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Semin Thromb Hemost 2009; 35(1): 003-008
DOI: 10.1055/s-0029-1214143
PREFACE

© Thieme Medical Publishers

Laboratory Diagnostics and Therapy in Thrombosis and Hemostasis: From Bedside to Bench to Bedside

Giuseppe Lippi1 , Emmanuel J. Favaloro2 , Massimo Franchini3
  • 1Sezione di Chimica Clinica, Università di Verona, Verona, Italy
  • 2Department of Haematology, ICPMR, Westmead Hospital, Westmead, NSW, Australia
  • 3Immunohematology and Transfusion Centre, Department of Pathology and Laboratory Medicine, University Hospital of Parma, Parma, Italy
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Publikationsverlauf

Publikationsdatum:
23. März 2009 (online)

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Welcome to the first issue of Seminars in Thrombosis and Hemostasis for 2009. The theme for this issue, “Laboratory Diagnostics and Therapy in Thrombosis and Hemostasis: From Bedside to Bench to Bedside,” arose from two main conceptual considerations. At a basic level, we reflected on the circular interactive process that the clinical-laboratory interface should reflect. Thus, the clinician, “at the bedside,” reflects on the best approach for the diagnosis of the patient's disease or wishes to monitor some form of patient therapy. Blood is drawn and sent to the laboratory, where tests are performed, and the results are relayed back to the clinicians, who then continue to manage the patient under their care. This process should follow a circular and interactive progression, as depicted in Fig. [1].

Figure 1 Cartoon showing the circular nature that the laboratory-clinical interface should represent. This would be applicable both to the basic day-day interactions and to the concept of translational medicine.

At a more complex level, we considered the concept of translational medicine, which is the emerging view of medical practice and interventional epidemiology, as a natural 21st century progression from evidence-based medicine. The main aim of this innovative science is the translation of basic research (“from the bench”) into practical clinical applications (“to the bedside”), a process that has incredible potential to develop and deliver new tools that may assist the prevention, diagnosis, and treatment of disease.[1] In the field of laboratory hemostasis, the effective relocation of promising research findings to daily laboratory practice is a challenge that may take several years and involve many sequential processes, including the discovery of new biological pathways, the development and validation of clinical assays to reliably measure innovative biomarkers, validation and release of reagents and diagnostic systems by commercial companies, evaluation of the clinical performance of commercial assays in the field, and reliable implementation into clinical practice through training of laboratory professionals and refining the interpretation and use of the newly derived information by all medical personnel.[2] [3]

Accordingly, this issue of Seminars in Thrombosis and Hemostasis is focused on the link between basic scientific endeavors and discoveries within the arena of clinical investigation and demonstrates how the results obtained at the bench can be used by, or usefully translated into, clinical practice. This issue is the second in a series of issues of Seminars in Thrombosis and Hemostasis that we are devoting to the laboratory-clinical interface and follows on from an issue we published late in 2008.[4]

The first article, by Lippi et al, takes us on a chronological journey of the history of laboratory testing in hemostasis, describing the continuous and often revolutionary scientific developments that have taken place from the ancient to the present times. Although this article can be seen in part as a progression of an article written by Tripodi in the previous issue,[5] Lippi and colleagues have now undertaken a more extensive appraisal of developments within the field of hemostasis. Basically, past and ongoing scientific developments have contributed to decoding several aspects of the intricate but essential physiologic phenomenon that is hemostasis, providing a reliable model to explain the leading mechanisms involved. Basic research, which yielded outstanding discoveries on the complex biology of primary hemostasis, secondary hemostasis, and fibrinolysis, has been eventually converted into reliable laboratory assays, which are now cornerstones in the diagnostic approach undertaken by clinicians for patients with hemostasis disturbances, both thrombotic or hemorrhagic. This article describes the discovery, as well as the biological function, of several hemostatic components including platelets, von Willebrand factor (VWF), factor VII, natural inhibitors of the coagulation cascade (antithrombin, protein C, and protein S), and factor V polymorphisms. This article also provides an overview on the development and standardization, as well as the clinical utility, of pivotal tests of laboratory hemostasis, including platelet count, platelet function assays, thromboelastography, thrombin generation, and thrombophilia testing.

