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Abstract

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  2. Abstract
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As the number of anticoagulant drugs increases and new ones are brought to market, the utility of the routine screening coagulation tests of today—namely the prothrombin time and activated partial thromboplastin time—will be significantly reduced in many clinical situations. Although the new anticoagulants are designed to require less frequent monitoring, it is imperative that the proper test is selected in situations where monitoring is needed. In addition, tests that are designed for the new generation of drugs may be informative in certain situations for monitoring the anticoagulants that have been in use for many years. Here, we present the chromogenic antifactor Xa assay and demonstrate its utility and its limitations in monitoring three anticoagulant drugs (unfractionated heparin, low molecular weight heparin, and fondaparinux) as well as one emerging anticoagulant, rivaroxaban. Am. J. Hematol. 2011. © 2011 Wiley-Liss, Inc.

The chromogenic antifactor Xa assay measures the concentration of anticoagulants that inhibit factor Xa. To generate drug concentration, the assay measures the extent to which exogenous factor Xa is inhibited by complexes of unfractionated heparin (UFH)-antithrombin, low-molecular weight heparin-antithrombin, or fondaparinux-antithrombin in patients being treated with UFH, low-molecular weight heparin (LMWH), or fondaparinux, respectively. The antifactor Xa assay may also be used to monitor the oral anticoagulant rivaroxaban, which was recently approved by the United States Food and Drug Administration for the prevention of deep vein thrombosis in surgical patients undergoing hip or knee replacement [1].

The antifactor Xa assay is sometimes referred to as the UFH assay (for UFH therapy) or the low molecular weight heparin assay (for LMWH therapy). It is important to note that the antifactor Xa assay is not the same test as the factor X activity assay or the chromogenic factor X assay. Factor Xa is the active form of factor X. The factor X activity assay is a clotting time–based assay that is used to diagnose a deficiency in factor X, which is an inherited condition that can result in bleeding. Because unfractionated and LMW heparins, fondaparinux, and rivaroxaban exert all or part of their anticoagulant effect by inhibiting factor Xa (not by causing a factor X deficiency), the factor X activity assay does not have a role in monitoring response to these therapies. Similarly, the chromogenic factor X assay is used to monitor warfarin therapy in patients who are also being treated with argatroban or other direct thrombin inhibitors (DTIs), because the chromogenic factor X assay is not affected by DTI, whereas the INR is prolonged by DTI. The chromogenic factor X assay can also be used to monitor warfarin in patients who have a lupus anticoagulant (LA), because the assay is not affected by LA, whereas occasionally the INR can be prolonged by LA. The chromogenic factor X assay cannot be used for heparin or fondaparinux monitoring [2]. Note that the factor X activity assay and the chromogenic factor X assay measure the amount of factor X activity in the patient, whereas the chromogenic antifactor Xa activity assay does not measure the patient's factor X, instead, it measures the ability of the patient plasma to inhibit exogenous factor Xa. Because of the similarity in name between these different tests, it is recommended that clinicians and coagulation laboratories be alert for errors in test selection.

The antifactor Xa assay is not routinely used to measure the response to UFH therapy because the activated partial thromboplastin time (aPTT) is more readily available, can be performed in small clinical laboratories or at the point of care, and because it has been used for this purpose for several decades. However, in circumstances where the aPTT is elevated by factors unrelated to UFH therapy, for example a deficiency of intrinsic clotting factors (i.e., VIII, IX, XI, XII, prekallikrein, high-molecular weight kininogen) or the presence of a LA, it is essential to use the antifactor Xa assay to monitor UFH therapy. Using the aPTT alone to determine heparin dosing in these patients is dangerous, because the elevated aPTT is due partly to heparin and partly to the factor deficiency or LA, and the therapeutic range is both elevated and unknown. If the aPTT is maintained within the usual therapeutic range, it may result in underdosage of UFH and progression or recurrence of thrombosis. Monitoring UFH therapy with antifactor Xa is also indicated in children less than 1-year-old [3]. In hospitals where access to antifactor Xa testing is limited or has a long turnaround time, a combination of sending simultaneous aPTT and antifactor Xa levels will give an estimate of what aPTT values are therapeutic for a particular patient on heparin, such that a subsequent aPTT that is obtained for that patient later that day when an antifactor Xa level is not rapidly available may still provide some information.

