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Background: Thrombelastography (TEG) is used to evaluate the viscoelastic properties of blood during clotting and provides a global assessment of hemostasis and clot lysis. TEG analysis initiated with recombinant human tissue factor (TF) has not been evaluated in clinically healthy horses.
Objectives: The purpose of this study was to determine whether TEG results are affected by the time elapsed between sampling and analysis (storage time) of equine blood samples and to establish a preliminary equine reference interval for a modified TEG assay, using recombinant human TF to initiate coagulation.
Methods: Citrated blood samples were obtained from 20 clinically healthy adult horses. Thirteen samples were stored for 30, 60, and 120 minutes at room temperature before TEG analysis. Coagulation was initiated by adding 20 μL of CaCl2 to 330 μL of blood and 10 μL of diluted recombinant TF for a final dilution of 1:3600. Reaction (R) and clotting (K) times, angle (α), and maximum amplitude (MA) were compared between time points. A preliminary reference interval (minimum–maximum values) was determined using data from all 20 horses after 30 minutes of sample storage.
Results: There was a significant effect of storage time on R, K, and α but not MA. Reference intervals were: R, 3.65–6.4 minutes; K, 1.8–5.45 minutes; α, 33.4–66.2°; MA, 41.2–64.1 mm; lysis at 30 minutes post-MA (LY30), <2.75%; and lysis at 60 minutes post-MA (LY60), 1.55–9.5%.
Conclusions: TEG can be performed on equine citrated blood samples using recombinant human TF to activate clot formation. TEG parameters were significantly affected by storage time, suggesting an incomplete inhibition of coagulation in citrated blood.
While conventional coagulation tests evaluate the soluble or plasmatic components of coagulation, in vivo coagulation is the sum of complex interactions among coagulation factors, platelets, endothelial cells, erythrocytes, and even leukocytes.1,2 Thrombelastography (TEG) assesses the mechanical properties of the blood clot during its formation and subsequent lysis.3–5 In human medicine, TEG is used to detect clinically significant coagulation disorders in patients perioperatively and in intensive care.6 TEG is commonly performed on native fresh whole blood within 5 minutes of collection.7 This constraint limits its use in clinical pathology laboratories and in many veterinary hospital facilities. More recently, the use of citrated whole blood has extended the time that may lapse between blood collection and initiation of coagulation.7,8
TEG has been validated and used to predict the risk of bleeding and assess hypercoagulability in dogs9–12 and has been used to monitor heparin therapy in cats.13 TEG also was used to evaluate a horse with a platelet dysfunction.14 Horses, like other species, can have congenital and acquired coagulation disorders15 and TEG could be useful in the rapid diagnosis of coagulopathies associated with gastrointestinal disorders or neonatal sepsis.16,17
The objectives of this study were to assess whether TEG results were affected by the time elapsed between blood collection and analysis (storage time) and to establish preliminary reference intervals for TEG parameters in healthy horses using modified recombinant human tissue factor (TF) to initiate coagulation. We hypothesized that storage time has an effect on the stability of TEG parameters in horses.
Blood samples were collected from 20 clinically healthy adult horses, including 19 mares and 1 gelding with a mean age of 12.3±3.9 years (range 6–19 years). Mares were neither pregnant nor in the postpartum period; their cycling stage was not known. Breeds included Standardbred (n=10), Quarter Horses (n=3), and crossbreeds (n=7). The horses were determined to be clinically healthy based on history and the results of physical examination, CBC, biochemistry profile, PT, and aPTT. The study protocol was approved by the Animal Ethics Committee of the Université de Montréal.
Blood samples were collected using direct jugular venipuncture with a double-pointed 18-G needle and vacuum tubes. In case of excessive needle trauma or animal movement, blood collection was repeated in the contralateral vein. Samples were collected in the following order: 1 dry tube, 1 EDTA tube, and 4 citrate tubes (BD Vacutainer, BD, Franklin Lakes, NJ, USA). The citrated tubes contained 3.2% sodium citrate (0.105 M) and were filled to obtain an anticoagulant to blood ratio of 1:9. Biochemistry profiles and CBCs were performed the same day. Citrated samples for PT and aPTT were handled and processed as previously described.18 The other (unspun) citrated tubes were stored at room temperature (22–25°C) for 30, 60, and 120 minutes to assess sample stability (n=13). Samples from the remaining 7 horses were stored for 30 minutes.
TEG analyses were run on duplicate samples of citrated blood with a computerized thrombelastograph (TEG 5000, Haemoscope Corporation, Niles, IL, USA). Coagulation was initiated using recombinant human TF (Innovin, Dade Behring, Marburg, Germany) diluted 1:100 in phosphate-buffered saline (PBS), pH 7.4, containing 4% bovine serum albumin. TEG assays were performed by mixing 330 μL of citrated whole blood with 20 μL of CaCl2 (0.2 M) and 10 μL of diluted TF that had been previously mixed in the reaction cup. The final TF dilution was 1:3600. In addition, duplicates were run without TF, using the same volume of diluent (10 μL of PBS with 4% bovine serum albumin). Reaction (R) and clotting (K) times, angle (α), maximum amplitude (MA), lysis at 30 minutes post-MA (LY30), and lysis at 30 minutes post-MA (LY60) were recorded. The program was set to run for 90 minutes; MA is usually reached within 20–35 minutes.
To evaluate the effect of storage time a linear model for repeated measures with time as within-subject factor was used. An a priori contrast test was used to evaluate the difference between each storage time. This model is robust to departures from normal distribution. A P-value <.05 was considered significant. Variance components analysis was used to assess the percentage of total variation that occurred at the duplicate level (n=13). Data were normally distributed based on a Shapiro–Wilk test. Reference intervals (minimum–maximum values) were established for samples analyzed after 30 minutes of storage. Statistical analysis was performed with SAS software (v. 9.1, SAS Institute Inc., Cary, NC, USA).
