A Novel Point-of-Care Assay for the Monitoring of Low-Molecular Weight Heparins in the Cardiac Catheterization Laboratory

ABSTRACT: Background. We designed a study to compare the novel point-of-care assay Hemonox clotting time (Hemonox-CT) with the activated clotting time (ACT) and anti-Xa activity to monitor the anticoagulation effects of enoxaparin and dalteparin during percutaneous coronary intervention (PCI). Methods. A total of 90 patients undergoing cardiac catheterization were assigned to intravenous (IV) enoxaparin 0.5 mg/kg, dalteparin 50 international units/kg or unfractionated heparin (UFH) 50 units/kg. We measured Hemonox-CT, ACT and plasma anti-Xa levels after serial sampling. Results. Baseline Hemonox-CT was similar in the enoxaparin (68 ± 9 sec) and dalteparin (68 ± 7 sec) groups with no detectable anti-Xa activity at baseline. Minutes after IV administration of enoxaparin and dalteparin, the mean Hemonox-CT increased to 171 ± 60 sec and 214 ± 70 sec for both groups, respectively. UFH induced a higher Hemonox-CT response (800 ± 243 sec). At all time points, Hemonox-CT was higher than the ACT. Peak Hemonox-CT was associated with a therapeutic anti-Xa activity ranging from 0.50 to 1.35 U/ml (mean 0.89 U/ml) for the enoxaparin group and 0.69 to 1.48 U/ml (mean 0.91 U/ml) for the dalteparin group. After reaching peak levels, there was a gradual and parallel decline in Hemonox-CT and anti-Xa activity. The enoxaparin and dalteparin-treated patients successfully underwent PCI without major hemorrhagic complications. Conclusions. The Hemonox-CT has better sensitivity to both enoxaparin and dalteparin than does the ACT. Also, Hemonox-CT correlates with anti-Xa activity after IV administration. Hemonox-CT may become an important tool to monitor the anticoagulation effects of low-molecular weight heparin during PCI. Additional studies are needed to determine the target Hemonox-CT.

J INVASIVE CARDIOL 2008;20:449–454

Low-molecular weight heparin (LMWH) has been safely and effectively used for the prophylaxis and treatment of venous thromboembolism,1–4 the management of patients with acute coronary syndromes5–8 and the anticoagulation of patients undergoing percutaneous coronary intervention (PCI) including high-risk patients managed with an early invasive treatment strategy.9–12
While routine LMWH prophylaxis does not require monitoring, in the interventional setting there may be a need to monitor the anticoagulant level of the LMWH to ensure safety and efficacy. Although the activated clotting time (ACT) is widely available and has been proposed as a method for monitoring the anticoagulant effect of LMWH in PCI, this method has not been validated and is not widely accepted in the interventional community at large.12,13,17

The Hemonox™ assay (International Technidyne Corp., Edison, New Jersey) has been developed for the purpose of monitoring the anticoagulant effects of LMWH (e.g., enoxaparin and dalteparin) in PCI. The purpose of this study is to compare this technology with the traditional measurement of the ACT and anti-Xa activity. Our data suggest that the Hemonox assay is highly sensitive to enoxaparin and dalteparin at low concentrations and that this assay may be ideal for monitoring intravenous (IV) LMWH in PCI.

Methods

Patient population. The Institutional Review Board of the State University of New York, Health Science Center at Brooklyn approved the protocol for this study. Eligible patients were men or non-pregnant women older than 18 years who were referred for cardiac catheterization and coronary angiography for a variety of indications such as stable angina pectoris, acute coronary syndrome, positive exercise or nuclear stress test or chest pain post myocardial infarction (MI). A total of 90 patients were enrolled who underwent diagnostic cardiac catheterization, and out of these, 46 patients underwent PCI. Informed consent was obtained from each patient before enrollment in this study. The demographics of the study population are shown in Table 1.

Exclusion criteria for the study included ST-segment elevation MI within 24 hours, active internal bleeding, bleeding diathesis, thrombocytopenia (< 100,000 per mm3), serum creatine > 2.0 mg/dl, and administration of an oral anticoagulant within 7 days.