The second article, by Montagnana and colleagues, can be seen as a logical continuum of the former and explores the circadian variation within hemostasis, trying to provide a reliable answer to the crucial question of whether circadian variation should be considered as an underrecognized link between biology and disease. Biological rhythms are a universal phenomenon in living organisms, with these serving to help organisms adapt within a circadian cycle to the 24-hour-oscillating environment. Not only should these biological rhythms be taken into account when assessing biological variation or normal reference ranges,[6] but also several lines of evidence now attest that cardiovascular disorders and venous thromboembolism (VTE) are both subject to circadian oscillations, which might be closely related to an internal biological clock. This article focuses on the highly repetitive rhythmic cycles that seem to modulate platelet and endothelial functions, as well as the concentration and activity of several proteins of the coagulation and fibrinolytic systems. Several hypotheses are proposed to explain the nature, the clinical significance, and the pathophysiologic consequences of this hemostatic clock, synthesizing the potential preventive and therapeutic approaches to a variety of conditions where either the severity of the illness or therapeutic efficacy exhibit circadian rhythmicity.

The third article of this issue of Seminars in Thrombosis and Hemostasis, by Tripodi and van den Besselaar, is devoted to the laboratory monitoring of traditional and innovative anticoagulant therapies, which are the gold standard for treatment of patients with VTE. This article can be seen in part as an extension of several earlier articles from Seminars in Thrombosis and Hemostasis, including that by Favaloro and Adcock on the standardization of the international normalized ratio (INR)[7] and that by Fareed and colleagues on the current and future status of therapeutic anticoagulants and antithrombotics.[8] Experience accumulated over decades clearly demonstrates that strict laboratory monitoring is required for patients administered unfractionated heparin and vitamin K antagonists, making the use of these traditional drugs challenging for both patients and physicians. However, our increased understanding of the intricate mechanisms regulating the coagulation pathway has allowed the formulation and development of new and promising anticoagulants such as orally active direct thrombin inhibitors and direct activated factor X inhibitors[9]; these appear to be equally effective as current anticoagulants, but (purportedly) do not require (strict) laboratory monitoring. Unfortunately, the number of clinical trials providing unequivocal results on the efficacy and safeness of these innovative pharmacologic agents is still limited, which makes their introduction into the routine anticoagulation of patients tricky in the real world. In addition to practical considerations on function and metabolism, Tripodi and van den Besselaar highlight that laboratory monitoring of direct thrombin and factor X inhibitors might be warranted under specific circumstances, thus requiring some laboratory methods to assess whether and to what extent anticoagulation with these drugs has been achieved. This would suggest that laboratory monitoring of therapeutic anticoagulation may still be with us for some years to come, although the extent and form of this monitoring is still evolving.

Indeed, medicine itself continues to evolve as a whole, and laboratory medicine also continues to be subjected to great change. Much emphasis is currently placed on personalized medicine, an attractive terminology, meaning an individualized approach to patients for either diagnosis or treatment using the most advanced and appropriate scientific and technological tools.[10] To be really effective, however, personalized medicine requires construction of a revolutionary health care framework, including a panel of laboratory tests adopted for each subject, which should be descriptive enough of the individual metabolic characteristics to be useful in a lifelong perspective, with any required adjustments due to peculiar genetic variations and/or diseases.[10] As a logic continuum in the theme of this issue, Rumilla et al provide an updated overview on pharmacogenetics in hemostasis, reinforcing the “outreach” concept of a “personalized” approach in several thrombotic and bleeding disorders.[11] It is clearly established that pharmacologic therapies are the mainstay of management for patients with both hemostatic and thrombotic diseases, as they would influence several components of primary and secondary hemostasis. However, it is also now known that the individual response to different drugs might vary substantially, so that the pharmacologic approach needs to be tailored to the patient, assessing risks and evaluating benefits on an individual basis. The recent exponential growth of pharmacogenomics in the field of hemostasis has contributed to spreading the concept of individualized/personalized medicine.[11] This in turn has revolutionized the current medical practice on the whole by allowing deeper understanding of disease pathways, improving diagnostic efficiency by the introduction of more sensitive and specific markers for risk stratification, and enhancing the great potential that lays in gene therapy, especially for single gene disorders. However, it is expected that in the very near future, pharmacogenomics as well as other sciences that share the suffix “-omics” (e.g., genomics, transcriptomics, proteomics, metabolomics, metallomics, lipidomics, glycomics, interactomics, spliceomics, exposomics, nutrigenomics, etc.) will also provide solid bases to permit better understanding and management of the intraindividual variability in response to medications.[12] The clinical management of drugs prescribed to treat thrombotic and hemostatic abnormalities, especially oral anticoagulants and antiplatelet medications, may in fact obtain great advantage from these emerging sciences, as long as the extent of variability in the individual response to therapy can be anticipated.