One sample was not analyzed at 120 minutes due to an error of manipulation. Duplicate analyses were obtained for all analyses except 3 (1 at 30 minutes and 2 at 60 minutes) because of technical problems, so a single value was used. Values for R, K, and α but not MA differed significantly depending on storage time (Figure 1). The time to reach MA was <30 minutes (range 17–29 minutes) for all samples. TEG performed without TF activation resulted in longer R and K values, and smaller MA and α values (Figure 2). Variance between duplicates was 7.1% for R, 5.9% for K, 8.5% for α, 17.9% for MA, and 49.1% for LY30. Preliminary reference intervals in samples stored 30 minutes (n=20) were as follows: R, 3.65–6.4 minutes; K, 1.8–5.45 minutes; α, 33.4–66.2°; MA, 41.2–64.1 mm; LY30, <2.75%; and LY60, 1.55–9.5%.
Figure 1. Effect of 3 different storage times (30, 60, and 120 minutes) on reaction time (R), clotting time (K), angle (α), and maximum amplitude (MA) in equine citrated blood samples (n=13 at 30 and 60 minutes, n=12 at 120 minutes). There is a significant effect of storage time on R, K, and α values (*P<.005). Δ, moderate outlier (< or >1.5 times the first or third quartile value).
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Figure 2. Thrombelastography tracing with and without recombinant tissue factor (TF) activation. Respective results (mean of duplicates) for reaction time (R), clotting time (K), angle (α), and maximum amplitude (MA) were as follows: 5.0 minutes, 3.55 minutes, 43.6°, and 53.1 mm (with TF activation); and 16.6 minutes, 6.05 minutes, 30.5°, and 42.35 mm (without TF activation). Inset standard tracing indicating R, K, α, and MA.
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Three of the 4 TEG parameters characterizing the clotting phase were influenced by storage time in citrated equine blood samples in this study. These results were similar to those reported for healthy dogs, in which R and K were shorter and MA and α significantly increased following prolonged (120-minutes) storage.9 Also in agreement with our results, human blood samples stored for 2 hours had a decreased K and an increased α,7 and a similar trend was seen in samples stored for >3 hours in another study.19 Storage times >30 minutes resulted in TEG tracings characterized by accelerated clot formation, suggesting in vitro activation secondary to incomplete inhibition by sodium citrate. In our study, a storage time <30 minutes was not investigated because there may be an initial period of instability in the first half hour after blood collection.20,21 Interestingly, TEG parameters were stable for up to 180 minutes when using a citrate-TF protocol on human blood20 and for 30 minutes to 2 hours19 or 1–8 hours21 when using a citrate–celite protocol. This may suggest that equine blood is less stable in vitro than human blood. Using a similar technique but with a different technology and reagents, Paltrinieri et al22 recently validated thromboelastometry in horses and found citrated blood samples to be relatively stable over 2, 4, and 20 of storage. However, many other factors could come into play, such as patient cooperation and vein trauma during blood sampling, as well as the type and concentration of activator used. Repeated sampling using the same blood tube can induce a falsely hypercoagulable tracing.19 This confounding factor was not present in our study or in the study of clinically healthy dogs,9 as a different tube was used for each time point to avoid repeated mixing and pipetting.
TEG on equine citrated blood samples can be performed using recombinant human TF. The biological activity of recombinant human TF on equine blood was not quantified, but without recombinant human TF the TEG tracing was prolonged, providing indirect evidence of its activity (Figure 2). TF, also known as factor III or cofactor VIIa, is a membrane-bound glycoprotein that functions as an initiator of coagulation in vivo through activation of factors VII and X, classically known as the extrinsic pathway. In addition to its classic tissue-bound distribution, blood-borne TF may also play a role in vivo.23 The recombinant TF used in this study was a mix of phospholipids and the extracellular portion of TF. The purpose of its use is to ensure that coagulation is initiated through the extrinsic pathway instead of through the contact phase (intrinsic pathway), which plays a lesser role in vivo. TF also accelerates the clotting process, allowing more rapid measurements. The TF concentration in our protocol allowed for a relatively short analytical time, as MA was reached in approximately 30 minutes. In dogs, it has been proposed that using very low TF concentrations would better reflect physiologic conditions than the relatively high concentration used here.9–11 It would be interesting to evaluate different dilutions of TF on normal equine blood and to determine their sensitivity and specificity in detecting hypercoagulable and hypocoagulable states.
The protocol used in the present study facilitated the evaluation of parameters of clot formation, stabilization, and initial lysis. Clinical cases with suspected or confirmed hyper- or hypocoagulable profiles have had TEG tracings that deviated from those of the healthy horses in this study (M.L. and C.B., unpublished data) but the ability to accurately detect clinically relevant alterations will require numerous well-documented cases in a prospective study.
In conclusion, we propose a protocol for TEG analysis of equine citrated blood samples with a fixed storage time of 30 minutes, suitable for most point-of-care settings. If the laboratory setting results in a longer delay between blood collection and analysis, our results suggest that a fixed storage time should be used and appropriate reference values for that storage time be established. However, it is unknown whether TEG parameters in horses with hypercoagulable or hypocoagulable conditions would be influenced to the same extent by time. TEG analysis in horses does not replace other coagulation tests but may be useful in emergency settings, for detecting hypercoagulable states, and possibly for monitoring anticoagulant therapy.