Monitoring of LMWH is advised in pregnant women receiving therapeutic doses of LMWH and in patients with a creatinine clearance reduced below 30 mL/min [4, 5]. Monitoring in obese patients is also sometimes recommended due to the possibility of overdosage or undosage using weight-based dosing formulas [5, 6]. However, the vast majority of patients receiving LMWH do not require monitoring.

The therapeutic range for antifactor Xa activity depends on which of these agents is used. In adults, the antifactor Xa therapeutic range is 0.3–0.7 IU/mL for UFH and 0.5–1.0 IU/mL for LMWH [7]. Because the reference ranges are different depending on the therapeutic agent, it is essential that the coagulation laboratory is aware of the anticoagulant being monitored before reporting the reference range. It is also important to consider the timing of antifactor Xa testing. For example, LMWH reaches its peak plasma concentration ∼4 hr after subcutaneous administration; thus, sample collection should be performed as close as possible to 4 hr after injection of LMWH [8].

The laboratory also needs to know the anticoagulant being monitored in order to select the appropriate standard curve to use for the antifactor Xa assay. For example, to monitor fondaparinux, fondaparinux should be used by the laboratory to construct the standard curve [9]. Using a heparin or LMWH standard curve to assay fondaparinux generates less accurate results [9]. Similarly, heparin should be used to generate the standard curve to assay heparin and LMWH should be used to generate the standard curve to assay LMWH, but “hybrid” curves may also be effective to measure either heparin or LMWH.

Fondaparinux is the first synthetic inhibitor of factor Xa. Similar to LMWH, this pentasaccharide exerts it anticoagulant effect by binding to antithrombin, and subsequently potentiating antithrombin-mediated inhibition of factor Xa. Unlike LMWH, fondaparinux molecules do not inhibit factor IIa. Because the anticoagulant effect is exerted exclusively by the inhibition of factor Xa, the antifactor Xa assay is the choice for therapeutic drug monitoring. There is not yet a consensus therapeutic reference range for fondaparinux therapy. However, the mean peak and minimum steady state concentrations have been determined. Healthy males receiving a single 2.5 mg dose of fondaparinux had an average peak steady state (3 hr) concentration of 0.39–0.5 mg/L [10]. In patients with deep vein thromboses or pulmonary embolism, dosing was determined by patient weight, with either 5 mg (weight < 50 kg), 7.5 mg (weight 50–100 kg), or 10 mg (weight > 100 kg) administered. The mean peak steady state concentrations for all three weight classes was 1.20–1.26 mg/L [10, 11].

Rivaroxaban is an oral drug that selectively inhibits factor Xa and is approved for use in many countries including the United States. Although regular monitoring is unnecessary, antifactor Xa activity levels are the assay of choice to assess potential overdose and/or compliance issues. The therapeutic ranges for rivaroxaban have not been well established. However, the peak plasma concentration is observed 2–4 hr [11, 12] after administration and the half-life is approximately 11–12 hr in the elderly and 5–9 hr in young patients [13]. The utility of the chromogenic antifactor Xa for UFH, LMWH, fondaparinux, and rivaroxaban are shown in Table I.

Table I. Utility of PTT and Chromogenic Antifactor Xa Assays in Anticoagulant Drug Monitoring
DrugFDA approved?Requires frequent monitoring?RoutePTT informative?Adult anti-FXa therapeutic range
  1. SC, subcutaneous administration; IV, intravenous administration; PO, oral administration.

