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Tumpa, Andrea; Barić Rafaj, Renata
Veterinarska stanica, 07/2020, Volume: 51, Issue: 5Journal Article
Hemostaza je proces zaustavljanja krvarenja. Spontana hemostaza uključuje složene biokemijske mehanizme zgrušavanja krvi, regulirane interakcije između ozlijeđenog tkiva, stijenke krvnih žila i cirkulirajućih trombocita, prokoagulacijskih, antikoagulacijskih te fibrinolitičkih plazmatskih proteina. Proces hemostaze dijelimo na tri faze: primarna, sekundarna te fibrinoliza. Nefunkcionalna primarna hemostaza (trombocitopenija, kao glavni uzrok) za posljedicu ima usporeno i otežano stvaranje trombocitnog ugruška. U slučaju ozljede krv će izlaziti iz krvnih žila nekoliko sekundi ili minuta dok se ne formira stabilan sekundarni hemostatski ugrušak. U slučaju nefunkcionalne sekundarne hemostaze krvarenje se ne primjećuje izravno, već s vremenskim odmakom, najčešće nakon nekoliko minuta. Imajući na umu postajanje različitih faza hemostaze te različite aktivacije koagulacijskih kaskada, razvijene su i različite metode laboratorijskog mjerenja. Ovaj rad navodi i objašnjava koagulacijske pretrage koje se danas primjenjuju u veterinarskoj praksi. Opisana je metoda i svrha svake pretrage uz glavne primjedbe vezane uz uzorkovanje i moguće interferencije. Uzorkovanje krvi za koagulacijske pretrage osjetljivije je od uzorkovanja za ostale pretrage krvi. Nužno je izbjeći aktivaciju trombocita te se iz tog razloga idealnim uzorkovanjem smatra ono kod kojeg je krv vađena bez staze. Većina koagulacijskih pretraga kao uzorak koristi citratnu plazmu siromašnu trombocitima koja se dobiva nakon centrifugiranja (15 minuta na 2000 g) pune krvi vađene uz antikoagulans 3-natrij-citrat-dihidrat (0,11 mol/L). Većina mjerenja osnovnih parametara hemostaze uključuje praćenje stvaranja ugruška (koaguluma), a prati se brzina i sposobnost ili nedostatak sposobnosti grušanja krvi. Danas su metode automatizirane, a vrijeme nastanka ugruška mjeri se elektromehaničkom ili foto-optičkom metodom. Osim mjerenja brzine nastanka ugruška moguće je mjerenje aktivnosti enzima i inhibitora koagulacije te mjerenje koncentracije pojedinih komponenti koagulacijskih kaskada i fibrinolize. U ovim slučajevima koriste se imunokemijske metode koje se temelje na specifičnoj reakciji antigen-antitijelo, pri čemu su od automatiziranih metoda u uporabi najčešće lateks imunoturbidimetrije, a od ručnih metoda ELISA-e. U novije se vrijeme sve više nastoji ispitati poremećaj zgrušavanja u punoj krvi pomoću tromboelastografije. Za razliku od standardnih koagulacijskih testova, tromboelastografija evaluira kinetiku cijelog procesa zgrušavanja, od početnog stvaranja ugruška, polimerizacije fibrina do konačne stabilnosti ugruška. U ispitivanju uzroka krvarenja laboratorijskim pretragama procjenjuje se: rezistencija krvnih žila, broj i funkcija trombocita, unutrašnji i vanjski put zgrušavanja, mehanizam fibrinolize i djelovanje inhibitora zgrušavanja. Laboratorijska dijagnostika poremećaja hemostaze započinje izvođenjem globalnih pretraga. Globalne koagulacijske pretrage su najjednostavnije te istovremeno obuhvaćaju i vanjski i unutarnji put zgrušavanja, a u njih ubrajamo: vrijeme krvarenja, protrombinsko vrijeme, aktivirano parcijalno tromboplastinsko vrijeme, trombinsko vrijeme, broj trombocita i fibrinogen. Nakon globalnih pretraga, u svrhu postavljanja konačne dijagnoze, slijede specifične pretrage aktivnosti i funkcije trombocita, analiza trombocitnih antitijela, a po potrebi i mjerenja koncentracije i aktivnosti pojedinih faktora zgrušavanja i von Willebrandovog faktora te inhibitora zgrušavanja (antitrombin i protein C). Laboratorijski je moguće pratiti i tijek fibrinolize mjerenjem koncentracije razgradnih produkata fibrina i fibrinogena te D-dimera. S obzirom na složenost koagulacijskog sustava, laboratorijsko ispitivanje hemostaze potrebno je provesti u više stupnjeva, počevši od nekoliko globalnih pretraga te barem jedne specifične. Važno je naglasiti da ne postoji ni jedan test koji samostalno daje uvid u funkciju cjelokupnog sustava zgrušavanja. Haemostasis is the process of stopping bleeding. Spontaneous haemostasis includes complex biochemical mechanisms of blood clotting, regulated interactions between injured tissue, blood vessels and circulating thrombocytes, procoagulants, anticoagulants, and fibrinolytic proteins. Haemostasis is divided into three phases: primary haemostasis, secondary haemostasis, and fibrinolysis. Nonfunctional primary haemostasis results in the slow and difficult formation of a platelet clot. In the event of an injury, blood will be released from the blood vessels for a few seconds or minutes until a stable secondary haemostatic clot is formed. In the case of nonfunctional secondary haemostasis, bleeding is not observed immediately, but after a few minutes. Given the different stages of haemostasis and various activation pathways, different laboratory measurement methods have been developed. This study outlines and describes the coagulation tests currently applied in veterinary practice. The method and the purpose of each test are described, along with major remarks regarding sampling and possible interferences. Blood sampling for coagulation tests is more sensitive than sampling for other blood analyses. It is essential to avoid platelet activation and for this reason, ideal sampling would be venepuncture without venous stasis. Most coagulation assays use platelet-poor plasma as a sample. The vast majority of haemostasis measurements include monitoring clot formation (coagulum), i.e. monitoring the speed and ability or lack of ability to form a blood clot. Today, methods are automated and the time of the clot formation is measured by electro-mechanical or photo-optical methods. Additionally, it is possible to measure the activity of enzymes and coagulation inhibitors, and the concentration of individual components of coagulation cascades and fibrinolysis. In these cases, immunochemical methods based on the specific antigen-antibody reaction are used. Thromboelastography is a relatively new method that is increasingly used. In contrast to standard coagulation tests, thromboelastography uses whole blood as a sample. This method evaluates the kinetics of the entire clotting process - from the initial formation of the clot, through polymerization of fibrin, to the final step of the formation of the stable secondary clot. Laboratory diagnostics begins with the global coagulation assays. Global coagulation assays are the simplest and they cover both the intrinsic and extrinsic pathway. Assays that are included are bleeding time, prothrombin time, activated partial thrombolytic time, thrombin time, platelet count, and fibrinogen. To set a definitive diagnosis, it is necessary to perform one or more specific coagulation tests. Specific tests include the analysis of activity and function of platelets, platelet antibodies, and measurement of the concentration and activity of von Willebrand factor and clotting inhibitors (antithrombin and protein C). It is also possible to follow the course of fibrinolysis by measuring the concentration of fibrin and fibrinogen degradation products. Due to the complexity of the coagulation system, laboratory testing of haemostasis needs to be carried out in several stages, starting from several global assays and at least one specific. It is important to emphasize that there is no single test that can independently provide insight into the function of the entire coagulation system.
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