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The clinical laboratory plays a very important role in monitoring anticoagulant therapy. In fact, monitoring anticoagulant therapy with the PT was the first form of therapeutic drug monitoring.


Heparin is a natural mammalian glycosaminoglycan. The manufacture of heparin involves extraction from bovine lung tissue and porcine or bovine intestinal mucosa. In the US, therapeutic use of heparin is limited to unfractionated preparations that are heterogeneous with respect to molecular weights varying from 5000 to 30,000 + daltons. Low molecular weight heparins, introduced in Europe in 1990, has revolutionized heparin anticoagulant therapy. Low molecular weight heparin preparations are manufactured by enzymatic degradation of unfractionated heparins or various other processes. In general, low molecular weight heparins have significant differences in pharmacologic properties as well as their effect on various coagulation assays.

Comparison of Unfractionated and Low Molecular Weight Heparins

Property Low Molecular Weight Heparin

Unfractionated Heparin

Molecular weight



Activated PTT ++ ++++
Thrombin time ++ ++++
Anti-Xa assay +++ ++
Platelet aggregation + ++++
Binding to endothelium ++ ++
Protamine neutralization + ++++
Lipoprotein lipase + ++++
Absorption (Sub Q) +++ ++
Clinical bleeding (?) ++ +++
Heparin induced thrombocytopenia + +++

Heparin requires a naturally occurring plasma constituent to express its anticoagulant activities. This plasma protein is antithrombin III, an a-2 globulin produced in the liver. Antithrombin III is a member of the SERPIN family of proteins. Thus, the name antithrombin III is something of a misnomer. AT III inhibits all of the serine proteases involved in hemostasis with the exception of factor VIIa. Recent studies suggest AT III may inhibit VIIa when complexed with tissue factor, The inhibitory properties of antithrombin III are timed-dependent in the absence of heparin. With the addition of heparin, the ability of antithrombin III heparin complex to inhibit serine proteases is markedly accelerated.

The half-life of injected heparin is in the range of 1 - 2 hours. However, the disappearance and clearance times of heparin are dose-related. Higher doses are associated with decreased clearance and longer disappearance times. Heparin may be cleared from the circulation by the liver and kidney and endothelial cells also play a role in the clearance of heparin. Heparin may be neutralized in circulation by Platelet factor 4, which is a platelet-specific protein found in the a granule. Heparin also interacts with other plasma proteins including von Willebrand factor, fibronectin and vitronectin.

Although historically heparin was given subcutaneously as well as intermittent or continuous IV infusion, most patients today receive heparin by continuous infusion. Continuous infusion appears to be the preferred mode of administration, not only because of the ease of obtaining samples for monitoring the heparin effect, but because there appears be less clinical bleeding. The decrease in clinical bleeding is primarily related to a decrease in the cumulative dose a patient receives over the course of treatment. In the treatment of acute deep vein thrombosis (DVT), usually a loading dose of 5,000 - 10,000 units of heparin is administered immediately followed by approximately 1000 -1500 units per hour. Dosage may be affected by a variety of factors including the acute nature of the thrombolytic process, body weight and other complicating disease states, such as renal and liver disease.

The laboratory monitoring of heparin began with the use of whole blood clotting times (Lee and White) and has progressed to the recent introduction of synthetic substrates, which allow quantitation of heparin in units per ml. Based on the CAP survey program, > 95% of laboratories utilize the APTT as a means of monitoring heparin therapy. However, the activated clotting time (ACT) is frequently used in settings where immediate answers are required, such as when performing bypass procedures and in dialysis units.

APTT is readily available in most laboratories and because it is a part of an ongoing quality control program, the APTT is the preferred test for monitoring heparin therapy. Historically, the therapeutic target for the prolongation of the APTT was 1 1/2 to 2 1/2 times the upper limit of normal range. Recently there has been a move to use the patient's baseline APTT to determine the therapeutic range. This provides a more realistic goal and recognizes the frequent occurrence of short APTTs in patients presenting with acute DVT or pulmonary emboli.

