James L Zehnder, MD
Lawrence LK Leung, MD
Stephen A Landaw, MD, PhD
INTRODUCTION — Routine tests of blood coagulation, such as the prothrombin time (PT), activated partial thromboplastin time (aPTT), and thrombin time (TT) are frequently ordered to assess clotting function in patients.
The nature of these three tests and the evaluation of abnormal test results will be reviewed here. These tests are sensitive for a deficiency in one or more of the known procoagulants, but not whether the deficiency is counterbalanced by a parallel deficiency of anti-coagulants (eg, proteins C and S), which might be relevant in the evaluation of acquired coagulation defects .
Use of these tests to manage anticoagulation is discussed briefly here, but is also presented separately, as are overviews of the coagulation pathway and the approach to the patient with a bleeding disorder. (See “Therapeutic use of warfarin” and “Therapeutic use of heparin and low molecular weight heparin” and “Overview of hemostasis” and “Approach to the adult patient with a bleeding diathesis” and “Approach to the child with bleeding symptoms”.)
OBTAINING THE SAMPLE — A properly drawn blood sample is the key to interpreting the results of clotting tests. The following are guidelines for obtaining the best possible sample:
- Whole blood is collected into citrated anticoagulant, using an evacuated sample tube containing a fixed amount of citrate as anticoagulant, in the ratio of one part citrate solution to nine parts of whole blood. If the patient is polycythemic (ie, hematocrit >55 percent), the amount of plasma in the removed sample will be reduced compared with the amount of plasma present in the same volume of whole blood in a patient with a normal hematocrit. As a result, the volume of citrated anticoagulant solution needs to be decreased proportionately for patients with high hematocrits. If these guidelines are not followed, clotting times may be artifactually prolonged due to overanticoagulation of the sample .
- If the pediatric sample tube (volume 2.7 mL) is not filled to within 90 percent of its full collection volume, or an adult sample tube (volume 5 mL) is not filled to at least 60 to 80 percent of its full collection volume, there will be excessive anticoagulation of the sample, leading to prolonged clotting times [3,4]. Accordingly, incompletely filled sample tubes should be discarded and the sample redrawn.
- The sample must be free of tissue fluids, intravenous solutions delivered through indwelling lines, and heparin. This is especially important for blood samples obtained from central venous catheters, such as those used for hemodialysis or administration of chemotherapy [5-8].
- For sampling from indwelling lines and for difficult-to-obtain samples, especially those obtained by inexperienced personnel, a two-syringe technique is often used. The first few mL drawn into the first syringe (or evacuated tube) are discarded and the required sample is obtained from the second syringe or tube.
- The anticoagulated blood should be mixed gently by inversion three or four times, sent to the laboratory in an expeditious manner, and tested within two hours if kept at room temperature (22 to 24ºC) or within four hours if kept cold (2 to 4ºC).
For some situations, the two-syringe technique is unnecessary [9,10]. As an example, blood was drawn by experienced phlebotomists in a series of 175 outpatients, using a two-syringe technique . The mean differences between tubes one and two for the prothrombin time (PT) and activated partial thromboplastin time (aPTT) were 0.1 and 0.48 seconds, respectively (p values >0.8 for both). Similar conclusions were reached for determination of INR (see below) in a study of 241 outpatients receiving warfarin; the differences between tubes one and two were either statistically or clinically insignificant .
It is helpful for the laboratory to know if the patient is taking a medication (eg, heparin, warfarin) which may affect one or more of the coagulation tests, as well as whether the test is being ordered to monitor such therapy, or has been obtained for diagnostic reasons. This information may affect the type of testing performed. Thus, if the patient is known to be taking warfarin and a prothrombin time is ordered, the laboratory may only measure the INR (see ‘Measurement of INR’ below). If, on the other hand, the patient is not taking warfarin and the prothrombin time is prolonged, the laboratory may do additional testing, such as a mixing test to determine whether the prolongation is due to a factor deficiency or the presence of an inhibitor (see ‘Mixing studies’ below).
