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Rocky Yang ; Muhammad Zubair ; Leila Moosavi .
Last Update: January 23, 2024 .
Prothrombin time is one of several blood tests routinely used in clinical practice to evaluate the coagulation status of patients. More specifically, prothrombin time is used to evaluate the extrinsic and common pathways of coagulation, which helps detect deficiencies of factors II, V, VII, and X and low fibrinogen concentrations.[1][2] Prothrombin time measures the time, in seconds, for plasma to clot after adding thromboplastin (a mixture of tissue factor, calcium, and phospholipid) to a patient's plasma sample.[1]
Different preparations of thromboplastin reagents are available but can give different prothrombin time results even when using the same plasma. Due to this variability, the World Health Organization (WHO) introduced the international normalized ratio (INR), the standard reporting format for prothrombin time results.[3][4] The INR represents the ratio of the patient's prothrombin time divided by a control prothrombin time value obtained using an international reference thromboplastin reagent developed by the WHO.[1]
Standard laboratory coagulation-based testing has traditionally been used to obtain measurements of prothrombin time to ensure reliable results. Due to the high turnaround time (up to 90 minutes), point-of-care (POC) devices (approximately 5 minutes) are becoming more desirable.[5] POC devices are of great value in emergency and operating room settings where clinical diagnosis and intervention are time-sensitive.[6]
With increased prescribing of vitamin K-antagonists like warfarin, point-of-care devices have also been more convenient for patients and clinicians to monitor medication effectiveness. With point-of-care devices, monitoring anticoagulation therapy can take place at thrombosis centers, primary care provider offices, and even by the patients themselves.[4] Although point-of-care devices have been shown to underestimate hemostatic abnormality, point-of-care devices are generally reliable in non-emergency settings.[7]
Test results are dependent on the specimen quality. Specimens and request slips must be labeled with the patient’s name, medical record number or date of birth, collection date, and specimen source, when applicable.[8] Coagulation tests must be performed using plasma samples, not serum, as clotting factors are removed in serum preparations. Standard percutaneous phlebotomy is the recommended method used to collect venous blood samples. However, blood samples may also be obtained from indwelling intravenous lines when necessary.[9]
Phlebotomists collect venous blood samples in plastic tubes with a light blue top that contains 3.2% sodium citrate.[10] Sodium citrate chelates the calcium in the blood sample and prevents the activation of the coagulation cascade.[11] This chelation keeps the blood sample in stasis until it is ready to be tested. Tube filling must be within 90% of the full collection volume with a blood-to-sodium citrate ratio of 9 to 1.[7] The tube is then gently inverted a few times to mix the sodium citrate solution with the blood. The tube should not be shaken to avoid hemolysis, which would lead to inaccurate results. Once the blood sample is ready to be tested, calcium chloride is added to restore the calcium required for coagulation activation.[11] Clot formation can be detected mechanically or optically, depending on the instrumentation used.[12]
Clot-based tests (eg, prothrombin time/INR, aPTT, and TT) detect the time interval from initiation of coagulation to clot formation. Detection of clot formation as an endpoint has been accomplished in several ways.[13] Early methods used a tilt-tube technique that depended on visually identifying clot formation in plasma samples. A water bath was necessary to keep the temperature at 37 °C. Currently, this time-intensive manual method is used only with international reference thromboplastins.[14]
As a result of high-volume testing, most coagulation testing is now performed on automated instruments that control the reaction’s temperature and detect endpoints using several methods. Most methods detect changes in physical/mechanical properties or the light transmission produced by polymerized fibrin.[15] Numerous approaches for mechanical endpoint detection have been developed. One mechanical method consists of a metal ball at the bottom of a sample cuvette sent into a back-and-forth motion by a magnet; the endpoint is detected when fibrin monomers polymerize into fibrin strands and impede the ball’s motion. Another mechanical detection system uses a magnet to hold a ball to the side of a rotating cuvette until fibrin strands physically displace the ball.[16] Optical methods (usually nephelometric but occasionally turbidimetric) use decreased light transmission or increased light scatter as fibrin monomers are polymerized into fibrin strands.[17][18]
Optical endpoints may occur at preset thresholds or use the kinetics, such as maximum acceleration of fibrin polymerization, to define endpoints. Light sources have traditionally been halogen lamps or lasers, but newer instruments may use light-emitting diodes that increase longevity and allow measurement at wavelengths that overlap less with interfering substances.[19] A potential advantage of mechanical over optical endpoint detection is reduced interference from substances that interfere with optical methods, such as hemoglobin, bilirubin, or lipids.[20]
As for any laboratory investigation, the accuracy of prothrombin time and advanced partial thromboplastin (aPTT) results must be monitored regularly using quality control materials.[21] The analytical examination process’s quality control (QC) monitors a measurement procedure to verify that it meets performance specifications appropriate for patient care or that an error condition must be corrected.[22] Automated hematology and coagulation test systems require 2 levels of controls every 8 hours of testing and each time a change in reagent occurs. If necessary, laboratories can assay QC samples more frequently to ensure accurate results.[23] For manual coagulation testing, each analyst must perform 2 levels of controls before testing patient samples and with each change in the reagent. In addition, patient and control samples must be tested in duplicate.[24]
The quality control for prothrombin time and aPTT testing may be assayed or unassayed.[25] Quality control that is deemed to be ‘assayed’ is supplied with target values. The target values assigned to a control are specific to the reagent and analyzer used to generate the test result. Ensure the correct target range is used. Unassayed control does not have target values assigned. If a laboratory chooses to use unassayed control, they must know they need to generate their target ranges.[26]
The acceptable range and rules for interpreting QC results are based on the probability of detecting a significant analytical error condition with an acceptably low false alert rate.[27] The desired process control performance characteristics must be established for each measurement before selecting the appropriate QC rules.[28] Westgard multi-rules are normally used to evaluate the quality control runs. If a run is declared out of control, the system is investigated (instrument, standards, controls, etc) to determine the cause of the problem. No analysis is performed until the issue has been resolved.[28]
Changing reagent lots can have an unexpected impact on QC results. Careful reagent lot crossover evaluation of QC target values is necessary. Because the matrix-related interaction between a QC material and a reagent can change with a different reagent lot, QC results may not be a reliable indicator of a measurement procedure’s performance for patient samples after a reagent lot change.[29] Use clinical patient samples to verify the consistency of results between old and new lots of reagents because of the unpredictability of a matrix-related bias being present for QC materials.[30]
The laboratory must participate in the external quality control or proficiency testing program because it is a regulatory requirement published by the Centers for Medicare and Medicaid Services (CMS) in the Clinical Laboratory Improvement Amendments regulations.[31] Ensuring the accuracy and reliability of the laboratory concerning other laboratories performing the same or comparable assays is helpful.[32] Required participation and scored CMS and voluntary accreditation organizations monitor results. The proficiency testing plan should be included in the quality QA plan and the laboratory's overall quality program.[33]
Indications for obtaining prothrombin time are as follows:
