Low-molecular-weight heparins have been used to alter coagulation in healthy dogs and dogs with signs of illness.1–3 Low-molecular-weight heparins are heparins with a short polysaccharide tail, which results in substantial inhibition of activated coagulation factor X activity with little activity against activated coagulation factor II (in contrast to UFH, which substantially impairs both activated coagulation factor X and activated coagulation factor II).4 In humans, use of LMWHs results in therapeutic anticoagulation with a pronounced decrease in bleeding complications, compared with results after use of UFH.5 There is also evidence that LMWHs are more effective at preventing postsurgical thrombotic complications in human patients.6
It is difficult to monitor heparin treatment (UFH or LMWH) in veterinary patients, primarily because of the wide individual variation in response to a single dose of heparin.7,8 Target prolongation of aPTT to 1.5 to 2.0 times the patient's baseline values has been accepted in clinical practice for anticoagulation with UFH, but this remains controversial and is not useful for monitoring treatment with LMWH. The aPTT is unaffected by LMWH treatment.9 A more accurate measure of circulating heparin concentration is determination of plasma AXA.10 However, results of the chromogenic AXA assay are not necessarily available for same-day therapeutic adjustments.
Viscoelastic coagulation monitoring has been evaluated as an option for monitoring after heparin treatment at institutions without same-day access to AXA assays. Thromboelastography and analysis via a dynamic viscoelastic coagulometer have been useful in guiding heparin treatment in humans.11,12 In a recent study13 in humans, investigators suggested that only those patients who had changes in thromboelastography tracings after treatment with LMWH benefited from that treatment, regardless of the AXA. In dogs, thromboelastography with recalcification alone as an activator for coagulation cannot be reliably used to distinguish plasma UFH concentrations > 0.075 U/mL (the therapeutic range for UFH in humans is at least 0.3 U/mL).14 The effect of LMWH (dalteparin) on a modified thromboelastography assay has been investigated in an in vitro study in dogs.15
Dynamic viscoelastic coagulometry measures changes in blood viscosity via a vibrating probe, whereas thromboelastography assesses clot formation by measuring torsional impedance to a fixed piston by a rotating cuvette. The differences in testing methods between thromboelastography and dynamic viscoelastic coagulometry may influence the sensitivity of each for the detection of anticoagulation induced by LMWH. Dynamic viscoelastic coagulometry is initiated by interaction of the sample with a mixture of glass beads, which activate the intrinsic pathway of coagulation. Thromboelastography may be activated by stimulating the intrinsic pathway (through recalcification16 or exposure of the sample to kaolin) or the extrinsic pathway (through the addition of tissue factor to the reaction). Both analyzers provide information on the entire hemostasis process via qualitative graphs and quantitative results.
The objectives of the study reported here were to evaluate the ability of dynamic viscoelastic coagulometry (activated with glass beads) and recalcified thromboelastography to detect coagulation alterations caused by both single and multiple doses of dalteparin administered SC to healthy adult dogs and to relate those changes to AXA. We hypothesized that both dynamic viscoelastic coagulometry and thromboelastography would be appropriate for monitoring LMWH treatment in dogs. An additional objective was to evaluate the pharmacodynamic effects of dalteparin in a population of dogs.
Materials and Methods
Animals—Six healthy adult dogs were included in the study. All dogs had results for a CBC, serum biochemical analysis, coagulation profile (consisting of PT, aPTT, and thrombin time), plasma AT activity, and platelet function (as measured by optical aggregometry) within the respective reference intervals. All aspects of the study were performed with the approval of the University of Georgia Animal Care and Use Committee.
