments confirmed this source, with Sb levels reaching up to 33.7 µg/L in low-volume conditions (0.5 mL) and 30.0 µg/L in higher-volume conditions (5 mL) for BD tubes rinsed with phosphate-buffered saline (PBS) (11). An inverse correlation between tube fill volume and Sb contamination further supported a surface area to volume effect. Although Greiner tubes are also plastic-based, they exhibited considerably lower Sb release (21.5 µg/L in nitric acid vs. 33.7 µg/L in BD tubes) and 13.4 µg/L in PBS, likely due to differences in polymer composition or catalyst residue (11).

The study’s strengths include the use of inductively coupled plasma MS for precise quantification and a robust control group (QC) that ensures the reliability of the results. However, notable limitations include the lack of tube material characterization and batch-level controls, accounting for variability in contamination levels across different production lots. The findings suggest that BD EDTA plastic tubes are unsuitable for evaluating blood Sb levels, and testing laboratories should consider validating alternative collection devices (11).

To address the material-related limitations of PET used in BCTs, including permeability and additive stability, Weikart et al. developed hybrid BCTs incorporating cyclic olefin polymer (COP) walls with silica-based internal barrier coatings (12). These hybrid tubes achieved a 10-fold reduction in the oxygen transmission rate (0.00015 vs. 0.00159 day⁻¹) and a 5-fold decrease in moisture vapor transmission, both compared to standard PET tubes (12). These enhancements conferred improved additive stability, reduced preservative gelation, and extended product shelf life, effectively minimizing the risk of alteration to the blood-to-preservative ratio. Hybrid BCTs also exhibited superior resistance to impact-induced breakage compared to glass (12). COP tolerates a broad range of temperatures (i.e., −70 to 121 °C) without mechanical fatigue, deformation, or breakage, unlike other plastics such as PET. Extreme centrifugation of water-filled hybrid BCTs did not result in any breakage. While these innovations hold promise, the authors noted the need to assess whether silica coatings might introduce their own assay interferences (12).

In summary, the wall material of BCTs (PET, PP, or hybrid COP with silica coatings) can negatively affect protein and trace element integrity. Research on Aβ peptides indicates that material properties cause notable variations in peptide recovery, with losses differing widely among tube brands. Similarly, research on Sb contamination has highlighted the potential for trace element interference from residual materials in PET-based tubes. Hybrid BCTs, which demonstrate reduced oxygen and moisture vapor transmission, improved additive stability, and enhanced resistance to breakage, are promising alternatives to BCTs made of traditional materials. However, further assessment of potential assay interferences introduced by silica coatings is necessary. Collectively, these findings emphasize the importance of rigorous validation and careful selection of BCTs tailored to specific clinical applications, particularly in high-precision settings such as neurodegenerative biomarker testing and trace element toxicology.

SFs

Surface-active agents, also known as SFs, are amphiphilic compounds incorporated into BCTs to improve blood flow dynamics, promote even distribution of clot activators, and prevent the adhesion of red blood cells (RBCs), platelets, and plasma proteins to tube surfaces (3,7). While these additives enhance sample handling and tube performance, emerging evidence suggests that they may interfere with the accuracy of immunoassays and contribute to preanalytical errors.

Dorokhin et al. provided foundational mechanistic insights into this issue by investigating the effects of Pluronic F-127, a nonionic SF, on antigen-antibody binding in cardiac troponin immunoassays (13). Using immunoassay techniques with magnetic particles as labels, they demonstrated that Pluronic F-127 increased the dissociation rate constant of the antigen-antibody complex in a concentration-dependent manner, rising from 1.4×10⁻³ s⁻¹ (no SF) to 3.5×10⁻³ s⁻¹ at a concentration of 0.04% (13). The researchers used magnetic tweezers to reveal a weakened binding strength, whereas circular dichroism spectroscopy indicated conformational changes in cardiac troponin, specifically an increase in the 222:208 nm ratio from 0.91 to 0.97, suggesting that the SF disrupted the antigen structure without affecting the antibody (13). Although the study employed advanced biophysical techniques and reported consistent results across multiple particles, it did not validate the observed effects in complex matrices such as serum, nor did it test SF concentrations representative of those commonly present in BCTs (13). The strengths of the study include its mechanistic depth, the use of sophisticated biophysical tools, and reproducibility across different particles. Its primary limitations are the absence of serum-based validation and testing for clinical relevance. Nonetheless, the study offers a plausible mechanistic explanation for how SFs may compromise immunoassay performance through structural modification of target analytes.

Building on this concept, Kim et al. explored an alternative strategy to mitigate SF-related assay interference by chemically modifying the surfaces of PET tubes (14). Using a guanidine base-catalyzed transesterification reaction with polyols such as ethylene glycol, they created chemoPET tubes that exhibit a reduced contact angle (from ~90° to ~30°), making the surface hydrophilic and functionally similar to glass (14).

Bowen et al. validated modified chemoPET tubes against conventional glass and plastic BCTs using immunoassays and MS platforms (15). They found that chemoPET tubes matched or surpassed analytical performance, particularly in MS-based methods where SFs are known to suppress ionization and confound detection of target analytes. In these evaluations, chemoPET tubes showed comparable performance to glass tubes for cortisol, total triiodothyronine (TT₃), total thyroxine (TT₄), and routine analytes such as sodium, alanine aminotransferase, and glucose on the Immulite 1000 and Siemens RxL platforms (15). While the study’s strength lies in its thorough platform comparison and clinical relevance, it did not evaluate scalability or manufacturing feasibility, limiting immediate clinical application.

In a follow-up study, Dil et al. characterized the chemoPET surface using X-ray photoelectron spectroscopy, a technique for analyzing surface elemental composition, and atomic force microscopy, which provides high-resolution topographical imaging at the nanoscale (16). Their analysis demonstrated enhanced hydrophilicity, reduced contact angle, and nanostructured surface roughness (16). Free triiodothyronine (T₃) and thyroxine (T₄) measurements showed no significant differences compared to those in glass tubes, and thromboelastography revealed equivalent clot formation kinetics, supporting both analytical and biological compatibility (16). This study’s strengths include long-term stability testing and comprehensive surface characterization, whereas its main limitation is the narrow range of analytes tested.

Evidence of SF-related interference in specific clinical analytes has also been documented. Yu et al. reported that additives in Vacuette tubes led to a 44% overestimation of 25-hydroxyvitamin D [25(OH)D] levels in immunoassays, highlighting the sensitivity of steroid hormone assays to BCT composition (17). This finding was further supported by liquid chromatography-tandem MS (LC-MS/MS) analysis, which demonstrated no significant bias across tube types and confirmed that the interference was specific to immunoassays. Yu et al. further demonstrated that the Siemens ADVIA Centaur XP platform exhibited a mean positive bias of 111.1% for 25(OH)D when using Vacuette tubes containing clot activator and gel, compared to LC-MS/MS, and a 27.1% bias when clot activator alone was used (17). A key strength of this study was the dual-platform comparison and inclusion of multiple tube types, whereas its main limitation was a modest sample size and a limited mechanistic investigation.

More recently, Chae et al. confirmed these findings using the Siemens Atellica IM platform, noting a 10 ng/mL increase in 25(OH)D levels in samples collected with Vacuette tubes compared to those without such additives (18). The study attributed the interference to potential SF-mediated desorption of antibodies from paramagnetic particles, which reduced the signal and produced a falsely elevated vitamin D result (18). The strengths of this study include its replication of previous findings using a modern platform and its verification with samples from healthy volunteers; however, it did not isolate the specific SF compounds responsible for the interference nor assess their chemical stability under varying conditions. The study raises concerns about assay specificity and the reliability of results exposed to SF.

Despite the growing awareness of SF-induced variability, a key barrier to systematic investigation is the lack of publicly available information regarding the specific SFs used in BCTs, including their identities, concentrations, and modes of application, such as coating vs. incorporation into stoppers or tube walls. This opacity hinders rigorous evaluation of their analytical impact and complicates efforts to establish harmonized standards.

In summary, SFs enhance the performance of BCTs by improving blood flow dynamics and preventing the adhesion of cellular components; however, emerging evidence highlights that these additives can considerably interfere with immunoassay accuracy. Studies show that SFs, such as Pluronic F-127, can alter the structural integrity of target analytes, resulting in increased dissociation rates in antigen-antibody complexes. Alternative approaches, such as developing chemically modified PET tubes, have shown promise in mitigating these interferences by creating hydrophilic surfaces that mimic glass. Comparative analyses indicate that these modified tubes can provide equivalent or improved analytical performance across various platforms, particularly in MS, where SFs are known to suppress ionization. The lack of transparency regarding the specific SFs used in BCTs, including their concentrations and modes of application, hinders efforts to understand their analytical impact. Therefore, future research should prioritize the characterization of commonly used SFs, such as Silwet L-720, and systematically evaluate their effects on a broader range of assays under clinically relevant conditions to enhance assay specificity and reliability (3).

Rubber stoppers

Rubber stoppers maintain internal pressure, prevent contamination, and facilitate vacuum-assisted blood draws (7). These stoppers are typically made from synthetic rubber materials, including polychloroprene, silicone, styrene-butadiene, and isobutylene-based elastomers. They are often color-coded to indicate tube additives, such as anticoagulants or separator gels (15,19). While essential for tube functionality, rubber stoppers can also introduce preanalytical variability through chemical leaching and analytical interference.

Early investigations revealed that certain rubber formulations contained plasticizers, such as tris (2-butoxyethyl) phosphate (TBEP), which can displace drugs from their plasma protein-binding sites, altering the free-to-bound ratio. Such displacement can result in the underestimation of drug concentrations in serum or plasma. Notably, Shah et al. and others documented this effect across multiple drugs, including quinidine, propranolol, lidocaine, alprenolol, imipramine, and various tricyclic antidepressants (15,20-23). In response to these findings, manufacturers have shifted toward low-extractable rubber formulations and phased out TBEP-containing stoppers.

In addition to leaching from organic plasticizers, inorganic metal ions—such as calcium, aluminum, magnesium, and zinc—used as curing agents or fillers in rubber manufacturing, can also enter blood specimens. Van den Besselaar et al. reported that magnesium ions leached from conventional rubber stoppers can interfere with coagulation assays by promoting phospholipid-mediated reactions (24-26). In one study, citrate plasma samples collected in standard Vacutainer tubes exhibited magnesium concentrations of 1.14 to 1.15 mmol/L, compared to 0.72 mmol/L in low-magnesium tubes. This resulted in significantly shorter prothrombin times across various thromboplastin reagents. For instance, prothrombin time values with Innovin, Recombiplastin 2G, and Neoplastin R reagents were 10.34, 10.64, and 13.85 s, respectively, in conventional tubes, vs. 10.77, 10.99, and 14.40 s in low-magnesium tubes (P<0.001). Similarly, the international normalized ratio (INR) was lower in standard tubes (e.g., 2.64 vs. 2.87 with Innovin; 2.68 vs. 2.89 with Recombiplastin 2G; P<0.001), highlighting the potential for stopper composition to introduce clinically significant variability in coagulation testing (26).

Sterilization processes can complicate the chemical stability of rubber stoppers. Kosecki et al. investigated the effects of gamma radiation sterilization on chlorobutyl stoppers and found that degradation products, particularly isobutylene, interfered with gas chromatography-based assays (27). In single-column gas chromatography systems, isobutylene peaks were misidentified as methanol, whereas dual-column setups were more effective at distinguishing and mitigating this interference (27).

Additional concerns arise from thiuram-containing rubber formulations, which release volatile sulfur-containing gases such as carbon disulfide and carbonyl sulfide. These compounds can bind to lead particles and result in falsely low blood lead level (BLL) readings. Mason et al. and Nakata et al. confirmed that the LeadCare II system, which uses anodic stripping voltammetry, underestimated BLL compared to inductively coupled plasma MS, particularly at elevated concentrations, with biases exceeding 29.25 µg/dL (28,29).

Sterilization processes, specifically gamma irradiation, can further compromise the chemical stability of stoppers by introducing degradation products that interfere with analytical methods. Therefore, careful selection and evaluation of rubber stopper materials, along with advancements in sterilization techniques and analytical safeguards, are important for ensuring the accuracy and reliability of clinical assays.

Stopper lubricants

Stopper lubricants are applied to BCT stoppers to facilitate smooth insertion and removal during manufacturing and use. Additionally, these compounds reduce friction and prevent the adherence of RBCs, clots, and other biological residues to the stopper surface (3). Despite their functional utility, certain lubricants have been identified as sources of analytical interference.

