Ann Thorac Surg 1996;62:533-537
© 1996 The Society of Thoracic Surgeons
Original Articles: Cardiovascular
In Vitro Effect of Hemodilution on Activated Clotting Time and High-Dose Thrombin Time During Cardiopulmonary Bypass
Robert J. Huyzen, MD,
Willem van Oeveren, PhD,
Feiyan Wei,
Pieter Stellingwerf,
Piet W. Boonstra, MD, PhD,
Y. J. Gu, MD, PhD
Departments of Anesthesiology and Cardiothoracic Surgery, University Hospital Groningen, Groningen, the Netherlands
Accepted for publication April 7, 1996.
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Abstract
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Background. Extreme dilution of clotting factors, as may occur during pediatric or neonatal cardiopulmonary bypass, often leads to inadequate monitoring of anticoagulation with activated clotting time (ACT). In this study we postulate that the high-dose thrombin time (HiTT) is less influenced by extreme dilution of clotting factors because it stimulates clotting through the common pathway.
Methods. Heparinized prebypass blood was obtained from 30 adult cardiac surgical patients and was diluted in a laboratory setting with saline solution to mimic the clinical clear prime solution (group I; n = 10), with saline solution containing similar heparin as in the prebypass blood (group II; n = 10), and with fresh frozen plasma to substitute clotting factors in the diluted blood (group III; n = 10). Blood was diluted to four different degrees: a control without dilution, 25%, 50%, and 75% dilution. The ACT and HiTT were measured and compared.
Results. In group I, significant prolongation of ACT was observed in blood diluted to 75% as compared with the nondiluted blood (p < 0.01). In contrast, HiTT was not prolonged at any degree of dilution but reduced proportionally to dilution up to 75%, reflecting the concomitant reduction of heparin. In group II, ACT increased at 25% dilution (p < 0.01) whereas HiTT increased at 50% dilution (p < 0.01). In group III, no prolongation of ACT or HiTT was found in any degree of dilution. Furthermore, adding fibrinogen to the diluted blood (n = 4) did not cause ACT to recover at 75% dilution, suggesting that dilution of other factors in the early clotting cascade rather than fibrinogen alone increases ACT.
Conclusions. These results imply that when blood is extremely diluted during cardiopulmonary bypass with a clear prime without substituted clotting factors, HiTT is a better test than ACT for anticoagulation monitoring.
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Introduction
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Activated clotting time (ACT) is widely accepted as a bedside monitoring test of anticoagulation with heparin during cardiopulmonary bypass (CPB) [1–3]. This test is based on activation with celite or kaolin as the contact activator of the intrinsic pathway of the coagulation system [4]. It is, however, stated that the ACT does not reflect heparin concentrations, but measures heparin function and is influenced by such factors as drugs, hypothermia, and hemodilution [4–6].
The high-dose thrombin time (HiTT) test is based on direct thrombin conversion of fibrinogen into insoluble fibrin in the final common pathway [7, 8]. This test is only dependent on functional fibrinogen and not on other clotting factors in the intrinsic clotting cascade. Because fibrinogen is excessively present in plasma, the HiTT seems more robust to hemodilution than ACT under circumstances where some other clotting factors are being limited [6, 8].
Clinically, extreme hemodilution is not uncommon during CPB in pediatric or neonatal cardiac operations [9]. During these operations red blood cell concentrate often is substituted for the prime solution to achieve the desired hematocrit, allowing further dilution of clotting factors. Therefore, the ACT may increase and fail to reflect the real anticoagulation state because of its dependence on a functional intrinsic pathway [10]. The prolongation in the ACT test may lead to reduced heparin use during CPB, thus giving patients an increased tendency to intravascular coagulation. As evidence of this, fibrinopeptide A, a subclinical plasma coagulation marker, was found inversely related to heparin concentration during CPB [3].