Procedures—Dogs were lightly sedated via administration of butorphanol tartratea (0.2 to 0.4 mg/kg, IV) and midazolamb (0.5 mg/kg, IV), and lidocaine hydrochloridec (1 to 2 mg/kg, SC) was injected over the right jugular vein to provide local anesthesia. An 18-gauge, 20-cm catheterd (priming volume, 0.7 mL) for collection of blood samples was inserted into the right jugular vein of each dog via a modified Seldinger technique. After placement, the catheter was flushed with sterile saline (0.9% NaCl) solutione and then with 50% dextrose solution.f
Food was withheld overnight, and baseline samples were collected the following morning for determination of a CBC, PT, aPTT, AT concentration, and AXA and for use in thromboelastography and dynamic viscoelastic coagulometry. All samples were collected via a standard 3-syringe technique, with an initial waste sample of 3 mL. Samples for use in coagulation analysis and for measurement of AXA were collected into a syringe, then transferred to an evacuated tube containing 3.2% citrate,g which was filled to achieve a final citrate-to-blood ratio of 1:9. Samples for PT, aPTT, AT concentration, and AXA analysis were immediately centrifuged at 1,500 × g for 10 minutes, and the plasma supernatant was subsequently removed and stored at −80°C. Samples for dynamic viscoelastic coagulometry and thromboelastography were kept at room temperature (20° to 22°C) for 30 minutes prior to analysis, and both were performed by a single operator on all samples collected during that day. Analysis of AXA was performed by personnel at the Cornell comparative coagulation laboratory via previously described techniques.10
Following the collection of baseline samples, dalteparinh (175 U/kg, SC) was administered in the right side of the thorax of each dog. After injection of this single dose of dalteparin (day 1), blood samples were collected from the jugular catheter at hourly intervals for dynamic viscoelastic coagulometry and thromboelastography. Samples were collected via a 3-syringe technique, and the jugular catheter was flushed with saline solution at the end of each sample collection. Samples were collected for 12 hours or until the ACT measured by dynamic viscoelastic coagulometry returned to ± 10 seconds of the baseline value. In addition to the samples collected for viscoelastic coagulation analysis, an additional sample of blood was collected at 2, 4, 8, and 12 hours after the dalteparin injection for measurement of AXA.
After the 12-hour sample collection period, all dogs received an additional dose of dalteparin (175 U/kg, SC) and subsequently received additional dalteparin doses every 12 hours for 3 additional days (days 2 through 4). Patency of the sample collection catheter was maintained by flushing it with 1 mL of 50% dextrose solution every 8 hours.14 After LMWH administration for 3 days, blood samples were again collected for a CBC, viscoelastic coagulation analysis, and evaluation of PT, aPTT, AT concentration, and AXA prior to administration of a final dose of dalteparin (day 4). Similar to the analysis after the single dose of dalteparin on day 1, blood samples for dynamic viscoelastic coagulometry and thromboelastography were collected at hourly intervals for 12 hours, and samples for AXA were collected at 1, 4, 8, and 10 hours after the dose of dalteparin was administered on day 4. Collection of samples for viscoelastic coagulation analysis was continued for 12 hours or until the ACT measured by dynamic viscoelastic coagulometry returned to ± 10 seconds of the baseline value. Samples for evaluation of trough AXAs were collected immediately before administration of the scheduled morning dose on days 2 and 3. Throughout the entire dosing interval, dogs were evaluated via physical examination at least twice daily, with a specific focus on any signs of hemorrhage or other adverse effects attributable to dalteparin administration.
Analysis of blood samples—Dynamic viscoelastic coagulometry was performed with glass bead–containing cuvettesi as described elsewhere.17 Briefly, 20 μL of 0.2M CaCl2j was added to each cuvette, and the solution was warmed to 37°C. After a 30-minute rest period, 340 μL of citrated whole blood was added to the cuvette and the analysis initiated. One channel of a dual-channel dynamic viscoelastic coagulometerk was used for analysis at each time point. Thromboelastography was also performed as described elsewhere.18 Briefly, 20 μL of 0.2M CaCl2j was added to a reaction cup that had been warmed to 37°C. Subsequently, 340 μL of citrated whole blood was added to the cup, and the analysis was initiated.
On day 1, thromboelastography assays were performed in duplicate. However, on day 4, assays were performed only on single samples. Thromboelastography analyses were conducted via 1 of 8 channels available on 4 thromboelastographsl; the same channel was used for the analysis of each dog. Regular quality-control procedures with lyophilized plasma productsm,n were performed on all viscoelastic coagulation machines. In addition, each thromboelastography column was tested for balance prior to the initiation of each sample test. During the study, the dynamic viscoelastic coagulometer was also tested once daily with an oilo of known viscosity to verify proper function.
Statistical analysis—Statistical analysis was performed with a commercially available statistics program.p Normality of data was assessed via the Kolmogorov-Smirnov method. Changes in AXA and variables for thromboelastography and dynamic viscoelastic coagulometry for individual dogs were evaluated via an ANOVA for repeated measures or an ANOVA on ranks (depending on distribution of the data), with post hoc corrections for multiple comparisons (eg, Holm-Sidak method) when indicated. Baseline values were compared via paired t tests or Wilcoxon signed rank tests as indicated. Correlations between AXA and variables for thromboelastography and dynamic viscoelastic coagulometry were performed via a Pearson correlation. A value of r ≥ 0.95 was considered to be an excellent correlation, r ≥ 0.85 to 0.94 was considered very good, r ≥ 0.75 to 0.84 was considered good, r ≥ 0.50 to 0.74 was considered fair, and r < 0.50 was considered poor. Values were considered significant at P < 0.05.
