Proper weight-based administration of unfractionated heparin has been shown to effectively reduce the morbidity and mortality associated with thromboembolic processes. In addition, it's a common medication for extracorporeal anticoagulation. The most common test for monitoring heparin therapy is the activated partial thromboplastin time (aPTT) test. However, this test does not directly measure heparin and is affected by physiologic and analytic variables. Anti-Xa testing offers improvements over aPTT testing for accurate measurement of heparin levels.
Presenters and Credentials:
Theresa Kinard, M.D., is an Instructor of Laboratory Medicine and Pathology and a Consultant in the Division of Transfusion Medicine at Mayo Clinic in Arizona.
Our speaker is Dr. Theresa Kinard, an Instructor in Laboratory Medicine and Pathology at Mayo Clinic, as well as a transfusion medicine consultant at Mayo Clinic Arizona. Dr. Kinard provides an overview of heparin anticoagulation monitoring and the use of the activated partial thromboplastin time and the chromogenic anti-Xa assay.Welcome to Mayo Medical Laboratories Hot Topics. These presentations provide short discussion of current topics and may be helpful to you in your practice.
Hello and thank you for viewing this Hot Topic on Heparin Monitoring by the Anti-Xa Assay. My name is Theresa Kinard and I am a practicing transfusion medicine consultant at Mayo Clinic in Phoenix, Arizona.
Our speaker has nothing to disclose.
As you review this presentation, consider the following important points regarding heparin anticoagulation monitoring:
- How and when should the testing be used in your practice?
- How will results impact patient management?
In this presentation, I will describe the structure and review the mechanism of action of unfractionated heparin. Next, I will describe the principle laboratory tests used to monitor heparin therapy–the activated partial thromboplastin time, or aPTT, and the chromogenic anti-Xa assay, and review how the therapeutic ranges were established with each testing methodology. Finally, I will discuss advantages and limitations of each test.
Unfractionated heparin was discovered in 1920 but did not reach clinical application until the 1940s. This anticoagulant is a popular and important inpatient medication because of the relatively short half-life, its ability to be quickly reversed, and its nonrenal elimination. It is administered intravenously or by subcutaneous injection for the treatment or prevention of thrombotic diseases. Proper weight-based administration of unfractionated heparin has been shown to effectively reduce morbidity and mortality associated with thromboembolic processes, such as venous thromboembolism, pulmonary embolism, acute coronary syndrome, and stroke. It is also a common medication for extracorporeal anticoagulation during procedures such as apheresis, hemodialysis, extracorporeal membrane oxygenation, and during invasive surgical interventions that utilize cardiopulmonary bypass.
Heparin is a negatively charged, sulfated polysaccharide, in which unfractionated heparin is a mixture of large and small heparin fractions weighing 3,000 to 30,000 daltons. Heparin is an indirect inhibitor of multiple procoagulant enzymes and its primary anticoagulation effect is mediated by antithrombin. Antithrombin is a naturally occurring anticoagulant protein found in blood, whose anticoagulant effect is magnified many-fold when interacting with heparin.
Heparin binds antithrombin through a high-affinity pentasaccharide, which produces a conformational change that converts antithrombin from a slow progressive thrombin inhibitor to a very rapid inhibitor. Interestingly, only one-third of heparin molecules contain the high-affinity pentasaccharide required for anticoagulant activity, which results in heterogeneous heparin response.
While heparin targets multiple factors in the contact activation pathway and tissue factor pathway, factors Xa and IIa are the most responsive to inhibition.
Binding of factor Xa or heparin to antithrombin causes a conformational change at the antithrombin’s reactive center that accelerates its interaction with factor Xa. However, antithrombin-mediated inactivation of thrombin, or factor IIa, requires the formation of a heparin-antithrombin-thrombin complex. This complex can be formed only by chains at least 18 saccharide units long. Antithrombin inhibition of factor IIa prevents fibrin formation and inhibits thrombin-induced activation of factors V and VIII. This is in contrast to low-molecular-weight heparin, which contains mostly smaller heparin fractions that are less than 18 saccharide units long and facilitates anticoagulation mainly by inhibiting factor Xa. Generally the aPPT responds to heparin therapy is exaggerated and will underestimate the anticoagulation level since the correlation with elevated aPPT values with incidence of bleeding is not established in accurate therapeutic PPTs may result in a higher risk of Thrombosis due to potential under anticoagulation. Heparin is unable to inactivate Xa in the prothrombinase complex or thrombin bound to fibrin or sub-endothelial surfaces.
