Coagulation Testing in the Core Laboratory

William E. Winter, MD; Sherri D. Flax, MD; Neil S. Harris, MD


Lab Med. 2017;48(4):295-313. 

In This Article

Coagulation Testing

Clinical tests of coagulation are functional assays that evaluate the rate of clot formation from the time that the coagulation cascade is activated. These tests are commonly used to identify defects of the extrinsic, intrinsic, and final common pathways of the coagulation cascade so that more advanced testing can be done to identify specific defects. The PT, aPTT, TT, and fibrinogen measurements are commonly used to detect abnormalities of secondary hemostasis. The measurement of fibrinogen is commonly used to detect fibrinogen deficiencies and defects in the fibrinogen activity of plasma.

Prothrombin Time

PT measures the function of the extrinsic and final common pathways of coagulation (Figure 15). Dr. Armand Quick, an American physician, described the test in 1935. The PT is sensitive to factors of the extrinsic (FVII) and common (factors II, V and X) pathways as well as fibrinogen (Table 5). The PT is commonly used to monitor warfarin anticoagulant therapy. This is often performed as a point of care test. Very high levels of heparin, high levels of antiphospholipid antibodies and elevated levels of fibrin split products may prolong the PT (Table 6).

The PT is performed as a 1-stage (quick) assay. The patient's plasma is added to a pre-warmed commercial thromboplastin/CaCl2 reagent. The thromboplastin/CaCl2 is pre-warmed to enhance the activation of the enzymatic coagulation factors. Sodium citrate is the preferred anticoagulation for patient sample collection. This places the coagulation cascade in a state of stasis until testing can be performed. The addition of calcium chloride restores the calcium required for coagulation and effectively replaces the calcium that was bound by sodium citrate. The resulting clot formation is detected by increased impedance or turbidity (mechanical clot detection) or decreased optical clarity (optical clot detection), based on the instrumentation used. The time to clot formation is then measured to the nearest 0.1 second. The reference interval for the PT is generally 10 to 13 seconds and varies with the type of thromboplastin used in the testing procedure and the method of clot detection.

Several point-of-care instruments are available for PT testing. Amperometric (electrochemical) clot detection system after the activation of coagulation with human recombinant thromboplastin is the basis of the CoaguChek (Roche Diagnostics, Indianapolis, IN) instrumentation. The Hemachron Signature Elite (Accriva Diagnostics San Diego, CA) uses a mechanical-optical method to detect a true end-point clot.

International Normalized Ratio (INR). Prior to the development of commercial thromboplastins, individual laboratories somewhat crudely prepared thromboplastin using a variety of techniques and animal sources, most often brain tissue. These preparations differed widely in their sensitivity to decreased levels of clotting factors. Commercial preparations of thromboplastin are derived from a variety of tissue sources, including rabbit brain, rabbit brain-lung mixtures, human placenta, and recombinant human tissue sources. The relative differences in the sensitivity of commercial thromboplastin reagents are indicated by the International Sensitivity Index (ISI). The World Health Organization (WHO) thromboplastin is assigned an ISI of 1.0. Commercial thromboplastins are calibrated using the WHO standard and assigned an ISI value to indicate their relative sensitivity. A commercial thromboplastin with a low ISI, near 1.0, is very sensitive to the presence or absence of functional clotting factors. A low ISI thromboplastin will be more useful in the detection of clotting factor deficiencies, in the extrinsic and common pathways, than a thromboplastin with a high ISI. The ISI also is specific for the instrument used for PT testing.

The PT is commonly used to monitor anticoagulation with warfarin therapy, which antagonizes vitamin K. The sensitivity of the PT varies according to the source of thromboplastin, but the assay also varies according to the instrument used. To correct for these differences, the international normalized ratio (INR) was developed to improve standardization of PT reporting globally. The INR represents the PT ratio that would be obtained if the international reference thromboplastin had been used to test the patient. The INR is calculated by plotting the logarithms of PT results obtained on normal individuals and patients on warfarin using a primary international reference thromboplastin, against the results the logarithms of PT results obtained using the testing laboratory's thromboplastin. The resulting slope of the comparison is the ISI for the particular thromboplastin-instrument combination. The PT ratio is derived by dividing the patient PT result by the geometric mean normal PT for the testing laboratory, raised to the power of the ISI of the thromboplastin/instrument combination used in the assay.