Normal values — All of the tests mentioned below have endpoints measured in seconds. The exception is the “INR”, which is dimensionless, since it is a ratio. For all of the tests described here, the normal ranges generally vary from one laboratory to another. Again, the exception is the INR, which takes sources of test variability (eg, reagents, laboratory equipment) into account (see ‘Measurement of INR’ below).
Normal values for each of the tests listed below (with the exception of the INR) are obtained by testing at least 30 normal, non-pregnant subjects using the same methodology as employed for the patient samples. The normal range (mean ± 2 standard deviations) for the testing laboratory should be stated whenever the laboratory reports its results.
PROTHROMBIN TIME — The prothrombin time (PT) (figure 1) is used to assess the extrinsic pathway of clotting, which consists of tissue factor and factor VII, and coagulation factors in the common pathway (factors II [prothrombin], V, X, and fibrinogen). (See “Overview of hemostasis”.)
In this test, clotting is initiated by recalcifying citrated patient plasma in the presence of thromboplastin (tissue factor). The endpoint for the PT (and also for the partial thromboplastin and thrombin times) is the time (in seconds) for the formation of a fibrin clot, which is detected by visual, optical, or electromechanical means. The sensitivity of the PT to reduced activity of the vitamin K-dependent factors within this pathway (ie, factors VII, X, and II; especially factor VII) comprises the rationale for the use of the PT to monitor warfarin therapy (see ‘Monitoring warfarin therapy’ below).
Results of the prothrombin time can be expressed in one of four different ways:
- PT with control value — The patient’s PT (in seconds) is reported along with the PT obtained from control (normal) plasma. The control value is needed since there can be significant inter-laboratory variability in the PT with different reagent/instrument combinations.
- PT expressed as INR — In order to promote standardization of the PT for monitoring oral anticoagulant therapy, the World Health Organization (WHO) developed an international reference thromboplastin, currently recombinant tissue factor, and recommended that the PT ratio be expressed as the International Normalized Ratio or INR . This allows values of the PT from various locations to be directly compared, as may happen when a patient taking warfarin has blood sampled at different laboratories. (See ‘Measurement of INR’ below.)
- Prothrombin time ratio (PTr) — The patient’s PT is expressed as a ratio, where PTr = (patient PT ÷ control PT). As an example, a PTr >1.2 was associated with a significantly increased risk of acute traumatic coagulopathy in a large multicenter retrospective study . In this study, reagents used had similar sensitivities (ISI range 1.03-1.09). A limitation of this method is that reagent/instrument variability may affect the results.
- Prothrombin time index (PTI) — The PTI is defined as PTI = (control PT)/(Patient PT) x 100. As the calculation is essentially the inverse of the PTr, the same limitations apply.
In the United States, most laboratories report the PT in seconds and also as the INR.
Causes of prolonged PT — In addition to the administration of warfarin, there are other causes of prothrombin time prolongation. These include (table 1):
- Vitamin K deficiency due, for example, to poor nutrition, or prolonged use of broad spectrum antibiotics. 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. (See “Overview of vitamin K”, section on ‘Deficiency’ and “Overview of the beta-lactam antibiotics”, section on ‘Hematologic reactions’.)
- Liver disease, which decreases the synthesis of both vitamin K-dependent and -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. (See “Acute liver failure: Prognosis and management”, section on ‘Coagulopathy’ and “Tests of the liver’s biosynthetic capacity (eg, albumin, coagulation factors, prothrombin time)”.)
- Deficiency or inhibition of factors VII , X, II (prothrombin), V, or fibrinogen (figure 1).
- The infrequent antiphospholipid antibodies (lupus anticoagulant phenomenon) with antiprothrombin activity. In such patients, the acquired prothrombin deficiency may be severe enough to cause clinical bleeding. (See “Pathogenesis of the antiphospholipid syndrome”.)
- While treatment with heparin does not normally prolong the PT (due to the addition of heparin-neutralizing materials to the PT reagent), the PT may be transiently elevated after bolus administration of heparin.
Monitoring warfarin therapy — The anticoagulant effect of warfarin is mediated by inhibition of the vitamin K-dependent gamma-carboxylation of coagulation factors II, VII, IX, and X . This results in the synthesis of immunologically detectable but biologically inactive forms of these coagulation proteins. (See “Vitamin K, gamma carboxyglutamic acid, and the function of coagulation and other proteins”.)