Results
Animals—Six mixed-breed dogs (2 neutered males and 4 spayed females) were included in the study. Dogs ranged from 5 to 8 years of age and had a mean ± SD body weight of 26.4 ± 5 kg. The single- and multiple-dose regimen was tolerated well by most dogs, and no evidence of inappropriate bleeding was observed. One dog was removed from the study because of persistent vomiting, which occurred at hour 6 of the sample collection period on day 1. The remainder of the blood samples were collected for day 1, and the dog was then removed from the study. The dog was treated with metoclopramide and SC administration of fluids. Consequently, data for 6 dogs were available for analysis on day 1 but data for only 5 dogs were available for analysis on day 4.
At all time points, AT values were within the reference interval (75% to 120%), although there was a significant (P = 0.024) decrease in the mean ± SD value between day 1 (110.0 ± 17.7%) and day 4 (93.2 ± 16.3%). Both PT and aPTT at time 0 on days 1 and 4 were within the reference intervals and did not differ significantly (P = 0.188 and P = 0.054, respectively) between days.
Dynamic viscoelastic coagulometry—Activated clotting time was progressively prolonged after the administration of dalteparin on day 1, and significant (P < 0.001) differences from the baseline value were detected at all time points, except for 12 hours (Table 1). Clot rate for the single-dose protocol progressively decreased and was significantly (P < 0.001) different from baseline values at 2 to 7 hours after dalteparin administration. The platelet function parameter was not significantly (P = 0.280) different among time points for the single-dose protocol. Activated clotting time, clot rate, and platelet function baseline values were not significantly (all values of P = 0.50) different on day 1 and 4. Activated clotting time on day 4 was significantly (P < 0.001) prolonged, compared with the baseline value on day 4, at 2 to 6 hours after dalteparin administration (Table 2). Clot rate was significantly (P < 0.001) lower than the baseline value at 3 and 4 hours after dalteparin administration. Platelet function was significantly (P < 0.001) lower than the baseline value at 5 and 6 hours after dalteparin administration.
Results for dynamic viscoelastic coagulometry and thromboelastography variables after administration of a single dose of dalteparin (175 U/kg, SC) to 6 healthy dogs.
| Time (h) | ACT (s) | Clot rate | Platelet function | R(min) | K(min) | α-angle (°) | MA (mm) | No. of MA |
|---|---|---|---|---|---|---|---|---|
| 0 | 112.8 ± 17 | 24.2 ± 7 | 3.1 ± 0.8 | 3.55 (2.8–4.4) | 1.75 (1.3–2.5) | 63.55 (58.2–69.8) | 57.28 (38.5–68.7) | 6 |
| 1 | 163.7 ± 41* | 18.0 ± 8 | 2.9 ± 0.5 | 23.50 (6.1–60.0)* | 8.83 (3.1–21.5)* | 21.20 (5.3–49.6)* | 36.50 (5.2–53.4) | 3 |
| 2 | 186.6 ± 30* | 13.8 ± 6* | 3.3 ± 0.5 | 51.05 (11.0–60.0)* | 8.30 (6.9–27.0)* | 28.30 (27.4–29.2)* | 37.38 (31.8–43.0)* | 2 |
| 3 | 208.3 ± 45* | 12.4 ± 5* | 2.5 ± 0.9 | 60.00 (36.9–60.0)* | — | 3.60* | 2.90* | 1 |
| 4 | 217.7 ± 49* | 10.4 ± 3* | 2.8 ± 1.0 | 46.40 (10.9–60.0)* | 8.45 (5.8–11.1) | 6.80 (1.9–33.9)* | 11.73 (2.3–47.7)* | 4 |
| 5 | 203.3 ± 36* | 12.7 ± 5* | 2.6 ± 0.6 | 54.85 (38.5–60.0)* | 23.0* | 6.60 (3.2–10.0)* | 3.90* | 1 |
| 6 | 207.3 ± 36* | 12.9 ± 5* | 2.5 ± 0.7 | 32.83 (11.2–60.0)* | 11.10 (5.7–16.6) | 18.20 (4.4–33.6)* | 40.0 (9.4–41.1)* | 3 |
