Ann Thorac Surg 1999;67:1012-1016
© 1999 The Society of Thoracic Surgeons
Original Articles
Assessment of heparin anticoagulation: comparison of two commercially available methods
Hanne I. Flom-Halvorsen, PhDb,
Eivind Øvrum, MD, PhDb,
Michel Abdelnoor, PhDa,
Stine Bjørnsena,
Frank Brosstad, MD, PhDa
a Research Institute for Internal Medicine, University of Oslo, Rikshospitalet, Oslo, Norway
b Oslo Heart Center, Oslo, Norway
Accepted for publication September 25, 1998.
Address reprint requests to Dr Øvrum, Oslo Heart Center, Pilestredet 32, N-0027 Oslo, Norway
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Abstract
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Background. The activated clotting time is a bedside method routinely used to monitor heparin anticoagulation during operations requiring cardiopulmonary bypass. The thrombolytic assessment system heparin management test is a new bedside method for monitoring heparin effect. We compared these methods with respect to their ability to reflect the actual heparin concentration in plasma determined by an anti-FXa method.
Methods. Two studies were done, an ex vivo study on ten patients who had coronary artery bypass using nonheparin-coated cardiopulmonary bypass circuits and full systemic heparinization and an in vitro study on single donor plasma spiked with heparin 0 to 10 IU/mL.
Results. Ex vivo study correlation coefficients of activated clotting time and the thrombolytic assessment system heparin management test clotting times versus anti-FXa-based heparin assay were low (r = 0.53, p = 0.002/r = 0.64, p < 0.001) in contrast with the corresponding correlation coefficients for the in vitro study (r = 0.98, p < 0.001/r = 0.99, p < 0.001). A substantial variability in duplicate activated clotting time determinations was noted, which was less pronounced with the thrombolytic assessment system heparin management test.
Conclusions. The thrombolytic assessment system method does not correlate better to the actual amount of heparin during cardiopulmonary bypass procedures than the activated clotting time method, which should be performed in duplicate.
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Introduction
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Operations requiring cardiopulmonary bypass (CPB) necessitate systemic heparinization of the patient. Because of considerable variation between patients in their anticoagulant response to heparin [1–5], monitoring of the heparin effect is mandatory before, during, and after surgical procedures. The most frequently used method for assessing heparin anticoagulation in this setting has been the activated clotting time (ACT), which reports the anticoagulant effect as ACT-seconds. However, a number of conditions such as hemodilution, platelet lysis, hypothermia, platelet dysfunction, thrombocytopenia, and hypercoagulation can influence the ACT [5–16], which might lead to incorrect estimation of heparin dosage in clinical situations where ACT is used for heparin assessment. Because the method is poorly sensitive to low heparin concentrations, it is often difficult to decide whether adequate heparin neutralization has been achieved postoperatively. Concerns of incomplete heparin reversal or heparin rebound often result in overdosage of protamine, which may give substantial side effects.
Many attempts have been made to develop bedside heparin assays that are as simple to perform as the ACT method but more sensitive and precise. Recently, the thrombolytic assessment system heparin management test (TAS HMT) has been introduced and found to be a rapid, simple, and more reliable method than the ACT for monitoring heparin anticoagulation in patients who had CPB [9, 17–19]. The aim of the present study was to evaluate this method in a direct comparison with ACT in patients who had coronary bypass operations (ex vivo study) and in whole-blood spiked with known amounts of heparin (in vitro study) using a chromogenic anti-factor Xa activity assay as the reference method.
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Material and methods
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Ex vivo study
Ten patients who had an elective coronary artery bypass procedure were included in the study. Cardiopulmonary bypass was done with standard uncoated CPB circuits (Baxter Healthcare Corp, Bentley Laboratories Division, Irvine, CA) for perfusion. Mild hypothermia (32°C) was instituted after start of CPB. Heparin (pig mucosa, 5000 IU/mL) (Nycomed Pharma AS, Oslo, Norway) was used for anticoagulation, and a standard bolus dose of 400 IU/kg was given intravenously. The activated clotting time (ACT) (High Range HemoTec Automated Coagulation Timer, Medtronic, Inc, Denver, CO) containing kaolin as the activating agent, was at least 480 seconds before starting CPB. Additional heparin was given if the ACT level was below this target level. Protamine sulphate (Leo, Løvens Kemiske Fabrik, Ballerup, Denmark) was used for neutralization of heparin at a dose of 1.3 mg per 100 IU heparin. The extracorporeal bypass circuit was disconnected before the administration of protamine. Supplemental doses of protamine were considered if the postoperative ACT was more than 130 seconds.
