Newer avenues in the monitoring of antithrombotic therapy: the role of automation

In the future, we may find that antithrombotic therapy provides its major effects other than by Factors Xa and IIa inhibition, in which case a panel of assays may be of clinical use to determine how a given patient is responding to therapy. All methods discussed previously for direct factor assays, global tests, or specific factor inhibition can be modified for synthetic substrates. With instruments such as the IL Multistat III centrifugal analyzer, which has detection capabilities for visible light, fluorescence and light scatter, it will be possible to panel profile synthetic substrate-based assays, immunologic assays (for example, platelet release products or circulating complexes of enzyme-inhibitor, such as thrombin-AT III) and possibly platelet aggregations/luminescence in one rotor. The field of coagulation is rapidly expanding in the development of newer tests to measure newly discovered proteins (protein C), development of new drugs and therapeutic regimens, and incorporating immunologic and enzymologic technologies and clinical chemistry standardization and quality control practices. Throughout this period of growth, automation has been in the forefront and is playing an ever-increasing role in every old and new aspect of coagulation evaluation. We are aware of the advantages that accompany any automated device over manual techniques, so there should be much hope and expectation for the new hemostasis and thrombosis laboratory.     

Monoclonal antibody-based plasma assays for fibrin(ogen) and derivatives, and their clinical relevance

The haemostatic balance can basically be described as the equilibrium between fibrin formation (coagulation) and fibrin lysis (fibrinolysis). The status of this balance may therefore be reflected by the products of these two processes. Until recently, the tests for assessment of fibrin(ogen) degradation products were performed in serum since they were based on polyclonal antibodies, which cross-react with fibrinogen. However, the use of serum introduces many artefacts so the utility of these serum tests is limited. New assays have now become available, which can be divided into quantitative enzyme immunoassays (EIAs) and semi-quantitative latex agglutination assays. The new assays can be carried out in plasma since they use highly specific monoclonal antibodies, the majority of which do not cross-react with fibrinogen. This makes it possible to avoid the serum artefacts. Furthermore, these plasma assays can discriminate between degradation products of fibrin and those of fibrinogen (FbDPs and FgDPs, respectively). The possible clinical utility of the new assays is discussed on the basis of literature data on the following clinical states: deep venous thrombosis (DVT) and pulmonary embolism, liver disease and liver transplantation, sickle cell disease, renal diseases, pregnancy and preeclampsia, disseminated intravascular coagulation (DIC), malignancy, coronary artery disease and thrombolytic therapy. Fibrinolysis appears to be accompanied by fibrinogenolysis. Detection of fibrin(ogen) derivatives may be used to rule out DVT and to monitor efficacy of anticoagulant treatment for DVT or DIC, and reflects severity of renal disease but not renal function. High levels of FgDPs were found during orthotopic liver transplantation and thrombolytic therapy. Fibrin(ogen) degradation products cannot be used to predict reperfusion following thrombolytic therapy. The fibrinolytic system remained active during normal and complicated pregnancy and in patients with malignancies. The new assays provide valuable information on fibrin(ogen)olysis in several diseases. More information on the haemostatic balance may be obtained by using these new assays for fibrin(ogen)olysis products in combination with assays for coagulation products.     

Hemostasis testing in patients with liver dysfunction: Advantages and caveats

Due to concomitant changes in pro- and anti-coagulant mechanisms, patients with liver dysfunction have a “rebalanced hemostasis”, which can easily be tipped toward either a hypo- or a hypercoagulable phenotype. Clinicians are often faced with the question whether patients with chronic liver disease undergoing invasive procedures or surgery and those having active bleeding require correction of the hemostasis abnormalities. Conventional coagulation screening tests, such as the prothrombin time/international normalized ratio and the activated partial thromboplastin time have been demonstrated to have numerous limitations in these patients and do not predict the risk of bleeding prior to high-risk procedures. The introduction of global coagulation assays, such as viscoelastic testing (VET), has been an important step forward in the assessment of the overall hemostasis profile. A growing body of evidence now suggests that the use of VET might be of significant clinical utility to prevent unnecessary infusion of blood products and to improve outcomes in numerous settings. The present review discusses the advantages and caveats of both conventional and global coagulation assays to assess the risk of bleeding in patients with chronic liver disease as well as the current role of transfusion and hemostatic agents to prevent or manage bleeding.     

