Karen A. Moffat

Comparison of 5-mg and 10-mg Loading Doses in Initiation of Warfarin Therapy

Warfarin levels are monitored by measuring the prothrombin time, which responds to reductions in levels of three vitamin K-dependent clotting factors: factors II, VII, and X [1, 2]. During the first 48 hours of treatment, the anticoagulant effect of warfarin is caused mainly by a reduction in the activity of factor VII, which has a half-life of 6 hours. In contrast, the antithrombotic effect of warfarin (which is thought to be caused primarily by a reduction in the activity of factor II) is delayed for as long as 60 hours [3, 4]. Therefore, during the first 48 hours of therapy, the anticoagulant and antithrombotic effects of warfarin may be dissociated. In addition, because the half-life of the vitamin K-dependent anticoagulant protein, protein C, is similar to that of factor VII [5], the early anticoagulant effect of warfarin (which results from reduction of factor VII) could be counteracted by a procoagulant effect (which results from reduction of protein C) [6]. Warfarin treatment is often initiated with a 10-mg loading dose and then reduced to a level that maintains the international normalized ratio (INR) within the therapeutic range. An alternative approach is to start warfarin therapy at a dose of 5 mg, which is the average dose required to maintain an INR of 2.0 to 3.0. Although a 10-mg loading dose produces a more rapid increase in the prothrombin time, this effect is caused largely by a decrease in factor VII levels and therefore may not produce a more rapid antithrombotic effect. The 10-mg loading dose has three potential short-comings. First, if heparin is discontinued as soon as the INR reaches the therapeutic range, thrombus extension may occur. This is because the antithrombotic effect of warfarin may not yet have manifested. Second, elderly or vitamin K-deficient patients may be exposed to an unnecessary risk for bleeding [7-12]. Third, protein C levels can be excessively decreased before the full antithrombotic effect of warfarin has been completely expressed through the reduction of factor II. On the basis of these considerations, we did a study to compare the relative effects of 5- and 10-mg loading doses of warfarin on four surrogate laboratory markers of efficacy and safety. Methods Patients with no contraindications to warfarin who required anticoagulation (target INR, 2.0 to 3.0) were invited to participate in the trial between June and November 1994 at The Hamilton Civic Hospitals (Ontario, Canada). Enrollment was targeted at 50 patients. All patients gave written, informed consent, and the study was approved by the local internal review board. Patients were assigned by random number table to receive warfarin at an initial dose of 5 or 10 mg. Subsequent doses were determined on the basis of nomograms that were developed before the study began through an iterative process. Because the study used laboratory outcomes, patients and health care professionals were not blinded to treatment assignments. Patients were followed for a maximum of 108 hours, during which time they received five doses of warfarin. Blood samples were taken before warfarin therapy was initiated, 12 hours after the first dose of warfarin, and at 24-hour intervals for 5 days. Blood was collected into vacuum-sealed containers that contained 0.102 mol of buffered sodium citrate per liter. To obtain platelet-free plasma, the sample was double centrifuged at 1700 g and frozen at 70C for batch assays [13]. The prothrombin time was measured using Thromborel S (International Sensitivity Index 1.06, Behring Diagnostics, Kanata, Ontario, Canada) and reported as an INR [14]. Heparin does not increase the prothrombin time in patients receiving both warfarin and heparin if this thromboplastin reagent is used. Factors II, VII, IX, and X were assayed by using the one-stage clotting method [15]. Protein C levels were measured by using a functional clotting assay (Dade International, Mississauga, Ontario, Canada) [16]. Treatment of supratherapeutic INRs with vitamin K was left to the discretion of the attending physician. The outcome measures were the time required to achieve an INR of 2.0 to 3.0, the proportion of INR values greater than 3.0, the time course for reductions in levels of factors II and X, and the time course for reduction in protein C levels. Additional data included levels of factors VII and