Prisco and Grifoni then provide a state-of-the-art on the clinical significance, technical features, and the role of D-dimer testing in patients with suspected venous thromboembolism (VTE). Deep vein thrombosis and pulmonary embolism represent to date the major causes of mortality and morbidity in hospitalized patients, but are also a diagnostic dilemma in emergency departments, where the timely and appropriate triage of patients is pivotal for preventing adverse outcomes. Several laboratory tests were made available for the initial assessment of patients with suspected VTE over the past decades, including fibrin/fibrinogen degradation products, thrombin-antithrombin complex, prothrombin fragment 1 + 2, fibrinopeptide A, soluble fibrin monomers, and cross-linked degradation products of fibrin.[13] However, at this time only D-dimer has remained as the “biochemical gold standard.” D-dimer is the final degradation product of cross-linked fibrin and is hence typically elevated in patients with acute VTE.[14] Because of the high negative predictive value, D-dimer testing has achieved a definitive role in the rule-out approach of patients with suspected deep vein thrombosis or pulmonary embolism, as well as in other thrombotic pathologies.[14] However, the clinical usefulness of D-dimer testing should be carefully evaluated in special clinical settings, and its measurement should be integrated at various steps in sequential diagnostic strategies, which include pretest clinical probability and imaging techniques. A major issue to be resolved as yet, however, is the large variability observed among different commercial assays, which in fact display heterogeneous sensitivity and specificity for the diagnosis of VTE. Some endeavors toward standardization have been attempted, but without any resolute outcome.[15]

Shifting from thrombosis to bleeding, Favaloro then drives us on an “emotional” trip on the most common inherited bleeding disorder, von Willebrand disease (VWD), once more linking laboratory findings with clinical outcomes. The diagnosis of VWD requires both clinical and laboratory evaluation. However, both the diagnosis and the functional characterization of this disorder are challenging due to the heterogeneous clinical presentation, the inherent limitations in the first-line and second-line diagnostic tests used by traditional and specialized laboratories, and especially because the current classification does not always reflect an unequivocal assignment for all the possible subtypes. Although laboratory investigation typically entails initial plasma testing of factor VIII coagulant (FVIII:C), VWF protein (antigen; VWF:Ag), and VWF activity (usually by the ristocetin cofactor assay [VWF:RCo]), new tests are emerging, including the collagen binding (VWF:CB) assay and other putative VWF “activity” assays, which are sometimes seen as possible alternatives to VWF:RCo, or otherwise used as supplementary tests of VWF activity. Furthermore, depending on local availability, further supplementary laboratory testing to achieve a definitive classification of the disorder might also include VWF multimers, ristocetin-induced platelet agglutination, VWF-factor VIII binding, and, under specific circumstances, genetic analysis. The use of these tests (as well as the long personal experience with several of these) is extensively reviewed in this article. The author's basic assertion is that the more extensive the investigation, the more likely the correct identification both of VWD and the likely type. Conversely, the use of limited test panels or poor test methodologies will compromise diagnostic accuracy and thus result in high likelihoods of an incorrect diagnosis and disease-type assignment leading to inappropriate managed care. Finally, the author discusses the potential emergence of a new paradigm for the identification and functional characterization of VWD, involving a desmopressin (DDAVP) challenge and evaluation of several tests for VWD, including FVIII:C, VWF:Ag, VWF:RCo, VWF:CB,[16] the PFA-100,[17] and the VWF propeptide assay. In particular, this approach should permit the more definitive discrimination and functional characterization of types 1, 2A, and 2M VWD, as well as those forms of VWD that show reduced VWF survival (or increased VWF clearance).