Current status of hemophilia patients and recombinant coagulation factor concentrates in Japan

The first recombinant factor VIII concentrate was introduced in 1987 to treat hemophilia A patients, and the product was licensed in the United States in 1992. More than 10 years have passed since the recombinant products have been used for treatment of hemophilia A. The new therapeutic options seem to be safe and effective through the gathered experiences. Recently, recombinant factor VIIa concentrate has become available to treat hemophilia patients with inhibitor and factor VII deficiency patients in Europe and also recombinant factor IX for the treatment of hemophilia B has been licensed in the United States and Europe. The usage of recombinant coagulation factors has expanded the routine therapy for hemophilia in many countries. In Japan, the consumption of recombinant factor VIII is increasing year by year, because many patients have started to think that the recombinant technology seems to be safe. Unfortunately, though, the factor VIIa and factor IX products have not been licensed yet in Japan. This article discusses the current status of patients with hemophilia and recombinant coagulation factor products in Japan.     

Clinical utility of viscoelastic testing (TEG and ROTEM analyzers) in the management of old and new therapies for hemophilia

Hemophilia A and B are rare inherited bleeding disorders resulting from deficiency of coagulation factors VIII and IX respectively. In the past few decades, the field of hemophilia has witnessed pivotal management challenges and therapeutic advances. Routine coagulation and factor assays, while useful in the classification of severity and treatment monitoring in hemophilia patients, have been shown to be of limited use in managing clinical presentations and outcomes. This prompted the investigation of viscoelastic studies in hemophilia care, which have established their utility in various bleeding and thrombotic states. In this review, we will discuss and critically assess the current literature highlighting the use of viscoelastic studies in various aspects of hemophilia including the determination of clinical phenotype, management of patients with inhibitors, perioperative management, and monitoring of novel agents.     

New strategies in the determination of fibrin and fibrin(ogen) derivatives by monoclonal antibodies

Summary Until recently only tests with a limited specificity were available for the assessment of the products of activated coagulation and/or fibrinolysis. Those assays were based on polyclonal antibodies, which crossreact with fibrinogen, and as a consequence they were performed on serum samples i.e. after removal of fibrinogen by clotting. Serum preparation, however, is a notorious source of artefactually high or low levels of fibrin(ogen) degradation products, and is not suitable for the determination of coagulation products. Recently, highly specific monoclonal antibodies (MoAb's) have been developed, the majority of which do not crossreact with fibrinogen. This has enabled new strategies to be developed, i.e. assays using these MoAb's on plasma samples. Furthermore, the new assays can discriminate between (individual) fibrin and fibrinogen degradation products, and coagulation products can be assessed in the same plasma samples.     

Chromogenic peptide substrate assays and their clinical applications

Chromogenic peptide substrates were first introduced into research laboratories in the early 1970s and were quickly utilised to develop assays for the determination of enzymes, proenzymes and inhibitors of the coagulation system. These assays were gradually introduced into coagulation and clinical chemistry laboratories as laboratory tools in the diagnosis and treatment of coagulation disorders. From the knowledge of the structures of the natural substrates attacked by enzymes other than those of the coagulation system or by synthesis and random screening, substrates for enzymes of the fibrinolytic, plasma and glandular kallikrein and complement systems were produced. These allowed various research groups to develop assays for components of these systems and subsequently led to the use of these assays in studies on various clinical conditions. Substrates for activated protein C ensured that assays for this enzyme and its inhibitors could be developed and introduced into the haematological routine. With the introduction of substrates for limulus lysate not only were assays for endotoxins in clinical samples produced but the control of all disposable products and injectables for endotoxin contamination can now be effected. Initially high costs and time-consuming manual assays were a hinderence to the general acceptance of the use of chromogenic peptide substrate assays and they were only used routinely in a few specialised laboratories. With the introduction of automated and microtitre plate methods however, these assays are are now available in most hospital laboratories. Since the first chromogenic peptide substrate was described thousands of articles have been published on the use of chromogenic substrate assays to measure proenzymes, enzyme activators, enzyme cofactors and inhibitors in blood and other body fluids in normal subjects and clinical material. We have endeavoured to cover as many of these as possible in this review.     

Recombinant coagulation factor products

Summary There are numerous new coagulation factor concentrates that are being manufactured using recombinant technology. Some are available for use currently for the treatment of bleeding disorders and others are in clinical trials. Recombinant factor VIII concentrates are licenced in most countries and one FVIII concentrate with the B domain deleted should be available in the near future. Recombinant VIIa concentrate is in advanced phase III testing for the treatment of patients with inhibitor antibodies. Recombinant factor IX has been used successfully in animals and will be tested in humans shortly. This paper reviews these products and discusses their uses and possible side-effects.     

