Introduction
With the introduction of the cell-based model of coagulation, it became clear that coagulation is a complex, simultaneously running process of initiation, amplification, and propagation between coagulation factors, platelets, and tissue factor–bearing cells.1 Conventional coagulation tests describe just a restricted part of this process,2 whereas thromboelastography is the only way to assess the global coagulation process.3,4
Susceptibility to preanalytical error is the most critical limitation of thromboelastography testing3; therefore, standardization of the test protocol itself is important.3 The Partnership on Rotational ViscoElastic Test Standardization published a detailed manifest of evidence-based guidelines3 on rotational viscoelastic assays in veterinary medicine. Different sampling methods have already been investigated.5,6,7,8,9,10 Although the use of evacuated tubes is suggested, no particular blood collection system has been recommended so far.11 For small animals, blood sampling collection into evacuated tubes is not routinely performed at our institution. Instead, blood collection via direct venipuncture of a saphenous vein and subsequent sampling of blood collected with a free-flow technique into an opened tube are common procedures in the clinical routine and spare other veins (eg forelegs) for the placement of IV catheters. To our knowledge, no comparison between open-tube and standardized evacuated tube–assisted sampling has been performed so far. Therefore, to establish a standardized protocol for thromboelastography at our institution, we compared open-tube sampling and the evacuated tube–assisted sampling method and investigated their impact on thromboelastography variables. Our hypothesis was that these 2 blood sampling methods would not lead to a clinically important difference in thromboelastography results.
Materials and Methods
Dogs
Protocols for this study were approved by the Institutional Ethics Committee and the Advisory Committee for Animal Experiments12 (reference No. BMWFW-68.205/0108-WF/V/3b/2017).
Ten adult male Beagles (6 neutered and 4 sexually intact) owned by the Clinic for Small Animal Internal Medicine of the University of Veterinary Medicine, Vienna, were examined during their regular blood donor health check. The body weight ranged from 12.2 to 18.9 kg (mean, 15.2 kg), and age ranged between 2.5 and 4 years (mean, 3.25 years). None of the participating dogs received either steroids, NSAIDs, or any antimicrobials within 10 days examination was performed by one of the authors (VS). A CBC including a microscopic blood smear examination and a full biochemical analysis including evaluation of renal function (urea and creatinine concentrations) and hepatic function (plasma concentrations of total protein, albumin, and glucose), liver enzymes (alanine aminotransferase, alkaline phosphatase, and glutamate dehydrogenase activities), electrolytes (sodium, potassium, total calcium, and phosphate), general inflammatory status (canine C-reactive protein), and screening for persistent hyperglycemia (fructosamine) were performed. Platelet concentration, fibrinogen concentration, prothrombin time, activated partial thromboplastin time, and thrombin time were determined to evaluate coagulation status. A dog was defined as healthy when clinical signs were absent and the laboratory values did not exceed the upper or lower reference limits more than the suggested recommended biological variance in the corresponding literature.13
Sampling methods
Blood samples were collected from the dogs during their blood donor training. To compare the 2 different blood sampling methods, two 2-mL blood samples were drawn in succession from each dog into plastic tubes containing 3.2% sodium citrate solution (Vacuette; Greiner Bio-One Premium) at a blood-to-anticoagulant ratio of 9:1. One evacuated tube was filled from the left jugular vein via an evacuated tube port and a 20-gauge needle (evacuated tube group [ETG] samples; Figure 1). A second tube was opened and filled by catching the blood as it flowed through a 20-gauge cannula from a lateral saphenous vein (free catch group [FCG] samples). The blood sample tubes for the health check were filled via an evacuated tube port in the recommended order (serum separator tubes followed by citrate-, heparin-, and EDTA-containing tubes) to prevent any contamination.3 All tubes were accurately filled by 1 operator (MP) and were gently inverted several times to ensure that blood was adequately mixed with anticoagulants.
To evaluate whether the quality of the blood sample collection (venipuncture technique) created a preanalytical confounder effect, the quality of blood sampling was assessed through a 4-part venipuncture scoring system (VPS) described elsewhere6 with minor modifications for the open-tube method (Appendix). This score considered the number of attempts needed to puncture the blood vessel, the required adjustments of the needle, the continuity of the blood flow and the eventual formation of a hematoma.
Thromboelastography protocol
A kaolin-activated traceby a thromboelastograph (TEG 5000 analyzer; Haemonetics) was performed from each citrated blood sample. After collection, the 2 citrate-containing tubes (1/method) were incubated in an upright position at room temperature for 60 minutes. One milliliter of citrated blood was transferred into a tube containing a kaolin activator (Haemonetics) and mixed gently by inverting 5 times. A plain cup was placed in the prewarmed (37 °C) cup holder of the analyzer, and 20 µL of 0.2M calcium chloride was added. Then, 340 µL of kaolin-activated, citrated blood was added rapidly, and the thromboelastography trace was started. The thromboelastography trace from each sample was assessed.
