von Willebrand factor disruption and continuous-flow circulatory devices.

Bleeding events remain a significant and frequent complication of continuous-flow left ventricular assist devices (VADs). von Willebrand factor (VWF) is critical to hemostasis by acting as a bridging molecule at sites of vascular injury for normal platelet adhesion as well as promoting platelet aggregation under conditions of high shear. Clinical and experimental data support a role for acquired von Willebrand disease in VAD bleeding episodes caused by shear-induced qualitative defects in VWF. Pathologic shear induces VWF unfolding and proteolysis of large multimers into smaller less hemostatic multimers via ADAMTS13 (a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13). This review outlines the pathobiology of VWF disruption in the context of VADs as well as current diagnostic and management strategies of the associated acquired von Willebrand disease.

An Investigation Into the Effects of In Vitro Dilution With Different Colloid Resuscitation Fluids on Clot Microstructure Formation.

BACKGROUND: Balancing the beneficial effects of resuscitation fluids against their detrimental effect on hemostasis is an important clinical issue. We aim to compare the in vitro effects of 3 different colloid resuscitation fluids (4.5% albumin, hydroxyethyl starch [Voluven 6%], and gelatin [Geloplasma]) on clot microstructure formation using a novel viscoelastic technique, the gel point. This novel hemorheologic technique measures the biophysical properties of the clot and provides an assessment of clot microstructure from its viscoelastic properties. Importantly, in contrast to many assays in routine clinical use, the measurement is performed using unadulterated whole blood in a near-patient setting and provides rapid assessment of coagulation. We hypothesized that different colloids will have a lesser or greater detrimental effect on clot microstructure formation when compared against each other. METHODS: Healthy volunteers were recruited into the study (n = 104), and a 20-mL sample of whole blood was obtained. Each volunteer was assigned to 1 of the 3 fluids, and the sample was diluted to 1 of 5 different dilutions (baseline, 10%, 20%, 40%, and 60%). The blood was tested using the gel point technique, which measures clot mechanical strength and quantifies clot microstructure (df) at the incipient stages of fibrin formation. RESULTS: df and clot mechanical strength decrease with progressive dilution for all 3 fluids. A significant reduction in df from baseline was recorded at dilutions of 20% for albumin (P < .0001), 40% for starch (P < .0001), and 60% for gelatin (P < .0001). We also observed significant differences, in terms of df, when comparing the different types of colloid (P < .0001). We found that albumin dilution produced the largest changes in clot microstructure, providing the lowest values of df (= 1.41 ± 0.061 at 60% dilution) compared with starch (1.52 ± 0.081) and gelatin (1.58 ± 0.063). CONCLUSIONS: We show that dilution with all 3 fluids has a significant effect on coagulation at even relatively low dilution volumes (20% and 40%). Furthermore, we quantify, using a novel viscoelastic technique, how the physiochemical properties of the 3 colloids exert individual changes on clot microstructure.

High Platelet Reactivity in Patients with Acute Coronary Syndromes Undergoing Percutaneous Coronary Intervention: Randomised Controlled Trial Comparing Prasugrel and Clopidogrel.

