Abstract
The objective of this study was to describe the results of thromboelastography platelet mapping (TEG-PM) carried out using 2 techniques in 20 healthy dogs. Maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), adenosine diphosphate (ADP) receptor activity (MAADP), and thromboxane A2 (TxA2) receptor activity (stimulated by arachidonic acid, MAAA) were recorded. Thromboelastography platelet mapping was carried out according to the manufacturer’s guidelines (2-analyzer technique) and using a variation of this method employing only 1 analyzer (1-analyzer technique) on 2 separate blood samples obtained from each dog. Mean [± standard deviation (SD)] MA values for the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4). Mean (± SD) percentage aggregation due to ADP receptor activity was 70.4% (± 32.8)/67.6% (± 33.7). Mean percentage aggregation due to TxA2 receptor activity was 77.3% (± 31.6)/78.1% (± 50.2). Results of TEG-PM were not significantly different for the 1-analyzer and 2-analyzer methods. High correlation was found between the 2 methods for MAfibrin [concordance correlation coefficient (r) = 0.930]; moderate correlation was found for MAthrombin (r = 0.70) and MAADP (r = 0.57); correlation between the 2 methods for MAAA was lower (r = 0.32). Thromboelastography platelet mapping (TEG-PM) should be further investigated to determine if it is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.
Résumé
Cette étude visait à décrire les résultats de la cartographie de la thromboélastographie des plaquettes (TEG-PM) effectuée à l’aide de deux techniques chez 20 chiens en santé. L’amplitude maximale (MA) générée par la thrombine (MAthrombine), la fibrine (MAfibrine), l’activité du récepteur de l’adénosine diphosphate (ADP) (MAADP), l’activité du récepteur de la thromboxane A2 (TxA2) (stimulée par l’acide arachidonique, MAAA) ont été mesurées. La TEG-PM a été effectuée selon les recommandations du manufacturier (technique à 2 analyseurs) ainsi qu’une variation de cette méthode en utilisant seulement un analyseur (technique à 1 analyseur) sur deux échantillons sanguins séparés obtenus de chaque chien. Les valeurs moyennes [± écart-type (SD)] de MA pour les techniques à 1 analyseur/2 analyseurs étaient: MAthrombine = 51,9 mm (± 7,1)/52,5 mm (± 8,0); MAfibrine = 20,7 mm (± 21,8)/23,0 mm (± 26,1); MAADP = 44,5 mm (± 15,6)/45,6 mm (± 17,0); et MAAA = 45,7 mm (± 11,6)/45,0 mm (± 15,4). La moyenne (± SD) du pourcentage d’agrégation due à l’activité du récepteur ADP était de 70,4 % (± 32,8)/67,6 % (± 33,7). La moyenne du pourcentage d’agrégation due à l’activité du récepteur TxA2 était de 77,3 % (± 31,6)/78,1 % (± 50,2). Il n’y avait pas de différence significative dans les résultats de TEG-PM entre les méthodes à 1 analyseur ou à 2 analyseurs. Une corrélation élevée a été trouvée entre les deux méthodes pour la MAfibrine [coefficient de concordance de corrélation (r) = 0,930]; une corrélation modérée a été trouvée pour MAthrombine (r = 0,70) et MAADP (r = 0,57); la corrélation entre les deux méthodes pour MAAA était plus faible (r = 0,32). Des études supplémentaires devraient être effectuées pour déterminer si la TEG-PM est une méthode qui convient pour mesurer le dysfonctionnement des plaquettes chez les chiens avec thrombopathie.
(Traduit par Docteur Serge Messier)
Thromboelastography (TEG) is a real-time hemostatic test of whole blood that analyzes the viscoelastic properties of thrombus formation and provides a thorough assessment of hemostasis by documenting the initiation, propagation, and lysis of the blood clot (1). While TEG has been used to document coagulation disorders in many species, it is not specific to platelet activity. Thrombin is a potent platelet agonist that is able to activate a major platelet receptor, glycoprotein alpha(IIb) beta(3), in addition to activating the coagulation cascade (2). While marked platelet dysfunction may lead to abnormal TEG tracings, thrombin activity is able to override the effects of milder platelet alterations, such as those induced by platelet-inhibiting drugs, which leads to normal TEG assays in these patients (3,4).
