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Severe coronavirus disease 2019 (COVID-19) is characterized by marked hypoxaemia and lung oedema, often accompanied by disordered blood coagulation and fibrinolytic systems, endothelial damage and intravascular fibrin deposition.We present a retrospective observational study of 104 patients admitted to hospital with COVID-19. Plasma samples were collected within 72 h of admission. In addition to routine coagulation and haematology testing, soluble thrombomodulin (sTM), thrombin-antithrombin (TAT), tissue plasminogen activator-plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin-α2 antiplasmin complex (PIC) were performed by automated chemiluminescent enzyme immunoassays.Significantly higher levels of D-dimer, TAT, sTM and tPAI-C were observed in non-survivors compared to survivors. To confirm which parameters were independent risk factors for mortality, multiple logistic regression was performed on D-dimer, TAT. sTM, tPAI-C and PIC data. Only increasing sTM was significantly associated with mortality, with an odds ratio of 1.065 for each 1.0 TU/mL increment (95% CI 1.025-1.115).Of the haemostatic variables measured, sTM, which can be rapidly assayed, is the best independent predictor of mortality in patients hospitalized with COVID-19, and this suggests that endothelial dysfunction plays an important role in disease progression.

BACKGROUND The Sysmex CN-6500 is a new haemostasis analyser with an integrated immunoassay module that performs chemiluminescence enzyme assay (CLEIA) in addition to coagulation, turbidimetric, chromogenic and platelet aggregation tests. AIMS To evaluate the analytical performance of the CN-6500 against the predicate device (Sysmex HISCL-800) for soluble thrombomodulin (TM), thrombin-antithrombin (TAT), tissue plasminogen activator/plasminogen activator inhibitor 1 complex (tPAI-C) and plasmin α2 plasmin inhibitor complex (PIC) assays. METHODS Imprecision was assessed by testing two levels of quality control plasmas 10 times on 5 separate days. Comparability was studied in 230 plasmas from normal donors (n = 30), patients with suspected disseminated intravascular coagulation (DIC, n = 100), sepsis (n = 20) or liver disease (n = 20), lipaemic (n = 20), haemolysed (n = 20) and icteric samples (n = 20). Limit of detection, limit of quantitation and linearity were determined by testing serial dilutions of normal plasma. Sample carryover was assessed by testing samples with high and low normal levels of the analytes concerned. RESULTS The CN-6500 performed 21 CLEIA tests per hour, while simultaneously performing coagulation tests. Acceptable between-run imprecision was obtained using commercial controls with normal and high activity for each analyte (%CV 0.99) across the measurement range. The lower limits of detection and quantitation were as follows: TM 0.1/

This guidance focuses on methodological aspects of lupus anticoagulant (LA) testing, as well as interpretation of results for clinicians. The main changes in how to test for LA compared with the International Society on Thrombosis and Haemostasis Scientific and Standardization Committee 2009 guidelines, in the preanalytical phase are more detailed recommendations on how to handle testing in anticoagulated patients, and the timing of testing. Also, routine coagulation tests are advised to obtain more information on the coagulation background of the patient, and when necessary, anti-Xa activity measurement for heparins or specific assays for direct oral anticoagulants should be performed. The three-step procedure with two test systems (diluted Russell's viper venom time and activated partial thromboplastin time [aPTT]) is essentially not changed. Silica remains the preferable activator in the aPTT assays, but ellagic acid is not excluded. We advise simultaneous performance of the mixing and confirmatory step, in each sample with a prolonged screening test. The confirmatory step can also be performed on a mixture of patient plasma and normal pooled plasma. Cutoff values should be established in-house on at least 120 normals, with transference of the manufacturer's cutoffs as an alternative. Reporting of results has not been changed, although more attention is focused on what clinicians should know. Patient selection for LA testing has been expanded.

