Anti-Xa Assay

The anti-Xa assay is a chromogenic test that measures the activity of drugs that inhibit factor Xa. Patient plasma is incubated with a fixed amount of factor Xa, with or without added antithrombin. The drug (heparin bound to antithrombin, or a direct inhibitor) neutralises part of the factor Xa, and the residual enzyme cleaves a chromogenic substrate. Colour intensity is inversely proportional to drug activity.

Results are read against a calibration curve specific to the drug being measured, so the assay is reported as IU/mL for unfractionated heparin and low molecular weight heparin, and in ng/mL for rivaroxaban, apixaban and edoxaban.

It is used when the aPTT is unreliable or the dose response is unpredictable. It measures drug activity. It does not measure bleeding or thrombotic risk, and therapeutic ranges rest on limited outcome data.

Indications.

• Unfractionated heparin monitoring when the baseline aPTT is prolonged (lupus anticoagulant, factor deficiency), when heparin resistance is suspected, or in pregnancy and extracorporeal circuits.
• Low molecular weight heparin monitoring in renal impairment (eGFR under 30 mL/min/1.73 m²), pregnancy, obesity (weight over 150 kg or body mass index over 40), low body weight, infants and children, recurrent events and bleeding.
• Fondaparinux in renal impairment or extremes of weight.
• Direct oral anticoagulant (rivaroxaban, apixaban, edoxaban) levels before urgent surgery or neuraxial procedures, before thrombolysis in stroke, in overdose, in major bleeding, in renal impairment and with interacting drugs.

Specimen. Citrated platelet poor plasma, centrifuged within 1 hour for unfractionated heparin because platelet factor 4 neutralises heparin, and within 4 hours for other drugs. Draw from the arm opposite the infusion.

Timing.

• Unfractionated heparin by infusion: 6 hours after starting or after a dose change.
• Low molecular weight heparin: peak 3 to 5 hours after the dose, once steady state is reached (after the third or fourth dose).
• Direct oral anticoagulants: peak 2 to 4 hours after the dose and trough just before the next dose.

Calibration. Heparin calibrated assays are used for heparins, and drug specific calibrators for each direct inhibitor. A heparin calibrated assay gives falsely high results with a direct factor Xa inhibitor. Antithrombin supplemented reagents mask antithrombin deficiency.

Usual ranges.

• Unfractionated heparin, treatment: 0.3 to 0.7 IU/mL.
• Low molecular weight heparin peak: twice daily treatment 0.6 to 1.0 IU/mL, once daily treatment 1.0 to 2.0 IU/mL, prophylaxis 0.2 to 0.5 IU/mL. A trough over about 0.5 IU/mL on twice daily treatment suggests accumulation.
• Children: unfractionated heparin about 0.35 to 0.7 IU/mL and low molecular weight heparin 0.5 to 1.0 IU/mL.
• Direct oral anticoagulants: expected peak and trough levels depend on the drug, dose and renal function, and are interpreted against a drug specific calibrator and local reference data.

Perioperative direct oral anticoagulant levels. A level under about 30 ng/mL is generally accepted for procedures with high bleeding risk and neuraxial anaesthesia, and under about 50 ng/mL for low risk procedures. For thrombolysis in acute ischaemic stroke, a level under about 50 ng/mL is used in many centres, and local policy applies.

Low anti-Xa on heparin (heparin resistance). Antithrombin deficiency (inherited or acquired), raised factor VIII and fibrinogen, sampling delay or poor timing, and underdosing.

High anti-Xa. Overdose, renal impairment with accumulation of low molecular weight heparin or fondaparinux, low body weight, and recent dose before sampling. A falsely high reading arises when a direct factor Xa inhibitor is measured with a heparin calibrated assay.

Critical findings needing immediate action. A markedly raised level in a patient with active bleeding, or in a patient with renal failure on treatment doses of low molecular weight heparin, needs urgent clinical review.

Interferences and pitfalls.

• Hyperbilirubinaemia, marked haemolysis and lipaemia falsely lower the result in some chromogenic assays.
• Delay before centrifugation falsely lowers unfractionated heparin levels.
• Sampling from a heparinised line causes spurious results.
• Different reagents give results that differ by up to 20 to 30%, so serial monitoring uses the same method.
• A level within range does not exclude bleeding or thrombosis, and therapeutic ranges are not validated by outcome data.
• Dabigatran does not affect anti-Xa assays, and needs a dilute thrombin time or ecarin based assay.
• Concurrent heparin and direct oral anticoagulant exposure, as during a switch, gives a mixed result.

Special groups.

• Pregnancy: clearance of low molecular weight heparin rises and volume of distribution increases, so routine monitoring is not needed, but it is used in extremes of weight, renal impairment and mechanical valves.
• Neonates and young infants: antithrombin is low, so response to heparin is reduced.
• Renal impairment: accumulation of low molecular weight heparin, fondaparinux and direct inhibitors.
• Obesity: dosing by total body weight overestimates exposure in some, and levels direct adjustment.
• Older adults: reduced clearance and bleeding risk.

Follow up tests. Antithrombin activity, creatinine and eGFR, platelet count, aPTT and thrombin time, dilute thrombin time for dabigatran, repeat anti-Xa level after dose adjustment, heparin induced thrombocytopenia testing if the platelet count falls.