| 7 | 196.5 ± 45* | 14.3 ± 7* | 2.7 ± 0.7 | 34.05 (11.0–60.0)* | 17.45 (5.6–20.5)* | 14.55 (2.1–34.9)* | 40.50 (2.8–45.4)* | 3 |
| 8 | 189.8 ± 40* | 17.1 ± 9 | 2.9 ± 0.8 | 14.58 (6.5–60.0)* | 5.60 (2.2–8.8) | 30.60 (2.2–60.8)* | 40.40 (2.7–63.5)* | 5 |
| 9 | 181.7 ± 35* | 19.6 ± 10 | 2.8 ± 0.8 | 7.73 (6.4–60.0) | 3.40 (2.1–10.2) | 49.20 (21.3–61.9)* | 46.45 (30.5–64.1) | 5 |
| 10 | 158.5 ± 30* | 22.8 ± 13 | 2.5 ± 0.5 | 7.25 (5.3–21.6) | 2.88 (1.7–14.6) | 53.08 (10.9–65.7) | 46.30 (23.7–65.8) | 6 |
| 11 | 164.3 ± 9* | 17.7 ± 2 | 2.5 ± 0.4 | 6.63 (3.3–10.3) | 2.55 (1.2–5.7) | 56.45 (34.5–72.2) | 49.95 (41.0–67.3) | 6 |
| 12 | 149.7 ± 30 | 20.7 ± 5 | 2.7 ± 1.0 | 5.33 (4.2–9.8) | 2.08 (1.3–4.2) | 61.65 (41.1–71.6) | 52.60 (41.3–67.5) | 6 |
The thromboelastography was activated via recalcification alone. Values reported are mean ± SD or median (range) for normally distributed and nonnormally distributed data, respectively.
Within a column, value differs significantly (P < 0.001) from the baseline (time 0) value.
— = Not determined. K = Clot formation time. No. of MA = Number of dogs for which measureable MA values were obtained. R = Reaction time.
Mean ± SD results for dynamic viscoelastic coagulometry analysis in 5 healthy adult dogs on day 4 of dalteparin treatment (175 U/kg, SC, q 12 h).
| Time (h) | ACT(s) | Clot rate | Platelet function |
|---|---|---|---|
| 0 | 152.6 ± 71 | 20.7 ± 11 | 3.2 ± 0.5 |
| 1 | 187.2 ± 67 | 17.7 ± 10 | 2.8 ± 0.6 |
| 2 | 208.8 ± 5* | 13.5 ± 7 | 3.1 ± 0.8 |
| 3 | 230.8 ± 46* | 12.1 ± 5* | 2.6 ± 1.0 |
| 4 | 227.8 ± 27* | 10.8 ± 3* | 2.9 ± 0.9 |
| 5 | 223.0 ± 44* | 12.7 ± 5 | 2.3 ± 0.8* |
| 6 | 218.0 ± 46* | 13.4 ± 5 | 2.1 ± 0.9* |
| 7 | 197.2 ± 50 | 15.5 ± 7 | 2.7 ± 0.7 |
| 8 | 174.0 ± 42 | 19.9 ± 8 | 3.3 ± 0.7 |
| 9 | 171.4 ± 34 | 21.3 ± 10 | 3.1 ± 0.8 |
| 10 | 148.4 ± 29 | 26.6 ± 12 | 3.4 ± 0.5 |
| 11 | 153.0 ± 14 | 21.0 ± 7 | 2.3 ± 0.4 |
See Table 1 for key.
Thromboelastography—The mean value for the duplicate samples from day 1 was calculated and used for analysis. A gradual prolongation in R (reaction time) was evident after the single injection of dalteparin, with R significantly (P < 0.001) different from the baseline value at hours 1 to 8 (Table 1). Because of the need to assay the subsequent sample, when R was not generated within 60 minutes, a value of 60 minutes was used for R for the purposes of statistical analysis.14 The most substantial prolongation was at 3 hours after dalteparin administration, when 4 of 6 dogs did not generate any clot activity (R was a flat line). Some statistical analysis was hampered by the lack of thromboelastography variables (eg, no K clot formation time) was determined at hour 3 for any of the dogs, and only 1 dog had an α-angle and MA value at hour 3); however, K was significantly (P < 0.001) different from the baseline value at 1, 2, 5, and 7 hours after dalteparin administration. The α-angle was significantly (P < 0.001) different from the baseline value at hours 1 to 9, and MA was also significantly (P < 0.001) different from the baseline value at hours 2 to 8, although only 1 dog had a tracing generated at 2 and 3 hours and only 2 dogs had a tracing generated at 4 hours. No attempt was made to assign a maximum or minimum value for K, α-angle, or MA to dogs with no generated values.