The next article, by Franchini and colleagues, is entirely devoted to extracorporeal immunoadsorption for the treatment of coagulation inhibitors, severe disorders caused by autoantibodies directed against clotting factors and associated with an increased morbidity and mortality.[18] This procedure is widely used for the removal of pathogenic antibodies in a variety of immunologic disorders but has also been recently included within immune tolerance protocols for both acquired and congenital hemophilia in particular in patients with high-titer inhibitors against coagulation factors. In this peculiar clinical setting, temporary removal of antibodies might be advisable before initiating replacement therapy to achieve hemostasis and stop acute bleeding or to cover a surgical procedure. Recent data confirm that extracorporeal immunoadsorption is a safe and useful technique for this purpose. However, further randomized clinical trials might be needed to finally establish its cost-effectiveness in patients with coagulation inhibitors.

In the article by Rumilla et al previously mentioned, an overview of the great advantages of introducing pharmacogenetics for the clinical management of thrombotic disorders was detailed. However, thrombosis is basically a multifactorial disorder, where several inherited or acquired risk factors contribute to propel the individual risk over a threshold that precipitates the development of disease. Hence, although valuable, the use of pharmacogenomics and gene therapy would be less useful in multifactorial, multigene disorders, as they would only cover a limited part of the managed care of patients with thrombosis. Instead, monogenic disorders like hemophilia A and B represent ideal candidates for treatment with gene therapy. In hemophiliacs, a therapeutic benefit by gene therapy should not require huge increases in the deficient factor, as even modest increases would be therapeutic, and the response to the treatment would be easily monitored. Two main approaches have been developed to replace endogenous production of factor VIII or factor IX (deficient in hemophilia A and B, respectively), which are mainly based on genetically modified cells or direct in vivo gene delivery using viral or plasmid vectors. The article by Viiala and colleagues is another great example of the sometimes frustrating process of translation of basic science into practical clinical outcomes. In this article, Viiala and colleagues provide a comprehensive overview on the progress of gene therapy for hemophilia in both preclinical and clinical models. Various approaches to enable delivery of the exogenous clotting factors (mainly by viral and nonviral vectors) are discussed in the context of current limitations, as emphasized in some preclinical and clinical trials. The conclusion of the authors is however appealing; despite some disappointing results in clinical trials to date, there are optimistic perspectives that the near future should deliver on the long-sought promise of a definite or long-lasting treatment for hemophilia besides convention replacement or bypassing therapies.

The next article by Isbister represents yet another change of focus, but still follows the theme of bench and bedside. Venomous snakes are present almost everywhere in this world, even within the oceans. Responsible for nearly 3 million bites a year, they pose a significant health problem in terms of both mortality and morbidity, causing as much as 100,000 deaths a year worldwide and severe pathologies in those who survive (e.g., amputations, renal failure, infections). The greatest problems are caused by the venom, a highly modified saliva produced by special glands of certain (but not all) species of snakes. Most venoms are “cocktails” of toxic components, including neurotoxins, hemorrhagins, coagulant toxins, nephrotoxins, myotoxins, and necrotoxins. Many of these proteins are harmless to humans, but some are “true” toxins, capable of causing serious harm when envenomed. What is the link between snake venoms and hemostasis, and between snake venoms and translational or personalized medicine? This is unveiled by Isbister, who provides an overview on laboratory studies, diagnosis, and pathophysiology of snakebite coagulopathy, using the Australian situation as a case example. Procoagulant toxins produced by some snakes, most of them living in Australia, are important hemotoxins that can result in a severe venom-induced consumption coagulopathy. Because of the previously mentioned heterogeneous mix of enzymes and proteins, the procoagulant activity of the various venoms cannot be easily characterized. Therefore, most studies have simply focused on identifying the in vitro activity of single isolated compounds, mainly by conversion of amidolytic substrates, such as the effect of a snake toxin on purified fibrinogen or on multiple single substrates. It follows, though, that the final effects of snake toxins on in vivo hemostasis are rarely known, and our understanding of the pathophysiology of envenoming is incomplete even in vitro. The challenging approach to the otherwise frequent pathology that is snakebite coagulopathy involves both diagnosis and therapy. The diagnosis is foremost for the treatment but still suffers from the lack of definitive clues on the pathogenesis, from the heterogeneous clinical presentation according to the type and amount of venom envenomed, and from the lack of universal agreement on which assays are the most informative in these circumstances. Therefore, the diagnostic approach to snakebite coagulopathy is still a search for the identification, optimization, and standardization of both diagnostic algorithms and tests. It is important to mention here that a better understanding of the coagulation effects arising from human envenoming not only will be helpful for a timely and accurate diagnosis but also for improving the treatment with antivenoms or adjuvant therapies such as factor replacement. Finally, reviewing the current knowledge on snakebite coagulopathy, Isbister provides a synthetic description of the snake toxins that have been most investigated and used for decades as laboratory reagents and as therapeutic agents and challenges the current therapeutic approach taken by emergency physicians.

