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© 2001 International Anesthesia Research Society
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Abstract |
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Activated clotting time (ACT) is a test used in the operating room for monitoring heparin effect. However, ACT does not correlate with heparin levels because of its lack of specificity for heparin and its variability during hypothermia and hemodilution on cardiopulmonary bypass (CPB). A modified ACT using maximal activation of Factor XII, MAX-ACT (Actalyke MAX-ACT; Array Medical, Somerville, NJ), may be less variable and more closely related to heparin levels. We compared MAX-ACT with ACT in 27 patients undergoing CPB. We measured ACT, MAX-ACT, temperature, and hematocrit at six time points: baseline; postheparin; on CPB 30, 60, and 90 min; and postprotamine. Additionally, we assessed anti-Factor Xa heparin activity and antithrombin III activity at four of these six time points. With institution of CPB and hemodilution, MAX-ACT and ACT did not change significantly but had a tendency to increase, whereas concomitant heparin levels decreased (P = 0.065). Neither test correlated with heparin levels. ACT and MAX-ACT did not differ during normothermia but did during hypothermia, and ACT was significantly longer than MAX-ACT (P = 0.009). At the postheparin time point, ACT-heparin sensitivity (defined as [ACT postheparin - ACT baseline]/[heparin concentration postheparin - heparin concentration baseline]) was greater than MAX-ACT-heparin sensitivity (analogous calculation for MAX-ACT; 520 [266 - 9366] s · U-1 · mL-1 vs 468 [203 - 8833] s · U-1 · mL-1; P = 0.022).
Implications: MAX-ACT (a new activated clotting time [ACT] test) uses more maximal clotting activation in vitro and, although it is less susceptible to increase because of hypothermia and hemodilution than ACT, lack of correlation with heparin levels remains a persistent limitation.
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Activated clotting time (ACT) is a test used in the operating room for monitoring heparin effect. However, ACT does not correlate with heparin levels because of prolongation of clotting times during conditions of hypothermia and hemodilution during cardiopulmonary bypass (CPB). This variability is a result of incomplete activation of the intrinsic coagulation cascade that occurs either during dilutional states or in the presence of the currently used activators. ACT increases upon initiation of CPB even in the presence of a decreased heparin level (1–3). With ACT monitoring, this therapeutic ACT would result in no additional heparin administration and smaller levels of circulating heparin. If the level of anticoagulation were inadequate, the result would be subclinical activation of coagulation, consumption of clotting factors, and increased postoperative bleeding. Further studies to identify a better method of coagulation monitoring therefore continue.
We studied a newly developed ACT test called MAX-ACT (Array Medical, Somerville, NJ). Tubes of MAX-ACT are oversaturated with a "cocktail" of activators (celite, kaolin, and glass) to maximally convert all Factor XII to XIIa. This is especially important in CPB, when the amount of Factor XII is smaller because of hemodilution. In theory, if all Factor XII can be activated, the prolongation of clot formation will depend less on the variability in intrinsic coagulation and more exclusively on the activity of the heparin-antithrombin III (AT-III) complex.
Our hypothesis was that MAX-ACT would correlate with heparin levels more accurately and reproducibly than the ACT and that MAX-ACT had less heparin sensitivity because of more adequate activation.
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Methods |
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The study was approved by our IRB, and an informed consent was obtained from all patients. We studied 27 patients scheduled for procedures requiring CPB. All patients who gave consent were included. Seven patients received heparin before surgery, two patients received aprotinin (for these patients, kaolin ACT tubes were used), and 10 patients received aminocaproic acid.
Anticoagulation for CPB was attained with bovine lung heparin (Fujisawa USA Inc, Deerfield, IL) 300 U/kg administered into the right atrium by the surgeon. ACT > 480 s was accepted as adequate anticoagulation for CPB. Additional heparin in 5000-U increments was administered to maintain the ACT > 480 s. After CPB, protamine was given in a ratio of 1 mg per 100 U of the total heparin dose. Coagulation was assessed with the celite-activated ACT test (Hemochron; International Technidyne Inc, Edison, NJ) and Actalyke MAX-ACT. For the ACT procedure, blood was drawn from an arterial catheter and 5 dead space volumes of blood were removed, after which 3 mL of blood was drawn. Of this, 2 mL was placed in celite ACT tubes, agitated, and measured in a Hemochron 8000 instrument (ITC, Edison, NJ). Blood (0.5 mL) was placed in Actalyke MAX-ACT tubes, agitated in a similar fashion, and measured in the second well of the same instrument. Values were measured at six time points: baseline (after the arterial catheter was inserted but before skin incision), postheparin (2 min after heparin administration), on CPB 30 min (CPB 30), on CPB 60 min (CPB 60), on CPB 90 min (CPB 90), and postprotamine (immediately after protamine administration). Hematocrit and esophageal temperature were monitored at the same time points.
