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*Institute for Laboratory Medicine and Pathobiochemistry, Campus Virchow Klinikum, Charité, Berlin;
Institute for Anesthesiology, Deutsches Herzzentrum Berlin, Berlin; and
Clinics for Anesthesiology and Intensive Care Medicine, University Clinics of the Saarland, Homburg Saar, Germany
Address correspondence and reprint requests to Andreas Koster, MD, Deutsches Herzzentrum Berlin, Augustenburgerplatz 1, 13353 Berlin, Germany. Address e-mail to Koster{at}DHZB.de
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Abstract |
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Implications: The ACT II anti-Xa-unfractionated heparin assay allows for reliable monitoring of large concentrations of UFH over a wide range of hematocrit, platelet, and coagulation factor levels. Further evaluation of this point-of-care device is indicated.
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Introduction |
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Large-dose heparinization requires frequent, point-of-care monitoring of the anticoagulant effect. Global anticoagulation tests, such as the commonly used activated clotting time (ACT), reflect the more general coagulation status of the patient than the actual heparin concentration (3–5). During longer perfusions, a continuing effect on the coagulation system can lead to a prolongation of the ACT, despite decreasing levels of UFHs (1,2). There is evidence that the maintenance of larger heparin levels inhibits the system of contact activation and contributes to a reduced postoperative blood loss (6). Moreover, the reversal agent protamine itself reveals an anticoagulant effect. Excessive protamine dosing, due to overestimation of actual heparin level after conclusion of CPB, contributes to further damage of the coagulation system (7). Therefore, an anticoagulation protocol for CPB, based on measurement of the concentration of UFHs, effectively contributes to reduced blood loss and reduced transfusion requirements (8–10) and appears to be desirable. However, the commercially available systems, such as the automated protamine titration assay (Hepcon HMS; Medtronic, Inc., Parker, CO), are controversial with reference to their reliability for precise measurement of heparin concentration (11,12). Values of the Hepcon HMS are only valid if the heparin concentration is measured in the central channels of the cartridge. Coagulation detection in the left or right margin chamber requires repetition of the test with a smaller or a larger range cartridge to avoid over- or underestimation of the heparin level. This prolongs the time for the measurement and also contributes to the increased costs of the system.
Assessment of anti-Xa activity is the laboratory reference method for the measurement of heparins. The available assays require time-consuming preanalytical procedures and are not appropriate for point-of-care monitoring. We adapted the Heptest® (AccuclotTM Heptest®, Sigma Diagnostics, Deisenhofen, Germany) for measurement of plasmatic anti-Xa activity for performance in whole blood using the ACT II (Medtronic) device. We assessed the reliability of this test in vitro and in vivo during CPB.
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Methods |
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Performance of Whole Blood Heptest® on the ACT II Device
Recalcification of the citrated blood was achieved by transferring 75 µL of the Recalmix solution of the Heptest® kit into the reaction chamber of the ACT II cartridges and inserting the flag. Then, 75 µL of the citrated whole blood sample was placed in the cartridge. Thereafter, 75 µL of the Factor Xa reagent was added, and the test was started after the automated incubation period.
Preliminary Investigations and Determination of the Optimal Reagent
Concentrations of UFHs, which are necessary to achieve sufficient anticoagulation during CPB, normally vary from patient to patient. Therefore, the initial goal was to design an assay that would reveal linearity over a wide range of UFHs. In preliminary investigations using the same method as described later, the exclusive use of the large-range Heptest® (Heptest® HI) demonstrated linearity up to 8 IU/mL UFH. However, spread of the coagulation times between 0 and 8 IU/mL UFH was reasonably low (130 s) and correlation to the chromogenic assay was poor (r = 0.56).
In the case of UFH concentrations below 4 IU/mL, this was attributed to so much excess available Factor Xa that the differences in UFH levels cannot be discriminated.
The optimal reagent for the assay was determined. The Heptest® HI reagent was diluted with predetermined volumes of the small-range test kit (Heptest®). A ratio of 1:3 of the Heptest® HI and Heptest® LOW revealed linearity to concentrations of up to 6 IU/mL UFH and spread the measuring range to a 300-s gap between concentrations of 0.5–8 IU/mL UFH. This was regarded as optimal for precise differentiation of the concentrations of UFH and fast acquisition of test results.