Early investigations into lubricant-associated assay interference focused on glycerol, which was once a commonly used compound for stopper lubrication. Baum et al. independently demonstrated that residual glycerol from rubber stoppers could leach into blood samples and interfere with biochemical assays (e.g., colorimetric assays) that directly or indirectly measure glycerol or triglycerides (30,31). Despite these early reports, systematic investigation into the broader effects of stopper lubricants has remained limited. The issue has become more salient with the increasing use of high-sensitivity technologies such as MS and nuclear magnetic resonance (NMR), both of which are particularly susceptible to chemical background noise. Lubricant-derived compounds may suppress ionization efficiency or introduce unidentified peaks, leading to inaccurate quantification or false-positive signals.

Given these vulnerabilities, the impact of stopper lubricants on contemporary analytical platforms warrants renewed attention. Modern BCTs may use a variety of proprietary lubricant formulations beyond glycerol, including silicones and synthetic polymers. However, few studies have explored their chemical stability, extraction potential, or interaction with common clinical assays. As analytical methods continue to evolve in sensitivity and complexity, even trace amounts of lubricant-related contaminants may contribute to variability in test results.

In summary, while early studies noted assay interference from glycerol-based lubricants, the broader implications of stopper lubricant composition are still underexplored. More research is needed to quantify these effects across various analytical platforms and assess the compatibility of lubricant formulations with new diagnostic technologies.

Gel separator tubes for therapeutic drug monitoring (TDM)

Accurate drug concentration measurement is essential for effective TDM, and the choice of BCTs plays an important role in minimizing preanalytical variability. Serum separator tubes (SSTs), plasma separator tubes (PSTs), and mechanical separator tubes offer practical benefits, including simplified sample processing and reduced cellular contamination. However, interactions between separator materials and drug compounds, such as lipophilic agents, have been shown to compromise analyte stability during storage, negatively impacting clinical and forensic toxicology.

SSTs and PSTs

SSTs and PSTs, developed by BD over 40 years ago, use polymer-based gels that create a physical barrier between serum or plasma and cellular components after centrifugation (3). These gels consist of viscous liquids, fillers, and tackifiers such as dibenzylidene sorbitol, tailored to match the densities of serum or plasma (1.026–1.031 g/cm³) and blood clots (1.092–1.095 g/cm³) (3). Although intended to improve preanalytical handling, early studies revealed that gel adsorption leads to the loss of lipophilic drugs such as lidocaine and phenytoin (32).

Wollmann et al. expanded on this concern by evaluating 28 psychoactive drugs and 13 metabolites using BD SSTs (33). They found significant concentration reductions at room temperature, ranging from 2% to 28% by day 2 (P<0.05) and up to 49% by day 6 (P<0.02) (33). Drug losses correlated strongly with lipophilicity, with correlation coefficients of r=−0.50 on day 2 and r=−0.42 on day 6 (33). This study contributed a robust analytical methodology using LC-MS/MS, adherence to FDA guidelines, and the use of patient-derived specimens; however, key limitations included the use of a single tube type and ambient storage, both of which limit the range of clinical applications.

Supporting these findings, Garza et al. evaluated SST and rapid serum tubes (containing thrombin as a clot activator) for 21 therapeutic drugs (34). While most analytes (such as acetaminophen, vancomycin, and levetiracetam) remained stable, lipophilic drugs (including lidocaine, free phenytoin, and tricyclic antidepressants) demonstrated greater than a 10% concentration loss under refrigeration over 48 h (34). Based on this evidence, SSTs were deemed acceptable for 17 of the 21 assays (34). The susceptibility of lipophilic and protein-bound drugs to gel adsorption remained a notable concern. A major limitation of the study was its reliance on calibrator-spiked instead of patient-derived samples.

Hepburn et al. investigated the impact of serum separator gel on sex steroid stability by comparing BD SST^™II Advance tubes with plain serum tubes in 47 healthy volunteers using LC-MS/MS (35). They found that androstenedione concentrations declined significantly in gel tubes over time, with a mean reduction of 13.9% on day 5 (P<0.001), possibly due to the adsorption of this hydrophobic analyte to the polyester-based gel matrix. Testosterone, 17-hydroxyprogesterone, and estradiol also showed modest but significant decreases in gel tubes on day 5 [2.1%, 3.3%, and 4.6%, respectively (P<0.01)], indicating that gel-mediated analyte loss affects multiple steroids (35). By contrast, plain tubes showed rising concentrations of estradiol and testosterone by day 5, with increases of 7.6% and 4.0%, respectively. These increases are hypothesized to result from ongoing enzymatic activity in unseparated serum, particularly by RBC or macrophage enzymes such as 17β-hydroxysteroid dehydrogenase, which may catalyze the conversion of androstenedione to testosterone or alter steroid hormone structures after collection. Additionally, the dissociation of steroids from carrier proteins, such as sex hormone-binding globulin, during refrigerated storage may increase the proportion of free hormone extracted during analysis. These mechanisms could contribute to falsely elevated hormone concentrations in samples that remain in contact with the cellular clot for extended periods (35). The observed decrease in androstenedione (13.9%) exceeded the allowable bias threshold of 10.5%, suggesting a risk of clinically significant underestimation. Strengths of this study include the use of a highly specific LC-MS/MS platform, application of real-world storage conditions, and direct relevance to preanalytical practices in endocrine testing. However, the modest sample size of 20 samples for testosterone, androstenedione, and 17-hydroxyprogesterone, and 27 for estradiol, along with the absence of diseased samples and potential gel-to-serum volume effects from repeated aliquoting—which may have amplified analyte loss—limit the applicability of the findings. Nonetheless, the study provides clear evidence that gel tubes may underestimate androstenedione, whereas unprocessed plain tubes risk overestimating estradiol and testosterone.

Consistent with these findings, Chen et al. investigated how polymer-based separator gels and clotting conditions affect the accurate quantification of synthetic cathinones in blood. Using a validated LC-MS/MS method, they evaluated the recovery of 71 structurally diverse synthetic cathinones across multiple BCTs, including plain serum tubes, plasma tubes with anticoagulants, and SSTs containing polymer gels (36). They found that hydrophobic compounds showed markedly reduced recoveries in SSTs compared to heparin plasma tubes, with a strong inverse correlation between LogP and recovery (r=−0.8) (36). For example, recoveries for 3,4-methylenedioxypyrovalerone (MDPV; LogP 3.79) and α-pyrrolidinohexiophenone (PV8; LogP 4.66) declined to approximately 55–60% in SSTs, whereas polar compounds such as butylone (LogP 1.77) retained near-complete recovery (36). Recovery improved in a dose-dependent manner with heparin supplementation, reaching over 85% for MDPV with 150 units of heparin. Conversely, thrombin added to washed PSTs induced a 20–30% recovery loss, mirroring SST performance (36). These results suggest that clotting and polymer gels synergistically promote the retention of lipophilic drugs, likely through fibrin-mediated gel adsorption. The study’s strengths include a wide range of analytes, thorough method validation—with intraday precision between 0.4% and 10.6% and strong calibration linearity (R² ≥0.994)—and well-controlled mechanistic experiments that examined the effects of clotting and anticoagulant use. However, its limitations include the use of spiked blood from healthy volunteers, which may not fully represent clinical or postmortem samples, and the inability to identify the specific chemical components of the proprietary gel. Overall, the findings emphasize the potential for substantial underestimation of hydrophobic analytes in gel-based tubes without anticoagulants, highlighting an important preanalytical risk in both forensic and clinical toxicology.

In line with the findings of Chen et al., Zungun et al. explored the stability of additional hormone analytes in gel-based vs. non-gel tubes (37). They evaluated the impact of serum separator gel and storage time on the stability of nine hormone immunoassays by comparing BD Vacutainer SST II Advance tubes and Improvacuter Gel and Clot Activator tubes with their respective gel-free counterparts using the UniCel^® DxI 800 Immunoassay System in samples from 36 healthy volunteers (37). They found that estradiol and testosterone concentrations were significantly lower in gel-containing tubes than in plain tubes, with median estradiol values at baseline of 135.8 pmol/L in Improvacuter gel tubes and 128.5 pmol/L in BD SSTs, compared to 157.8 pmol/L in the BD Clot Activator reference tube (P<0.01 for both) (37). Similarly, testosterone concentrations measured 1.24 and 1.21 nmol/L in Improvacuter and BD gel tubes, respectively, versus 1.31 nmol/L in the reference tube (P<0.01), suggesting analyte adsorption to the gel matrix (37). Contrastingly, other hormones such as free T₃, progesterone, and follicle-stimulating hormone remained stable across all tube types, with differences within allowable analytical variation (37). Over a 48 h storage period at 2–8 °C, the concentrations of all analytes fluctuated slightly, but changes remained within the total allowable error (TAE) limits, indicating that clinical stability was maintained (37). Conversely, estradiol and testosterone levels measured in gel tubes consistently fell below the lower analytical limits established using the BD Clot Activator tube, suggesting a risk of clinically important underestimations in these measures (37). Strengths of the study included the evaluation of multiple hormones, direct comparison of gel and non-gel tubes from two manufacturers, and use of TAE criteria to assess clinical impact. Limitations included reliance on a single analyzer platform and the lack of biochemical or compositional analysis of the gel matrix, which hinders understanding of the underlying mechanisms. The findings align with prior literature indicating that separator gels may adsorb hydrophobic steroid hormones and underscore the importance of validating tube type before their use in hormone assays. Thus, while most hormones remain stable in gel tubes over time, estradiol and testosterone are susceptible to gel-mediated losses that may lead to clinically significant underestimations (37).

Tokudome et al. provided further evidence of gel-induced analyte loss in SSTs (38). After 168 h of room temperature storage, amitriptyline concentrations in Vacutainer tubes fell to just 2.5% of baseline values, compared to 75.2% in gel-free controls (38). At 4 °C, concentrations dropped to 14.0% in gel tubes vs. 101.2% in controls. Additional lipophilic drugs, including mirtazapine, chlorpromazine, and flunitrazepam, showed similar losses. Analyte adsorption correlated strongly with lipophilicity (r=0.96). Although the study used controlled experiments, limitations included a small sample size (n=3) and the absence of authentic clinical specimens (38).

The same vulnerabilities observed in SSTs have also been reported for PSTs, which utilize similar gel barriers and are frequently used for plasma-based testing. Jordan et al. assessed PSTs against serum for TDM of 11 drugs using the Roche Cobas 8000 platform (39). PSTs exhibited a notable negative bias for lipophilic drugs, particularly phenytoin (−6.8%) and carbamazepine (−7.6%), with 7 and 11 out of 40 specimens, respectively, exceeding the TAE goal of 10% (39).

Seven-day refrigerated stability studies further highlighted these differences. In PSTs, carbamazepine concentrations declined considerably, showing a −10.7% bias on day 1 and −13.6% by day 7, whereas concentrations in serum remained within acceptable limits (39). Phenytoin concentrations in PSTs also exceeded the TAE threshold on day 1 and exhibited inconsistent recovery over the study period. Similarly, phenobarbital demonstrated a sustained negative bias in PSTs while remaining stable in serum tubes throughout the 7 days (39).

This study adhered to the Clinical and Laboratory Standards Institute (CLSI) EP09 and EP35 guidelines, utilized both spiked and patient samples, and conducted duplicate testing. Limitations include the inability to definitively distinguish gel adsorption from chemical degradation and the use of spiked specimens for certain analytes.

Shepard and Bliumkin conducted a comprehensive stability assessment of 53 drugs and metabolites relevant to forensic toxicology using BD PSTs (40). After 3 months of refrigerated storage, lipophilic analytes such as fentanyl, carfentanil, mirtazapine, nortriptyline, fluoxetine, and citalopram exhibited greater than 60% loss (40). Conversely, more hydrophilic compounds such as morphine, hydromorphone, and methamphetamine retained greater than 90% of their initial concentrations (40). Substantial losses were observed within 24 h, indicating both rapid onset and cumulative adsorption (40). While the study included a wide drug panel and rigorous storage conditions, it was limited by the exclusive use of drug-spiked plasma and a single plasma volume.

In summary, the use of gel separator tubes (SSTs and PSTs) in TDM presents notable challenges, particularly for lipophilic drugs. While these tubes provide practical benefits such as simplified sample processing and reduced cellular contamination, they have been found to selectively adsorb lipophilic analytes, leading to substantial concentration losses during storage. Studies indicate that drugs such as lidocaine, phenytoin, and various psychoactive substances experience considerable reductions in concentration due to gel adsorption, with losses ranging from 10% to over 60%, depending on the drug and storage conditions. This adsorption is particularly pronounced at room temperature and can compromise the accuracy of drug quantification. Given these findings, laboratories should reconsider the routine use of gel separator tubes for monitoring lipophilic therapeutic agents. Instead, gel-free tubes or mechanically separated alternatives should be prioritized to ensure diagnostic reliability and patient safety, particularly for drugs that are susceptible to adsorption.