Thromboelastography results on day 4 were similar to those on day 1; many dogs were missing data because of the inability to generate a thromboelastogram. The R was significantly (P < 0.001) different from the baseline value at 1 to 7 hours after dalteparin injection, and all dogs had an R of 60 minutes at hour 4 (Table 3). No significant (P = 0.058) differences from the baseline value were detected for K. The α-angle was significantly (P < 0.001) different from the baseline value at hours 1 to 8, although no dogs had a value for α-angle at hour 4. Similar findings were also seen for MA, although the MA differed significantly (P < 0.001) from the baseline value only for hours 2 and 5. Notably, no MA value was generated for most dogs between 2 and 7 hours after the dalteparin injection.
Values for thromboelastography in 5 healthy adult dogs on day 4 of dalteparin treatment (175 U/kg, SC, q 12 h).
| Time (h) | R(min) | K(min) | α-angle (°) | MA (mm) | No. of MA |
|---|---|---|---|---|---|
| 0 | 3.33 ± 0.3 | 3.18 ± 2.8 | 55.90 ± 16.7 | 54.47 ± 2.2 | 4 |
| 1 | 39.86 ± 27.6* | 6.20 ± 3.7 | 35.65 ± 15.9* | 46.50 ± 5.9 | 2 |
| 2 | 57.52 ± 5.5* | — | 1.20* | 2.20 | 1 |
| 3 | 52.02 ± 17.8* | 21.90 | 10.50* | — | 0 |
| 4 | 60.00 ± 0* | — | — | — | 0 |
| 5 | 44.66 ± 21.3* | 12.50 | 10.20 ± 7.4* | 28.70* | 1 |
| 6 | 43.46 ± 23.0* | 9.80 | 21.00* | 41.00 | 1 |
| 7 | 40.32 ± 21.1* | 18.40 ± 13.6 | 15.85 ± 10.7* | 34.20 | 1 |
| 8 | 24.64 ± 20.7 | 12.97 ± 13.6 | 22.40 ± 17.4* | 49.25 ± 2.1 | 2 |
| 9 | 20.60 ± 22.3 | 9.50 ± 1.5 | 37.55 ± 21.4 | 44.43 ± 14.9 | 5 |
| 10 | 7.26 ± 3.0 | 4.08 ± 2.8 | 48.44 ± 19.9 | 47.84 ± 12.1 | 4 |
| 11 | 9.65 ± 6.6 | 6.8 ± 7.9 | 43.15 ± 21.9 | 42.00 ± 13.1 | 5 |
| 12 | 6.57 ± 0.8 | 3.23 ± 0.5 | 50.67 ± 5.3 | 45.83 ± 4.2 | 3 |
See Table 1 for key.
AXA analysis—The AXA was significantly (P < 0.001) increased from the baseline value at 3 to 9 hours after the single dose of dalteparin but was not significantly (P = 1.00) different from the baseline value at 12 hours after dalteparin administration (Table 4). On day 1, all dogs had an AXA that was within the proposed therapeutic range (0.5 to 1 U/mL)18 for at least 1 time point. On day 4, 3 of 5 dogs had a detectable AXA at baseline (although the values did not differ significantly [P = 0.08] from the baseline value on day 1), and significant (P < 0.001) increases were detected at 3 and 6 hours after the dalteparin injection on day 4. All dogs had an AXA that would be considered within the therapeutic range (0.5 to 1.0 U/mL)19 at 2 and 4 hours after the dalteparin injection on day 4.
Mean ± SD values for AXA at specific times after administration of a single dose of dalteparin (175 U/kg, SC) on day 1 (n = 6 healthy adult dogs) or multiple doses of dalteparin (175 U/kg, SC, q 12 h) on day 4 (5 healthy adult dogs).
| Time (h) | Single dose (U/mL) | Multiple dose (U/mL) |
|---|---|---|
| 0 | < 0.10 ± 0 | 0.14 ± 0.13 |
| 2 | 0.67 ± 0.2* | 0.94 ± 0.23* |
| 4 | 0.77 ± 0.16* | 1.00 ± 0.16* |
| 8 | 0.42 ± 0.07* | 0.60 ± 0.16 |
| 10 | — | 0.38 ± 0.18 |
| 12 | <0.10 ± 0 | — |
See Table 1 for key.