How to minimize blood loss during liver surgery in patients with cirrhosis

Patients with liver disease frequently have substantial changes in their haemostatic system. This is reflected in abnormal test results on routine coagulation screening assays such as the prothrombin time (PT), activated thromboplastin time (APTT) and platelet count. Traditionally, attempts were made to correct abnormalities in the haemostatic system as measured by routine coagulation assays prior to invasive procedures by infusion of platelets or fresh frozen plasma (FFP). Recent laboratory and clinical data have indicated that the haemostatic reserve in cirrhotic patients is relatively well maintained although the coagulation screening assays suggest otherwise. Pre-procedural correction of coagulation tests with blood products may therefore not be necessary, and may even have harmful side-effects. In particular, fluid overload resulting in exacerbation of portal hypertension by infusion of blood products may in fact promote bleeding. In recent years, it has become clear that reduction of the central and portal venous pressure by fluid restriction and avoidance of blood product transfusion is a beneficial strategy in minimizing bleeding during liver surgery in cirrhotic patients. Some investigators have even taken this a step further and suggested pre-procedural phlebotomy in liver transplant recipients. The aim of this review is to provide an overview of recent studies and developments which have changed our understanding of the clinical relevance of abnormal coagulation tests in patients with cirrhosis, and which have contributed to a reduction in blood loss and transfusion requirements when liver surgery is needed in these patients.     

New products for managing inhibitors to coagulation factors: a focus on recombinant factor VIIa concentrate

The treatment of alloantibody and autoantibody inhibitors directed against the factor VIII coagulant protein is one of the most challenging and expensive problems in hematology. Because the currently available plasma replacement products used in this context do not control the bleeding complications in all patients, and because of the usual emergent quality of the bleeding complications, there has been a definite need to have a uniformly reliable product for instant use, which possesses a high degree of hemostatic reliability and safety. The recent introduction of recombinant factor VIIa (rFVIIa) has been a welcome addition to the pharmacologic armamentarium for the treatment of neutralizing antibodies against coagulation factors. The mechanisms of action of rFVIIa have also been interesting and have provided insight into how the coagulation pathway accomplishes adequate hemostasis. This review will discuss this new medication and place into the context of coagulation inhibitor therapy.     

Laboratory measurement of thrombin activity—What every clinician scientist needs to know

Advances in our knowledge of the biochemistry of coagulation and fibrinolysis have facilitated the development of sensitive and specific assays that detect platelet activation, the generation of coagulation enzymes, and products of intravascular fibrin formation and dissolution. This review focuses on activation markers of blood coagulation and, in particular, on mechanistic information on the pathophysiology of blood coagulation they have provided. The methodological problems faced in employing these moieties in clinical studies are examined. Only the proper use of coagulation activation markers will enable us to establish their real clinical usefulness.     

The introduction of high-purity products in Canada

Summary . In October 1993, the Canadian Blood Agency, the agent for the provinces/territories in Canada, agreed to the introduction of high-purity coagulation products, either recombinant or highly purified factor concentrates, for the management of coagulation deficiencies. This represented a cost increase of approximately $30 million (40%) for the national coagulation product inventory.
Representatives of the relevant recipients of the products, some of the treaters, the distributor and the funders met on a regular basis in order to monitor the impact of these new products. The Association of Hemophilia Clinic Directors of Canada also agreed to include some specific outcome studies over a longer period of time to include evaluation of inhibitor formation, prophylaxis regimens, immune status and the incidence of thrombosis. The 'Hemophilia and Von Willebrand's Disease' clinical practice guidelines were also developed under their auspices. A usage monitoring system has been implemented and has been continuously managed by the Canadian Blood Agency. This resulted in trends of practice and rationales for unexpected use being identified early for planning and funding purposes.
The Working Group set up under the auspices of the Canadian Blood Agency was an effective vehicle to evaluate the conversion and the impact of these new products in the country and can serve as a model for future endeavours.     

