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A Practical Guide to Laboratory Haemostasis

 

Anti-Xa Assays



Introduction

The plasma Anti-Xa assay is an test that is used for monitoring patients on LMWHs or UFH.  UFH is commonly monitored by means of the APTT but in some cases [e.g. in patients with a high FVIII level] - the APTT can underestimate 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 LMWHs is dependent upon binding to antithrombin (AT).  Binding induces a conformational change in the molecule which accelerates its inhibitory activity. LMWHs have primarily anti-Xa activity whilst UFH 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.
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 antithrombin) and then adding a fixed amount of Xa.  This results in the formation of an inactive AT-Xa complex and 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 in the sample and may be quantitated from a calibration curve

 

Method

Chromogenic Assay: A standard curve is constructed using dilutions of the relevant heparin or heparinoid – see Comments.  Alternatively a series of plasma calibrants of known LMWH concentration can be used.
In the example shown below – a series of commercial plasma samples of known LMWH concentration have been used to construct a standard curve.  Residual anti-Xa activity is measured using a chromogenic assay and a Xa-specific substrate.

Reference [LMWH] Absorbance at 405nm
0.00 IU/mL 1017
0.4 5IU/mL 564
0.82 IU/mL 368
1.54 IU/mL 154

Nb.  The absorbance is inversely proportional to the concentration of Xa and as a result the greater the absorbance, the lower the plasma Xa activity.
The figure below shows a graph of the concentration of heparin plotted on the X axis using a linear scale against the corresponding absorbance again using a linear scale. A linear or 2nd-order polynomial reference curve is generated from which specimen results are interpolated.  A heparin reference curve is most linear between 0.1-0.6 IU/mL, but varies since anti-Xa activity varies significantly for different heparins and heparinoids.

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. 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 concentration on the Log axis.

Interpretation

Reference ranges for anti-Xa levels depend on heparin type, dose, schedule and indication. When a person is not taking heparin, anti-Xa concentrations 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.

The formula below is useful for calculating the dose of protamine sulphate required to neutralise UFH based upon the anti-Xa level i.e. the concentration of FH.

Remember - protamine sulphate in overdose can act as an anticoagulant.

Reference Ranges

See references for reading regarding anti-Xa reference ranges - it is a 'hot' topic.

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

 

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.