Anesth Analg 2004;99:1290-1294
© 2004 International Anesthesia Research Society
doi: 10.1213/01.ANE.0000133909.66768.C8
CARDIOVASCULAR ANESTHESIA
A Novel Thrombelastograph® Tissue Factor/Kaolin Assay of Activated Clotting Times for Monitoring Heparin Anticoagulation During Cardiopulmonary Bypass
Jack J. Chavez, MD*,
Donald E. Foley, MD*,
Carolyn C. Snider, MT*,
James C. Howell, CCP*,
Eli Cohen, PhD
,
Robert A. Muenchen, MS
, and
Roger C. Carroll, PhD*
*Department of Anesthesiology, University of Tennessee Graduate School of Medicine, Knoxville, Tennessee;
Haemoscope Corporation, Niles, Illinois; and the
Statistical Consulting Center, University of Tennessee at Knoxville
Address correspondence to Roger C. Carroll, PhD, Department of Anesthesiology, University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920. Address e-mail to RCarrol1{at}utk.edu Reprints will not be available from the authors.
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Abstract
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We used a thrombelastograph (TEG®) assay with tissue factor and kaolin (TEG® TF/K) to measure activated clotting time (ACT) in 31 patients during cardiopulmonary bypass. For comparison, ACTs were also determined by a Hemochron Jr. Signature® and a Hepcon® HMS. The TEG® TF/K correlated with both the Hepcon (r2 = 0.789) and Hemochron (r2 = 0.743) ACTs. The average ACT after heparin was 319 ± 119 s (mean ± SD) for the TEG® TF/K compared with 624 ± 118 s for the Hepcon instrument. To evaluate the effects of hemodilution on TEG® TF/K and Hemochron assays, ACT assays were performed on blood diluted to 50% and titrated with heparin from 0 to 6 U/mL. Both instruments showed significant (P < 0.01) changes in the ACT-versus-heparin slope, but the 0 heparin intercept for the TEG® TF/K ACTs was not significantly changed (P = 0.292), in contrast to that for the Hemochron device (P = 0.041). Both instruments also indicated the same 1.3:1 ratio of protamine to heparin for optimum heparin neutralization, with increasing ACTs at ratios >2.6:1. The TEG® TF/K ACT assay rapidly monitors heparin anticoagulation, in addition to the capabilities of this instrument to monitor platelet function, clotting factors, and fibrinolysis.
IMPLICATIONS: This study evaluates the thrombelastograph as a new assay of blood clotting times; it is activated by the addition of tissue clotting factor and kaolin and used to monitor heparin anticoagulation during cardiopulmonary bypass. The thrombelastograph assay correlates with standard assays, is faster, is less sensitive to hemodilution, and can provide more information.
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Introduction
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Standard activated clotting time (ACT) assays, which use either celite or kaolin to activate clotting factors, correlate poorly with heparin levels during cardiopulmonary bypass (CPB) (1) because of hemodilution and depletion of coagulation factors on extracorporeal surfaces (2). Hemodilution is suggested to be the most prominent factor in prolongation of ACTs using standard monitoring devices unless volumes exceed 1 mL; then the sample temperature has an effect (3). Previous studies (1,2) have augmented the coagulation cascade either by replacing or activating clotting factors that are depleted during bypass to attempt to improve the correlation of ACTs with heparin dose. Methods of augmentation include maximal activation of Factor XII with supersaturating levels of celite, kaolin, and glass (1) or mixing with normal plasma (2).
Tissue factor (TF) is a cell membrane-bound glycoprotein present on subendothelial cells (4). Its function after blood vessel injury is to bind Factor VIIa in blood, activating the extrinsic blood coagulation pathway to activate the common pathway for blood coagulation. Investigators have supplemented the coagulation cascade with TF to overcome the effects of aprotinin, often used during bypass surgery, on clot formation as monitored by the thrombelastograph hemostasis analyzer (TEG®) (5). TF-supplemented TEG® has also been used to monitor coagulation in children undergoing cardiac surgery (6) and to monitor platelet glycoprotein IIb/IIIa inhibitors (7).
The TEG® has proven to be a rapid, simple, and inexpensive viscoelastic test of overall coagulation that reflects both the quantity and quality of clotting factors and platelet function (8,9). TEG® measures the reaction time (R), clot formation time (K), rate of clot growth (ANGLE), clot strength maximum amplitude (MA), coagulation index (CI), and rate of fibrinolysis. Because it provides dynamic information on whole-blood coagulation, TEG® has been extensively used to assess hemostasis in surgical and obstetric patients (5–12).
