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Laboratory Monitoring for Heparins, Fondaparinux, Direct Thrombin Inhibitors, and Oral Anti-Xa Medications
Laboratory testing modalities and recommendations for monitoring heparin, heparin-like medications, direct thrombin inhibitors (DTIs), and direct anti-Xa inhibitors (DXIs), including indications, testing recommendations, testing modalities, and test limitations are discussed below.
There are several options available for laboratory monitoring of these drugs, all with varying levels of utility. New DTI’s and DXI’s are in clinical trials and the number of patients undergoing direct oral anticoagulant therapy will continue to expand.
Unfractionated Heparin
Testing Recommendations
Therapeutic heparin is usually administered as a continuous IV infusion. Response to heparin is highly variable between patients, and heparin activity requires monitoring to prevent both over and under coagulation. Therapeutic monitoring should be performed every 6 hours after initiating or changing IV infusion until therapeutic levels are reached and every 24 hours thereafter. Low-dose subcutaneous heparin administration is usually not monitored.
Testing Modalities
In the United States, activated partial thromboplastin time (PTT) is the predominant modality for monitoring unfractionated heparin (UFH) therapy. This is primarily due to low cost and wide availability considerations and not to the specific advantages of the method. It is recommended that PTT-based tests be correlated to anti-Xa activity in each laboratory that performs PTT-based UFH monitoring. PTT has a number of limitations due to the highly variable response to UFH that is dependent on PTT reagents, instrumentation, patient-to-patient variation, lupus anticoagulant interference, factor deficiencies, high levels of factor (FVIII), and the heparin preparation itself. Alternatively, direct measures of heparin activity using Factor (FXa)–based assays or protamine titration can be used.
Partial Thromboplastin Time Correlation/Calibration for Heparin Monitoring
Most laboratories performing PTT-based testing calibrate the assay using anti-Xa activity. To do so at least 40 samples from patients receiving IV heparin are assayed in parallel for PTT and anti-Xa activity. The therapeutic PTT range is then established on the basis of the lower cutoff of 0.3 units (U) and upper cutoff of 0.7 U of anti-FXa activity. Because heparin has a variable effect in vivo, the calibration should be done on samples taken from patients receiving the specific preparation of UFH rather than samples spiked with heparin in vitro. A linear regression curve fitting is used to establish the cutoffs. Only about 50%–60% of variability of PTT is attributable directly to the concentration of heparin in ex vivo samples. Thus r 2 values of 0.5–0.6 are to be expected.
Anti-Xa Assays for Direct Measurement of Heparin Activity
Chromogenic antithrombin (AT)–dependent FXa inhibition offers an alternative to PTT and has substantially fewer interferences. There are slight variations in these assays among different manufacturers. In a typical assay, the following sequence is used: (1) add fixed amount of FXa to patient plasma sample, (2) short incubation period, and (3) add chromogenic substrate to assay residual activity. Reduction in the FXa activity due to test plasma heparin can then be plotted on the standard curve using standardized heparin activity as a calibrator. Either kinetic or endpoint measurements of color change due to FXa activity in cleaving the chromogenic substrate can be taken. Readout is performed at the wavelength of 405 nm. The relationship between color change or development and heparin level is indirectly proportional in that higher levels of heparin result in lower levels of color formation and vice versa. This assay can be performed either with or without addition of excess exogenous AT. The assays relying on endogenous AT only are affected by low AT concentration. This variant may be advantageous if there is a concern for heparin resistance due to low AT.
Preanalytical Considerations and Result Interpretation
The specimen should be drawn from a different extremity than the one used for infusion. Timely testing is critical to obtain a proper heparin activity result. Heparin can be bound and inactivated by platelet factor 4 released from platelets during prolonged storage. Thus, it is recommended that plasma be separated from cellular components and platelets within 1 hour of collection to minimize this effect. Testing should be completed within 4 hours of collection.
Result Interpretation
Lupus anticoagulant, factor deficiencies, factor inhibitors, and pharmacological coagulation inhibitors other than heparin affect the PTT. The presence of those variables can cause overestimation of PTT-based heparin activity, leading to underdosing. Elevated levels of FVIII activity cause underestimation of the effect and possible overdosing. Direct anti-Xa-based heparin activity tests suffer from fewer interferences. Nonetheless simultaneous presence of other anti-factor Xa active medications such as low-molecular-weight heparin (LMWH), fondaparinux, and DXIs can produce overestimation of UFH activity. Lack of standardization between different anti-Xa tests has been shown to cause up to 30% difference in UFH concentration reporting between different kits.
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