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A Practical Guide to Haemostasis


Anti-Xa Assays


Introduction

The plasma Anti-Xa assay is a functional test that is used for monitoring patients on Low Molecular Weight Heparins [LMWHs], Unfractionated Heparin [UFH] and the Direct Oral Anticoagulants [DOACs] that have anti-Xa activity.  UFH is commonly monitored by means of the APTT but in some cases [e.g. in patients with a high FVIII level or a prolonged APTT before instituting UFH therapy] - the APTT can underestimate or overestimate the degree of anticoagulation induced by the UFH and the measurement of a plasma anti-Xa level may provide a more accurate assessment of anticoagulation.

Principles

The activity of both UFH and the LMWHs is dependent upon their binding to Antithrombin (AT). Binding of Heparin to Antithrombin induces a conformational change in the molecule which accelerates its inhibitory activity.  LMWHs bound to Antithrombin have primarily Anti-Xa activity whilst UFH bound to Antithrombin has both Anti-Xa and Anti-IIa activity.

In patients receiving either LMWHs or UFH, the Xa inhibitory activity of Antithrombin is increased and this can be measured using with a clotting-based assay or more commonly a chromogenic assay.  The test involves patient citrated platelet poor plasma which is then mixed with a known amount of Factor Xa.  Depending upon the degree of Xa inhibition [and this is related directly to the UFH/LMWH concentration] the residual Xa is assayed using either a clotting-based Factor X assay but more commonly using a chromogenic substrate specific for Factor Xa. 
A standard curve is constructed by adding known amounts of LMWH or UFH to plasma (which provides a source of Antithrombin although some assays may add exogenous Antithrombin) and then adding a fixed amount of Factor Xa.  This results in the formation of an inactive AT-Xa complex and the residual Xa is measured using either a clotting-based assay or more commonly a chromogenic assay. 
The residual Xa activity is inversely proportional to the concentration of heparin or DOAC in the plasma sample and may be quantitated from the relevant calibration curve.

The DOACs that inhibit Factor Xa - Apixaban, Edoxaban, Rivaroxaban, Betrixaban or Fondaparinux can be measured using a similar approach as their concentration is inversely proportional to the residual Xa concentration and the residual plasma levels of Xa can be determined by comparing the result with a reference curve generated from calibration standards specific for each DOAC and from which the DOAC concentration can be derived.

When used for LMWH/UFH, the results from the heparin-specific reference curves are expressed in U/mL and when used for DOACs the results from the reference curve are expressed as ng/mL.

 

Method

1. Chromogenic Assay: A Test plasma sample from a patient on either UFH, a LMWH or a DOAC is incubated with Factor Xa.  The Xa is inactivated by the Antithrombin-Heparin complex  or DOAC and residual Xa is assayed using a chromogenic substrate specific for Xa.  A standard curve is constructed using dilutions of the relevant heparin, heparinoid or DOAC added to a normal plasma pool.

2. Clotting-based Assay: This is similar to the chromogenic assay but the anti-Xa activity is measured by performing an APTT-based Factor X assay on each dilution rather than using a chromogenic substrate. A standard curve is constructed by using serial dilutions of a known concentration of the relevant heparin i.e. the heparin the patient is receiving or DOAC. The clotting time for each of these heparin concentrations is determined with the FX assay. The clotting times for each plasma dilution are then plotted on log-lin paper with the clotting times on the linear axis and the heparin or DOAC concentration on the Log axis.

Interpretation

Reference ranges for anti-Xa levels depend on the anticoagulant in use, the type, dose, schedule and indication. When an individual is not taking heparin or a DOAC, the anti-Xa concentration should be zero or undetectable.

When it is used as to monitor LMWHs, anti-Xa levels are usually ordered as a 'peak' test. It is collected 3-4 hours after a LMWH dose is given, when the concentration of LMWH in the blood is expected to be at its highest level. Random and “trough” anti-Xa tests may also be ordered when there are concerns that a LMWH may be accumulating e.g. in renal failure.

When the anti-Xa level is used to monitor a DOAC, the recommendations regarding the time to check peak and through levels depend upon the DOAC in use. 

 

  Apixaban Edoxaban Rivaroxaban Betrixaban
Plasma peak level 1-4 hrs after ingestion 1-2 hrs after ingestion 2-4 hrs after ingestion 3-4 hrs after ingestion
Plasma trough level 12-24 hrs after ingestion 12-24 hrs after ingestion 16-24 hrs after ingestion 37 hrs after ingestion


Reference Ranges

See references for anti-Xa reference ranges.

Category Anti-Xa level
Patients not on heparin 0 U/mL
Therapeutic range for the treatment of a DVT UFH:             0.3-0.7 U/mL
LMWH:          0.4-1.1 U/mL bd
LMWH:          1-2 U/mL
Danaparoid:   0.5-0.8 U/mL
DVT Prophylaxis No defined range but generally in the region of 0.1-0.3 U/mL measured 3-4 hours after a subcutaneous injection