The high-negative charge surrounding large-molecular-weight heparins results in binding to positively charged proteins and surfaces. The nonselective binding to cells and proteins reduces the anticoagulant response and pharmacokinetics. At therapeutic doses, the response to heparin is nonlinear, meaning the intensity and duration of effects rises disportionately at increasing doses. So, for patients receiving a 25 U/kg bolus, the biologic half-life is about 30 minutes, versus 60 minutes after a 100 U/kg bolus, and 150 minutes after a 400 U/kg bolus.
A major portion of unfractionated heparin is cleared by a rapid, dose-dependent mechanism. Large fractions are rapidly cleared by depolymerization by endothelial cells and macrophages and this clearance is saturable. Low-molecular-weight fractions accumulate and are slowly cleared by the kidneys.
UFH: Therapeutic Monitoring
Therapeutic monitoring of anticoagulation activity of unfractionated heparin should be measured 6 hours after a bolus and 6 hours after each rate change. At least 6 hours is required to achieve steady state kinetics, so monitoring heparin therapy more frequently than 6 hours results in non-steady state analysis, which can lead to erroneous dosing calculations.
Laboratory monitoring is generally performed by the activated partial thromboplastin time, or aPTT, or the chromogenic anti-Xa assay. Protamine titration is the gold standard to which the other 2 methods are compared to, but this assay is not widely available and is labor intensive, making it only available in research laboratories.
Activated Partial Thromboplastin Time
The PTT continues to be the principle method by which most laboratories monitor IV and fractionated heparin (UFH) therapy. It is a clot-based test that requires citrated patient blood to produce platelet-poor plasma, or PPP, for testing. Phospholipid and an activator are added to the patient’s PPP and this mixture is allowed to incubate. Calcium is added to reverse the citrate and the clotting time is measured.
The PTT offers many advantages. It is quick, cheap, and widely available. However, it does not directly measure heparin. Numerous PTT reagents exist and each demonstrates a variable response to heparin therapy. Furthermore, several physiologic factors can impact the results of this clotting test.
Physiologic Limitations of the PTT
The PTT can be impacted by many patient-dependent physiologic variables, as well as analytic variables related to laboratory testing, as mentioned previously. Clinical conditions can alter the PTT, but do not correlate with bleeding or protection from thrombosis. A common situation is concurrent administration of anticoagulation, usually a vitamin K antagonist. Patients with INR greater than 1.3 due to warfarin may have unreliable PTT monitoring of heparin. Another commonly encountered clinical condition is patients with antiphospholipid antibodies, which interfere with the clotting test. Furthermore, factor deficiencies, for example, as a result of liver disease or consumptive coagulopathy in disseminated intravascular coagulopathy, will alter the PTT results, which will also make it a poor reflection of heparin anticoagulation. All of the just mentioned clinical situations produce an elevated baseline PTT and can potentially result in underanticoagulation with heparin.
On the opposite side of the spectrum, clinical conditions may also result in a blunted PTT response. Acute-phase reactants factors VIII and fibrinogen are the most important confounders, and may be dramatically elevated and shorten the baseline PTT. At the same time, acutely ill patients are also known to have antithrombin deficiency. These conditions can result in potential overanticoagulation when monitoring heparin by the PTT.
Preanalytical variables can also play a role in the variable heparin PTT response. The variable laboratory response to heparin therapy is primarily reagent dependent, but may also be instrument and procedure dependent. Additionally, erroneous baseline PTTs may result from improper blood collection leading to premature activated samples and underfilled tubes resulting in relatively higher concentrations of sodium citrate in the collection tube.
Generally, the PTT response to heparin therapy is exaggerated and will underestimate the anticoagulation level. Since the correlation with elevated PTT levels with incidence of bleeding is not established, inaccurate therapeutic PTTs may result in a higher risk of thrombosis due to potential underanticoagulation.
So, when do we know that our patients are “therapeutic”?
In 1972, Basu and colleagues did a prospective study in patients with venous thromboembolism who were treated with heparin and found that the risk for recurrent thromboembolic disease is associated with failure to obtain a PTT ratio of approximately 1.5 to 2.5 times the control value. The control value is the mean PTT obtained by testing a minimum number of plasma samples from healthy persons. In addition, early clinical studies showed evidence to support bringing the PTT to the therapeutic range within 24 hours to prevent thrombosis.
Thus, the PTT ratio of 1.5 to 2.5 times the control became the standard for the therapeutic range. Based on these studies, the authors reported that the corresponding heparin level was 0.2 to 0.4 U/mL based on protamine titration, which is equivalent to anti-Xa level of 0.3 to 0.7 U/mL.