BACKGROUND: Prasugrel is more effective than clopidogrel in reducing platelet aggregation in acute coronary syndromes. Data available on prasugrel reloading in clopidogrel treated patients with high residual platelet reactivity (HRPR) i.e. poor responders, is limited. OBJECTIVES: To determine the effects of prasugrel loading on platelet function in patients on clopidogrel and high platelet reactivity undergoing percutaneous coronary intervention for acute coronary syndrome (ACS). PATIENTS: Patients with ACS on clopidogrel who were scheduled for PCI found to have a platelet reactivity ≥40 AUC with the Multiplate Analyzer, i.e. "poor responders" were randomised to prasugrel (60 mg loading and 10 mg maintenance dose) or clopidogrel (600 mg reloading and 150 mg maintenance dose). The primary outcome measure was proportion of patients with platelet reactivity <40 AUC 4 hours after loading with study medication, and also at one hour (secondary outcome). 44 patients were enrolled and the study was terminated early as clopidogrel use decreased sharply due to introduction of newer P2Y12 inhibitors. RESULTS: At 4 hours after study medication 100% of patients treated with prasugrel compared to 91% of those treated with clopidogrel had platelet reactivity <40 AUC (p = 0.49), while at 1 hour the proportions were 95% and 64% respectively (p = 0.02). Mean platelet reactivity at 4 and 1 hours after study medication in prasugrel and clopidogrel groups respectively were 12 versus 22 (p = 0.005) and 19 versus 34 (p = 0.01) respectively. CONCLUSIONS: Routine platelet function testing identifies patients with high residual platelet reactivity ("poor responders") on clopidogrel. A strategy of prasugrel rather than clopidogrel reloading results in earlier and more sustained suppression of platelet reactivity. Future trials need to identify if this translates into clinical benefit. TRIAL REGISTRATION: ClinicalTrials.gov NCT01339026.

The changes in clot microstructure in patients with ischaemic stroke and the effects of therapeutic intervention: a prospective observational study.

BACKGROUND: Stroke is the second largest cause of death worldwide. Hypercoagulability is a key feature in ischaemic stroke due to the development of an abnormally dense clot structure but techniques assessing the mechanics and quality of clot microstructure have limited clinical use. We have previously validated a new haemorheological technique using three parameters to reflect clot microstructure (Fractal Dimension (d f )) ex-vivo, real-time clot formation time (T GP ) and blood clot strength (elasticity at the gel point (G'GP)). We aimed to evaluate these novel clotting biomarkers in ischaemic stroke and changes of clot structure following therapeutic intervention. METHODS: In a prospective cohort study clot microstructure was compared in ischaemic stroke patients and a control group of healthy volunteers. Further assessment took place at 2-4 hours and at 24 hours after therapeutic intervention in the stroke group to assess the effects of thrombolysis and anti-platelet therapy. RESULTS: 75 patients (mean age 72.8 years [SD 13.1]; 47 male, 28 female) with ischaemic stroke were recruited. Of the 75 patients, 32 were thrombolysed with t-PA and 43 were loaded with 300 mg aspirin. The following parameters were significantly different between patients with stroke and the 74 healthy subjects: d f (1.760 ± .053 versus 1.735 ± 0.048, p = 0.003), TGP (208 ± 67 versus 231 ± 75, p = 0.05), G'GP (0.056 ± 0.017 versus 0.045 ± 0.014, p < 0.0001) and fibrinogen (3.7 ± 0.8 versus 3.2 ± 0.5, p < 0.00001). There was a significant decrease in d f (p = 0.02), G'GP (p = 0.01) and fibrinogen (p = 0.01) following the administration of aspirin and for d f (p = 0.003) and fibrinogen (p < 0.001) following thrombolysis as compared to baseline values. CONCLUSION: Patients with ischaemic stroke have denser and stronger clot structure as detected by d f and G'GP. The effect of thrombolysis on clot microstructure (d f ) was more prominent than antiplatelet therapy. Further work is needed to assess the clinical and therapeutic implications of these novel biomarkers.

Fractal dimension: a novel clot microstructure biomarker use in ST elevation myocardial infarction patients.