Heparin-associated thrombocytopenia (HAT) is one of the most threatening complications of heparin therapy. The incidence of HAT has been reported as varying form less than 1% of patients to as high as 30%. There are two types of HAT. Type I is seen frequently after the initiation of heparin therapy and consists of a drop in the platelet count into the range of 100,000 to 150,000 /ml. This decrease in platelets may persist for several days; however, even though the patient continues to receive heparin, the platelet count subsequently returns to the original value prior to the initiation of heparin therapy. The exact mechanism for this type of HAT is still speculative, although many suggest it is the result of in vivo heparin-induced platelet aggregation with clearance of the aggregates in the liver and spleen.

Type II HAT is associated with antibodies directed against heparin. This antibody recognizes a repeating epitope in heparin. The complex of antiheparin and heparin then binds to a platelet membrane Fc receptor. Subsequent immune destruction results in a profound thrombocytopenia (less than 100,000 /ml) and often associated thromboembolic events. This form of HAT usually occurs after 6 days of heparin administration; however, it may be seen earlier in patients who have previously been exposed to heparin. Type II HAT is seen regardless of the route of administration or the cumulative dose. Consequently, this form of HAT may be seen in the ICU patients who are receiving heparin flushes through indwelling access catheters.

When Type II HAT is suspected, heparin should be discontinued immediately. Typically, in the patient receiving heparin for DVT, oral anticoagulants have already been initiated, and the transition to this form of anticoagulant therapy is relatively easy. In the setting where HAT develops in a patient who requires bypass surgery or renal dialysis, management is considerably more difficult. In some instances it is possible to use a different type of heparin (species of origin) and successfully perform the surgery, provided there is no evidence of cross-reactivity between the patient's antiheparin antibody and the second source of heparin. Other forms of therapy have included the use of dextran, low molecular weight heparin preparations or heparinoids, snake venoms to defibrinate the patient's plasma (Ancrod) and the use of Iloprost, a prostacyclin analog. There is no consensus as to the optimal approach.

The laboratory confirmation of the presence of Type II HAT is based on screening assays that are rather crude. Platelet aggregation studies should be performed in the presence of the heparin being utilized for the patient's treatment as well as at least two sources of platelet-rich plasma obtained from normal donors. The aggregation studies consist of heparin, normal platelet-rich plasma, and the patient's platelet-poor plasma mixed in the aggregation cuvette. The presence of aggregation greater than 20% suggests the possibility of HAT. It is important to appreciate that the absence of a positive result does not rule out HAT. A more specific test is the serotonin platelet release assay. In this assay, complexes of the heparin and IgG bind to the platelet Fc receptor, resulting in the release of serotonin, which can be quantitated. The presence of a positive serotonin release assay in considered diagnostic of HAT.

Oral Anticoagulant Therapy:

Oral anticoagulant therapy is based on the administration of coumarin or its derivatives. This class of drugs block the reductase enzymes in the vitamin K pathway, resulting in increased levels of nonfunctional vitamin K epoxide. Vitamin K acts as a cofactor in posttranslational step converting various vitamin-K dependent protein precursors into functional procoagulant proteins. This vitamin K-driven reaction involves the carboxylation of glutamic acid to give rise to g carboxyglutamic acid (GLA). These GLA residues function to localize the vitamin K-dependent proteins to phospholipid membrane surfaces. Typically, activated platelets provide this surface; however, other cells such as endothelial cells, monocytes, and tumor cells may function in a similar fashion.

In the presence of oral anticoagulants, formation of GLA residues is impeded. As a result, there is diminished surface localization of the vitamin K-dependent complexes, resulting in an anticoagulant effect. Early reports also suggested that in the presence of proteins induced by vitamin K antagonist (PIVKAs) serves an anticoagulant role as well. The PIVKA proteins represent the precursors of the functional procoagulants. The role in the therapeutic effect of oral anticoagulants remains unresolved.

The monitoring of oral anticoagulant therapy has relied upon the PT and variants of the PT. For many years there has been an international controversy regarding the optimal test system to monitor oral anticoagulants. In the U.S., tissue thromboplastins from various animal species have been used for the PT determination. Today, virtually, all of the thromboplastins marketed in the U.S. are of rabbit tissue origin (rabbit brain or rabbit brain/lung). Recombinant human tissue factor preparations are now available in the U.S. However, in Europe a number of different types of thromboplastins have been utilized including bovine brain, monkey brain, and human brain. Dr. Leon Poller championed the human brain thromboplastin as a sensitive reagent that would reflect the anticoagulant status of a patient more accurately. His efforts resulted in the concept of an international normalized ratio (INR) to express PT.

