Blood coagulation is a complex system of zymogen clotting factors, serine proteases, cofactors, platelets and phospholipids. The classic view of these all following separate coagulation mechanisms of platelet haemostasis, intrinsic and extrinsic cascading pathways has given way to the current understanding that these processes are tightly integrated. Many factors can affect how blood coagulates, and this study focuses on the role of storage media.
Coagulation can be measured in a number of different ways, from point of care devices, through routine laboratory assays such as prothrombin and partial thromboplastin times, to the high end tests of factors, cofactors and genetic variability. One point of care device, the thrombelastograph (TEG®; Haemoscope® Corp, Skokie, IL, USA) is used for monitoring of coagulation (especially cardiac surgery and liver transplantation), in addition to its established track record for research purposes. Its basic variables are r-time, k-time, α-angle and maximum amplitude (MA). The r-time reflects the time (measured in mm or min) for the first signs of coagulation (fibrin formation) to appear, in other words the time to initiation of clot formation. This point is marked when the amplitude of the trace reaches 2 mm, and is determined mostly by circulating coagulation factors. The k-time is measured (in mm or min) from the point at which the r-time is marked to the point at which the amplitude reaches 20 mm, and represents the speed of polymerization of fibrin once coagulation has started. The α-angle, which is measured from the r-time as a tangent of the developing curve. It, too, represents polymerization of fibrin and strengthening of the clot. The MA reflects the total strength of the clot, and is measured as the widest amplitude of the trace obtained, i.e. the point at which the clot reaches its greatest visco-elastic strength (a function of mostly platelets and fibrin). The benefit is that it provides a quick assessment of global coagulation and the interplay of coagulation systems and pathways . Activation of these pathways can be attained by adding specific activators (e.g. kaolin or tissue factor). Alternatively the fresh whole blood can interact with the initial container in which the blood was stored, albeit for only a few minutes, or with the TEG® cuvette itself; therefore the coagulation activation or inhibition by the initial storage media is of utmost importance in the interpretation of the data obtained .
Polypropylene (PP) and polycarbonate (PC) containers are commonly encountered in the clinical and laboratory settings. PP is a long-lasting implantable polymer, commonly used for medical disposable syringes, as well as in non-resorbable sutures due to its minimal effect on the inflammatory response . PC, a hard thermoplastic, is also widely used in medical containers and tubes, haemodialysis filters, and blood oxygenators.
The impact of these polymer surface effects become especially important when choice of storage containers is considered. It is recommended that PP containers should be used for handling of blood samples, as glass containers cause contact activation of coagulation, and increase leucocyte and platelet adhesion [4,5]. PP reduces surface activation of factor XII (Hageman factor) , and therefore also causes a minimal thrombogenic response . PC, however, has a highly polar, negatively charged, wettable (hydrophilic) surface and this could interfere with the effects on coagulation.
Two of our previous studies of in vitro haemodilution revealed interesting, paradoxical TEG® results to each other. This first was a pilot study, making use of PC containers for storage and haemodilution of the whole blood by 50% with various intravenous (i.v.) fluids, whereas the second study used only PP containers . A hypothesis was formulated that varied surface compositions and characteristics of containers would produce different coagulation profiles.
The purpose of this study was to determine the TEG® coagulation effects of PP and PC containers on fresh whole blood, thereby testing the hypothesis that surface properties of the different types of containers induced significantly different effects on coagulation.
University of Cape Town Ethical review board approval was obtained prior to commencement of the study. Informed consent was obtained from all volunteers. The study was divided into two limbs, with eight separate volunteers in each limb (i.e. volunteers were their own controls within groups (either PP or PC), but not between groups). Exclusion criteria were individuals with a history of bleeding disorders, renal disease, hepatic disease, those who were taking coagulation-altering drugs, or those who had received an i.v. infusion of any type of fluid in the preceding 3 months.
First limb (PP)
Blood was taken by a two-syringe technique from a free-flowing forearm vein (opposite arm for second sampling) into PP syringes (Curity®, South Africa) using 21-G butterfly needles (Techno Med cc, South Africa). The first syringe of blood was discarded (as it may contain tissue thromboplastins from venepuncture, thereby falsely activating coagulation), and blood from the second syringe was deposited into a 5 mL PP container (Infopac, South Africa), from where 360 μL was pipetted into calibrated TEG® model 3000 analysers (Haemoscope® Corp, Skokie, IL, USA) containing proprietary PP cuvettes. The samples were not activated prior to analysis, thereby allowing spontaneous activation of coagulation via the unstimulated intrinsic pathway (‘Native’ analysis as described in the TEG 3000® manufacturer's manual). TEG® analysis was commenced at 5 min after venepuncture. Each TEG® sample surface was then covered with mineral oil as recommended by the manufacturers. Automated pipettes with single-use PP tips were used on all occasions.
Second limb (PC)
The methods for obtaining blood were the same as in first limb, except that the blood was first placed in a PC reservoir (Bibby Sterilin Ltd, Staffordshire, UK). The handling of samples was otherwise the same as in first limb.
TEG® analysis was also commenced at 5 min from venepuncture, and continued for a minimum period of 90 min to allow a complete analysis of all TEG® variables. r-times, k-times, α-angles and MA were measured and analysed statistically.
One-way analysis of variance (ANOVA) and unpaired t-tests were used to analyse differences between the limbs. Post hoc least significant difference (LSD) testing was used to identify individual differences between limbs where these were demonstrated to exist by the ANOVA technique. Pilot data and previous studies, indicated that eight volunteers per limb were estimated to give sufficient power (80%) to display differences at the 5% significance level if, in fact, they were present.