IX. Results Fifty-one consecutive patients were enrolled in the trial. Two were excluded after one dose of warfarin: One died, and one required cardiac catheterization. Twenty-five patients were randomly assigned to receive a 10-mg loading dose of warfarin; 24 were assigned to receive a 5-mg loading dose. The two groups did not differ in age; weight; or frequency of acute thromboembolism, cancer, or surgery during or immediately before the study period. Forty-eight patients received concomitant heparin therapy for all or part of the study period. Beginning on study day 2, the dose of warfarin was adjusted using a nomogram. Warfarin doses in the two groups were similar except on days 1 and 2. The 10-mg group achieved an INR greater than 2.0 statistically significantly sooner than did the 5-mg group (Table 1). At 36 hours, 11 of 25 patients (44% [CI, 34% to 54%]) in the 10-mg group had INRs of 2.0 or greater compared with 2 of 24 patients (8% [CI, 3% to 14%]) in the 5-mg group (P = 0.005). At 60 hours, 9 of 25 patients in the 10-mg group (36% [CI, 17% to 54%]) had INRs greater than 3.0 compared with none of 23 patients in the 5-mg group (P = 0.002). Five patients (4 in the 10-mg group and 1 in the 5-mg group) received vitamin K. These patients had INRs of 4.8 to 9.3 and received 0.5 to 2.0 mg of vitamin K subcutaneously. No patient bled. Table 1. International Normalized Ratios for Patients Assigned to Receive a 5- or 10-mg Loading Dose of Warfarin* Levels of factors II and X declined slowly, and no substantial differences were seen between the 5-and 10-mg groups. In contrast, levels of factor VII and protein C decreased more rapidly and were statistically significantly lower in the 10-mg group than in the 5-mg group at 36 and 60 hours (Figure 1). Figure 1. Plasma levels of coagulation factors in patients receiving a 10-mg (striped bars) or 5-mg (white bars) loading dose of warfarin for each of six time points assessed. top middle bottom Discussion We compared the relative effects of 5- and 10-mg loading doses of warfarin. The time course of reduction in levels of factor II was used as a surrogate end point for clinical efficacy, and excessive elevation of the prothrombin time and unopposed reduction in protein C levels were used as surrogate end points for safety. Patients who received a 10-mg loading dose of warfarin achieved INRs greater than 2.0 more rapidly than did patients who received a 5-mg loading dose. However, because this change in the INR was entirely caused by the early reduction of factor VII levels in the 10-mg group, it may not reflect an antithrombotic effect of warfarin, which is thought to result from a reduction in factor II levels. Two separate experimental observations in rabbits support this idea: The first is the early report of a 5-day delay in achieving an antithrombotic effect with warfarin, although the prothrombin time was first prolonged into the therapeutic range and the level of factor VII was markedly reduced after 2 days of treatment [17]. This observation provides the rationale for overlapping heparin and warfarin therapy for 5 days during the initiation of anticoagulant therapy [17, 18]. The second is the finding that the antithrombotic effect of warfarin is abrogated by the infusion of factor II and (to a lesser extent) factor X [18]. In our study, the rate of reduction in levels of factor II and factor X activity were similar in the 5- and 10-mg groups. On the other hand, the 10-mg loading dose was associated with a significantly more rapid decrease in protein C activity (which decreased before levels of factors X and II were substantially reduced) than that seen in the 5-mg group and an excessive prolongation of the INR. The combination of markedly reduced protein C levels and near-normal levels of factors II and X over the first 2 days of warfarin therapy could produce a hypercoagulable state, and excessive prolongation of the INR could create a higher risk for bleeding. Our study is limited because we used surrogate markers for efficacy and safety. A much larger trial using clinical outcome measures is needed to determine whether the surrogate markers that we used are clinically relevant. Nevertheless, our findings suggest that both regimens result in a therapeutic INR in most patients by day 5 of treatment. From Hamilton Civic Hospital, McMaster University, and The Hamilton Civic Hospitals Research Centre, Hamilton, Ontario, Canada.