In this study, we modified the TEG® by adding both kaolin and TF (TF/K) to obtain ACT values for heparinized blood before, during, and after CPB. We chose to supplement the coagulation cascade with saturating levels of TF/K to maximally activate both arms of the coagulation cascade and to minimize the effects of clotting factor depletion. The TEG® TF/K R values were correlated with ACTs measured by the Hemochron Signature Jr. and Hepcon HMS instruments. We also determined the effect of hemodilution on ACTs versus heparin titration curves as measured by the TEG® TF/K assay and by the Hemochron instrument.
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Methods
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The IRB of the University of Tennessee Medical Center in Knoxville approved the research protocol. Thirty-one patients undergoing elective CPB and giving informed consent were enrolled from May to July 2003. Sample size was determined from a literature survey of similar studies of CPB patients and ACT measurements. We excluded patients receiving prophylactic heparin or antiplatelet drugs and those who had a history of clotting or bleeding. The average age of this patient population was 65 yr (range, 43–83 yr) and included 10 women. The average body mass index was 24.5 kg/m2 (range, 17.3–34.8 kg/m2). There were 24 coronary artery bypass grafting procedures, 4 valve replacements, and 3 patients who had both procedures. The mean cross-clamp time was 51 min (range, 28–100 min). The average extracorporeal circulation time was 102 min (range, 55–394 min).
No modifications to our clinical anesthesia care were made for this study. Anesthesia was induced with etomidate, midazolam, fentanyl, and vecuronium and was maintained by IV opioid, volatile anesthetic, and vecuronium. Blood samples were taken from the arterial catheter in the operating room before heparin administration, every 30 min after heparin administration during bypass, and 15 min after heparin reversal with protamine. At each blood sampling, 10 mL of blood and IV fluid was drawn and discarded. By using a new syringe, 3 mL of blood was drawn for ACT tests. Initial heparin dosing (300 U/kg) followed a standard procedure guided by the Hepcon HMS, with a kaolin ACT target of >450 s for the Heparin Dose Response cartridge. Heparin maintenance was guided by six-channel heparin/protamine-titration cartridges. Normothermic to mildly hypothermic CPB (bladder temperatures averaged 34°C ± 2.7°C; mean ± SD) was conducted with non-heparin-coated systems, and the pump was primed with 200 mL of aprotinin (10,000 KIU/mL). Each patient was initially given a bolus of 100 mL of aprotinin, and infusion was continued at 50 mL/h during CPB. Additional heparin was administered to maintain the Hepcon HMS kaolin ACT at more than 450 s. Vasoactive and inotropic drugs were administered as necessary during the procedures. Protamine reversal at the end of bypass was monitored by Hepcon HMS protamine-titration cartridges.
ACT assays were run during surgery on three devices: 1) the modified TEG® TF/K (Haemoscope Corporation, Niles, IL), 2) another point-of-care device (Hemochron Jr. Signature; International Technidyne, Edison, NJ), and 3) our routine in-surgery monitoring device (Hepcon HMS; Medtronic Inc., Minneapolis, MN). The last two assays were performed according to the manufacturers’ instructions by using ACT cartridges with dried silica, kaolin, and phospholipids. The TEG® TF/K ACT assay was performed on blood samples within 3 min of sampling. To a standard TEG® cup was added 0.01 mL of a mixture of TF in a phospholipid liposomal preparation and 0.8% (wt/vol) kaolin (the reagent mixture was provided by Haemoscope Corporation). A 0.35-mL aliquot of blood was added, and TEG® monitoring was started. The R time (in seconds) was recorded for each sample as the ACT. Repeat assays of 25 blood samples drawn from the same healthy donor by using 5 different lots of reagent showed a 2% coefficient of variation in ACTs (45 ± 8 s; mean ± SD). Preliminary experiments with healthy donor blood activated with just TF also gave good reproducible R values for blood titrated with heparin ex vivo by the procedure below. However, when assaying patients’ blood while they were on-pump during CPB, we found that TF alone was insufficient to clot the samples in a reasonable time (<10 min) unless kaolin was added.
A medical technologist performed testing with the TEG® TF/K and Hemochron Jr. Signature devices while the perfusionist, blinded to the other results, assayed ACTs with the Hepcon HMS device. The patients received our standard care with the Hepcon device without any effect from the results with the other devices.