Correlations—Correlations were determined between AXA and the measured viscoelastic variables. Activated clotting time had a good correlation with AXA (r = 0.761; P < 0.001). Clot rate had a negative correlation with AXA (r = −0.678; P < 0.001). Platelet function had a poor negative correlation with AXA (r = −0.317; P = 0.026). Thromboelastography values were correlated with AXA; there was a significant (P < 0.001) positive correlation for R (r = 0.810) and K (r = 0.602), and there was a significant (P < 0.001) negative correlation for α-angle (r = −0.795) and MA (r = −0.599). Despite the good correlations, R could not be used to distinguish AXA > 0.7 U/mL. Because of the lack of a clot, R was not obtained for 17 of 52 recalcified samples, but ACT was obtained for all samples. Samples for thromboelastography that did not generate an R also did not generate values for K, α-angle, or MA, and some additional samples that had an extremely prolonged R or a shallow tracing also did not yield values for K, α-angle, or MA (20/52 samples missing for each variable).
The degree of prolongation of ACT, compared with the baseline value, was also correlated with AXA (r = 0.723; P < 0.001). Linear regression analysis of these data revealed that a prolongation of ACT of 1.7 times the baseline value on day 1 was the minimum prolongation associated with an AXA of 0.5 U/mL, although 2 dogs had an AXA of 0.4 U/mL, despite the fact both dogs had prolongations of 1.78 and 1.9 times the baseline ACT. Eight dogs also had an AXA ≥ 0.5 U/mL with ACT prolongations of 1.16 to 1.5 times the baseline values. The same linear regression analysis for R revealed a significant correlation (r = 0.347; P = 0.012).
Discussion
Investigators should consider the use of an activator for viscoelastic coagulation analysis when the analysis is to be used for therapeutic monitoring of heparin effects. This study revealed good correlations of the ACT (glass bead–activated coagulometry) with measured AXA. Although a good correlation was observed for R, a third of the samples with a higher AXA could not be determined within the time period (60 minutes), and no samples with an AXA > 0.7 U/mL generated usable thromboelastography tracings. Given that the proposed therapeutic AXA range for LMWH is between 0.5 and 1.0 U/mL, recalcified thromboelastography is not a good tool to use for monitoring anticoagulation with LMWH, except to suggest that adequate AXAs have not been obtained. This is similar to the results for monitoring of UFH treatment via recalcified thromboelastography.14 Although R was generated at AXAs up to 0.7 U/mL in the present study, R was not generated at AXAs > 0.1 U/mL for healthy adult dogs treated with UFH.14
The difference in the generation of thromboelastography variables between UFH treatment and LMWH treatment may relate to the specificity of each molecule; LMWH does not have an appreciable effect on generation of activated coagulation factor II, whereas UFH is a potent inhibitor of both activated coagulation factor X and activated coagulation factor II. Recalcified thromboelastography relies almost exclusively on the intrinsic (contact) pathway for initiation of clot formation,16 and although there is evidence for inhibition of activated coagulation factor XII by surface-immobilized heparin and AT, this does not appear to be a mechanism for heparin in solution.20 It is more likely that the inhibition of activated coagulation factor II by UFH results in a stronger inhibition of fibrin formation in an in vitro system,21 which prevents association of the thromboelastography pin with the cup. By providing a strong activator such as tissue factor, the relative effects of heparin in solution may be minimized and allow adequate fibrin formation to proceed. It follows from this hypothesis that tissue factor–activated thromboelastography may be useful for monitoring UFH treatment in dogs, and this has been reported in a recent study.q
Investigators of in vitro studies15,22 have evaluated the use of thromboelastography cups containing heparinase to determine LMWH effects in dogs and humans. These special cups contain an enzyme that degrades heparin in the sample, which allows thrombin generation to proceed. Because there is the exact same dose of heparinase in each of the cups, the same amount of heparin will be neutralized, which allows differentiation among plasma heparin concentrations (eg, higher AXA will have more residual heparin after exposure to heparinase).20 Thromboelastography cups containing heparinase are more expensive than plain thromboelastography cups and require refrigeration for storage. Results of in vitro experiments indicated that recalcified thromboelastography alone could be used to determine dalteparin concentrations in the therapeutic range (data not shown), but this was not observed in the tested samples of the present study. The use of an activator such as tissue factor or kaolin minimizes ex vivo or preanalytic effects on variables and may allow monitoring of heparinized canine samples with thromboelastography by providing a robust initiator of coagulation.23 Notably, the use of kaolin to activate canine thromboelastography samples with in vitro addition of dalteparin (activation of coagulation with a strong intrinsic or contact activator) and analyzed with heparinase cups does not result in reliable prediction of AXA.15 A different contact activator (glass beads) was used in the present study, and results of dynamic viscoelastic coagulometry had reasonable correlations with AXA.