Objectives Risk factors for thromboembolism in SLE are poorly understood. We hypothesized a possible role for protein C, based on its dual activity in inflammation and haemostasis and on the evidence of an association between acquired activated protein C (APC) resistance (APCR) and high-avidity anti-protein C antibodies (anti-PC) with a severe thrombotic phenotype in venous thrombosis APS patients. Methods In a cross-sectional study of 156 SLE patients, the presence and avidity of IgG anti-PC was established by in house-ELISA, and APCR to exogenous recombinant human APC (rhAPC) and Protac (which activates endogenous protein C) was assessed by thrombin generation-based assays. Associations with aPL profile, thrombotic history and disease activity (BILAG and SLEDAI-2K) were also established. Results Anti-PC were detected in 54.5% of patients and APCR in 59%. Anti-PC positivity was associated with APCR to both rhAPC (P Conclusion Anti-PC occur in patients with SLE, independently of aPL profile, and are associated with APCR. High-avidity anti-PC are associated with thrombosis and with active disease and might prove a novel marker to monitor the risk of thrombosis and disease progression in SLE.

BACKGROUND The CN-6000 (Sysmex Corp.) is a new haemostasis analyser with blood coagulation, amidolytic, immuno-turbidometric and light transmission aggregometry (LTA) capabilities. Transmitted light is monitored at multiple wavelengths (340, 405, 575, 660, 800 nm), from an LED light source. AIMS To evaluate the performance of the CN-6000 against a predicate device. METHODS The CN-6000 was evaluated against the CS-5100 (Sysmex) for 14 different tests, using 880 samples from normal subjects, anticoagulated patients, critically ill patients, plasmas with high or low fibrinogen content or abnormal levels of interfering substances. Between-day assay imprecision was assessed using commercial QC materials (n = 10 replicates on each of 5 days). RESULTS Acceptable levels of imprecision were obtained for all assays. Agreement between the two analysers was excellent for all assays. Throughput was 35% higher using the CN-6000 (337 vs 250 tests per hour for PT, aPTT and fibrinogen). The CN-6000 also demonstrated improved clot detection in plasmas with high levels of interfering substances as demonstrated by a 29% reduction in "vote-outs" due to low light transmission (24 vs 34). CONCLUSIONS The CN-6000 demonstrated excellent comparability with the predicate instrument and acceptable levels of imprecision in all assays. Improvements in throughput and clot detection in the presence of interfering substances were also shown.

Background: Acquired activated protein C resistance (APCr) has been identified in antiphospholipid syndrome (APS) and systemic lupus erythematosus (SLE). Objective: To assess agreement between the ST-Genesia® and CAT analysers in identifying APCr prevalence in APS/SLE patients, using three thrombin generation (TG) methods. Methods: APCr was assessed with the ST-Genesia using STG-ThromboScreen and with the CAT using recombinant human activated protein C and Protac® in 105 APS, 53 SLE patients and 36 thrombotic controls. Agreement was expressed in % and by Cohen’s kappa coefficient. Results: APCr values were consistently lower with the ST-Genesia® compared to the CAT, using either method, in both APS and SLE patients. Agreement between the two analysers in identifying APS and SLE patients with APCr was poor (≤65.9%, ≤0.20) or fair (≤68.5%, ≥0.29), regardless of TG method, respectively; no agreement was observed in thrombotic controls. APCr with both the ST Genesia and the CAT using Protac®, but not the CAT using rhAPC, was significantly greater in triple antiphospholipid antibody (aPL) APS patients compared to double/single aPL patients (p < 0.04) and in thrombotic SLE patients compared to non-thrombotic SLE patients (p < 0.05). Notably, the ST-Genesia®, unlike the CAT, with either method, identified significantly greater APCr in pregnancy morbidity (median, confidence intervals; 36.9%, 21.9–49.0%) compared to thrombotic (45.7%, 39.6–55.5%) APS patients (p = 0.03). Conclusion: Despite the broadly similar methodology used by CAT and ST-Genesia®, agreement in APCr was poor/fair, with results not being interchangeable. This may reflect differences in the TG method, use of different reagents, and analyser data handling.

