The prothrombin time (PT) and the activated partial thromboplastin time (APTT) are the most requested tests to investigate patients with congenital or acquired coagulopathies and drug monitoring. They are defined as the time (seconds) needed to clot platelet-poor plasma upon addition of coagulation triggers, such as tissue factor in complex with phospholipids and calcium chloride (in the PT), or negatively charged phospholipids-activators and calcium chloride (in the APTT). They can hence be considered as global coagulation tests sensitive to deficiencies of many coagulation factors. In brief, they are both sensitive to factor X, V, II and fibrinogen, whereas the PT is only sensitive to factor VII deficiency and the APTT is sensitive to pre-kallikrein, high molecular weight kininogen (HMWK), factor XII, XI, IX and VIII deficiencies. Importantly, none of the two is sensitive to factor XIII deficiency (Figure 1). Furthermore, the PT and APTT are variably sensitive to the presence of circulating anticoagulants directed against specific coagulation factors or against negatively charged phospholipids in complex with proteins, such as the lupus anticoagulants (LAC). The APTT is also sensitive to the presence of unfractionated heparin (UFH), whereas the PT not substantially influenced by the presence of this drug in plasma due to the fact that commercial thromboplastins are added with exogenous heparin inhibitors. This addition is required for assessing the therapeutic international normalized ratio (INR) during co-administration of vitamin K antagonists (VKA) and UFH in the treatment of acute venous thromboembolism (VTE). Low molecular weight heparin (LMWH) does not influence the PT. However, and at variance with widespread opinion, the APTT may be variably prolonged, depending on the brand of LMWH used.
A comprehensive history of the development of the two tests is beyond the scope of this article, and can be found elsewhere .
The PT was designed by Armand Quick  in 1935, who needed a simple test to investigate patients with jaundice. The name “prothrombin time” is a misnomer as it implies that the PT is sensitive to factor II (prothrombin), which was probably one the few coagulation factors known at that time. The PT evolved rapidly (upon the introduction of VKA treatment) with new thromboplastins derived from animal tissues (mostly brain) and recombinant preparations. The test also evolved from the original manual-visual clot detection technique to the more sophisticated coagulometers.
The APTT (formerly known as PTT) was designed in the 1950s by Langdell et al. , who needed a test to assess the progress in the purification of factor VIII concentrates to be used for hemophiliacs. The PTT was essentially a modification of the re-calcification time by removing platelets from plasma and by adding negatively charged phospholipids to speed up coagulation. In 1961 Proctor and Rapaport  improved the design of the PTT by adding particulate substances (kaolin) in order to activate massively the contact factors (this is where the APTT name comes from) of the coagulation cascade and to shorten the coagulation time. Like the PT, the APTT also underwent many changes over years, mainly entailing the standardization of phospholipids and activators to make it more suitable for investigating patients with hemophilia and allied disorders, to monitor UFH and to screen for LACs.
As their names clearly imply, the results of the two tests have been expressed at the beginning as clotting time (seconds). Over the years other results reporting have been introduced, however, making the scenario rather complicated. Some of these expressions can only be used for specific clinical conditions for which the PT/APTT are used. Without a clear knowledge of their meaning and limitations test reporting not only may create confusion among laboratory operators and (especially) clinicians, but can also negatively impact on the diagnostic reasoning and the clinical decision making when used outside their formal setting. This article is aimed to review the main results reporting and give guidance on how to use them.
Prothrombin time (PT)
Normal plasma in the PT clots approximately within 10–12 s upon triggering coagulation, and the normal reference range is (usually) narrow. This result reporting was used at the very beginning, but it soon became clear that clinicians were not very familiar with such a scale, so that it was decided to express results in terms of “percentage activity” (also known as prothrombin activity). The percentage is intuitive and based on the observation that diluting pooled normal plasma (arbitrarily considered as 100%) produces percentage activities to decrease with increasing dilutions and clotting times to be prolonged. Plots of paired data (percentage activities vs. clotting times) allow the preparation of a calibration curve that can be used to interpolate the percentage activity from the patient PT (Figure 2). This expression, although also intuitive for clinicians, has some limitations. First, the relationship between clotting times and percentage activities is not linearly related unless some sort of modification (e.g. log-scale) is applied to the data. Second, the slope and intercept of the calibration curve (even within the same thromboplastin/coagulometer combination) depend on the pooled normal plasma and on the type of diluent used, thus making standardization of the assay challenging. The shape of the curve (hyperbolic) that tends to parallel the axes at low and high dilutions also makes the percentage activity scarcely responsive to the variation of the clotting times at low percentage activity and excessively responsive at high percentage activity, respectively. Such an effect implies that relatively large clotting time variations in patients on VKA may translate into relatively small percentage activity variations, thus complicating dose-adjustment of patients using these drugs. Conversely, small clotting time variations in healthy subjects translate into large variation of percentage activity, which may occasionally lead to values far higher than 100%, which might be erroneously interpreted as an index of hypercoagulability. Third, the percentage activity does not account for the different sensitivity of commercial thromboplastins to the defect induced by VKA, as earlier shown by Palareti et al. . Therefore, it became rather clear that the PT percentage activity could not be used to monitor patients on VKA and that scale was used for some years to express results for patients other than oral anticoagulation. Although still used in some European countries, our opinion is that this approach should be now abandoned.
