CE Update. Heparins: Clinical Use and Laboratory Monitoring

Transcription

1 Heparins: Clinical Use and Laboratory Monitoring Maureane Hoffman, MD, PhD (Chief, Transfusion Service and Hematology Laboratories, Pathology & Laboratory Medicine Service, Durham VA Medical Center, Durham, NC) DOI: /LMSXWC3A4LBIJP2B Submitted Revision Received Accepted Abstract Heparin has long been used as an antithrombotic to treat and prevent thromboembolic events, as well as for systemic anti-coagulation during cardiopulmonary bypass and dialysis. Heparin continues to have distinct advantages when intense anti-coagulation is needed, but newer low molecular weight derivatives of heparin (LMWH) have advantages in the management of thromboembolism. One advantage of LMWH is that they often do not require laboratory monitoring; when monitoring is indicated it can be done with an anti-factor Xa activity assay. Monitoring is recommended for heparin because the patient response to a given dose is highly variable. The aptt and anti-xa assays are used for monitoring. However, the therapeutic ranges have not been rigorously validated, and current monitoring is not highly predictive of patient outcome. Keywords: thrombosis, blood coagulation, heparin, anti-thrombin, coagulation, clinical pathology, hematology After reading this article, readers should be able to discuss what heparin and low molecular weight heparins (LMWH) are, how they act as anticoagulants, the clinical settings in which they are used, the rationale for laboratory monitoring, and limitations of current monitoring strategies. Coagulation exam 51001questions and corresponding answer form are located after this CE Update on page 627. Blood coagulation is the process by which liquid blood is converted to a more or less solid form, principally by the formation and stabilization of a fibrin polymer. Blood coagulation can occur at the site of a wound, in a blood vessel, after the death of an animal/human, in a test tube, or in an automated analyzer. Hemostasis is the normal, protective process that stops bleeding. It involves contributions from the vasculature, formed elements of blood, and the blood coagulation proteins. Hemostasis can occur only in a living, bleeding organism. Thrombosis is the formation of a blood clot within a blood vessel of a living creature. Thrombosis is a pathological process that interferes with the blood supply to tissues and causes problems such as heart attacks and strokes. The mechanisms involved in thrombosis can vary by site and are not necessarily identical to the mechanism of hemostasis. An anti-coagulant stops blood from clotting. An Corresponding Author Maureane Hoffman maureane.hoffman@med.va.gov Abbreviations LMWH, low molecular weight heparin; UFH, unfractionated heparin; AT, anti-thrombin; SERPIN, serine protease inhibitor; RCL, reactive center loop; HIT, heparin-induced thrombocytopenia; aptt, activated partial thromboplastin time; ACT, Activated Clotting Time; PTT, partial thromboplastin time; CAP, College of American Pathologists anti-thrombotic agent stops blood from clotting within the vessels of a living organism. Since thrombosis of different types or sites can have different mechanisms, different anti-thrombotics are indicated for different types of thrombosis. Until very recently the 3 mainstays of anti-thrombotic therapy were heparin, warfarin, and aspirin: all old drugs from biological sources. 1 All are still very widely used, although they are beginning to be supplanted by newer, engineered replacements. The word heparin is from the Greek root meaning liver and refers to the tissue from which it was first prepared. The term is usually used to refer to preparations that are more accurately termed unfractionated heparin (UFH). A heparin is a type of carbohydrate termed glycosaminoglycan. It is a heterogeneous mixture of polymers with a variable number of sulfated saccharides (sugars). The different molecules comprising UFH differ in length, in the pattern of sugars, and in the extent and type of modifications of the sugars. The molecular weight of the constituent molecules in UFH ranges from approximately 3,000 to 30,000. Unfractionated heparin is prepared from biological sources, usually porcine intestine or bovine lung. Heparin prepared from dog liver was described by Howell and McLean in 1918, and heparin came into medical use as an anti-thrombotic agent in the 1930s. 2 How Heparin Works Biochemically The coagulation cascade consists of a series of steps in which a protease cleaves and thereby activates the next protease in the sequence. This process acts as a biological amplifier because each protease can activate many molecules of the next labmedicine.com October 2010 Volume 41 Number 10 LABMEDICINE 621