Thromboelastography platelet mapping (TEG-PM) is an adaptation of TEG that documents the contribution of adenosine diphosphate (ADP) or arachidonic acid (AA)-mediated platelets to clot formation (3–5). Platelet mapping has been used to describe platelet function in healthy humans as well as in humans receiving platelet-inhibiting drugs and those with high platelet reactivity (3,5–7). Further studies using TEG-PM to monitor platelet function should be conducted in dogs to determine if the assay has similar predictive value.
The first objective of this study was to describe maximum amplitudes (MA) generated by thrombin (MAthrombin), fibrin (MAfibrin), ADP receptor activity (MAADP), arachidonic acid (MAAA), and percentage platelet aggregation produced by TEG-PM in healthy dogs. According to the manufacturer’s guidelines, TEG-PM analysis should be carried out within 2 h of blood sample collection. As the guidelines involve 4 blood samples running simultaneously, 2 TEG instruments are required, each with 2 sample channels.
The second objective of the study was to describe the results of TEG-PM performed using the 2 techniques. Thromboelastography platelet mapping was carried out with 1 instrument by analyzing pairs of samples consecutively within 2 h of collection. Additionally, TEG-PM was done on a separate blood sample from the same individual using 2 instruments in parallel, as recommended by the manufacturer.
Twenty healthy dogs owned by staff and students at the Ontario Veterinary College were recruited for this study. Nine of the dogs were spayed females, 10 were neutered males, and 1 was an intact male. The mean age of the dogs was 4.5 y (from 1 to 10 y) and the mean weight was 30.6 kg (from 3.0 to 61.0 kg). Dogs were determined to be healthy on the basis of a normal physical examination, complete blood cell count, serum biochemical profile, coagulation profile (prothrombin time, activated partial thromboplastin time, and fibrinogen concentration), and urinalysis. The dogs were not on any medications, excluding parasite prophylaxis, in the 6 wk before the study. This study was performed with client consent and in accordance with the standards of the Canadian Council on Animal Care and the Ontario Animals for Research Act and was approved by the University of Guelph Animal Care Committee.
Blood was obtained from a single, atraumatic jugular venipuncture using a 22-ga needle and 12-mL syringe. Immediately after collection, blood was transferred into a 1.8-mL 3.2% sodium citrate tube (9:1 blood-to-citrate ratio) and a 4-mL heparin blood collection tube (17 IU heparin/mL blood). To minimize turbulence associated with sample handling, needles were removed from the syringes and stoppers were removed from the tubes before transferring blood. The tubes were gently inverted 5 times to allow the blood and anticoagulant to mix. The procedure was repeated 3 h later using the opposite jugular vein.
Thromboelastography platelet mapping was performed using a computerized thromboelastography analyzer (TEG 5000 Thrombelastograph Hemostasis Analyzer; Haemonetics, Braintree, Massachusetts, USA) and data recorded from each channel were transferred electronically to a computer. For all assays, the cup was prewarmed to 37°C before blood or reagents were added. The blood and reagents were mixed before analysis by pipetting the sample up and down 3 times in the cup. Kaolin-activated TEG was used to measure the maximum amplitude of clot formation mediated by thrombin (MAthrombin). One mL of citrated whole blood was added to a kaolin-coated vial (Haemonetics). After gently inverting the vial 5 times, 360 μL of this sample was added to a TEG cup containing 20 μL of 0.2M calcium chloride. The contribution of cross-linked fibrin alone to the clot (MAfibrin) was assessed by adding 10 μL of Activator F (Haemonetics), a factor XIIIa and reptilase reagent, to a TEG cup, followed by 360 μL of heparinized blood. The contribution of ADP and thromboxane A2 (TxA2) platelet receptors to clot formation was measured using ADP and AA reagents, respectively. To measure MAADP, 360 μL of heparinized blood was added to a cup containing 10 μL of Activator F and 10 μL of ADP (Haemonetics), to yield a final concentration of 2 μM of ADP. A similar procedure was used to measure MAAA, except that 10 μL of AA reagent (Haemonetics) was added to the cup, which yielded a final concentration of 1 mM of AA.
Thromboelastography platelet mapping was conducted using both a 1-analyzer and a 2-analyzer technique on each dog. Before the study, a randomized list was created to determine which TEG-PM technique would be performed first for each dog. For the 1-analyzer technique, blood was collected and allowed to equilibrate to room temperature for 30 min. After this equilibration period, the Activator F reagent was reconstituted and the MAthrombin and MAfibrin samples were analyzed. As soon as the initial samples were completed (approximately 90 min after initial blood collection), the ADP and AA reagents were reconstituted and the MAADP and MAAA samples were analyzed. For the 2-analyzer technique, blood was collected and allowed to equilibrate to room temperature for 30 min, after which reagents were reconstituted and all 4 samples were run in parallel.
The mean, median, range, standard deviation (SD), and coefficient of variation (CV) for the population tested were calculated for MAthrombin, MAfibrin, MAADP, and MAAA generated by each technique. Intra-individual CVs were calculated between the 2 samples per dog for each MA variable. Platelet aggregation in response to each agonist was calculated using the formula: % aggregation = [(MAADP or AA − MAfibrin)/(MAthrombin − MAfibrin) × 100] (4). Data were analyzed using a computerized software program (GraphPad Prism 5; GraphPad Software, La Jolla, California, USA). Data distribution was tested for normality using a D’Agostino-Pearson test. Data that were not normally distributed were log-transformed to assess for normality. A paired t-test (normally distributed data) or a Mann-Whitney test (non-normally distributed data) was performed to detect a significant difference between the 2 methods. Concordance correlations were calculated using a computerized software program (SAS; SAS Institute, Cary, North Carolina, USA) to test for agreement between the 1-analyzer and 2-analyzer methods. Statistical significance was set at P < 0.05.
The mean (± SD) TEG values generated by the 1-analyzer/2-analyzer techniques were: MAthrombin = 51.9 mm (± 7.1)/52.5 mm (± 8.0); MAfibrin = 20.7 mm (± 21.8)/23.0 mm (± 26.1); MAADP = 44.5 mm (± 15.6)/45.6 mm (± 17.0); and MAAA = 45.7 mm (± 11.6)/45.0 mm (± 15.4) (Table I, Figure 1). Mean (± SD) percentage aggregation in response to ADP was 74.4% (± 37.2)/75.3% (± 43.1); mean percentage aggregation in response to AA was 88.6% (± 47.4)/81.5% (± 52.3) (Table II). There were no significant differences between the 1-analyzer and 2-analyzer methods for MAthrombin (P = 0.87), MAfibrin (P = 0.37), MAADP (P = 0.71), and MAAA (P = 0.39). Concordance correlation coefficients (r) between the 1-analyzer and 2-analyzer methods were: MAthrombin = 0.70; MAfibrin = 0.93; MAADP = 0.57; and MAAA = 0.32 (Figure 2). The mean (median) intra-individual CVs between the 1-analyzer and 2-analyzer methods were MAthrombin = 6.1% (3.9); MAfibrin = 33.3% (28.7); MAADP = 13.9% (6.2); and MAAA = 23.2% (12.3).
Table I.
