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.

Index of Tests

Heparin Neutralization [CO005100]

Related Information

Synonyms Heparinase; Hepzyme®

Applies to ACT; Heparin; PTT

Abstract Heparin contamination of specimens is a common cause of an unexpected PTT prolongation. Heparinase (Hepzyme®) can be used to determine if the PTT prolongation is due to heparin. In addition, while patients are receiving heparin, it is sometimes necessary to perform coagulation tests that are affected by heparin. In such cases, heparinase can be used to remove heparin from the specimen so that coagulation tests can be performed without heparin interference.

Specimen Plasma

Container Blue top (sodium citrate) tube

Collection Routine venipuncture. 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. Deliver tubes immediately to the laboratory.

Storage Instructions Separate plasma from cells as soon as possible, ideally within 1 hour of collection. Store plasma according to the guidelines for the individual postheparinase coagulation tests that will be performed.

Causes for Rejection Specimen received more than 4 hours after collection, tube not full, specimen clotted

Turnaround Time 1 day, unless testing is batched less frequently

Reference Interval If heparin is the explanation for the prolonged PTT, the PTT will become normal after treatment of the specimen with heparinase.

Use Determine if an unexpected PTT prolongation is due to heparin contamination; remove known heparin from a specimen so that coagulation tests can be performed without heparin interference

Methodology To determine if a prolonged PTT is due to heparin, measure the PTT before and after heparinase treatment. 1 mL of patient plasma is added to one vial of heparinase and kept at room temperature for 15 minutes. Heparinase is an enzyme that degrades unfractionated heparin (and low-molecular weight heparin) by cleaving it at multiple sites including within the pentasaccharide sequence. The pentasaccharide sequence is the antithrombin binding site and therefore is required for heparin anticoagulation. Heparinase degradation yields small fragments of about 1000 daltons that lack anticoagulant activity.1 Heparinase is produced by the bacterium Flavobacterium heparinum. Up to 2 units/mL heparin can be degraded. As an alternative to heparinase, heparin-binding cellulose can be used to remove heparin from specimens. The cellulose material is added to the specimen, where it binds to heparin. The specimen is then centrifuged, bringing cellulose and heparin into the pellet. The supernatant plasma is then free of heparin.

Additional Information Some laboratories use thrombin time to detect heparin. The thrombin time is very sensitive to heparin, therefore, if the thrombin time is normal, heparin cannot account for a PTT prolongation.

In one study, heparin contamination accounted for 39% of unexpected PTT prolongations in patients who were not receiving heparin or Coumadin® treatments.2 Heparin contamination may account for an unexplained PTT prolongation even when a heparinized line is carefully flushed to remove heparin prior to specimen collection.

If the PTT shortens significantly but remains prolonged after heparinase, a coagulation abnormality may be present in addition to heparin contamination. If a markedly prolonged PTT (eg, >150 seconds) shortens significantly but remains slightly prolonged after heparinase, a small amount of residual heparin is a possible explanation, because the initial amount of heparin contamination was very high. If a second heparinase treatment of the specimen produces a normal PTT, heparin is the confirmed explanation.


1. Lindhardt R, Grant A, Cooney CL, et al, "Differential Anticoagulant Activity of Heparin Fragments Prepared Using Microbial Heparinase,"J Biol Chem, 1982, 257:7310-3.

2. Newman RS and Fagin AR, "Heparin Contamination in Coagulation Testing and a Protocol to Avoid It and the Risk of Inappropriate FFP Transfusion,"Am J Clin Pathol, 1995, 104(4):447-9.


Ameer GA, Barabino G, Sasisekharan R, et al, "Ex Vivo Evaluation of a Taylor-Couette Flow, Immobilized Heparinase I Device for Clinical Application,"Proc Natl Acad Sci U S A, 1999, 96(5):2350-5.

Hutt ED and Kingdon HS, "Use of Heparinase to Eliminate Heparin Inhibition in Routine Coagulation Assays,"J Lab Clin Med, 1972, 79(6):1027-34.

Michelsen LG, Kikura M, Levy JH, et al, "Heparinase I (Neutralase) Reversal of Systemic Anticoagulation,"Anesthesiology, 1996, 85(2):339-46.


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