2 Figure 1_Interactions of antithrombin (AT, green ribbon backbone), protease (aqua ribbon backbone), and heparins (blue space-filling spheres). The left panel shows the structures of the protease and AT. The middle panel shows the conformational change in the RCL of antithrombin induced by the binding of the pentasaccharide (based on coordinates from the crystal structure of the AT-pentasaccharide complex). The right panel shows the structure of the thrombin- AT-heparin complex. Images were constructed using the computer modeling program PyMOL ( from coordinates deposited in the Protein Data Bank. protease in the series. Anti-thrombin (AT) is a serine protease inhibitor (SERPIN) that is the major plasma inhibitor of coagulation proteases. It can inhibit all of the procoagulant proteases to some extent. The simplest explanation of how heparin works is that heparin makes AT a better inhibitor of these proteases. Most protease inhibitors act by a mechanism in which the protease and inhibitor bind together very tightly like a lock and key. Serine protease inhibitors, including AT, act by a different mechanism, in which the SERPIN is a suicide substrate. 3 The target protease binds to a portion of the SERPIN called the reactive center loop (RCL) and cleaves a bond in the RCL. As soon as the bond is cleaved, it triggers a dramatic conformational change in the SERPIN. 4 The RCL, with the protease still attached, swings from one side to the other of the SERPIN molecule like the closing of an old-fashioned mouse trap. The protease is trapped in a complex with the SERPIN and thereby is irreversibly inactivated. An animation of this remarkable process is available as online supplemental material to the review article by Huntington. 3 Heparin molecules are negatively charged and bind to positively charged patches on AT as well as on some of the coagulation proteases. Heparin binding enhances AT inhibition of its target proteases in 2 ways. First, binding of heparin to AT causes a change in the conformation of AT making the RCL more accessible to proteases in what is known as an allosteric mechanism. The term allosteric refers to a change in the shape and activity of a protein (in this case, AT) resulting from the binding of another substance (in this case heparin) at a point other than the chemically active site. In other words, heparin binds to AT at a site other than the RCL and causes a change in the shape of the AT molecule improving its effectiveness as an inhibitor of proteases. The allosteric effect of heparin only requires a heparin molecule of 5 saccharides in length. The heparin-induced conformational change is illustrated in Figure 1. The amino acid where the protease will cleave is shown as red spheres and labeled P1. Think of it as the bait in the mousetrap. It is located in the middle of the RCL. Access to it is usually impeded because it is very close to the body of the AT molecule (left panel, Figure 1). However, once a heparin molecule binds to the specific pentasaccharide binding site, the RCL shifts and now points out away from the rest of the AT molecule (center panel, Figure 1). Since the protease can now more easily attack the RCL, it can be more readily trapped (inhibited). Second, a single, longer heparin molecule can bind to AT and to the target protease, tethering them together and facilitating their interaction (right panel, Figure 1). This is called a template mechanism. The exact length of the heparin molecule required to bind both proteins is different for different proteases, but it is approximately saccharides for thrombin, for coagulation factor XIa, 6 and 23 for coagulation factor IXa. 7 When the mousetrap snaps shut, the heparin molecule is released and can then go on to promote inhibition of more protease molecules. Some proteases, such as factor Xa, do not bind heparins very well. Thus, factor Xa inhibition is accelerated primarily by the allosteric mechanism. The many different molecules that make up a preparation of UFH each have different effects on AT inhibition of coagulation factors. In fact, only about one-third of the molecules in UFH can even bind to AT. 8 Thus, there is a lot of inactive junk in UFH under the best of conditions. The active molecules do not each promote inhibition of the same proteases. However, in the aggregate the mixture of molecules in UFH promotes inhibition of all of the coagulant proteases. The heterogeneity of UFH gives it the ability to promote inhibition of more proteases than any single heparin molecule could. However, this heterogeneity also gives UFH some undesirable characteristics, especially that its pharmacokinetics are extremely complicated and the response of any given individual is not completely predictable. Unfractionated heparin is often given intravenously, so the mixture of molecules in the blood starts out being exactly like the mixture present in the bottle. However, the different-sized molecules are cleared from the circulation at different rates. Since the different-sized molecules have different anti-coagulant properties, the ability of the mixture of heparin 622 LABMEDICINE Volume 41 Number 10 October 2010 labmedicine.com

