Assays of different aspects of haemostasis – what do they measure?

In addition to the classical light transmission aggregometry that requires preparation of platelet-rich plasma, thus limiting its usefulness in the acute clinical setting, commercial tests for assessment of aggregation in whole blood have been launched. In general, the tests have been designed to monitor treatment response to the common classes of anti-platelet drugs, aspirin, P2Y₁₂ (ADP- receptor) - antagonists and GPIIb/IIIa antagonists by adding arachidonic acid, ADP or a strong platelet agonist such as the thrombin PAR1 receptor-activating peptide (TRAP, amino acid sequence SFLLRN) or collagen, respectively.

In the Multiplate® (Roche, Switzerland) [3] the blood sample (anticoagulated with citrate or hirudin) is added to a disposable cuvette containing electrodes to which platelets adhere and aggregate following addition of collagen, TRAP, ADP, arachidonic acid or ristocetin. This causes a change in impedance which is registered. Multiplate is designed to measure aggregation in whole blood but can be used on platelet suspensions [4].

VerifyNow® (Accumetrics, USA) is a fully automated cartridge-based instrument for assessment of antiplatelet medications, with three types of test cartridges, containing ADP, arachidonic acid or TRAP. Citrated whole blood is mixed with the platelet agonist and fibrinogen-coated beads. Activated platelets will bind to the beads and agglutinate and the increase in light transmittance due to this will be recorded [5].

The Plateletworks® (Helena Laboratories, USA) aggregation kits are based upon comparing platelet counts within a control EDTA tube and after aggregation with ADP, arachidonic acid or collagen within citrated tubes. Results are expressed as % aggregation or % inhibition [6].

Over the past years a number of studies have been trying to establish the optimal test and range of platelet reactivity associated with the highest protection against thrombosis and the lowest risk of bleeding. The reported correlation between on-treatment platelet reactivity and outcome also suggested the use of platelet function testing to personalize antiplatelet therapy. Several studies have also been conducted in this field, but major clinical trials have failed to demonstrate a benefit of such a strategy in improving clinical outcomes (recently reviewed by [7,8]).

The classical adhesion test is to count platelets before and after passage of heparinised blood through a column filled with glass beads. Commercially available instruments include the platelet function analyzer 100 (PFA-100®, Siemens Healthcare Diagnostics Inc., USA) and the cone and plate(let) analyzer (CPA) called Impact-R® (DiaMed, Switzerland). Both of these tests measure platelet adhesion and aggregation under conditions of high shear and require anti-coagulated whole blood [9,10]. The PFA-100 measures the time to occlusion (CT, closure time) of blood flow through a collagen coated membrane in the presence of epinephrine or ADP. In the CPA system the sample is added to a polystyrene well and plasma proteins adheres to the surface of the well. Shear stress is applied by rotating a conical device immersed in the sample, leading to adhesion and aggregation of platelets. The platelets are visualized and quantified by staining. The results are expressed as percentage of surface covered by aggregates (SC) and average aggregate size (AS) [11]. The PFA-100 and the CPA have been shown to detect wWF disorders [3,9,12] and GPIIb/IIIa inhibitors [6,10]. There are also other instruments where shear can be applied, either using the cone-and-plate technology or the coaxial cylinder coquette, where the blood is placed between two coaxial cylinders where the inner one rotates to generate the shear [13].

Platelet adhesion can also be analysed using perfusion chambers [14]. The perfusion system consists of two parallel plates. One of the plates accepts a glass coverslip that can be coated with proteins, e.g. fibrinogen, collagen, vWF, laminin, or with endothelial cells. Platelet surface coverage, aggregate size and platelet rolling velocities can be evaluated by video microscopy or confocal microscopy. The shear can be varied which allows the coagulation process to be studied under physiological flow conditions. Ready-made flow chambers, sometimes also offering integrated pump-, imaging- and/or analysis systems are now entering the market [15]. The use of flow-based assays for assessment of haemostasis has been recently reviewed by the ISTH SSC Biorheology Subcommittee [13,16]. As platelet adhesion in flow-based devices require the presence of red blood cells, platelet suspensions such as platelet concentrates for transfusion cannot be easily evaluated. Therefore, we have recently proposed a novel assay where platelet activation upon adhesion to protein-coated beads can be studied using flow cytometry [17].

