INTRODUCTION — Several tests of the coagulation system are available, including the prothrombin time (PT), activated partial thromboplastin time (aPTT), and others; these may be ordered in a variety of clinical settings.

This topic reviews the principles and interpretation of coagulation tests that are routinely available for clinical use.

Additional information regarding the use of these tests in specific clinical settings is presented separately:

Unexplained bleeding
Preoperative testing
Monitoring anticoagulation:
Warfarin
Heparin
Direct oral anticoagulants
Platelet function testing

ENSURING ACCURACY

Sample collection and handling — Coagulation tests must be performed on plasma rather than serum, because clotting factors are removed during serum preparation, along with the clotted cellular elements.

Accurate coagulation testing requires that the blood sample be collected and handled appropriately. The following parameters are important for ensuring accuracy:

Collection tube – Samples for testing coagulation must be drawn into a tube containing an inhibitor of coagulation that can be removed at the start of the assay. A sodium citrate solution (3.2 percent sodium citrate) in a tube with a light blue top is most commonly used. The amount of citrate solution in the tube is fixed to provide an appropriate ratio of one part citrate solution to nine parts whole blood when the tube is properly filled. Patients with polycythemia require removal of some of the citrate due to a reduced plasma volume. (See ‘Sources of interference’ below.)
Blood volume – The tube must be filled with enough blood to provide an appropriate ratio of citrate to whole blood. Underfilled tubes can result in artificially prolonged coagulation times. Tubes should not be uncapped as it will result in an incorrect volume of blood being added [1]. Tubes must be filled to within 90 percent of the full collection volume. If the tube is underfilled it may lead to inaccurate results. Improperly filled tubes should be discarded and a new draw requested [2]. Polycythemia effectively reduces the amount of plasma in the tube. (See ‘Sources of interference’ below.)
Mixing – Because blue top tubes contain a liquid sodium citrate solution, they should be inverted gently a few times as soon as possible after phlebotomy, in order to mix the citrate solution with the blood. The tube should not be shaken as this can cause hemolysis and may lead to inaccurate results.
Elapsed time and temperature – The sample should be tested in a timely manner to avoid degradation of the labile coagulation factors (especially factors V and VIII and protein S); substantial degradation of coagulation factors could lead to artificial prolongation of coagulation times [3]. The total time between phlebotomy and testing should not exceed 24 hours. Primary coagulations tubes cannot be frozen prior to separation of plasma from cells.

Sources of interference — Inaccurate results may occur if the following are present:

Intravenous solutions – Ideally, coagulation specimens should be obtained by percutaneous phlebotomy. A discard tube is not required when drawing blood from percutaneous phlebotomy [4,5]. However, in intensive care unit settings coagulation testing is often obtained from indwelling catheters. The sample must be free of solutions delivered through indwelling intravenous lines, which may dilute the sample and/or introduce heparin. This is especially important for blood obtained from central venous catheters or ports, which are often flushed with heparin or citrate solutions that could lead to artificial prolongation of coagulation times [6-9]. When drawing samples from indwelling lines, the first few milliliters withdrawn are discarded, and the required sample is obtained from a second syringe or tube to avoid contamination with solution in the line.
Anticoagulants – Good medical practice dictates awareness by the laboratory of anticoagulant therapy as this may greatly impact test interpretation and patient care. This may be done by the physician as part of the order entry procedure, by the laboratory personnel checking patient medications in the electronic medical record, or by contacting the ordering physician directly.
Other substances – Other substances present in the sample such as lipids or bilirubin due to lipemia, hyperbilirubinemia, and hemolysis can interfere with determination of clotting times. If it is not possible to avoid such an interference, dilution of the sample may allow an estimate of the clotting time. The need for sample dilution can be assessed by the laboratory at the time of testing.

Polycythemia (eg, hematocrit >55 percent) causes a corresponding reduction of plasma volume in the blood collection tube. Thus, patients with polycythemia require removal of some of the citrate solution to maintain the correct ratio of citrate to whole blood and to prevent artificial prolongation of coagulation times [10]. There are no corresponding recommendations for severe anemia. The best approach to such situations is to be aware of the potential interference and to contact the coagulation laboratory for guidance in proper collection if accurate clotting times are required for patient care.