OBJECTIVES: Changes in clot microstructure are increasingly implicated in the pathology of atherosclerosis although most data are from techniques in the remote laboratory using altered blood. We validate the novel biomarker Gel Point in STEMI patients and assess therapeutic interventions. Gel Point marks the transition of blood from a visco-elastic liquid to visco-elastic solid and is rapidly measured using unadulterated blood. The Gel Point provides measurements of three parameters to reflect clot microstructure (fractal dimension (df)), real-time clot formation time (TGP) and blood clot strength (elasticity at the Gel Point (G'GP)). METHODS: We prospectively recruited 38 consecutive patients with STEMI undergoing primary percutaneous coronary intervention (pPCI). Venous blood samples were collected on admission, after pPCI and 24 h after admission for assessment of the new biomarkers, standard coagulation tests and scanning electron microscopy (SEM). RESULTS: df after pPCI was lower than df on admission (mean 1.631 [SD 0.063] vs 1.751 [0.052], p < 0.000001) whereas df at 24 h was similar to that on admission. G'GP also showed similar trend to df (p < 0.001). TGP was prolonged at after-PCI measurement compared with admission (median 854 s [IQR 581-1801] vs 217 [179-305], p < 0.00001). Changes in the values of df and G'GP were consistent with changes in the SEM images of the mature clot. CONCLUSIONS: We characterise Gel Point derived markers of clot microstructure in patients admitted with emergency arterial thrombosis. This point of care test can potentially be used to assess the efficacy of therapeutic interventions by measuring changes in clot microstructure.

A new biomarker quantifies differences in clot microstructure in patients with venous thromboembolism.

This study compared patients with venous thromboembolism (VTE) to non-VTE patients using a biomarker of clot microstructure (df ) and clot formation time (TGP ). df was the only marker that identified a significant difference (P < 0·001) between the VTE (n = 60) and non-VTE cohorts (n = 69). The 'abnormal' clot microstructures in the VTE patients suggests either inadequate response to anticoagulant therapy or the presence of a procoagulant state not detected by other markers of coagulation (i.e., International Normalized Ratio). Furthermore, elevated values of df in first time VTE patients who later develop a secondary event indicates that df may identify those at risk of recurrence.

A new structural biomarker that quantifies and predicts changes in clot strength and quality in a model of progressive haemodilution.

INTRODUCTION: We investigated the effect of progressive haemodilution on the dynamics of fibrin clot formation and clot microstructure using a novel rheological method. The technique measures clotting time (TGP), clot strength (G`GP), and quantifies clot microstructure (df) at the incipient stages of fibrin formation. We use computational modelling to examine the relationship between structure and mass, as well as helium ion microscopy (HIM) to compare morphological changes in the fully formed clot to that of the incipient clot. METHODS: This is an in vitro study; 90 healthy volunteers were recruited with informed consent and a 20ml sample of whole blood obtained from each volunteer. Five clinically relevant dilutions were investigated using 0.9w.v isotonic saline (0, 10, 20, 40 and 60%, n=18 for each dilution). The rheological method of assessing structural clot changes was compared against conventional coagulation screen and fibrinogen estimation. RESULTS: Fractal dimension (df) and final clot microstructure both decreased with progressive dilution (significant at a dilution of 20%) with similar relationships observed for final clot characteristics in HIM images. Significant correlations were observed between df and G`GP (clot strength) (0.345, p=0.02), as well as clotting time (PT: -0.690, p>0.001; APTT: -0.672, p>0.001; TGP: -0.385, p=0.006). CONCLUSIONS: This study provides new insight into the effects of haemodilution by isotonic saline on clotting time (TGP), clot strength (G'GP) and clot microstructure (df). Previous studies have attempted to link clot microstructure to clot quality/strength, however this study provides a significant step in quantifying these relationships.

Performance of a new, rapid, automated immunoassay for the detection of anti-platelet factor 4/heparin complex antibodies.

