Anti-Xa Assay (Heparin Assay)

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The anti–factor Xa assay is designed to measure plasma heparin (unfractionated heparin [UH] and low molecular weight heparin [LMWH]) levels and to monitor anticoagulant therapy.

Therapeutic ranges of heparin are as follows:

LMWH: 0.5-1.2 IU/mL^[1]

UH: 0.3-0.7 IU/mL^[2]

Prophylactic ranges of heparin are as follows:

LMWH: 0.2-0.5 IU/mL^[1]

UH: 0.1-0.4 IU/mL^[3]

The most commonly used methodology is the chromogenic assay, which uses a chromophore-linked substrate of factor Xa. Upon cleavage of the substrate by the active enzyme (factor Xa), a colored compound is released.

This methodology incorporates the addition of known amounts of factor Xa and antithrombin to the sample. LMWH or UH forms an inhibitory complex with antithrombin and inactivates factor Xa. Therefore, the excess amount of factor Xa remaining in the sample is inversely proportional to the original amount of LMWH or UH. Consequently, higher levels of LMWH or UH in the sample lead to lower chromogenic intensity. The results are then compared to a standard curve^[4]and are provided in concentration of anti–factor Xa (units/mL).

Some reagents for the anti-Xa assay use a patient’s own antithrombin, and other reagents add antithrombin exogenously to make a complex of heparin and antithrombin.

A low level of anti-Xa may be seen if the specimen is not collected at the right time or if there was a delay in separation of the plasma from the cellular component of the blood.

A high level of anti-Xa may be seen if the patient has renal impairment (in the case of LMWH)or if the specimen is contaminated with heparin (specimen drawn from lines containing heparin).

Specimen: Citrated plasma

Timing of monitoring LMWH: Blood must be drawn 4 hours after subcutaneous injection unless trough level needs to be assessed.

Timing of monitoring danaparoid: Blood must be drawn 6 hours after subcutaneous injection.

Plasma must be separated from cellular components within 1 hour (platelet factor 4, released by platelets, neutralizes the effect of heparin).^[1]

Panels: The anti-Xa assay is usually ordered separately. It may be ordered with partial thromboplastin time (PTT) to measure UH.

The anti–factor Xa assay is designed to measure plasma heparin (UH and LMWH) levels and to monitor anticoagulant therapy. Heparin is a mixture of negatively charged glycosaminoglycans (sulfated mucopolysaccharides) that have anticoagulant properties due to their interaction with the natural anticoagulant antithrombin. This interaction leads to the conformational change of antithrombin, increasing the time of its anticoagulation activity several-fold. Antithrombin’s anticoagulant effect results from its inactivation of certain coagulation factors, for the most part IIa and Xa, by enzymatic cleavage (serine protease).^{[2, 4]}

Two heparin preparations are available—UH and LMWH.

UH contains heparin molecules with a wide range of molecular weight (3,000-30,000 Dalton [an average of 15,000-18,000 Dalton, which corresponds with polymers of 45-50 monosaccharide]).^{[2, 4]}

LMWH is prepared from UH via filtration and enzymatic or chemical depolymerization. LMWH contains only smaller molecular weight polymers (mean molecular weight of 5,000 Dalton, which approximately equals polymers of 17-18^[1]monosaccharides, including the specific pentasaccharide sequence required for binding to antithrombin). While the specific binding of LMWH (specific pentasaccharide) to antithrombin is enough for the inactivation of factor Xa, nonspecific binding of longer polysaccharide chains (>18 monosaccharides) is required for the inactivation of thrombin. Therefore, LMWH more specifically inhibits coagulation by inactivating factor Xa more than UH. LMWH also inhibits thrombin (factor IIa); however, this depends on the proportion of higher molecular weight components.^[2]

Factor Xa assay is indicated to monitor anticoagulant effects.^{[2, 4, 1]}Monitoring anticoagulant therapy is required for intravenous administration but is not essential for subcutaneous therapy.

For monitoring LMWH, the anti-Xa assay plays an essential role since it is the only assay offered for this purpose, since LMWH has minimal effect on PTT. Furthermore, the anti-Xa assay is the most accurate methodology for monitoring UH, especially in the presence of a circulating inhibitor or when the baseline PTT is already prolonged (as in factor XII deficiency).

Unfractionated heparin

The anticoagulant response with heparin is less predictable than with LMWH (nonspecific binding to plasma proteins, endothelial cells, and monocytes). Monitoring is indicated if greater than a prophylactic dose is given.

Low molecular weight heparin

The anticoagulant response with LMWH is more predictable than with heparin (less nonspecific binding). Monitoring is indicated if LMWH is administered to patients with renal dysfunction, those at the extremes of weight, infants, pregnant woman, or patients at high risk for thrombosis/bleeding, as well as if it is used for a prolonged period.^{[5, 6]}

If the collection sample is hemolyzed, icteric, or lipemic, interpret the results with caution. Hemolysis, bilirubin, and lipids may interfere with the chromogenic assay.

The anti-Xa assay is suitable to monitor fondaparinux and danaparoid if the appropriate standard curve is used.^[3]

Protamine sulphate is ineffective for reversing the anticoagulant effect of LMWH.

In the monitoring of UH, the anti-Xa assay (chromogenic assay) is not affected by acute-phase reaction (elevated factor VIII, fibrinogen), unlike the PTT assay. The anti-Xa assay is also unaffected by factor deficiencies, with the exception of antithrombin deficiency (the heparin concentration is underestimated) if the reagent does not contain antithrombin.

Weitz JI. Antithrombotic Drugs. In: Hoffman F, Benz EJ, Shattil SJ, eds. Hematology: Basic Principles and Practice. 5th ed. Philadelpha, PA: Churchill Livingstone; 2009: Chapter 137.

Lehman CM, Frank EL. Laboratory Monitoring of Heparin Therapy: Partial Thromboplastin Time or Anti-Xa Assay? LabMedicine. 2009; Volume 40, Number 1.

Hirsh J, Warkentin TE, Shaughnessy SG et al. Heparin and Low-Molecular-Weight Heparin: Mechanisms of Action, Pharmacokinetics, dosing, Monitoring, Efficacy, and Safety. Chest. 2001; Jan; 119(Suppl): 64S-94S.

Bates SM, Weitz JI. Coagulation Assays. Circulation. 2005; 112:e53-e60.

Castellucci LA, Cameron C, Le Gal G, Rodger MA, Coyle D, Wells PS, et al. Clinical and Safety Outcomes Associated With Treatment of Acute Venous Thromboembolism: A Systematic Review and Meta-analysis. JAMA. 2014 Sep 17. 312(11):1122-1135. [Medline].

Sardar P, Chatterjee S, Herzog E, Nairooz R, Mukherjee D, Halperin JL. Novel oral anticoagulants in patients with renal insufficiency: a meta-analysis of randomized trials. Can J Cardiol. 2014 Aug. 30(8):888-97. [Medline].

Reka G Szigeti, MD, PhD Assistant Professor, Department of Pathology and Immunology, Baylor College of Medicine

Disclosure: Nothing to disclose.

Eric B Staros, MD Associate Professor of Pathology, St Louis University School of Medicine; Director of Clinical Laboratories, Director of Cytopathology, Department of Pathology, St Louis University Hospital

Eric B Staros, MD is a member of the following medical societies: American Medical Association, American Society for Clinical Pathology, College of American Pathologists, Association for Molecular Pathology

Disclosure: Nothing to disclose.

Anti-Xa Assay (Heparin Assay)

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