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Thrombin time

The thrombin time (TT), also known as the thrombin clotting time (TCT), is a blood coagulation test that measures the time required for the formation of a fibrin clot in platelet-poor plasma after the addition of exogenous thrombin. This assay specifically evaluates the efficiency of the final step in the coagulation cascade, where thrombin converts soluble fibrinogen into insoluble fibrin strands, bypassing earlier pathways such as prothrombin activation.^[1]^[2] Clinically, the TT is ordered to investigate unexplained bleeding or thrombotic episodes, recurrent miscarriages, or prolonged results from other coagulation tests like prothrombin time (PT) or activated partial thromboplastin time (aPTT). It is particularly useful for detecting abnormalities in fibrinogen quantity or function, such as hypofibrinogenemia, dysfibrinogenemia, or the presence of inhibitors like heparin, fibrin degradation products, or direct thrombin inhibitors (e.g., dabigatran).^[2]^[1] The test is performed by mixing citrated plasma with a standardized thrombin reagent at 37°C and recording the clotting time using optical or mechanical detection methods, typically without the need for calcium ions. Normal TT values generally range from 15 to 20 seconds, though reference intervals may vary by laboratory and reagent; prolonged times (>20 seconds) often necessitate mixing studies or reptilase time assays to differentiate between fibrinogen deficiencies and inhibitory effects.^[2]^[1] In practice, the TT plays a key role in diagnosing acquired coagulopathies, such as those seen in disseminated intravascular coagulation (DIC), liver disease, or massive transfusion, as well as inherited disorders like afibrinogenemia. It also aids in monitoring unfractionated heparin therapy, where elevated TT correlates with anticoagulant levels, though it is less sensitive to low-molecular-weight heparin or other agents. Recent guidelines emphasize its utility in specialized settings, such as evaluating direct oral anticoagulants, but note limitations like reagent variability and insensitivity to upstream coagulation defects.^[2]^[1]

Fundamentals

Definition

The thrombin time (TT), also known as thrombin clotting time (TCT), is a blood test that measures the time required for thrombin to convert fibrinogen in plasma to fibrin, thereby forming a clot.^[3]^[1] This assay directly evaluates the rate of this enzymatic reaction by adding a standardized amount of exogenous thrombin to patient plasma, allowing for the quantification of clot formation in seconds.^[1] It specifically assesses the final common pathway of the coagulation cascade, focusing on the enzymatic activity of thrombin on fibrinogen without involvement of upstream clotting factors such as those in the intrinsic or extrinsic pathways.^[4] By bypassing earlier stages of the cascade, the TT isolates potential abnormalities in fibrinogen structure, concentration, or thrombin inhibition, providing a targeted measure of the terminal phase of hemostasis.^[1] The test was developed in the mid-20th century, building on foundational research from 1950–1952 that elucidated the fibrinogen-to-fibrin conversion mechanism, as part of broader coagulation screening panels to isolate fibrinogen-related issues.^[5]

Biochemical Principle

Thrombin, also known as factor IIa, plays a central role in the final stage of the coagulation cascade by catalyzing the conversion of soluble fibrinogen into insoluble fibrin, the primary structural component of blood clots. This process begins with thrombin binding to specific sites on the fibrinogen molecule, a 340 kDa glycoprotein composed of two sets of three polypeptide chains (Aα, Bβ, and γ). Thrombin preferentially cleaves fibrinopeptide A (FPA) from the Aα chains, exposing polymerization sites (known as "knobs") that enable fibrin monomers to self-assemble into protofibrils; subsequent cleavage of fibrinopeptide B (FPB) from the Bβ chains further stabilizes the fibrin network by promoting lateral aggregation and cross-linking.^[6]^[7] The thrombin time (TT) test isolates and directly assesses this fibrinogen-to-fibrin conversion by adding a standardized concentration of exogenous thrombin to citrated patient plasma, bypassing upstream coagulation factors and platelet involvement. This isolates the reaction to the thrombin-fibrinogen interaction, measuring the time from thrombin addition until visible fibrin formation, typically via clot detection methods. The simplified biochemical reaction is represented as:

\text{Fibrinogen} + \text{[Thrombin](/page/Thrombin)} \rightarrow \text{[Fibrin](/page/Fibrin)} + \text{Fibrinopeptides (A and B)}