Background: Current guidelines have contributed to more uniformity in the performance and interpretation of lupus anticoagulant (LA ) testing. However, points to reconsider include testing for LA in patients on anticoagulation, cut‐off values, and interpretation of results. Objectives: The aim of this International Society of Thrombosis and Haemostasis Scientific and Standardization committee (ISTH SSC ) questionnaire was to capture the spectrum of clinical and laboratory practice in LA detection, focusing on variability in practice, so that the responses could inform further ISTH SSC recommendations. Methods: Members of the ISTH SSC on Lupus Anticoagulant/Antiphospholipid Antibodies and participants of the Lupus Anticoagulant/Antiphospholipid Antibodies Programme of the External quality Control of diagnostic Assays and Tests Foundation were invited to complete a questionnaire on LA testing that was placed on the ISTH website using RedCap, with data tallied using simple descriptive statistics. Results: There was good agreement on several key recommendations in the ISTH and other guidelines on LA testing, such as sample processing, principles of testing, choice of tests, repeat testing to confirm persistent positivity and the use of interpretative reporting. However, the results highlight that there is less agreement on some other aspects, including the timing of testing in relation to thrombosis or pregnancy, testing in patients on anticoagulation, cut‐off values, and calculation and interpretation of results. Conclusions: Although some of the variability in practice in LA testing reflects the lack of substantive data to underpin evidence‐based recommendations, a more uniform approach, based on further guidance, should reduce the inter‐center variability of LA testing.

This guidance document has been prepared on behalf of the International Council for Standardization in Haematology (ICSH). The aim of the document is to provide guidance and recommendations for the processing of citrated blood samples for coagulation tests in clinical laboratories in all regions of the world. The following areas are included in this document: Sample transport including use of pneumatic tubes systems; clots in citrated samples; centrifugation; primary tube storage and stability; interfering substances including haemolysis, icterus and lipaemia; secondary aliquots-transport, storage and processing; preanalytical variables for platelet function testing. The following areas are excluded from this document, but are included in an associated ICSH document addressing collection of samples for coagulation tests in clinical laboratories; ordering tests; sample collection tube and anticoagulant; preparation of the patient; sample collection device; venous stasis before sample collection; order of draw when different sample types are collected; sample labelling; blood-to-anticoagulant ratio (tube filling); influence of haematocrit. The recommendations are based on published data in peer-reviewed literature and expert opinion.

Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca2+). Therefore, we measured alterations in Ca2+ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca2+ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects’ IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NFκB signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca2+ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca2+ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca2+ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.

This guidance document was prepared on behalf of the International Council for Standardization in Haematology (ICSH), by the ADAMTS13 Assay Working Group, which comprises an international group of both clinical and laboratory experts. The document provides recommendations on best practice for the performance of ADAMTS13 assays in clinical laboratories. ADAMTS13 assays support the differential diagnosis of thrombotic microangiopathies and have utility in the management of thrombotic thrombocytopenic purpura (TTP). There are three types of assay: activity, antigen and autoantibody/inhibitor assays. Methods for activity assays differ in terms of sensitivity, specificity, precision and turnaround time. The most widely used assays involve VWF peptide substrates and either chromogenic ELISA or FRET techniques, although chemiluminescence assays and rapid screening tests have recently become available. Tests for autoantibodies and inhibitors allow confirmation of acquired, immune-mediated TTP, while antigen assays may be useful in congenital TTP and as prognostic markers. In this document, we have attempted to describe ADAMTS13 assays and the conditions that affect them, as well as: blood collection, sample processing, quality control, standardization and clinical utility; recognizing that laboratories in different parts of the world have varying levels of sophistication. The recommendations are based on expert opinion, published literature and good clinical laboratory practice.