Mechanical separator tubes: a gel-free alternative

Mechanical separator tubes, such as the BD Vacutainer Barricor, employ a high-density mechanical barrier instead of polymer gel to separate plasma from cellular components after centrifugation (41-43). This design minimizes the risk of drug adsorption and addresses many limitations associated with polymer gel-based separator tubes, making them a promising alternative for TDM and various diagnostic testing procedures (41).

Schrapp et al. conducted one of the most comprehensive comparisons between gel-based and mechanical separator tubes (44). In their evaluation of 167 therapeutic compounds, plasma separator gel tubes considerably reduced measured concentrations of 40 drugs, most notably neuroleptics (69.2%), antidepressants (42.3%), and cardiovascular agents (19.2%) (44). By contrast, only six drugs, including two antidepressants (imipramine, trimipramine), two neuroleptics (alimemazine, promethazine), one analgesic (sufentanil), and one anxiolytic (clobazam), showed decreased levels in Barricor tubes (44). These findings highlight the reduced analyte interaction potential of gel-free barriers as well as the benefits of mechanical separator tubes for preserving lipophilic drugs (44).

Similarly, Arslan et al. found that digoxin levels measured in Barricor tubes were higher than those in conventional serum tubes, suggesting improved analyte recovery and reduced surface interaction (45). Subsequently, Hegstad et al. demonstrated poor long-term stability for lipophilic drugs such as verapamil when stored in SSTs, reinforcing the need for gel-free alternatives such as Barricor, particularly for extended storage protocols (46).

Morosyuk et al. further validated the performance of Barricor tubes in preserving drug concentrations during long-term refrigerated storage (47). Hydrophobic drugs such as phenytoin and carbamazepine remained stable throughout the study period, in contrast to the declines typically observed in gel-containing tubes (47). These findings underscore the material advantage of mechanical barriers in preventing analyte adsorption and ensuring storage stability (47).

Jordan et al. conducted a comprehensive evaluation of Barricor tube performance for TDM of 11 drugs using the Roche Cobas^® 8000 platform (39). Minimal bias was observed for gentamicin (1.5%), methotrexate (0.8%), phenobarbital (0.2%), and theophylline (0.2%) compared to serum, supporting the suitability of Barricor as an alternative matrix for these analytes. This study was the first to assess Barricor tubes for TDM of gentamicin, methotrexate, phenobarbital, and theophylline using the Roche platform.

Refrigerated stability over 7 days demonstrated that most drugs, including hydrophobic compounds such as carbamazepine and valproic acid, remained within ±10% of baseline concentrations in Barricor tubes (39). However, phenytoin exhibited notable variability: although the average bias between Barricor and serum was low (0.8%), concentrations in both tube types exceeded the TAE threshold by days 4 and 5, respectively, and wide error bars limited definitive conclusions.

In PSTs, carbamazepine showed a −7.6% bias, with 11 of 40 samples exceeding the TAE limit, whereas phenytoin demonstrated a −6.8% bias, with 7 of 40 samples outside the TAE (39). PSTs also exhibited a considerable decline in carbamazepine concentrations over time, with a −10.7% bias on day 1 and −13.6% by day 7 (39). Similarly, phenobarbital exhibited a persistent negative bias throughout the 7-day stability period in PSTs but remained stable in Barricor and serum tubes (39). The study adhered to CLSI EP09 and EP35 protocols and included both spiked and patient-derived samples, although the number of patient samples was limited for less frequently prescribed drugs such as salicylate and theophylline. Limitations included the inability to distinguish drug loss due to gel adsorption from chemical degradation and potential variability introduced by matrix effects. Overall, the study demonstrated that Barricor tubes are a reliable alternative to serum tubes for most drugs evaluated, though caution may be warranted when interpreting phenytoin concentrations owing to its inconsistent stability profile.

Separator tubes: beyond TDM—balancing analytical stability and operational performance

Beyond TDM, mechanical separator tubes offer distinct advantages in diagnostic and research settings. Padoan et al. reported that Barricor tubes considerably reduced residual white blood cell counts by 63.9–82.7% and RBC counts by 29.7–66.4% compared to BD PSTs (41). These reductions varied based on centrifugation speed and duration (41). Although higher centrifugation velocities increased hemolysis in Barricor tubes, all samples remained below clinically significant thresholds (≤0.5 g/L), emphasizing the need for optimized centrifugation protocols (41).

Building on these findings, Fournier et al. demonstrated that aspartate aminotransferase, potassium, glucose, and lactate dehydrogenase remained stable for 10 days in Barricor tubes, compared to fewer than 5 days in PST tubes, whereas phosphate was stable for 4 vs. 2 days, respectively, demonstrating Barricor’s superior analyte stability during prolonged storage (42). Similarly, Gawria et al. found that phosphate exceeded the maximal allowable bias after 68 h in Barricor tubes compared to 29–35 h in gel tubes, and potassium after 40 vs. 9–12 h, confirming Barricor’s extended stability and potential to reduce preanalytical errors during transport and delayed processing (43).

Knutti et al. demonstrated that Barricor tubes preserve biochemical markers such as taurine, glutamic acid, interleukin 8, and soluble CD40 ligand more effectively than gel tubes under biobanking conditions (48). These findings highlight the potential of mechanical separator tubes in translational research, particularly where biomolecular stability is critical (48).

Jordan et al. also emphasized the operational benefits of Barricor tubes, noting enhanced laboratory workflow efficiency and reduced sample-handling requirements (39). These features make Barricor tubes particularly advantageous in high-throughput clinical settings. Similarly, Cembrowski et al. validated the use of Barricor tubes in routine diagnostics, demonstrating a reduction in preanalytical errors for high-sensitivity troponin T and other important analytes (49).

Despite their advantages, rare yet significant preanalytical errors have been documented. Allard and Bowen reported cases of improper gel-barrier migration in separator tubes, which obstructed automated analyzers and resulted in delayed or erroneous test results (50). Although such issues are more prevalent in gel-based tubes, the findings emphasize the importance of implementing visual inspection protocols and routine tube integrity checks as part of preanalytical quality assurance (QA) (50).

In a related investigation of gel-based separator tubes, Bao et al. evaluated two batches of BCTs manufactured by K.S. Medical to assess the potential for chemical interference from separator gels (51). Tubes from the new batch (s2005030), which contained a transparent gel, produced markedly elevated calcium concentrations when filled with deionized water: 0.50 mmol/L for 1 mL, 0.31 mmol/L for 2 mL, and 0.15 mmol/L for 3 mL on the AU5400 and AU5800 analyzers (Beckman Coulter) (51). By contrast, tubes from the old batch (s2001006), containing a milky white gel, yielded consistently low calcium concentrations (<0.03 mmol/L) across all test volumes (51). The inverse relationship between calcium concentration and water volume in the new batch suggested a dilution-dependent leaching effect from the separator gel matrix (51). These findings provide strong evidence that separator gels may not only adsorb analytes but also introduce exogenous contaminants, resulting in falsely elevated test results. The authors recommended lot-specific verification of new BCT batches to ensure analytical accuracy, although the lack of chemical identification of the leached substance and limited evaluation across other platforms were noted limitations (51). Nevertheless, the results highlight the notable preanalytical risks associated with compositional differences between old and new gel formulations as well as the need for enhanced QA procedures for newly introduced BCT lots, despite the practical challenges laboratories may face in routinely implementing lot-specific BCT protocols.

Given these concerns, recent studies have focused on validating mechanical separator tubes in routine outpatient settings. Orhan et al. compared BD Barricor lithium-heparin tubes with mechanical separators to BD SST II Advance tubes in 52 outpatient and oncology patients, evaluating 18 routine biochemistry analytes using Beckman Coulter platforms (52). They found that potassium levels in Barricor tubes were significantly lower than those in SSTs, with mean reductions of 4.94% in outpatients and 5.95% in oncology patients (P<0.001 and P=0.002, respectively) (52). In both cohorts, the total error for potassium exceeded the TAE threshold of 5.61%, reaching 6.44% and 7.45% (52). These findings align with previous reports attributing higher potassium values in serum tubes to platelet degranulation during clot formation, supporting the known risk of pseudohyperkalemia in SSTs. All other analytes remained within desirable analytical limits, and the authors highlighted practical benefits of Barricor tubes, including shorter centrifugation times and improved turnaround in oncology workflows (52). Strengths of the study included adherence to CLSI guidelines, analyte-specific bias evaluation, and inclusion of both general and high-priority clinical populations. Study limitations include its small oncology cohort (n=16), single-center design, and absence of certain analytes such as phosphorus and high-density lipoprotein (HDL). Nonetheless, the findings support the broader adoption of mechanical separator tubes while emphasizing the need for potassium-specific reference adjustments and ongoing analyte validation.

In summary, mechanical separator tubes, such as BD Barricor, provide a gel-free alternative that markedly enhances analyte stability, particularly for lipophilic drugs susceptible to gel adsorption. These tubes demonstrate superior preservation of therapeutic and routine analytes in both TDM and broader clinical applications while also reducing preanalytical variability and improving operational efficiencies in high-throughput environments. As with any BCD, it is crucial to maintain ongoing performance monitoring, adhere to proper handling protocols, and select tubes tailored to specific analytes to optimize laboratory outcomes and ensure diagnostic reliability.

Clot activators

Advancements in clotting agents, such as silica and thrombin-based technologies, have improved the speed and efficiency of serum preparation for clinical laboratory testing. These agents facilitate coagulation by activating intrinsic and extrinsic pathways, both of which converge at the activation of factor X, leading to thrombin generation, fibrinogen cleavage, and stable fibrin clot formation (5,53). While these innovations enhance serum yield and reduce turnaround times, concerns persist regarding their compatibility with various analytical platforms and their potential to introduce preanalytical variability (5,54,55).

Silica-based clot activators, which initiate coagulation through factor XII in the intrinsic pathway, are widely used in BCTs due to their reliability and ease of integration into routine workflows (5,53). However, mounting evidence suggests that certain clot activator formulations, particularly when combined with silicone SFs, can interfere with select assays such as ion-selective electrode-based measurements.

Naznin et al. demonstrated that silica clot activators and silicone SFs in standard vacutainer tubes caused significant positive interference in serum lithium measurements using ion-selective electrode methods (56). In lithium-free volunteers, clot activator tubes produced false lithium readings with a mean concentration of 1.78 mmol/L, whereas patients undergoing lithium therapy showed inflated values with a mean of 2.80 mmol/L (56). Although this study identified a clear preanalytical source of error, its limitations included a relatively small sample size (n=40) and the absence of a manufacturer comparison (56).

Supporting these findings, Ikkurthi et al. observed a mean positive bias of 0.18 mmol/L in lithium concentrations measured from clot activator tubes compared to those from glass vials (57). These discrepancies were attributed to clot activators or associated additives that interfere with the ion-selective electrode matrix (57). Saharia et al. further confirmed this interference, reporting significant variation in lithium measurements across different serum tubes (58). The authors proposed that residual clot activators and SFs may not completely sediment with the clot after centrifugation, remaining in the serum phase and contributing to measurement inaccuracies (58). Collectively, these findings emphasize the necessity for method-specific validation of BCTs, particularly for assays sensitive to ionic or SF interferences.

In response to limitations associated with traditional clot activators, researchers have explored alternative prothrombin activators derived from snake venom. Zhao et al. introduced outer surface protein A (OsPA), a novel prothrombin activator complex isolated from the venom of Oxyuranus scutellatus, also known as the Coastal Taipan (59). OsPA tubes generated thrombin levels up to 20 times higher than those in commercially available serum tubes, considerably reducing clotting time while maintaining analyte stability for up to 7 days (59). Building upon this work, Zhao et al. evaluated recombinant ecarin, a prothrombin activator derived from the saw-scaled viper, Echis carinatus (60). Recombinant ecarin effectively facilitated clot formation in anticoagulated samples, including those treated with heparin, warfarin, or dabigatran, revealing its potential utility in challenging clinical scenarios (60).

Despite their promising clotting efficiency, venom-derived activators face several key limitations. Zhao et al. noted challenges related to reduced enzymatic activity following sterilization, limited clinical validation, and small sample sizes (59,60). These issues must be addressed before widespread clinical adoption is feasible.

Overall, while clot activators are essential for efficient serum preparation, they can introduce assay-specific biases, particularly in methods sensitive to ionic or SF interferences. Their effect on lithium testing underscores the need for method-specific validation, as silica- and silicone-based additives may cause spurious results. Novel clotting agents such as OsPA and recombinant ecarin show promise for anticoagulated specimens by improving thrombin generation and reducing clotting times, though clinical use is limited by post-sterilization enzymatic activity, suggesting the need for further validation. Therefore, careful selection and rigorous evaluation of clot activator-containing BCTs are essential to minimize preanalytical variability and ensure assay reliability across different analytical platforms.

