Conventional Coagulation Assays Research Articles

Massive haemorrhage: a current perspective

Trauma and massive haemorrhage are associated with coagulopathy secondary to tissue injury, hypoperfusion, dilution, and consumption of clotting factors and platelets occurring already at the scene of the accident. Concepts of damage control surgery have evolved prioritizing early control of the cause of bleeding by non-definitive means, while haemostatic control resuscitation seeks early control of coagulopathy. Haemostatic resuscitation provides transfusions with plasma and platelets in addition to red blood cells in an immediate and sustained manner as part of the transfusion protocol for massively bleeding patients and consequently limiting the amount of crystalloids and colloids are crucial for avoiding further impairment of haemostatic competence. Transfusion of red blood cells, plasma and platelets in a similar proportion as in whole blood prevents both hypovolaemia and coagulopathy. Results from recent before-and-after studies in massively bleeding patients indicate that trauma exsanguination protocols involving early administration of plasma and platelets are associated with improved survival. Furthermore, viscoelastic whole blood assays, such as thrombelastography (TEG) appear advantageous for identifying coagulopathy in trauma patients with severe haemorrhage as opposed the conventional coagulation assays. In our view, patients with uncontrolled bleeding, including trauma patients, should be treated with goal-directed haemostatic control resuscitation involving early administration of plasma and platelets and based on the results of TEG analysis. The aim of the goal-directed therapy should be to maintain a normal haemostatic competence until surgical haemostasis is achieved, as this appears to be associated with reduced mortality.

Recombinant factor VIIa is effective at reversing coagulopathy in a lactic acidosis model

The interplay of coagulopathy, acidosis, and hypothermia contributes to the death of the most seriously injured trauma patients. Because of in vitro testing and retrospective series, current recommendations advise correcting acidosis before administering recombinant factor VII (rFVIIa). A lactic acidosis was induced in 40 kg swine, and 10 blood samples were withdrawn for testing. rFVIIa was added to the samples with and without bicarbonate correction of the pH. Conventional coagulation assays as well as rotational thromboelastography (ROTEM) were performed on these in vitro samples. Additionally, 10 acidotic and coagulopathic animals had rFVIIa administered after randomization to pH correction with bicarbonate, or remaining acidotic. Conventional coagulation and ROTEM assays were performed on the animals. Ex-vivo samples had a mean pH of 7.14 and International Normalized Ratio (INR) of 1.46. Addition of rFVIIa to these samples corrected the INR to 0.98 (p < 0.05). A similar effect was seen for the ROTEM extem Clotting Time (extem CT). Bicarbonate correction alone of these samples had only a modest effect on INR (1.36, p < 0.05). When administered in vivo to acidotic animals (pH, 7.15), rFVIIa lowered the INR from 1.49 to 1.01 (p < 0.05). Similar improvements in extem CT were seen. rFVIIa is effective at reversing the coagulopathy from lactic acidosis in a large animal model. Recommendations against its use in acidotic patients may not be valid.

Thromboelastography in equine medicine: Technique and use in clinical research

SummaryThromboelastography (TEG) is a viscoelastic, whole blood‐based assay that integrates information from both the cellular and soluble components of coagulation, providing a global evaluation of the haemostatic system. This contrasts with the conventional coagulation assays (i.e. platelet count, prothrombin time [PT], activated partial thromboplastin time [aPTT] and fibrinogen concentration [FIB]), which only provide information about one component (e.g. clotting factors in the case of PT and aPTT) of the haemostatic process, requiring the combination of several assays for a complete evaluation of haemostasis. Thromboelastography is an old technology that has been used in human medicine for over 50 years. However, it is relatively new in veterinary medicine and has only been applied to horses in the last 5 years. Clinical applications in human medicine include diagnosis and monitoring of coagulopathies. Currently, extensive research is being carried out to expand the use of TEG in dogs and cats. Therefore, it is expected that the use of this technique will also further expand in horses in the near future. To date, the available studies in the equine species have evaluated TEG in healthy horses, horses with gastrointestinal disease, septic foals, horses with exercise‐induced pulmonary haemorrhage (EIPH) and a filly with Glanzmann's thrombasthenia. The main objective of this review is to introduce the TEG technique to equine clinicians, providing information on how the TEG functions, blood sample collection and processing, variables measured and their interpretations, normal reference values and areas of potential clinical application.