The quest for alternative results reporting led to the development of the PT-ratio (patient-to-normal clotting time) in the early 1960s. This scale is as intuitive as the percentage activity, but with a different meaning. Although low or high percentage activity respectively mean defective or heightened coagulation, lower or higher ratios than the unity respectively mean heightened or defective coagulation. Although the PT-ratio is more robust than the percentage activity, it is unable to account for the different responsiveness of commercial thromboplastins to the defect induced by VKA, and cannot therefore be used for reliable dose-adjustment. Some attempts were made in order to standardize this results reporting for VKA dose-adjustment, mainly based on the so-called Manchester ratio . Unfortunately, this approach could only be applicable to a specific brand of thromboplastin.
International normalized ratio (INR)
The awareness of the efficacy/safety of VKA for treatment and prophylaxis of VTE in the early 1970s contributed to sharply increase the use of these drugs, so that many clinicians become more confident and started treating their patients with VKAs. The number of patients treated increased quite rapidly, and earlier clinical observations also provided specific therapeutic intervals within which patients should be maintained during anticoagulation to limit the risk of bleeding or thrombosis. Unfortunately, these therapeutic intervals (even when expressed in term of PT-ratio) depended heavily on the thromboplastin used for testing and could not be generalized . This situation made the development, validation and implementation of a robust system of standardization no longer deferrable. In the early 1980s a new system of standardization, called INR, was developed following the statistical approach devised by Kirkwood , who took advantage of earlier observations on the PT-ratio made by Biggs and Denson . PT results were corrected mathematically into INR by raising the PT-ratio to a power equal to the international sensitivity index (ISI). Briefly, the ISI is the slope of the calibration line of the paired PT obtained with the working thromboplastin and a thromboplastin designated as international standard (i.e. the World Health Organization [WHO] reference thromboplastin preparation, originally identified with the “67/40” material) when the PT is measured in healthy subjects plasma and from patients stabilized on VKA (Figure 3). Therefore, the ISI represents a reliable measure of responsiveness of the working thromboplastin to the defect induced by VKA relatively to the international standard, and can be used to convert PT-ratio into INR according to the equation:
By definition, the INR represents the PT-ratio that would have been obtained if the patient plasma had been tested with the international standard instead of the working thromboplastin. In 1983 the Kirkwood calibration model was endorsed by the Expert Committee on Biological Standardization of WHO, which issued guidelines for its application . Ensuing modification were meant to resolve practical issues that emerged over the years, and led the way to the present model detailed in the last revised guidelines issued by WHO in 2013 . Manufacturers of commercial thromboplastins are encouraged to provide the ISI of their thromboplastins by calibration versus the existing international standards on a like-to-like (same species) basis. Caregivers (clinical laboratories and physicians prescribing VKA) are asked to adopt the new scale for drug dose-adjustment. The mainstay of the model is the preparation and provision of thromboplastin standards from different species, which are interrelated by iterative calibration (predecessor-to-successor) by means of international collaborative studies, which secure continuity of the system over time. Presently, there are two international standards available from WHO: RBT/05 from rabbit brain  and rTF/09 from human recombinant relipidated tissue factor . The system of calibration based on the revised Kirkwood model is still in place, and is the focus of continuous attention aimed at improving its reliability. One may guess that it will be the system of choice to monitor patients on treatment, at least until coumarin drugs will be replaced by the new generation of direct oral anticoagulants (DOAC) [14–17] that (allegedly) do not require dose-adjustment by laboratory testing.