UFHYesYesSC/IVUsually0.3–0.7 IU/mL
LMWHYesNoSCNo0.5–1.0 IU/mL
FondaparinuxYesNoSCNoUnknown
RivaroxabanYesNoPONoUnknown

There are several commercially available chromogenic antifactor Xa assays, all of which determine the extent of factor Xa inhibition by measuring the ability of patient plasma to cleave a chromophore off of a synthetic factor Xa substrate [14]. Endogenous factor Xa that is not complexed with anticoagulant molecules is able to cleave the chromophore off of the substrate and produce a colorimetric change that can be detected by a spectrophotometer [14]. On the other hand, if a patient is using UFH, LMWH, fondaparinux, or rivaroxaban, then less factor Xa is available to cleave the chromophore off the synthetic substrate, resulting in a decreased color change. Thus, the strength of the color change is inversely proportional to the concentration of anticoagulant in the sample [12]. A standard curve, using known amounts of anticoagulant, is used by the analyzer to convert the color reading into drug concentration. Results are reported in drug concentration, such that a high antifactor Xa level indicates high or supratherapeutic anticoagulation, and low antifactor Xa levels indicate low or subtherapeutic anticoagulation.

When using the antifactor Xa assay to monitor UFH, LMWH, or fondaparinux, it is important to consider which test method is utilized by the coagulation laboratory. The commercial tests for antifactor Xa differ in whether they add exogenous dextran sulfate and/or antithrombin [15]. Interpreting the clinical significance of these results can be challenging because unbound heparins—that do not exert an anticoagulant effect in vivo—may exert an anticoagulant effect in vitro if exogenous AT or dextran sulfate is present in the testing reagents.

Adding excess dextran sulfate is intended to reduce the impact of proteins other than AT that bind UFH in vivo. This test may be useful in clinical scenarios where excess heparin binding proteins are suspected to interfere with the ability of UFH to bind to AT, resulting in an unexpectedly low anticoagulant effect. This may especially be true in situations where heparin is administered at high doses [15]. However, this testing strategy could also overestimate in vivo anticoagulation status.

Assays that add exogenous AT are intended to correct for conditions that cause low in vivo antithrombin levels, such as inherited antithrombin deficiency, neonatal age [16], and treatment in the pediatric ICU [14]. Because UFH, LMWH, and fondaparinux all potentiate their effects via AT, this assay is relevant to the interpretation of all three drug assays. These assays may overestimate in vivo heparin activity by allowing drug that is unbound to AT in vivo—and therefore inactive—to bind to exogenous AT added to the assay and exert an anticoagulant effect in vitro. It is unlikely that this assay will effect testing for rivaroxaban, as rivaroxaban does not exert an effect on AT.

Thus, assays that add exogenous AT or dextran sulfate may overestimate the actual in vivo activity of UFH, LMWH, or fondaparinux in patients with excess plasma proteins or deficient levels of AT [15]. In order to avoid an interpretive error of the true antifactor Xa activity, preanalytical consultations with physicians ordering the antifactor Xa assay may be beneficial in some cases. The benefits and drawbacks of each testing strategy are described in Table II.

Table II. Chromogenic Antifactor Xa Assays for the Measurement of UFH, LMWH, and Fondaparinux
Test descriptionPossible benefitsPossible drawbacks
No exogenous AT or dextran sulfateIn vitro effect closely emulates in vivo effect.May underestimate anticoagulation
Exogenous AT onlyIdentifies scenarios in which heparin effect is limited by AT availabilityMay overestimate in vivo anticoagulation status
Exogenous dextran sulfate onlyIdentifies scenarios where binding proteins are competing extensively with AT for heparinMay overestimate in vivo anticoagulation status

As a class, anticoagulants with significant antifactor Xa activity feature predictable pharmacology and, therefore, do not typically require monitoring. The antifactor Xa assay therefore is only indicated in specialized clinical situations, and it must be interpreted in the context of the clinical situation. Nonetheless, as more anticoagulants which are factor X inhibitors come to market, the role of antifactor Xa monitoring will likely increase to ensure therapeutic compliance as well as to diagnose the cause for bleeding in a patient being treated with these anticoagulants [17]. Widespread use of oral factor Xa inhibitors may soon require more hospital laboratories to perform the antifactor Xa assay on site, even though use of these agents should reduce the need for anticoagulation monitoring with the aPTT.

References

  1. Top of page
  2. Abstract
  3. References