Because of the above-mentioned problem and characteristics of the ACT and HiTT, we performed a study in a laboratory setting using heparinized blood obtained from patients undergoing coronary artery bypass grafting to examine the effect of dilution of clotting factors on the performance of both ACT and HiTT.
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Material and Methods
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Study Design
After institutional approval by the University Hospital Groningen and informed consent from patients, heparinized blood was obtained from 30 adult patients who were scheduled for elective coronary artery bypass grafting. Patients with the use of any medication known to affect the clotting cascade and who had a tendency toward heparin resistance were excluded. From each patient, 20 mL of blood was collected 5 minutes after systemic heparinization with bovine heparin, 300 IU/kg (LEO, Emmen, the Netherlands), before the institution of CPB.
The collected blood was allocated to the following study groups in the laboratory: group I (n = 10), dilution with saline solution to mimic the clinical clear prime solution; group II (n = 10), dilution with saline solution containing as many units of heparin as determined in the prebypass blood; and group III (n = 10), dilution with fresh frozen plasma to substitute the clotting factors in the diluted blood without additional heparin. For each experiment, blood was diluted to four different concentrations: a control without dilution, 25%, 50%, and 75% dilution.
In blood diluted with saline solution, additional tests were performed by adding fibrinogen (2 mg/mL) to examine whether the recovery of fibrinogen in the diluted blood may normalize the ACT (n = 4), and by adding antithrombin III (1 U/mL) to examine whether the reduction of HiTT at a high degree of dilution was due to the reduction of antithrombin III (n = 4). Furthermore, tubes containing high dose of thromboplastin were used to stimulate clotting through the extrinsic pathway (n = 4) in comparison with ACT in the saline solution, diluted blood. For each test, 0.3 mL of rabbit brain thromboplastin (4 mg/mL) was used for 1.7 mL of blood, either diluted or nondiluted.
Measurements
The ACT was measured in tubes containing 12 mg of celite (CA 510; International Technidyne Co, Edison, NJ) with 2 mL of blood. Measurements of HiTT were performed in tubes containing 18 U of thrombin (A 501; International Technidyne Co) with 1.5 mL of blood after the thrombin was dissolved with 0.5 mL of distilled water and prewarmed for 3 minutes. All measurements were performed using a Hemochron 8000 (International Technidyne Co). Heparin level from the prebypass samples was estimated by the Hemochron 8000, based on computerized calculation from the value of HiTT. Values of ACT exceeding 1,000 seconds were interrupted, and the value was noted as 1,000 seconds.
Statistical Analysis
Statistical analysis was performed with one-way analysis of variance using the SPSS statistical package. The Mann-Whitney U test was performed to identify differences in clotting times between the groups, and the paired t test was used for differences between the different dilutions in each group. Results are expressed as mean ± standard error of the mean, and a p value of less than 0.05 was considered significant.
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Results
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Dilution With Saline Solution Alone
In blood diluted with saline solution alone, ACT was more than twice as long as the nondiluted controls at 75% dilution (from baseline 459 ± 24 seconds to 951 ± 49 seconds; p < 0.01), whereas at 50% dilution ACTs were prolonged only slightly (488 ± 30 seconds) (Fig 1
). In contrast, HiTT was not prolonged at any degree of dilution but decreased gradually toward 75% dilution (from baseline 165 ± 16 seconds to 55 ± 6 seconds; p < 0.01). Thus, there was a significant difference between ACT and HiTT at 75% dilution with saline solution alone (p < 0.05) (Table 1).
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Fig 1. . Activated clotting time (ACT) and high-dose thrombin time (HiTT) measured from prebypass heparinized blood diluted with saline solution in different degrees (n = 10). Significant prolongation of ACT was noticed in samples with 75% dilution in comparison with 0% dilution (p < 0.01). The HiTT was reduced proportionally to dilution up to 75%.