The blood samples in PC revealed consistently longer r-times and k-times, as well as decreased α-angles and MAs than those in PP. The values obtained from samples stored in PP fell within the TEG® manufacturer's specified normal values, whereas the results from PC-stored samples for k-time, α-angle and MA did not (see Fig. 1 and Table 1).
The data in this trial shows that just the presence of a PC tube (in place of a PP tube) causes a significantly slower initiation of clot formation, as well as impaired rate of clot formation.
Standardization of blood sampling and handling thereafter is important in producing reliable, reproducible results. Within an institution, where local reference values are determined and used, it is especially relevant to follow the standard methods for the handling of samples. Where methods and techniques vary between institutions and laboratories for the same investigations, caution must be exercised when results are compared out of the context of their own reference limits . For the TEG® to provide reliable, robust data, care must be taken when using the instrument both for research and in the clinical setting. Blood may incorrectly be placed in the nearest tube available for TEG® analysis; this could often be a PP or PC material. We have shown that this may increase variability of results, which could lead to incorrect interpretation and actions. The main concern clinically would be the interpretation of a normal coagulable state as being hypocoagulable, thereby potentially increasing the possibility of drugs or blood products unnecessarily being administered to correct the obtained TEG® variables. Correct handling of this blood is essential for reliable laboratory and near-patient assessments to be made. Contact activation by use of a non-siliconized glass tube, for example (such as a vacuum-containing automatic blood sampling tube), will cause activation of coagulation , thereby producing false results in many tests of coagulation. The TEG® is highly susceptible to these effects.
PC tubes (usually clear, hard screw-top plastic tubes) are widely used in medicine. PC is a strong, hard, amorphous thermoplastic material ; its uses include urine collection for microscopy and culture, storage of small pathology samples, as well as storage of tissues and body fluids for microbiological investigation, and is one of the most commonly found types of plastic tube on the wards, as well as in operating room. PC has a highly polar, negatively charged, wettable (hydrophilic) surface. Besides its use in medical containers and tubes, it is widely used in haemodialysis filters, as well as in blood oxygenators. PP surfaces are non-wettable (i.e. hydrophobic), and minimal contact and platelet activation occurs on its surfaces .
Previous work into biocompatibility implicates leucocyte and platelet adhesion, and activation on bio-incompatible surfaces . This would most certainly translate to a coagulation effect if whole blood coagulation is measured. Further work revealed that different inflammatory mediators are released during blood coagulation in contact with different materials . Interestingly, glass surfaces not only activate coagulation, but also enhance fibrinolysis by plasmin activation . The type of glass is probably of less importance .
Plastics become electrically charged during the manufacturing process, forming electrets on the surface, which are analogous to magnets . This surface charge of the container may be responsible for so-called specific ion adsorption, where certain ions are adsorbed out of solution by the container, providing an altered surface milieu . The surface charge is also responsible for the formation of an electric double-layer (or bi-layer), which leads to adsorption and accumulation of counter-ions at the interfacial surface. The electrokinetic forces can enhance or inhibit platelet and coagulation effects, depending on specific ion effects . It is known that a negative surface charge of a container partially resembles the negative charge of vascular endothelium, thereby also inhibiting coagulation .
Hydrophilic and hydrophobic surfaces selectively adsorb different proteins, also affecting the platelet and coagulation responses. Initial fibrin mono-layer formation mediates platelet adhesion, whereas albumin-coated surfaces reduce platelet adhesion . Nygren and colleagues found that, not only are proteins more highly bound to hydrophobic than hydrophilic glass surfaces, but platelets adhere and aggregate more readily on hydrophobic glass surfaces . Proteins adhere to the surface of most polymers within 3 min. It has been shown that a good microdomain is one where hydrophilic and hydrophobic surfaces are tightly alternated, leading to differential protein adsorption, with albumin adhering to hydrophilic microdomains, and fibrinogen and γ-globulin selectively adhering to hydrophobic microdomains. Inhibition of platelet adhesion and/or activation follows, depending on the polymer composition .
Other considerations such as the contact angle of fluid on a surface, interfacial free energy, work of adhesion, critical surface tensions (or ‘wettability’), and the optimum polar/apolar ratio, as well as porosity and texture of the surface, are known to have complex and dynamic effects on coagulation . Water content of plastics may also cause the paradoxical effects on coagulation, where it is apparent that polymers with a high water content inhibit platelet activation, but stimulate the coagulation system, with the low-water polymers seeming to produce inverse effects . Various combinations observed, certainly in our study, can therefore be responsible for the variation observed in coagulation responses. Most of these actions can be expected to have effects on the contact activation pathway, which is less reliant on direct tissue factor activation. ‘Native’ TEG® traces, as used in this study, rely on this subtle contact activation between the fresh whole blood and the cuvettes or storage containers.
The drawback of this study is that it is an in vitro study, therefore the question is how should these data be interpreted clinically. The benefits of in vitro investigations are apparent in often providing a larger signal in a more controlled setting, assisting mechanistic answers for clinical questions. Care should be taken, however, when interpreting these results back to a clinical setting. Interestingly, the values obtained from the PC-stored samples reflected k-times, α-angles and MA falling outside normal reference ranges, indicating a mild hypocoagulability.
In summary, PP and PC tubes produce different responses on in vitro coagulation testing, with PC causing the most variation in results. It is likely that a marked effect occurs due to the interfacial charges, considering the electric bi-layer specifically on the contact activation, or intrinsic, coagulation pathway. The results of this investigation suggest that TEG® results obtained from blood stored in a PC tube could in certain as yet undefined circumstances lead a clinician to believe that a hypocoagulable state existed due to TEG® values outside reference ranges.
Considering the fact that the samples placed in different polymers produced different TEG® results, we believe that care should be exercised in the choice of containers for storage of blood clinically, or in laboratory trials.
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