Anticoagulants

Anticoagulants are important additives in BCTs, designed to prevent clot formation and preserve analyte integrity in hematology, chemistry, and coagulation testing. Inappropriate selection, improper handling, or incorrect tube filling can introduce considerable preanalytical variability and compromise assay accuracy (5).

Heparin

Heparin is one of the most commonly used anticoagulants in clinical laboratories, typically employed at concentrations of 10–30 USP/mL of blood (5). Heparin exerts its anticoagulant effect by activating antithrombin III, which in turn inhibits thrombin and other key coagulation factors. Despite its widespread use, heparin can interfere with various assays, particularly those measuring metal ions, such as calcium, magnesium, and potassium, due to its high affinity for cationic species.

Mahler et al. demonstrated that underfilled heparinized tubes introduced measurable analytical bias, including a 0.05-unit potential of hydrogen (pH) shift and 0.3 mmol/L reduction in ionized calcium levels (61). While the paired-sample design provided direct evidence of volume-related effects, the small sample size of eight healthy volunteers limits the generalizability to broader patient populations.

Jacobs et al. further investigated the assay-specific impact of heparin on 25(OH)D measurements (62). Using 34 paired patient samples collected in serum-gel and lithium-heparin tubes, the authors compared results across two immunoassay platforms: the Liaison XL (DiaSorin) and Cobas e801 (Roche) (62). A significant positive bias of 57.5% (P<0.001) was observed in heparin plasma compared to serum on the Liaison XL, resulting in a higher proportion of patients classified as vitamin D sufficient (79% vs. 64%) (62). By contrast, the Cobas e801 showed negligible bias (−0.2%), suggesting that the interference is platform-specific (62). The study’s strengths include its within-subject design and multiplatform comparison, whereas limitations included the use of a single tube type, a relatively small sample size, and reliance on thawed specimens. The mechanism of interference remains unresolved.

In neurodegenerative disease research, Jiang et al. found that heparin can influence measurements of Alzheimer’s biomarkers (63). Plasma tau and phosphorylated tau 181 (p-tau181) concentrations were considerably higher in lithium-heparin (4.87 pg/mL) and sodium-heparin (4.41 pg/mL) tubes compared to dipotassium ethylenediaminetetraacetate (K₂EDTA) tubes (3.15 pg/mL) (63). Similarly, p-tau181 levels were elevated in lithium-heparin and sodium-heparin samples relative to EDTA. No significant differences were found for Aβ peptides (63). The selective elevation of tau-related biomarkers suggests that heparin may interfere with antibody detection or promote tau aggregation. Although the findings are compelling, the study was limited to young, healthy individuals and a single analytical platform.

The formulation of heparin (liquid vs. dry) also impacts analyte accuracy. Chhapola et al. compared liquid and dry balanced heparin in pediatric blood gas testing (64). Liquid heparin considerably compromised analyte accuracy, with a high proportion of samples exceeding TAE limits for sodium (77%), pCO₂ (40%), chloride (54%), potassium (34%), and bicarbonate (23%) (64). Only pH (0%) and lactate (3%) remained within TAE thresholds (64). These findings suggest that dilution effects and ionic binding from liquid heparin markedly distort blood gas and electrolyte measurements.

Dukić et al., representing the Croatian Society of Medical Biochemistry and Laboratory Medicine, voiced these concerns in formal recommendations against the use of liquid heparin in blood gas analysis (65). They highlighted its potential to distort pH, partial pressure of oxygen (pO₂), and partial pressure of carbon dioxide (pCO₂) values and emphasized the necessity of standardizing the use of dry balanced heparin to minimize matrix-related bias (65).

EDTA

K₂EDTA, typically used at 1.5–2.0 mg/mL of blood, is preferred in hematology owing to its ability to chelate divalent cations such as calcium and magnesium, thereby preserving cellular morphology. However, improper handling, including underfilling, can alter test results. EDTA contamination is a known source of spurious hyperkalemia, hypocalcemia, hypomagnesemia, and hypozincemia in chemistry assays (5,66).

Toprak et al. demonstrated that inadequate fill volumes in EDTA tubes affect endocrine measurements (67). A 50% filled EDTA tube caused a 19% reduction in adrenocorticotropic hormone (ACTH) levels, whereas a 25% filled tube resulted in a 50% decrease (67). These errors are clinically significant, particularly in diagnosing Cushing’s syndrome, where ACTH quantification is essential for distinguishing between ACTH-dependent and ACTH-independent forms (67). The study did not directly quantify EDTA concentration, a limitation that prevents the determination of a clinically relevant interference threshold.

Oxalates

Oxalates, specifically potassium oxalate (~2 mg/mL of blood), are primarily used as anticoagulants for glucose preservation. They function by chelating calcium to inhibit coagulation; however, they adversely affect RBC morphology. As discussed in a review by Lima-Oliveira et al., potassium oxalate can cause a 10% reduction in RBC size, making it unsuitable for tests requiring intact cellular morphology, such as peripheral blood smears or erythrocyte sedimentation rates (5). Contrastingly, ammonium oxalate induces RBC swelling (5). Although double oxalate mixtures are designed to balance these opposing effects, they remain inadequate for preserving leukocyte morphology and can inhibit key enzymatic activities, including those of amylase, lactate dehydrogenase, and various phosphatases (3,5).

Sodium citrate

Sodium citrate, used at 105–129 mM (3.2% or 3.8%), is the anticoagulant of choice for coagulation assays. It chelates calcium to preserve clotting factor activity, enabling accurate prothrombin time and activated partial thromboplastin time (aPTT) measurements (5). Cornes noted that underfilled citrate tubes can cause up to a 12% deviation in aPTT results, although this was based on secondary data rather than controlled experiments (68). Calcoen et al. reported clotting artifacts on automated analyzers associated with a faulty lot of citrate tubes (69). Visual inspection revealed clotted or partially filled samples, emphasizing the need for consistent additive volume. The authors recommended that laboratories monitor the frequency of clotted specimens as a quality indicator and investigate anomalies in additive volume (69).

Gros et al. documented substantial variability in citrate concentrations across manufacturers (3.2–23.0%) and detected trace contaminants, including potassium, calcium, magnesium, and lithium (70). Their use of purified water rather than whole blood introduces uncertainty regarding clinical impact due to the absence of protein-binding and cellular matrix interactions (70). The analysis also revealed anticoagulant volume deficiencies ranging from −11.6% to −91.1%, with draw-volume concentration biases up to 25.7%, stressing the need for lot-specific QC (70).

In response to global supply shortages during the coronavirus disease 2019 (COVID-19) pandemic, Gosselin et al. issued guidance for laboratories transitioning to alternative or in-house citrate tubes (71). They argued that even slight deviations in citrate concentration or draw volume can considerably affect coagulation parameters (71). The authors recommended validating new tubes with a minimum of 40 paired patient samples covering prothrombin time/INR, aPTT, fibrinogen, D-dimer, and anti-Xa assays (71).

Anticoagulants are essential additives in BCTs as they preserve analyte integrity across various testing modalities. However, factors such as formulation, concentration, fill volume, and compatibility with specific assays can considerably influence test results. For instance, heparin, while widely used, can introduce dilutional and ionic effects that compromise blood gas and electrolyte measurements, as well as interfere with immunoassays and neurodegenerative biomarkers. Similarly, EDTA and oxalates can distort electrolyte and hormone measurements, whereas sodium citrate requires strict adherence to volume control to ensure reliable coagulation results. Collectively, these findings emphasize the importance of selecting and validating anticoagulant-containing BCTs based on analyte-specific requirements, preanalytical conditions, and intended clinical use.

Glycolysis inhibitors

Certain BCTs include glycolysis inhibitors to preserve labile analytes such as glucose. These additives are important for ensuring accurate glucose measurements in the diagnosis and management of diabetes mellitus, prediabetes, and gestational diabetes mellitus (GDM) (72-74). Glycolysis begins immediately after blood collection, resulting in progressive decreases in glucose concentrations that may lead to underdiagnosis and misinformed clinical decisions (75-78).

In the absence of glycolysis inhibitors, studies have shown that glucose concentrations may decline by approximately 10% at room temperature within 24 h, particularly in serum tubes (79). Even lithium-heparin tubes with separator gels are affected, showing average glucose declines of 8.4 mg/dL owing to glycolytic activity by entrapped RBCs (79).

Traditionally, sodium fluoride (NaF) has been used to inhibit glycolysis at a concentration of approximately 2–3 mg/mL. NaF alone does not exert immediate inhibition and may require up to 4 h to fully suppress glycolytic enzyme activity (80). To address this limitation, citrate-buffered NaF tubes have been developed to enhance glycolysis inhibition by lowering sample pH.

Szoke et al. demonstrated that Venosafe^® Glycaemia tubes containing citrate buffer maintained glucose stability within a ±2% bias over 4 h (81). Dimeski et al. confirmed these findings, reporting glucose concentrations remained stable within ±1.8% bias for up to 24 h (82). Saracevic et al. raised concerns about overestimation, reporting glucose levels up to 7.3% higher in citrate-buffered tubes compared to lithium-heparin tubes, suggesting limited interchangeability and the need for method-specific validation (83).

Alternative inhibitors have also been explored. Banerjee et al. evaluated D-mannose as a glycolysis inhibitor and found that glucose declined by only 3 mg/dL over 3 h, compared to a 16 mg/dL drop in NaF-treated samples (84). While promising, D-mannose requires further validation before routine clinical application.

Coward et al. compared several BCTs, including Barricor and sodium fluoride/citrate/Na2EDTA (FC-Mix) tubes, and found that both maintained glucose stability for up to 24 h at 4 °C, outperforming fluoride-EDTA tubes (85). These findings suggest that tube type and additive composition play a key role in sample preservation, even under refrigeration.

Szoke et al. expanded their earlier work, reporting that citrate-buffered tubes detected 25% more GDM cases than NaF-only tubes, reinforcing the clinical relevance of improved glycolytic inhibition (86). Nevraumont et al. similarly observed a 20% increase in impaired fasting glucose and GDM detection rates when using citrate-buffered tubes compared to standard NaF tubes (87). These studies also reported a 25% increase in GDM case identification compared to NaF tubes (86,87). While these results suggest improved diagnostic sensitivity, the clinical implications remain uncertain.

Kume et al. introduced NaF-adenosine triphosphate (ATP) tubes, incorporating ATP to enhance inhibition (88). Glucose declined by only 5.8±2.9 mg/dL after 24 h, compared to 9.0±2.7 mg/dL in standard NaF tubes (88). Similarly, Heilmann et al. found that citrate-buffered tubes improved sensitivity in detecting diabetes and prediabetes compared to NaF tubes (89). While these innovations show promise, most studies were limited by small sample sizes, single-center designs, and a lack of long-term clinical outcome data.

Therefore, glycolysis inhibitors are necessary for accurate glucose testing, particularly in the context of diabetes and GDM screening. While conventional NaF provides delayed inhibition, newer formulations such as citrate-buffered NaF and NaF-ATP tubes offer improved glucose stability and may enhance diagnostic sensitivity. However, concerns about overestimation, lack of standardization, and potential overdiagnosis persist. Until validated by larger multicenter outcome studies, clinical laboratories should cautiously adopt advanced inhibitors, balancing diagnostic accuracy with clinical utility.

Importance of order of draw (OOD) in blood collection

Adhering to the recommended OOD is essential for proper phlebotomy technique and helps prevent additive carryover that can compromise laboratory test results. The CLSI GP41-ED7 guidelines, along with recommendations from the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group for the Preanalytical Phase (WG-PRE), provide a standardized sequence for blood collection: blood culture tube, sodium citrate tube, serum tube (with or without clot activator and gel), heparin tube (with or without gel), EDTA tube (with or without gel), and glycolysis inhibitor tube (e.g., sodium fluoride or potassium oxalate) (90,91). Deviating from this sequence can result in additive contamination, particularly from EDTA and citrate, which may considerably alter analytical results and impact patient care.

Cadamuro et al. provided early experimental evidence of additive carryover effects. Their study demonstrated that even minimal EDTA contamination could result in a 20% decrease in calcium levels and a 35% increase in potassium levels (92). At higher levels of contamination, calcium and magnesium were nearly undetectable, revealing the potential for substantial analytical interference when OOD is not properly followed (92).

Ercan et al. confirmed the clinical relevance of these findings by showing that collecting blood in EDTA tubes prior to serum or plasma tubes led to spurious hyperkalemia (93). This resulted in false diagnoses and unnecessary clinical interventions, including electrolyte management protocols and additional testing. The study emphasized the need for vigilant adherence to OOD even in routine outpatient settings (93).

Keppel et al. reported widespread noncompliance with established OOD protocols in both inpatient and outpatient phlebotomy services, particularly in high-volume laboratories (94). They noted that phlebotomists often rely on tube color rather than additive type, which can vary between manufacturers, increasing the likelihood of incorrect tube sequencing (94). Simundic et al. also highlighted this issue, pointing to non-standardized color coding and inconsistent tube labeling as notable contributors to procedural error (19).

