Which additive prevents coagulation by binding calcium

Thus, new technologies applied in the clinical laboratory to determine analyte concentrations can be significantly affected by BCTs components such as the separator gel.

Ideally, separator gels should maintain uniform chemical and physical properties for the intended period of use and be inert to the specimens collected in BCTs. Plastic tubes require clot activators that use either intrinsic or extrinsic pathways to ensure rapid and dense clot formation Clot activation by the intrinsic pathway is surface-dependent and a greater density of activating surface sites speeds clotting time.

Siliceous substances e. The amount of clot activator varies by manufacturer Clot activators also diminish latent fibrin formation in the separated serum Clot activation by the extrinsic pathway, coagulation initiated by adding substances extrinsic to blood, is biochemical e.

Although these clot activators produce rapid clotting 10 to 20 minutes , the clots formed are gelatinous and do not easily separate from serum Clot activators can be added to tubes by adding small beads or paper coated discs, or they can be sprayed on interior tube surfaces with a carrier e.

These carriers allow rapid clot activator suspension into blood so that the carriers dissolve into both serum and clots as the clotting is initiated PVP and water-soluble SFs also release clot activators into blood specimens to reduce the need for mixing BD has recently released a serum tube containing thrombin rapid serum tubes RST TM ; Table 1 , orange stopper for rapid clot activation within 5 minutes Dimeski et al.

Based on these findings, it is clear that additional studies are needed to ensure that the RST tubes give clinically equivalent results to other commercially available serum tubes, especially for partially filled tubes of blood.

Some clot activators are problematic in that they must be thoroughly mixed to allow complete pelleting with the clot. If soluble fibrin clots form, they can interfere with pipetting device accuracy or in solid-phase binding in immunoassays To limit these problems, plasma gas may be used to introduce heteroatoms non-carbon and hydrogen atoms in the backbone of the molecular structure to the tube wall surface to accelerate clotting without contaminating the serum or clot with binders or activators Various studies have revealed the impact of clot activators on laboratory test performance.

Sampson et al. The clot activators or silicone SFs can interact with ion-specific analyzer membranes, which increase the measured voltage and falsely elevate serum lithium ion concentration. Clot activators can also falsely elevate serum testosterone measurements 69 , but changing the ion pair eliminates this problem Proteome analysis by MS may also be altered by clot activators Silica and silicate clot activators, when sprayed onto plastic tubes, induces the release of pro-, active, and complexed matrix metalloproteinases Recently, it was shown that ficolin-1 and ficolin-2 levels, a group of proteins that can activate the complement pathway, and their binding capacities were significantly affected, presumably, by the silicate material in SST tubes Thus, it is critical that the optimal amounts and composition of clot activators and water-soluble agents be determined and consistently added to different types and sizes of BCTs in order for these substances to function properly without adversely affecting the quality of the blood specimens and test results.

SFs are commonly used to decrease non-specific adsorption, but they must be carefully selected and optimized for immunoassays since, at high concentrations, they may cause the loss of antibodies passively adsorbed onto the solid support beads used in immunoassays Commercially available tubes contain a variety of SFs 7 , 8 , 10 that improve blood flow, distribute clot activator, and prevent proteins, RBCs, and platelets from adsorbing to tube walls Silicone SF-coated tubes have been shown to interfere with ion-specific electrode measurement of ionized magnesium and lithium 7 , Silicone SFs seems to interact with ion-specific electrode membranes to increase the measured voltage during magnesium and lithium determinations 7 , In addition, water-soluble silicone polymer coatings in separator tubes can physically mask antibodies and alter avidin-biotin binding reactions in immunoradiometric assays Bowen et al.

Competitive immunoassays e. A general molecular structure and B schematic structure with polyether polyethylene oxide and polypropylene oxide attached via hydrosilation reaction to the polydimethylsiloxane backbone; x, y, m, n are integers independently greater than zero; z can be hydrogen or alkyl radical Due to immunoassay interference, BD reformulated their tubes to reduce SF levels 7.

