The determination of circulating heparin activity is of significant interest in numerous clinical situations in which anticoagulant or antithrombotic therapeutic strategies are used. With regard to monitoring techniques, both exogenously administered heparin (1–3) and endogenously released heparin (4–8) have been detected with thrombelastography (TEG®). In particular, use of heparinase-modified TEG® data (3–8) specifically implicate heparin as the inhibitor responsible for changes in coagulation in both clinical (3–6) and laboratory (7,8) settings. Thus, on first consideration, a TEG®-based technique seems a facile method to detect heparin activity and modify heparin-based therapy in the perioperative period.
However, before embracing TEG® as a method to determine adequate heparin-mediated anticoagulation clinically or as a technique to determine quantitative heparin activity in the laboratory, it would be appropriate to determine whether changes in TEG® variables significantly correlate with other well established methods used to determine heparin activity. Such methods include the measurement of anti-Xa activity (1,9,10) as well as of activated partial thromboplastin time (aPTT) and activated coagulation time (ACT). Thus, one purpose of this study was to determine the sensitivity of the different heparin detection methods. A second goal was to determine whether changes in anti-Xa activity secondary to exogenous heparin administration would correlate with changes in TEG® variables, aPTT values, and ACT values. A conscious rabbit model was used to achieve these goals.
The study was approved by our Animal Review Committee. Conscious male New Zealand White rabbits (Myrtle’s Rabbits, Thompson Station, TN) weighing 2.96 ± 0.18 kg (mean ± sd;n = 11) were briefly restrained (<2 min) and had 22-gauge catheters placed in the central ear artery and the marginal ear vein. On the basis of preliminary data, a sample size of at least 9 rabbits was required to achieve a β of 0.8. Animals subsequently had 1.5 mL of arterial blood withdrawn before the administration of heparin and after every injection of 10 U/kg heparin (derived from beef lung; American Pharmaceutical Partners, Inc., Los Angeles, CA). Animals were administered heparin 10 U/kg 3 times at 5-min intervals for a total dose of 30 U/kg. A 450-μL aliquot of whole blood was immediately anticoagulated with 50 μL of 129 mM sodium citrate. Fifty microliters of citrated blood was used to determine aPTT (SCA 2000® Veterinary Coagulation Analyzer; Synbiotics, San Diego, CA). The remainder of the citrated sample was centrifuged at 2500 g for 15 min, with the plasma then removed and centrifuged a second time at 2500 g for 15 min to obtain platelet-poor plasma. Plasma samples were immediately stored at −85°C before anti-Xa activity determination with a commercially available kit (Biopool International, Ventura, CA) by using a modified chromogenic assay (9,10). Another 400 μL of blood was used to determine ACT (Hemochron Response®; International Technidyne Corp., Edison, NJ). The remaining blood samples at each time point were subjected to the TEG®-based analysis subsequently described.
TEG® analyses were performed with two computer-controlled Thrombelastographs® (Model 5000; Haemoscope Corp., Niles, IL), with a total of four channels available for concurrent data collection. The proper functioning of the TEG® was confirmed daily with quality control standards purchased from Haemoscope. All blood samples destined for TEG® analysis were placed in disposable plastic cups in the thrombelastograph® within 1 min after collection. A 360-μL blood sample was placed in a standard cup after 0, 10, 20, and 30 U/kg of heparin was administered. The following TEG® variables were measured for each sample over a 2-h period at 39°C (the normal temperature of the rabbit): reaction time (R; min), angle (α; degrees), maximum amplitude (MA; mm), and shear elastic modulus (G; dynes/cm2). A detailed description of the methodology of TEG® has been presented elsewhere (11,12). In brief, R is defined as the time from when the blood sample is placed into the TEG® cup until initial fibrin formation occurs, as noted by a signal of 2-mm amplitude. The α is the angle formed from R to the inflection point of the TEG® signal as clot strength stabilizes; it is a measure of the speed of clot formation. MA is the largest amplitude of the TEG® signal and is a measure of clot strength. Finally, G is a measure of clot strength (11) calculated from MA as follows: G = (5000 × MA)/(100 − MA). The relationship between MA and G is curvilinear—as MA varies from 0 to 100, G concordantly varies from 0 to infinity.
All parametric variables are expressed as mean ± sd. Analysis of the effects of heparin administration on anti-Xa activity, aPTT, and ACT was conducted by one-way measures of analysis of variance with repeated measures. TEG® variables were expressed as median and first through third quartiles. Nonparametric methods were used for analyses of TEG® variables, because it was possible to have no detectable clot formation after heparin administration. In such a case, the observation was assigned an R value of 120 min, an α value of 0°, an MA value of 0 mm, and a G value of 0 dynes/cm2. Analyses of TEG® variables were conducted with Friedman repeated-measures analysis of variance on ranks. The Student-Newman-Keuls test was used for post hoc comparisons. Linear regression analysis was performed to compare the change in anti-Xa activity with the dose of heparin administered. Further, linear regression analysis was performed to compare the change in aPTT values, ACT values, and TEG® variable values with the changes in anti-Xa activity. An α error of <0.05 was considered significant.
