Daria Pašalić
Department of Medical Chemistry, Biochemistry and Clinical Chemistry
Zagreb University School of Medicine
Šalata ul 2.
10 000 Zagreb, Croatia
Phone +385 (1) 4590 205; +385 (1) 4566 940
E-mail: dariapasalic [at] gmail [dot] com

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Muszbek L.S08-1: Factor XIII – the state of the art. Biochemia Medica 2009;19(Suppl 1):S62-S63.
University of Debrecen, Medical and Health Science Center, Clinical Research Center, Debrecen, Hungary
Corresponding author:muszbek [at] med [dot] unideb [dot] hu
Factor XIII (FXIII) circulates in the plasma as a tetrameric zymogen (pFXIII; FXIII-A2B2). Its potentially active A subunit (FXIII-A) is synthesized in cells of bone marrow origin; it is also present in platelets and monocytes/macrophages in dimeric form (cFXIII; FXIII-A2). In the plasma the protective/carrier B subunit (FXIII-B) is in excess. pFXIII is converted into an active transglutaminase (FXIIIa) by thrombin and Ca2+ in the terminal phase of clotting cascade. The main function of FXIIIa is to cross-link fibrin chains and a2 plasmin inhibitor to fibrin through glutaminyl-lysyl isopeptide bonds. This way it mechanically stabilizes fibrin and protects from fibrinolysis. FXIII, a multifunctional protein, is also essential for maintaining pregnancy and plays an important role in wound healing and angiogenesis.
Inherited FXIII deficiencies are classified as FXIII-A and FXIII-B deficiencies. FXIII-A deficiency is a rare (1:2,000,000), but severe hemorrhagic diathesis. Delayed umbilical stump bleeding is characteristic and in non-supplemented patients subcutaneous, intramuscular and intracranial bleeding occurs with relatively high frequency. Impaired wound healing and spontaneous abortion in women also are features of FXIII-A deficiency. Type 1 deficiency (low activity and antigen) is much more frequent than type 2 (low activity with normal or moderately decreased antigen concentration). The rarely reported FXIII-B deficiency results in mild bleeding diathesis. Various forms of severe acquired deficiencies due to neutralizing or non-neutralizing auto-antibody against FXIII-A have been described. A significant portion of patients with an autoantibody against FXIII suffers from autoimmune disease. Virus inactivated pFXIII concentrate is now available for treatment and prophylaxis of FXIII deficiency.
A quantitative FXIII activity assay is to be used as first line (screening) test for the diagnosis of FXIII deficiency. The traditional qualitative clot solubility assay is now obsolete and should not be used as screening test. Quantitative FXIII assays are based on two principles: 1) measurement of ammonia released during the transglutaminase reaction, 2) the incorporation of labeled substrate amine into a glutamine donor substrate protein. The former methods are easy to perform, quick kinetic assays, while the latter ones are more sensitive, but time-consuming laborious methods. For classification determination of FXIII-A2B2 and FXIII subunit antigen in plasma and FXIII-A in platelets are recommended. Mixing studies and binding assays are used to detect neutralizing and non-neutralizing autoantibodies against FXIII subunits.
The involvement of FXIII in thrombotic diseases has been investigated in two aspects. 1) Elevated FXIII level has been shown to be a risk factor of myocardial infarction and peripheral artery disease in women. 2) During the last decade a common polymorphism in the FXIII-A gene, resulting in Val34Leu replacement, has been intensively investigated in this respect. The proteolytic activation of the Leu34 FXIII-A variant is accelerated and the polymorphism also influences the structure of fibrin. Although there are contradictory results, meta-analysis of the reported studies demonstrated the protective effect of FXIII-A Val34Leu polymorphism against venous thrombosis and against coronary artery disease. Gene-gene and gene-environment interactions, like insulin resistance or fibrinogen concentration, considerably modify the effect off the polymorphism.
Zupančić Šalek S. S08-2: von Willebrand disease – link between genotype and phenotype. Biochemia Medica 2009;19(Suppl 1):S63-S64.