3 molecules in the blood to promote inhibition of the different coagulation factors changes over time. For example, the relative ability to promote inhibition of factor Xa and of thrombin is 1:1 initially. However, the larger molecules promoting thrombin inhibition clear more rapidly than the small molecules promoting factor Xa inhibition. Thus, the ratio of anti-factor Xa to antithrombin activity increases over time. This is one of the features complicating laboratory monitoring of UFH. Another undesirable feature of UFH is that it can elicit an immune response leading to heparin-induced thrombocytopenia (HIT). This condition can lead to serious thrombosis in some patients. Thus, it is necessary to check the platelet count before and during therapy with UFH. If the platelet count drops significantly, the possibility of HIT should be considered, and heparin may have to be replaced with a different anti-thrombotic. These difficulties have led to a search for anti-thrombotics with more desirable characteristics. Can Something Better Than UFH Be Created? Many newer, engineered versions of heparin have been developed and are now on the market. In general, these are classified as LMWHs. With 1 exception, they are produced from UFH by chemical or enzymatic cleavage followed by purification. This creates preparations that are much more homogeneous than the UFH from which they were made. They have less inactive junk in them and are less likely to cause HIT. Even though they are lumped together as a class of drugs, the LMWHs differ from one another. They vary in polysaccharide length and chemical modification and, thus, vary in their abilities to enhance AT inhibition of the different coagulation proteases. Since they are much smaller than UFH, they depend more on the allosteric mechanism rather than the template mechanism. Since FXa is primarily inhibited by the allosteric mechanism and thrombin by the template mechanism, we consider LMWHs as predominantly enhancing factor Xa inhibition (anti-fxa activity), with different abilities to enhance thrombin inhibition (AT activity). For example, enoxaparin (Lovenox) is an LMWH that has the significant ability to enhance thrombin inhibition. At the other end of the spectrum is fondaparinux (Arixtra), a synthetic pentasaccharide that only enhances AT activity by the allosteric mechanism and thus has almost pure anti-fxa activity. Keep in mind that UFH and all LMWHs, including fondaparinux, do not inhibit any of the proteases. They all act by enhancing AT inhibition. Thus, a patient who is AT deficient may be resistant to the effects of any of the heparin-like anti-thrombotics. schematically in Figure 2, the rate of AT inhibition of each of the proteases is enhanced by UFH. By contrast, the LMWHs (the example of Lovenox or enoxaparin shown in Figure 2) predominantly enhance inhibition of FXa and thrombin. Because it promotes inhibition at multiple steps, heparin can profoundly inhibit the aptt. The synergistic effect of inhibition at multiple steps in the intrinsic pathway is reflected in the upward curve of the line when the aptt is plotted against the concentration of heparin present (Figure 3). This is in marked contrast to enoxaparin, which barely prolongs the aptt. The curves in Figure 3 were obtained by adding heparins directly into normal plasma. While you might think the aptt would directly reflect the effects UFH has in the body, this unfortunately is not exactly true. Unfractionated heparin has some molecules promoting inhibition of factors very near the start of the intrinsic pathway, especially factor XIa. Inhibition of FXIa has a major effect on the aptt, but it is not thought to play a major role in the anti-thrombotic effects of UFH. As can be seen from the example of enoxaparin, inhibition of factor Xa and thrombin have relatively small effects on the aptt, even though they are thought to have significant anti-thrombotic effects. Thus, while UFH clearly has anti-coagulant effects in plasma, those effects do not completely parallel the anti-thrombotic effects of administering UFH to patients. How UFH and LMWH Work as Anti-Coagulants in Plasma Anti-thrombin can inhibit each of the proteases in the intrinsic pathway of coagulation, the pathway assayed by the activated partial thromboplastin time (aptt). As indicated Figure 2_Enhanced inhibition of proteases in the intrinsic pathway by heparin compared to an LMWH, enoxaparin (Lovenox). Heparin increases AT inhibition of all of the proteases in this pathway, though to different degrees. Lovenox and most of the LMWHs primarily enhance inhibition of thrombin and FXa. Fondaparinux (pentasaccharide) primarily enhances inhibition of FXa only. labmedicine.com October 2010 Volume 41 Number 10 LABMEDICINE 623