The viscosity of the blood increases as the fibrin network forms during blood coagulation. Viscoelastic assays enables analysis of clot formation, clot elasticity development and the fibrinolysis process in real time. The elasticity of the clot depends on several factors; the contractile force exerted by the platelets during clot retraction, platelet concentration [18-20], haematocrit [18,21], fibrinogen concentration [18-20,22], FXIII [23,24] and the thrombin generation during coagulation [25]. However, it is important to emphasize that the major signal comes from platelet contractile forces and not from the fibrin network [26,27].

Thromboelastography was first described by Hartert in 1948 [28]. There are two commercial thromboelastographs, TEG® (Haemonetics, USA) and ROTEM® (Pentapharm, Switzerland). The measuring unit of both instruments consists of a cylindrical cup, made of disposable plastic. A pin is suspended into the cup, and the pin is connected to a detector. The cup and the pin will make a forced oscillation relative each other with an angle of 4.75° (Figure 1) [25,29]. It is not possible to measure the blood viscosity or to detect the initiation of coagulation, as the first signals appear when the pin is connected via the first fibrin fibres spanning the whole distance to the wall of the cup. The clot elasticity is expressed in mm in the tracing (Figure 2). The major difference between the instruments lies in the oscillation. In the TEG instrument the cup oscillates and in the ROTEM instrument the pin oscillates. The ROTEM instrument has an electronic pipette connected to the instrument to simplify pipetting of the different reagents provided, and the software provides exact step-by-step instructions to make the instrument easy to use.

Free oscillation rheometry (FOR) (MediRox AB, Sweden) is a newer technology which makes it possible to measure changes in viscosity and elasticity in clotting whole blood and in dissolving clots and to obtain results in SI-units. In this instrument (ReoRox®), oscillation is initiated by a forced turn of the sample cup every 2.5 seconds (Figure 1). After a brief hold time, the sample cup is released, allowing free rotational oscillation around the longitudinal axis. An optic angular sensor records the frequency and damping of the oscillation as a function of time. FOR analysis includes simultaneous measurement of blood viscosity and thus allows detection even of the initial phases of coagulation, before connection of the cup and bob by fibrin fibres. Gold-plating of the cylindrical cup and the bob immersed in the centre is used to ensure strong, covalent binding of fibrinogen to the cup and bob. This prevents detachment when platelet contractile forces are increasing during clot retraction, which has been suggested as a potential problem in TEG and ROTEM [30]. Potential detachment, as well as differences in oscillation and detection strategies may be partial explanations to why the difference between platelet-free plasma and whole blood is smaller in ROTEM than in FOR (see Figure 3).

All VHA systems have a panel of different tests focusing on different aspects of the coagulation process (Table 2). The tests commercially available for TEG use kaolin or a combination of kaolin and tissue factor (TF) to activate intrinsic or extrinsic pathway of coagulation, respectively [31]. ROTEM also has reagents that can activate the intrinsic or extrinsic pathway. Both systems have tests to monitor treatment with heparin and protamine [32] as well as for evaluation of the contribution of fibrin to the clot strength [31,33]. None of the above tests can be used to monitor anti-platelet treatment with ADP-receptor inhibitors or aspirin. The PlateletMapping™ assay was introduced to monitor anti-platelet therapy by TEG. A native blood sample activated by kaolin is compared with heparinised samples where the fibrin network is formed by adding reptilase and FXIIIa and the platelets are activated by ADP or arachidonic acid [34]. This test predicted a first ischemic event after elective stenting in patients with coronary artery disease on clopidogrel and aspirin [35]. TEG has been used to monitor blood component therapy in patients undergoing cardiac, liver, or trauma surgery [36-39]. Treatment algorithms have been developed to interpret the results and guide treatment with blood components [31,33,40]. Low clot strength in trauma patients is associated with increased mortality [39]. For the FOR, there is a panel of tests available including tests to monitor heparin treatment and for evaluation of the contribution of fibrin to the clot strength. The coagulation is activated via the extrinsic pathway or by addition of a specific PAR1-activating peptide in the TRAP test to activate platelets via thrombin receptor PAR1.The instrument has mainly been used for studies of the platelet contribution to whole blood coagulation and quality control of platelet concentrates [18,41-44]. The clinical studies published so far have been limited in number [45-48]. FOR reports a wider measuring range for elasticity as compared to thromboelastography [18,49]. As an example, in pregnancy the increase in clot elasticity was significant already in the first trimester utilising FOR, with an gradual increase to 52% in the third trimester [47], while no significant changes in maximal amplitude between trimesters were detected using TEG, and the maximum increase during pregnancy was less than 8% [50].