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Table 1. . Comparison of Activated Clotting Time and High-Dose Thrombin Time Under Different Degrees of Dilutiona
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Dilution With Saline Solution Containing Heparin
In this group, in which heparin concentration in each diluted sample was maintained as high as the predilution level, the ACT increased already at 25% dilution (from 498 ± 64 to 667 ± 92 seconds; p < 0.01) (Fig 2). Further, the ACT was prolonged to 904 ± 59 seconds at 50% dilution (p < 0.01) and 1000 ± 0 seconds at 75% dilution (p < 0.01). The HiTT did not change significantly at 25% dilution in this group (from 170 ± 13 to 184 ± 10 seconds), but increased to 253 ± 26 seconds at 50% dilution (p < 0.01) and 307 ± 27 seconds at 75% dilution (p < 0.01) (see Fig 2).
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Fig 2. . Activated clotting time (ACT) and high-dose thrombin time (HiTT) measured from prebypass heparinized blood diluted with saline solution containing similar heparin as the prebypass blood (n = 10). The ACT was prolonged significantly at 25% (p < 0.05), 50% (p < 0.01), and 75% (p < 0.01) dilution in comparison with 0% dilution. The HiTT also was significantly prolonged at the 50% (p < 0.01) and 75% (p < 0.01) dilutions.
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Dilution With Fresh Frozen Plasma
In samples where dilution was made with fresh frozen plasma to recover the clotting factors, no prolongation of ACT was found at any degree of dilution (Fig 3). In contrast, the ACT decreased from 503 ± 44 to 394 ± 33 seconds at 75% dilution (p < 0.01) as the heparin concentration was reduced in the diluted blood. The HiTT also decreased (from baseline 166 ± 12 seconds to 85 ± 13 seconds; p < 0.01) and maintained a pattern similar to that found in the saline solution-diluted group.
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Fig 3. . Activated clotting time (ACT) and high-dose thrombin time (HiTT) measured from prebypass heparinized blood diluted with fresh frozen plasma (FFP) to recover the clotting factors (n = 10). No prolongation of ACT was noticed with a 75% dilution with FFP.
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Dilution With Saline Solution Containing Fibrinogen
In blood samples diluted with saline solution and fibrinogen, ACT did not return to the baseline but was still prolonged to more than 1,000 seconds at 75% dilution (Fig 4).
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Fig 4. . Activated clotting time (ACT) determined in blood samples diluted with either saline solution or saline solution plus fibrinogen (2 mg/mL).
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Dilution With Saline Solution Containing Antithrombin III
In blood samples diluted with saline solution and antithrombin III, HiTT remained slightly higher than in samples diluted with saline solution alone at different dilution levels (Fig 5).
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Fig 5. . High-dose thrombin time (HiTT) determined in blood samples diluted with either saline solution or saline solution plus antithrombin III (AT III, 1 U/mL).
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Clotting Time From the Extrinsic Pathway
The extrinsic pathway-associated clotting time stimulated with a high dose of thromboplastin was not significantly prolonged at 75% dilution, in contrast to the intrinsic pathway-associated ACT (Fig 6).
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Fig 6. . Comparison of the intrinsic and extrinsic pathway-associated activated clotting time (ACT) under different degree of dilution with saline solution (for details, see Material and Methods). Clotting times are expressed as percent change compared with control samples without dilution. (*p < 0.05 between groups.)
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There was no significant difference among groups regarding heparin level in the nondiluted pre-CPB blood (3.4 ± 0.4 IU/mL in group I, 3.5 ± 0.3 IU/mL in group I, and 3.5 ± 0.6 IU/mL in group III).
