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Assays of Different Aspects of Haemostasis Ł What Do They

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Assays of Different Aspects of Haemostasis Ł What Do They Tynngård et al. Thrombosis Journal (2015) 13:8 DOI 10.1186/s12959-015-0036-2 REVIEW Open Access Assays of different aspects of haemostasis – what do they measure? Nahreen Tynngård1,2, Tomas L Lindahl1 and Sofia Ramström3* Abstract Haemostasis is a complex process affected by many factors including both cellular and plasma components. It is a multistep process starting with platelet adhesion to damaged endothelium and ending in clot fibrinolysis. There are several methods available to study different aspects of haemostasis including adhesion, aggregation, coagulation and fibrinolysis. This review describes the different methods, what aspects of haemostasis they measure and their limitations. Methods discussed include methods to study adhesion (e.g. PFA-100, cone and platelet(let) analyzer and perfusion chambers) and aggregation (e.g. Multiplate, VerifyNow and Plateletworks). Furthermore the principles behind viscoelastic haemostatic assays are presented as well as methods that can analyse aspects of haemostasis in plasma or platelet-rich-plasma samples (thrombin generation, overall haemostasis potential and Thrombodynamics Analyzer). Keywords: Coagulation, Haemostasis, Platelets, Coagulation assays, Platelet function testing Introduction and cannot measure, with conclusions summarized in Haemostasis is a complex process involving adhesion of Table 1. platelets to damaged endothelium, formation of a plate- let plug (aggregation), formation of a fibrin network to stabilise the plug, clot retraction and finally fibrinolysis. Tests of primary haemostasis In addition all of these processes take place under flow Aggregometry and is controlled and regulated by factors released from In addition to the classical light transmission aggrego- the surrounding endothelium. Surgery may cause coagu- metry that requires preparation of platelet-rich plasma, lopathy due to acidosis, hypothermia and haemodilution thus limiting its usefulness in the acute clinical setting, and is associated with increased fibrinolytic activity which commercial tests for assessment of aggregation in whole warrants monitoring to guide transfusion therapy [1,2]. blood have been launched. In general, the tests have Thus an “ideal” haemostasis assay should preferably been designed to monitor treatment response to the measure all these processes at relevant shear conditions common classes of anti-platelet drugs, aspirin, P2Y12 and provide results in a timely manner. Indeed, there are (ADP- receptor) - antagonists and GPIIb/IIIa antagonists many different methods available for measuring one or by adding arachidonic acid, ADP or a strong platelet more of the many diverse events of the haemostatic agonist such as the thrombin PAR1 receptor-activating process and platelet function. However, none of the peptide (TRAP, amino acid sequence SFLLRN) or colla- methods currently available measures all these processes gen, respectively. and they all have their pros and cons. This review will In the Multiplate® (Roche, Switzerland) [3] the blood describe a number of assays used for evaluation of differ- sample (anticoagulated with citrate or hirudin) is added ent aspects of haemostasis from a technical point of to a disposable cuvette containing electrodes to which view, highlighting their methodology and what they can platelets adhere and aggregate following addition of col- lagen, TRAP, ADP, arachidonic acid or ristocetin. This causes a change in impedance which is registered. Multi- * Correspondence: [email protected] 3Department of Clinical and Experimental Medicine, Linköping University, plate is designed to measure aggregation in whole blood Linköping, Sweden but can be used on platelet suspensions [4]. Full list of author information is available at the end of the article © 2015 Tynngård et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Tynngård et al. Thrombosis Journal (2015) 13:8 Page 2 of 10 Table 1 Aspects of global haemostasis assays Assay Sample type Adhesion Aggregation Coagulation Endothelium Shear No. of citations Plasma PRP WB Init. Prop. Elast. Lysis Multiplate -++-+ ----- - 250 VerifyNow --+-+ ----- - 476 Plateletworks --+-+ ----- - 26 Impact-R --+++ ----- + 121 PFA-100 --+++ ----- + 745 Perfusion chambers --+++ (+) (+) --(+) + 630 TEG (+) ++- - - + + + - - 4016 ROTEM (+) ++- - - + + + - - 3932 ReoRox (+) ++- - ++ + + - - 28 Sonoclot (+) ++- - ++ + + - - 113 Thrombin generation ++− -- ++−−--118 OHP + −−-- + (+) -+- - 23 Thrombodynamics +++- - ++−−-- 