Zagreb University Hospital Centre, Zagreb, Croatia
Corresponding author:silva [dot] zupancic-salek [at] zg [dot] t-com [dot] hr
Von Willebrand disease (vWd) is the most common inherited bleeding disorder and is due to quantitative (types 1 and 3) or qualitative (type 2) defects of von Willebrand factor (vWF). VW disease is inherited by autosomal dominant or recessive patterns, but women with mild forms are more symptomatic. Reported prevalence of vW disease derived from epidemiological studies of up to 1% in the general population although only approximately 1 in 10,000 individuals has clinically significant bleeding. vW factor is a large glycoprotein which is essential to platelet-dependent primary haemostasis, particularly in the microvasculature where high fluid shear forces are present. vWF also acts as a carrier for procoagulant factor VIII (FVIII) in the circulation, protecting FVIII from proteolytic degradation and transporting it to the site of vascular injury. The VWF gene is located on the short arm of chromosome 12 at 12 p13.3. VWD is classified into three main sub-types: Type 1 – a partial quantitative deficiency of VWF, type 2 – a qualitative abnormality of VWF, and type 3 – a virtually complete absence of VWF. Type 2 is further divided into four sub-categories with specific functional defects: type 2A shows a selective loss of high molecular weight (HMW) vWF multimers and an associated decrease in platelet-vWF interaction; Type 2B is associated with and increased affinity of VWF for platelet glycoprotein Ib; type 2M is associated with a VWF multimers pattern that is similar to normal, but with reduced vWF-platelet interaction. An enormous diversity of mutations has been characterized in VWD.
The initial diagnosis of vW disease is based on clinical and phenotypic information. Genetic analysis may provide information to confirm or support the initial diagnosis and additionally to permit family studies and counseling which are of particular value in type 3 vWD. Genetic investigation also plays a role in differential diagnosis. Genetic investigation also plays a role in differential diagnosis, distinguishing vWD type 2N and mild hemophilia.
Identification of the genetic basis of the disorder may inform treatment choices and improve our understanding of the mechanism underlying different vWD subtypes. It is apparent that genetic testing is likely to have limited clinical utility in cases where the phenotype clearly reveals the VWD sub-type.
Characterization of the phenotype and identification of mutations in VWF gene in patients with vW disease contribute of understanding of the genetics and biochemistry of VWF and VWD.
Coen Herak D.S08-3: Hemostatic system in children. Biochemia Medica 2009;19(Suppl 1):S64-S66.
Clinical Institute of Laboratory Diagnosis, Zagreb University School of Medicine and Clinical Hospital Center, Zagreb, Croatia
Corresponding author:desireecoen [at] yahoo [dot] com
For many years, understanding and characterization of hemostatic system in children has lagged behind those of adults, primarily due to technical difficulties in blood sampling in small children and the requirement of large amounts of blood volume. The greatest contribution in this field has been done by Maureen Andrew who first published age-dependent reference intervals for premature infants, healthy full-term newborns, and children 1-16 years of age. The knowledge that the levels of multiple hemostatic parameters differ between children and adults, and vary over the pediatric age range, has led to introduction of the.concept of developmental hemostasis.
Hemostasis is a dynamic process which begins in-utero. Fetal liver begins to synthesize fibrinogen at 5.5 weeks of gestation whereas blood clotting and fibrinolytic activity can be detected at 11 weeks of gestation. Physiologic concentrations of coagulation proteins gradually increase during fetal and neonatal life. The molecular structure of the majority of fetal coagulation proteins is identical to adult structures, except fibrinogen and plasminogen which contain increased amounts of sialic acid and von Willebrand factor (VWF) which is made up of ultra-large molecular weight multimers and changes to the adult plasma form postnatally.
In newborns, plasma concentration of vitamin K-dependent proteins (II, VII, IX, X) and contact factors (XI, XII prekallikrein, high molecular weight kininogen) are approximately 50% of adult values, increase gradually during the first 6 months of life to about 80% of adult normal values, but remain decreased throughout childhood. Furthermore plasma levels of almost all coagulation inhibitors are reduced, from approximately 20% for tissue factor pathway inhibitor, 35% for protein C (PC) and protein S (PS) to 50% of adult levels for antithrombin and heparin cofactor II. Although adult levels are usually reached by 6 months of age, plasma concentrations of PC remain decreased through childhood until adolescence. In addition PS circulates in newborns only in its free active form due to the absence of C4b binding protein.