Study design and medications. Enrolled patients were consecutively assigned to three different groups to receive intravenous (IV) enoxaparin (0.5 mg/kg), dalteparin (50 IU/kg), or unfractionated heparin (UFH) (50 Units/kg). The treatment dose was given in 2 boluses 5 minutes apart. Bolus doses were: (a) 0.1 mg/kg followed by 0.4 mg/kg enoxaparin; (b) 20 IU/kg followed by 30 IU/kg dalteparin; and (c) 10 U/kg followed by 40 U/kg UFH, respectively. Aspirin, clopidogrel and glycoprotein (GP) IIb/IIIa inhibitors were given to all patients who underwent PCI following diagnostic angiography with the choice of which particular GP IIb/IIIa inhibitor (eptifibatide, tirofiban or abciximab) left to the discretion of the operating physician.

Blood samples. Blood samples were drawn at baseline following insertion of the femoral sheath 5 minutes following each bolus dose and then 15, 30, 60, 90 and up to 120 minutes after the first bolus dose administration of the anticoagulant medication. All the blood samples were drawn after a 5 ml discard and were collected in duplicate in sodium citrate vacutainers. Arterial access sheaths were removed when the ACT had fallen below 160 seconds.

Standard blue-top tubes containing 3.2% sodium citrate were used for the collection of blood samples for plasma separation. The collection tubes were centrifuged at 3,000 g for 15 minutes at 22ºC within 45 minutes for plasma separation and immediately stored at -70ºC; frozen samples were transported in dry ice for anti-Xa measurement.

Determination of the Hemonox in clinical samples from PCI patients. The Hemochron Jr. Signature+ Whole Blood Microcoagulation System with software version 2.1 or higher (International Technidyne Corp.) was used to perform the Hemonox clotting time (Hemonox-CT) assay and the ACT tests (cuvette cat # JACT-LR). The Hemonox method uses a lipidated recombinant rabbit brain tissue factor-based reagent and formulation buffer (Pel-Freez Corp., Rogers, Arkansas). The test is used for evaluation of anticoagulation in whole blood samples where the expected enoxaparin or dalteparin concentration is 0–2.5 units per ml of blood. Following whole blood sample introduction, the instrument measures 15 µl of whole blood, automatically moves it into the test channel within the Hemonox cuvette, and the remainder of the blood sample not needed for testing is automatically drawn into the waste channel. After mixing with the reagent, the sample is then moved back and forth within the test channel and monitored for clot formation. The instrument recognizes that a clot endpoint has been achieved when the movement decreases below a predetermined rate. The instrument reports the whole blood clotting time value in seconds.

Determination of plasma anti-Xa activity. Plasma anti-Xa activity levels were determined by using a chromogenic assay at Loyola University Medical Center, Maywood, Illinois. The assay provides a measurement of absolute heparin/LMWH concentration utilizing its inhibitory effects against bovine factor Xa and quantitation by a chromogenic substrate for factor Xa.

In Vitro analyses of the effect of LMWH on Hemonox and ACT coagulation tests. Enoxaparin or dalteparin was added at increasing concentrations to fresh whole blood samples obtained from 4 healthy donors. Testing was performed in duplicate on the blood samples from each individual donor and evaluation was done simultaneously for Hemonox-CT and ACT coagulation tests. Clotting time results were compared to the amount of LMWH added to the whole blood sample. All point-of-care tests were performed using the Hemochron Jr. Signature+ Microcoagulation System.

Assessment of safety. A new MI was defined as an elevation in CK-MB (or total creatine kinase in absence of CK-MB) more than three times the upper limit of normal. Blood for CK-MB analysis was drawn before PCI and every 8 hours twice post PCI. Severe thrombocytopenia was defined by a platelet count below 50,000/µl. Mild thrombocytopenia was defined as a platelet count below 100,000/µl or a count 50% of the baseline value. Minor and major bleeding were defined according to the criteria used by the Thrombolysis in Myocardial Infarction (TIMI) trial.17 Major hemorrhage was defined as a decrease in hemoglobin > 5 g/dL (hematocrit decrease > 15% when hemoglobin is not available) or intracranial bleeding. Minor hemorrhage was defined as spontaneous and observed gross hematuria, hematemesis or observed blood loss associated with a decrease in hemoglobin ≥ 3 g/dL or hematocrit decrease ≥ 10% when hemoglobin is not available.

Statistical analysis. Results are expressed as mean ± standard deviation. Simple regression analysis and analysis of variance (ANOVA) were used to test the association between continuous variables; the a level was set at 0.05.