The recorded trace is described by 5 main values. Clot initiation is described by the reaction time (R) represented by the first fibrin polymers. The clot kinetics are reflected by clot formation time (K) (the time needed to reach 20-mm clot amplitude) and the α angle (line tangent to the clot curve). The maximal clot strength is described by maximum amplitude (MA) and the global clot rigidity (G), which is the modification of MA to physical units.4,6
Statistical analysis
The data were processed with a commercial statistic analytic program (IBM Corp) and with consultation from the statistical service of the University of Veterinary Medicine, Vienna. Normality of the results was assessed by the Kolmogorov-Smirnov test. The α angle, R, MA, and G data had normal distribution, and the K data had nonnormal distribution. The variables were graphically depicted as box-and-whisker plots. As not all variables were normally distributed, the nonparametric Wilcoxon signed rank test was used to compare the thromboelastography results between the ETG and FCG. Potential differences in preanalytical sampling quality that might influence the thromboelastography results were determined by comparison of VPS scores between the ETG and FCG with a Kruskal-Wallis test. Values of P < 0.05 were considered significant for all comparisons.
Results
Dogs
All dogs were clinically healthy and had just minimal variations in hematologic and biochemistry results and coagulation status, which were in line with the aforementioned inclusion criteria. Three dogs showed a macroscopic mild to moderate hemolysis in the plasma samples for the health check, which did not lead to exclusion because it was considered a method-induced preanalytical artifact. One dog was excluded from the thromboelastography results because of a low platelet count (92 × 103/µL; reference range, 150 × 103 to 500 × 103/µL) with an extraordinary amount of platelet aggregates. All other coagulation tests were within the respective reference ranges.
VPS data
A total of 18 venipunctures in 9 dogs were accounted for the VPS. Ten dogs were classified by use of the VPS with a score of 0, 7 with score 1, 1 with score 2, and none with score 3. Regarding the different sampling methods, score 0 was equally distributed (5 samples/group), score 1 was observed 3 times for the ETG, and 4 times for the FCG. Only 1 sample was categorized with score 2 in the ETG. Comparison of results between the ETG and FCG with the Kruskal-Wallis test revealed no significant (P = 0.606) differences.
Thromboelastography
For the ETG, results showed an R of 3.43 ± 0.84 minutes (mean ± SD), K of 1.60 minutes (1.3 to 1.75 minutes; median, interpercentile range), α of 67.70 ± 5.03 degrees (mean ± SD), MA of 58.68 ± 7.04 mm (mean ± SD), and G of 7,370 ± 1,836 dynes/sec (mean ± SD). For the FCG, the results were an R of 4.53 minutes ± 0.62 (mean ± SD), K of 1.60 minutes (1.35 to 2.05; median and interpercentile range), α of 65.94 ± 4.92 degrees (mean ± SD), MA of 58.19 ± 3.36 mm (mean ± SD), and G of 7,022 ± 915 dynes/sec (mean ± SD).
Inferential statistics from the Wilcoxon signed rank test showed a small but statistically significant difference in R (P = 0.016) between the ETG and FCG (Figure 2). The other variables did not show any statistically significant different.
Discussion
Our data revealed a significantly longer R when venous blood samples were collected by an open-tube sampling in comparison to sampling with evacuated tubes. Our findings of a shorter R with evacuated tubes that have a negative pressure component agreed with the results of other studies, where blood samples were collected via negative pressure by syringe aspiration or evacuated tube.6,8,14
A shorter R represents an earlier initiation of plasmatic coagulation. The 2 different blood sampling methods used in our study create different mechanical stress on a blood sample. An open-tube sampling initiates only low shear stress,5 whereas the vacuum of an evacuated tube creates a negative pressure, which leads to higher shear stress.15 Therefore, higher shear stress during blood sampling seems to accelerate coagulation.8 One possible explanation for this could be a higher amount of cell-free RNA. Assuming that higher mechanical stress leads to a higher amount of cell lysis, the amount of free cellular RNA would be higher with the evacuated tube sampling method. The cell-free RNA leads to a procoagulable state by activating the factor VII–activating protease and the contact pathway through the prekallikrein–factor XII interaction.16,17,18 Furthermore, hemolysis could be representative of cell lysis in vitro.19,20 In our study, 3 dogs showed a macroscopic mild to moderate hemolysis in EDTA-treated blood samples obtained by the evacuated tube method for the health check, and a slight increase of the mean corpuscular hemoglobin concentration indicated a slight tendency to microscopic hemolysis.19 Additionally, one of these dogs was excluded due to a low platelet count (92 × 103/µL) with an extraordinary amount of platelet aggregates, which might be linked to the higher mechanical stress.
The venipuncture quality per se can also influence the procoagulable state of the blood sample, as revealed by a shorter R in a sample following suboptimal venipuncture.7 It is assumed that a suboptimal puncture causes higher tissue damage and greater release of tissue factor in comparison to a clean venipuncture. Therefore, it is recommended to collect blood in a specific order, beginning with a discharge alias serum tube to avoid contamination of the subsequently collected citrated sample with tissue factor.3 Interestingly, when the evacuated tube–assisted sampling method was used, a blood sample was collected through the same port into a serum separator tube just before the sample was collected into the citrate-containing tube for thromboelastography, and this could have led to an underestimation in the samples of the tissue trauma caused by the jugular venipuncture for ETG sample collection. In our study, the VPS scores between both groups were nearly equally distributed. The scores also indicated that the venipuncture was of high quality. This might explain why no effect of venipuncture quality was detected.