This step requires no additional clotting factors or cellular components, making the TT highly specific for evaluating the terminal phase of hemostasis.^[1]^[8] The TT is particularly sensitive to both the quantity and quality of fibrinogen in plasma. Low fibrinogen levels (hypofibrinogenemia, typically below 100 mg/dL) prolong the clotting time due to insufficient substrate for thrombin, while qualitative defects such as dysfibrinogenemia—caused by genetic mutations altering fibrinogen structure—can impair thrombin binding or cleavage efficiency, also resulting in extended TT values. These abnormalities directly reflect disruptions in the enzymatic kinetics of fibrinopeptide release and subsequent fibrin polymerization, without influence from other hemostatic elements.^[1]^[6]

Clinical Applications

Indications

The thrombin time (TT) test is primarily indicated to investigate unexplained prolongation of the prothrombin time (PT) or activated partial thromboplastin time (aPTT), where fibrinogen dysfunction or other defects in the final common pathway of coagulation are suspected. It serves as a targeted assay to confirm or rule out fibrinogen-related disorders when initial screening tests suggest abnormalities in fibrin formation. Additionally, the TT is employed in comprehensive coagulation panels for patients presenting with bleeding tendencies or thrombotic events, helping to pinpoint the underlying cause without broader diagnostic delays.^[9]^[10] In clinical practice, the TT plays a key role in diagnosing hypofibrinogenemia, dysfibrinogenemia, and afibrinogenemia, both congenital and acquired forms, by assessing the functional integrity of fibrinogen conversion to fibrin. It is particularly useful in evaluating patients with suspected inherited fibrinogen deficiencies or acquired reductions due to conditions such as liver disease, disseminated intravascular coagulation (DIC), or malignancy. The test also aids in monitoring fibrinolytic therapy during thrombolysis or in managing DIC, where rapid assessment of fibrinogen status can guide therapeutic interventions to prevent excessive bleeding. It is also used to monitor therapy with unfractionated heparin and direct thrombin inhibitors.^[11]^[9]^[10]^[10] Furthermore, the TT helps differentiate heparin contamination in plasma samples from genuine fibrinogen defects, as even low levels of unfractionated heparin can prolong the test result, prompting further investigation with agents like protamine sulfate. It is routinely ordered as part of bleeding diathesis workups in patients with unexplained hemorrhage or recurrent thrombotic episodes and in pre-surgical coagulation screening when fibrinogen abnormalities are anticipated, ensuring safer procedural outcomes.^[9]^[11]^[10]

Interpretation

A prolonged thrombin time (TT), typically exceeding 22 seconds, signifies potential abnormalities in the final stage of the coagulation cascade, specifically impaired fibrin formation from fibrinogen. This prolongation commonly arises from low fibrinogen levels (below 100 mg/dL, or 1 g/L), dysfunctional fibrinogen (such as in dysfibrinogenemia), or the presence of thrombin inhibitors like heparin, direct thrombin inhibitors (e.g., dabigatran or hirudin), or rare thrombin inhibitors such as antibodies against thrombin.^[9]^[12]^[10] In clinical practice, a prolonged TT prompts correlation with fibrinogen assays to confirm whether the issue stems from quantitative deficiency (e.g., hypofibrinogenemia in disseminated intravascular coagulation or liver disease) or qualitative defects (e.g., abnormal fibrinogen structure).^[13]^[10] A normal TT result generally indicates intact fibrinogen quantity and function, effectively ruling out fibrinogen-related disorders as a primary cause of bleeding tendencies or unexplained prolongation in other coagulation tests like prothrombin time (PT) or activated partial thromboplastin time (aPTT).^[9]^[12] Shortened TT values are uncommon and may reflect elevated fibrinogen concentrations or sample contaminants such as dextran or hydroxyethyl starch infusions, though these scenarios rarely impact routine diagnostics.^[9]^[13] Interpretation of TT often requires integration with complementary assays for differential diagnosis. For instance, a prolonged TT alongside a normal reptilase time strongly suggests the influence of heparin or similar thrombin inhibitors, as reptilase is unaffected by these agents.^[12]^[10] Additionally, combining TT with fibrinogen concentration measurements helps distinguish between quantitative fibrinogen deficits and qualitative dysfunctions, guiding targeted therapeutic interventions in bleeding or thrombotic disorders.^[9]^[13]