Mean, median, range, standard deviation (SD), and coefficient of variation (CV) for each TEG-PM parameter using the 1-analyzer or 2-analyzer method
| MAthrombin | MAfibrin | MAADP | MAAA | |||||
|---|---|---|---|---|---|---|---|---|
|
|
|
|
|
|||||
| 1-analyzer | 2-analyzer | 1-analyzer | 2-analyzer | 1-analyzer | 2-analyzer | 1-analyzer | 2-analyzer | |
| Mean (mm) | 51.9 | 52.5 | 20.7 | 23.0 | 44.5 | 45.6 | 45.7 | 45.0 |
| Median (mm) | 51.0 | 50.8 | 9.4 | 8.3 | 46.9 | 50.4 | 47.2 | 50.6 |
| Range (mm) | 42.6 to 63.5 | 38.0 to 63.5 | 2.5 to 61.4 | 3.0 to 71.1 | 7.5 to 66.3 | 10.9 to 66.2 | 20.5 to 61.6 | 12.0 to 61.4 |
| SD | 7.1 | 8.0 | 21.8 | 26.1 | 15.6 | 17.0 | 11.6 | 15.4 |
| CV (%) | 13.7 | 15.2 | 105.3 | 113.5 | 35.1 | 37.3 | 25.4 | 33.8 |
Figure 1.
Scatter plot of thromboelastography maximal amplitude (TEG MA) data of the MAthrombin, MAfibrin, MAADP, and MAAA for the 1-analyzer and 2-analyzer techniques of thromboelastography platelet mapping. All individual data points are shown and the horizontal line represents the mean.
Table II.
Mean, median, standard deviation (SD), and coefficient of variation (CV) for calculated percentage aggregation ADP and percentage aggregation AA for the 1-analyzer or 2-analyzer method
| Percentage aggregation ADP | Percentage aggregation AA | |||
|---|---|---|---|---|
|
|
|
|||
| 1-analyzer | 2-analyzer | 1-analyzer | 2-analyzer | |
| Mean (mm) | 74.4 | 75.3 | 88.6 | 81.5 |
| Median (mm) | 82.0 | 75.8 | 92.1 | 93.8 |
| SD | 37.2 | 43.1 | 47.4 | 52.3 |
| CV (%) | 50.0 | 57.3 | 53.5 | 64.1 |
ADP — Adenosine diphosphate.
AA — Arachidonic acid.
Figure 2.
Plot comparison of data from individual dogs for MAthrombin, MAfibrin, MAADP, and MAAA using the 1-analyzer and 2-analyzer techniques of thromboelastography platelet mapping.
To date, TEG-PM has been described in only a few studies on dogs. A recent study conducted ADP-induced platelet mapping to evaluate the effects of clopidogrel therapy. The study identified a decreased MAADP in dogs treated with clopidogrel, while MAthrombin was not affected, which highlighted the usefulness of TEG-PM in detecting platelet abnormalities (8). Another study reported that administering acepromazine did not significantly change MAADP or MAAA in healthy dogs (9). These canine TEG-PM studies documented lower mean MA values [mean MAADP of 31.8 mm and 38.1 mm in 2 groups of normal dogs (8) and mean MAADP of 38.3 mm and mean MAAA of 21.3 mm in normal dogs before placebo treatment (9)]. In the present study, however, the mean MAADP and MAAA values were lower than those reported for healthy humans (MAADP = 51.1 mm; MAAA = 64.6 mm) (5). These differences may be due to variations in sample-handling techniques, differences in agonist concentrations used, differences in platelet reactivity among the canine population, and variations in platelet reactivity across species (9–12).