Heparin administration resulted in a linear increase in anti-Xa activity (r = 0.94, P < 0.0001), with an increase of 3.2 mU of anti-Xa activity in plasma observed for every unit of heparin administrated. The coefficient of variation of the anti-Xa activity assay was 3.8% ± 3.5%. The anti-Xa activity observed after each bolus of heparin was significantly different from all other doses (Table 1). Compared with the 0 U/kg heparin dose, heparin administration significantly increased ACT values; however, there were no significant differences in ACT values between the 10, 20, and 30 U/kg doses. In contrast, the aPTT values observed after the 10 U/kg heparin dose were not significantly different from 0 U/kg, whereas the aPTT values noted after 20 and 30 U/kg were different from each other and the 0 U/kg aPTT values. With regard to TEG® variables, both R and α demonstrated values significantly different from the 0 U/kg heparin dose after 10, 20, and 30 U/kg of heparin was administered. Further, there were significant differences in R and α values between the 10 and 20 U/kg heparin doses; however, there were no significant differences between the 20 and 30 U/kg heparin doses. MA and G demonstrated a heparin dose response similar to that of aPTT.
Linear regression analyses of anti-Xa activity, ACT, aPTT, and TEG® variables are displayed in Table 2. Although significant, the relationship between anti-Xa activity and ACT values was the weakest. The relationship between anti-Xa activity and aPTT was much stronger, similar to the relationship between anti-Xa activity and G. Finally, R and α had the strongest relationship with anti-Xa activity.
In vitro, varying activities of both low-molecular-weight heparin (1,2) and standard heparin (7) have been demonstrated to correlate with changes in the TEG® variables R (1,7) and/or α(2). Indeed, r = 0.89, with P < 0.0001, in a previous study comparing changes in R with varying in vitro heparin activity in rabbit blood (7). It has also been demonstrated that the administration of low-molecular-weight heparin to orthopedic patients resulted in R values that significantly correlated with anti-Xa activity with a generalized estimating equation (1). Although it was clear that R changed concordantly with anti-Xa activity, no data or equations were presented that indicated in absolute values how R and anti-Xa activity correlated (1). This study does demonstrate that α and R are the TEG® variables most sensitive to heparin effects in the rabbit. These findings in rabbits are in agreement with those seen in humans (13), where the addition of trace amounts of exogenous heparin in vitro (80 mU/mL) or exposure to heparin flush in vivo resulted in significantly increased R and decreased α values, as determined by heparinase-modified TEG®. Further, the addition of 100–200 mU of heparin in vitro can abolish discernible clotting measured by TEG®(14). Thus, at the heparin doses administered and anti-Xa activities observed, changes in α and R may well serve to detect changes in circulating heparin activity in the 40–100 mU/mL range in rabbits or humans (1).
This study demonstrates important differences between the sensitivity of TEG®, aPTT, and ACT to increases in circulating heparin activity after small- to moderate-dose heparin administration. Although TEG® appears to be the most sensitive of the techniques used, the ability to discern increases of more than ∼60 mU/mL is poor because no discernible clot may be observed. This was not the case in a previous human study (1), wherein a nearly 200 mU/mL increase in heparin activity was still measurable by TEG®; however, citrated whole-blood samples were analyzed, and it was unclear how much calcium was used to initiate clotting. The methodological difference between this human study (1) and the present study, wherein fresh, unstimulated (e.g., celite, tissue factor) blood was analyzed, likely explains these different sensitivities to increases in anti-Xa activity. With regard to aPTT, an average increase of 67 mU/mL anti-Xa activity was required to discern a difference from the 0 U/kg dosage. Finally, although always able to discern an increase in anti-Xa activity from the 0 U/kg heparin dose, ACT analysis could not discern a difference between the 10, 20, or 30 U/kg doses of heparin. Thus, given the devices used, the ability of TEG® to discern changes in circulating heparin became limited secondary to loss of clot formation at anti-Xa activities at which aPTT and ACT just began to detect changes.
In conclusion, it was demonstrated the TEG®-based methodology can more sensitively detect changes in heparin activity in vivo in the rabbit compared with aPTT or ACT. Further, changes in the TEG® variables α and R demonstrated the strongest relationship to changes in anti-Xa activities in this rabbit model. Thus, changes in α and R values, especially when compared with heparin-naïve values, can easily alert both the clinician and scientist to the presence of changes in heparin activity in vivo.
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