In Vitro Investigations
With written informed consent, citrated whole blood samples (10 ml) were obtained from 10 healthy volunteers (7 male and 3 female students, mean age 25 yr). Samples from 10 volunteers were used to establish a standard calibration curve for UFHs (Liquemin, Hoffman-La Roche, Grenzach-Wyhlen, Germany).
The relationship between ACT II anti-Xa-UFH assay measurement and each heparin concentration was assessed under the following conditions:
The within-assay variance was measured using two different concentrations (2 and 6 IU/mL) of UFHs. Each measurement was performed 10 times.
Accordingly, the influence of storage time at room temperature (18°–24°C) was evaluated by repetition of the measurements (mean value of measurement in duplicates) after 30, 60, 120, 240, and 480 min.
In Vivo Investigations
After written informed consent and approval by the local ethics committee, samples were collected from 15 patients who underwent cardiovascular surgery that involved CPB.
According to our departmental protocol, preoperative anticoagulation with salicylic acid was stopped 10 days before surgery and replaced with either a single subcutaneous bolus injection (7500 IU) of UFH (n = 8 patients) or continuous infusion of UFH (n = 5 patients), according to values of the activated partial thromboplastin time of 40–60 s. Two patients had been treated with warfarin to an international normalized ratio value of 1.8.
Anesthesia was performed according to the departmental standard using a total IV technique (propofol, sufentanil, and pancuronium bromide). CPB was performed with standard equipment, such as roller pumps and membrane oxygenators. All components were nonheparin-coated for the CPB system.
The platelet count, fibrinogen level, and hematocrit were performed in parallel to each measurement.
Samples were collected before and after the initial UFH bolus. Thereafter, samples were collected at intervals of 30 min during perfusion and after protamine administration. The anticoagulation protocol was performed according to the departmental standard: an ACT value of 480 s was the predetermined target, and the necessary individual heparin concentration to achieve this ACT value was calculated by means of the heparin titration cartridge of the Hepcon HMS device. Five minutes after injection of the initial individual bolus of UFH, both the ACT and heparin level (as provided by the Hepcon HMS device) were measured. During a further course of surgery, heparin concentration was measured at intervals of 30 min and adjusted to the individual target value according to the calculations of the Hepcon HMS device. Anticoagulation was exclusively guided by maintenance of heparin concentration without further measurements of ACT. By the end of CPB, the protamine dose was based on the residual heparin concentration. In all patients, aprotinin (Antagosan; Hoechst, Frankfurt, Germany) was administered with a bolus of 2 x 106 kallikrein inhibiting units (KIU) for the patient, 2 x 106 KIU into the priming solution of the CPB, and a constant infusion of 0.5 x 106 KIU/h during extracorporeal circulation.
Chromogenic Anti-Xa Assay
The chromogenic assay was performed on an STA analyzer (Roche Diagnostics, Mannheim, Germany) that used the STA LMWH test kit (Roche Diagnostics). Fifty microliters of the sample were diluted with 50 µL of diluent buffer and 100 µL of Factor Xa, and 100 µL of substrate was added. For kinetic measurement of the remaining Factor Xa, the change in optical density was assessed at 405 nm.
Statistical Analysis
Correlation between the results of the plasmatic chromogenic test and the ACT II anti-Xa assay was analyzed using Pearson’s correlation coefficient. In vitro data were analyzed using analysis of variance and the Scheffé test. A P < 0.01 was considered significant.
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Results |
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The duration of CPB ranged from 85 to 321 min, with a mean of 137 ± 43 min. Surgery was performed in normothermia (n = 4), moderate hypothermia (32°–30°C, n = 9), and deep hypothermia (16°C, n = 2).
Platelet counts ranged from 243 to 24 ± 35 x 106/µL with a mean of 129 ± 23 x 106/µL the fibrinogen levels ranged from 54 to 250 mg/dL with a mean of 154 ± 31 mg/dL and the hematocrit values from 37% to 22% with a mean of 27% ± 3.6%.