The concept that thrombin plays a central role in hemostasis through its multiple functions across blood coagulation, platelet activation, and fibrinolysis is now clearly acknowledged, and is the subject of the next article by Adams. In particular, the measurement of thrombin generation can be seen as a potentially useful test that could be applied to the screening, monitoring, and/or diagnosis of hemostatic abnormalities. Advances in thrombin generation assays have created significant interest and debate as to whether they may provide a more physiologically relevant testing system than those of traditional coagulation tests. To date, a variety of thrombin generation assays has been developed, which have been suggested for investigation both hypocoagulable and hypercoagulable states. Several previous articles have addressed the clinical applications of thrombin generation assays, as well as their drawbacks, which mainly include poor standardization of the tests and the lack of reliable studies that demonstrate clear relationships between thrombin generation with bleeding and thrombosis phenotypes, as well as with monitoring anticoagulation (reviewed in Ref. [19]). Ideally, thrombin generation assays represent one typical situation in which the findings of the bench might be effectively translated to the bedside, for improving both the decision-making and the managed care. Accordingly, Adams briefly compares the process of thrombin generation in vivo versus in vitro, discusses potential applications of thrombin generation assays, and explores whether thrombin generation tests are already “useful” or, to this point in time, predominately represent “hype.”

In the last article of this issue of Seminars in Thrombosis and Hemostasis, Brand-Miller and colleagues explore the challenging relationship between thrombogenesis and glycemic index (an in vivo measure of the blood glucose response to a standard amount of carbohydrate from a food relative to a reference food) and glycemic load (which reflects both quality and quantity of carbohydrate and is defined as the product of the amount of carbohydrate and its glycemic index). Although it is traditionally accepted that hyperglycemia and insulin resistance are independent risk factors for cardiovascular disease, as major components of the metabolic syndrome, the definitive mechanisms linking glycemic imbalance with cardiovascular events are still poorly recognized and most likely multifaceted. Brand-Miller et al suggest that postprandial glycemic “spikes” might adversely affect vascular structure and function via multiple mechanisms including inflammation, oxidative stress, oxidation of atherogenic lipoproteins, glycation of protein, and, least but not last, a supposed procoagulant activity (increasing plasminogen activator inhibitor-1 activity and other cardiovascular risk factors). This association has now been confirmed in randomized and prospective trials, where both glycemic index and and/or glycemic load independently predicted cardiovascular disease, with relative risk ratios between 1.2 to 1.9 comparing highest and lowest quartiles. Taken together, the findings presented in this article are consistent with the hypothesis that clinicians may be able to improve cardiovascular outcomes by recommending the judicious use of foods with low glycemic index and/or glycemic load. Although both glycemic index and glycemic load should be considered, there is evidence that glycemic index per se (i.e., independent of glycemic load) is particularly important. Accordingly, focusing on glycemic load alone might lead to inappropriate overrestriction of all carbohydrates. Further practical guidance is available at the Web site http://www.glycemicindex.com/. In addition, it needs to be recognized that this article is necessarily focused on carbohydrate and thrombogenesis, and there are other important components to a well-balanced diet including high-quality protein, particularly as we age.[20]

The guest editors of this issue of Seminars in Thrombosis and Hemostasis would like to sincerely thank all the authors for their interesting and timely contributions. We hope that you as readers enjoy the collation of articles and the second of this series of issues related to the laboratory-clinical interface, and the first of the series devoted to translational research in hemostasis.

REFERENCES