Morovat et al. Yet, in this study, the control tubes were coated with the problematic SF. Wang et al. Interestingly, Silwet L and other types of Silwet surfactants can be used in separator gel formulations, which may account for the clinically significant biased thyroid hormone test results with the reformulated tubes stated above Further studies have examined whether additives supply molecules that may interfere with MS peaks.

Drake et al. Tube additives may also affect the ionization process during liquid chromatography-MS analysis, thereby suppressing metabolite ionization. Yin et al. SF detergent properties can also alter cell membrane permeability and lipophilic structures.

A study showed that SFs in tubes affected free fatty acid concentrations in specimens rather than interfere with their analytical detection Thus, producing BCTs with SF that do not contaminate the blood specimens and cause assay interferences would be ideal. The importance of the order of draw in obtaining accurate laboratory tests has been known for many decades. Calam and Cooper 80 demonstrated that the initial drawing of blood into potassium-EDTA tubes falsely decreased and increased the calcium and potassium values, respectively, in blood collected into subsequent tubes containing no anticoagulants These findings prompted the development of Clinical and Laboratory Standards Institute CLSI guidelines to standardize tube sequence and syringe use for blood collection to minimize carryover of tube additives When laboratories switched from glass to plastic tubes, the CLSI order of draw guideline changed because plastic serum tubes were considered equivalent to gel separator tubes with clot activators The current CLSI guideline for glass and plastic tubes order of draw is as follows: blood culture tubes; sodium citrate tubes; serum tubes with and without clot activator and with or without gel separator; heparin tubes with or without gel separator; EDTA tubes; acid citrate dextrose containing tubes; and glycolytic inhibitor fluoride, iodoacetate tubes However, the use of order of draw has recently been questioned and studies by Salvagno et al.

Nevertheless, an extensive study with more analytes is warranted. Tube manufacturers color-code tube closures for easy identification of tube additives. Laboratorians must understand associated additives, proper order of draw, and carryover effects of additives on clinical assays. Protease inhibitors are among the most abundant plasma protein components 83 , far outnumbering active proteases except where activation occurs by surfaces or other stimuli.

Chelating agents, such as EDTA and citrate, do not directly inhibit serine proteases, but they do limit the activation of proteases in the coagulation system by interfering with calcium-mediated surface binding and by allowing inhibitors to dominate. Direct inhibitors of thrombin or coagulation factor Xa serve as alternative anticoagulants, but they have not achieved widespread use because of cost Such products, however, can increase protein stability and allow chemistry and hematology tests on a single specimen.

Small bioactive peptides such as parathyroid hormone and insulin are more stable in EDTA-anticoagulated plasma compared to citrate-anticoagulated plasma or serum Aprotinin increases the stability of brain-type natriuretic peptides 86 ; some reference laboratories recommend the collection of specimens for bioactive peptide analysis in tubes containing aprotinin or other protease inhibitors.

Many peptides, such as glucagon-like peptide 1, undergo rapid cleavage by the exopeptidase dipeptidyl peptidase IV 87 , and thus collection tubes must contain exopeptidase inhibitors to recover the intact peptide. EDTA-containing tubes are generally recommended for proteomic analyses to minimize protein component changes 88 ; small peptide components can also undergo rapid degradation by exopeptidases However, addition of chemically reactive protease inhibitors, such as sulfonyl halides, can covalently modify proteins An alternative approach is to inhibit protease activity by decreasing pH Although endogenous protease inhibitors are quite abundant in plasma, most are mainly against serine dependent endoproteases and exhibit relatively little activity against exopeptidases.

Therefore, the addition of exogenous, low molecular weight protein inhibitors or small synthetic compounds to a blood specimen is often used to stabilize samples. Protease activity may be accentuated by the release of intracellular proteases from white or RBCs.

For example, insulin is substantially less stable in hemolyzed blood because of the thiol proteases from RBCs The use of protease inhibitors has a limited effect on the recovery of chemokines and cytokines from plasma, but the rapid processing of blood can limit this problem because most cytokines and chemokines are degraded by intracellular protease The addition of exogenous protease inhibitors depends on the intended use of specimens.

Because there is wide variability in protein and peptide stability, each laboratory should analyze the stability of components of interest; where protein or peptide stability problems are identified, protease inhibitors should be considered.

Blood collection systems e. Prevention of pre-analytical errors from BCT additives remains an ongoing problem for tube and assay manufacturers and ultimately affects the ability of clinical laboratories to produce accurate results.

Any new or modified blood collection product should ideally be thoroughly evaluated for any potential problems inherently caused in the downstream processing and analysis of specimens. BCT manufacturers should also consider evaluating their products under conditions of reduced specimen volumes, extended contact times, and long-term storage.

Because it is not possible for manufacturers to assess the impact of their tubes on all assay platforms, it is important that they establish close working relationships with their customers and should consider developing a surveillance program to quickly identify problems. Similarly, manufacturers of assays and instrument platforms should ideally verify the performance of their assays with a wide variety of BCTs on the market and on different lots of the same tube type.

Reference interval studies performed on older instruments or tubes no longer in use should be repeated using materials and conditions that are consistent with current use. Blood collection device problems may go unnoticed by laboratorians since routine quality control QC practice typically does not assess all aspects of laboratory testing from blood collection, including specimen processing, analytical testing, and test reporting 7 , 8.

Proficiency testing programs, which do not require blood collection, also fail to detect blood collection device problems 7 , 8. Hence, QC and proficiency testing specimens in clinical laboratories are analyzed but not processed as patient specimens are.

Although QC specimens are typically non-commutable with native patient specimens because the QC matrices are usually altered by manufacturing processes from that of native specimens, the comparison of control sera results from specimens exposed and non-exposed to BCTs could potentially reveal the adverse effects of additives 7 , 8 , This could be done by clinical laboratories or perhaps by tube manufacturers by exposing QC sera to BCTs on a lot-by-lot basis.

When laboratorians change the tubes they use, they should also perform a comparative tube evaluation 94 ; this tube comparison study should be similar to the one described for method comparison studies, using the CLSI EP9-A guideline In addition to contacting tube manufacturers, tube-related issues should also be reported to regulatory agencies i.

Finally, the routine evaluation of BCTs by clinical laboratories should be incorporated into QC plans based on risk management to help prevent or detect tube-related errors and enhance the quality of the test results A CLSI guideline is available for tube manufacturers, in vitro diagnostic manufacturers, and clinical laboratories for verification and validation of venous and capillary BCTs for chemistry, immunochemistry, hematology, and coagulation 99 , Although current BCTs largely work as designed and are therefore often taken for granted, it is important that laboratorians become aware of the potential problems that they can cause in the analysis of specimens.

BCTs are medical devices and, as such, have inherent limitations. When improperly used or because of problems related to their manufacturing, BCT-related interferences in test results can adversely influence patient outcomes, decrease laboratory efficiency, delay test results, and increase the cost per test due to recollection and retesting.

Thus, optimization and standardization of BCTs are vital for the reliable test analysis. Because laboratory test result quality ultimately depends on specimen integrity, tube manufacturers, in vitro diagnostic companies, and laboratorians should all remain vigilant in protecting against the adverse effects of BCT problems on clinical laboratory assays.

The authors would like to thank Ms. Krista Tanquary for editing and reviewing of the manuscript. Potential conflict of interest.

National Center for Biotechnology Information , U. Journal List Biochem Med Zagreb v. Biochem Med Zagreb. Published online Feb Raffick A. Remaley 2. Alan T. Author information Article notes Copyright and License information Disclaimer. Received Oct 22; Accepted Jan 3. This article has been cited by other articles in PMC. Abstract Improper design or use of blood collection devices can adversely affect the accuracy of laboratory test results.