Plasma concentrations of coagulation factors V, VIII, XIII and fibrinogen are close to adult values. Moreover, the levels of VWF are increased through the first 2 months of life, while the levels of alpha 2 macroglobulin that are nearly 2-fold higher than adult values, remain increased through childhood.
Fibrinolytic system in children is an evolving age-dependent process similar to coagulation system. Plasminogen levels are decreased to 50% and plasmin inhibitor to 80% of adult values until 6 months of age. Plasma concentrations of tissue-type plasminogen activator antigen are significantly increased at birth, progressively decrease after the first day of life, and remain decreased at approximately 50% of adult values throughout childhood. On the contrary, concentrations of plasminogen activator inhibitor-1 antigen at birth and throughout childhood are significantly increased compared to adult values. Therefore, the immaturity of hemostatic system in newborns results in decreased thrombin generation and hipofibrinolytic activity during childhood.
Since hemostatic system in children is profoundly different compared to adults, the knowledge of developmental hemostasis and applying age-dependent reference ranges are essential for diagnosis, optimal prevention and treatment of hemostatic disorders in children.
Woods T.S08-4:Quality assessment of haemostatic assays and EQA schemes. Biochemia Medica 2009;19(Suppl 1):S66-S67.
UK NEQAS for Blood Coagulation, Sheffield, United Kingdom
Corresponding author:tim [dot] woods [at] coageqa [dot] org [dot] uk
Quality assurance may be used as an overall term to describe all measures that are taken to ensure the reliability of laboratory testing and reporting. Internal quality control (IQC) and external quality assessment (EQA) are two distinct, yet complementary components of a laboratory quality assurance programme. IQC is used to establish whether a series of techniques and procedures are performing consistently over a period of time, therefore ensuring day to day laboratory consistency. EQA is used to identify the degree of agreement between one laboratory’s results and those obtained by other centres. In larger EQA schemes, such as UK NEQAS for Blood Coagulation with over 1000 registered centres for the laboratory based programme and over 2000 centres for the Point of Care Testing (POCT) programme, retrospective analysis of results obtained by participating laboratories permits the identification, not only of poor individual laboratory performance, but also those reagents and methods that produce unreliable or misleading results.
In order to determine laboratory performance, target values must be established for each test sample. There are few established reference methods in haemostasis, and it is difficult to identify expert laboratories for the wide range of methods and techniques used for each test. Consequently, UK NEQAS for Blood Coagulation uses the median value of all results returned by participants as the target value. For screening tests, where different reagent sensitivities are apparent, reagent specific median values are determined.
EQA programmes for Blood Coagulation have been established in a number of countries and in all of these programmes, samples are distributed to participating centres on a periodic or cyclical basis. Individual programmes vary considerably in the numbers of tests or analytes offered, registered participants, the source of samples and frequency of distribution. It is generally accepted that the larger the participant database, the more robust may be the comparisons between reagent groups, and therefore the analysis of results for individual participants. The scope of these comparisons by UK NEQAS for Blood Coagulation is possibly wider than many other Blood Coagulation EQA schemes, due to the inclusion of at least 30 different tests in the laboratory based programme alone.
The primary function of EQA is proficiency testing of individual laboratories. This should include all aspects of coagulation and analytes offered should represent the current “state of the art”. In conjunction with their primary function, well established EQA programmes can also provide information concerning the relative performance of analytical procedures, including the method principle, reagents and instruments. EQA schemes may also identify method-dependent variability of results, a point that will be covered in this presentation.
Confidentiality is an important feature of UK NEQA Schemes, and information regarding individual laboratory performance is not divulged to anyone other than the nominated head of the department or their deputy.
Improved performance has been clearly linked to ongoing participation by a laboratory in EQA programmes. This has been seen not only in overall performance, evidenced by a reduction of the variability of results between laboratories, but also in respect of individual laboratories.