SPECIFIC TESTS

Clotting times — Clotting times measure the time it takes plasma to clot when various substances are added. The citrate in the blue top collection tube chelates calcium in the collection tube so that coagulation is unable to proceed, because calcium is required for assembly of coagulation factor complexes on activated cell surfaces or phospholipids. Sufficient calcium to overcome the chelator is added back to the sample at the time of test initiation, along with a source of phospholipid and an initiator (tissue factor for the prothrombin time [PT]; silica or diatomaceous earth for the activated thromboplastin time [aPTT]). The precise composition of PT and aPTT reagents is proprietary and generally not disclosed. PT instrument reagent systems are standardized using the international normalized ratio (INR). (See ‘Prothrombin time (PT) and INR’ below.)

While the PT and aPTT provide an overall assessment of clot formation, they do not provide information about fibrin crosslinking or clot dissolution and will thus be insensitive to abnormalities of factor XIII function or abnormal fibrinolysis.

Prothrombin time (PT) and INR — The prothrombin time (PT) measures the time it takes plasma to clot when exposed to tissue factor, which assesses the extrinsic and common pathways of coagulation

The PT test is performed by recalcifying citrated patient plasma in the presence of tissue factor and phospholipid and determining the time it takes to form a fibrin clot. The formation of a fibrin clot is detected by visual, optical, or electromechanical methods. The result is measured in seconds and reported along with a control value and/or an INR.

The normal range for the PT varies by laboratory and reagent/instrument combination, and local institutional ranges should be used. In most laboratories, the normal range is approximately 11 to 13 seconds.

The INR is dimensionless. It is calculated as a ratio of the patient’s PT to a control PT obtained using an international reference thromboplastin reagent developed by the World Health Organization (WHO), using the following formula [11]:

INR = [Patient PT ÷ Control PT]^ISI

The control value for the PT is the mean normal PT for the laboratory determined from ≥30 fresh, normal plasmas handled identically to patient material. The ISI (international sensitivity index) is based on an international reference thromboplastin reagent; however, it is useful to have the ISI value confirmed within each laboratory for each PT reagent and instrument to account for effects of handling and equipment performance [12,13].

Unlike the PT, the results of the INR will be similar on a blood sample tested in any laboratory using any thromboplastin reagent/instrument system when calibrated correctly. This allows comparison of the patient’s testing performed at different times and/or locations, which is of great benefit for warfarin monitoring .Use of the INR is also extremely valuable for research studies because it allows investigators to compare the degree of anticoagulation of patients from different institutions.

Uses of the PT/INR — Clinical uses of the PT include the following:

Evaluation of unexplained bleeding
Diagnosing disseminated intravascular coagulation
Obtaining a baseline value prior to initiating anticoagulation ●Monitoring warfarintherapy –●Assessment of liver synthetic function

As noted above, the INR was developed to allow patients receiving warfarin at steady state to compare values obtained at different times and from different laboratories. The INR is also commonly used as a surrogate for the PT in assessing integrity of the extrinsic and common pathways in bleeding patients and to assess end-stage liver disease as part of the model for end-stage liver diseases (MELD) score.

Causes of prolonged PT — Causes of a prolonged PT include the following

Vitamin K antagonists – Vitamin K antagonists such as warfarininterfere with post-translational modifications of procoagulant factors II, VII, IX, and X, resulting in a prolonged PT
Other anticoagulants – Heparins (unfractionated or low molecular weight) and fondaparinuxshould in theory prolong the PT because they inhibit thrombin and/or factor Xa. However, most PT reagents contain heparin-binding chemicals (eg, heparinase, polybrene) that block this effect [14]. The PT may become elevated at heparin concentrations above 1 unit/mL, such as after a heparin bolus, due to saturation of the heparin binders. The increase in PT should not be used to guide therapy; monitoring of heparins is discussed separately

All of the available direct acting anticoagulants prolong the PT, including the parenteral direct thrombin inhibitor argatroban and the direct oral anticoagulants (DOACs) dabigatran, rivaroxaban, apixaban, and edoxaban. However, the degree of prolongation varies by the particular drug and the PT reagent used, and therefore the PT is not reliable for monitoring drug effect. All of the DOACs are approved for use without monitoring. We do not check the PT in an individual receiving a DOAC and we do not make changes to dosing or monitoring of these agents based on the result of the PT, with the possible exception of an individual who has serious bleeding for whom a prolongation of the PT may be taken as evidence of persistent drug effect. These subjects are addressed in separate topic reviews.