Keywords: blood collection devices, blood collection sample tube, clinical assays, clinical chemistry, interference, pre-analytical, surfactant. Introduction Proper blood collection and timely processing are critical pre-analytical steps required for the integrity of laboratory results. Blood collection device history Reusable glass syringes with steel hypodermic needles and a hard rubber hub were the first devices used to collect blood 1. Blood collection tubes BCTs consist of tube walls, rubber stoppers, lubricants, anticoagulants, separator gels, clot activators, and SFs, all of which can affect the quality of the specimens, accuracy and precision of laboratory tests Figure 1.

Open in a separate window. Figure 1. Tube walls Evacuated BCTs are generally cylindrical, measuring 50 mm to mm in length and 10 mm to 20 mm in diameter 9. Rubber stoppers Rubber stoppers are routinely color-coded according to anticoagulant type and the presence of a separator gel. Stopper lubricants Lubricants, such as silicone oils, fluids, and glycerol, facilitate the insertion and removal of stoppers 6 , 9 , Anticoagulants Although serum is used for most assays, plasma is a useful alternative due to its rapid processing time.

Table 1. Evacuated blood collection tube stopper color and additives. Separator gels Separator gels are used to separate serum from clotted whole blood or plasma from cells Clot activators and water-soluble agents Plastic tubes require clot activators that use either intrinsic or extrinsic pathways to ensure rapid and dense clot formation Surfactants SFs are commonly used to decrease non-specific adsorption, but they must be carefully selected and optimized for immunoassays since, at high concentrations, they may cause the loss of antibodies passively adsorbed onto the solid support beads used in immunoassays Figure 2.

Order of draw The importance of the order of draw in obtaining accurate laboratory tests has been known for many decades.

Protease inhibitors Protease inhibitors are among the most abundant plasma protein components 83 , far outnumbering active proteases except where activation occurs by surfaces or other stimuli. Recommendations Prevention of pre-analytical errors from BCT additives remains an ongoing problem for tube and assay manufacturers and ultimately affects the ability of clinical laboratories to produce accurate results. Conclusions Although current BCTs largely work as designed and are therefore often taken for granted, it is important that laboratorians become aware of the potential problems that they can cause in the analysis of specimens.

Acknowledgments The authors would like to thank Ms. Footnotes Potential conflict of interest None declared. References 1. Observations on an outbreak of infectious hepatitis in Baltimore during Amer J Pub Health. Funderburk JV. Patent No. Serum-separating tubes, also known as serum separator tubes or SSTs, are used in medical clinical chemistry tests requiring blood serum.

What is the first step in routine blood collection? Venipuncture Procedure: A phlebotomist must have a professional, courteous, and understanding manner in all contact with all patients. What is a yellow top blood tube for? Yellow-top tube ACD : Tube contains acid citrate dextrose as an anticoagulant. This tube is used for the collection of whole blood for special studies. Royal blue-top tube: There are 2 types of tubes; one contains the anticoagulant EDTA and the other does not contain an anticoagulant.

What color is a discard tube? If a coag tube light blue is the only tube or the first tube to be drawn, a 5 mL discard tube must be drawn first. What does sodium citrate do to blood? Anticoagulation of blood without chelation can be achieved by inhibition of the contact pathway by corn trypsin inhibitor CTI. What is EDTA tube? EDTA stands for Ethylenediaminetetraacetic acid. EDTA functions by binding calcium in the blood and keeping the blood from clotting. How does EDTA work as an anticoagulant?

Ethylenediaminetetraacetic acid EDTA strongly and irreversibly chelates binds calcium ions, preventing blood from clotting. Citrate is in liquid form in the tube and is used for coagulation tests, as well as in blood transfusion bags.

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