Results

The Hemonox-CT in patients treated with enoxaparin or dalteparin during cardiac catheterization. Patients were treated with either enoxaparin (n = 42), dalteparin (n = 34) or UFH (n = 14), and the point-of-care Hemonox-CT was determined at baseline and at up to seven time points after the baseline blood sample collection for each patient. Parallel chromogenic plasma anti-Xa activity was determined for patients in the enoxaparin and dalteparin groups. Hemonox-CT was compared to anti-Xa activity at each time point in both treatment groups (Figure 1).

The mean Hemonox-CT was similar in the enoxaparin (68.9 ± 9 seconds) and dalteparin (68 ± 7 seconds) treatment groups at baseline; no detectable anti-Xa activity was observed at baseline. After the first IV bolus of 0.1 mg/kg enoxaparin or 20 IU/kg of dalteparin, the Hemonox-CT increased 0.30 ± 0.11 U/ml in the enoxaparin group and 0.61 ± 0.0.22 U/ml in the dalteparin group (p < 0.05) compared to baseline values for both groups. Additional boluses of 0.4 mg/kg of enoxaparin and 30 IU/kg dalteparin resulted in an increase in Hemonox-CT to 171 ± 60 seconds (range: 101–314 seconds) and 214 ± 70 seconds (range: 125–351 seconds) for the enoxaparin and dalteparin treatment groups, respectively. Peak Hemonox-CT was associated with a therapeutic anti-Xa activity level (for PCI have been reported > 0.5 U/ml) ranging from 0.50–1.35 U/ml (mean 0.89 U/ml) in the enoxaparin group and 0.69 to 1.48 U/ml (mean 0.91 U/ml) in the dalteparin group.16,22,23 Following the second bolus after reaching the peak levels, there was a gradual decline in Hemonox-CT and anti-Xa levels; the latter dropped to a mean value of 0.48 ± 0.15 U/ml at 60 minutes post second bolus for both LMWH-treatment groups (Figure 1).

Patients in the UFH treatment group showed Hemonox-CT baseline value similar to those observed in the LMWH treatment groups (mean 71 ± 8). After the first bolus dose of 10 U/kg of UFH, the mean Hemonox-CT rose to 118 ± 36 seconds. The second bolus dose of 40 U/kg of UFH induced a mean Hemonox response of 800 ± 243 seconds. The Hemonox-CT gradually decreased at 30–60 minutes, yielding a mean value of 110 ± 28 seconds at 120 minutes after the second dose; no anti-Xa activity level was evaluated for patients treated with UFH.

Comparison of the effect of enoxaparin and dalteparin on coagulation tests Hemonox-CT and ACT. We compared the sensitivity of the ACT and the Hemonox-CT in monitoring the anticoagulant effects of enoxaparin and dalteparin both in vitro and in clinical samples derived from patients undergoing cardiac catheterization.

In Vitro data:  Enoxaparin or dalteparin was added at increasing concentrations to fresh whole blood samples obtained from healthy volunteers (n = 4). Samples were evaluated in duplicate from individual donors for Hemonox-CT and ACT coagulation tests. Clotting time results were compared to the amount of LMWH added to the whole blood sample (Figure 2). Both the Hemonox-CT and the ACT demonstrated a linear correlation of LMWH activity to clotting time. The Hemonox-CT showed a greater sensitivity to the LMWH concentration with increases of 2- to 8-fold from baseline at LMWH levels of 1 and 2 IU/ml blood compared to 1.2- to 1.8-fold for the ACT.

Clinical data: Whole blood samples from patients undergoing cardiac catheterization were obtained at baseline and at seven time points after drug administration. Hemonox-CT and ACT tests were simultaneously performed. The increases in clotting time from the baseline for both coagulation tests were calculated at each time point after drug administration (Figures 3A, B and C). All three anticoagulants induced higher clotting times for Hemonox than ACT (p < 0.05) at peak response (5 minutes after the second bolus). Enoxaparin induced a 2-fold increase (range of 1.5- to 4.1-fold increase) in Hemonox-CT and a 1.2-fold increase (with a range of 0.76 to 1.7-fold increase) in ACT from baseline. Dalteparin raised the Hemonox-CT by 2.9-fold (range of 1.0- to 4.3-fold increase) and ACT by 1.3-fold (range: 1.2- to 1.8-fold increase) from baseline. UFH induced a 7.8-fold increase in Hemonox-CT compared to 1.3-fold increase with the ACT test.