TEG and ReoRox uses abciximab, which blocks the binding of fibrinogen to the GPIIb receptor, to evaluate the contribution of fibrin to the clot strength whereas ROTEM uses cytochalasin D which inhibits the reorganisation of the platelet cytoskeleton. However, it has been shown that these substances alone might not completely block the platelet contribution to the clot strength [26] and that a combination of cytochalasin D and abciximab or tirofiban (similar mechanism of action as abciximab) more effectively blocks platelets [51].

The VHA instruments usually operate at 37°C but the temperature can be adjusted. This allows coagulation to be monitored in patients with hypothermia which is important since hypothermia affects coagulation [24]. Hyperfibrinolysis during trauma or major surgery is an important therapeutic target in bleeding management. VHA can be used to detect fibrinolysis by measuring the reduction in elasticity (Figure 2). However, it is important to recognise that under normal conditions, the “lysis” parameter in these instruments does not reflect ongoing fibrinolytic activity in the sample, but instead mirrors the decrease in platelet contractile force, probably due to exhaustion, as this decrease is also seen in samples treated with the fibrinolysis inhibiting drug tranexamic acid (Figure 3). In addition, the test used in the VHA can influence the ability of the VHA to detect fibrinolysis [52,53]. For example, RapidTEG was shown to be less sensitive than kaolin TEG to low fibrinolytic activity [52]. It has been pointed out that currently available assays to TEG and ROTEM are insufficient to detect low increase in fibrinolytic activity [54]. FOR can also detect increasing fibrinolytic activity by measuring changes in viscosity when the blood reverts back into liquid form (Figure 2) [53]. VHAs have been used to assess the effects of haemodilution with Ringer’s actetate, hydroxylethyl starch (HES) and albumin on coagulation and treatment with fibrinogen and FXIII concentrates in vitro [23,24,27,55,56].

The Sonoclot® analyser (Sienco, Inc., USA) has a tubular probe that oscillates up and down in the whole blood sample. The resistance to movement is measured and recorded during the coagulation process [57]. Since the introduction in the 70’s [58], the use of Sonoclot® has been relatively limited as compared to TEG and ROTEM.

A number of other approaches to evaluate “global haemostasis” have been described and commercialised, but not gained widespread interest. However, as it may be interesting to discuss their methodology, we will describe them briefly in this section.

The Clot Signature Analyzer™ (CSA™; Xylum Corporation, USA) is a global haemostasis screening instrument intended for use with native whole blood [59]. The blood is passed through a thin plastic tube under pressure. Two holes are then punched in the tube, causing a fall in pressure. The platelet and fibrin clot will plug the holes and this time is recorded as the “platelet hemostasis time” (PHT). The CSA also records the clot time (CT) as the coagulation spreads though the lumen of the tube. A subsample of the blood is also passed through a second tube with collagen fibrils and the time to reach a 50% pressure reduction in this tube is called collagen-induced thrombus formation time (CITF). Despite a relatively good sensitivity in picking out patients with known bleeding disorders in a multi-centre trial [60], the test could not help in distinguishing between platelet and coagulation factor defects.

The Gorog Thrombosis Test (GTT) [61] or thrombotic status analyser (TSA) [62] (Montrose Diagnostics, UK) adds native blood to a vertical conical tube with a hole in the bottom. The tube also contains two steel balls. Platelets are activated by shear stress (175 dyne/cm²) when they pass the first large ball, and will then aggregate and initiate coagulation in the area between the balls. A light sensor records the time between blood droplets leaving the end of the tube to determine the time for occlusion, and the time for thrombolysis when blood flow starts again.

The Hemostasis Analysis System (HAS; Hemodyne, Inc., USA) is measuring the force developed by platelets as they undergo cellular contraction (“platelet contractile force”, PCF™), and speed of clot formation in whole blood between a cup and parallel upper plate at 37°C [63]. The time between assay start and PCF onset is termed the thrombin generation time (TGT™) and is used as a surrogate marker for thrombin generation [64].

The HemoSTATUS™ test or “platelet-activated clotting time” (Medtronic Blood Management, USA) measures acceleration of kaolin-activated clotting time (ACT) by different concentrations of platelet activating factor (PAF). The test has mainly been used for the evaluation of patients during cardiac surgery [65,66], even though its usefulness for predicting blood loss has been questioned [67,68].