Mahato et al. further investigated the impact of OOD deviations on commonly ordered chemistry and coagulation tests (95). Their study showed that reversing the tube sequence led to considerable alterations in potassium levels and coagulation parameters. In particular, prothrombin time and aPTT were prolonged in specimens contaminated with EDTA or citrate. Similarly, Mahto et al. reported that contamination with EDTA or potassium oxalate prolonged coagulation times by up to 20 s, which could lead to inappropriate anticoagulation management decisions (96).

Although some authors have questioned the necessity of strict OOD adherence in closed blood collection systems, Bazzano et al., in a systematic review, emphasized that real-world compliance remains inconsistent (97). Their review highlighted that many studies evaluating OOD were limited by small sample sizes, non-standardized protocols, and a lack of rigorous assessment of downstream clinical outcomes. These methodological limitations, combined with inconsistent staff training and variable institutional policies, reduce confidence in dismissing the importance of draw order (97).

In conclusion, adhering to the correct OOD is crucial for minimizing additive carryover, reducing test inaccuracies, and preventing misdiagnoses in laboratory testing. The CLSI GP41-ED7 guidelines provide a standardized sequence for blood collection that helps prevent contamination from additives, particularly EDTA and citrate, which can considerably alter analytical results. Evidence from various studies highlights the detrimental effects of deviations from the OOD, including substantial reductions in calcium levels and increases in potassium levels, leading to false diagnoses and unnecessary clinical interventions. Furthermore, widespread noncompliance with established OOD protocols often results from reliance on tube color rather than the specific additive type, underscoring the need for consistent practices and adequate training for phlebotomy personnel.

Effects of BCT components on metabolomic and proteomic studies

Metabolomics and proteomics are key areas in clinical research. Metabolomics focuses on small molecules (<1,500 Da) that provide a snapshot of physiological and disease-related metabolic pathways (98,99), whereas proteomics investigates proteins by examining their structure, function, and interactions to identify disease-specific biomarkers and monitor therapeutic responses (100,101).

Both areas employ sensitive analytical technologies, such as MS and NMR spectroscopy, for which preanalytical variability remains a significant source of error (102). Variables such as the choice of blood matrix (serum or plasma), anticoagulants, clot activators, and storage conditions directly impact the reproducibility and reliability of metabolomic and proteomic data (103-110).

In metabolomics, the choice of serum or plasma considerably affects the metabolite profile (111). Serum typically contains higher concentrations of amino acids, carbohydrates, and lipids due to glycolysis and proteolytic activity during clotting (112). Consequently, serum is often preferable for NMR-based metabolomics because it reduces spectral interference from anticoagulants. By contrast, plasma is typically favored for proteomics applications (102). EDTA-stabilized plasma, for example, minimizes enzymatic degradation and enhances reproducibility in MS-based proteomic analyses (113).

Beyond matrix type and anticoagulants, tube additives such as clot activators and separator gels can considerably influence metabolomic and proteomic data quality. López-Bascón et al. demonstrated that polymeric gel separator tubes remarkably alter serum metabolite profiles compared to conventional tubes (114). Particularly, serum collected in gel tubes showed decreases in several key metabolites, including alanine (−16.0%, P=0.0018), proline (−16.0%, P=0.0166), threonine (−7.46%, P=0.0134), aconitic acid (−38.2%, P=0.0166), and lactic acid (−1.63%, P=0.0143), alongside increases in monopalmitin (9.93%, P=0.0222) and dodecanol (1.95%, P=0.0166).

These metabolites are integral to major metabolic pathways, including amino acid biosynthesis, glycolysis, and the citric acid cycle. The study also found that plasma samples collected in gel tubes exhibited minimal metabolic variation, suggesting a matrix-dependent effect. Although constrained by a small, predominantly female cohort and a focus on gas-liquid chromatography-time-of-flight MS, the findings highlight the importance of validating tube selection in metabolomics workflows (114).

In addition to gel separators, other tube additives can introduce analytical variability. MS-based analyses are particularly susceptible to interference from SFs and polymer residues found in BCTs. These substances may introduce chemical noise, ion suppression, or enhancement, impairing the detection of low-abundance analytes. Salvagno et al. and Canez et al. investigated labware contamination in lipidomics and found that PP microcentrifuge tubes contributed numerous contaminant signals, some misidentified as lipids based on mass-to-charge (m/z) ratios (110,115). Particularly, Eppendorf tubes produced 485 extraneous signals, resulting in severe ion suppression in 40 low-abundance lipids and moderate suppression in more abundant analytes (110).

Recent studies also demonstrate preanalytical challenges in vibrational spectroscopy. Da Silva et al. examined the impact of separator gels on Fourier-transform infrared (FTIR) spectroscopy-based diagnostics for chronic kidney disease using Vacuette^® tubes (116). Gel tubes reduced classification accuracy from 85% to 65% when air served as the background reference. While accuracy improved with diluted gel as background, spectral contamination from gel components (e.g., olefins and carbohydrates) remained evident. Vibrational bands corresponding to critical disease-associated metabolites such as galactose-4-sulfate, phenolic derivatives, and tryptophan were obscured, limiting diagnostic utility. These findings call attention to the need for careful material selection or signal correction strategies when using FTIR-based methods (116).

In proteomics, anticoagulant selection has a measurable impact. Halvey et al. found that plasma proteome profiles generated using LC-MS/MS shotgun proteomics varied depending on whether EDTA or heparin BCTs were used (101). A total of 36 proteins showed considerably different abundance (false discovery rate <0.05), with many proteins underrepresented in heparin-treated samples, possibly due to heparin interference with spectral counting methods. Approximately 11% of proteins differed by more than twofold between the two anticoagulants, supporting the call for standardized selection of tube types in proteomic workflows.

Thus, the selection of appropriate BCT components, including matrix type, additives, anticoagulants, and separator materials, is crucial for ensuring reproducible and accurate data in metabolomic and proteomic applications. The choice between serum and plasma, as well as the specific anticoagulants employed, can considerably influence metabolite and protein profiles, impacting the reliability of analytical results. Additionally, tube additives such as clot activators and separator gels can introduce analytical variability, affecting the detection of low-abundance analytes and leading to misinterpretation of data. By minimizing preanalytical variability due to BCDs and preserving analytical accuracy, researchers can enhance the quality of findings in these vital fields of clinical research.

Validation and verification of BCTs

The COVID-19 pandemic exacerbated supply chain disruptions, considerably impacting the availability of BCTs (71). In response to these shortages, suppliers implemented protective allocation measures and prioritized high-demand tubes; however, these actions did not provide immediate relief. Healthcare providers were encouraged to adopt conservation strategies, including limiting unnecessary blood tests, reducing the frequency of complete blood count draws, and utilizing alternative tube types or brands. The adoption of alternative tubes required rigorous validation and verification by clinical laboratories to prevent erroneous test results, as different types or brands may contain varying additives that can compromise sample integrity. Laboratory professionals must understand these validation and verification processes to ensure the ongoing reliability of BCTs in clinical settings. The steps for validating and verifying new or reformulated BCTs in a typical clinical laboratory will be discussed in the following sections. More details on tube validation and verification studies can be found in the CLSI GP34-A guideline.

Overview of validation and verification processes

Validation and verification are essential QA processes that ensure BCTs perform reliably within clinical laboratories. Validation is a systematic, ongoing process used to confirm that a device consistently fulfills its intended clinical purpose, whereas verification is a one-time, laboratory-specific assessment confirming that a manufacturer’s performance claims hold under local conditions (15,117). Both processes are vital for minimizing preanalytical variability and ensuring test accuracy, particularly when implementing new or alternative BCTs.

Limitations of regulatory oversight and the need for local protocol

Despite international standards such as the international organization for standardization (ISO) 9001 and ISO 15189, financial constraints often drive laboratories to adopt lower-cost BCTs that may lack comprehensive regulatory oversight (117). Thus, rigorous, locally developed validation protocols are required. These should be scientifically robust, ethically approved, and thoroughly documented. Validation should encompass the entire blood collection system, including tubes, needles, holders, and transfer devices, and must follow a written protocol reviewed by laboratory leadership or an institutional review board (7). Ethics committee approval is recommended, particularly when studies involve patient specimens (117). The lot number and expiration date of BCTs must also be documented.

Technical validation requirements

Technical validation is essential to ensure that BCTs meet the manufacturer’s claims regarding their structure, functionality, and safety under local practices (117). To achieve this, technical validation should include at least 240 randomized blood collections, with 120 per tube type, as advised by CLSI EP28-A3 (117). This process involves assessing various performance issues, including physical defects, vacuum integrity, fill volume, cap sealing, and sample quality. Additionally, lot rejection criteria, such as failure rates exceeding 1%, may also be applied (117).

Traditional validation approaches typically involve comparing candidate tubes with a reference tube by measuring differences in defect rates (117). However, this method assumes that the reference tube is free of error. In practice, reference tubes may have unrecognized defects or inconsistent performance, which can obscure the true quality of a candidate tube and result in misleading conclusions.

To address this limitation, sigma metrics may be used as a more objective, reproducible, and statistically robust alternative. Sigma metrics quantify performance by converting observed defect rates into a standardized index expressed as defects per one million opportunities (118). This approach enables laboratories to evaluate BCTs based on absolute performance rather than relative performance. By eliminating reliance on potentially flawed comparator tubes, sigma-based validation enhances the accuracy, transparency, and fairness of performance assessments. Furthermore, it allows for the application of consistent, predefined acceptance thresholds across different sites and BCTs. The use of sigma metrics supports long-term monitoring, enhances reproducibility, and promotes continuous quality improvement in the preanalytical phase (118). Given these advantages, sigma-based technical validation should be employed when introducing new or reformulated BCTs into clinical practice.

Clinical validation for analytical and clinical equivalence

Clinical validation, as outlined in the review by Bowen and Adcock, refers to the process of evaluating whether new BCTs introduce clinically significant bias when compared to existing tubes, using routine laboratory instruments and methods under real-world conditions (7). This process typically includes paired comparisons across a representative range of analytes and patient populations to determine whether any observed differences fall within acceptable error limits and do not compromise clinical decision-making.

Methodological guidelines for BCT comparison studies

BCT comparison studies should follow the CLSI EP9-A3 guideline, using a minimum of 40 patient samples per analyte within the clinically relevant range. Statistical tools include Deming or Passing-Bablok regression, Bland-Altman analysis, and paired t-tests or Wilcoxon signed-rank tests. Precision studies should follow the CLSI EP5-A guideline, which recommends a 20-day protocol involving at least two reagent lots, two daily runs, and duplicate testing at two control levels. For analytes present at low concentrations in healthy individuals, spiking with known quantities is recommended. Validation strategies should reflect the BCT’s intended clinical application and encompass evaluation across various analytical platforms, such as ion-selective electrodes, immunoassays, enzymatic spectrophotometry, and MS (7,117,119).

Imprecision and lot-to-lot variability studies

Imprecision should be reported as the coefficient of variation [CV (%)] or standard deviation, with at least 20 replicates at two or more clinical decision levels. A Gaussian distribution should be confirmed, and outliers should be identified using methods such as Dixon’s or Tukey’s test. Lot-to-lot variability should be evaluated across at least three lots using analysis of variance to detect significant differences (7). Risk analysis techniques such as failure mode and effects analysis can further support the identification of potential failure modes and their impact (117).

Additional validation considerations

Additional validation parameters include evaluating the effects of tube materials, separator gels, stoppers, and SFs. Particular attention should be paid to interactions with platforms such as LC-MS or ion-selective electrodes. Studies should include clinically relevant sample types such as hemolyzed, lipemic, and icteric specimens and assess the impact of delayed centrifugation, extended storage, and tube-to-tube transfers, specifically for labile analytes such as potassium, calcium, lactate dehydrogenase, and drug concentrations. Verification must be repeated following any change in the manufacturer, tube composition, or supplier, even when products are marketed as functionally equivalent (7,117). This is particularly important for sodium citrate tubes used in coagulation testing, where manufacturer-dependent variability can considerably alter clotting results, as documented in recent guidance during supply shortages (71).

Stability and acceptance criteria

Stability studies should measure analyte concentrations over defined intervals, such as 4, 8, and 24 h, under routine storage conditions and be analyzed using repeated-measures analysis of variance or Friedman’s tests. Observed changes should be interpreted using bias specifications derived from biological variation (7). Clinical acceptance criteria should be determined in advance based on biological variation, medical decision limits, or PT thresholds. If acceptance criteria are not met, the medical significance of non-equivalence must be evaluated. Manufacturers can cover the cost of tubes and reagents needed for as part of the procurement process (117).