4 Figure 3_Comparison of the effects of unfractionated heparin and enoxaparin (Lovenox) on the aptt. The drugs were added directly to normal pooled plasma at the indicated concentrations and then assayed for the aptt. How UFH and LMWH Work as Anti-Thrombotics in Patients Unfractionated heparin is primarily used in 3 clinical settings. 1) Very high intensity but short-lived (hours) of anticoagulation required for situations with an intense stimulus for clotting; principally cardiopulmonary bypass, percutaneous coronary interventions, like angioplasty and stent placement, and hemodialysis. In this setting UFH is given intravenously. 2) Low intensity over a period of days for prevention of thrombosis. In this setting UFH is often given subcutaneously. 3) Moderate intensity anticoagulation over a period of days to treat thromboembolism. This usually means deep vein thrombosis and pulmonary embolism; however, UFH is sometimes also used to prevent further thrombosis in the coronary arteries of someone who has suffered a heart attack or an impending heart attack. In these settings UFH is given intravenously. Unfractionated heparin can profoundly inhibit coagulation in vivo and block both hemostasis and thrombosis if given in a high enough dose. This is a desirable characteristic when UFH is used to prevent thrombosis in settings where the stimulus for clotting is very strong, as in setting 1. In the case of cardiopulmonary bypass during open heart surgery, the blood is run through a pump and oxygenator before being returned to the body. If suppression of coagulation is not profound, the blood will clot in the pump circuit, which is, of course, very dire for the patient. A big advantage of UFH in this setting is that its effects are reversible by the administration of protamine. Protamine is a highly positively charged molecule that binds to the negatively charged sites of heparin even more tightly than AT does. Thus, the intense anticoagulation needed during open heart surgery can be reversed at the end of the case by administration of protamine. Since the LMWHs promote inhibition of the coagulation process in only 1 or 2 steps, the anti-coagulant response to increasing doses is more linear than with UFH. This is important because it means the anti-thrombotic effect of the LMWHs can be more precisely titrated than that of UFH. In the case of UFH, a very small error in dosing or small difference in responsiveness of the patient can lead to a big difference in the intensity of anti-coagulation. Their more precise control makes LMWHs safer drugs to use when a sub-maximal level of anticoagulant effect is desired. A good example is the prophylaxis or treatment of deep vein thrombosis, as in settings 2 and 3. In these settings, it is desirable to tune down, but not completely block, the coagulation process. This produces a sufficient effect to prevent thrombosis but (hopefully) still allows enough hemostatic function to prevent most bleeding side effects. A more predictable dose/response relationship is not the only advantage of LMWHs. They also have a more predictable pharmacokinetic profile, allowing once or twice daily weight-adjusted dosing without laboratory monitoring. Several randomized trials and meta-analyses have shown equivalence of subcutaneous LMWH and intravenous UFH in terms of bleeding risk and prevention of recurrent venous thromboembolism. While UFH is clearly effective in reducing the development or extension of thromboembolic disease, LMWHs are becoming preferred for these indications because they tend to be associated with fewer bleeding complications and do not require monitoring in most patients. Even though the drug itself is much more expensive than UFH, the use of LMWHs allows outpatient treatment, thus saving the considerable expense of keeping a patient in the hospital for anticoagulant therapy. Monitoring UFH and LMWH Levels and Anti-Thrombotic Effects Because LMWHs have predominantly anti-fxa activity, it is appropriate to monitor their levels when monitoring is needed by an anti-fxa assay. In other words, the test used for monitoring reflects the activity responsible for their therapeutic effects in the body. Low molecular weight heparins do require monitoring in patients with renal failure (because these drugs are cleared by the kidneys) as well as in obese patients, young children, and pregnant patients where the pharmacokinetics and volume of distribution are different from otherwise healthy adults. Monitoring is also indicated when the patient does not get the expected response, for example, when a patient on a LMWH continues to experience thrombosis. The situation is different with UFH. Laboratory monitoring is more essential for good patient outcomes, yet it is more difficult to accomplish than with LMWH. In the setting of intense anti-coagulation, such as cardiopulmonary bypass, monitoring is required to 1) make certain a sufficient level of anti-coagulation is achieved before beginning the invasive procedure, and 2) ensure anti-coagulation is reversed at the end of the procedure. The intense level of anticoagulation required in this setting renders the aptt nearly unclottable and useless for monitoring. An Activated Clotting Time (ACT) point-of-care test is usually used for this purpose in the operating room or angiography suite. It can be thought of as a whole-blood partial thromboplastin time (PTT) that has been adapted to reflect an intense degree of anti-coagulation. Since the blood is already anti-coagulated in the patient, the sample is not drawn into an additional anticoagulant. The ACT is initiated with similar reagents to those used for the aptt (such as kaolin), and clotting is monitored for a longer period of time. Target values are usually defined locally on an empirical basis, and the clinical laboratory rarely performs ACT assays. The treatment of venous thromboembolism is the setting in which most monitoring of heparin therapy occurs. The aptt and anti-fxa assays are generally used for monitoring. 624 LABMEDICINE Volume 41 Number 10 October 2010 labmedicine.com