Invited Commentary

Invited Commentary

The prognostic value of thrombelastography in identifying neurosurgical patients with worse prognosis

Coagulopathy in patients with intracranial haemorrhage or traumatic brain injury (TBI) is associated with clinical deterioration and worse outcome. Whole blood viscoelastic haemostatic assays, like thrombelastography (TEG), might aid conventional coagulation assays in identification of patients with worse prognosis. We performed a review of patients (totalling 78 patients) with primary acute intracranial haemorrhage or isolated TBI admitted to a neurointensive care unit (NICU) for more than 24 h during a period of 9 months, who had TEG analysis performed at admission. Primary outcome was all-cause 30-day mortality, whereas decline in Glasgow Coma Scale (GCS) score at 24 h after admission or death due to cerebral incarceration were secondary outcomes. Patients were defined as hypocoaguable if TEG reaction time was more than 8 min, angle less than 55° and/or maximal amplitude less than 51 mm. Patients were defined hypocoaguable according to conventional coagulation assays if international normalized ratio was more than 1.3, platelet counts less than 100×10/l and/or activated partial thromboplastine time more than 35 s. Eight patients were hypocoaguable by TEG on admission to NICU and had higher 30-day mortality (63% vs. 16%, P=0.008), more often declined in GCS (57% vs. 16%, P=0.02) and expired due to cerebral incarceration (50% vs. 6%, P=0.02). Hypocoagulability by TEG, lower admission GCS and subarachnoid haemorrhage were independently associated with higher 30-day mortality [TEG: odds ratio (OR) 14.8 (2.2-100.1), P=0.006; GCS: OR 1.3 (1.1-1.5), P=0.006; subarachnoid haemorrhage: OR: 5.3 (1.3-22.3), P=0.02]. Only two patients were hypocoaguable by both conventional coagulation assays and TEG. The current data indicate that hypocoagulability by TEG at admission to NICU predicts worse prognosis. Low concordance with conventional coagulation assays indicates that TEG might be valuable in identifying patients with clinically relevant coagulopathy.

Thromboelastography in Veterinary Medicine

Thromboelastography (TEG) has been used in experimental animal studies since the early 1960s and in a routine clinical setting for the past decade. From the data currently available, it is clear that both the scope and limitations of TEG in animals resemble those observed in humans. TEG has been used to diagnose hypercoagulability in animals with disseminated intravascular coagulation, various types of cancer, and critical illness. Its ability to detect and monitor animals with various types of coagulopathies has been well established, both clinically and in experimental studies. TEG is often used in animals to monitor the effect of different pro- and anticoagulant drugs and often performs better at this task than conventional coagulation assays. TEG is already well established in veterinary medicine, and with the rapid dissemination of the technique currently taking place, we can expect to see a wide variety of interesting animal data published in the near future.

Management of major blood loss: An update

Haemorrhage remains a major cause of potentially preventable deaths. Trauma and massive transfusion are associated with coagulopathy secondary to tissue injury, hypoperfusion, dilution and consumption of clotting factors and platelets. Concepts of damage control surgery have evolved, prioritizing the early control of the cause of bleeding by non-definitive means, while haemostatic control resuscitation seeks early control of coagulopathy. Haemostatic resuscitation provides transfusions with plasma and platelets in addition to red blood cells (RBCs) in an immediate and sustained manner as part of the transfusion protocol for massively bleeding patients. Transfusion of RBCs, plasma and platelets in a similar proportion as in whole blood prevents both hypovolaemia and coagulopathy. Although an early and effective reversal of coagulopathy is documented, the most effective means of preventing coagulopathy of massive transfusion remains debated and randomized controlled studies are lacking. Results from recent before-and-after studies in massively bleeding patients indicate that trauma exsanguination protocols involving the early administration of plasma and platelets are associated with improved survival. Furthermore, viscoelastic whole blood assays, such as thrombelastography (TEG)/rotation thromboelastometry (ROTEM), appear advantageous for identifying coagulopathy in patients with severe haemorrhage, as opposed to conventional coagulation assays. In our view, patients with uncontrolled bleeding, regardless of its cause, should be treated with goal-directed haemostatic control resuscitation involving the early administration of plasma and platelets and based on the results of the TEG/ROTEM analysis. The aim of the goal-directed therapy should be to maintain a normal haemostatic competence until surgical haemostasis is achieved, as this appears to be associated with reduced mortality.

Hypocoagulability, as evaluated by thrombelastography, at admission to the ICU is associated with increased 30-day mortality