The dose of dalteparin chosen for the study reported here was slightly different from other reported doses.12 On the basis of a study3 of another LMWH (enoxaparin) in dogs, in which higher doses resulted in more sustained AXA, we evaluated the effect of a higher dose of dalteparin administered less frequently (175 U/kg, q 12 h). In contrast to results for a dose of 150 U of dalteparin/kg, SC, every 8 hours,1 we were unable to detect prolonged AXA in dogs for the duration of the dosing interval in the present study, although some residual AXA was detected in the baseline samples on day 4 (2 dogs had an AXA of 0.2 U/mL and 1 dog had an AXA of 0.3 U/mL) for the present study. For a clinical goal of maintaining plasma AXA close to the target range of 0.5 to 1.0 U/mL, it appears that a dosing interval of 8 hours is required. Dalteparin doses > 175 U/kg may result in residual activity for an entire 12-hour dosing interval, and some dogs in the present study had a relatively high AXA (up to 1.3 U/mL) at 2 hours after SC administration of the dose on day 4. Although bleeding complications were not evident in these dogs, higher dalteparin doses may predispose animals (especially ill animals) to hemorrhagic complications.
To accurately monitor the effect of the dose of dalteparin used in the present study, AXA ideally would have been assessed hourly to determine the peak effect and decay of this effect. Because the pharmacokinetics of dalteparin in healthy dogs has been reported in 2 studies12 that involved the use of doses similar to that used in the present study, we opted for intermittent sampling for monitoring of AXA, which may have caused us to miss the peak effect of the drug. In addition, the small number of dogs in the study and the small number of data points limited the power of the study. We also used healthy dogs, and the effects of dalteparin in critically ill animals may be different, although at least one study2 indicates that the pharmacokinetics may be similar in critically ill animals. These aspects must be considered in the interpretation and use of the data.
Viscoelastic coagulation monitoring with strong activators of the intrinsic or extrinsic pathway appears to be adequate for monitoring dalteparin treatment in healthy adult dogs. The maximum dalteparin effect (as measured by AXA) in the present study was detected 4 hours after dalteparin administration, which is a time frame similar to recommendations to identify the peak heparin effect of UFH treatment in dogs.14
ABBREVIATIONS
| ACT | Activated clotting time |
| aPTT | Activated partial thromboplastin time |
| AT | Antithrombin |
| AXA | Anti–activated coagulation factor X activity |
| LMWH | Low-molecular-weight heparin |
| MA | Maximum amplitude |
| PT | Prothrombin time |
| UFH | Unfractionated heparin |
Butorphanol 1%, Fort Dodge, Fort Dodge, Iowa.
Midazolam 0.5%, Baxter Healthcare Corp, Deerfield, Ill.
Lidocaine 2%, Hospira Inc, North Chicago, Ill.
Arrow Teleflex, Reading, Pa.
Baxter Healthcare Corp, Deerfield, Ill.
Hospira Inc, North Chicago, Ill.
Citrate tubes, Becton Dickinson, Franklin Lakes, NJ.
Fragmin, 25,000 U/mL, Pfizer Canada, Montreal, QC, Canada.
gbACT+, Sienco Inc, Arvada, Colo.
Haemoscope, Niles, Ill.
SCP-2 Sonoclot analyzer, Sienco Inc, Arvada, Colo.
TEG 5000, Haemoscope, Niles, Ill.
TEG Level I control solution, Haemoscope, Niles, Ill.
TEG Level II control solution, Haemoscope, Niles, Ill.
SonOil, Sienco Arvada, Colo.
SigmaStat, version 3.5, Systat Software Inc, Chicago, Ill.
Brainard BM, Koenig AK, Pittman JR, et al. Evaluation of high-dose tissue-factor activated TEG for assessment of unfractionated and low molecular weight heparin therapy in dogs (abstr). J Vet Emerg Crit Care 2011;21(suppl 1):S10.
References
- 1.↑
Mischke RHSchuttert CGrebe SI. Anticoagulant effects of repeated subcutaneous injections of high doses of unfractionated heparin in healthy dogs. Am J Vet Res 2001; 62:1887–1891.
- 2.↑
Scott KCHansen BDDeFrancesco TC. Coagulation effects of low molecular weight heparin compared with heparin in dogs considered to be at risk for clinically significant venous thrombosis. J Vet Emerg Crit Care 2009; 19:74–80.
- 3.↑
Lunsford KVMackin AJLangston VC, et al. Pharmacokinetics of subcutaneous low molecular weight heparin (enoxaparin) in dogs. J Am Anim Hosp Assoc 2009; 45:261–267.