Blood samples were collected with a syringe, containing no anticoagulant, from the central venous cannula, discarding the first 10 mL, at the following intervals: (1) after induction of anesthesia but before systemic heparinization, (2) 3 minutes after administration of heparin, (3) 10 minutes after establishing CPB, (4) 30 minutes after establishing CPB, (5) 10 minutes after administration of protamine, and (6) 2 hours postoperatively. Samples of whole blood were then (1) directly analyzed simultaneously (in dual-channel) for ACT determinations and in accordance with the manufacturer’s instructions, and (2) after addition of 1/10 volume of 0.129 mol/L sodium citrate, sequentially analyzed using the TAS HMT equipment (Cardiovascular Diagnostics, Inc, Raleigh, NC) in accordance with the instructions given by the manufacturer, and (3) centrifuged at 2,700 g for 20 minutes at 4°C to obtain plasma for subsequent heparin determination by chromogenic anti-FXa assay (Coatest Heparin; Chromogenix, Mölndal, Sweden). The assay did not involve addition of excess antithrombin to plasma samples. The standard curve was determined by using the same heparin (and lot number) used for patients during cardiac operations. The plasma samples for heparin analysis were stored at -70°C until assayed in one batch.
In vitro study
A venous catheter was placed in the cubital vein of one healthy donor, filled with saline, and stoppered. Blood samples were drawn at intervals with a syringe, discarding the first 3 mL and added to tubes containing final concentration of either 0, 0.15, 0.25, 0.5, 1.0, 1.5, 2.5, 3.5, 4.5, 5.5, 7.5, or 10.0 IU/mL of unfractionated heparin (pig mucosa, Nycomed Pharma AS, Oslo, Norway) for simultaneous ACT determinations (dual-channel) or the same amount of heparin plus sodium citrate (0.129 mol/L) to 1/10 volume of total volume for sequential TAS HMT determinations. Plasma for heparin analysis was prepared from the remaining volume of the latter specimens by centrifugation for 20 minutes at 2,700 g at 4°C and stored at -70°C until assayed in one batch.
Statistics
Linear regression analysis was used on data with and without log10 transformation to analyze the association between the measured heparin concentration and the ACT and TAS HMT clotting times in seconds. The data used were corrected by substraction of baseline values (no heparin present), which constituted the preoperative level in the ex vivo study, and for the in vitro study the first level where heparin could be detected by the anti-FXa assay. The two last time points in the ex vivo study were excluded (no heparin present). Statistical correlation was also done between spiked and anti-FXa-measured levels of heparin using data substracted from baseline values (no heparin measured). A p value less than 0.05 was considered significant for the linear regression analysis. The mean values of the double determinations were used throughout for the in vitro and the ex vivo studies. For the ex vivo study, percent change values during heparinization were calculated using the following formula: CPB value minus pre-CPB value (3 minutes after heparin) divided by baseline value x 100.
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Results
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The results of the ACT, TAS HMT, and heparin assays on the ten patients who had CPB are shown in Figure 1 and Table 1. Three minutes after intravenous heparin bolus injection, a maximum peak concentration of heparin averaging 7.8 IU/mL plasma was obtained, which decreased to an average of 4.2 IU/mL plasma between CPB time points 10 and 30 minutes as measured by the anti-FXa assay. No heparin was detected in plasma 10 minutes after protamine was given, which was also the case 2 hours later. This pattern of variation in the heparin concentration occurred with both the ACT and the TAS HMT methods. Although the curves showed that the ACT and TAS HMT methods reflected changes in heparin concentration during heparinization, the data in Table 1 indicate that while the heparin concentration dropped to 50% (average between the time points 10 and 30 minutes after CPB) of baseline value (ie, pre-CPB after heparin), the ACT and TAS HMT decreased by an average of only 15% and 10%, respectively, during CPB.
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Fig 1. The relationship between the clotting time in seconds of activated clotting time (ACT) and thrombolytic assessment system heparin management test (TAS HMT) methods and the plasma heparin concentration (IU/mL) measured by anti-FXa assay in ten patients who had cardiopulmonary bypass (CPB). The data are presented as mean values.
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Another observation from Table 1 was the higher variability (as expressed by the standard deviation) for the ACT method compared with the TAS HMT assay during heparinization, ie, the mean standard deviations were 179 and 28, respectively, from the three time points in which heparin could be measured in the patients (before CPB and 10 and 30 minutes after CPB).
The statistical correlations between the ACT and TAS HMT versus the anti-FXa method for heparin determination were done with linear regression analysis. For the ex vivo study both methods demonstrated a statistically significant but marginal correlation, as evidenced by the Pearson correlation for ACT-anti-FXa (r = 0.53, p = 0.002) and TAS HMT-anti-FXa (r = 0.64, p < 0.001) (Fig 2, Table 2). For the in vitro study, good statistical correlations were obtained between both ACT values and TAS HMT values when compared with the anti-FXa assay for heparin determination (ACT versus heparin, r = 0.98, p < 0.001; TAS HMT versus heparin, r = 0.99, p < 0.001) (Fig 3, Table 2). Log10-transformation of the data which corrected for their baseline values verified a normal distribution for the measured values (not shown).