5 However, neither reflects the anti-thrombotic mechanism of UFH in the patient or directly reflects the level of active drugs in the blood. Monitoring is recommended by professional and accrediting organizations to ascertain that the UFH level is in a therapeutic range and presumably minimizes the risk of recurrence or extension of thrombosis. However, the idea that the aptt value or ratio predicts the response to heparin therapy is not supported by a wealth of data. In 1972, Basu and colleagues reported a retrospective analysis of a prospective study suggesting an aptt of times the control value reduced the risk of recurrent thromboembolism. 9 Despite the limited evidence, titrating heparin dosing to an aptt of times control became an accepted practice. However, it soon became clear that due to variability in the sensitivity of aptt reagents to heparin, the use of a fixed aptt ratio for all reagents was inappropriate. That range was determined to correspond to a heparin level of IU/mL as measured by a protamine titration assay of heparin. 10 It was subsequently determined that IU/mL by protamine titration corresponded to U/mL by an anti-xa activity assay. This limited information formed the basis for assigning the heparin therapeutic range to be anti-fxa units of heparin/ml. Some, 16 but not all, 17 retrospective studies have found that a subtherapeutic aptt is associated with subsequent thrombosis. However, a randomized trial showed that exceeding the therapeutic range during treatment with UFH for venous thrombosis was not associated with a greater risk of bleeding. 15 There is evidence that weight-based dosing of heparin is safer and more effective than using a fixed-dose regimen, 18 with or without laboratory monitoring. Thus, there are no direct clinical data demonstrating that the recommended target range is optimal for heparin anti-coagulation or even that the aptt is very good at predicting the safety and efficacy of heparin therapy. In an effort to improve interlaboratory agreement in the monitoring of UFH, the College of American Pathologists (CAP) 11 and the American College of Chest Physicians 12 recommend the therapeutic range of the aptt be defined in each laboratory by correlation with a direct measure of heparin activity such as the factor Xa inhibition or the protamine titration assay. The relevant section of the 2009 Hematology and Coagulation Checklist reads as follows: HEM.23476: Is there documentation that the aptt-based heparin therapeutic range is established and validated using an appropriate technique? 1) The aptt and heparin activity is measured for each sample and the aptt range is calculated by comparison to heparin activity or 2) the aptt of patient samples using the new lot or aptt method is compared to the prior aptt lot. It is recommended that the first method be used initially to establish the therapeutic range before starting patient testing with a new instrument or new reagent, while the second method can be used for validation of the therapeutic range with subsequent reagent lot changes. It is not best practice to use plasma samples spiked with heparin in vitro to calculate the therapeutic range, as differences in heparin binding proteins in vitro may lead to overestimation of the therapeutic range. It would be convenient if we could simply add UFH to plasma (as we did to obtain the data shown in Figure 3) and use the samples to determine the therapeutic range. However, using plasma spiked with UFH gives different results from Figure 4_Determination of the anti-fxa-correlated aptt therapeutic range. The aptt values corresponding to anti-factor Xa activity of U/mL are the therapeutic range of management of venous thrombosis using UFH. This is data from our clinical laboratory. those using plasma samples from patients who have been given the drug (compare the curves in Figure 3 and Figure 4). Not only is the aptt at any given anti-fxa level higher when UFH is just added into plasma, but the scatter of the results is much less. Therefore, plasma samples from patients who have received intravenous UFH are used for a therapeutic range study. The samples are assayed for both the aptt and a measure of heparin activity. Since an anti-fxa assay is available for many coagulation analyzers, it is the most commonly used activity assay. The aptt versus anti-xa results are plotted for each sample as shown in Figure 4. The linear correlation between the 2 is calculated, and aptt values corresponding to the therapeutic range by the activity assay are determined. A recent study suggested, however, that determination of the therapeutic range by correlation of the aptt with an anti- FXa assay did not improve the consistency of results or dosing decisions among 4 CAP-accredited laboratories that participated in the study. 