The full anticoagulant effect of warfarin is delayed until the normal clotting factors are cleared from the circulation, and the peak effect does not occur until 36 to 72 hours after drug administration . During the first few days of warfarin therapy, prolongation of the PT mainly reflects depression of factor VII, which has a half-life of only five to seven hours; thus, the intrinsic coagulation pathway that does not require factor VII remains intact (figure 2). Equilibrium levels of factors II, IX, and X are not reached until about one week after the initiation of therapy. (See “Therapeutic use of warfarin”, section on ‘Mechanism of action’.)
Measurement of INR — The INR, which compensates for differences in sensitivity of various PT reagents to the effects of warfarin, is used to monitor warfarin therapy . The INR is calculated from the following formula:
INR = [Patient PT ÷ Control PT]ISI
The ISI (international sensitivity index) should be determined for each PT reagent and instrument combination. Although the ISI is traceable to an international reference thromboplastin reagent, it is useful to have the ISI value confirmed within each laboratory, since this may be affected by differences in handling of the reagents and the type of equipment used [15,16]. The control value for the PT is the mean normal prothrombin time for the laboratory, and should be determined from ≥20 fresh normal plasmas handled identically to patient material. Apparatus that allows the patient to monitor warfarin therapy at home is now available (figure 3). (See “Outpatient management of oral anticoagulation”.)
Using warfarin, the therapeutic range for the INR varies with the clinical indication. For most indications the recommended range is 2.0 to 3.0 . A higher (or lower) INR range may be recommended in certain specific clinical settings. (See “Therapeutic use of warfarin”, section on ‘Initial dose’ and “Therapeutic use of warfarin”, section on ‘Maintenance therapy’ and “Antithrombotic therapy in patients with prosthetic heart valves” and “Treatment of the antiphospholipid syndrome”, section on ‘Specific clinical settings’ and “Treatment of lower extremity deep vein thrombosis”, section on ‘Idiopathic VTE’.)
Appropriate treatment of a patient with an INR that is prolonged beyond the therapeutic range (ie, a prolonged or excessive INR) is discussed separately. (See “Correcting excess anticoagulation after warfarin”.)
The INR in liver disease — The INR was specifically devised for monitoring patients undergoing treatment with vitamin K antagonists. Accordingly, the INR may not be directly applicable to patients with liver disease, whose pattern of changes in both vitamin K-dependent and vitamin K-independent procoagulant and anticoagulant factors are dissimilar . This subject is discussed in detail separately. (See “Tests of the liver’s biosynthetic capacity (eg, albumin, coagulation factors, prothrombin time)”, section on ‘International normalized ratio’ and “Coagulation abnormalities in patients with liver disease”, section on ‘Prothrombin time (PT) and the International Normalized Ratio (INR)’.)
Monitoring warfarin therapy in the antiphospholipid syndrome — Several reports have challenged the reproducibility and validity of the PT(INR) for monitoring patients with lupus anticoagulants who are taking warfarin [18,19]. If this is a problem, alternative methods of monitoring warfarin effect which are insensitive to the presence of a lupus anticoagulant are required. There are a number of possible solutions to this problem:
- Use of a chromogenic factor Xa assay
- Use of the prothrombin-proconvertin time instead of the INR (not available in the United States)
- Use of a commercial reagent insensitive to the patient’s particular antiphospholipid antibody.
This problem is discussed in detail elsewhere. (See “Treatment of the antiphospholipid syndrome”, section on ‘Problems in monitoring INR’.)
ACTIVATED PARTIAL THROMBOPLASTIN TIME — The activated partial thromboplastin time (aPTT or PTT) (figure 1) is used to assess the integrity of the intrinsic coagulation pathway (prekallikrein, high molecular weight kininogen, factors XII, XI, IX, VIII) and final common pathway (factors II, V, X, and fibrinogen), and to monitor heparin therapy. (See “Overview of hemostasis”.)
The 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, silica), which results in contact factor activation, thereby initiating coagulation via the intrinsic clotting pathway .