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Purpose Serine protease (SP) enzymes play a critical role in both the coagulation and complement cascades. Anti-SP antibodies are found in approximately 40% of patients systemic lupus erythematosus (SLE) and antiphospholipid Syndrome (APS). Complement activation is important in SLE and APS. However, little is known about the effects of anti-SP antibodies on complement activation in these diseases. We sought to investigate whether affinity-purified antibodies to the SP Factor(F)Xa and Thrombin (Thr) alter the effects of these SP on complement cleavage in the presence or absence of the physiological inhibitor anti-thrombin (AT). Methods Serum was obtained by informed consent from patients with SLE (n=10) and/or APS (n=2) under long-term follow-up at University College London Hospital. A novel method was developed to affinity purify anti-FXa and anti-Thr IgG separately. We incubated FXa (2 µM) and Thr (2.7 µM) separately with complement component C3 to determine baseline C3 cleavage. We then repeated the experiment in the presence of AT3, the appropriate anti-SP (anti-FXa or anti-Thr) or both. Finally we reviewed medical records of 40 patients with SLE to determine whether low C3 was associated with seropositivity for anti-FXa and/or anti-Thr in these patients. Results Both FXa and Thr cleaved C3 into C3a/C3 b. This was inhibited by AT, though both cleavage and inhibition by AT were stronger for Thr than FXa. Conversely anti-SP antibodies enhanced C3 cleavage by SP. Anti-FXa IgG (n=3) increased FXa-mediated cleavage of C3 by 1.3-fold. Anti-Thr IgG (n=8) increased Thr-mediated C3 cleavage by 1.8-fold. AT mediated inhibition was prevented by addition of anti-FXa IgG (n=2) but not anti-Thr IgG. Analysis of clinical serology records for the last 10 consecutive clinic visits of 28 anti-SP-positive patients showed a lower level of C3 (0.92 g/L) in the patients double positive for anti-FXa and anti-Thr than for anti-Thr alone (1.12 g/L) or anti-FXa alone (1.16 g/L). Conclusions Anti-FXa and anti-Thr enhance cleavage of C3 by FXa and Thr respectively. Presence of these antibodies in-vivo in patients with SLE and/or APS may promote increased complement activation and disease activity. This finding may have potential translational implications for future treatment of these diseases.

Reduced ADAMTS13 activity is seen in thrombotic thrombocytopenic purpura (TTP), and may lead to accumulation of prothrombotic ultra-large von Willebrand factor (ULVWF) multimers in vivo. ADAMTS13 activity and its relationship with VWF antigen (VWF:Ag) levels and platelet function in 'non-TTP related' TIA or ischaemic stroke has not been comprehensively studied.In this prospective pilot observational analytical case-control study, ADAMTS13 activity and VWF:Ag levels were quantified in platelet poor plasma in 53 patients in the early phase (≤ 4 weeks) and 34 of these patients in the late phase (≥ 3 months) after TIA or ischaemic stroke on aspirin. Data were compared with those from 22 controls not on aspirin. The impact of ADAMTS13 on platelet function in whole blood was quantified by measuring Collagen-ADP (C-ADP) and Collagen-Epinephrine closure times on a platelet function analyser (PFA-100(®)).Median ADAMTS13 activity was significantly reduced in the early phase (71.96% vs. 95.5%, P0.01) but not in the late phase after TIA or stroke compared with controls (86.3% vs. 95.5%, P=0.19). There was a significant inverse relationship between ADAMTS13 activity and VWF:Ag levels in the early phase (r=-0.31; P=0.024), but not in the late phase after TIA or stroke (P=0.74). There was a positive correlation between ADAMTS13 activity and C-ADP closure times in early phase patients only, likely mediated via VWF:Ag levels.ADAMTS13 activity is reduced and VWF:Ag expression is increased within 4 weeks of TIA or ischaemic stroke onset, and can promote enhanced platelet adhesion and aggregation in response to stimulation with collagen and ADP via VWF-mediated pathways. These data improve our understanding of the dynamic haemostatic and thrombotic profiles of ischaemic cerebrovascular disease (CVD) patients, and are important in view of the potential future role that ADAMTS13 may have to play as an anti-thrombotic agent in CVD.