By definition the INR (as an harmonization scale) is only valid for patients stabilized on VKA. In all the other circumstances it can be used for results reporting, but it may fail to harmonize results obtained with different commercial thromboplastins. For instance, it was demonstrated that the INR fails to harmonize results across different thromboplastins when used to express PT results for patients with chronic liver disease, patients with disseminated intravascular coagulation (DIC) congenital deficiency of pro-coagulant factors, or for those on DOAC . This is not surprising if one considers that the ISI (which is the mainstay of the calibration model) is determined by using plasmas from patients stabilized on VKA, and is therefore only dependent on the coagulation defect(s) induced by these drugs. The clotting abnormalities induced by VKA have features that are different from other defects that prolong PT (e.g. chronic liver disease, DIC, hypo-coagulability due to single factor deficiency or DOAC). Therefore, it can be concluded that it will fail to harmonize results stemming from different thromboplastins.
The failure of harmonizing results across thromboplastins for patients with chronic liver disease might have a practical impact as the PT is used for the calculation of the model for end-stage liver disease (MELD) score used to prioritize patients for liver transplantation . The MELD score includes in its calculation bilirubin, serum creatinine and INR. Taken for granted that the measurement of bilirubin and creatinine should not substantially vary across laboratories , the INR measurement may instead vary and the MELD may hence vary accordingly. These variations are likely to change the priority system to allocate organs . Recently, Tripodi et al.  and Bellest et al.  independently proposed an alternative calibration model tentatively called INRLIVER (as opposed to the INRVKA) aimed at solving this problem. The modified system calls for the ISILIVER being determined by using in the calibration plot the PT for plasmas from patients with chronic liver disease instead of patient stabilized on VKA. It has been shown that the ISILIVER can then be reliably used to convert and harmonize results into the new scale INRLIVER. This calibration model, although considered by the International Society for Thrombosis and Haemostasis (ISTH)  and by a multidisciplinary study group , has not yet been endorsed by the relevant standardization authorities and the MELD score is currently (erroneously) calculated by the INRVKA.
The results of PT are also incorporated into a score system for DIC diagnosis and monitoring . The score, devised by the ISTH, assigned to the PT prolongation (see Table 1) one or two points depending on whether the prolongation above the upper limit of the reference range is 3 or more seconds, respectively. In order to improve the accuracy of the score system and to make it universally applicable to clinical trials investigating DIC, results reporting for the PT should be harmonized across laboratories. Recently, attempts have been made to this end, and it was shown that the INR system may be modified by determining an ISI valid for DIC by replacing in the calibration plot patients with VKA for patients with DIC . However, this system has not been implemented so far to the best of our knowledge. Until then, result reporting in patients with DIC should be clotting time (seconds) if the ISTH DIC score is applied .
Patients on DOAC display PT or APTT that are variably prolonged over the upper limit of the reference range . The PT prolongation is more evident with rivaroxaban (Xarelto) and much less with dabigatran (Pradaxa) or apixaban (Eliquis) and the prolongation of the APTT is more evident with dabigratran than with rivaroxaban or apixaban. However, the PT or APTT prolongations are heavily dependent on the reagent composition used for testing. Tripodi et al.  have recently shown that the adoption of the INRVKA to report PT results for patients on rivaroxaban rather than minimizing, increases the between-thromboplastin variability and should therefore not be used to report PT results in these patients. Attempts have been made to devise a PT-ratio valid for rivaroxaban in which the rivaroxaban sensitivity index (called rivaroxaban-SI) is calculated by substituting in the calibration plot (working thromboplastin vs. standard thromboplastin) plasmas from patients on VKA for plasmas from patients on rivaroxaban. The patient PT-ratio is then converted into the so-called rivaroxaban-PT-ratio by the equation:
This model of calibration proved feasible and effective to minimize the between-thromboplastin variability of PT-ratio induced by rivaroxaban . However, the system has not yet been implemented.
All in all, the above observations underscore the strength and weakness of the INR as a system of standardization across laboratories measuring the PT. The strength is its reliability to minimize result differences stemming from different thromboplastins when the test material is plasma from patients stabilized on VKA and the possibility to introduce modifications to make it suitable to harmonize PT results in other clinical settings. The weakness is the indiscriminate application of the INRVKA for conditions other than anticoagulation with VKA. In the latter settings, the expectation that results are harmonized across laboratories is not plausible, may lead to erroneous conclusions and may impact negatively the management of specific categories of patients.