INR = (patient PT in seconds/mean normal PT in seconds)ISI

The ISI must be specified for each new lot of thromboplastin, in addition to identifying the laboratory instrument used for PT testing.

Preanalytical Variables. Factor VII can be activated by prolonged cold storage (4ºC or lower). Therefore, PT results can be shortened with prolonged plasma storage.

The PT is often prolonged with patients with polycythemia as a result of the change in the ratio of the anticoagulant to plasma. The blood to sodium citrate ratio is 9:1 for most coagulation tests. Elevation in the polycythemic patient's hemoglobin concentration lowers the relative amount of plasma in the patient's blood, resulting in a relative increase in the anticoagulant in the collection tube. The increase in citrate binds the CaCl2 added to the system during the testing process, resulting in an increase in the PT. The anticoagulant should be adjusted, according to the patient's hematocrit, prior to collection to prevent false elevation of the PT. Similarly, underfilled tubes in other patients can result in prolongation of the PT. Severe anemia has not been demonstrated to commonly interfere with the PT.

If heparin is present in the patient sample, in addition to warfarin, then the PT will reflect the combined effect of heparin and warfarin. Heparin can be either be removed or neutralized, prior to testing, to improve test accuracy.

Activated Partial Thromboplastin Time

aPTT is used to evaluate the intrinsic and common pathways of coagulation (Table 5; Figure 15). The aPTT is useful clinically as a screening test for inherited and acquired factor deficiencies as well as to monitor unfractionated heparin therapy, although the anti-Xa assay (see below) is the preferred measure of the effects of unfractionated heparin.

The assay was originally developed as a modification of the PT. The partial thromboplastin time (PTT) was described by Langdell, Wagner, and Brinkous in 1953 at the University of North Carolina. When thromboplastin was added to the PT, available at the time, the plasma from hemophiliac patients clotted as rapidly as normal plasma. By altering the thromboplastin used in the assay, Langdell and his associates were able to develop an assay that was sensitive to the defect in hemophiliac plasma. The altered thromboplastin or partial thromboplastin was prepared by ultracentrifugation of tissue extracts. In 1961, Rapaport and colleagues described the addition of kaolin to stabilize and shorten the PTT, which maximally activates plasma. This assay was described as the aPTT.

Current commercial aPTT reagents contain a partial thromoplastin, which is a contact activator and a platelet phospholipid substitute. Thromboplastins, such as cephalin or phosphatide, are combined with a contact activator such as kaolin, clite, micronized silica and ellagic acid, which eliminate the variability associated with glass activators. A platelet phospholipid substitute is included in commercial aPTT reagents as well to eliminate variability caused by altered platelet number or function.

The aPTT is performed by adding the commercial aPTT reagent to the citrated patient plasma and incubating for several minutes, which results in factor activation in the extrinsic and common pathways. Calcium chloride is added, resulting in clotting with the time from activation to clot detection measured in tenths of seconds. The aPTT reference interval will vary according to the type of instrumentation, anticoagulant, tube type, reagent type and reagent lot.

Pre-analytic variables. Pre-analytic variables that may affect the aPTT include difficult venipuncture, which leads to in vivo activation of the extrinsic pathway and results in a shortened aPTT. Of interest, a persistently shortened aPTT result with repeat venipuncture may be a risk factor for hypercoagulability.

Prolongation of the aPTT may result when blood is obtained from intravenous catheters that have been flushed with heparin. In order to prevent heparin contamination of the sample, an initial volume of blood is typically discarded. Other medications, if infused in the same catheter as a blood sample for the aPTT is obtained, may have variable effects on the aPTT results. Similar to the PT, aPTT results may be adversely prolonged by alterations of the ratio of blood to anticoagulant in polycythemic patients or with underfilled blood tubes, leading to an excess of anticoagulant. Causes of a prolonged aPTT are listed in Table 7.

Thrombin Time

The TT is used to evaluate the conversion of fibrin to fibrinogen, in the final common pathway of the coagulation cascade (Figure 17). The TT was first developed by Jim and Goldfein in 1957. It is used clinically to detect hypofibrinogenemia, dysfibrinogenemia (abnormal fibrinogens), and the presence of thrombin inhibitors, such as certain therapeutic drugs (eg, heparin) and antibodies (Table 8).