In order to report an INR value, it is necessary to know the international sensitivity index (ISI) for the thromboplastin being used as well as the mean of the PT range. The INR merely expresses the ratio of the patient PT to the mean of the normal range raised to the power of the ISI. The INT, therefore, represents the PT that would have been obtained on the patient plasma if the international reference preparation (IRP) of thromboplastin (human brain) had been utilized. Thus, it provides a means of comparing PT results from laboratory to laboratory correcting for the differences in thromboplastin reagents. The concept of the INR has been well received in Europe and is now being implemented in the

U.S. Keep in mind that the INR is only appropriate for patients who are stably anticoagulated. Patients who are in the early phases of oral anticoagulant therapy or patients in who the PT is being obtained for other diagnostic purposes should not have their results reported as an INR value.

The introduction of the INR has emphasized the need for lower doses of oral anticoagulants. In the past, when PT results were reported as ratios, the therapeutic range was typically quoted as PT ratios of 1.5 2.5. With the use of the INR, the majority of patients are satisfactorily anticoagulated with ratios of 1.3 1.6. As a consequence, the incidence of bleeding in patients receiving oral anticoagulant therapy will most likely significantly decrease.

College of American Pathologists Conference XXXI On Laboratory Monitoring of Anticoagulant Therapy

Laboratory Monitoring of Oral Anticoagulant Therapy:

Objective: To review the state of the art laboratory monitoring of oral anticoagulant therapy, as reflected by the medical literature and the consensus opinion of recognized experts in the field, and to make recommendations for improvement in laboratory monitoring of oral anticoagulant therapy.

Data Sources:

Review of the medical literature, primarily from the last 10 years, and current laboratory practice by a panel of 8 international experts in the field of oral anticoagulant monitoring.

Summary of Recommendations Pertaining to Clinical Monitoring:

During the initial phase of oral anticoagulant therapy, it is prudent to monitor patient status daily or at least 4 5 times per week, until some degree of consistency of the INR response to a stable dose is noted.

The frequency of testing in stabilized anticoagulated patients should be determined on an individual patient basis. In general, the INR should be determined at interval not exceeding 4 weeks.

Lupus anticoagulants can alter the PT. The effect may give rise to clinically important differences in INR values that may result in incorrect dosing. A normal baseline PT does not rule out an effect of a lupus anticoagulant on the INR during therapy.

CVMC uses the PT to monitor oral anticoagulation therapy. The mean population normal range for the PT is 11.2 - 12.7 seconds.

Laboratory Monitoring of Unfractionated Heparin Therapy:

Objective: To review the state of the art as reflected in the medical literature and the consensus opinion of recognized experts in the field regarding the laboratory monitoring of unfractionated heparin therapy.

Data Sources, Extraction and Synthesis:

The authors made an extensive review of the literature. The draft manuscript was circulated to every participant in the consensus prior to the convening of the conference. Extensive discussion concerning all of the issues addressed in the manuscript as well as the resulting recommendations occurred. This information was then used to revise the manuscript into its final form.

Summary of Recommendations Pertaining to Clinical Monitoring:

Fixed, low-dose heparin does not require anticoagulant monitoring.

Adjusted dose and therapeutic heparin require monitoring.

Upon initiation of therapy or after altering the dose of the continuous infusion of heparin, anticoagulant monitoring should occur at 6 hour intervals until a stable therapeutic response is reached.

Monitoring heparin therapy given by continuous infusion that has reached a stable therapeutic response should be monitored daily, at the same time of the day, preferably prior to 10 am.

CVMC uses the aPTT to monitor heparin therapy. The mean population normal range for the aPTT is 28.0 - 32.2 seconds.


College of American Pathologists Conference XXXI: Laboratory Monitoring of Anticoagulant Therapy, Archives of Pathology and Laboratory Medicine, September 1998, pages 765 822.

Clinical Laboratory Medicine, Edited by Kenneth D. McClatchey, M.D., D.D.S., Williams & Wilkins, 1994, pages 1088 - 1091.

Clinical Diagnosis and Management by Laboratory Methods, John Bernard Henry, M.D., 19th edition, W.B. Saunders Company, 1996, pages 740 - 743.