Manufacturer compatibility and component integration

BCTs and their components should be obtained from the same manufacturer unless compatibility and integration with components from other manufacturers have been validated by regulatory authorities. The use of unvalidated component combinations is strongly discouraged (117).

Recent validation and verification studies

Recent studies reinforce the importance of rigorous BCT validation. Ucar et al. conducted a technical validation of BD Barricor lithium-heparin plasma tubes vs. BD SST II Advance Plus tubes in 150 healthy volunteers (120). Comparative evaluation across 21 analytes in clinical chemistry and immunoassay platforms employed paired t-tests, Bland-Altman plots, and percentage bias estimation. While most results met predefined performance goals, white particulate matter was observed in 24.6% of Barricor tubes, raising concerns about potential downstream interference (120).

Abusoglu et al. performed an end-user verification study comparing VacuSEL and BD SSTs across 23 routine chemistry analytes in 50 individuals (121). Following CLSI GP34-A and GP41-A6 protocols, they applied Deming regression, bias plots, and TAE thresholds based on biological variation (121). Lactate dehydrogenase bias exceeded desirable specifications at 6.41% but remained within TAE (121). Sodium and total protein exhibited storage-related shifts, but these remained within acceptable limits (121). VacuSEL tubes demonstrated lower hemolysis indices and higher serum yields; however, performance concerning chloride and creatinine, combined with the exclusive use of healthy participants, indicates a need for broader multicenter validation (121).

Nell et al. conducted a multicenter verification study across three institutions, evaluating four alternative BCT brands: Vacucare, Vacuette, Vacutest, and V-TUBE compared to BD Vacutainer tubes (122). Paired samples from at least 40 individuals per tube type were analyzed for routine hematology and coagulation assays (122). K₂EDTA tubes from Vacucare, Vacuette, and Vacutest showed comparable performance against BD tubes (122). By contrast, V-TUBE displayed clinically unacceptable mean cell volume and mean cell hemoglobin concentration biases. Mean cell volume ranged from 1.15% to 1.47% vs. an allowable limit of ±0.95%, whereas mean cell hemoglobin concentration ranged from −1.65% to −0.93% vs. an allowable limit of ±0.43% (122). For sodium citrate tubes, none of the alternative brands met criteria for prothrombin time or aPTT (122). Vacucare’s aPTT bias ranged from 2.78% to 4.59%, exceeding the desirable allowable limit of ±2.30% (122). These findings align with the observations by Gosselin et al., who warned that unvalidated citrate tubes from unknown or inconsistent manufacturing sources pose considerable risks to coagulation testing accuracy and patient safety (71).

Limitations in current validation practices

These findings demonstrate that even when following CLSI guidance, performance differences can still arise due to tube and analyzer interactions, lot variability, and limited population diversity. Validation results may also be influenced by tube composition, SF interference, and preanalytical handling conditions, factors often overlooked in routine verification protocols (7). Reliance on healthy volunteers and small sample sizes can reduce generalizability. Furthermore, in the U.S., many laboratories rely on a single BCT manufacturer for routine testing, which restricts access to comparative performance data and heightens vulnerability to supply chain disruptions (7). Therefore, statistically rigorous, site-specific validation that encompasses multiple lots, diverse specimens, and analyzer-specific assessments will ensure the safe implementation of new BCTs, particularly in regulated or high-risk testing domains.

Overall, the validation and verification of BCTs are critical processes for ensuring consistent analytical performance and maintaining diagnostic accuracy. Laboratories must establish site-specific protocols that align with CLSI guidance and incorporate comprehensive evaluations of BCTs into their internal QA and external PT frameworks.

Integrating BCTs into QC and PT

Limitations of traditional QC approaches

QC programs are used to monitor analytical accuracy in clinical laboratories. While QC materials are typically tested against established performance thresholds, conventional protocols often do not replicate the full range of preanalytical conditions encountered in routine practice. Particularly, QC samples are processed independently of the BCTs used for patient specimens, limiting the ability of QC procedures to detect BCT-related interferences.

BCT-related interferences in QC materials

This disconnect is problematic because BCT-related factors, such as additive leaching, adsorption, and pH shifts, can alter test results in ways that standard QC procedures may not detect. To address this issue, laboratories are encouraged to conduct paired assessments in which QC materials are processed both with and without exposure to BCTs. These evaluations can help uncover subtle but clinically relevant tube effects; however, implementation is often challenged by the wide variety of tube types and variability between lots. Bowen et al. demonstrated statistically and clinically significant changes in TT₃, TT₄, and cortisol concentrations in QC materials when poured into commonly used plastic serum tubes, such as plain red top, rapid serum tubes, and SST, compared to glass tubes. Particularly, TT₃ levels increased by up to 24.9% and TT₄ by up to 29.0% at certain QC levels (15). These differences frequently exceeded both the significant change limit and maximum desirable bias based on biological variation, particularly when measured using the Immulite^™ 1000 platform (15).

Utility of QC materials in detecting tube effects

Although introducing QC materials into BCTs may not fully replicate patient specimens due to differences in matrix composition, viscosity, and the absence of cellular components, this approach remains a practical strategy for detecting tube-related artifacts. Bowen et al. further showed that even when serum specimens from healthy volunteers did not display clinically significant alterations in TT₃ or cortisol levels across tube types, TT₄ values were considerably impacted by both Vacuette tubes and SSTs (3). This discrepancy suggests that QC materials may be more sensitive to BCT-induced assay interference, possibly due to the absence of cellular buffering or the smaller volume used. Given this sensitivity, evaluating QC materials in the context of BCT exposure can serve as an early warning for potential analytical issues that may not be apparent in standard QC workflows or external PT.

Standardizing QC practices through manufacturer support and workflow alignment

To improve feasibility, manufacturers could support this process by pre-exposing control sera to BCTs under standardized conditions during lot release testing. This strategy could help laboratories detect potential interferences during internal verification or routine QC processes.

In addition to manufacturer-led testing, laboratories can enhance the diagnostic relevance of QC programs by aligning them more closely with actual patient workflows. This alignment includes processing QC materials using the same BCTs, centrifugation protocols, and storage conditions applied to clinical samples. Such mirroring facilitates better detection of BCT-related variability and strengthens the robustness of internal QA.

BCT integration in PT

PT is a cornerstone of external QA, allowing laboratories to benchmark their performance against that of peers using comparable methods. Most PT programs rely on pre-aliquoted vials that bypass preanalytical variables such as BCTs; consequently, tube-related effects are unlikely to be detected during PT. To replicate real-world conditions, PT materials should be transferred into the candidate BCTs before analysis (7). This recommendation is based on the principle that PT samples, such as QC materials, should undergo the same preanalytical exposures as patient samples to uncover BCT-specific interferences that would otherwise remain undetected. This approach integrates BCT-associated variables into the total testing process, enhancing the clinical relevance of PT results and improving the ability to detect preanalytical biases.

Overall, integrating BCT-specific considerations into QC and PT frameworks is essential for developing a comprehensive QA approach in clinical laboratories. By simulating real-world preanalytical conditions, laboratories can more effectively identify tube-related interferences and improve internal monitoring. This alignment not only improves the detection of preanalytical biases but also strengthens interlaboratory comparability. Ultimately, this strategy promotes both diagnostic accuracy and patient safety.

Integrated vs. combined venous blood collection systems

Venous blood collection systems can considerably influence specimen quality and diagnostic accuracy. Over the past decade, a growing body of evidence has underscored the advantages of using integrated systems, in which tubes, needles, holders, and transfer devices are provided by a single manufacturer and validated for mutual compatibility (123).

Early observations by Plebani et al. warned that assembling venous blood collection systems from different manufacturers, referred to as combined systems, can result in unverified combinations that compromise both safety and analytical performance (123,124). These concerns are reinforced by the CLSI GP41 and GP39 A6 standards, which caution that mismatched components may cause hemolysis, underfilling, component disengagement, and other technical failures (90,123).

In response, the EFLM WG-PRE emphasized the importance of using integrated systems validated by the manufacturer. Lippi et al. stressed that only systems with demonstrated safety and performance should be used and that laboratories are responsible for verifying local compatibility whenever integrated configurations are modified (117,123).

Building on this, the joint EFLM and Latin American Confederation of Clinical Biochemistry recommendation by Simundic et al. stated that all blood collection components, including needle, holder, and tube, should originate from the same manufacturer (125). Deviations from this standard are discouraged unless formal risk assessments are conducted and documented in accordance with ISO 15189 (123).

Despite strong guidance, empirical comparisons between integrated and combined systems remain limited. Most studies are observational, small in scale, and rely heavily on manufacturer-provided data, which often lack generalizability to routine clinical practice (123). No randomized controlled trials or meta-analyses have directly compared the two configurations (123). To address this gap, Rigoni and Tessarolo conducted a systematic review that synthesized 20 documents published between 2010 and 2021 (123). Their analysis encompassed three key domains: analytical quality, patient and user safety, and cost-effectiveness. The review confirmed that integrated systems are consistently endorsed by international authorities, including the EFLM, CLSI, and the World Health Organization (WHO). These systems were associated with reduced hemolysis, dilutional effects, and analytical interference and were shown to simplify training and minimize specimen handling errors. This highlights the importance of adopting integrated systems to enhance laboratory efficiency and diagnostic accuracy. Future research should continue to explore the benefits of these systems in various clinical settings.

Conversely, unvalidated combined systems were found to introduce considerable risks, including incompatible additives, unstable anticoagulant concentrations, and inconsistent separator gels. Regulatory ambiguities within the European Union regarding post-market surveillance and legal responsibility were also highlighted (123). Although these systems may appear cost-effective, they can ultimately result in increased clinical and financial burdens.

Supporting evidence cited in the review, such as Chung et al., demonstrated that improper tube selection can lead to sample rejection, delayed turnaround times, increased healthcare costs, and reduced diagnostic confidence (123,126). Similarly, Bowen and Adcock advised that laboratories cannot assume safety and efficacy based solely on manufacturer claims; local verification is recommended (7,123).

In summary, a decade of evolving evidence and international consensus supports the use of integrated venous blood collection systems as the preferred standard in clinical laboratories. These systems, which consist of tubes, needles, holders, and transfer devices from a single manufacturer, have been shown to enhance specimen quality and diagnostic accuracy by minimizing risks associated with mismatched components (123). When supply limitations or procurement policies necessitate alternative configurations, laboratories must conduct comprehensive local validation and risk assessments to ensure clinical safety and performance (123). Until robust, high-quality comparative data become available, integrated systems remain the most reliable option for ensuring accurate, efficient, and safe laboratory testing (123).

BCD components: catheters, syringes, and needles as sources of preanalytical errors

In addition to BCTs, other BCD components such as catheters, syringes, and various needle types are key contributors to preanalytical variability. These devices can impact the integrity of the blood sample at the point of collection and may introduce hemolysis, contamination, or changes in blood volume. This section explores how these components can affect sample quality and laboratory test results.

Catheters

Catheters, as illustrated in Figure 1B, are frequently employed in emergency and intensive care settings for both fluid administration and blood sampling. Commonly constructed from materials such as polytetrafluoroethylene, polyurethane, silicone, polyvinyl chloride, and polyolefin, these devices offer practical advantages in critically ill patients but pose several preanalytical risks (127). Blood collected through catheters is subject to mechanical shear stress, distorting blood cells, activating platelets, and promoting hemolysis. This may result in artificially elevated concentrations of analytes such as potassium, lactate dehydrogenase, and aspartate aminotransferase (125).

Hemolysis is among the most prevalent complications associated with catheter-based sampling. In a critical review and meta-analysis, Lippi et al. reported that hemolyzed specimens are considerably more common when collected via catheters than in those obtained through standard venipuncture, with an overall odds ratio of 3.4 and relative risk of 1.07 (128). In emergency settings, catheter-drawn samples show hemolysis rates of up to 12%, whereas rates from standard venipuncture are generally reported to be approximately 2–3%. Hemolysis accounts for 40–70% of all rejected samples in clinical laboratories. These findings highlight the potential for spurious laboratory results that may lead to unnecessary diagnostic workups and clinical interventions.

In a large retrospective study, Mrazek et al. analyzed 19,001 emergency department samples to investigate the relationship between collection technique and hemolysis (129). They found a strong positive correlation between the level of vacuum applied during sampling and extent of the hemolysis as measured by free hemoglobin (129). Particularly, high vacuum produced considerably greater hemolysis than low vacuum or manual aspiration methods (129). A highly predictive pressure-dependent model was developed to estimate free hemoglobin levels based on the vacuum force applied (R²=0.99) (129). Catheter use and collection tube design must be considered to minimize preanalytical variability (129).