Correlation between anti-Xa activity level and Hemonox-CT and ACT coagulation tests. The correlation between anti-Xa activity and Hemonox-CT was highly significant for individual patients (Figure 4 A), but was less significant for the entire study population (Figure 4 B). There was a correlation (r = 0.7) between the plasma anti-Xa level and Hemonox-CT in the enoxaparin treatment group. Similar results were obtained with the dalteparin treatment group (r = 0.7, not shown in Figure); anti-Xa activity level was not evaluated in the UFH treatment group. There was a poor correlation between ACT and anti-Xa level for the enoxaparin treatment group (r = 0.45 as seen in Figure 4C) as well as for the dalteparin treatment group (r = 0.4 not shown in Figure).

Safety outcomes. PCI was successful in all patients undergoing intervention. There were no deaths, abrupt closures or urgent revascularization for the study population during the hospital course. One of the 46 PCI patients (2%) had a periprocedural non-Q-wave MI. None of the patients suffered major bleeding, and no patient received a transfusion. There were no groin complications, nor were there any episodes of thrombocytopenia.

Discussion

This is the first report of a novel LMWH monitoring device, the Hemonox-CT, for the two commonly used LMWHs, enoxaparin and dalteparin. This device has a better sensitivity and an improved signal-to-noise ratio compared to the traditional methods used to monitor anticoagulation in the cardiac catheterization laboratory. The Hemonox-CT is not affected by concurrent GP IIb/IIIa use.24 Furthermore, our data show a correlation between the Hemonox-CT and anti-Xa activity, which is considered the gold standard to monitor LMWH.

Although LMWH is commonly prescribed in noninvasive settings, it is not commonly used in the context of PCI.5–7 In a large randomized clinical trial, enoxaparin did not show benefit over UFH in high-risk patients undergoing an early invasive approach.10 In this trial, however, monitoring of the anticoagulant effect was performed for UFH, but not for enoxaparin. This raised the question that although routine monitoring of the anticoagulant activity of LMWH appears to be unnecessary in the noninvasive setting, it may be important to monitor in the setting of PCI.

Although aPTT and ACT are widely used for monitoring the anticoagulant effect of UFH in noninvasive and invasive settings, respectively, these tests are not broadly accepted as a means to monitor LMWH during PCI. The ACT and aPTT are not significantly prolonged by enoxaparin.14

Anti-Xa activity monitoring of LMWH is prudent in certain clinical circumstances such as in patients with weight > 150 kg and creatine clearance < 25 ml/minute.15 Also, an anti-Xa level of at least 0.5 IU/ml has been recommended in patients with acute coronary syndromes treated with subcutaneous enoxaparin.16 However, a point-of-care anti-Xa assay is not currently available.

Hemonox-CT is sensitive to low doses of intravenously administered LMWH (Figure 1). Peak responses are reached within minutes and returned to baseline in a time course that appears favorable for current interventional practice. The anti-Xa activity paralleled this response.

The ACT is widely used for monitoring anticoagulation effects of UFH during PCI. Recent data have demonstrated the sensitivity of the ACT to the anticoagulant effect of LMWH in PCI patients.12,13,17 The Hemonox-CT is more sensitive and produces a greater signal-to-noise ratio than does the ACT to LMWH activity. Thus, the Hemonox-CT may be an ideal means of monitoring IV LMWH activity during PCI. The importance of LMWH monitoring may grow over time given the demonstrated benefit of enoxaparin during PCI in the STEEPLE trial.19 Although bivalirudin appears to be an excellent molecule for PCI, the use of IV LMWH during PCI may have several advantages including lower cost and partial reversibility with protamine in the event of complications such as coronary perforation.20,21 Furthermore, heparin-induced platelet activation is reduced with LMWH and thus may reduce the need for routine adjunctive GP IIb/IIIa inhibitors during PCI.25

These theoretical and practical advantages of LMWH, along with its demonstrated benefits in the STEEPLE trial, point to the potential for a growing use of IV LMWH in PCI. Monitoring devices, such as the Hemonox-CT may therefore be of increasing importance over time.