Heparin-induced thrombocytopenia (HIT) is a life-threatening adverse reaction to heparin that must be identified quickly to determine appropriate anticoagulant therapy strategies. The most common antibodies involved in HIT are directed against platelet factor 4/heparin (PF4/H) complexes. Many methods for anti-PF4/H detection exist such as enzyme immunoassays (EIAs), which have been shown to exhibit high-negative predictive value allowing for the exclusion of HIT in the majority of suspected patients; however, most EIAs are performed in a batch mode, thereby delaying results to the physician. HemosIL HIT-Ab(PF4-H) is a new, rapid method for the detection of total immunoglobulin against PF4/H complexes on ACL TOP Family systems. The assay was evaluated in a multicentre study at three sites with 414 HIT-suspected patients. Using a cut-off value of 1.0 U/ml for HemosIL HIT-Ab(PF4-H), the new test was compared with Asserachrom HPIA. Results showed a co-positivity of 60.2% [95% confidence interval (CI) 48.9-70.8], co-negativity of 94.6% (95% CI 91.5-96.7), and overall agreement of 87.7% (95% CI 84.1-90.7). These results are comparable to other PF4/H antibody assays available; with the added benefit of full automation and on-demand testing which provides results at the critical moment when physicians are required to make clinical decisions regarding anticoagulant therapy.

Biomarkers of lung injury after one-lung ventilation for lung resection.

BACKGROUND AND OBJECTIVE: Acute lung injury contributes to the mortality of patients after lung resection and one-lung ventilation (OLV). The objective of this study was to characterise the effect of lung resection and OLV on proposed biomarkers of lung injury in exhaled breath condensate (EBC) and plasma. METHODS: In adults undergoing lung resection, EBC was collected before and at 30-min intervals during OLV. Inflammatory mediators were assayed in plasma samples taken preoperatively, immediately postoperatively and 24 h postoperatively. RESULTS: EBC pH decreased from 6.51 ± 0.43 preoperatively, to 6.17 ± 0.78 and 6.09 ± 0.83 at 30 and 60 min, respectively (mean ± SD, P = 0.034, n = 20). Plasma concentrations of the receptor for advanced glycation end-products, von Willebrand factor and interleukin-6 increased comparing preoperative and postoperative samples (all P < 0.001, n = 30). By contrast, levels of Krebs von den Lungen-6 and surfactant protein-D decreased (P < 0.001, n=30), and correlated inversely with the extent of lung resected. CONCLUSIONS: Lung resection and OLV was associated with a rapid reduction in EBC pH and differential changes in plasma biomarkers of lung injury. Further investigation of EBC pH as a marker of ventilator-induced lung injury is warranted.

Anticoagulation of a patient with hypertrophic cardiomyopathy and factor VII deficiency.

A 50-year-old male patient with hypertrophic cardiomyopathy and atrial fibrillation was anticoagulated, with warfarin following insertion of a cardioverter defibrillator. He became markedly over anticoagulated after standard moderate induction doses of warfarin. His baseline prothrombin time was prolonged and further investigation showed the patient to have a mild factor VII deficiency. He was restarted on low-dose warfarin and successfully stabilized with a target international normalized ratio (INR) of 3.0 (range 2.5-3.5). We used the data from factor VII levels and thrombin generation studies before and after anticoagulation to control dosage and to decide on a suitable therapeutic range for the INR. Molecular studies showed him to have two separate mutations in the factor VII gene. This report highlights the importance of noting the baseline prothrombin time before initiating oral anticoagulation and describes how well tolerated anticoagulation can be achieved in a patient with congenital factor VII deficiency.

Heparin induced thrombocytopenia in a patient with factor V Leiden following cardiac surgery.

We report a patient who died as a result of heparin induced thrombocytopenia (HIT) and arterial thromboses following cardiac surgery. The onset was three days after exposure to low molecular weight heparin on the eighth postoperative day. The patient was heterozygous for the factor V Leiden mutation. We have reviewed 15 patients previously diagnosed as HIT on clinical and laboratory criteria and found an incidence of 6.7% (1/15) activated protein C resistance. This second patient had a pulmonary embolus and HIT after only three days exposure to low molecular weight heparin. We postulate that factor V Leiden hastens the onset and magnifies the severity of HIT.

Adult cystic fibrosis patients with and without infective exacerbations and their factor XII levels.