Key points Finding an inherited complement abnormality in HSCT-associated TMA provides a rationale for the use of a complement inhibitor. Alternative complement inhibitors such as Coversin should be considered in patients who are resistant to eculizumab.

Secondary hemophagocytic lymphohistiocytosis (sHLH) may be responsible for some of the deaths in adult patients with severe COVID-19. We present our experience of low-volume plasma exchange (PLEX) with low-dose steroid in the treatment of adult patients with sHLH and acute liver failure caused by dengue virus and other nonviral triggers and discuss how this may be effective in the management of severe COVID-19. sHLH is poorly understood and without effective treatment. Endothelium of the capillaries of the lungs and kidneys and of liver sinusoids does not express von Willebrand factor (VWF) in health and is where most macrophages are located. Plasma VWF levels are high in sHLH and require clearance by macrophages, which when activated enlarge and likely block the lumen. Current histology studies neither appreciate microcirculatory sludge nor display endothelial–macrophage interactions. We hypothesize that low-volume PLEX and low-dose steroid may reverse sHLH and improve survival in severe COVID-19 patients with acute lung injury.

Objectives: The aim of this study is to assess the utility of plasma von-Willebrand factor (VWF) levels in predicting in-hospital survival for patients with severe alcoholic hepatitis. Methods: From a prospectively collected database of acute-on-chronic liver failure (ACLF) patients, we retrospectively selected all severe alcoholic hepatitis (discriminant function [DF] ≥32) patients and correlated baseline plasma VWF (antigen, Ag and collagen-binding activity [CBA]) levels with disease severity. In-hospital survival was classified as discharged alive (or) died/discharged in a terminal condition (poor outcome). Results: Of 34 consecutive severe alcoholic hepatitis patients (age: 40.5, 30–63 years; median, range, hospital stay: 6, 2–15 days) studied, 15 had a poor outcome. Plasma VWF-CBA was higher in patients with poor outcome (736 [396–1157] IU/dL) as compared to patients discharged in stable state (492 [97–986] IU/dL; P = 0.03). VWF-CBA correlated well with VWF-Ag, model for end-stage liver disease (MELD) score, sequential organ failure assessment score and ACLF grades. AUROC to predict composite poor outcome was similar for plasma VWF-CBA (0.72, 0.54–0.89) and MELD score (0.71, 0.53–0.89). Plasma VWF-CBA was higher in 23 “very severe” alcoholic hepatitis (DF >60 or MELD >30) patients. Combining plasma VWF-CBA (>750 IU/dL) and criteria for “very severe” alcoholic hepatitis had a sensitivity of 100% (81%–100%) and negative predictive value of 100% (68%–100%) for predicting poor outcome. Conclusions: Plasma VWF levels are markedly elevated, correlate with organ failure, and predict in-hospital survival in severe alcoholic hepatitis, and also in “very severe” alcoholic hepatitis, patients.

Steve Kitchen, Elaine Gray, Ian Mackie, Trevor Baglin and Mike Makris on behalf of the BCSH committee Sheffield Haemophilia and Thrombosis Centre, Sheffield Teaching Hospitals NHS Trust, Sheffield, Haemostasis section, Biotherapeutics Group, National Institute for Biological Standards and Control, Potters Bar, Haemostasis Research Unit, Department of Haematology, University College London, London, Department of Haematology, Addenbrooke’s Hospital, Cambridge, and Department of Cardiovascular Science, University of Sheffield, Sheffield, UK