We also obtained ACTs versus heparin titration, as well as protamine neutralization curves, for two of the instruments: the TEG® TF/K and the Hemochron Jr. Signature. From an individual volunteer donor who gave informed consent, blood was drawn into a plastic syringe, and 2.5-mL aliquots were immediately added to tubes in an ice bath. These tubes contained aliquots of 50 U/mL heparin stock in amounts calculated to provide final heparin concentrations from 0 to 6 U/mL. After mixing, the blood was kept on ice until it was transferred to a 37°C bath for 3 min to warm the blood before assay. The blood was then immediately assayed for ACTs by using both the Hemochron Signature and TEG® TF/K tests. Samples were assayed sequentially from smallest to largest heparin concentrations, and all assays were completed within 1 h of obtaining blood. To determine the effect of hemodilution on these ACTs versus heparin titration curves, blood was drawn from the same donor and added to an equal volume of ice-cold balanced salt solution used in CPB (Normosol®; Abbott Laboratories, North Chicago, IL). The diluted blood was then added to the iced heparin-containing tubes, and ACTs versus heparin titration curves were obtained by following the same procedure as described above for undiluted blood. For protamine neutralization curves, whole blood from a healthy donor, either with or without 50% hemodilution as above, was anticoagulated with heparin 3.3 U/mL. After adding protamine at increment ratios from 0 to 2.6:1, just before the sample was warmed for 3 min at 37°C, the samples were assayed for ACT as above. Fresh blood from the same donor was drawn in the morning for each set of heparin titrations or protamine neutralization assays on consecutive days.
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Results
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Initial titrations of heparin with healthy donor blood showed a linear response of TEG® TF/K ACTs with heparin concentrations up to 6 U/mL (Fig. 1). There was also a good linear regression correlation of TEG® TF/K ACTs with the Hemochron Jr. Signature ACTs over this same range of heparin concentrations (Fig. 2).
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Figure 1. Thrombelastograph (TEG®) tissue factor/kaolin (TF/K) activated clotting times (ACTs) versus heparin concentration linear regression determined for healthy donor blood. The formula for intercept and slope and the linear regression R2 value are given beneath the figure.
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Figure 2. Linear regression of thrombelastograph (TEG®) tissue factor/kaolin (TF/K) versus Hemochron Jr. Signature activated clotting times (ACTs) at different heparin concentrations for healthy donor blood. The formula for intercept and slope and the linear regression R2 value are given beneath the figure.
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Titrations of heparin with donor blood, undiluted or at 50% hemodilution, show a linear ACT response of both the TEG® TF/K and Hemochron Jr. Signature versus heparin concentration (Fig. 3). The Hemochron Jr. Signature heparin titrations showed significant changes in both intercept (P = 0.041) and slope (P = 0.002) at 50% hemodilution by group-indicator (0, 1) multiple regression analysis. The TEG® TF/K also showed a significant effect of hemodilution on slope (P = 0.009), but not on intercept (P = 0.292). Because the intercept is the value in the absence of heparin, this indicates that the effects of hemodilution alone are significantly greater for the Hemochron Jr. Signature. However, both assays are affected by hemodilution in conjunction with increasing heparin concentrations.
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Figure 3. Effect of 50% hemodilution on activated clotting time (ACT) versus heparin concentration as determined by Hemochron Jr. Signature or thrombelastograph (TEG®) tissue factor/kaolin (TF/K). Formulas for intercepts and slopes, as well as linear regression R2 values, are given beneath the figure.
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The neutralization of heparin by protamine is another important use of the ACT assay. We compared the neutralization curves generated by titration of protamine against 3.3 U/mL heparin in both undiluted and 50% hemodiluted blood; the results are shown in Figure 4. The optimum ratio was observed at 1.3:1 by both the Hemochron and TEG® TF/K, and both showed increases in ACT at ratios >2.6, as previously reported (13). The same optimum ratio was observed with blood anticoagulated with heparin 6.6 U/mL (data not shown). Whereas the 50% hemodilution lengthened the ACT for both instruments, the optimum ratio of 1.3:1 and increased ACT at ratios >2.6 were still observed with both instruments.
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Figure 4. Effect of the protamine/heparin ratio and 50% hemodilution on activated clotting times (ACTs) as determined by Hemochron Jr. Signature or thrombelastograph (TEG®) tissue factor/kaolin (TF/K). Whole blood from a healthy donor was anticoagulated with heparin 3.3 U/mL, either without dilution (closed symbols) or with 50% dilution with Normosol (open symbols). The indicated ratio of protamine was added just before warming the sample for 3 min at 37°C, and samples were assayed in duplicate for ACT by Hemochron Jr. Signature (A) or TEG® TF/K (B). Curve fitting was performed by a locally weighted least-squares regression method with the StatView 4.0 Lowess option.