Plasma and plasma products in the treatment of massive haemorrhage

Massive haemorrhage requires the use of plasma products when it is accompanied by a coagulopathy or when the more than one blood volume has been lost and intractable bleeding continues. The coagulopathy results from haemorrhagic shock, hypothermia, and activation, consumption and dilution of coagulation factors. Plasma products have a critical role in maintaining sufficient levels of coagulation proteins to ensure haemostasis can occur. Fresh frozen plasma is a source of all coagulation proteins and is required when the prothrombin time and activated partial thromboplastin time exceed 1.5 times the normal control. Cryoprecipitate is the plasma product of choice if fibrinogen, the most critical coagulation protein, is required rapidly and to maintain levels at >1g/L. Prothrombin complex concentrates, monocomponent factor therapy and fibrin sealants each have a role in specific clinical settings. Recombinant factor VIIa has now been shown to have a role in massive haemorrhage. Randomised controlled trials are currently underway to determine the optimal dose and timing of its administration. The physiology and management of the coagulation disturbance using plasma products in the massive haemorrhage of specific clinical situations are described.     

Use of pharmacokinetics in the coagulation factor treatment of patients with haemophilia

Dosing decisions for replacement coagulation factors in patients with haemophilia should be made on an individual patient basis, with the required dose dependent on factors including the clinical situation, the severity of the factor deficiency, and the location and extent of bleeding. Moreover, there is considerable variability in the pharmacokinetics of coagulation products that needs to be considered; in particular, with both factor (F) IX and FVIII products, there is considerable inter-patient variability in in vivo recovery and terminal half-life values. In the present report, we provide a practical guide to calculating and applying pharmacokinetic parameters relevant to the optimal dosing of coagulation products. We discuss the conduct of a pharmacokinetic study in an individual patient, how to calculate pharmacokinetic values from raw data and clinical situations where an individual pharmacokinetic study is helpful. We highlight the importance of considering an individual pharmacokinetic study in all patients starting a new coagulation product.     

Predictive Value of Inflammatory and Coagulation Parameters in the Course of Severe Ulcerative Colitis

Alterations in markers of coagulation have been found in patients with inflammatory bowel disease. Our aim was to study the predictive value of coagulation and inflammatory parameters in the course of severe ulcerative colitis. Twenty-seven patients were included. The disease course was followed for one year. Sensitivity, specificity, negative predictive value, positive predictive value, and likelihood ratio, as well as the clinical predictive value of laboratory variables were calculated. Inflammatory variables, such as ESR, CRP, and leukocyte and platelet count showed poor diagnostic accuracy. Several coagulation parameters, such as fibrinogen and fibrin(ogen) degradation products, were increased in patients with active ulcerative colitis, whereas coagulation factor XIII was decreased. No significant relationship between clinical course and coagulation parameters was demonstrated, though both inflammatory and coagulation parameters were useful in the assessment of disease activity in patients with active ulcerative colitis.     

Advances in the treatment of inherited coagulation disorders

Inherited coagulation disorders constitute a broad spectrum of coagulation factor deficiencies that include X‐linked factor (F)VIII or FIX deficiency that causes haemophilia, and autosomal recessive disorders producing heterogeneous deficiencies in fibrinogen (FI), prothrombin (FII), FV, FVII, FX, FXI, FXIII and combined FV+FVIII. Significant advances in treatments for patients with congenital haemophilia A (FVIII deficiency) and B (FIX deficiency) over the last two decades have resulted from improvements in the production, availability and patient access to factor replacement products. Translation of advances in biotechnology, namely recombinant protein technology, targeted protein modifications to improve function and potentially reduce immunogenicity, and advanced formulations to optimize bioavailability and sustain activity offer promisingly new treatments for haemophilia as well as recessively inherited bleeding disorders in patients who otherwise have few therapeutic options. Though a theoretical risk remains for blood‐borne viral infections with pooled plasma‐derived products, this concern has diminished with breakthroughs in purification and viral inactivation methods. Development of inhibitory antibodies is still the most daunting problem for patients with inherited bleeding disorders, complicating treatment approaches to control and prevent bleeding, and posing risks for allergic and anaphylactic reactions in susceptible patients. The objectives of this review are to (i) highlight emerging advances in hemostatic therapies that are bioengineered to improve pharmacokinetic properties and bioavailability, sustain functional activity, and possibly eliminate immunogenicity of recombinant factor proteins; and (ii) present an overview of key clinical trials of novel factor products currently in the development pipeline.     

The role of factor XI in coagulation: a matter of revision.

In 1991 it was demonstrated that, besides factor XII, thrombin is capable of activating factor XI in vitro. Thrombin-dependent activation of factor XI is an integral part of the revised theoretical model of coagulation in which coagulation is initiated by the extrinsic pathway and maintained by thrombin-induced activation of clotting factors V, VIII, and XI. In this review, special interest is given to the new role of factor XI in coagulation, with emphasise on data supporting the concept of thrombin-mediated factor XI activation in vivo. Furthermore, activation of factor XI in human disease, especially atherosclerotic disease, measured by newly developed immunologic assays, is discussed. The relation of factor XI to fibrinolysis through activation of the carboxypeptidase, thrombin-activatable fibrinolysis inhibitor (TAFI) by thrombin provides an explanation for the bleeding tendency observed in factor XI-deficient patients. The probable link with factor XI-mediated TAFI activation may have clinical and therapeutic consequences and deserves further study.     

