State-of-the-art coagulation testing

Giuseppe Lippi MD
Associate Professor
Istituto di Chimica e Microscopia Clinica
University of Verona Italy
E: [email protected]

Massimo Franchini MD
Senior Assistant
Servizio di Immunoematologia e Trasfusione
University Hospital Verona
Italy

Haemostasis is a delicate equilibrium between anti- and prothrombotic factors, which promote either blood in a fluid state within vascular compartments or blood clot formation following endothelial injury. Imbalance between haemorrhagic or thrombotic forces lead to delayed repair of vessel injury (haemorrhage) or excessive blood clotting (thrombosis), producing clinically meaningful disturbances.(1) Revolutionary scientific discoveries have allowed thorough comprehension and characterisation of the intricate mechanisms that regulate such a delicate balance.

Primary haemostasis is defined as the formation of the primary platelet plug, which involves platelets, blood vessel components and the von Willebrand factor. After endothelial damage following immediate activation of primary haemostasis, secondary haemostasis (blood coagulation) develops.

The intricate model of blood coagulation was initially described by two independent groups in the early 1960s.(2,3) It involves a series of coordinated and calcium-dependent conversions of proenzymes to the respective serine proteases, culminating in the conversion (activation) of prothrombin to thrombin. Activation, amplification of the coagulation cascade and stabilisation of the blood clot are modulated by a balanced activity of specific proteases and allosteric or enzymatic inhibitors.(1) Each reaction within the coagulation cascade is catalysed by a single enzymatic molecule, which is able to activate in sequence several specific substrates. It is therefore easy to comprehend the enormous potential of this model, which is reflected by its intrinsic amplificatory potential. The success of the coagulation cascade depends on the ideal representation that the two basic coagulation tests provide for this model, the prothrombin time (PT) and the activated partial thromboplastin time (APTT), which respectively reflect the traditional extrinsic and intrinsic pathways.

Although such tests adequately reflect the function of the coagulation system in vitro, they are not suited to represent the intricate physiology ex vivo. Severe deficiencies of clotting factor XII (FXII), high-molecular weight kininogen and kallikrein are not associated with any haemorrhagic diathesis. Therefore, activation of the extrinsic pathway does not provide a definite contribution to the haemostatic process ex vivo. Nonetheless, activation of coagulation factor X (FX) by the extrinsic pathway is not sufficient to balance moderate to severe deficits of clotting factors VIII (FVIII) and IX (FIX), which are associated with the traditional haemophilia A and B disorders.

These observations have provided the basis for an innovative view of the coagulation system, which places thrombin generation at the core of the haemostatic process. Therefore, the coagulation system might be ideally represented in sequential steps, in which the extrinsic pathway is the key mechanism to initiate the system, while factors of the intrinsic pathway are responsible for the following propagation and amplification. Such phases involve:

• Activation of the extrinsic pathway and initiation of blood coagulation by tissue factor (TF) exposure on specific surfaces.
• Preliminary thrombin generation, catalysed by an enzymatically active complex between activated factor VII (FVIIa) and TF.
• Propagation and amplification of the enzymatic reactions (“thrombin burst”). This phase is promoted by the small amount of thrombin generated during the initial step. It initiates a feedback activation of the intrinsic pathway, characterised by a pronounced formation of numerous active enzymatic complexes.
• Conversion of fibrinogen to fibrin.
• Fibrin polymerisation and stabilisation by coagulation factor XIII (FXIII).
• Attenuation and termination of the blood coagulation by natural inhibitors, which hamper excessive blood clot formation.
• Dissolution and elimination of the blood clot (fibrinolysis),
• Tissue repair and healing.(1)

Traditional coagulation testing
Regardless their scope, coagulation tests can be divided into two categories: first-line (screening) and second-line (specific) tests (see Table 1).(4) First-line or routine coagulation tests are traditionally PT, APTT, fibrinogen and D-dimer. They are employed in the diagnostic and therapeutic approach to either haemorrhagic or thrombotic pathologies. Tests widely used to screen for qualitative or quantitative deficiencies of the coagulation cascade are APTT, PT and fibrinogen (functional assay). Although clinical evidence has unmasked the minor role played by the intrinsic pathway in the activation of the haemostatic system ex vivo, the APTT is as yet essential to screen for haemostatic disturbances arising from deficiencies of the intrinsic pathway, namely FVIII, FIX and factor XI (FXI), which might lead to haemophilia A, B and C.

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In fact, the APTT test is based on primary activation of FXII by various substrates (eg, ellagic acid, kaolin, silica), followed by sequential activation of FIX and FXI in the presence of FVIII. The test is employed to adjust and monitor anticoagulant therapy with unfractionated heparin, as well as for investigating the intrinsic pathway. Additionally, there is emerging evidence that shortened APTT is independently associated with venous thromboembolism (VTE) and this test might be hypothetically requested to detect hypercoagulability.(5) The PT reagent contains an activator of FVII (purified or recombinant thromboplastin) and calcium chloride, and the test is employed to screen for FVII deficiencies and monitor oral anticoagulant therapy (OAT) with coumarins. Additionally, the test might be useful to monitor therapy with recombinant activated FVII as a reliable surrogate for the FVIIa functional assay.(6) Isolated APTT prolongations usually reflect abnormalities of the intrinsic pathway, specific (acquired haemophilia) or aspecific (lupus-like anticoagulants) inhibitors of clotting factors, heparin therapy or contamination. Isolated PT prolongations reflect abnormalities of the extrinsic pathway or OAT, while combined prolongations of PT and APTT should unmask deficiencies in factors of the common pathway.