Causes of prolonged aPTT — Prolongation of the aPTT can occur with a deficiency of, or an inhibitor to, any of the clotting factors except for factor VII (figure 1 and table 1). In addition, certain lupus anticoagulants, which are antibodies directed against plasma proteins bound to anionic phospholipids, cause aPTT prolongation by interfering with the in vitro assembly of the prothrombinase complex. This in vitro event is paradoxically associated with an increased risk of venous and arterial thrombosis. (See “Clinical manifestations of the antiphospholipid syndrome”.)
At present, aPTT methods are not standardized in a manner analogous to that used to determine the INR from the prothrombin time (see ‘Measurement of INR’ above). As a result, different reagent and instrument combinations result in variations in test results . Of importance, some commercial reagents have reduced sensitivity to heparin and may not be suitable for monitoring the effect of heparin.
Monitoring heparin therapy — Heparin is an indirect thrombin inhibitor which complexes with antithrombin (AT), converting this circulating cofactor from a slow to a rapid inactivator of thrombin, factor Xa, and to a lesser extent, factors XIIa, XIa, and IXa. In patients with venous thromboembolism, adequate heparin anticoagulation must be achieved within the first 24 hours to reduce the risk of recurrence . (See “Treatment of lower extremity deep vein thrombosis”, section on ‘Unfractionated heparin’.)
The critical therapeutic level of heparin to be reached within 24 hours (as measured by the aPTT) is 1.5 times the mean of the control value or the upper limit of the normal aPTT range. The goal of maintenance heparin therapy is to maintain the aPTT in the range of 1.5 to 2.5 times the patient’s aPTT baseline value. This level of anticoagulation corresponds roughly to a heparin blood concentration of 0.2 to 0.4 units/mL by the protamine sulfate titration assay, and 0.3 to 0.7 units/mL by the chromogenic anti-factor Xa heparin assay. (See “Therapeutic use of heparin and low molecular weight heparin”, section on ‘Heparin monitoring’.)
There is, however, a wide variability in the aPTT and heparin blood levels with different reagents and even with different batches of the same reagent. As a result, it is recommended for each laboratory to establish the minimal therapeutic level of heparin, as measured by the aPTT, that will provide a heparin blood level of at least 0.2 units/mL by the protamine titration assay for each batch of thromboplastin reagent being used; this is particularly important if the reagent is provided by a different manufacturer.
As with unfractionated heparin, low molecular weight heparins (LMWHs) inactivate factor Xa, but have a lesser effect on thrombin because most of the molecules are not long enough to bind to thrombin and AT simultaneously. As a result, LMWHs often do not prolong the aPTT. However, laboratory monitoring is not necessary in nonpregnant patients because the anticoagulant response (anti-Xa activity) to a fixed dose of LMW heparin is highly correlated with the patient’s body weight, and there is little correlation between anti-Xa activity and either bleeding or recurrent thrombosis. If necessary, monitoring can be performed by testing for anti-factor Xa activity. (See “Therapeutic use of heparin and low molecular weight heparin” and “Anticoagulation during pregnancy”, section on ‘LMW heparin’.)
THROMBIN TIME — The thrombin time (TT) measures the final step of the clotting pathway, the conversion of fibrinogen to fibrin. The test is performed by recalcifying citrated plasma in the presence of dilute bovine or human thrombin and recording the time (in seconds) for a clot to form .
Causes of prolonged TT — The following conditions cause a prolongation of the TT :
- The presence of heparin, a direct thrombin inhibitor such as hirudin, hirulog, or argatroban, or heparan-like compounds (eg, danaparoid). (See “Anticoagulants other than heparin and warfarin”, section on ‘Direct thrombin inhibitors’.)
- The presence of fibrin/fibrinogen degradation products
- Hypofibrinogenemia (<100 mg/dL), dysfibrinogenemia, or hyperfibrinogenemia (>400 mg/dL). (See “Disorders of fibrinogen”.)
- Bovine thrombin antibodies from prior exposure to bovine thrombin (only when tested using bovine thrombin reagent; TT will be normal if tested using human thrombin reagent)
- High concentrations of serum proteins, as in multiple myeloma and amyloidosis.