Factor XIII (FXIII) has an important role in the control of bleeding through fibrin cross-linking; however, its effect within the menstrual cycle is not fully understood. The aim of this study was to examine changes in FXIII activity during the normal menstrual cycle and correlate FXIII activity with menstrual blood loss. A total of 32 healthy normal women of reproductive age were recruited. Menstrual blood loss was measured using the pictorial blood-assessment chart (PBAC). A bleeding score questionnaire was also completed. Blood samples were taken during the menstrual, proliferative, periovulatory, secretory and premenstrual phase for assessment of FXIII level. The mean ± SD FXIII level was lowest during menstrual and periovulatory phases (114 ± 23 and 114 ± 21 IU/dl, respectively). Mean FXIII level during the secretory and premenstrual phases were higher than the menstrual phase (P = 0.036). Mean secretory phase FXIII was also significantly higher compared with the periovulatory phase (P = 0.02). There was no significant correlation between FXIII level during the menstrual phase and age (P = 0.53) or PBAC score (P = 0.53). There were no significant differences in FXIII level during the menstrual phase between women with PBAC scores of at least 100 (n = 14; mean 116 IU/dl) and women with PBAC scores less than 100 (n = 18; mean 113 IU/dl). There was no correlation between FXIII level and bleeding score. FXIII activity was lower during menstrual and periovulatory phases of the cycle. However, the small difference between mean values (8 IU/dl) would be unlikely to have a significant impact on diagnosis of FXIII deficiency and clinical management.

Over 150 mutations have been identified in the ADAMTS13 gene in patients with congenital thrombotic thrombocytopenic purpura (TTP). The majority of these (86%), lead to reduced (50%) secretion of mutant recombinant ADAMTS13. The mechanism by which this occurs has not been investigated in vitro. Two novel ADAMTS13 mutations (p.I143T and p.Y570C) identified in two congenital adolescence onset TTP patients were studied, to investigate their effects on ADAMTS13 secretion and subcellular localisation.HEK293T cells were transiently transfected with wild type or mutant ADAMTS13 cDNA. Immunofluorescence and confocal microscopy were used to study localisation within the endoplasmic reticulum (ER) and Golgi. The cell proteasome and lysosomes were inhibited in cells stably expressing ADAMTS13 to investigate degradation of ADAMTS13 by either organelle.Both mutations severely impaired secretion and both mutants localised within the ER and Golgi. Proteasome inhibition led to the intracellular accumulation of both mutants, suggesting proteasome degradation. Lysosome inhibition on the other hand did not lead to increased intracellular accumulation of the mutants.Proteasome degradation of these ADAMTS13 mutants contributed to their reduced secretion.

Rivaroxaban is established for the treatment and secondary prevention of venous thromboembolism, but whether it is useful in patients with antiphospholipid syndrome is uncertain.This randomised, controlled, open-label, phase 2/3, non-inferiority trial, done in two UK hospitals, included patients with antiphospholipid syndrome who were taking warfarin for previous venous thromboembolism, with a target international normalised ratio of 2·5. Patients were randomly assigned 1:1 to continue with warfarin or receive 20 mg oral rivaroxaban daily. Randomisation was done centrally, stratified by centre and patient type (with vs without systemic lupus erythematosus). The primary outcome was percentage change in endogenous thrombin potential (ETP) from randomisation to day 42, with non-inferiority set at less than 20% difference from warfarin in mean percentage change. Analysis was by modified intention to treat. Other thrombin generation parameters, thrombosis, and bleeding were also assessed. Treatment effect was measured as the ratio of rivaroxaban to warfarin for thrombin generation. This trial is registered with the ISRCTN registry, number ISRCTN68222801.Of 116 patients randomised between June 5, 2013, and Nov 11, 2014, 54 who received rivaroxaban and 56 who received warfarin were assessed. At day 42, ETP was higher in the rivaroxaban than in the warfarin group (geometric mean 1086 nmol/L per min, 95% CI 957-1233 vs 548, 484-621, treatment effect 2·0, 95% CI 1·7-2·4, p0·0001). Peak thrombin generation was lower in the rivaroxaban group (56 nmol/L, 95% CI 47-66 vs 86 nmol/L, 72-102, treatment effect 0·6, 95% CI 0·5-0·8, p=0·0006). No thrombosis or major bleeding were seen. Serious adverse events occurred in four patients in each group.ETP for rivaroxaban did not reach the non-inferiority threshold, but as there was no increase in thrombotic risk compared with standard-intensity warfarin, this drug could be an effective and safe alternative in patients with antiphospholipid syndrome and previous venous thromboembolism.Arthritis Research UK, Comprehensive Clinical Trials Unit at UCL, LUPUS UK, Bayer, National Institute for Health Research Biomedical Research Centre.