- 4.↑
Hirsh JRaschke R. Heparin and low-molecular-weight heparin: the seventh ACCP Conference on Antithrombotic and Thrombolytic Therapy. Chest 2004; 126:188S–203S.
- 5.↑
Kamphuisen PWAgnelli G. What is the optimal pharmacological prophylaxis for the prevention of deep-vein thrombosis and pulmonary embolism in patients with acute ischemic stroke? Thromb Res 2007; 119:265–274.
- 6.↑
Petersen JLMahaffey KWHasselblad V, et al. Efficacy and bleeding complications among patients randomized to enoxaparin or unfractionated heparin for antithrombin therapy in non-ST-segment elevation acute coronary syndromes: a systematic overview. JAMA 2004; 292:89–96.
- 7.↑
Diquelou ABarbaste CGabaig AM, et al. Pharmacokinetics and pharmacodynamics of a therapeutic dose of unfractionated heparin (200 U/kg) administered subcutaneously or intravenously to healthy dogs. Vet Clin Pathol 2005; 34:237–242.
- 8.↑
Helmond SEPolzin DJArmstrong PJ, et al. Treatment of immune-mediated hemolytic anemia with individually adjusted heparin dosing in dogs. J Vet Intern Med 2010; 24:597–605.
- 9.↑
Mischke RJacobs C. The monitoring of heparin administration by screening tests in experimental dogs. Res Vet Sci 2001; 70:101–108.
- 10.↑
Brooks MB. Evaluation of a chromogenic assay to measure the factor Xa inhibitory activity of unfractionated heparin in canine plasma. Vet Clin Pathol 2004; 33:208–214.
- 11.
Shinoda TArakura HKatakura M, et al. Usefulness of thrombelastography for dosage monitoring of low molecular weight heparin and unfractionated heparin during hemodialysis. Artif Organs 1990; 14:413–415.
- 12.↑
Lee BAl-Waili NButler G, et al. Assessment of heparin anticoagulation by Sonoclot analyzer in arterial reconstruction surgery. Technol Health Care 2011; 19:109–114.
- 13.↑
Van PYCho SDUnderwood SJ, et al. Thrombelastography versus antifactor Xa levels in the assessment of prophylactic-dose enoxaparin in critically ill patients. J Trauma 2009; 66:1509–1515.
- 14.↑
Pittman JRKoenig ABrainard BM. The effect of unfractionated heparin on thrombelastographic analysis in healthy dogs. J Vet Emerg Crit Care 2010; 20:216–223.
- 15.↑
Jessen LRWiinberg BJensen AL, et al. In vitro heparinization of canine whole blood with low molecular weight heparin (dalteparin) significantly and dose-dependently prolongs heparinase-modified tissue factor-activated thromboelastography parameters and prothrombinase-induced clotting time. Vet Clin Pathol 2008; 37:363–372.
- 16.↑
Ralph AGBrainard BMKoenig A. Effects of rest temperature, contact activation, and sample collection technique on canine thrombelastography. J Vet Emerg Crit Care 2012; 22:320–326.
- 17.↑
Babski DMKoenig APittman JR, et al. Sonoclot evaluation of whole blood coagulation in healthy adult dogs. J Vet Emerg Crit Care 2012; 23:in press.
- 18.↑
Epstein KLBrainard BMLopes MA, et al. Thrombelastography in 26 healthy horses with and without activation by recombinant human tissue factor. J Vet Emerg Crit Care 2009; 19:96–101.
- 19.↑
Monagle PChalmers EChan A, et al. Antithrombotic therapy in neonates and children: American College of Chest Physicians evidence-based clinical practice guidelines (8th edition). Chest 2008; 133:887S–968S.
- 20.↑
Sanchez JElgue GRiesenfeld J, et al. Studies of adsorption, activation, and inhibition of factor XII on immobilized heparin. Thromb Res 1998; 89:41–50.
- 21.↑
Mischke RGrebe S. The correlation between plasma anti-factor Xa activity and haemostatic tests in healthy dogs, following the administration of a low molecular weight heparin. Res Vet Sci 2000; 69:241–247.
- 22.
Cvirn GTafeit EHoerl G, et al. Heparinase-modified thrombelastometry: inactivation of heparin in plasma samples. Clin Lab 2010; 56:585–589.
- 23.↑
Smith SAMcMichael MGalligan A, et al. Clot formation in canine whole blood as measured by rotational thromboelastometry is influenced by sample handling and coagulation activator. Blood Coagul Fibrinolysis 2010; 21:692–702.