Results of a worldwide survey on the assessment of platelet function by light transmission aggregometry: a report from the platelet physiology subcommittee of the SSC of the ISTH

Background: Light transmission aggregometry (LTA) is the most common method used in clinical and research laboratories to assess platelet function. However, the method has never been standardized. Objectives: As the first step towards development of methodological guidelines, the Platelet Physiology Subcommittee of the Scientific and Standardization Committee of the International Society on Thrombosis and Haemostasis (ISTH) undertook a large, detailed, global survey of LTA practices. Methods: Members of ISTH and of External Quality Assurance in Thrombosis and Haemostasis organizations were invited to complete a 129 item, online questionnaire. Results were analyzed anonymously to participant identities. Results: The online supplement for this article (http://www.isth.org/Publications/OfficialCommunications/PlateletPhysiology/LightTransmissionAggregometry/tabid/201/Default.aspx) contains the full details of the study findings. 359 (244 clinical, 115 research) laboratories from 48 countries participated in the survey. LTA was widely used to assess inherited or acquired bleeding disorders. Common practices were identified in sample collection, processing and analysis and although some are generally considered acceptable, others are not ideal. The agonist concentrations used for LTA varied, and many laboratories used ADP, collagen, epinephrine and Ristocetin, at more than one concentration, in addition to arachidonic acid. The parameters commonly used to assess LTA responses were maximal amplitude or % aggregation, which was considered particularly important, in addition to the presence of a ‘secondary wave’, deaggregation, shape change and a measure of the lag phase. However, many laboratories did not have appropriate reference intervals. Conclusions: This is the largest and most detailed survey of LTA practices ever undertaken. It shows a very high variability in LTA practices worldwide, and, as a consequence, methodological standardization is necessary. The information gathered in this survey will be helpful in the development of ISTH methodological guidelines for LTA.

Diagnostic utility of light transmission platelet aggregometry: results from a prospective study of individuals referred for bleeding disorder assessments.

Summary.  Background: Light transmission aggregometry (LTA) is commonly performed to assess individuals for bleeding disorders. Objectives: The goal was to evaluate the incidence and spectrum of platelet function abnormalities in a prospective cohort of individuals referred for bleeding disorder assessments after exclusion of thrombocytopenia and von Willebrand disease. Patients/methods: Subjects were healthy controls and patients from a prospective cohort of individuals referred for bleeding disorder assessments after exclusion of thrombocytopenia and von Willebrand disease. LTA was performed by standardized methods using platelet‐rich plasma adjusted to 250 × 109 platelets L−1. Maximal aggregation data were analyzed to determine the likelihood of detecting a platelet function disorder by LTA, and the sensitivity and specificity of LTA for platelet disorders. Results: The incidence of false positive LTA among subjects excluded of having bleeding disorders was similar to healthy controls. Abnormal LTA was more common in subjects with bleeding disorders and the likelihood of a bleeding disorder was significantly increased (odds ratio 32) when maximal aggregation was reduced with two or more agonists. Receiver operator curve analyses indicated that LTA had high specificity and moderate sensitivity for detecting inherited defects in platelet function and that the LTA agonists 1.25 μg mL−1 collagen, 6 μM epinephrine, 1.6 mM arachidonic acid and 1.0 μM thromboxane analogue U44619 detected most inherited disorders with abnormal LTA. Conclusions: LTA is valuable for detecting platelet function abnormalities among individuals referred for bleeding problems, particularly when the test indicates abnormal responses to multiple agonists.