Vitamin K deficiency – Potential causes include impaired nutrition, prolonged use of broad spectrum antibiotics, or fat malabsorption syndromes. When vitamin K deficiency is mild, only the PT may be prolonged due to a predominant effect on factor VII. However, in severe vitamin K deficiency, both the PT and aPTT may be prolonged.
Liver disease – Liver disease may be associated with decreased production of both vitamin K-dependent and vitamin K-independent clotting factors. When liver disease is mild, only the PT may be prolonged due to a predominant effect on factor VII. However, in severe and/or chronic liver disease, both the PT and aPTT may be prolonged. Importantly, liver disease is also associated with decreased production of anticoagulant factors. Thus, a prolonged PT does not reflect the overall hemostatic picture
DIC – In disseminated intravascular coagulation (DIC), coagulation factors become consumed and depleted. This may result in prolonged PT and aPTT. Importantly, anticoagulant factors may also be depleted, and the PT does not reflect the overall hemostatic picture
Factor deficiency – Deficient activity of coagulation factors in the extrinsic pathway may be due to inherited disorders or acquired inhibitors (eg, autoantibodies). This includes deficiency of fibrinogen and factors II, V, VII, or X, or a combined deficiency involving one of these factors
Antiphospholipid antibodies – Lupus anticoagulants or antiphospholipid antibodies with specificity for prothrombin (factor II) may cause hypoprothrombinemia and prolongation of the PT; occasionally patients with such antibodies and high antibody titers present with hypoprothrombinemia and bleeding. The combination of high antiprothrombin antibody titers and low prothrombin levels can result in false-negative lupus anticoagulant tests because of a prozone effect . However, isolated prolongation of the aPTT is more common. (See ‘Causes of prolonged aPTT’ below and

As noted above, polycythemia (hematocrit >55 percent) can artificially prolong the PT if the amount of citrated anticoagulant solution in the collection tube is not appropriately decreased. (See ‘Sample collection and handling’ above.)

Activated partial thromboplastin time (aPTT) — The activated partial thromboplastin time (aPTT, PTT) measures the time it takes plasma to clot when exposed to substances that activate the contact factors, which assesses the intrinsic and common pathways of coagulation

The aPTT test is performed by recalcifying citrated plasma in the presence of a thromboplastic material that does not have tissue factor activity (hence the term partial thromboplastin) and a negatively charged substance (eg, celite, kaolin [aluminum silicate], silica), which results in contact factor activation, thereby initiating coagulation via the intrinsic clotting pathway . The thromboplastic material provides a source of phospholipids.

The normal range for the aPTT varies by laboratory and reagent/instrument combination, and local institutional ranges should be used. In most laboratories, the normal range is approximately 25 to 35 seconds.

There is no standardization of the aPTT test across different reagent/instrument systems analogous to the INR for the PT. Thus, aPTT values from different laboratories cannot be compared directly. For heparin monitoring, it is recommended that each laboratory establish the therapeutic range by determining the aPTT range that corresponds to 0.2 to 0.4 units/mL by protamine titration or 0.3 to 0.7 anti-factor Xa units/mL. (See ‘Monitoring heparins’ below.)

Uses of the aPTT — Clinical uses of the aPTT include the following:

Evaluation of unexplained bleeding
Diagnosing disseminated intravascular coagulation (DIC)
Obtaining a baseline value prior to initiating anticoagulation
Monitoring therapy with unfractionated heparin(for individuals with a normal baseline aPTT) –●Monitoring therapy with parenteral direct thrombin inhibitors (eg, argatroban, hirudin) –

Of note, low molecular weight (LMW) heparins often do not prolong the aPTT. If necessary, monitoring can be performed by testing for anti-factor Xa activity. However, laboratory monitoring is generally not necessary in nonpregnant patients, because the anticoagulant response to a fixed dose of LMW heparin is highly correlated with the patient’s body weight. Causes of prolonged aPTT — Causes of a prolonged aPTT include the following

Heparin – Heparin is an indirect thrombin inhibitor that complexes with antithrombin (AT), converting AT from a slow to a rapid inactivator of thrombin (factor IIa), factor Xa, and, to a lesser extent, factors IXa, XIa, and XIIa.