We investigated haemostatic and inflammatory parameters in patients with cystic fibrosis in an attempt to understand a previous finding of low factor XII levels in this patient population. We selected two groups of patients, adults attending outpatient annual review clinic who were well, chronically inflammed and adult patients with an infective exacerbation requiring antibiotics or admission to hospital, acutely inflammed. We measured known positive acute phase haemostatic factors, fibrinogen and factor VIII. Antithrombin and factor XII were also measured as both these factors have been proposed to be negative acute phase proteins in in-vitro cell models. Interleukin-6 was also measured as the proposed modulator of these factors during inflammation. Activated factor XII was measured to exclude XII activation as a cause of the low XII activity levels. Cystic fibrosis patients admitted to hospital with infective exacerbations, showed significantly more evidence of inflammation than the annual review patients. Fibrinogen and factor VIII were higher and factor XII was lower in these patients. This work suggests that factor XII behaves as a negative acute phase protein with no signs of elevated activated XII levels in either group. This supports similar findings from in-vitro cell culture. This study also shows low antithrombin levels in both patient populations, although there was no statistical difference between groups, which is probably related to their liver disorder.

Can ROTEM thromboelastometry predict postoperative bleeding after cardiac surgery?

OBJECTIVE: To evaluate the predictive ability of ROTEM thromboelastometry (Pentapharm, Basel, Switzerland) to identify patients bleeding more than 200 mL/h in the early postoperative period after cardiac surgery. DESIGN: A prospective observational study. SETTING: A single university hospital. PARTICIPANTS: Fifty-eight adult male and female patients undergoing primary coronary artery revascularization. INTERVENTIONS: Blood samples taken preoperatively and at 1, 2, and 3 hours after surgery. MEASUREMENTS AND MAIN RESULTS: Eight patients bled at least 200 mL/h in the study period. All (100%) had at least 1 abnormal ROTEM result in the study period. Of the 49 patients not found to be bleeding more than 200 mL/h in any of the first 4 postoperative hours, 46 (94%) had at least 1 abnormal ROTEM result. The positive and negative predictive values were 14.8% and 100%, respectively. CONCLUSIONS: ROTEM thromboelastometry has poor predictive utility to identify patients who bleed more than 200 mL/h in the early postoperative period after cardiac surgery. However, its negative predictive value was good.

Pulmonary arterial thrombosis in eisenmenger syndrome is associated with biventricular dysfunction and decreased pulmonary flow velocity.

OBJECTIVES: This study sought to determine what factors are associated with pulmonary artery thrombi in Eisenmenger patients. BACKGROUND: Pulmonary artery thrombosis is common in Eisenmenger syndrome, although its underlying pathophysiology is poorly understood. METHODS: Adult patients with Eisenmenger syndrome underwent computed tomography pulmonary angiography, cardiac magnetic resonance imaging, and echocardiography. Measurement of ventricular function, pulmonary artery size, and pulmonary artery blood flow were obtained. Hypercoagulability screening and platelet function assays were performed. RESULTS: Of 55 consecutive patients, 11 (20%) had a detectable thrombus. These patients were older (p = 0.032), but did not differ in oxygen saturation, hemoglobin, or hematocrit from those without thrombus. Right ventricular ejection fraction by magnetic resonance imaging was lower in those with thrombus (0.41 +/- 0.15 vs. 0.53 +/- 0.13, p = 0.017), as was left ventricular ejection fraction (0.48 +/- 0.12 vs. 0.60 +/- 0.09, p = 0.002), a finding corroborated by tissue Doppler and increased brain natriuretic peptide. Those with thrombus also had a larger main pulmonary artery diameter (48 +/- 14 mm vs. 38 +/- 9 mm, p = 0.007) and a lower peak systolic velocity in the pulmonary artery (p = 0.003). There were no differences in clotting factors, platelet function, or bronchial arteries between groups. Logistic regression showed pulmonary artery velocity to be independently associated with thrombosis. CONCLUSIONS: Pulmonary arterial thrombosis among adults with Eisenmenger syndrome is common and relates to older age, biventricular dysfunction, and slow pulmonary artery blood flow rather than degree of cyanosis or coagulation abnormalities. Further work to define treatment efficacy is needed.