Patients who have been exposed to bovine thrombin during surgery may develop antibodies to bovine thrombin. In general, such antibodies are specific for bovine thrombin and are usually not associated with an increased bleeding risk unless they crossreact with human thrombin or the patient has coexisting inhibitors to other clotting factors.
Occasional patients exposed to topical bovine thrombin develop antibodies to bovine factor V present in the topical thrombin preparation, which crossreact with human factor V, causing prolongation of the PT and aPTT and a significant bleeding risk [25,26]. (See “Acquired inhibitors of coagulation”, section on ‘Thrombin inhibitors’.)
Reptilase time — Reptilase is an enzyme similar to thrombin that is found in the venom of Bothrops snakes. However, it differs from thrombin by generating fibrinopeptide A but not fibrinopeptide B from fibrinogen and by resisting inhibition by heparin via antithrombin.
The reptilase time (RT) is similar to the TT in measuring the conversion of fibrinogen to fibrin . The reptilase time is useful for detecting abnormalities in fibrinogen (in which case the TT is also prolonged) and in detecting the presence of heparin (heparin will cause prolongation of the TT but not reptilase time). Thus, the RT is most useful for determining if heparin is the cause of a prolonged TT (see ‘Heparin in the sample’ below).
Thrombin inhibitors — Direct thrombin inhibitors such as hirudin, hirulog, and argatroban are associated with prolongation of the PT, aPTT, and TT, but will not prolong the reptilase time. Argatroban, and probably the other direct thrombin inhibitors, can be effectively monitored with use of the aPTT or activated clotting times, but it interferes with the INR and aPTT for monitoring concomitant warfarin and heparin therapy, respectively, as well as routine clot-based assays for fibrinogen and protein C . Specialized tests are available for monitoring such patients . (See “Anticoagulants other than heparin and warfarin”.)
ANTI FACTOR XA ACTIVITY — Anti factor Xa activity is often measured in order to determine adequacy of anticoagulation in patients receiving agents that directly or indirectly interfere with factor Xa activity (eg, heparin, low molecular weight heparins, direct factor Xa inhibitors). This subject is discussed separately. (See “Therapeutic use of heparin and low molecular weight heparin”, section on ‘Heparin monitoring’ and “Therapeutic use of heparin and low molecular weight heparin”, section on ‘Anti-factor Xa testing’.)
ACTIVATED WHOLE BLOOD CLOTTING TIME — The activated whole blood clotting time (ACT) is performed by addition of an activating agent (eg, celite, kaolin) to a sample of freshly-drawn whole blood and measuring the time (in seconds) for formation of a clot. This test has been generally replaced by the activated partial thromboplastin time (aPTT) for the clinical evaluation of coagulation defects, as well as for monitoring heparin therapy (see ‘Activated partial thromboplastin time’ above).
However, the aPTT often becomes infinitely prolonged when heparin concentrations exceed 1.0 U/mL, as employed in coronary artery bypass surgery or percutaneous coronary artery interventions. For such procedures, heparin monitoring is usually performed via the ACT, since this test shows a graded response to heparin concentrations in the range of 1 to 5 U/mL . Point-of-care instruments are available for this purpose, and have been successfully employed in the operating room, cardiac catheterization laboratory, and dialysis suites [30,31]. (See “Hemodialysis anticoagulation”, section on ‘Standard anticoagulation’ and “Antithrombotic therapy for intracoronary stent implantation: Clinical trials”, section on ‘Heparin’.)
The ACT is insensitive to the heparin pentasaccharide fondaparinux, and may be less sensitive to some low molecular weight heparins such as enoxaparin . However, the test has been successfully used to monitor treatment with dalteparin . As with the aPTT, test results vary with the system employed, and need to be rigorously standardized in order to prevent over- or under-anticoagulation with these agents [30,32].
FIBRIN D-DIMER — Plasmin cleaves fibrin at multiple sites and releases fibrin degradation products (FDPs). One of the major FDPs is D-dimer, which consists of two D domains from adjacent fibrin monomers that have been crosslinked by activated factor XIII. (See “Thrombotic and hemorrhagic disorders due to abnormal fibrinolysis”, section on ‘Fibrin degradation products and fibrin D-dimer’.)