Circulating levels of von Willebrand factor (vWF) predict mortality in patients with cirrhosis. We hypothesized that systemic inflammation in acute-on-chronic liver failure (ACLF) will stimulate endothelium, increase vWF levels, and promote platelet microthrombi causing organ failure. In this prospective study, we correlated plasma vWF levels with organ failure, liver disease severity, sepsis, and systemic inflammatory response syndrome (SIRS) and also analyzed if vWF levels predicted in-hospital composite poor outcome (i.e. death/discharged in terminal condition/liver transplantation) in consecutive ACLF patients. Twenty-one of the 50 ACLF patients studied had composite poor outcome. ACLF patients had markedly elevated vWF antigen and activity (sevenfold and fivefold median increase, respectively) on days 1 and 3. Median ratio of vWF to a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS13) activity on day 1 was significantly higher in ACLF patients (11.2) compared to 20 compensated cirrhosis patients (3.3) and healthy volunteers (0.9). On day 1, area under ROC curve (AUROC) to predict composite poor outcome of hospital stay for ACLF patients for vWF antigen, vWF activity, and model for end-stage liver disease (MELD) score were 0.63, 0.68, and 0.74, respectively. vWF activity correlated better with liver disease severity (MELD score, ACLF grade) and organ failure (Sequential Organ Failure Assessment [SOFA] score) than vWF antigen; in contrast, neither vWF antigen nor activity correlated with platelet count, sepsis, or SIRS. vWF levels are markedly elevated, correlate with organ failure, and predict in-hospital survival in ACLF patients. This data provides a mechanistic basis for postulating that vWF-reducing treatments such as plasma exchange may benefit ACLF patients.

Activated protein C resistance (APCR) within a thrombin generation test (TGT) system is associated with an increased risk of venous thromboembolism (VTE). However, application of the TGT is restricted by the analytical platforms used to monitor thrombin generation. Using a routine coagulation analyser we have developed an automated chromogenic TGT that is sensitive to defects in the protein C pathway.The TGT was performed on a TOP500 analyser, in the presence and absence of Protac. The reaction was monitored using a substrate with slow kinetics for thrombin (S-2444). Results were expressed as the area under the curve normalised ratio (AUCnr). Assay results were compared with Coatest APCR (expressed as APC-ratio [CoAPCr]).Samples were obtained from 35 healthy subjects and 91 patients with previous history of VTE. Of these patients, 19, 17, and 9 had heterozygous factor V Leiden (FVL), antiphospholipid syndrome (APS), and protein C/protein S deficiencies (PC/PS) respectively.Inter-assay imprecision in the presence and absence of Protac were 20% and5% respectively. There was a significant difference between the AUCnr of normals (median [IQR]: 2.8 [2.4-4.7]) compared to: FVL (1.0 [0.7-1.2]); PC/PS (1.1 [0.9-1.2]); and APS (1.1 [0.8-1.4]); p0.001 for each comparison. No significant difference was seen between the AUCnr of normals and other VTE patients. The detection rate of AUCnr and CoAPCr were: 100% and 56% for FVL; 88% and 44% for PC/PS; and 64% and 45% for APS respectively.The automated TGT exhibited good sensitivity to defects in the protein C pathway.