Laboratory Methodology

Specimen Requirements

The specimen for thrombin time (TT) testing is collected as whole blood using a light blue-top tube containing 3.2% buffered sodium citrate anticoagulant, with a standard ratio of 9:1 (blood to anticoagulant) to inhibit clotting during collection and transport.^[11]^[14] This concentration is preferred over 3.8% trisodium citrate for optimal results in coagulation assays.^[15] The tube must be filled to the appropriate volume mark to ensure the correct ratio, and venipuncture should be atraumatic to minimize activation of coagulation factors.^[16] Following collection, the blood is centrifuged promptly to prepare platelet-poor plasma, typically via double centrifugation: first at 1500-2000 × g for 10-15 minutes to separate plasma, followed by a second spin of the supernatant at the same conditions to remove residual platelets.^[17]^[11] The resulting plasma should be free of cells and platelets (platelet count <10 × 10⁹/L) for accurate testing. A minimum plasma volume of 1-2 mL is required, depending on the analyzer.^[18] Storage conditions are critical to preserve sample integrity: platelet-poor plasma is stable at room temperature (20-25°C) for up to 4 hours if testing is imminent, but for longer delays, it should be refrigerated (2-8°C) for up to 8 hours or frozen at -20°C or lower for extended periods (up to 6 months at -70°C), with only one freeze-thaw cycle permitted.^[19]^[14] Samples must be transported to the laboratory within 3-4 hours to avoid deterioration.^[20] Hemolysis and lipemia must be avoided during collection and processing, as these can cause spurious results in optical-based coagulation assays by interfering with light detection or enzyme activity.^[21]^[22] For pediatric patients, particularly infants with elevated hematocrit (>55%), the citrate volume in the tube should be adjusted (e.g., by removing excess anticoagulant) to maintain the 9:1 ratio and prevent over-anticoagulation.^[23]^[24] This adjustment is calculated based on the patient's hematocrit to ensure accurate plasma citrate concentration.^[25]

Test Procedure

The thrombin time assay involves the addition of a standardized thrombin reagent to citrated patient plasma to initiate fibrin clot formation, with the elapsed time measured from reagent addition to clot detection. In the standard manual procedure, 0.1 mL of patient plasma is pipetted into a reaction vessel, such as a glass or plastic tube, and pre-incubated at 37°C for 1 minute to ensure optimal temperature equilibrium. Subsequently, 0.1 mL of thrombin reagent—derived from bovine or human sources and standardized to 1-10 NIH units/mL, depending on the reagent—is rapidly added while starting a timer, and the mixture is gently agitated if required by the method.^[26]^[1] Clot formation is detected visually or mechanically in manual setups, where the operator observes the appearance of fibrin strands or a solid gel by periodically tilting the tube, recording the time in seconds via stopwatch when the endpoint is reached. Automated systems, commonly used in clinical laboratories, employ photo-optical detection principles, monitoring changes in light transmission or turbidity within the sample cuvette to precisely determine the clotting endpoint without manual intervention. These analyzers, such as the Sysmex CA series or Instrumentation Laboratory ACL platforms, maintain the reaction at 37°C and process multiple samples concurrently for efficiency.^[27]^[12] To ensure assay accuracy and reproducibility, quality control measures are implemented daily, including the testing of normal and abnormal control plasmas alongside patient samples to verify reagent performance and instrument calibration. The thrombin reagent, once reconstituted from lyophilized form with distilled water, exhibits stability for 4-6 hours when stored at 2-8°C, after which it must be discarded to prevent degradation that could affect results. This step directly evaluates the thrombin-fibrinogen interaction central to the coagulation cascade.^[28]^[29]

Reference Ranges

The reference range for thrombin time (TT) is laboratory-specific and depends on the reagent and methodology employed, but typical values for adults are often 17 to 23 seconds.^[9] These ranges are established by individual laboratories through validation studies on healthy adult populations, following guidelines such as those from the Clinical and Laboratory Standards Institute (CLSI), which recommend testing at least 120 apparently healthy reference individuals to define the central 95% interval.^[30] To ensure consistency across different methods or reagents, results may be reported as a ratio of the patient's TT to the mean normal plasma TT, where a ratio approaching 1.0 indicates normality.^[31] In pediatric populations, reference ranges are slightly longer due to developmental hemostasis; for example, newborns may exhibit TT values of approximately 19 to 26 seconds.^[9] These age-specific variations are determined similarly through studies of healthy children stratified by age groups, accounting for physiological differences in coagulation factors.^[32] TT results are influenced by plasma fibrinogen levels, with normal adult concentrations ranging from 200 to 400 mg/dL (or 2 to 4 g/L), though the test is not designed for quantitative fibrinogen measurement and prolongation occurs primarily at extremes (e.g., below 100 mg/dL).^[33]^[34]