Results of a previous study8 suggested that delayed exposure to anticoagulant results in a more coagulable-appearing sample. It could be assumed that open-tube sampling, compared with the evacuated tube–assisted sampling method, would lead to a delayed mixture with anticoagulant and to a more coagulable-appearing sample in the open-tube sample. Nevertheless, our results do not confirm this finding and suggest that gentle blood collection by open-tube sampling with a short delay in mixture with the anticoagulant does not lead to an activation of coagulation. In contrast, samples collected with the evacuated tube–assisted sampling method were mixed without any delay and had a significantly shorter R than samples collected with the open-tube method, so that the assumed effect of delayed anticoagulation does not have an impact in open-tube sampling.
The exact impact of the blood-to-anticoagulant ratio for thromboelastography remains unclear.3 Results of studies3,21 for conventional coagulation tests comparing the impact of different sampling volumes in different citrate concentrations emphasize that an underfilling of the tube may create an artificial prolongation. With the method used to obtain FCG samples, it is not possible to fill the tubes as accurately as in the evacuated tube–assisted method. Thus, that slight variation in the blood-to-anticoagulant ratio in the FCG samples in our study could be possible. However, in the aforementioned study, the impact on the results through underfilling was less prominent with 3.2% citrate concentrations. Additionally, underfilled samples would have a significant impact when > 30% to 40% were underfilled, which should be clearly visible.3,21 In the present study, we used 3.2% citrate solution–containing tubes as recommended,21 and every tube was filled to the fixed indicator mark and gently mixed by inverting the tube to keep any possible variation as small as possible. Therefore, it is very unlikely that slight variations in sampling volume between both sampling methods would have had an impact. Although our data revealed a significant difference in R between the 2 sampling methods, the changes were relatively small and our data for the FCG samples are comparable with the reference interval of healthy dogs in another study.22 It is questionable whether this difference would have an impact on the clinical decision for an individual patient. The R is known as the value that is most affected by different venipuncture methods.11 If a standardized protocol regarding blood sampling is used, the difference can be considered negligible, so that we can conclude that open-tube sampling is a practical, reliable procedure for a standardized thromboelastography protocol.
Based on previous results regarding the negligible effect of the sampling site (jugular vs saphenous vein),6,13 we did not include the venipuncture site as a separate variable in our investigations for practical reasons. Therefore, one of the limitations of our study design is the indistinguishability between the effect of the vessel and the effect of the sampling method. Whether the puncture of a bigger and more deeply situated vein potentially causes greater tissue trauma5,6,7,14 could not be determined on the basis of our results. Additionally, because of the small sample size, it is possible that the effect of the sampling method could be underestimated. Because only Beagles were used in the present study, it is unknown whether breed-specific variations in thromboelastography results (such as those found for Greyhounds vs non-Greyhounds)23 would increase or decrease the differences identified between methods. As most studies comparing sampling methods use a single breed, mostly Beagles,5,6 further studies are needed to investigate a possible effect of blood sampling in different dog breeds. Furthermore, the sampling methods were compared in healthy dogs. Thus, it remains unclear whether a significant difference between the sampling methods would be found when assessing a critically ill dog and, if so, whether the difference would be of the same magnitude.
The open-tube sampling method was associated with a small but statistically significant difference in R for healthy dogs in the study reported here; because this variable has limited clinical significance,11 the clinical impact of this difference can be considered negligible. Our study results supported that open-tube sampling is an appropriate blood sampling method in healthy dogs undergoing thromboelastography assessment with a standardized test protocol.
Acknowledgments
No third-party funding was received in connection with this study. The authors declare that there were no conflicts of interest.
References
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Appendix 1
Description of a venipuncture score system used to assess whether the venipuncture technique created a preanalytical confounding effect in a study to evaluate the effects of open tube and evacuated tube assisted sampling methods on thromboelastography variables for blood samples from healthy dogs.
Score | Feature | Description |
---|---|---|
0 | Attempts | Venipuncture successfully performed with 1 attempt |
Readjustments | No readjustments of the needle within the vessel | |
Blood flow | Constant blood flow into the evacuated tube or constant flow or dripping of blood into the open tube | |
1 | Attempts | Venipuncture successfully performed with 1 attempt |
Readjustments | 1–2 readjustments of the needle within the vessel | |
Blood flow | Blood flows into the evacuated tube or drips into the open tube slowly but nearly constantly | |
2 | Attempts | Venipuncture performed with 1 or 2 attempts |
Readjustments | Multiple readjustments of needle position required | |
Blood flow | Moderate interruption of the blood flow into the tube | |
3 | Attempts | Numerous attempts at venipuncture |
Readjustments | Multiple readjustments of needle position required | |
Blood flow | Marked interruption of blood flow or hematoma occurred during sample collection |
The scoring system was described elsewhere6; minor modifications were made for use in the present study.