From: Elizabeth M. Van Cott, M.D., and Michael Laposata, M.D., Ph.D., “Coagulation.” In: Jacobs DS et al, ed. The Laboratory Test Handbook, 5th Edition. Lexi-Comp, Cleveland, 2001; 327-358.
Related Information
Activated Clotting TimeActivated Partial Thromboplastin Time
Antithrombin
Heparin Neutralization
Thrombin Time
Synonyms Antifactor Xa Assay; Anti-Xa Assay; Heparin Assay
Applies to Danaparoid; Heparin; LMWH; Low-Molecular Weight Heparin; Orgaran®; PF4; Platelet Factor 4; PTT
Abstract Two relatively new anticoagulants, low-molecular weight heparin (LMWH) and danaparoid (Orgaran®), when present at therapeutic levels, usually do not significantly prolong the activated partial thromboplastin time (PTT). Therefore, when laboratory tests are used to monitor therapeutic anticoagulant levels of LMWH or danaparoid, antifactor Xa assays are necessary. In addition, in some instances the PTT cannot be used to monitor unfractionated heparin. For example, lupus anticoagulants* or certain factor deficiencies (eg, factor XII deficiencies) may prolong the baseline PTT and/or accentuate the PTT prolongation when heparin is added. In these cases, unfractionated heparin may be monitored with antifactor Xa assays (*Note: if the antifactor Xa assay demonstrates that the heparinized PTT is not affected by the lupus anticoagulant, cautious use of the PTT may be tried in that patient).
Specimen Plasma
Container One blue top (sodium citrate) tube
Sampling Time Draw specimen 4 hours after subcutaneous injection of LMWH or 6 hours after subcutaneous injection of danaparoid, otherwise, falsely low values may occur. The therapeutic antifactor Xa ranges with subcutaneous LMWH and danaparoid are defined for the peak levels.
Collection Routine venipuncture. Deliver tube to laboratory immediately, otherwise falsely low values may occur (because platelets release platelet factor 4 (PF4) which can neutralize heparin, LMWH, or danaparoid). If multiple tests are being drawn, draw blue top tubes after any red top tubes but before any lavender top (EDTA), green top (heparin), or gray top (oxalate/fluoride) tubes. Immediately invert tube gently at least 4 times to mix. Tubes must be appropriately filled.
Storage Instructions Separate plasma from cells as soon as possible, ideally within 1 hour of specimen collection. Otherwise, falsely low values may occur (because platelets release PF4, which can neutralize heparin, LMWH, or danaparoid). Plasma can be stored for 2 hours at room temperature or on ice; otherwise, store frozen.
Turnaround Time 1 day, unless testing is batched less frequently
Special Instructions Notify the laboratory specifically as to which drug should be measured (heparin, LMWH, or danaparoid), because the laboratory must construct a standard curve using the same drug that the patient is receiving.
Reference Interval
Patients not on anticoagulants: 0 units/mL
Therapeutic range for treatment of existing deep venous thrombosis (DVT):
* heparin 0.3-0.7 units/mL
* LMWH: 0.4-1.1 units/mL for twice daily subcutaneous dosing. For once daily subcutaneous LMWH dosing, the therapeutic range is less certain but is approximately 1-2 units/mL.
* danaparoid: 0.5-0.8 units/mL
Target range for deep vein thrombosis (DVT) prophylaxis (prevention): There is no defined target range for prophylaxis of deep vein thrombosis (DVT) because such anticoagulation is not usually monitored. When anti-Xa levels have been measured, mean values have been <0.45 units/mL.
Use Determine if the patient is at the desired level of anticoagulation with therapeutic doses of heparin, LMWH, or danaparoid
Limitations More expensive and less readily available than the PTT for heparin monitoring
Methodology Chromogenic.1 Patient plasma is added to a known amount of excess factor Xa with excess antithrombin. If heparin (or LMWH or danaparoid) is present in the patient plasma, it will bind to antithrombin and inhibit factor Xa. The amount of residual factor Xa is inversely proportional to the amount of heparin in the plasma. The amount of residual factor Xa is detected by adding a chromogenic substrate that resembles the natural substrate of factor Xa. Factor Xa cleaves the chromogenic substrate, releasing a colored compound that can be detected by a spectrophotometer. Results are reported as anticoagulant concentration in antifactor Xa units/mL, such that high antifactor Xa values indicate high levels of anticoagulation and low antifactor Xa values indicate low levels of anticoagulation. Deficiencies of antithrombin in the patient do not affect the assay, because excess antithrombin is provided in the reaction.
Additional Information Therapeutic doses of unfractionated heparin require intense laboratory monitoring, because the amount of in vivo anticoagulation for a given dose is variable. That is, the dose-response for heparin is unpredictable. In contrast, LMWH and danaparoid do have a predictable dose-response, therefore, laboratory monitoring is usually not essential. In fact, if a LMWH or danaparoid antifactor Xa level is subtherapeutic, the most common causes are drawing the specimen at the wrong time (see below) or specimen transportation was longer than 2 hours. Most of the time, LMWH and danaparoid antifactor Xa levels are in the appropriate range when specimens are drawn correctly. Occasions in which periodic monitoring of LMWH might be considered include renal failure, pregnancy (increased dosage requirement in the third trimester), pediatric patients (increased dosage requirement in newborns), obesity, underweight patients, prolonged use, or patients at high risk for bleeding or thrombosis. It is probably also advisable to periodically monitor danaparoid in these same conditions.
The dose-response for unfractionated heparin is unpredictable because many of the heparin chains are long. The long chains can bind nonspecifically to a variety of proteins and cells, and the amounts of these heparin-binding proteins in particular vary considerably among patients, and even vary within the same patient at different times. In contrast, LMWH and danaparoid consist of shorter chains (ie, low-molecular weight) that have much less nonspecific binding.
Causes of subtherapeutic antifactor Xa level:
* specimen drawn at incorrect time (collection times are 4 hours after injection of LMWH, 6 hours after injection of danaparoid)
* specimen transportation longer than 2 hours
* patient receiving prophylactic dose, therefore, therapeutic range is not applicable and anti-Xa level is actually appropriate for dose
* higher dose needed (uncommon with LMWH or danaparoid, more common with heparin, eg, an acute phase state often increases the heparin dose requirement)
Causes of supratherapeutic antifactor Xa level:
* renal failure (with LMWH or danaparoid) (decreased renal clearance)
* heparin contamination, if specimen was drawn from a line
* lower dose needed (uncommon with LMWH or danaparoid, more common with heparin)
Footnotes
1. Teien AN and Lie M, “Evaluation of an Amidolytic Heparin Assay Method: Increased Sensitivity by Adding Purified Antithrombin III,”Thromb Res, 1977, 10:399-410.
References
Hirsh J, Warkentin TE, Raschke R, et al, “Heparin and Low-Molecular Weight Heparin. Mechanisms of Action, Pharmacokinetics, Dosing Considerations, Monitoring, Efficacy and Safety,”Chest, 1998, 114(5 Suppl):489-510.
Laposata M, Green D, Van Cott EM, et al, “College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy. The Clinical Use and Laboratory Monitoring of Low-Molecular Weight Heparin, Danaparoid, Hirudin and Related Compounds, and Argatroban,”Arch Pathol Lab Med, 1998, 122(9):799-807.
Olson JD, Arkin CF, Brandt JT, et al, “College of American Pathologists Conference XXXI on Laboratory Monitoring of Anticoagulant Therapy. Laboratory Monitoring of Unfractionated Heparin Therapy,”Arch Pathol Lab Med, 1998, 122(9):782-98.