Limitations and Variants

Interferences

Several anticoagulants interfere with the thrombin time (TT) assay by inhibiting thrombin activity, resulting in falsely prolonged clotting times. Unfractionated heparin binds to antithrombin III, enhancing its inhibition of thrombin and thereby extending the TT. Low-molecular-weight heparins generally do not prolong the TT at therapeutic doses. Direct thrombin inhibitors, such as dabigatran and argatroban, directly block thrombin's catalytic site, causing marked prolongation of the TT that is often dose-dependent and exceeds typical therapeutic ranges.^[35] To mitigate these effects, laboratories may pretreat samples with protamine sulfate to neutralize heparin or employ alternative assays like the reptilase time, which is unaffected by these inhibitors.^[36] Fibrin degradation products (FDPs) and fibrinogenolysis products, generated during fibrinolysis, can falsely prolong the TT by competitively binding to fibrinogen or interfering with fibrin polymerization, thereby reducing the availability of functional fibrinogen for thrombin-mediated clotting.^[37] Sample aging exacerbates this issue, as plasma stored for more than 8 hours at room temperature undergoes degradation of coagulation factors and accumulation of breakdown products, leading to unreliable TT results.^[38] Mitigation involves prompt processing of samples within 4-8 hours of collection and, if FDPs are suspected, confirming with fibrinogen assays or dilution studies to assess the degree of interference.^[39] Pre-analytical errors, including an improper blood-to-citrate ratio, can alter TT outcomes; excess citrate can prolong the TT through plasma dilution or direct effects on the clotting process. Contamination of the sample with EDTA or heparin from improper tube handling similarly extends the TT by mimicking anticoagulant effects.^[40] Therapeutic agents like ancrod and batroxobin, thrombin-like snake venom enzymes used for defibrinogenation in certain treatments, interfere by rapidly depleting fibrinogen levels, resulting in prolonged TT.^[41] These interferences can be minimized through standardized phlebotomy protocols ensuring correct anticoagulant ratios and tube types, along with visual inspection for contamination; differentiation from true hypofibrinogenemia may require related tests such as reptilase time.^[42] The reptilase time is a functional clotting assay that serves as a key complementary test to the thrombin time (TT), employing batroxobin—a thrombin-like enzyme derived from the venom of the snake Bothrops atrox—to cleave fibrinopeptide A from fibrinogen, thereby initiating fibrin clot formation without activating factor XIII.^[43] Unlike the standard TT, the reptilase time is resistant to prolongation by heparin or direct thrombin inhibitors, making it useful for distinguishing between fibrinogen abnormalities (such as hypofibrinogenemia or dysfibrinogenemia) and the presence of thrombin inhibitors. However, it is also prolonged by FDPs.^[44] Normal reptilase time values typically range from 12 to 21 seconds, depending on the reagent and laboratory method, with prolongation indicating fibrinogen defects while a normal result in the face of an abnormal TT points to inhibitors or anticoagulants.^[45] Fibrinogen assays using the Clauss method provide a direct quantitative measure of functional fibrinogen levels, complementing the TT by assessing the substrate availability for thrombin-mediated clotting without being confounded by thrombin-specific inhibitors.^[46] In this assay, patient plasma is diluted and clotted with a high concentration of thrombin (typically 20-100 IU/mL), and the clotting time is compared against a standard curve to estimate fibrinogen concentration, where shorter times correspond to higher fibrinogen levels. This method is insensitive to thrombin inhibitors but sensitive to fibrinogen quantity and quality, allowing it to confirm whether TT prolongation stems from low fibrinogen rather than inhibitory factors; reference ranges are generally 200-400 mg/dL for adults.^[46] Modifications to the standard TT, such as the addition of protamine sulfate, address heparin interference by neutralizing unfractionated heparin in the sample, enabling accurate assessment of residual anticoagulant activity post-administration.^[47] In this variant, protamine (typically 50-500 µg/mL) is added to the plasma before thrombin, shortening the clotting time if heparin was the cause of prolongation, as seen in cardiac surgery monitoring where it helps verify adequate heparin reversal.^[48] Additionally, assays involving eptacog alfa (recombinant activated factor VII) are utilized in hemophilia A or B patients with inhibitors to evaluate thrombin generation and detect specific bypassing agent efficacy, often through modified clotting or generation tests that bypass factor VIII/IX deficiencies.^[49] These eptacog-based assays quantify peak thrombin levels in inhibitor-containing plasma, aiding in dose optimization and inhibitor characterization in therapeutic contexts.^[50]

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