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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 molecular which accelerates its inhibitory activity significantly. 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), 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

A standard curve is constructed using dilutions of the relevant heparin or heparinoid – see Comments below.  Alternatively a series of plasma calibrants of known LMWH concentration can be used.

a. Using a chromogenic assay:

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 was measured using a chromogenic assay and a Xa-specific substrate.

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.

Reference Ranges

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 about 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.

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

Comments

1. A standard curve should be constructed for UFH and the standard curve for a specific LMWH should not be used i.e. the heparin used for preparation of the calibration curve should be the same heparin as used for patient therapy. 

2. Unfractionated heparin activity is reported in international units per mL (IU/mL) whereas LMWHs are reported in anti-Xa units per mL (anti-Xa U/mL).

3. Elevated levels of haemoglobin, bilirubin, or lipids may interfere with the assay and results from haemolysed, icteric, or lipaemic specimens should be interpreted with caution.

4. Fondaparinux is a synthetic direct Xa-inhibitor and even in prophylactic doses may prolong the PT and APTT and can interfere with factor VIII assays. It has no effect upon the Clauss fibrinogen assay or the thrombin time. A fondaparinux standard curve should be used for reporting fondaparinux levels when using an anti-Xa assay.

5. Danaparoid is a mixture of heparan sulphate, dermatan sulphate and chondroitin sulphate.  Again its activity should be monitored using an anti-Xa assay with Danaparoid used to construct the standard curve.

6. Protamine sulphate rapidly reverses the anticoagulant effects of UFH but has less effect upon LMWH. The following formula can be used to calculate the amount of protamine sulphate required to neutralise heparin.

Useful Links & References

1. American Society for Clinical Pathology. Laboratory Monitoring of Heparin Therapy: Partial Thromboplastin Time or Anti-Xa Assay

2. Bounameaux, H. and P. de Moerloose, Is laboratory monitoring of low-molecular-weight heparin therapy necessary? No. J Thromb Haemost, 2004. 2(4): p. 551-4.

3. Favaloro, E.J., et al., How useful is the monitoring of (low molecular weight) heparin therapy by anti-Xa assay? A laboratory perspective. Lab Hematol, 2005. 11(3): p. 157-62.

4. Harenberg, J., Is laboratory monitoring of low-molecular-weight heparin therapy necessary? Yes. J Thromb Haemost, 2004. 2(4): p. 547-50.

5. Hirsh, J., et al., Parenteral anticoagulants: American College of Chest Physicians Evidence-Based Clinical Practice Guidelines (8th Edition). Chest, 2008. 133(6 Suppl): p. 141S-159S.

6. Smythe, M.A., J.C. Mattson, and J.M. Koerber, The heparin anti-Xa therapeutic range: are we there yet? Chest, 2002. 121(1): p. 303-4.

Data Interpretation

Click HERE to go to the Data Interpretation Exercises.