Margetić S.S08-5: Disorders of hemostasis in obesity. Biochemia Medica 2009;19(Suppl 1):S67-S69.
University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia
Corresponding author:sandra [dot] margetic1 [at] zg [dot] t-com [dot] hr
Obesity is a known metabolic risk factor for atherosclerosis, cardiovascular disease (CVD) and consequent arterial thrombosis. Pathophysiology of arterial thrombosis in CVD is complex and multifactorial, in whose development and progression the hemostatic system has an important role.
The results of investigations to date provide more and more data on procoagulant (prothrombotic) state that is a consequence of disorders in hemostatic system in obese subjects. Disorders of hemostatic system by synergistic interactions with other metabolic risk factors (insulin resistance, hyperinsulinemia, dyslipidemia, hypertension), often present in obese subjects, additionally contribute to the overall risk for CVD. The connection of metabolic (atherosclerotic) and prothrombotic risk factors in development and progression of CVD largely explaines complex pathophysiological processes that cause atherosclerotic plaque, plaque rupture and consequent arterial thrombosis.
Disorders of coagulation and fibrinolytic systems in obese subjects include all constituents of hemostatic system: dysfunction of vascular endotehelial cells, alterations of platelet function, alterations of plasma coagulation phase and abnormality of fibrinolytic system. The shift of physiological hemostasis toward prothrombotic state is the result of enhanced platelet aggregability, hypercoagulability and decreased fibrinolytic activity.
Important feature of obesity is a chronic subclinic inflammatory state caused by action of proinflammatory cytokines (TNF-alfa, IL-6) from adipose tissue. Inflammation and hemostatic system are close related pathophysiological processes that sinergistically act and modulate the activity of each other. Chronic inflammatory state in obese subjects turned out to be a significant factor of disorders in hemostatic system. Proinflammatory cytokines from adipose tissue directly cause endothelial dysfunction by disturbing a balance in the production and action of vasoactive substances with proinflammatory and procoagulant and antiinflammatory and anticoagulant properties. In endothelial dysfunction factors that promote vasoconstriction, proinflammatory, procoagulant and antifibrinolytic state (thromboxan A2 (TXA2), von Willebrand factor (vWF), platelet activating factor (PAF), plasminogen activator inhibitor-1 (PAI-1)) prevail the impact of factors with opposite actions. Endothelial dysfunction is also stimulated by other accompaning metabolic disorders (hyperinsulinemia and dyslipidemia) often present in obese subjects.
Disorder of platelet function in obesity manifests as enhanced platelet activation and consequent aggregation, that additionally contribute to the prothrombotic state. It has been shown that a physiological inhibitory effect of insulin on platelet aggregation is absent in obese subjects and in those with insulin resistance there is an increased platelet susceptibility with physiological aggregation agonists (ADP, thrombin, collagen). Dysfunction of endothelial cells contribute also to strengthened activation of platelets by increased synthesis of factors that promote platelet activation and aggregation (vWF, PAF, TXA2) with simultaneous reduced synthesis of inhibitors of aggregation (prostacyclin, nitric oxide).
Alterations of plasma coagulation cascade in obesity are characterized by increased values of certain coagulation factors: fibrinogen, tissue factor (TF), factor VII (FVII) and factor VIII (FVIII). Elevated fibrinogen level is primarily the result of chronic inflammatory state stimulated by action of proinflammatory cytokines from adipose tissue on enhanced fibrinogen synthesis in liver.
Dysfuntion of endothelial cells and activation of inflammatory cells cause increased expression of TF at the cell membranes, with exposing large amounts of TF into blood and increasing activation of coagulation through FVII activation.
Well established disorder of the hemostatic system in obese subjects is diminished fibrinolytic activity characterized by systemic hypofibrinolysis, primarily due to increased synthesis of plasminogen activator inhibitor-1 (PAI-1). Increased PAI-1 plasma level in obese subjects is mostly the result of excess production in adipocytes mediated by proinflammatory cytokines, and partly of endothelial dysfunction that cause increased synthesis of PAI-1 in endothelial cells.
The results of investigations to date about disorders of hemostatic system in obesity will be presented in the lecture.