Since D-dimer is generated from cross-linked fibrin, but not from fibrinogen, an elevated plasma concentration of D-dimer indicates recent or ongoing intravascular blood coagulation. A listing of the various conditions that lead to an increased D-dimer level is shown in the table (table 2).
ECARIN CLOTTING TIME — The ecarin clotting time (ECT) is a coagulation test that has been used to monitor treatment with direct thrombin inhibitors . Ecarin is a metalloproteinase derived from the venom of the saw-scaled viper Echis carinatus, which activates prothrombin to meizothrombin, an intermediate step in the conversion of prothrombin to thrombin. Meizothrombin has markedly reduced fibrinogen clotting activity compared with thrombin, and is not susceptible to inhibition by antithrombin or the heparin-antithrombin complex (due to steric hindrance), but it can readily complex with direct thrombin inhibitors (DTIs; eg, hirudin, lepirudin, argatroban, dabigatran). Accordingly, the ECT is prolonged with increasing amounts of these DTIs [35-37]. (See “Anticoagulants other than heparin and warfarin”, section on ‘Direct thrombin inhibitors’.)
Although it has been suggested that the ECT be used to assess drug levels in patient taking the new DTI dabigatran, this test is not currently available in most coagulation laboratories. The aPTT and TT can also be used to assess dabigatran effect and are much more widely available .
EVALUATION OF ABNORMAL CLOTTING TIMES — The above clotting tests are primarily used to evaluate a patient with a possible bleeding diathesis or for monitoring therapy with warfarin or heparin. (See “Approach to the adult patient with a bleeding diathesis”.) Prolonged values are abnormal in the untreated patient, while shortened values may be predictive of an increase in coagulability (see ‘Shortened clotting times’ below).
Prolonged clotting times — Simultaneously drawn PT (INR) and aPTT are needed in order to properly evaluate the coagulation system (table 1).
- If the aPTT is prolonged and the PT (INR) is normal, the problem is localized to the intrinsic pathway. Although the aPTT is abnormal in deficiencies of factors XII, prekallikrein, or high molecular weight kininogen, these disorders are not associated with clinical bleeding. The most common inherited bleeding disorders giving this picture are von Willebrand disease and isolated deficiencies of factors VIII, IX, and XI, while common acquired causes of this pattern are heparin therapy (see ‘Heparin in the sample’ below) and the lupus anticoagulant phenomenon caused by the presence of antiphospholipid antibodies (see ‘Antiphospholipid antibodies’ below).
- If the aPTT is normal and the PT (INR) is prolonged, the problem lies in the extrinsic pathway (factor VII). The most common acquired causes are the use of warfarin, chronic liver disease, and vitamin K deficiency. An acquired inhibitor of factor VII and congenital factor VII deficiency are rare causes. (See “Acquired inhibitors of coagulation”, section on ‘Factor VII inhibitors’ and “Rare (recessively inherited) coagulation disorders”, section on ‘Factor VII deficiency’.)
- If both the aPTT and PT (INR) are prolonged, the problem is likely to be in the final common pathway. In this situation, the thrombin time (TT) can be used to assess the last step in the pathway, conversion of fibrinogen to fibrin. If the TT is normal, the problem resides in the common pathway, due to abnormalities in factors II (prothrombin), V, or X. Common acquired conditions giving a prolonged PT and aPTT and a normal TT are liver disease, disseminated intravascular coagulation, and overanticoagulation with coumadin. Conditions that prolong the TT are noted above (see ‘Causes of prolonged TT’ above).
- If both the aPTT and the PT (INR) are normal in a patient with a bleeding diathesis, then thrombocytopenia, mild deficiency of von Willebrand factor, platelet dysfunction, vascular disorders, and, rarely, factor XIII deficiency or a disorder of the fibrinolytic system should be considered. (See “Approach to the adult patient with a bleeding diathesis”, section on ‘Normal PT and aPTT’.)