Whole blood samples were obtained at baseline, postheparin, CPB 30, and postprotamine and were centrifuged at 4°C to obtain plasma for storage. This plasma would later be used to measure anti-Factor Xa heparin activity and AT-III activity. Anti-Xa was measured with the ChromoScreen Chromogenic Heparin Assay (Fisher Scientific, Pittsburgh, PA) (4,5). Factor Xa inhibition test was used to assay the wide range of therapeutic heparin concentrations. In this method, both Factor Xa and AT-III are present in excess, and the rate of Factor Xa inhibition is directly proportional to the heparin concentration. Residual Factor Xa activity is measured with a Factor Xa-specific chromogenic substrate, inversely proportional to the heparin concentration. Sensitivity is between 0.0 and 0.05 U/mL (4,5), and the coefficient of variation is between 3.7% and 8.9%.
We analyzed ACT-heparin sensitivity and MAX-ACT-heparin sensitivity after heparinization and 30 min on CPB. The sensitivity was defined as (ACT postheparin - ACT baseline)/(anti-Xa heparin concentration postheparin - anti-Xa heparin concentration baseline). Our calculation was based on the work of Culliford et al. (6), in which sensitivity to heparin was measured as the increase in ACT per unit of plasma heparin. Sensitivity is equivalent to the slope of the dose-response curve in which the heparin level is plotted against the ACT. In contrast to Culliford et al., we calculated sensitivity as an increase in ACT from preheparin levels and divided by the increase in plasma heparin concentration from baseline (because a sizable fraction of patients received heparin before surgery, heparin levels at baseline were different from zero).
The percentage of AT-III activity (ACCUCOLOR; Sigma Diagnostics; St. Louis, MOAQ) was measured by chromogenic assay at the same time points as anti-Xa. In this two-stage method (7), thrombin was added to plasma containing AT-III in the presence of heparin. After an initial incubation period (Stage I), residual thrombin was determined with thrombin-specific chromogenic substrate (Stage II). The residual thrombin activity is inversely proportional to the AT-III concentration. Commercial AT-III control was used. AT-III concentration in the patient specimen was adjusted for the AT-III concentration in the control (7) as follows:
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The standard curve for AT-III was established by the serial dilution of a normal human plasma pool, and the results are given as a percentage of this pool (normal range, 79%–125%) (8). A normal population range determined for Sigma Diagnostics (n = 70) and supported in the literature is 83%–119% (9–11). The ACCUCOLOR AT-III procedure is sensitive to 10% AT-III with a coefficient of variation <10% (7).
MAX-ACT and ACT were compared at all time points and during conditions of normothermia versus hypothermia. Normothermia was defined as the following time points: baseline, postheparin, and postprotamine. Hypothermia was defined as time points CPB 30 and CPB 60. CPB 90 was not included in the temperature analysis because temperature varied at this time point depending on the duration of hypothermic CPB. Data for ACT, MAX-ACT, and heparin levels are presented as mean ± SD. However, because of the nonparametric nature of the data, all statistical analyses were conducted with Wilcoxon’s two-sample test. Change in AT-III levels was determined by using paired Student’s t-tests and was corrected for multiple comparisons. We sought correlation of heparin sensitivity, by both tests, with bleeding and blood product transfusion. P values <0.05 were considered significant.
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Results |
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Twenty-seven patients were enrolled. Surgery included 17 primary coronary artery bypass grafts, 3 reoperations, 5 valvular surgeries, and 5 thoracic aortic surgery procedures. Two patients received aprotinin, and 10 patients received aminocaproic acid. Three patients were excluded after enrollment. Two were excluded because of the administration of a nonstandard heparin dose for the conduct of partial bypass. One patient was excluded because of abnormal baseline coagulation studies secondary to liver congestion. This patient had significantly increased ACT and MAX-ACT throughout the surgery, received multiple transfusions of blood and blood products after surgery, and was one of two patients who received aprotinin. Another aprotinin patient had ACT analyzed in kaolin tubes with MAX-ACT values consistently below ACT values. Seven patients were receiving an infusion of heparin before surgery. Temperature, hematocrit, ACT, MAX-ACT values, heparin level mea-sured by anti-Xa activity, and percentage of AT-III levels in all patients are shown in Table 1 and in Figures 1 and 2.