Thrombelastography (TEG), a cell-based whole blood assay, may better reflect haemostatic competence than conventional coagulation assays and this was therefore evaluated including the clot forming parameters: R, angle and maximal amplitude in patients at ICU admission. This was a prospective, observational study of patients admitted to a general ICU at a tertiary care university hospital with an expected stay of more than 24 h. Blood samples for TEG and standard coagulation analysis were obtained at admission. The APACHE II and sequential organ failure assessment (SOFA) scores and 30-day mortality were recorded. At ICU admission, 106 patients (42%) showed hypocoagulability as evaluated by TEG and these patients had higher first day SOFA score (P < 0.0001) and higher 30-days (42 vs. 13%, P < 0.0001) mortality than patients presenting with a normal TEG. In 30-day survivors, admission platelet count (P = 0.05), angle (P < 0.001) and maximal amplitude (P = 0.001) were higher and R decreased (P = 0.0013) compared with nonsurvivors. Hypocoagulability at admission as evaluated by TEG was an independent risk factor for 30-day mortality [adjusted odds ratio (OR) 3.5; 95% confidence interval (CI) 1.7-7.1]. Hypocoagulability as evaluated by TEG was frequent at admission in general ICU patients and associated with a higher rate of ventilator treatment, higher rate of renal replacement therapy and a higher use of blood products. Hypocoagulability is an independent risk factor for 30-day mortality.

Hemostatic strategies for minimizing mortality in surgery with major blood loss

Continued hemorrhage remains a major contributor of mortality in massively transfused patients and controversy regarding their optimal management exists. This article reviews recent advances that impact the use and effectiveness of massive transfusion. In the past 18 months, nine retrospective studies and three before and after studies have evaluated the implementation of massive transfusion protocols in massively transfused patients receiving more than 10 units of red blood cells (RBCs) within 24 h from arrival. All studies demonstrate that patients receiving a high fresh frozen plasma (FFP):RBC or platelet:RBC ratio have improved survival, with patients receiving both high FFP:RBC and platelet:RBC ratios exhibiting the highest survival rate. When whole blood thrombelastography is used to guide transfusion therapy in massively bleeding patients, an increase in FFP and platelet to RBC ratio is also seen, and this is associated with improved survival. This indicates that thrombelastography is better than conventional coagulation assays to monitor coagulopathy and predict transfusion requirements in massive bleeders. Implementation of more aggressive hemostatic resuscitation strategies in massively bleeding patients seems reasonable, and optimally, thrombelastography should be used to monitor coagulopathy and guide FFP and platelet transfusions.

Point of Care Coagulation Tests in Critically Ill Patients

Point of care assays for various analytes have been established in critical care, including blood gas analysis, glucose, electrolytes, and markers for cardiac ischemia. Coagulation assays can also be adapted to the critical care environment by using whole blood as sample material and instruments optimized for point of care analysis. Available assays include the conventional coagulation assays, such as prothrombin time and activated partial thromboplastin time, fibrinogen, assays for monitoring of anticoagulant drugs, global coagulation assays based on thrombelastography and viscosimetry, platelet function assays, and D-dimer assays. The main problem in point of care coagulation diagnostics is quality control. Point of care coagulation assays help in rapidly establishing a diagnosis, clarifying causes of bleeding, and monitoring therapy. Thrombelastography and similar assays extend the scope of coagulation diagnostics by visualizing the process of clot formation and extending the observation period to provide an estimate of clot stability versus mechanical and proteolytic attack.

Association between prothrombin activation fragment (F1.2), cerebral ischemia (S-100 ) and international normalized ratio (INR) in patients with ventricular assisted devices

Prothrombin time, expressed as international normalized ratio (INR) and activated partial thromboplastin time (aPTT), are standard methods of monitoring coumadin and heparin administration. Prothrombin activation fragment (F1.2) is an index of in vivo thrombin generation. We hypothesized that F1.2 would provide a better surrogate of thromboembolism risk than standard coagulation assays during ventricular assist device (VAD) support. INR, PTT and F1.2 were analyzed in 31 patients after implantation of a left-sided VAD daily during hospitalization and weekly after discharge. Thromboembolic events (TE) were defined by evidence of neurological injury revealed by plasma levels of S-100beta. The relationships between F1.2, INR for patients on coumadin and aPTT for patients on heparin were evaluated from 1250 observations of blood samples. S-100beta was positively correlated with F1.2, but not with INR and aPTT. Correlation between S-100beta and F1.2 is significantly higher than with the other two markers (P<0.0001). Higher values of aPTT and INR were not associated with TE. Compared to conventional coagulation assays, the F1.2 level provides a single endpoint that is a more accurate predictor of TE after VAD implantation. Further trials that incorporate the F1.2 marker into anticoagulation algorithms may help reduce adverse events in this high-risk population.