Benzalkonium chloride, a quaternary ammonium compound, is used as a broad-spectrum antimicrobial agent to coat both the internal and external surfaces of central venous catheters (130). Its primary function is to prevent microbial colonization of the catheter, which is a key risk factor for catheter-related infections. Benzalkonium chloride is entrapped in a hydrophilic hydromer layer composed of polyvinylpyrrolidone, which is cross-linked and bound to the polyurethane catheter surface. Upon contact with aqueous solutions such as blood, benzalkonium chloride is released slowly, ensuring sustained antiseptic activity on catheter surfaces. Despite its antiseptic benefits, chemical interference remains a notable concern during catheter-based blood collection. A seminal report by Gaylord et al. identified benzalkonium chloride, incorporated into certain heparin-bound catheter formulations, as a potential interferent (131). When leached into blood samples, this compound can disrupt ion-selective electrode assays, leading to falsely elevated sodium and potassium concentrations. The underlying mechanism of interference is hypothesized to involve structural interactions between benzalkonium chloride and the ion-selective membrane or sodium-specific ionophores, particularly those utilizing methyl monensin, as employed in Vitros analyzers (132). These interactions can change the electrochemical properties of the membrane, resulting in inaccurate sodium measurements. Therefore, catheter materials that minimize the risk of chemical leaching into specimens meant for electrolyte analysis must be prioritized.

Another underrecognized preanalytical issue involves the adsorption of lipophilic drugs such as tacrolimus and cyclosporine to catheter surfaces. Hacker et al. demonstrated both in vitro and in clinical cases that even after a single saline flush, substantial residual concentrations of tacrolimus (55 µg/L) and cyclosporine A (132 µg/L) remained in catheter lumens (133). In one patient, tacrolimus concentrations from a catheter-drawn sample measured 86 µg/L, whereas those from a venipuncture performed 2 days after infusion cessation measured only 2.9 µg/L. Notably, drug release persisted even after flushing with more than 24 L of saline, indicating that residual drug may continue to leach into blood samples over extended periods. These findings challenge the assumption that unused catheter lumens are free from contamination and raise important concerns about falsely elevated drug levels during TDM (133).

More recently, Marschner et al. reported a pediatric case involving a peripherally inserted polyurethane central catheter in which tacrolimus contamination persisted for 27 days following intravenous administration (134). Falsely elevated tacrolimus concentrations were observed in blood samples collected from both the drug administration and blood-draw lumens (134). This case identifies the persistent and unpredictable nature of drug adherence to catheter surfaces. Particularly, avoidance of previously accessed catheter lumens for TDM of calcineurin inhibitors such as tacrolimus is recommended.

In response to these challenges, professional organizations have issued formal recommendations. The EFLM WG-PRE, in collaboration with the Latin American WG-PRE LATAM of COLABIOCLI, advises against routine blood collection through catheters (125). These guidelines cite increased risks of hemolysis, contamination, and analytical interference, emphasizing the need for standardized procedures when catheter sampling is unavoidable (125). Additional concerns include the lack of universally accepted protocols for flushing, waiting times, and discard volumes prior to sample collection.

To reduce catheter-related preanalytical error, new technologies have emerged. One such innovation is the PIVO^™ needle-free blood collection system, which utilizes a flexible polymer cannula to collect blood through existing peripheral intravenous catheters (135). Compatible with both standard vacuum tubes and syringes, PIVO™ offers a minimally invasive alternative to traditional catheter draws (135). In a controlled study involving healthy volunteers, Natali et al. reported a hemolysis rate of 1.8% with PIVO^™, compared to 3.3% with standard collection methods, using a free hemoglobin threshold of 50 mg/dL (136). While promising, these findings require further validation in acutely ill populations, where vascular integrity and flow dynamics are often compromised.

While catheter-based blood collection can be necessary in acute care settings, it introduces multiple preanalytical vulnerabilities that can notably affect test accuracy. These include hemolysis, chemical interference, and drug adsorption. Additionally, the use of catheters can cause mechanical shear stress, distorting blood cells and activating platelets, which may lead to elevated analyte concentrations and higher hemolysis rates compared to standard venipuncture (125,128). Additionally, the presence of antimicrobial agents such as benzalkonium chloride can interfere with ion-selective assays, leading to falsely elevated sodium and potassium levels (131,132). When catheter use is unavoidable, strict adherence to standardized protocols, appropriate device selection, and ongoing QA are essential to safeguard result reliability. Innovative technologies, such as the PIVO^™ needle-free blood collection system, offer potential solutions to minimize hemolysis and improve specimen quality (135,136); however, venipuncture remains the reference standard for obtaining high-quality laboratory specimens.

Syringes

A closed blood collection system is generally recommended for venous sampling to enhance safety and preserve specimen integrity (125). Despite this, syringes, as displayed in Figure 1C, remain widely used in clinical practice, particularly for patients with small or fragile veins and in settings requiring rapid testing of labile analytes such as arterial blood gas (ABG) analysis (125). Structurally, syringes consist of a cylindrical barrel with millimeter gradations and a plunger that generates negative pressure upon retraction. Most blood collection syringes are manufactured from PP or polyethylene and may contain various additives, including plasticizers, stabilizers, lubricants, antistatic agents, and antioxidants to enhance performance and manufacturability (3,15).

Historically, glass syringes were preferred for ABG collection owing to their low gas permeability; however, they have largely been replaced by plastic alternatives to improve compliance with the Occupational Safety and Health Administration regulations and reduce the breakage risk (137,138). While plastic syringes offer logistical and safety advantages, their higher gas permeability poses a risk to the stability of oxygen- and carbon dioxide-sensitive analytes, particularly when processing delays occur.

In one of the earliest evaluations of syringe-dependent variability, Lima-Oliveira et al. compared four brands of heparin-coated syringes and identified statistically and clinically significant differences across multiple analytes, including pO₂, potassium, calcium, and glucose (139). Notably, potassium concentrations were considerably lower in samples drawn using an in-house prepared syringe than those drawn using a commercial product (3.56 vs. 3.80 mmol/L, P<0.01), surpassing allowable analytical bias limits (139). These differences were attributed to variations in syringe composition, such as heparin formulation, plastic type, and manufacturing methods, emphasizing that material-related properties can introduce preanalytical bias even under standardized handling conditions (139).

More recently, Çuhadar et al. assessed ABG stability in plastic syringes stored under various conditions (137). Samples free from air contamination remained stable for up to 1 h at room temperature, with pO₂ remaining stable for 120 min and pH and glucose levels stable for 90 min. By contrast, samples exposed to air exhibited deviations exceeding 55%, whereas refrigerated samples showed a pO₂ increase of up to 26.7% (137). Although storage conditions were controlled during the study, it employed only a single syringe type, limiting the generalizability of the findings. Nevertheless, the results highlight the critical interplay between syringe material properties—specifically gas permeability—and storage environment (137).

Zavorsky and van Wijk applied survival analysis to estimate storage time thresholds for ABG and CO oximetry analytes collected in plastic syringes (138). They found that 95% of samples remained analytically stable for up to 40 min at room temperature. Interestingly, pO₂ stability was diminished in samples stored on slushed ice, a result attributed to increased gas diffusion through the syringe wall at lower temperatures. Although this study did not investigate cellular oxygen consumption, the findings reinforce earlier observations that syringe wall permeability is a primary factor affecting analyte integrity, particularly for gas-sensitive parameters (138).

Overall, variations in syringe composition (e.g., level of gas permeability and type of heparin coating) can lead to clinically meaningful differences in analyte concentrations, even under standardized collection and storage conditions (137-139). Furthermore, the stability of analytes such as O₂ and glucose can be negatively impacted by exposure to air and the storage environment, highlighting the critical interplay between syringe material properties and sample integrity (137,138). Therefore, clinical laboratories must carefully evaluate and validate the types of syringes used in their workflows to mitigate preanalytical errors and enhance the reliability of test results.

Needles

Needles, as illustrated in Figure 1D, are hollow metallic devices typically made of stainless steel, titanium, or other metal alloys, designed to access veins and facilitate blood flow into collection tubes. The gauge size determines the diameter of the needle, with higher gauge numbers corresponding to smaller internal diameters (140,141). These devices may introduce preanalytical errors by releasing trace metals such as chromium, nickel, or manganese from their surfaces into the collected sample. This risk is particularly relevant for ultra-trace element testing, where even minimal contamination may considerably skew results.

Laur et al. emphasized the potential for trace metal contamination during venipuncture with stainless steel needles, citing the presence of chromium, nickel, and manganese in standard steel alloys (140). Although their study did not experimentally quantify the leaching of metals from the needle, elevated imprecision was reported at low concentrations for several trace elements, most notably manganese (CV of 22.1% at 2.01 µg/L) and nickel (CV of 24.8% at 0.81 µg/L) (140). The study design did not control for needle composition, limiting the ability to differentiate analytical variability from material-based contamination. Nevertheless, the findings underscore the importance of using standardized, contaminant-free materials, such as plastic cannulas, when collecting specimens for trace metal analysis (140). In a more rigorous experimental study, Sommer et al. investigated both in vivo and in vitro contamination potential from stainless steel needles (141). Involving 100 healthy volunteers and 100 simulated collections, they found no significant clinical differences in chromium or cobalt concentrations between sequentially collected tubes (0.191 and 0.024 µg/L, respectively) (141). However, broader lot screening of the same needle model revealed that 8% of batches failed contamination criteria, with chromium levels reaching as high as 4.62 µg/L (141). These results demonstrate that, although contamination may not be universally present, lot-to-lot variability can introduce sporadic risks, reinforcing the need for QC screening during device procurement (141).

Needle gauge and internal geometry are critical determinants of hemolysis and sample quality. Smaller gauge needles (e.g., 23- to 27-gauge) are associated with increased shear forces that elevate hemolysis risk, whereas larger gauge needles (13- to 17-gauge) may induce turbulent flow, potentially compromising analyte stability (142,143). Scanning electron microscopy conducted by Lippi et al. identified substantial variability in internal diameters among nominally identical 21-gauge needles, with differences of up to 35% (ranging from 393.8±2.6 to 605.4±9.6 µm) (144). Although the clinical significance of such variability remains to be fully elucidated, these findings suggest that uncontrolled manufacturing tolerances may influence flow dynamics, shear stress, and susceptibility to preanalytical error.

Moreover, Lippi et al. reported that variations in needle wall thickness and internal roughness can alter flow resistance and cause RBC deformation, particularly when smaller gauges are used (144). These structural inconsistencies may amplify hemolysis by increasing the interaction force between the erythrocytes and the needle wall. From a fluid dynamics perspective, blood flow inside the needle is governed by Bernoulli’s principle and Euler equations, whereby a reduction in internal diameter exponentially increases the velocity and shear stress (144). A 20% reduction in inner diameter can generate a 44% increase in interaction force, thereby raising the risk of RBC lysis (144).

Careful selection of needle gauge, alloy composition, bevel geometry, and batch-level QA is crucial for minimizing variability and protecting specimen integrity. Additionally, incorporating scanning electron microscopy and mechanical characterization into QC procedures can further enhance reliability and reduce preanalytical errors related to the device.

Butterfly needles

Butterfly needles, also known as winged infusion sets, as illustrated in Figure 1E, are commonly used for patients with fragile veins due to their ease of use and comfort. However, the gauge and bore geometry of these needles can greatly impact specimen quality and the likelihood of hemolysis.

Umemura et al. retrospectively analyzed 53,189 venipunctures to compare complication rates and hemolysis incidence between 22-gauge and finer 23- to 25-gauge butterfly needles (145). Whereas finer gauges reduced the complication frequency from one in 10,825 (22-gauge) to one in 29,747 (23- to 25-gauge), the rate of gross hemolysis increased from 0.10% to 0.28% (145). In hemolyzed samples, serum potassium levels increased from 4.1 to 6.2 mmol/L, aspartate aminotransferase rose from 28 to 86 U/L, and lactate dehydrogenase increased from 198 to 412 U/L (145). These findings suggest substantial RBC lysis and potential for spurious laboratory results; however, the lack of paired sampling between gauges limits inferences about cause (145).

To address whether needle design can mitigate hemolysis despite small gauge size, Rosada et al. compared a 25-gauge UltraTouch Push Button butterfly needle, featuring a five-bevel tip and ultra-thin wall, to a standard 23-gauge Safety Lok device in 161 elderly inpatients (146). Although the hemolysis index was lower with the UltraTouch needle (6.16 vs. 8.60 mg/dL, a 28% reduction), this difference did not reach statistical significance (P=0.76) (146). In a paired sub-cohort of 36 patients, the UltraTouch device yielded a 47% reduction in hemolysis index (P=0.57), again without statistical significance (146). Importantly, serum potassium was significantly lower with the UltraTouch needle (3.85 vs. 3.96 mmol/L, P=0.002), suggesting improved preservation of RBC integrity (146).