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Fig 2. Ex vivo study. (A) Regression line between the clotting time (in seconds) of whole blood activated clotting time (ACT). (B) Thrombolytic assessment system heparin management test (TAS HMT) and plasma heparin concentration (IU/mL) measured in samples from ten patients who had cardiopulmonary bypass. The data are corrected for their respective baseline values, and the mean values of the double determinations are used.
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Table 2. Linear Regression Analysis of the Two Methods and Plasma Heparin Concentration in the In Vitro and Ex Vivo Studiesa
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Fig 3. In vitro study. (A) Regression line between the activated clotting time (ACT). (B) thrombolytic assessment system heparin management test (TAS HMT) seconds and measured plasma heparin concentration (IU/mL) in samples from donor whole-blood spiked with known amounts of heparin. The data are corrected for their baseline values and presented as mean values.
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When the in vitro results of the whole-blood heparin spiked values were compared with those of the plasma derived anti-FXa measurements converted into whole-blood equivalent values, a good accordance was noted, as evidenced by linear regression analysis (r = 1.00, p < 0.001). However, the heparin assay kit used as the reference method was not able to detect heparin in samples supplemented with 0.25 IU heparin/mL or less, whereas for ACT and TAS HMT a definite increase in clotting time was registered even in this low heparin range (Table 3). A heparin kit from another manufacturer (Rotachrom Heparin; Diagnostica Stago, Asnieres-sur-Seine, France) had the same results.
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Comment
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The ACT is traditionally used to assess heparin anticoagulation in whole blood during CPB-based procedures, even though most surgeons have been aware of its limitations and inaccuracy. Recently, a new device, the TAS HMT, has been introduced for bedside heparin assay in citrated whole blood and has been reported to be superior to ACT [9, 17–19]. The aim of the present study was to compare the ACT and TAS HMT methods with respect to their ability to reflect the actual heparin concentration in plasma, as determined by an anti-FXa method.
For the two studies, both the ACT and TAS HMT correlated statistically with the reference method anti-FXa assay in linear regression analysis. The in vitro study found good correlation coefficients for ACT and TAS HMT, whereas in the ex vivo study marginal correlations were obtained for both systems (Table 2). Similar to our results, previous reports have also documented weak or no correlation between ACT measurements and plasma heparin concentration during CPB [5, 7, 8]. The few data available for the TAS HMT method for whole-blood heparin assay show that the TAS HMT, in contrast to our results, correlated well with heparin levels ex vivo and better than ACT during CPB [17, 19]. It should be noted, however, that these studies are not exactly comparable to ours with regard to the patient group [17] and the type of ACT device used (dual-channel Hemochrome) [19].
The low ex vivo correlations found in the present study between the heparin concentration and the ACT or TAS HMT indicate that the new TAS HMT system seems to have the same disadvantages and limitations as ACT values in that they seem to be similarly affected by CPB-related hemodilution and hypothermia. In addition, neither ACT nor TAS HMT reflected changes in heparin concentration during heparinization, as measurements of the heparin level showed a pronounced decrease despite almost unchanged ACT or TAS HMT values (Table 1). On this basis and the low ex vivo correlations between anti-FXa assay and ACT or TAS HMT, neither ACT nor TAS HMT is suited for precise assessment of heparin concentration. Further, the inaccurate detection of plasma heparin changes during CPB by ACT and TAS HMT might lead to incorrect estimation of the actual heparin concentration and make exact neutralization of heparin by protamine sulphate difficult. The practical solution to this problem is based on empiric data. Most cardiac centers use a neutralization dose of protamine equal to 1.3 x initial heparin concentration, an amount that should far exceed the actual heparin concentration after cessation of CPB.
The correlation coefficients of the ex vivo study contrast with the findings of the in vitro study, where higher correlations between heparin and ACT or TAS HMT were found (Table 2). This discrepancy might be caused by ex vivo conditions known to influence the ACT during CPB, such as hypothermia and hemodilution [7]; the same conditions are likely to influence the TAS HMT system as well.
The ACT method showed a substantially higher variability between duplicate determinations than TAS HMT for heparin activity at 10 and 30 minutes on CPB (Table 1). This finding strengthens the importance of performing duplicate ACT measurements, as single measurements would lead to either overestimation or underestimation of heparin activity and consequently to an increase or decrease of heparin dosage. Previous studies using celite-activated ACT tubes also support the use of duplicate ACT measurements [20, 21]. The present study had good in vitro concordance between the measured and expected heparin values as determined by the anti-FXa method (Table 3).
In conclusion, we compared ACT and TAS HMT with respect to their ability to determine actual heparin concentration in plasma and found that the TAS HMT system seems to have the same disadvantages during CPB as the traditional ACT method. This conclusion is based on the low ex vivo correlation between ACT or TAS HMT value and the heparin concentration and indicates that both methods are poor estimates of the actual heparin level during CPB. Further, on the basis of the substantial variability noted for the ACT determinations, we suggest that duplicate measurements be done when using the ACT method for monitoring heparin effect.
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Abstract
Introduction