13 Using the anti-fxa-correlation method, the 4 laboratories agreed whether a patient was above, below, or within the therapeutic range only 16% of the time. Poor correlation between the results of anti-fxa and aptt assays contributed significantly to the discrepancies. The authors of that study suggested anti-fxa assays might be better for monitoring UFH therapy. However, few clinical data are available to support use of the anti-fxa assays in UFH monitoring. In one trial patients with acute venous thromboembolism requiring large daily doses of UFH were randomized to monitoring by either direct anti-fxa level or protamine titrationcorrelated aptt. The 2 groups showed roughly equal rates of recurrent thrombosis and bleeding. 14 This is really the only prospective trial aimed at determining the best method of monitoring. Thus, opinions on the clinical value of various monitoring techniques have been based largely on observational and laboratory-based data. The best method for monitoring UFH is not known and randomized, controlled clinical trials would be necessary to determine the optimal approach. In summary, the current evidence suggests that the heparin dosing strategy (prompt initiation of weight-based dosing) may be more important than laboratory monitoring in determining the outcome of UFH therapy. At the present time LMWHs are replacing UFH as the treatment of choice labmedicine.com October 2010 Volume 41 Number 10 LABMEDICINE 625

6 for thromboembolism, since LMWHs are as effective as UFH, and most likely safer, without the need for laboratory monitoring. The debate on monitoring UFH therapy may thus become moot. In the meantime, the clinical laboratory must decide whether to use the aptt or anti-fxa assay for UFH monitoring. There are no data suggesting the anti-fxa assay is better, and the aptt is certainly cheaper. If a laboratory opts for the aptt, the aptt therapeutic range for the specific reagent/instrument combination used must be determined by correlation with a direct assay of heparin activity, with the anti-fxa assay being the most convenient. LM 1. Mueller RL, Scheidt S. History of drugs for thrombotic disease. Discovery, development, and directions for the future. Circulation. 1994;89: Crafoord C. Preliminary report on post-operative treatment with heparin as a preventive of thrombosis. Acta Chir Scand. 1937;79: Huntington JA. Shape-shifting serpins Advantages of a mobile mechanism. Trends Biochem Sci. 2006;31: Stratikos E, Gettins PG. Formation of the covalent serpin-proteinase complex involves translocation of the proteinase by more than 70 Å and full insertion of the reactive center loop into beta-sheet A. Proc Natl Acad Sci USA. 1999;96: Pratt CW, Whinna HC, Church FC. A comparison of three heparin-binding serine proteinase inhibitors. J Biol Chem. 1992;267: Zhao M, Abdel-Razek T, Sun MF, et al. Characterization of a heparin binding site on the heavy chain of factor XI. J Biol Chem. 1998;273: Johnson DJ, Langdown J, Huntington JA. Molecular basis of factor IXa recognition by heparin-activated antithrombin revealed by a 1.7-Å structure of the ternary complex. Proc Natl Acad Sci USA. 2010;107: Lam LH, Silbert JE, Rosenberg RD. The separation of active and inactive forms of heparin. Biochem Biophys Res Commun. 1976;69: Basu D, Gallus A, Hirsh J, et al. A prospective study of the value of monitoring heparin treatment with the activated partial thromboplastin time. N Engl J Med. 1972;287: Chiu HM, Hirsh J, Yung WL, et al. Relationship between the anticoagulant and antithrombotic effects of heparin in experimental venous thrombosis. Blood. 1977;49: Olson JD, Arkin CF, Brandt JT, et al. College of American Pathologists Conference XXXI on laboratory monitoring of anticoagulant therapy: Laboratory monitoring of unfractionated heparin therapy. Arch Pathol Lab Med. 1998;122: Hirsh J, Bauer KA, Donati MB, et al. Parenteral anticoagulants: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines 8th ed. Chest. 2008;133(suppl 6):141S-159S. 13. Cuker A, Ptashkin B, Konkle BA, et al. Interlaboratory agreement in the monitoring of unfractionated heparin using the anti-factor Xa-correlated activated partial thromboplastin time. J Thromb Haemost. 2009;7: Levine MN, Hirsh J, Gent M, et al. A randomized trial comparing activated thromboplastin time with heparin assay in patients with acute venous thromboembolism requiring large daily doses of heparin. Arch Intern Med. 1994;154: Hull RD, Raskob GE, Rosenbloom D, et al. Optimal therapeutic level of heparin therapy in patients with venous thrombosis. Arch Intern Med. 1992;152: Hull RD, Raskob GE, Brant RF, et al. Relation between the time to achieve the lower limit of the APTT therapeutic range and recurrent venous thromboembolism during heparin treatment for deep vein thrombosis. Arch Intern Med. 1997;157: Zidane M, Schram MT, Planken EW, et al. Frequency of major hemorrhage in patients treated with unfractionated intravenous heparin for deep venous thrombosis or pulmonary embolism: A study in routine clinical practice. Arch Intern Med. 2000;160: Raschke RA, Reilly BM, Guidry JR, et al. The weight-based heparin dosing nomogram compared with a standard care nomogram. A randomized controlled trial. Ann Intern Med. 1993;119: LABMEDICINE Volume 41 Number 10 October 2010 labmedicine.com