Shortened clotting times — The primary utility of coagulation tests is to detect clotting factor deficiencies or inhibitors, which result in prolonged clotting times. Under most circumstances, shortening of clotting times (PT, aPTT, or TT) reflects poor sample collection or preparation techniques. However, clotting factors may be increased or activated in vivo, as in malignancy, disseminated intravascular coagulation, or following short-term exercise, resulting in shortening of clotting times, especially the aPTT .
A shortened clotting time that does not appear to reflect technical error has been associated with an increased risk of thrombosis, recurrent thrombosis, recurrent miscarriage, or bleeding, and may increase the risk of thrombosis associated with other common thrombotic risk factors (eg, factor V Leiden, obesity, increased levels of D-Dimer) [40-47].
Mixing studies — After an abnormality in a clotting test has been detected, it is important to differentiate between a clotting factor deficiency and an inhibitor (eg, an antibody or other interfering substance (eg, heparin) directed against the clotting factor). This is accomplished by performing a mixing study, in which the patient’s plasma is mixed in a 1:1 ratio with normal pooled plasma, and the abnormal tests are repeated.
There are three important principles underlying such mixing tests:
- As a general rule, clotting tests will give normal values when 50 percent activity of the involved coagulation factors are present. Thus, if the clotting test returns to normal after a 1:1 dilution with normal pooled plasma, a factor deficiency was the cause of the abnormal test.
- Most agents that inhibit clotting factor activity (such as antibodies) will not be effectively diluted out after addition of an equal volume of normal pooled plasma. Thus, if the test remains abnormal after 1:1 dilution, an inhibitor was the cause of the abnormal test.
- Some inhibitors may give normal results when tested immediately after 1:1 dilution; incubation of the diluted sample for up to two hours at 37ºC may resolve this issue. As an example, delayed reactivity is characteristic of factor VIII inhibitors . (See “Acquired inhibitors of coagulation”, section on ‘Factor VIII inhibitors’.)
If the 1:1 dilution corrects the abnormal test, the deficient factor(s) can be determined by individual clotting factor assays. If the test is not corrected by dilution, the most common inhibiting factors are:
- Heparin in the sample (see ‘Heparin in the sample’ below).
- Antiphospholipid antibodies; these are more commonly associated with a hypercoagulable state rather than bleeding. (See “Pathogenesis of the antiphospholipid syndrome”.)
- Inhibitors directed against factors VIII, IX, or X. These inhibitors can be associated with life-threatening bleeding and require urgent attention by a hematologist and the involved laboratory. (See “Acquired inhibitors of coagulation”.)
- Inhibitors of thrombin, such as fibrin or fibrinogen degradation products. These can be detected using a variety of assays (such as fibrin degradation products or D-dimer).
- Inhibitors to other factors. These are less common and are detected by individual factor assays. (See “Acquired inhibitors of coagulation”.)
Antiphospholipid antibodies — The presence of an antiphospholipid antibody (aPL) producing the lupus anticoagulant phenomenon is suggested by the presence of a prolonged aPTT which does not correct after 1:1 dilution with normal pooled plasma in a nonbleeding patient with or without a known rheumatologic disorder. Demonstrating that addition of phospholipid corrects the clotting time confirms the presence of the lupus anticoagulant phenomenon.
Clotting tests can be made more sensitive to the presence of aPL by reducing the concentration of lipid material (dilute PT or PTT), increasing preincubation times, or activating coagulation factors in vitro with kaolin (kaolin plasma clotting time).
The dilute Russell viper venom test employs a 1:1 mixture of patient and control plasma along with limiting quantities of thromboplastic material. In this way it is sensitive to the amount of phospholipid available for assembly of the prothrombinase complex, and thus for the presence of antiphospholipid antibodies.
The dilute Russell’s viper venom time (dRVVT) is particularly sensitive to the presence of anti-ß2-glycoprotein I antibodies (which are most closely correlated with thrombotic events), while the kaolin plasma clotting time is more sensitive to antiprothrombin antibodies . (See “Pathogenesis of the antiphospholipid syndrome”.)
However, there is currently no standardized test for the lupus anticoagulant phenomenon, and no internationally accepted reference or control materials. Furthermore, the test chosen, the source of the reagents, and instruments used in the assay all affect the ability to detect a lupus anticoagulant [50,51].