Activated partial thromboplastin time (APTT)
The APTT is currently used as a global test to investigate patients suspected of (or having) bleeding diathesis, for dose-adjustment of UFH, to search for LACs or for the detection of activated protein C (APC) resistance .
Historically APTT results have been reported as clotting times (seconds), but not as percentage activity. However, like for the PT, the ratio of patient-to-normal clotting time is used by many laboratories, especially when the APTT is employed for dose-adjustment of UFH. The pioneer work of Basu et al.  established rather empirically that the therapeutic interval for an effective/safe treatment with UFH of acute VTE should be such to prolong the APTT from 1.5 to 2.5 times over the baseline value. It was much later realized that the 1.5–2.5 APTT prolongation does not fit to all the variety of APTT reagents commercially available, and it was also demonstrated that the indiscriminate application of this therapeutic interval regardless of the reagent used might lead to the patient being under- or over-anticoagulated when specific brand of APTT reagents poorly or excessively responsive to UFH are used for dose-adjustment . Despite this evidence, the reference interval of 1.5–2.5 APTT prolongation is still quoted in many review articles and text books. Attempts have been made to translate the concept of the INR also to the APTT, with provision of an international APTT standard against which to calibrate commercial APTT reagents . However, the model of calibration proved excessively complex to be applied in practice, and was abandoned also due to the fact that UFH has been subsequently and gradually replaced by LMWH, a drug that does not usually requires strict laboratory control for dose-adjustment. However, there are still clinical conditions where UFH is prescribed, so that clinical laboratories measuring the APTT and clinicians prescribing UFH must be aware of these pitfalls and determine local therapeutic intervals which should be validated for their APTT reagents. This can be empirically achieved by testing plasmas from patients on treatment with UFH by protamine titration and by the local APTT, with results expressed as APTT-ratio (patient-to-normal clotting time). Paired data are then plotted and the best fit line is drawn. The therapeutic APTT-ratio interval with the local reagent is eventually determined by graphical interpolation to correspond to 0.2–0.4 UFH units (protamine titration) . When protamine titration is unavailable, the anti-factor Xa activity with UFH titer of 0.3–0.7 units should be regarded as the alternative assay with which the APTT could be compared . The use of a pooled normal plasma spiked with graded amounts of UFH instead of in vivo heparinized plasmas, although more convenient, should not be used to determine APTT therapeutic interval because it is not representative of heparinization in vivo.
The most recent ISTH guidelines for the APTT to be used for laboratory diagnosis of LAC recommend expressing the APTT as ratio (patient-to-normal coagulation time) .
More than 20 years ago, Dahlback et al.  proposed that the APTT could be used (with and without exogenous addition of activated protein C, APC) to identify patients with poor anticoagulant response to this naturally occurring anticoagulant. Results are expressed as APTT-ratio with-to-without APC (low ratios identify resistance to APC).
Finally, there are no specific recommendations on how to express results for the APTT when used to investigate patients suspected of (or having) bleeding diathesis. In our opinion, however, the APTT-ratio should also be recommended in this context. Besides being intuitive, the APTT-ratio has additional advantages over the clotting time in seconds, as it contributes to minimize within-laboratory variability of the measurement. This is inherently attributable to the fact that the normal plasma used for the ratio calculation is concomitantly tested with the patient plasma.
The history of laboratory medicine is plenty of false myths and legends, some of which became firmly rooted in daily and routine laboratory practice . The PT and APTT are time-honored global tests, used for many purposes in the clinical laboratory. The varied result reporting that have been introduced over time, especially for the PT, made their application rather confusing for clinicians in many clinical circumstances. As an example, clinicians are particularly impressed by the INR which is the “universal” expression for PT results in their mind regardless of the context where it is used. They (very often) do not inform the laboratory on the reason(s) why they are prescribing the test. The logical consequence is that the (unaware) laboratory professional does not know if results should be reported as INRVKAfor patients on anticoagulation with these drugs, or as other result expressions for other categories of patients.
Specific recommendations on how to express PT and APTT are lacking and this makes the current situation even more confusing. Until the standardization authorities make decision on this issue we propose that clinical laboratories adopt the proposal summarized in Table 2. Comments on the significance of results and how to proceed with further investigations should also be provided together with analytical results whenever deemed useful .
Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.
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About the article
Published Online: 2015-08-06
Published in Print: 2016-02-01