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The TEG® TF/K ACTs were then compared with the Hemochron Jr. Signature and Hepcon HMS ACTs for patients undergoing CPB. Of the 31 patients enrolled in this study, 21 were male. Their average age was 65 ± 11 yr (mean ± SD), with a range of 43–83 yr. Weights averaged 85 ± 17 kg, with a range of 57–124 kg. The average time on the bypass pump was 102 ± 60 min (range, 55–394 min). Preoperative platelet counts averaged 204,400 ± 55,000/µL (range, 116,000–364,000/µL). Postoperative platelet counts averaged 160,400 ± 67,000/µL (range, 84,000–418,000/µL). Hematocrits before bypass averaged 35% ± 6% (range, 22%–45%) and 23% ± 5% postbypass (range, 13%–36%). On the basis of the hematocrits, hemodilution averaged 33% ± 11% (range, 3%–54%).
The TEG® TF/K ACT correlates well with both the Hepcon HMS and the Hemochron Jr. Signature ACT (Fig. 5). Table 1 gives the average ACTs before heparin, after heparin administration while on bypass, and after protamine administration for the three instruments. The absolute TEG® TF/K ACTs were approximately half the ACTs for the other devices.
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Figure 5. Cardiopulmonary bypass patients’ activated clotting times (ACTs) correlated by linear regression comparing (A) thrombelastograph (TEG®) tissue factor/kaolin (TF/K) with Hepcon HMS and (B) TEG® TF/K with Hemochron Jr. Signature. Formulas for TEG® TF/K intercepts and slopes, as well as linear regression R2 values, are given beneath the figure.
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Discussion
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The TEG® TF/K ACT correlates with more established ACT measurements, both in the laboratory and during CPB. Because volumes for all these devices are <1 mL, the sample temperature should not have an effect (3). The TEG® TF/K has the advantage of giving results faster, with an average ACT of 319 seconds after heparin administration (versus 624 seconds for the Hepcon HMS and 523 seconds for the Hemochron Jr. Signature). In the laboratory, the TEG® TF/K ACTs are linear over an effective range of heparin concentrations up to 6 U/mL and are less influenced by 50% hemodilution compared with the Hemochron Jr. Signature device. This is likely due to the supplementation with an excess of TF/K to maximally augment blood coagulation through both intrinsic and extrinsic pathways. Similar decreases in ACTs and a better correlation with heparin levels were reported by Leyvi et al. (1), who used an oversaturating level of celite, kaolin, and glass to maximally activate Factor VII, and by Koster et al. (2), who used normal plasma as a supplementing agent.
The ACT-versus-heparin titrations shown in Figure 3 cannot duplicate the combined effects of hemodilution, aprotinin, and/or clotting factor consumption. With these limitations in mind, we calculate that the Hemochron Jr. Signature ACT gives a mean heparin value of 4.2 vs 2.1 U/mL at 50% hemodilution. TEG® TF/K ACTs for the same patient population give heparin 8.3 U/mL without dilution versus 5.4 U/mL at 50% hemodilution. The primary concern of the clinician is to maintain ACT within a certain range. Although this range is well established for the Hepcon HMS, further clinical studies are required to determine corresponding variables for the TEG® TF/K.
Although heparin does not directly affect the extrinsic coagulation pathway, it does inhibit Factor X and thrombin in the common pathway and inhibits Factors XII, XI, and IX in the intrinsic pathway. It has been suggested that during bypass surgery, the main mechanism of thrombin generation in vivo is through the TF/Factor VIIa extrinsic pathway (14). Ex vivo, TF enhancement of the common pathway through maximal Factor VII activation may facilitate measurement of heparin’s direct effects on the common pathway and, by feedback activation by thrombin, the effects of heparin on the intrinsic pathway.
Variables of the TEG® TF/K clot profile, in addition to R, used as indicators for ACT in this study were K, ANGLE, MA, and CI. These were not evaluated in this study, but the TEG® standard assay has already proven useful in surgical settings to monitor intraoperative changes in blood coagulation (15) and the return of normal hemostasis after heparin neutralization (16). The addition of this TF/K ACT assay to the TEG®’s capabilities allows for monitoring heparin anticoagulation during bypass, as well as postbypass protamine neutralization, and it offers a unique opportunity to monitor multiple clotting functions in a single instrument. Until there are more data regarding its use in clinical settings, we do not suggest complete reliance on this assay in lieu of systems such as the Hepcon HMS.
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Acknowledgments
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Supported by the Department of Anesthesiology, T. K. Beene Research Gift Fund, and Haemoscope Corporation, which manufacturers the TEG® and might benefit from this publication.
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References
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Accepted for publication May 11, 2004.
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Abstract
Introduction