Clinical utility and impact of the use of the chromogenic vs one-stage factor activity assays in haemophilia A and B

Treatment of haemophilia A/B patients comprises factor VIII (FVIII) or factor IX (FIX) concentrate replacement therapy, respectively. FVIII and FIX activity levels can be measured in clinical laboratories using one-stage activated partial thromboplastin time (aPTT)-based clotting or two-stage chromogenic factor activity assays. We discuss strengths and limitations of these assays, providing examples of clinical scenarios to highlight some of the challenges associated with their current use for diagnostic and monitoring purposes. Substantial inter-laboratory variability has been reported for one-stage assays when measuring the activity of factor replacement products due to the wide range of currently available aPTT reagents, calibration standards, factor-deficient plasmas, assay conditions and instruments. Chromogenic activity assays may avoid some limitations associated with one-stage assays, but their regulatory status, perceived higher cost, and lack of laboratory expertise may influence their use. Haemophilia management guidelines recommend the differential application of one or both assays for initial diagnosis and disease severity characterisation, post-infusion monitoring and replacement factor potency labelling. Efficient communication between clinical and laboratory staff is crucial to ensure application of the most appropriate assay to each clinical situation, correct interpretation of assay results and, ultimately, accurate diagnosis and optimal and safe treatment of haemophilia A or B patients.     

Hemostasis and fibrinolysis in renal transplantation

Results of histologic, biochemical, and immunologic studies suggest that coagulation and fibrinolysis are possibly important in kidney graft rejection, but there is no information about how or why they are involved. The touchstones of their importance are histologic reports of platelet-fibrin thrombi in glomerular vessels of rejecting kidneys. Most of the nonhistologic studies have made use of functional assays aimed at measuring coagulation changes in blood. Little use has been made of immunoassays, and there are almost no studies of recently described anticoagulant systems, such as the protein C pathway. Endothelial cells form the interface between donor and recipient tissues. Antibody or thrombin-stimulated endothelium produces PAF, and thrombin-stimulated endothelium produces both plasminogen activators and plasminogen-activator inhibitors. As important as these reactions might be, information about them has been obtained with the use of either in vitro experiments or artificial systems, and there is no direct evidence that they are relevant to transplantation. However, these observations do show that tissue factor, PAF and fibrinolytic activities can be immunologically triggered. This opens the possibility that allogeneic recognition may initiate the triggering process. The clotting and fibrinolytic phenomena discussed in this overview were consequences of allogeneic recognition. Such recognition reactions cause monocytes and macrophages to produce tissue factor, which is a potent initiator of coagulation. Endothelial cells also can be stimulated to elaborate tissue factor by immune complexes, interleukin-1, or endotoxin. These observations give cause to speculate that the link between immunity and coagulation in kidney transplantation could be products of allogeneic recognition that activate Factor VII and lead to fibrin deposition. If true, this suggests new approaches to the old problems of diagnosis and treatment of rejection reactions in organ transplantation.     

Maintenance control of oral anticoagulant therapy by an automated chromogenic substrate assay of factor X

Summary An amidolytic assay of factor X based on the new chromogenic peptide substrate S 2337 (Kabi Diagnostica) was adapted for use with the Kem-o-Mat (Coulter Electronics) automated substrate analyser. Factor X was assayed in 25 healthy controls and in 375 patients on Warfarin therapy. The results in the control group correlated well with a one stage coagulation factor X assay. A good correlation was also found when the S 2337 factor X assay was compared with Thrombotest (Nyegaard & Co.) results in the patients. From the regression line of the S 2337 factor X assay on the Thrombotest results, the comparable range for factor X amidolytic activity in well controlled anticoagulated patients was found to be 22.5-37.5% with this method. Concordant classification of patients by both tests according to proposed therapeutic ranges demonstrated fully concordant information in 76% and fully discordant information in none. This study demonstrates that chromogenic substrate assays for anticoagulant control can be readily automated. Amidolytic assays of factor X based on the S 2337 substrate, therefore, warrant further clinical investigation as a potential method for controlling maintenance oral anticoagulant therapy.