Volume 48, Issue s1

Apart from the central beneficial role platelets play in hemostasis, they are also involved in atherothrombotic diseases. Here, we review the current knowledge of platelet intracellular signal transduction pathways involved in platelet adhesion, activation, amplification of the activation signal and aggregation, as well as pathways limiting platelet aggregation. A thorough understanding of these pathways allows explanation of the mechanism of action of existing antiplatelet agents, but also helps to identify targets for novel drug development. Clin Chem Lab Med 2010;48:S3–13.

Hypercoagulable state is not a uniform disease. It is a complex condition with an abnormal propensity for thrombosis that may or may not lead to thrombosis, depending on complex gene-gene and gene-environment interactions. The prevalence of the hypercoagulable state depends on the ethnicity and clinical history of the population being studied. The consequences of a hypercoagulable state due to thrombosis of veins and arteries are the most important cause of sickness and death in developed countries at present. Primary hypercoagulable state is an inherited condition caused by the reduced level of natural anticoagulants due to a qualitative defect or quantitative deficiency of an antithrombotic protein, or increased concentrations or function of coagulation factors. Most of the inherited abnormalities recognized to date have little or no effect on arterial thrombosis and are associated primarily with venous thromboembolism. Arterial thrombosis usually develops as a complication of atherosclerosis and patients usually have more than one traditional risk factor. Secondary hypercoagulable states generally occur as a result of a large number of transient or permanent acquired conditions that increase the tendency for formation of blood clots. New epidemiological data and clinical trials suggest that many acquired risk factors in the pathophysiology of arterial and venous thrombosis overlap and coexist for both disorders. Clin Chem Lab Med 2010;48:S15–26.

Thrombophilia screening is aimed at detecting the most frequent and well-defined causes of venous thrombosis, such as activated protein C resistance/factor V Leiden mutation, prothrombin G20210A gene mutation, deficiencies of natural anticoagulants, such as antithrombin, protein C and protein S, the presence of antiphospholipid antibodies, hyperhomocysteinemia and increased factor VIII activity. At this time, thrombophilia screening is not recommended for those possible congenital or acquired risk factors, whose association with increased risk of thrombosis has not been proven sufficiently. Laboratory investigations should include a step-wise approach to the diagnosis of thrombotic disorders with respect to the assays and methods of analysis that are used. The assays recommended for the first diagnostic step of screening should establish, whether the subject has one of the common causes of thrombophilia. If one or more abnormal results are obtained, the second diagnostic step includes the assays recommended for confirmation and/or characterization of the defect. When performing the investigation of thrombophilia, it is important to consider all pre-analytical and other variables that may affect the results of thrombophilia testing, including time of testing, age, gender, liver function, hormonal status, pregnancy or the acute phase response to inflammatory diseases. This is necessary, in order to avoid, any misinterpretation of the results. This review summarizes the current knowledge concerning thrombophilia investigations, with special focus on the diagnostic algorithm regarding patient selection, the assays and methods of analysis used and all the variables that should be considered when employing tests for the diagnosis of thrombophilia. Clin Chem Lab Med 2010;48:S27–39.

Venous thrombosis (VT) represents a common and serious disorder that occurs as the result of clotting of the blood in the venous system and venous obstruction. Environmental risk factors and genetic predisposition play an important role in the development of thrombosis. It is therefore seen as a classic example of a complex common disease. We have focused on the role of genetic risk factors, primarily related to the hemostatic system, in triggering thrombotic events. Since the identification of antithrombin deficiency in 1965, major efforts have been made during the past 15 years to identify other genetic entities that lead to increased thrombotic risk. Results of early genetic studies demonstrated that two types of genetic defects cause VT: loss of function mutations in the natural anticoagulants antithrombin, protein C and protein S and gain of function mutations in procoagulant factors V (FV Leiden) and II (prothrombin G20210A). The high incidence of these mutations in Caucasians induced a shift from family studies to case-control association studies. Several investigations have been performed on the role of other candidate genetic risk factors predisposing to VT, including such variants in FXIII , FIX and fibrinogen genes. Moreover, the contribution of genetic variation in genes encoding less-well studied proteins that are part of the anticoagulant pathways has been evaluated. Recently, different genome-wide association studies have been performed in which several single nucleotide polymorphisms were investigated and related to the risk of VT. However, further studies are needed to identify additional genetic causes of thrombosis and to assess functional molecular mechanisms. Clin Chem Lab Med 2010;48:S41–51.

Protein C (PC) and protein S (PS) are vitamin K-dependent glycoproteins that play an important role in the regulation of blood coagulation as natural anticoagulants. PC is activated by thrombin and the resulting activated PC (APC) inactivates membrane-bound activated factor VIII and factor V. The free form of PS is an important cofactor of APC. Deficiencies in these proteins lead to an increased risk of venous thromboembolism; a few reports have also associated these deficiencies with arterial diseases. The degree of risk and the prevalence of PC and PS deficiency among patients with thrombosis and in those in the general population have been examined by several population studies with conflicting results, primarily due to methodological variability. The molecular genetic background of PC and PS deficiencies is heterogeneous. Most of the mutations cause type I deficiency (quantitative disorder). Type II deficiency (dysfunctional molecule) is diagnosed in approximately 5%–15% of cases. The diagnosis of PC and PS deficiencies is challenging; functional tests are influenced by several pre-analytical and analytical factors, and the diagnosis using molecular genetics also has special difficulties. Large gene segment deletions often remain undetected by DNA sequencing methods. The presence of the PS pseudogene makes genetic diagnosis even more complicated. Clin Chem Lab Med 2010;48:S53–66.