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Comment
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During pediatric or neonatal cardiac operations the ACT often increases from approximately 480 to more than 1,000 seconds after the onset of CPB [10, 11]. Clearly this is not related to increased heparin concentration. Extreme dilution of the circulating blood, along with other factors such as hypothermia and platelet dysfunction, is considered the cause of ACT prolongation, but there is little information in the literature to clarify the underlying mechanism. In this study, by using heparinized blood in the laboratory under different degrees of dilution, we demonstrated that a dilution of 50% to 75% of the prebypass blood significantly prolonged the ACT. We suggest that in these situations concentrations of contact factors such as XI or XII are too low for sufficient thrombin formation, which may result in prolonged values of ACT. In neonates the concentrations of contact factors XI and XII are already very low, being less than 50% of concentrations in adults [12]. These contact factors are further diluted by the prime volume once bypass begins [9]. Whole blood added to the prime may offset the dilution of clotting factors to some degree but is often insufficient for correcting the coagulation deficit [13, 14], shown by ACT values of more than 700 seconds. Also, antithrombin III substitution did not significantly change the ACT values [15].
In this study we found, in contrast with ACT, that the HiTT gradually decreased as levels of heparin and clotting factors were reduced in the diluted samples. In plasma, a linear relationship between thrombin time and plasma heparin levels has been demonstrated [7]. Also, in whole blood the HiTT was reported to be correlated with heparin concentration after administration of heparin before and during CPB [8]. However, in diluted blood samples where heparin concentration was kept as high as in the nondiluted blood (group II), HiTT increased significantly at 50% and 75% dilution, which suggests that the HiTT may still be influenced by dilution under high heparin concentrations. On the other hand, in samples with antithrombin III diluted, HiTT remained higher than the saline solution-diluted samples, which further suggests that the HiTT determination of heparin is partially dependent on antithrombin III. Although in the HiTT test fibrinogen is directly converted by thrombin, additional thrombin generation is dependent on the positive feedback of thrombin on factor X and V of the clotting cascade [16]. This positive feedback is probably inhibited by dilution with high concentrations of heparin.
After restoration of the coagulation insufficiency with fresh frozen plasma in the diluted blood we observed that the ACT recovered even under the high degree of hemodilution, which indicates that the main cause of prolongation of ACT under extreme hemodilution is the lack of clotting factors and not blood cells. Fibrinogen is a key component for clot formation in the test tube. However, supplementing fibrinogen alone in the diluted blood did not recover the ACT at 75% dilution, which further supports the hypothesis that severe dilution of the clotting factors from the clotting cascade rather than fibrinogen increases the ACT.
Further, it seems that the lack of clotting factors under extreme hemodilution is mainly due to the intrinsic pathway rather than the extrinsic pathway. This is supported by the additional comparison of high-dose thromboplastin time with ACT, the former is known to stimulate clotting through the extrinsic pathway, which was significantly less influenced at 75% dilution.
The selective intrinsic pathway inhibition during hemodilution and measurement of the ACT may result in inadequate heparinization, which has been shown to increase coagulation activity [17], probably due to extrinsic pathway activation [18]. More recently Dietrich and associates [19] found a relation between inadequate anticoagulation and clotting activity, platelet release, and fibrinolysis in patients with heparin tachyphylaxis. They recommended the use of a higher heparin dose and adjuvant anticoagulation drugs to prevent these negative effects on hemostasis.
We conclude that ACT was prolonged significantly when whole blood was diluted up to 75% in vitro with saline solution, and that this prolongation was independent of heparin. The failure of ACT was mainly due to its dependence on an intact intrinsic clotting system. The HiTT test minimized the effect of hemodilution on anticoagulation monitoring, because this test depends mainly on fibrinogen concentration, which is not limiting under 75% dilution. For clinical monitoring at high hemodilution and at stable heparinization, it is important to realize that HiTT can also be slightly increased because the positive feedback of thrombin on the clotting system is ineffective. Maintaining a higher HiTT is thus recommended to prevent any activity of the extrinsic or common coagulation pathway under a high degree of hemodilution during CPB.
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Footnotes
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Address reprint requests to Dr van Oeveren, Blood Interaction Research, Department of Cardiothoracic Surgery, University Hospital, 59 Oostersingel, 9713 EZ Groningen, the Netherlands.
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References
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Abstract
Introduction