The presence of an aPL is confirmed by demonstrating correction of the clotting time by added phospholipid. This can be in the form of hexagonal phospholipid, or phospholipid from freeze-thawed platelets . (See “Pathogenesis of the antiphospholipid syndrome”, section on ‘Antiphospholipid antibodies’.)
Heparin in the sample — A first step in evaluating an isolated prolonged aPTT or TT is to exclude heparin as the etiology. This is especially important in hospitalized patients, in whom blood is often drawn from heparinized venous access devices. The simplest approach is to redraw a blood sample using an uncontaminated peripheral vein. Alternatively, one can use one of the following two methods:
- Reptilase time method: perform a thrombin time (TT) and reptilase time (RT). Heparin is present if the TT is prolonged and the RT is normal.
- Heparin reversal method: if a prolonged TT is normalized after the addition of protamine, a commercially available ion exchange resin which absorbs heparin in the sample (Heparsorb), or a heparinase (Hepzyme), heparin or a heparin-like material is present.
POINT OF CARE TESTING — A number of coagulation tests can be performed at or near the patient (ie, at the point of care), rather than in a central laboratory, with the rationale that this will result in the rapid generation of results and improve patient care. Such testing includes the following :
- Prothrombin time and INR (see “Outpatient management of oral anticoagulation”, section on ‘Self-monitoring and self-management’)
- Activated partial thromboplastin time
- Activated clotting time (see ‘Activated whole blood clotting time’ above)
- Thrombin time
- Thromboelastography (see “Platelet function testing”, section on ‘Thromboelastography’)
- Platelet function tests (see “Platelet function testing”, section on ‘Platelet aggregometry’)
Point of care testing is subject to the same strict quality control procedures that exist in central laboratories; one challenge with POCT is to enforce strict adherence to quality standards outside of the clinical laboratory setting. A discussion of the currently available tests and their limitations has been presented .
The blood sample — A properly drawn blood sample is the key to interpreting the results of clotting tests. The sample must be free of tissue fluids, intravenous solutions delivered through indwelling lines, and heparin. For sampling from indwelling lines and for difficult-to-obtain samples, especially those obtained by inexperienced personnel, a two-syringe technique is often used (see ‘Obtaining the sample’ above).
The prothrombin time — The prothrombin time is used to assess the extrinsic pathway of clotting (figure 1), (see ‘Prothrombin time’ above), which consists of tissue factor and factor VII, and coagulation factors in the common pathway (factors II [prothrombin], V, X, and fibrinogen).
The prothrombin time may be used to monitor therapy with coumadin and other vitamin K antagonists. This function has been standardized through use of the INR (see ‘Measurement of INR’ above)
The activated partial thromboplastin time — The activated partial thromboplastin time (aPTT or PTT) (figure 1) is used to assess the integrity of the intrinsic coagulation pathway (prekallikrein, high molecular weight kininogen, factors XII, XI, IX, VIII) and final common pathway (factors II, V, X, and fibrinogen), as well as to monitor heparin therapy (see ‘Activated partial thromboplastin time’ above).
The thrombin time — The thrombin time (TT) measures the final step of the clotting pathway, the conversion of fibrinogen to fibrin through the addition of exogenous thrombin (figure 1), (see ‘Thrombin time’ above).
Evaluating abnormal clotting times — Simultaneously drawn prothrombin time (PT) and activated partial thromboplastin time (aPTT) are needed in order to properly evaluate the coagulation system. Under appropriate circumstances, the thrombin time may also need to be obtained. The table outlines the various combinations of test results that may be obtained, and the conditions most likely to cause these changes (table 1). (See ‘Evaluation of abnormal clotting times’ above.)
For each abnormal test result obtained, mixing the patient’s sample in a 1:1 ratio with normal pooled plasma will help to determine if the abnormality is due to a factor deficiency or an inhibitor (see ‘Mixing studies’ above):
- If the test is corrected, a factor deficiency is present
- If the test remains abnormal, an inhibitory substance is present
The most common inhibitory factor is heparin, which can be determined by simultaneous thrombin and reptilase times. Heparin is present if the thrombin time is prolonged and the reptilase time is normal (see ‘Heparin in the sample’ above).
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