Further corroborating the importance of design over gauge alone, Giussani et al. evaluated butterfly needle performance in 577 oncology outpatients with difficult venous access (143). They reported a significantly lower hemolysis rate with 23- to 25-gauge UltraTouch devices (0.7%) compared to Safety Lok sets (3.3%) (P<0.001), despite the use of finer needles (143). Analytical performance remained uncompromised, indicating that optimized structural features such as bevel configuration, internal bore diameter, and wall thickness can mitigate the hemolysis typically associated with smaller gauge needles (143).

Giussani et al. also found that patients experienced reduced procedural pain and anxiety when the UltraTouch Push Button system was used. Phlebotomists also reported lower perceived difficulty during venipuncture with UltraTouch devices (143). These improvements suggest that ergonomic enhancements and material innovation improve sample quality, streamline clinical workflows, and enhance patient satisfaction.

Early studies linked finer butterfly needles to increased hemolysis and erroneous laboratory results (145). However, recent evidence suggests that these risks can be effectively mitigated through better device design. Needle gauge is not the sole factor influencing preanalytical performance; rather, a combination of engineering features, including bevel structure, material flexibility, and internal geometry, collectively determines specimen quality. By adopting structurally optimized butterfly needles, healthcare providers can simultaneously reduce preanalytical errors and enhance result reliability, particularly in patients with fragile or compromised veins.

Recommendations for clinical laboratories and manufacturers

To minimize preanalytical variability and enhance the reliability of laboratory testing, a series of recommendations is proposed for BCT components, catheters, syringes, and needles. These recommendations underscore the necessity for coordinated efforts between clinical laboratories and manufacturers and are summarized in Table 2. We strongly encourage tube manufacturers to adopt Lean, Six Sigma, and failure mode and effects analysis methodologies in the design and development of BCDs. This approach should prioritize minimizing interference with clinical laboratory test results. By implementing evidence-based practices and fostering collaboration between laboratories and manufacturers, we can considerably enhance the overall quality of laboratory testing.

Tube wall materials

The choice of specialized BCT materials should be based on specific assay requirements. For instance, low-binding polymers are preferred for Alzheimer’s biomarkers, such as Aβ peptides. Manufacturers should consider creating hybrid tubes with surface-modified materials that reduce analyte adsorption. Concurrently, laboratories must validate how tube materials affect target analytes and identify any leachable compounds that could interfere with assays.

SFs

Clinical laboratories must validate the compatibility of low SF or SF-free tubes for immunoassay workflows. Manufacturers should clearly label the type and concentration of SF and provide SF-free alternatives to reduce assay interference. Enhanced transparency in labeling will facilitate informed tube selection and promote test standardization.

Rubber stoppers

The choice of rubber stoppers is critical. For coagulation assays, low-magnesium stoppers should be utilized to prevent falsely shortened clotting times. Thiuram-free stoppers are recommended for BLL testing to eliminate reactive gas contamination, whereas silicone-coated stoppers should be avoided in workflows involving immunoassays or MS. Manufacturers are urged to develop low-interference stopper materials and provide comprehensive assay compatibility data.

BCT separators

Attention must also be given to BCT separators, as their selection can substantially influence the accuracy of TDM. For immunosuppressant drug monitoring, EDTA tubes are preferred, whereas heparin tubes are generally suitable for most therapeutic drug assays. SSTs should be avoided for lipophilic drugs that are susceptible to gel adsorption, particularly during extended storage. Best practices include prompt centrifugation within 2–3 h and refrigerated storage at 2–8 °C. Manufacturers should continue to refine gel formulations and develop mechanical separator tubes, such as BD Barricor, to minimize analyte loss. Comprehensive QC and clear labeling of additives will further support the accurate interpretation of results.

Clot activators

Standardized clotting protocols are essential for ensuring consistent serum preparation. For analytes such as lithium, using clot activator-free BCTs or those without residual particulates is preferred. Rapid-acting clotting agents are advantageous for time-sensitive tests, and emerging activators such as recombinant ecarin and OsPA show promise but require further clinical validation. Manufacturers should aim for scalable and cost-effective production to facilitate the widespread adoption of these innovations.

Anticoagulants

Strict adherence to blood-to-additive ratios is essential, and assay-specific validation of anticoagulants, particularly heparin and citrate, should be conducted to assess potential interference. Manufacturers must implement robust QC measures to ensure consistent additive concentrations and explore refined formulations that minimize binding or matrix effects.

Glycolysis inhibitors

Glucose stabilization tubes are essential for maintaining sample integrity, particularly in preventing glycolysis during transport and storage. Citrate-buffered or NaF-ATP tubes are recommended for their effectiveness in inhibiting glycolysis. Prompt sample processing is crucial, particularly for tubes with limited stability. Additionally, further research on emerging inhibitors such as D-mannose is needed. Educating laboratory staff about the performance characteristics of these tubes and standardizing glucose tube effectiveness across different platforms should both be prioritized.

OOD

The correct OOD is important to prevent additive carryover and ensure the accuracy of test results. Institutions must conduct regular audits and training to ensure compliance with OOD protocols. Manufacturers can assist in these efforts by improving additive deposition techniques and designing tubes to minimize carryover risk.

BCTs for metabolomics and proteomics

In metabolomics, EDTA plasma is preferred for MS-based assays, whereas serum is generally favored for NMR analyses. Heparinized plasma is recommended in proteomics to minimize ion suppression. Serum samples should be allowed to clot for 30–60 min, whereas plasma samples should be cooled immediately and stored at −80 °C for long-term stability. Laboratories should use blank tube controls and implement rigorous QC procedures to detect additive contamination. Manufacturers must also produce high-purity, low-interference tubes suitable for high-sensitivity applications.

Integrated blood collection systems

Given the diverse requirements for metabolomics and proteomics, clinical laboratories should prioritize integrated blood collection systems to minimize compatibility-related preanalytical errors across various assays. By doing so, these systems can use components from a single manufacturer to reduce variability. If combined systems are necessary, compatibility must be demonstrated through institutional validation studies. Manufacturers should focus on reducing batch-to-batch variability, ensuring consistent additive application, and disclosing potential interactions between components and assays. Collaborative efforts will enhance reliability and ensure compliance with regulatory standards.

Catheters

The selection of appropriate catheters is crucial for minimizing hemolysis and ensuring sample integrity. To minimize hemolysis and sample dilution, catheter-based blood collection should utilize larger-gauge catheters (at least 22 gauge) when clinically feasible. Standardized flushing protocols and adherence to post-flush wait times are necessary to prevent dilutional artifacts. Blood sampling should be performed from drug-free sites to avoid contamination from adsorbed medications. Clinical personnel must be trained in catheter-specific collection techniques, with hemolysis monitoring integrated into QA workflows. Manufacturers should focus on catheter designs that minimize hemolysis and improve performance across populations, including pediatric and small-gauge applications, while incorporating sustainable materials to support environmental goals.

Syringes and needles

Clinical laboratories must develop standardized protocols for syringes and needles to minimize preanalytical errors in blood collection. Rapid processing of specimens collected in plastic syringes is essential for preserving the integrity of gas-sensitive analytes. Deliberate needle selection should balance patient comfort with hemolysis risk, favoring designs that minimize shear stress. QA programs, including routine monitoring of hemolysis rates and contamination, will help ensure diagnostic accuracy. Ongoing staff education on blood collection techniques and adherence to protocols will further reduce variability. Manufacturers should prioritize innovations in syringe and needle design, such as multi-bevel tips to reduce hemolysis, coatings to limit trace metal contamination, and advanced gas-impermeable materials. Uniformity in material composition and device performance must be maintained through stringent QC. Collaborative research between laboratories and manufacturers will optimize device performance and enhance diagnostic reliability.

Conclusions

Components of BCDs, including tube constituents such as wall materials, SFs, rubber stoppers, separator gels, mechanical barriers, clot activators, anticoagulants, and glycolysis inhibitors, along with collection devices such as catheters, syringes, and needles, all play crucial roles in laboratory test accuracy. This is particularly relevant in advanced diagnostic domains such as proteomics and metabolomics, where increased assay sensitivity requires careful tube selection, thorough validation, and meticulous handling.

In this review, we examined BCTs and their components, identified known limitations, and provided recommendations to reduce errors associated with blood specimen collection and testing. Although most BCTs generally function as designed, their impact on research findings and clinical test results is often underestimated. BCTs are frequently regarded as “taken-for-granted” medical devices, and their influence on assay performance is commonly overlooked. However, as regulated medical devices, they possess inherent limitations. When misused, poorly understood, or affected by manufacturer inconsistencies, BCTs can adversely affect laboratory results. Inaccuracies arising from such preanalytical errors may reduce laboratory efficiency, delay result reporting, increase testing costs owing to recollection and retesting, and, most importantly, delay clinical decision-making and patient care.

These findings highlight the importance of moving from method-focused QC approaches to validation frameworks that incorporate device-specific factors. BCDs should be recognized as active components in the testing process rather than merely inert containers. This shift underscores the need to consider interactions among BCDs, analytes, assay chemistries, and laboratory workflows, as these can considerably influence diagnostic accuracy and patient outcomes.

While this narrative review provides a comprehensive synthesis of recent findings, it has several limitations. The analysis was restricted to English-language studies published between January 1, 2014, and January 31, 2025, potentially omitting relevant non-indexed or foreign-language literature. Studies focusing exclusively on microcollection devices used primarily for pediatric blood collection were excluded, as these introduce distinct preanalytical variables beyond the scope of this review. The increasing adoption of blood self-sampling devices and the variability introduced by international regulatory differences were also not comprehensively explored, although some information presented here may be relevant to these devices. Additionally, no formal quality appraisal of the included studies was conducted, and the review does not address the full spectrum of BCD-related impacts across all laboratory specialties. Furthermore, quantitative data linking BCD-related errors to clinical outcomes remains limited.

Improving accuracy in clinical assays requires enhanced regulatory oversight and global standardization of BCD manufacturing practices. Continued innovation in tube materials and additives may help mitigate analyte instability and analytical interference. Routine implementation of robust validation and verification protocols is strongly recommended. Validation ensures that BCDs meet intended clinical performance standards, whereas verification confirms their suitability under local laboratory conditions. Incorporating BCD-specific variables into routine QC and PT workflows will help reduce the risk of undetected interferences. To prevent rare but impactful tube-related failures, such as barrier migration, additive precipitation, or clotting defects, laboratories should adopt visual inspection protocols and routine integrity checks prior to sample processing. Additionally, engaging in staff education and competency assessment regarding BCD limitations is critical for reducing preventable preanalytical errors.

Future research should focus on optimizing BCD formulations, investigating real-world failure rates, evaluating the impact of regulatory policies on diagnostic reliability, and integrating novel technologies to reduce preanalytical variability. Emerging tools, such as artificial intelligence, specifically, machine learning, hold promise for modeling and predicting BCD-associated interferences by analyzing large-scale chromatographic, spectral, and clinical datasets, which can enable data-driven improvements in device design, assay selection, and workflow standardization. To the authors’ knowledge, no published studies to date have directly applied artificial intelligence to this specific application; such approaches could hypothetically detect complex interference patterns related to SFs, anticoagulants, or polymer-based gels, and enable the development of algorithms for automated detection, classification, and correction of aberrant analytical signals. Large-scale, multicenter studies are also needed to establish generalizable validation frameworks that reflect real-world clinical complexity. These studies should involve diverse patient populations and various clinical settings to ensure that the findings are broadly applicable. Moreover, cross-industry collaborations between clinical laboratories, academic institutions, and manufacturers could accelerate the development and implementation of next-generation blood collection systems. In the future, AI may also support longitudinal tracking of tube performance, facilitate early detection of problematic lots or manufacturing inconsistencies, and enhance QC efforts, ultimately contributing to improved regulatory oversight and diagnostic reliability.

In conclusion, as laboratory medicine continues to evolve, a thorough understanding of BCD design, material composition, and performance characteristics is crucial for ensuring diagnostic accuracy and optimal patient outcomes. Collaboration among clinical laboratories, device manufacturers, and regulatory agencies will be essential in reducing preanalytical variability and advancing the development of high-performance blood collection systems that support reliable, high-quality testing and patient care.

Acknowledgments

The authors would like to thank Dr. Daryl Henderson, Dr. Michael Hoffman, and Dr. Raven Bowen for their assistance with editing and reviewing the manuscript. We also sincerely thank Krista Tanquary for organizing the references in EndNote™, Natasha Bowen for creating the figures, and Ms. Boglarka Huddleston for her work in assisting with the literature search strategy.

Footnote

Reporting Checklist: The authors have completed the Narrative Review reporting checklist. Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-5/rc

Peer Review File: Available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-5/prf

Funding: None.

Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at https://jlpm.amegroups.com/article/view/10.21037/jlpm-25-5/coif). R.A.R.B. serves as an unpaid editorial board member of Journal of Laboratory and Precision Medicine from July 2023 to June 2027. The other author has no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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