Heparin in the blood sample (eg, due to testing from an indwelling venous catheter) can falsely elevate the aPTT. Re-testing is indicated if this is thought to be the reason for the aPTT prolongation. In more complex cases (eg, unable to obtain peripheral sample, suspected surreptitious use of heparin), the reptilase time can be used to determine if heparin is the cause of the aPTT prolongation. (See ‘Reptilase time (RT)’ below.)

Direct thrombin inhibitors and direct factor Xa inhibitors – Direct thrombin inhibitors and direct factor Xa inhibitors both can cause prolongation of the aPTT, although there is not a well-defined correlation between the degree of prolongation and the degree of anticoagulation for the oral agents.
Other anticoagulants – Fondaparinuxmay cause mild prolongation of the aPTT.

Warfarin has a weak effect on most aPTT reagents, but supratherapeutic warfarin doses may increase the aPTT, and warfarin will increase the sensitivity of the aPTT to heparin effect .

Liver disease – When liver disease is mild, only the PT may be prolonged due to a predominant effect on factor VII. However, in severe and/or chronic liver disease, both the PT and aPTT may be prolonged. Importantly, liver disease is also associated with decreased production of anticoagulant factors. Thus, a prolonged aPTT does not reflect the overall hemostatic picture. ●DIC – As noted above, coagulation factors become consumed and depleted in patients with disseminated intravascular coagulation (DIC). This may result in prolonged PT and aPTT. Importantly, anticoagulant factors may also be depleted, and the aPTT does not reflect the overall hemostatic picture.
von Willebrand disease – von Willebrand disease (VWD) can cause prolongation of the aPTT because von Willebrand factor is the carrier (and stabilizer) of factor VIII. If factor VIII levels are sufficiently low, the aPTT may become prolonged. Other patients with VWD may have a normal aPTT.
Hemophilia A or B – Hemophilia A (hereditary deficiency of factor VIII) and hemophilia B (hereditary deficiency of factor IX) cause prolongation of the aPTT in individuals with severe or moderate factor deficiencies (eg, ≤15 percent activity). Some individuals with mild disease may have a normal aPTT.
Other inherited factor deficiencies – Additional inherited factor deficiencies that can cause a prolonged aPTT include the following:
Hereditary factor XI deficiency (sometimes called hemophilia C), which is common in Ashkenazi Jews.
Hereditary factor XII deficiency, which is not associated with clinical bleeding•Hereditary deficiencies of factors X, V, prothrombin (factor II), fibrinogen, or combined vitamin K-dependent factor deficiency.
Factor inhibitors – The most common factor inhibitors are to factor VIII. These may be alloantibodies (eg, in patients with severe hemophilia A who develop an immune response to transfused human factor VIII) or autoantibodies. Autoantibodies to factor VIII may be associated with autoimmune disease, other systemic illnesses, or present with bleeding with no apparent precipitant. It is important to distinguish between factor VIII inhibitors and other inhibitors in the aPTT assay such as lupus anticoagulants because factor VIII inhibitors may be associated with life-threatening bleeding whereas lupus anticoagulants may be associated with thrombosis. A distinguishing characteristic of factor VIII inhibitors is increased prolongation of the aPTT after one to two hours of incubation at 37°C relative to the degree of prolongation at five minutes of incubation
Lupus anticoagulant-type inhibitors – Some antiphospholipid antibodies (aPLs) can act as in vitro anticoagulants and cause prolongation of the aPTT. The effect is due to interference with assembly of the prothrombinase complex on phospholipids in the in vitro assay; in vivo, these antibodies do not increase bleeding risk, and they may be prothrombotic. Lupus anticoagulants are defined by demonstrating prolongation of a phospholipid-dependent assay that does not correct with addition of normal plasma but does correct with addition of excess phospholipid. (See ‘Lupus anticoagulant tests’ below.)

In a series of 55 children with a prolonged aPTT on preoperative testing, 39 (71 percent) had a lupus anticoagulant [18]. Causes included a variety of diagnoses (mostly benign but some potentially requiring treatment). Although lupus anticoagulants cause aPTT prolongation, the most common phenotype is increased risk of thrombosis rather than bleeding.

Lupus anticoagulants occasionally affect prothrombin and prolong the PT. (See ‘Causes of prolonged PT’ above.)

Author:

James L Zehnder, MD

Clinical use of coagulation tests