Endothelial cell damage in heparin-induced thrombocytopenia.

Heparin-induced thrombocytopenia type II is a severe complication of heparin treatment that may result in thrombosis. When thrombosis occurs it carries a 50% mortality rate. The exact pathophysiology is not fully understood but in the majority of cases it is associated with the production of heparin/platelet factor 4 antibodies. The endothelium provides a protective anticoagulant surface over which blood flows. Perturbation of the endothelial cells causes a reversal of the anticoagulant properties of the cells to that of a procoagulant surface. This is often due to release or down-regulation of the anticoagulant membrane proteins such as thrombomodulin and up-regulation of procoagulant factors such as tissue factor. We studied 10 patients in our cardiothoracic institute with clinically and laboratory-confirmed heparin-induced thrombocytopenia type II for evidence of endothelial cell damage. There was a statistically significant rise in the concentrations of von Willebrand factor (P < 0.0001) and soluble thrombomodulin (P = 0.004) when patients with heparin-induced thrombocytopenia type II were compared with healthy laboratory controls and patients having had cardiopulmonary bypass surgery (von Willebrand factor 324 versus 103 versus 108 U/dl and soluble thrombomodulin 9.5 versus 2.3 versus 1.2 ng/ml, respectively). Our findings suggest that endothelial cell damage is a major factor in the pathophysiology of heparin-induced thrombocytopenia.

Blood viscosity and its relationship to iron deficiency, symptoms, and exercise capacity in adults with cyanotic congenital heart disease.

OBJECTIVES: This study sought to determine the relationship between blood viscosity and iron deficiency and their impact on symptoms and exercise function in adults with cyanotic congenital heart disease. BACKGROUND: Iron deficiency is believed to raise whole blood viscosity in cyanotic congenital heart disease, although available data are inconsistent. METHODS: Thirty-nine cyanotic adults were prospectively assessed for iron deficiency (transferrin saturation < or =5%), hyperviscosity symptoms, and exercise capacity. Same-day measurement of whole blood viscosity and hematocrit (Hct) adjusted viscosity (cells resuspended in autologous plasma to Hct of 45%) was performed at shear rates ranging from 0.277 s(-1) to 128.5 s(-1). RESULTS: Viscosity did not differ between patients with iron deficiency (n = 14) and those without (n = 25). Whole blood viscosity correlated with Hct (r = 0.63, p < 0.001 at low shear and r = 0.84, p < 0.001 at high shear) but not with red blood cell size or iron indices. Hyperviscosity symptoms were independent of iron indices but directly correlated with increased Hct-adjusted viscosity (r = 0.41, p = 0.01). Exercise capacity did not differ in iron-deficient patients. However, peak oxygen consumption was higher in those with Hct > or = 65% (12.6 +/- 3.4 ml/kg/m2 vs. 9.8 +/- 2.6 ml/kg/m2, mean +/- SD, p = 0.036) despite higher whole blood viscosity in these same individuals (p < 0.01 for all shear rates). CONCLUSIONS: Iron deficiency is common in cyanotic adults but does not alter viscosity. Hyperviscosity symptoms are associated with a higher Hct-adjusted viscosity independent of cell size or iron stores. Higher Hct is associated with better exercise capacity. Further work to understand the origin of hyperviscosity symptoms is warranted.

Role of nuclear receptor signaling in platelets: antithrombotic effects of PPARbeta.