The anti-Xa assay range is higher because of the variable clearance rates of heparin molecules. As mentioned previously, smaller heparin molecules are cleared more slowly; thus, these low-molecular-weight heparins have a greater inhibitory effect on factor Xa than on thrombin. Tests that measure antifactor Xa activity, such as the anti-Xa assay, will detect higher levels than tests that measure antithrombin activity, such as the protamine titration, which utilizes the thrombin time.
Other studies that evaluated the upper limits associated with bleeding used the anti-Xa for heparin monitoring. These studies found that anti-Xa levels greater than 0.74 to 0.88 were associated with more bleeding complications, supporting the avoidance of exceeding a Xa greater than 0.7 to 0.8 to minimize bleeding risk.
Using a fixed ratio for the therapeutic range is problematic because the responsiveness of different PTT reagents to heparin is variable. Researchers in the sentinel study found that the PTT corresponded to heparin concentrations of 0.2 to 0.4 U/mL by protamine titration, based on their single PTT reagent. However, different PTT reagents and laboratory instruments demonstrate variable sensitivities to heparin, and a 1.5 to 2.5 times the mean control does not necessarily correlate with the established therapeutic heparin concentrations. Laboratory and reagent variability may result in PTT values ranging from 1.6 to 3.7 times the control, which corresponds to a heparin level of 0.3 U/mL to 2.4 to 6.2 times the control for heparin levels of 0.7 U/mL by anti-Xa.
Attempts to standardize the reagent variability to a reference reagent, like the thromboplastin reference reagent, which produces the international sensitivity index for the international normalized ratio (INR), have been unsuccessful.
Rather than using a fixed ratio for therapeutic ranges, a better method to determine aPTT therapeutic ranges can be established by calibrating to plasma heparin concentrations using 0.3 to 0.7 U/ml on anti-Xa, or 0.2 to 0.4 U/mL by protamine titration, which compensates for the variable response of PTT reagents to heparin.
In the 1990s, the American College of Chest Physicians and College of American Pathologists recommended that specific therapeutic goal ranges of PTTs for individual institutions should be determined according to a corresponding heparin level.
These recommendations came after a study that showed patients managed by anti-Xa had fewer recurrent venous thromboembolism and fewer bleeding complications than patients monitored by the PTT, despite receiving significantly less heparin. Additional benefits to support monitoring heparin therapy by anti-Xa include fewer monitoring tests and dosage changes, achieving the therapeutic range sooner, and maintaining the range longer.
CAP requires revalidation of the therapeutic ranges with each change in reagent lot or instrument, and validation requires obtaining ex vivo sampling from real patients on heparin therapy.
As a consequence, laboratories must monitor lot changes and perform the difficult task of establishing new reference ranges regularly. The laboratory must identify patients for ex vivo testing by working with clinical teams, samples must be collected at the right time, and testing must performed in a timely manner. Many patients will have a small window of opportunity if a transition to warfarin is anticipated. Furthermore, providers must be continuously educated on the changing therapeutic ranges.
Therefore, it may seem more practical to use the anti-Xa to monitor heparin therapy directly.
Transitioning to an anti-Xa assay is easier now because the assay is available on many automated analyzers, which can be performed 24/7, and the expense of this test has decreased.
Variability does exist between anti-Xa assays when compared with the protamine titration as the reference, but much less than with the PTT. Studies suggest that the interlaboratory correlation and agreement among Xa assays are reasonable, and quantitatively less than demonstrated by the PTT. Therefore, the anti-Xa assay will have the same therapeutic range of 0.3 to 0.7 U/mL regardless of the instrument or reagent. Furthermore, the range will not change with future changes in equipment or reagents. Unlike the PTT, the anti-Xa result will not be affected by underfilled collection tubes, will not be susceptible to interference by acute phase reactants factor VIII or fibrinogen, and will not be influenced by factor deficiencies found in liver disease and consumptive coagulopathy.
Although the reagents needed to perform the Xa assay are more costly, fewer tests are needed when the anti-Xa is used. When reduced testing frequency, reduced dose adjustments, and the associated reduced labor for such activities are taken into consideration, the overall institutional cost may be equivalent to the PTT. There is strong data to support that anti-Xa is at least cost neutral.
Finally, laboratories may also be in favor of this transition to eliminate the need to establish PTT therapeutic ranges.
What is the anti-Xa assay and how does it determine plasma heparin levels?