Fibrinogen testing is usually prescribed to identify afibrinogenemias and dysfibrinogenemias, as an aid to diagnose disseminated intravascular coagulation (DIC), and sometimes to help evaluate the risk of developing cardiovascular disorders. D-dimer testing is the biochemical gold standard in the diagnostic approach to VTE. The rationale of placing D-dimer in the diagnostic workout for VTE relies on its high negative predictive value, which allows reliable exclusion of thrombosis in patients displaying nondiagnostic values.(7)

Innovative coagulation tests
Although first-line coagulation tests are consolidated in the routine practice of clinical laboratories, second-line test panels are constantly evolving. The major innovations concern the so-called thrombophilia screening.(8) VTE is the leading cause of morbidity and mortality in general hospitals. Therefore, a comprehensive thrombophilia screening disclosed the great potential for identifying patients at major risk of thromboembolic events, providing valuable information on either type or duration of therapy, with substantial clinical and economical revenues. Thrombophilic factors are continuously identified and relative tests are made available to clinical laboratories (see Table 1).

The diagnostic approach to abnormalities of primary haemostasis is still a major challenge. In clinical practice, the investigation of unexplained bleeding ideally uses easy, reliable, and inexpensive screening tests, eventually followed by second-line analyses. The bleeding time test has been widely used for decades for this purpose. Nevertheless, this test is relatively insensitive and non-specific for identifying abnormalities of primary haemostasis and its diagnostic efficiency has been critically questioned. The platelet function analyser, PFA-100, is a relatively new method that has been developed as a quantitative, simple and rapid tool of assessing primary haemostasis.(9) PFA-100 is a useful and minimally invasive screening test for the investigation of von Willebrand disease, and various acquired and congenital intrinsic platelet function disorders. It is also useful for evaluating primary haemostasis before surgical procedures and for monitoring desmopressin therapy in patients with Type 1 von Willebrand’s disease. Moreover, recent studies have shown its potential for therapeutically monitoring the effectiveness of antiplatelet medications in cardiovascular disease management.(10)

Besides consolidated routine coagulation tests, there is increasing evidence that identification of decrease or increase thrombin generation is pivotal, as they promote either haemorrhage or thrombosis. Current coagulation tests provides little information on the whole coagulation potential of the blood. Therefore, innovative tests capable of mirroring the whole haemostatic potential would be suitable to screen for haemorrhagic or prothrombotic disturbances. Therefore, emerging thrombin-generation assays provide an accurate reflection of in vivo biology, covering initiation, development and final clot strength during clot formation.(11) The ensemble of the thrombin generation assays parameters hold the potential for the laboratory assessment of a large spectrum of clotting abnormalities, including acquired or congenital hypohaemostatic (ie, Von Willebrand disease, haemophilia, chronic liver failure, platelet dysfunctions) and hyperhaemostatic states (ie, detection of lupus anticoagulant, various forms of thrombomodulin and activated protein C resistance), as well as monitoring both haemostatic (ie, monitoring the treatment of haemophiliacs by FVIII or recombinant FVIIa) and anticoagulant agents (ie, low-molecular-weight heparin, antiplatelet agents).(11,12) Recent evidence indicates that thrombin generation assays would also be suitable to screen patients needing further specific thrombophilia testing.(13)

Innovative scenarios are emerging in the challenging issue of long-term management of OAT. Commercial availability and use of portable coagulation monitors (PCMs) for PT measurement is spreading worldwide. PCMs are easy of use, have shorter test duration, improved turnaround time and increased patient convenience. Several studies have investigated the analytical performance and overall reliability of these testing devices. The slightly lower precision of PT measured with PCMs is outweighed by the clinical advantages.(14) For patients who have been appropriately trained to their use, self-management by PCMs might turn out to be at least as effective as the routine care provided by traditional oral anticoagulation clinics, improving patient’s convenience, optimising resources utilisation and quality.(15)

Conclusions
Specialised haemostasis laboratories offer a series of coagulation tests. The important tests for the general physician are the so-called first-line screening tests. Depending on results of these traditional tests, specific assays may be indicated in a second phase of investigation. Nonetheless, innovative coagulation tests are emerging as reliable tools to monitor therapy and screen patients with haemostasis disturbances. Among these, PFA-100, thrombin generation assays and PCMs are gaining relevance in clinical decision-making. These are valuable aids for increasing convenience for patients and improving clinical outcomes, and decreasing the costs of unnecessary or redundant investigations.

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References

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15. Available from: http://bmj.bmjjournals.com/cgi/eletters/331/7524/1057#120787