Development of North American Consensus Guidelines for Medical Laboratories That Perform and Interpret Platelet Function Testing Using Light Transmission Aggregometry

Platelet function testing is important for the diagnostic evaluation of common and rare bleeding disorders. Our study goals were to promote best practices and reduce unnecessary testing variances by developing North American guidelines on platelet function testing. Guidelines were developed by consensus for expert recommendations (minimum level for approval, 70%) that included recommendations on the evaluation and interpretation of light transmission platelet aggregometry (LTA). To assess consensus, medical opinions on recommendations were gathered from diagnostic laboratories that perform LTA, in collaboration with the Quality Management Program-Laboratory Services (QMP-LS) in Ontario, Canada (10 laboratories), and the North American Specialized Coagulation Laboratory Association (NASCOLA; 47 laboratories, 5 overlapping the QMP-LS group). Adequate consensus was achieved for all and 89% of recommendations for the QMP-LS and NASCOLA groups, respectively. The recommendations adopted provide North American laboratories with additional guidance on platelet function testing, including how to interpret LTA abnormalities.

Variability in clinical laboratory practice in testing for disorders of platelet function Results of two surveys of the North American Specialized Coagulation Laboratory Association

Disorders of platelet function are important causes of abnormal bleeding that require laboratory tests for diagnosis. Currently there are limited guidelines on how to perform clinical testing for these disorders. The goal of our study was to obtain information on how disorders of platelet function are currently evaluated in clinical laboratories. Two patterns-of-practice surveys were distributed to laboratories of the North American Specialized Coagulation Laboratory Association (NASCOLA). The information collected was analyzed to determine practices and common problems. Forty-seven NASCOLA laboratories participated and 54% completed both surveys. The majority of the laboratories that responded performed more than 50 aggregation tests per year, mainly using platelet rich plasma based methodologies. A minority performed testing for platelet secretion and dense granule abnormalities. While platelet aggregation results were reviewed in various ways, laboratories most commonly issued a combined report containing quantitative values (% aggregation and/or slope) and a qualitative interpretation. Although laboratories used similar agonists for aggregation testing, the final agonist concentrations varied widely. Several approaches were also used to obtain reference intervals. Comments offered by the participants indicated that performing, and interpreting platelet function tests were challenging for many clinical laboratories. Although common practices have evolved, there is considerable variability in the diagnostic test procedures used by clinical laboratories to evaluate disorders of platelet function. These patterns-of-practice surveys illustrate a need for guidelines and recommendations for clinical laboratories performing tests of platelet function.

Laboratory testing for heparin-induced thrombocytopenia is inconsistent in North America: A survey of North American specialized coagulation laboratories

Heparin-induced thrombocytopenia (HIT) is a serious complication of heparin therapy. As HIT is considered a clinico-pathologic entity, laboratory practices have an important role in diagnosing or excluding HIT. It was the objective of this study to assess the current status of laboratory testing for HIT in North America. An online survey consisting of 67 questions related to laboratory testing for HIT was developed by the North American Specialized Coagulation Laboratory Association (NASCOLA), and distributed to its 59 members. The survey included queries about HIT test ordering practices, HIT immunoassay and activation assays performed, and reporting practices. Data was collected from the 44 NASCOLA laboratories who responded. Of these sites, 88% performed immunoassays for HIT, commonly using commercial assays. However, sites varied in practices related to use of controls, immunoglobulin class of antibody detected, and in result interpretation and reporting. Platelet activation assays for HIT were performed by 36% of sites, commonly using assays of serotonin release (50%) or heparin-induced platelet aggregation (43%). Sites varied in the use of washed platelets versus platelet-rich plasma, controls, and heparin concentrations. This survey is the first comprehensive assessment of patterns of practice in HIT testing among diagnostic coagulation laboratories in North America. We observed site-specific variability of testing methods encompassing all stages of testing, including pre-analytical handling, testing methodologies, and result interpretation and reporting. The variability in HIT platelet activation assay methods among institutions indicates a need for proficiency testing to assess assay performance, and for consensus guidelines on HIT laboratory testing.