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that heterodimerize with the retinoid X receptor and then modulate at the transcriptional level the function of many target genes. Three PPARs are known: alpha, beta (sometimes called delta), and gamma. The better studied are PPARalpha and PPARgamma, which are activated by fibrates and thiazolidinediones/glitazones, respectively. It is now believed that activation of the PPARs could be associated with the prevention of heart attack and stroke in humans. Here we report, for the first time, that human platelets contain PPARbeta and that its selective activation inhibits platelet aggregation. PPARbeta is a putative receptor for prostacyclin. Prostacyclin is an important antithrombotic hormone that synergizes with nitric oxide to inhibit platelet aggregation. In the current study, we show that PPARbeta ligands similarly synergize with nitric oxide to inhibit platelet aggregation. These observations challenge our view of a nuclear receptor because PPARbeta is present and active in nonnucleated platelets. Furthermore, these data suggest that some of the antithrombotic actions of prostacyclin may be mediated via activation of PPARs. Thus, our results identify PPARbeta as a novel antiplatelet target that may mediate some of the effects of prostacyclin in blood.

Factor XII auto-antibodies present in a patient with a B-cell lymphoma.

A 64-year-old woman was transferred for investigation of a mediastinal mass, biopsy of which showed a diffuse large B-cell lymphoma. She was also found to have an antiphospholipid antibody. The pre-operative coagulation screen showed a prolonged activated partial thromboplastin time, 71.3 s (normal range, 26-36 s), which was not corrected by the addition of normal plasma. The dilute Russell's viper venom time was positive. Anti-cardiolipin assay was strongly positive, immunoglobulin M was 153 AU; immunoglobulin G was normal, 3.1 AU. Assays of factors VIII, IX and XI showed higher concentrations with increasing dilutions in one-stage factor assays from 1: 10 to 1: 80 suggestive of an inhibitor. Factor XII was 9 U/dl and results were unaffected by increasing dilution, suggesting specific antibodies to factor XII. The factor XII antigen was 40 U/dl. The patient had immunoglobulin M auto-antibodies to factor XII.

Teicoplanin-dependent antibodies: detection and characterization.

There are only a few reports of thrombocytopenia associated with clinical doses of teicoplanin, a glycopeptide antibiotic used against Gram-positive bacteria. We investigated 39 patients receiving teicoplanin; 31 were thrombocytopenic with platelet counts between 1-105 x 10(9)/l and 8 were not thrombocytopenic. We identified 14 thrombocytopenic cases (45%) and two (25%) non-thrombocytopenic cases with IgG teicoplanin-dependent platelet-reactive antibodies. Use of glycoprotein (GP) capture enzyme-linked immunosorbent assay with platelets and GPIIb/IIIa transfected Chinese Hamster Ovary cells as well as flow cytometry with GP-deficient platelets indicated that the GPIIb/IIIa complex is a major target antigen of these antibodies.

A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique.

This study assessed the inter-laboratory imprecision associated with the measurement of the endogenous thrombin potential (ETP). The initial studies used techniques that had evolved in each of the participating laboratories. Samples from normal healthy subjects (n = 10), two patients receiving coumarin therapy [International Normalized Ratio approximately 2.0 and approximately 4.0] and a further two subjects receiving treatment with unfractionated heparin (anti-Xa 0.07 i.u./ml and 0.31 i.u./ml) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation in potency estimates was observed between the centres, although individual laboratories using fully automated techniques achieved acceptable levels of imprecision as assessed by the coefficient of variation (CV) (intra-assay CV < 9.5%, inter-assay CV < 12.5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating appraisal of two chromogenic substrates, CBS.0068 or Pefachrom TG, demonstrated markedly improved agreement in potency estimates between centres and good correlation (r > 0.96) between the chromogenic substrates. Our data demonstrates that an automated ETP method can be standardized between laboratories and suitable levels of imprecision achieved, using different analysers (COBAS Mira at two centres and an ACL-300R) and two thrombin substrates. This indicates that more widespread use of ETP measurements in clinical laboratories is feasible.