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]
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
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
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,
Michelsen LG, Kikura M, Levy JH, et al, "Heparinase I (Neutralase)
Reversal of Systemic Anticoagulation,"Anesthesiology, 1996,