The effects of unfractionated heparin, low molecular weight heparin and danaparoid on the thromboelastogram (TEG): an in-vitro comparison of standard and heparinase-modified TEGs with conventional coagulation assays

To investigate the effects of unfractionated heparin (UFH), low molecular weight heparin (LMWH) and danaparoid (DPD) added to whole blood in vitro on standard and heparinase-modified thromboelastogram (TEG) parameters compared with conventional assays of coagulation. The effects of UFH, LMWH and DPD on standard TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-activated factor X (anti-FXa) activity, at concentrations of these anticoagulants ranging from 0.025 to 1 U/ml. In the second part of the study, the effects of very low concentrations (0.005-0.05 U/ml) of UFH, LMWH and DPD on the difference between standard and heparinase-modified TEG parameters were compared with the prothrombin time, activated partial thromboplastin time, thrombin time and anti-FXa activity. Standard TEG parameters were outside the reference range at lower concentrations of UFH, LMWH and DPD than most conventional coagulation assays were able to detect. Only anti-FXa activity was more sensitive to the presence of these anticoagulants than the standard TEG alone. The lowest concentration of UFH, LMWH and DPD used in this study (0.005 U/ml) caused significant differences between the standard and heparinase-modified alpha-angles of the TEG. In addition, the difference between standard and heparinase-modified TEG parameters distinguished between low concentrations (0.005-0.05 U/ml) of UFH with greater sensitivity than anti-FXa activity, but were less sensitive to LMWH and DPD. The standard TEG is more sensitive to UFH, LMWH and DPD than most conventional coagulation tests, with the exception of anti-FXa activity. Calculation of the difference between standard and heparinase-modified TEG parameters greatly increases the sensitivity of the assay for the effects of these anticoagulants, and is more sensitive to very low quantities of UFH than anti-FXa activity.

EVALUATION OF BEDSIDE PROTHROMBIN TIME AND ACTIVATED PARTIAL THROMBOPLASTIN TIME MEASUREMENT BY COAGULATION ANALYZER COAGUCHECK PLUS® IN VARIOUS CLINICAL SETTINGS

In the present study CoaguChek Plus ® (CCP ®), a coagulation test system using whole blood, was evaluated with respect to its comparability with widely distributed conventional routine coagulation assays. A correlation of r=0.997 (p<0.0001) was found between INR of CCP ®-prothrombin time (CCP ®-PT) and Thrombotest ® (KC-1 analyzer) in patients on oral anticoagulant therapy. A correlation of r=0.899 (p<0.001) between CCP ®-aPTT and Actin FS ® aPTT (STA analyzer) was found in heparinized patients. Impaired hepatic coagulation factor synthesis in liver cirrhosis patients was detected by CCP ®-PT with a sensitivity of 0.75 and by Normotest ® (STA analyzer) with a sensitivity of 0.92. Those patients with normal CCP ®-PT values and liver disease had, only mild reductions (> 30% of normals) in coagulation factors II, V, VII or X. CCP ®-aPTT was also performed in patients with a deficiency in the so called endogenous coagulation factors VIII, IX, XI and XII. CCP ®-aPTT showed a sensitivity similar to that of Actin FS ® aPTT in the detection even of mild deficiencies in factors VIII, IX and XII; factor XI deficiency was however detected only in patients with severe (<12% of normals) disease; lupus anticoagulants were detected with a high sensitivity. © 1997 Elsevier Science Ltd

Low dose heparin therapy: In vitro verification of antithrombotic effect

Low dose heparin therapy has been used routinely for prophylaxis of deep venous thrombosis, yet in vitro data regarding its antithrombotic effects are sparse. The effects of heparin on venous thrombus formation were studied in an in vitro perfusion system. Fresh blood collected from human volunteers was treated with varying heparin doses and perfused at a shear rate of 100 sec −1 over everted, injured porcine vein segments, simulating conditions in the venous circulation. Platelet and fibrin deposition were measured by use of indium 111 and iodine 125 radiolabels, respectively. The effects of heparin on the intrinsic coagulation cascade were monitored by the activated clotting time. Increasing doses of heparin resulted in significant reductions in fibrin and platelet deposition (ANOVA F = 2.67 and 3.17, respectively, p < 0.05). At a dose of only 0.19 USP units/ml blood, equivalent to a 1000 unit bolus of heparin in a 70 kg man, a noticeable reduction in both fibrin and platelet deposition was observed without an increase in the activated clotting time. These data confirm the antithrombotic effects of heparin at low dose ranges and may explain the clinically observed phenomenon of deep venous prophylaxis without an appreciable alteration in the conventional coagulation assays.

Control of Anticoagulant Therapy with a Chromogenic Substrate

Prothrombin is determined with the aid of a recently developed assay, based on the amidolysis of a chromogenic substrate. The assay proved to be reliable when it was compared with more conventional coagulation assays in the control of oral anticoagulant therapy, both in the therapeutic range and in a case of overdosage. As is the case in coagulation tests, heparin therapy remains a disturbing circumstance. The prothrombin concentration was measured (a) in the plasma of 50 long-term anticoagulated patients, and the results were compared with those obtained with a one-stage coagulation assay and with those obtained with Thrombotest determinations, and (b) during vitamin K administration in the plasma of a patient with a severe intoxication of a vitamin K antagonist.