First of all, it is very important to educate providers on how to order the anti-Xa. While each institution may have slightly different nomenclature, the anti-Xa assay is not synonymous with the factor X activity or the factor X antigen level. These are different tests. The anti-Xa assay may also be called the antifactor Xa test or functional heparin assay. However, laboratories may offer various anti-Xa assays because this testing principle can be used to monitor other indirect Xa inhibitors, such as low-molecular-weight heparin and fondaparinux, or oral direct Xa inhibitors, such as rivaroxaban. These anticoagulants may require monitoring for certain special patient populations and in certain clinical conditions. Each anticoagulant requires its own anti-Xa calibration curve, and the appropriate anti-Xa assay must be requested from the laboratory. While the principles of the test remain the same, this presentation will only refer to the anti-Xa assay for unfractionated heparin monitoring.
Chromgenic Anti-Xa Assay
The antifactor Xa assay is a chromogenic assay. A known amount of factor Xa is added to platelet-poor plasma, which includes therapeutically administered heparin. The heparin enhances factor Xa inhibition by antithrombin and the remaining uninhibited Xa cleaves an added chromogenic substrate. This process creates a colored compound that can be detected by a spectrophotometer and is directly proportional to the amount of factor Xa present. The amount of color is correlated to a plasma heparin level with the appropriate heparin standard curve. In other words, the amount of unbound residual factor Xa is negatively proportional to the heparin concentration in the plasma sample, and by measuring the activity of only Xa, the anti-Xa assay is more specific than the PTT for measuring heparin activity.
Limitations of Chromogenic Anti-Xa Assay
However, the anti-Xa assay is not perfect. Being a chromogenic assay, the anti-X assay will be affected by hemolysis, icterus, and hypertriglyceridemia, which affects the instrument’s ability to measure and discriminate the chromogenic reaction. Patients with significantly elevated hemolysis, total bilirubin, and triglycerides, should have their heparin monitored by PTT.
Furthermore, anti-Xa reagents that do not supplement with antithrombin may underestimate heparin concentrations in the presence of a significant antithrombin deficiency. Conversely, the suspicion or diagnosis of antithrombin deficiency may be missed if antithrombin is supplemented in the reagent. From the laboratory perspective, the anti-Xa assay is expensive and requires prompt attention for processing.
Therapeutic Heparin Titration
While heparin therapy was initially administered based on a one-size-fits-all dosing nomogram, weight-based dosing protocols published in the early 1990s proved to be effective, safe, and superior to the previous standard, and avoid the risk of overanticoagulation. Weight-based heparin protocols are the current standard of care.
Presented is a recognized protocol to titrate IV unfractionated heparin therapy to the targeted goal of 0.3 to 0.7 U/mL. These protocols may be modified to the desired targeted therapeutic range. For example, patients whose risk of bleeding is greater than the need or benefit to have rapid therapeutic anticoagulation may consider omitting or reducing the bolus or having a narrow, reduced intensity therapeutic goal. The heparin protocol can also be modified to achieve prophylactic plasma heparin concentrations of 0.1 to 0.3 U/mL.
Implementation Anti-Xa Monitoring of Heparin
For most large institutions, transition to anti-Xa monitoring may be simpler than expected. Most coagulometers used by medical centers already have the photo-optic capability to perform a chromogenic Xa; thus, new instruments may not need to be purchased. However, like all other tests performed, the laboratory must perform validations, but the long-term calibration needs are not increased, and directly monitoring heparin therapy with the anti-Xa assay would eliminate PTT correlations. Testing automation results in the same turnaround times as the PTT and with no increase in laboratory labor.
Furthermore, a change in practice will have to be reviewed and accepted by the clinicians, and supported by the institution’s administration. A successful transition will require close collaboration between the providers, laboratory, and information technology to provide the proper clinical and management tools and education.
In summary, the PTT is affected by many biologic factors unrelated to the heparin concentration. It is not a standardized test and the results may be significantly different with the same patient and between patients due to biologic and analytic variables. The historical goal of 1.5 to 2.5 times the control is antiquated and unsafe, and may potentially lead the clinician to over-, or most commonly, underanticoagulate his or her patient. If the PTT is the institution’s standard for monitoring heparin, it should be calibrated, at least annually, and with each lot change, to 0.3 to 0.7 U/mL by the anti-Xa assay or 0.2 to 0.4 by protamine titration.
When the anti-Xa assay is used to monitor heparin, the therapeutic ranges will remain unchanged, despite differences in the instruments, reagents, and reagent lots. The variability in actual patient plasma heparin levels are less when using an anti-Xa protocol than a PTT-based protocol. Direct heparin level monitoring with the anti-Xa has been shown to achieve therapeutic levels faster and maintain them longer, while reducing testing and dose adjustments. While the anti-Xa is more expensive, reduction in testing, dose adjustments, and associated labor make it a cost-effective method that improves patient care.
I have provided clinical references on this topic.