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ACT and MAX-ACT did not differ significantly at baseline (P = 0.803) and postprotamine (P = 0.139). ACT values were statistically greater than MAX-ACT at postheparin (P = 0.043), CPB 30 (P = 0.038), and CPB 60 (P = 0.013). At CPB 90 (n = 18), the test for the hypothesis that ACT and MAX-ACT were different reached borderline significance (P = 0.06). By using the previously defined time points for hypothermia and normothermia, the ACT - MAX-ACT difference (
) was significantly different during hypothermia compared with normothermia (P = 0.009) (Fig. 1). The change in MAX-ACT from postheparin (509 [303 - 873]) to CPB 30 (501 [380 - 755]) was not significantly different from the change in ACT (502 [376 - 923] to 579 [399 - 755]) at the same time points. However, ACT values were significantly greater than MAX-ACT values upon initiation of CPB (see Fig. 1). During the same interval, heparin level was statistically unchanged but trended toward a decrease (P = 0.065). At postheparin, ACT-heparin sensitivity was greater than MAX-ACT-heparin sensitivity (P = 0.022) ( Table 2). At CPB 30, this difference in heparin sensitivity was no longer statistically significant (P = 0.088).
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ACT-heparin sensitivity and MAX-ACT-heparin sensitivity values were unchanged from postheparin to the CPB 30 time point (P = 0.3). Heparin sensitivity by either ACT or MAX-ACT did not correlate with postoperative chest tube drainage or transfusion of blood products.
AT-III levels decreased significantly from baseline to postheparin (P = 0.0057) and did not change further with institution of CPB or with protamine administration (Fig. 2). The reduction in the percentage of AT-III levels also did not correlate with chest tube drainage or transfusion requirements.
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Activation of coagulation occurs during CPB because of contact with the nonendothelial surfaces of the extracorporeal circuit (Factor XII activation) and because of tissue factor release in the late phase of CPB (1–3). Systemic anticoagulation is required to inhibit the coagulation cascade to prevent the occurrence of gross thrombus formation and the microvascular consumption of coagulation factors (12).
ACT is currently the most commonly used test for monitoring the anticoagulant effect of heparin in the operating room but correlates rather poorly with laboratory assessment of heparin activity and concentration, especially during periods of hypothermia and hemodilution (1,3,6,13,14). Methods of monitoring heparin concentration are available but also have limitations. Whole blood heparin concentration can be performed at bedside with an automated heparin-protamine titration method (15), but this requires the use of standard algorithms to calculate blood volume, which is highly variable. Heparin concentration correlates well with anti-Xa plasma heparin measurements (1,16), with the latter considered as a "gold standard" performed only in special laboratories. Measurement of heparin concentration does not replace measurement of heparin antithrombotic properties, which can be altered in patients with heparin resistance.
Despite many institutions now using normothermic CPB or mild hypothermia, a number of cases require hypothermic CPBs that are more complex, with longer duration of CPB. This increases the need for a monitoring test that correlates with heparin levels and prevents subclinical coagulation during hypothermia. Comparison of ACT and MAX-ACT during warm bypass or with different levels of hypothermia was not done in this work and requires further study.
Even though visible clots or microthrombus are not detected during standard ACT management, the increase in markers of coagulation such as prothrombin factor 1.2 and fibrinopeptide A and in markers of fibrinolysis (such as D-dimer) indicate some degree of coagulation process during CPB (16). In the study of Despotis et al. (16), the mentioned markers were significantly reduced in a group of patients managed by whole blood heparin concentration and kaolin ACT, with post-CPB bleeding and transfusion requirements decreased compared with a celite ACT-managed group. However, other authors did not find heparin dose management with use of whole blood heparin concentration to improve postoperative bleeding (17,18).
A potential explanation for the variability in the ACT measurement is that activation of the coagulation cascade is submaximal with a single activator. In theory, if more complete saturation of Factor XII activation sites could be achieved, then the resultant clotting times would potentially be both shorter and less susceptible to variation. The MAX-ACT is a monitor of heparin anticoagulation that more fully saturates Factor XII activation because it uses three activators concurrently. We compared MAX-ACT with ACT with respect to duration of clotting times and their respective variabilities during conditions of hypothermia.