11 The assays ability to measure adhesion, aggregation, coagulation in terms of initiation (Init.), propagation (Prop.), clot elasticity (Elast) and fibrinolysis (Lysis). The table shows the type of sample (plasma, platelet-rich-plasma (PRP) or whole blood (WB)) that can be assessed in each assay. The table also shows if the measurement can include the contribution by endothelium and shear components. Plus (+) means yes and minus (−) means no, signs within parentheses means possible in theory, but not commonly used. For the propagation component, the perfusion chambers and the Thrombodynamics detects spatial clot propagation, whereas the other techniques detects other processes occurring after the initial clotting and first fibrin fibres have formed as propagation. To reflect how common these different assays are, we present the number of citations found on PubMed on the 30th of November 2014. VerifyNow® (Accumetrics, USA) is a fully automated include the platelet function analyzer 100 (PFA-100®, cartridge-based instrument for assessment of antiplatelet Siemens Healthcare Diagnostics Inc., USA) and the medications, with three types of test cartridges, contain- cone and plate(let) analyzer (CPA) called Impact-R® ing ADP, arachidonic acid or TRAP. Citrated whole (DiaMed, Switzerland). Both of these tests measure blood is mixed with the platelet agonist and fibrinogen- platelet adhesion and aggregation under conditions of coated beads. Activated platelets will bind to the beads high shear and require anti-coagulated whole blood and agglutinate and the increase in light transmittance [9,10]. The PFA-100 measures the time to occlusion due to this will be recorded [5]. (CT, closure time) of blood flow through a collagen The Plateletworks® (Helena Laboratories, USA) ag- coated membrane in the presence of epinephrine or gregation kits are based upon comparing platelet ADP.IntheCPAsystemthesampleisaddedtoa counts within a control EDTA tube and after aggrega- polystyrene well and plasma proteins adheres to the tion with ADP, arachidonic acid or collagen within surface of the well. Shear stress is applied by rotating citrated tubes. Results are expressed as % aggregation a conical device immersed in the sample, leading to or % inhibition [6]. adhesion and aggregation of platelets. The platelets Over the past years a number of studies have been try- are visualized and quantified by staining. The results ing to establish the optimal test and range of platelet re- are expressed as percentage of surface covered by activity associated with the highest protection against aggregates (SC) and average aggregate size (AS) [11]. thrombosis and the lowest risk of bleeding. The reported The PFA-100 and the CPA have been shown to detect correlation between on-treatment platelet reactivity and wWF disorders [3,9,12] and GPIIb/IIIa inhibitors outcome also suggested the use of platelet function [6,10]. There are also other instruments where shear testing to personalize antiplatelet therapy. Several can be applied, either using the cone-and-plate tech- studies have also been conducted in this field, but nology or the coaxial cylinder coquette, where the major clinical trials have failed to demonstrate a bene- blood is placed between two coaxial cylinders where fit of such a strategy in improving clinical outcomes the inner one rotates to generate the shear [13]. (recently reviewed by [7,8]). Platelet adhesion can also be analysed using perfusion chambers [14]. The perfusion system consists of two Adhesion assays parallel plates. One of the plates accepts a glass coverslip The classical adhesion test is to count platelets before and that can be coated with proteins, e.g. fibrinogen, collagen, after passage of heparinised blood through a column filled vWF, laminin, or with endothelial cells. Platelet surface with glass beads. Commercially available instruments coverage, aggregate size and platelet rolling velocities can Tynngård et al. Thrombosis Journal (2015) 13:8 Page 3 of 10 be evaluated by video microscopy or confocal microscopy. TEG® (Haemonetics, USA) and ROTEM® (Pentapharm, The shear can be varied which allows the coagulation Switzerland). The measuring unit of both instruments con- process to be studied under physiological flow conditions. sists of a cylindrical cup, made of disposable plastic. A pin Ready-made flow chambers, sometimes also offering inte- is suspended into the cup, and the pin is connected to a de- grated pump-, imaging- and/or analysis systems are now tector. The cup and the pin will make a forced oscillation entering the market [15]. The use of flow-based assays for relative each
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