Heparin concentrations obtained by the chromogenic method varied from 0.0 to 8.9 IU/mL with a mean of 3.2 ± 2.70 IU/mL. Values of the ACT anti-Xa UFH assay ranged from 22 to 425 s with a mean of 161 ± 104 s. The correlation coefficient of the ACT II anti-Xa UFH assay to the chromogenic method was 0.9 (Fig. 3).
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Discussion |
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In the in vitro part of the investigation, the test revealed excellent reproducibility and linearity up to concentrations of 6 IU/mL UFH. The interindividual variation of the coagulation times for defined heparin concentrations exceeding 5% suggests that a preoperative individual calibration is mandatory. However, further investigations with a larger number of patients are necessary for the final evaluation of this subject.
Test results were nearly uninfluenced by the dilution to a hematocrit of 20% of platelets and procoagulants as often observed during CPB. However, an increase of the hematocrit to 60% led to a prolongation of the coagulation time. This effect was pronounced in the larger concentrations of UFH and can be explained by the larger concentration of UFH in the correspondingly smaller plasma volume.
In the in vivo part of the investigation, the test provided a close correlation (r = 0.90) to the values of the chromogenic reference method. This is noteworthy because the CPB procedures included extreme conditions of perfusion, such as profound deep hypothermia (16°C) and long-term CPB (>3 h) with depletion of platelets (<20,000/µL) and fibrinogen (<50 mg/dL), and confirmed the results obtained in the in vitro set-up. In order to avoid further imprecision by additional measurement of the hematocrit value, which would have been necessary, the values of the plasmatic chromogenic assay were not corrected to the hematocrit values of whole blood. Therefore, the values of the plasmatic chromogenic assay, in contrast to the constant volume of the whole blood, are influenced by the hematocrit as it determines the volume in which UFHs are dissolved. Apart from the individual response to heparins as influenced, for example, by cell binding or binding to platelet factor 4 in the sample, these variations of the hematocrit (37%–22%) explain the scatter along the correlation curve in Fig. 3. Consequently, a correlation coefficient of 0.9 can be considered as satisfactory.
The ACT II device appears to be preferable to other single channel options for the adaptation of other more specific coagulation tests because: first, the tests are performed in double cartridges so that duplicate measurements can be performed in parallel. Second, the tests can be easily performed because of the air-tight reaction chamber and stability of the reagent allows for prepreparation of the cartridges that effectively reduces the operating steps in the operation room. Moreover, the automated incubation period contributes to the further ease of handling. Third, the small-range cartridge only needs a volume of approximately 150–200 µL in comparison with the 2 mL needed for most other devices. Because the reagents are major contributors to the cost of the assay, the reduction of the volumes necessary for the test effectively reduced the costs.
The ACT II anti-Xa-UFH assay is in the series of tests transformed to the ACT II device for monitoring inhibitors of plasmatic coagulation, such as low molecular weight heparins, heparinoids, and direct thrombin inhibitors like recombinant-hirudin (13,14). Therefore, current clinically important anticoagulants can be monitored with one device at the site of patient care.
However, although the test is easy and quick to perform, the need for preanalytical construction of an individual calibration curve and for precise pipetting steps in the operating room limit the value of the assay in its current form. Therefore, before routine use in clinical practice is practical, further automation of the assay is needed. In addition, further clinical investigations with a larger number of patients are necessary for the final evaluation of the assay.
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Acknowledgments |
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References |
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This article has been cited by other articles:
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  W. C. Oliver Jr Overview of Heparin and Protamine Management and Dosing Regimens in Pediatric Cardiac Surgical Patients Seminars in Cardiothoracic and Vascular Anesthesia, December 1, 2003; 7(4): 387 - 410. [Abstract] [PDF]
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 A. Koster, G. Despotis, M. Gruendel, T. Fischer, M. Praus, H. Kuppe, and J. H. Levy The Plasma Supplemented Modified Activated Clotting Time for Monitoring of Heparinization During Cardiopulmonary Bypass: A Pilot Investigation Anesth. Analg., July 1, 2002; 95(1): 26 - 30. [Abstract] [Full Text] [PDF]
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