Our data demonstrate that when ACT and MAX-ACT were statistically different from each other, ACT was of longer duration. This reflects more complete activation of the intrinsic coagulation cascade by using the combination of activators present in the MAX-ACT. The difference between these two tests was not demonstrated during nonheparinized conditions but was statistically significant during conditions of hypothermia while the patient was on CPB. This suggests that MAX-ACT is less susceptible to the factors that increase ACT during conditions of hypothermia.
Culliford et al. (6) found that ACT is not significantly prolonged by hypothermia in the absence of added heparin but is prolonged by hypothermia in the presence of heparin. By using the anti-Xa neutralization method of heparin concentration monitoring, they found that heparin sensitivity was increased during CPB as compared with baseline values (6), which is consistent with our findings. ACT-heparin sensitivity is enhanced under conditions of hypothermia and hemodilution. Cohen et al. (3) examined the ACT-heparin concentration index and found that it changed from 92 ± 17 s · U-1 · mL-1 to 139 ± 34 s · U-1 · mL-1 upon initiation of CPB, and Culliford et al. (6) showed that ACT per unit of heparin nearly doubled (from 95 to 180 s/U) with initiation of CPB. In this study, ACT-heparin sensitivity was greater than MAX-ACT-heparin sensitivity at the postheparin time point, demonstrating a larger change per unit of heparin with ACT monitoring than with MAX-ACT. This most likely reflects more complete activation of the coagulation cascade and shorter clotting times with MAX-ACT. Initiation of CPB resulted in an increase in ACT-heparin sensitivity, consistent with previous investigations (3,6), and no change in the MAX-ACT-heparin sensitivity.
In an attempt to eliminate alterations in the percentage of AT-III activity level as an explanation for the variability in ACT, percentage of AT-III activity was measured at multiple time points. AT-III is the cofactor necessary for heparin’s action and, if deficient, may result in a decrease in heparin sensitivity (19). Acquired AT-III deficiency in the preoperative cardiac surgical period may be related to the preoperative use of heparin or nitroglycerin infusion or the effect of hemodilution (20). Kesteven et al. (14) showed that in the range in which AT-III levels decreased during CPB, the Hemochron ACT was not affected. Despotis et al. (20) later found that a decrease in AT-III level to <80 U/dL reduces the responsiveness of the whole blood ACT to large doses of heparin in vitro, but only a weak effect was found in vivo.
In this study, the percentage of AT-III activity decreased significantly before bypass, similar to the study of Savidge et al. (21). This is explained by hemodilution. It did not change further from the postheparin to the CPB 30 time point or for the duration of the procedure. This is in contrast to previous investigations (14,21), in which the percentage of AT-III activity level continued to decline. The finding that percentage of AT-III values were below baseline values and did not change during CPB indicates that variability in AT-III activity would not be a cause of the altered ACT-heparin sensitivity on CPB (21). Evaluation of percentage of AT-III activity did not explain ACT or MAX-ACT variability.
One of the limitations of this study is that we did not measure heparin levels at all time points because of cost issues and because heparin levels measured by anti-Xa activity were smaller than those typically reported during CPB (1,3,6,17). This may be caused by actual small anti-Xa levels in the presence of adequate anti-IIa activity (seen with bovine heparin) or may be the result of heparin degradation during storage or conduct of the laboratory assay. However, the variances in the reported values were small, and the trend for heparin concentration to decrease during CPB was evident, thus allowing us to perform limited statistical analyses with these results.
In conclusion, MAX-ACT is a test of heparin anticoagulation that uses near-maximal activation of intrinsic coagulation, resulting in shorter clotting times and hence reduced time to acquisition of results. For this reason, this novel test is also less affected by conditions of hypothermia than is the standard celite ACT. The MAX-ACT clotting time, like the celite ACT, does not correlate with heparin concentration during CPB and remains a limitation of all current ACT tests used during cardiac surgery.
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Acknowledgments |
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Array Medical provided Actalyke MAX-ACT tubes and sponsored measurement of heparin levels by anti-Factor Xa activity and AT-III levels.
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References |
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Despotis GJ, Summerfield AL, Joist JH, et al. Comparison of activated coagulation time and whole blood heparin measurements with laboratory plasma anti-Xa heparin concentration in patients having cardiac operations. J Thorac Cardiovasc Surg 1994; 108: 1076–82.
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