Figure 17.

In the measurement of the thrombin time, thrombin and calcium are added to citrated plasma. The time to the appearance of a clot in the reaction tube is measured to tenths of a second.

The TT measures the time for clot formation when thrombin is added to citrated plasma and is measured in tenths of seconds similar to the PT and aPTT. Thrombin catalyzes the conversion of fibrinogen to fibrin, in the last stage of the final clot formation, by cleaving fibrinopeptides A and B. Polymerization of fibrin to from a clot occurs. By adding exogenous thrombin, the phospholipid-dependent pathways (extrinsic, intrinsic, and common) are bypassed. Sources of reagent thrombin include human and bovine sources, which vary in thrombin concentration and heparin sensitivity. Lower concentrations of thrombin are more sensitive to heparin, dysfibrinogenemias, and other abnormalities than are preparations containing higher amounts of thrombin. The TT normal reference interval will thus vary according to the type and concentration of the thrombin preparation used in the assay and must be established by the testing laboratory.

Preanalytic Variables. Reagents containing bovine thrombin may result in elevated TTs in patients who develop bovine thrombin antibodies, often following exposure to topical hemostatic agents. The TT may be prolonged in patients receiving thrombin inhibitors, such as hirudins, or in patients with elevated fibrin-degradation products (FDPs). The presence of FDPs inhibits the conversion of fibrinogen to fibrin. The TT is sensitive to the presence of heparin and is often used to screen samples with a prolonged aPTT result for heparin contamination. High levels of immunoglobulin paraproteins can interfere with fibrin formation and result in prolongation of the TT.

Tests to Measure Fibrin Formation

Fibrin formation can be evaluateted by the fibrinogen assay and the reptilase time.

Fibrinogen Assay

Activation of fibrinogen by thrombin leads to the formation of an effective clot. Fibrinogen assays are useful for the diagnosis of hypofibrinogenemia, dysfibrinogenemia, disseminated intravascular coagulation, primary fibrinolysis, and other clinical conditions. Several fibrinogen assays are available, including gravimetric, immunological (antigen-based), optical, and functional assays.

The Clauss assay is the most commonly performed fibrinogen assay and is a modified TT. The assay uses diluted plasma where clotting is initiated with a high concentration of reagent thrombin. A calibration curve is plotted using serial dilutions of a reference plasma standard. The test plasma is diluted, incubated, and then phospholipid and thrombin are added, followed by calcium. Timing begins with the addition of calcium. The time taken for a clot to form is compared to the reference plasma calibration curve to derive the fibrinogen concentration (eg, mg/dL) of the test sample. Most laboratories use an automated method in which the optical density of the mixture exceeds a determined threshold, as a means of clot detection.

Immunological mass assays are based on enzyme linked immunoabsorbant (ELISA), radial immunodiffusion and electrophoresis techniques. The immunological assays measure protein (antigen) concentration rather than functional activity. Discrepancy between the fibrinogen activity and antigen level are characteristic of dysfibrinogenemias.

Gravimetric assays are performed by adding thrombin and calcium to dilute patient plasma. The resultant clot is compressed to extrude plasma and unused reagent, dried and weighed. This assay is technically difficult.

Preanalytical Variables. Increased turbidity of patient plasma may produce falsely low results for assays that use optical density to detect clot formation. Hyperbilirubinemia and hyperlipidemia are 2 such conditions that can result in increased plasma turbidity.

Very high concentrations of unfractionated heparin may lead to underestimation of the true fibrinogen level.

Reptilase Time

The reptilase time is similar to the TT except that snake venom is used to activate the coagulation cascade. Snake venom, from Bothrops atox, a South American pit viper, contains reptilase, an enzyme that is thrombin-like in nature and hydrolyzes fibrinopeptide A from intact fibrinogen molecules, in contrast with thrombin, which cleaves fibrinopeptides A and B from fibrinogen. The resulting clot is weaker than a clot formed by the action of thrombin on fibrinogen. The advantage of the reptilase time is that is not affected by the presence of heparin and only minimally affected by fibrin-degradation products. The reptilase time is useful, when compared to the TT for a given patient, to detect the presence of thrombin inhibitors.