Values for MAfibrin were highly variable in this study population. In humans, TEG-PM analysis normally results in a small fibrin-mediated blood clot (mean MAfibrin 7.5 mm, most values < 10 mm) (5). In this study, only 11/20 dogs had MAfibrin values of < 10 mm for both blood samples acquired. High MAfibrin values may have resulted from in-vitro activation of platelets. Although each sample was obtained atraumatically and samples were allowed to equilibrate for 30 min before analysis, platelet sensitization cannot be ruled out. A brief delay in contact of blood with anticoagulant may result in hemostatic pathway initiation prior to TEG recording (10). Interestingly, 8/9 patients with MAfibrin measurements of > 10 mm had this finding on both the first and second samples. An additional consideration is that there may be a difference in responsiveness to Activator F reagent among dogs. The earlier studies on canine TEG-PM did not report MAfibrin values, but rather reported results as MAADP, MAAA, or used a calculation to describe the MA change attributed to ADP or AA platelet activation (MAADP/MAthrombin or MAAA/MAthrombin, respectively) (8,9). Calculated platelet aggregation using the formula previously described or calculated platelet receptor inhibition (100% to percentage aggregation) is commonly reported in human medicine (3–5,7). The occurrence of some extreme MAfibrin values in the present study led to high variability in calculated platelet aggregation. Further study of TEG-PM is needed in larger numbers of dogs to define normal MAfibrin in dogs and to determine the best way to report canine TEG-PM results.
Results for MAADP and MAAA in this study varied widely. Concentrations of 5 to 20 μM of ADP and 1 mM of AA are reported to induce maximal canine platelet aggregation in whole blood aggregometry studies (8,12). In comparison, the TEG-PM kit used yields final concentrations of 2 μM of ADP and 1 mM of AA. While testing conditions produced by TEG differ from those obtained in an aggregometer, it is possible that the lower ADP concentration led to submaximal platelet stimulation and subsequent MAADP variability. Platelet mapping results are also reported to have high variability among humans (7).
Breed and inter-individual variations in platelet function among dogs have been reported and may account for the variability in the TEG-PM parameters observed herein (11). The analytic variation in MAthrombin was low in this study, as shown by the low intra-individual CVs calculated, which indicates that the differences observed in the results of the study population are due mostly to biological variability. However, higher intra-individual CVs were observed with MAADP and MAAA, but especially with MAfibrin. This may indicate that the variability observed in these results is due largely to analytical variation. Such analytical variation may limit the clinical usefulness of TEG-PM in testing canine platelet function. This technique should be evaluated on larger numbers of dogs to further investigate the biological and analytical variability of TEG-PM. The impact of the time between collecting blood samples and measuring MAADP and MAAA in the 1-analyzer method is not known. When the 1-analyzer technique was used, these variables were always measured in the second pair approximately 90 min after blood collection compared to 30 min after blood collection when the 2-analyzer technique was used. The manufacturer recommends that TEG-PM analysis be initiated within 120 min of sample collection, which was achieved in this study. A slight trend towards hypercoagulability was observed for tissue-factor activated MAthrombin in citrated canine blood samples analyzed at 120 min compared to 30 min (13). The effect of time on TEG-PM parameters has not been described. It is possible that the 90-min lag time between collection of blood and performing these assays led to high variation in these samples.
Because this study was a preliminary investigation of the feasibility and potential shortcomings of TEG-PM in dogs, only 20 dogs were used, which is a limitation of the study. Another limitation was the lack of comparison to other methods of analyzing platelet function. Previous studies in humans showed that TEG-PM analysis correlated well with other techniques of analyzing platelet function, including optical platelet aggregometry and the Platelet Function Analyzer 100 (14,15). A recent canine study that examined the effects of clopidogrel on platelet function reported similar results from ADP-induced TEG-PM and impedance aggregometry (8). It is recommended that TEG-PM assays should be performed on larger numbers of dogs to determine the inter-individual and intra-individual variability of this assay and that TEG-PM should be correlated with other measures of platelet function such as aggregometry in healthy and diseased dogs.
This study was unable to directly compare the 1-analyzer and 2-analyzer techniques using the same blood sample. To do so would require 3 TEG analyzers operating in tandem. An alternative method using 2 TEG analyzers would be to run each method consecutively on the same sample. If this was done, however, the second round of testing would take place well beyond the recommended 120 min after blood collection.
In conclusion, this study found that TEG-PM results obtained using a 1-analyzer and a 2-analyzer technique in healthy dogs were not significantly different. Further validation studies are needed to determine if TEG-PM is a suitable method for measuring platelet dysfunction in dogs with thrombopathy.
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