Antithrombin (AT) belongs to the serpin family and is a key regulator of the coagulation system. AT inhibits active clotting factors, particularly thrombin and factor Xa; its absence is incompatible with life. This review gives an overview of the protein and gene structure of AT, and attempts to explain how glucosaminoglycans, such as heparin and heparan sulfate accelerate the inhibitory reaction that is accompanied by drastic conformational change. Hypotheses on the regulation of blood coagulation by AT in physiological conditions are discussed. Epidemiology of inherited thrombophilia caused by AT deficiency and its molecular genetic background with genotype-phenotype correlations are summarized. The importance of the classification of AT deficiencies and the phenotypic differences of various subtypes are emphasized. The causes of acquired AT deficiency are also included in the review. Particular attention is devoted to the laboratory diagnosis of AT deficiency. The assay principles of functional first line laboratory tests and tests required for classification are discussed critically, and test results expected in various AT deficiency subtypes are summarized. The reader is provided with a clinically oriented algorithm for the correct diagnosis and classification of AT deficiency, which could be useful in the practice of routine diagnosis of thrombophilia. Clin Chem Lab Med 2010;48:S67–78.

Factor V Leiden and prothrombin ( F2 ) c.20210G>A mutation detection are very important in order to define the increased relative risk for venous thromboembolism in selected patients. Use of DNA-based methods to detect both mutations has become widely available in clinical diagnostic laboratories, including fluorescence-based quantitative real-time PCR (qPCR). The latter is a rapid, simple, robust and reliable method to identify genotypes of interest. There are several chemistries used for qPCR; this article describes their principles and applicability for Factor V Leiden and prothrombin ( F2 ) c.20210G>A mutation detection. Clin Chem Lab Med 2010;48:S79–87.

It is now widely accepted that hyperhomocysteinemia (HHC) is a risk factor for thrombophilia. HHC is the result of either impaired enzyme function or a deficiency of vitamin B (folate, B 6 , B 12 ), or both, and can be treated with vitamin supplements. Measuring plasma total homocysteine (tHcy) is included in the routine thrombophilia panel in many laboratories, despite having a limited value to the clinician. Many methods are available for tHcy measurements. High-pressure liquid chromatography (HPLC) with fluorescence detection is a widely used method, but is being replaced by more convenient immuno- or enzyme assays. In this paper a general overview on homocysteine is given, with an emphasis on laboratory methods. Clin Chem Lab Med 2010;48:S89–95.

Thrombotic risk factors and thrombosis in children has been receiving increased attention. True idiopathic thrombosis is extremely rare in children. Most patients have a significant underlying medical condition and the presence of a central catheter is the most important risk factor. Children are more likely than adults to have one or more significant genetic abnormality or coagulation deficiency. This review discusses problems concerning the heterogeneity of thrombotic states in children and highlights the importance of understanding the concept of developmental hemostasis. Issues regarding step-wise test selection and the interpretation of results are addressed, as well as basic monitoring of anticoagulant drug effects. Clin Chem Lab Med 2010;48:S97–104.

Thrombophilia can be identified in about half of all patients presenting with venous thromboembolism (VTE). Thrombophilia screening for various indications has increased tremendously, but whether the results of such tests help in the clinical management of patients is uncertain. Here, current recommendations for thrombophilia screening in selected groups of patients, and considerations whether other high-risk subjects should be tested are reviewed. The methods for determination of the most common thrombophilic defects (antithrombin, protein C, protein S deficiencies, Factor V Leiden and prothrombin G20210A) associated with strong to moderate risk of VTE are described, indicating the timing and location of thrombophilia screening. Circumstances when a positive result of thrombophilia screening helps clinicians decide if adjustments of the anticoagulant regime are needed are discussed. Finally, psychological, social and ethical dilemmas associated with thrombophilia screening are indicated. Clin Chem Lab Med 2010;48:S105–13.

Genetic association studies explore the association between genetic polymorphisms and a certain trait, disease or predisposition to disease. It has long been acknowledged that many genetic association studies fail to replicate their initial positive findings. This raises concern about the methodological quality of these reports. Case-control genetic association studies often suffer from various methodological flaws in study design and data analysis, and are often reported poorly. Flawed methodology and poor reporting leads to distorted results and incorrect conclusions. Many journals have adopted guidelines for reporting genetic association studies. In this review, some major methodological determinants of genetic association studies will be discussed. Clin Chem Lab Med 2010;48:S115–8.

Advances in the field of human genetics has made it possible to develop prevention strategies for rare genetic disorders and to tailor pharmacotherapeutic approaches to anticoagulation and certain cancers. However, it is still not clear how genetic variations influence the risk and outcome of common diseases. Data from genome-wide association studies is just beginning to answer these questions. In contrast, pharmacogenetic knowledge is frequently not yet translated into clinical practice, even though in some cases, particularly regarding drugs used in treatment of thrombotic diseases (e.g., coumarines, platelet aggregation inhibitors), it is already known that testing for genetic variants prior to pharmacotherapy may help to prevent severe adverse drug reactions or avoid therapeutic failure. In this review, we address the potential impact that genetic alterations in the genes for vitamin K epoxide reductase and some cytochrome P450 variants may have on therapeutic strategies in anticoagulation. Clin Chem Lab Med 2010;48:S119–27.