An evaluation of methods for determining reference intervals for light transmission platelet aggregation tests on samples with normal or reduced platelet counts

Light transmission platelet aggregation tests are important for diagnosing platelet function defects. However, uncertainties exist about the best procedures to determine aggregation reference intervals. We investigated methods for determining reference intervals for light transmission aggregation tests, using the % maximal aggregation values for prospectively collected data on healthy control samples. Reference intervals for samples tested at 250 x 10(9) platelets/l were determined by mean +/- 2 standard deviations and non-parametric analyses. To establish reference intervals for tests on thrombocytopenic subjects, regression analyses were used to estimate 95% confidence limits for % maximal aggregation, according to sample platelet counts, using data for control samples diluted to match the platelet count of undiluted thrombocytopenic patient platelet-rich plasma samples. For samples tested at 250 x 10(9) platelets/l, non-parametric analyses described 95% of data for healthy control samples better than mean +/- 2 standard deviations. For samples tested at lower counts, to match thrombocytopenic samples, the % maximal aggregation was influenced by platelet count and derived limits were wider at very low platelet counts for almost all agonists. With ristocetin, it proved feasible to test samples with very low platelet counts to exclude Bernard-Soulier syndrome and type 2B von Willebrand disease. Non-parametric analyses should be the preferred method to establish light transmission aggregation reference intervals for samples tested at normal platelet counts. The derived limits for thrombocytopenic samples provide guidance for evaluating thrombocytopenic platelet function disorders, including which agonists to test, based on the sample platelet count.

Diagnostic Usefulness of a Lumi-Aggregometer Adenosine Triphosphate Release Assay for the Assessment of Platelet Function Disorders

Platelet dense granule release assays are recommended for diagnosing platelet function disorders and are commonly performed by Lumi-Aggregometer (Chrono-Log, Havertown, PA) assays of adenosine triphosphate (ATP) release. We conducted a prospective cohort study of people tested for ATP release defects to assess bleeding symptoms. Reduced release, with 1 or more agonists, was more common among patients with bleeding disorders than among healthy control subjects (P < .001). The respective likelihood (odds ratio [95% confidence interval]) of a bleeding disorder or an inherited platelet function disorder were high when release was reduced with 1 or more agonists (17 [6-46]; 128 [30-545]), even if aggregation was normal (12 [4-34]; 105 [20-565]). ATP release had high specificity and moderate sensitivity for inherited platelet function disorders, with most abnormalities detected by the combination of 6 μmol/L epinephrine, 5.0 μg/mL collagen, and 1 μmol/L U46619. Platelet ATP release assays are useful for evaluating common bleeding disorders, regardless of aggregation findings.

Results of an external proficiency testing exercise on platelet dense-granule deficiency testing by whole mount electron microscopy.

Performance on specialized diagnostic tests for platelet disorders, including dense-granule deficiency, is rarely evaluated by external quality assessment (EQA). Members of the North American Specialized Coagulation Laboratory Association that evaluate platelet dense-granule deficiency commonly use whole-mount electron microscopy (EM) methods. This observation led us to develop a pilot EQA survey with standardized EM images and clinical samples on grids from a healthy control subject and a subject with dense-granule deficiency. The survey participants were 8 centers, including 2 with no experience in platelet whole mount EM. All participants, including inexperienced sites, correctly interpreted findings for the normal and dense-granule-deficient platelets. Among experienced sites, agreement was excellent (>82%) on platelet structures to count or not count as dense granules. Participants indicated that future EQA challenges should include clinical samples on grids and standardized images. This is the first report that platelet EM can be assessed by EQA.

Hemodialysis for the treatment of dabigatran-associated bleeding: a case report and systematic review

Dabigatran, a direct thrombin inhibitor, is effective for the treatment of venous thromboembolism and the prevention of stroke and systemic embolism resulting from atrial fibrillation. The most effective way of reversing the anticoagulant effect of dabigatran in patients who have bleeding complications is unknown.