Introduction Total hip/knee replacement surgery (THR/TKR respectively) is associated with an increased risk of venous thromboembolism. Dabigatran is recommended as a thromboprophylactic agent post orthopaedic surgery. The aim of this study was to assess the post-operative (Day-1 and Day-2) effect of prophylactic Dabigatran on: the thrombin generation (TG) assay; prothrombin fragment 1.2 (F1.2); thrombin-antithrombin complexes (TAT); D-dimer (D-D); and other coagulation parameters. Methods and samples Nineteen patients (12 THR, 7 TKR) who received 110 mg dabigatran 4 hours post-operatively, then 220 mg the following day, were recruited. Blood was collected: pre-operatively (Pre-); peri-operatively (Peri-); 19 hours after 110 mg dabigatran (Day-1); and 17 hours after 220 mg dabigatran (Day-2). The TG assay was measured using the Calibrated Automated Thrombogram and a low concentration of tissue factor. Other coagulation parameters measured included activated partial thromboplastin time (APTT), thrombin-time (TT), ecarin-clotting time (ECT) and Hemoclot tests. Results From Pre- to Peri-, ETP/peak-thrombin, F1.2, TAT and D-D increased significantly. From Peri- to Day-1 and Day-2: TAT reduced progressively; D-D increased; F1.2 did not change significantly; lag-time and time-to-peak prolonged; ETP/Peak-thrombin increased spuriously, due to Dabigatran interfering with the α-2 macroglobulin:thrombin complex in the TG assay. APTT, TT, ECT and Hemoclot increased progressively post-operatively; good correlations were seen between these tests. Conclusion The effect of dabigatran on the TG assay, showed a spurious increase in ETP and Peak-thrombin due to its interference with the TG assay. Dabigatran reduced TAT, but not F1.2, suggesting that thrombin was still being generated after surgery, but was blocked by Dabigatran.

The guideline writing group was selected to be representative of UK-based medical experts. MEDLINE was systematically searched for publications in English up to the Summer of 2010 using key words platelet, platelet function testing and platelet aggregometry. Relevant references generated from initial papers and published guidelines/reviews were also examined. Meeting abstracts were not included. The writing group produced the draft guideline, which was subsequently revised and agreed by consensus. Further comment was made by members of the Haemostasis and Thrombosis Task Force of the British Committee for Standards in Haematology. The guideline was then reviewed by a sounding board of approximately 40 UK haematologists, the British Committee for Standards in Haematology (BCSH) and the British Society for Haematology Committee and comments incorporated where appropriate. Criteria used to quote levels and grades of evidence are as outlined in appendix 7 of the Procedure for Guidelines Commissioned by the BCSH [http://www.bcshguidelines.com/BCSH_PROCESS/EVIDENCE_LEVELS_AND_GRADES_OF_RECOMMENDATION/43_GRADE.html]. The objective of this guideline is to provide healthcare professionals with clear guidance on platelet function testing in patients with suspected bleeding disorders. The guidance may not be appropriate to patients receiving antiplatelet therapy and in all cases individual patient circumstances may dictate an alternative approach.

ADAMTS13 deficiency leading to excess ultralarge von Willebrand factor (VWF) multimers and platelet clumping is typically found in thrombotic thrombocytopenic purpura (a type of thrombotic microangiopathy). Idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH) is a microangiopathy of portal venules associated with significant thrombocytopenia and predisposing gut disorders. To determine whether the portal microangiopathy in NCIPH is associated with ADAMTS13 deficiency. Plasma levels of ADAMTS13, anti-ADAMTS13 antibodies, and VWF were compared between cases (NCIPH patients) and controls (with chronic liver diseases of other etiology) matched for severity of liver dysfunction. Eighteen NCIPH patients [median (range) MELD score 12 (7–25)] and 25 controls [MELD score 11 (4–26)] were studied. ADAMTS13 activity was reduced in all 18 NCIPH patients and significantly lower than controls (median, IQR: 12.5%, 5–25% and 59.0%, 44–84%, respectively, P

P>Total hip/knee replacement surgeries are associated with an increased risk of venous thromboembolism and post-operative thromboprophylaxis has become standard treatment. This study aimed to: (i) assess the impact of hip/knee replacement surgery on ex vivo thrombin generation (TG), prothrombin fragments 1 + 2 (F1 + 2), thrombin-antithrombin complexes (TAT) and D-dimer; (ii) compare the anticoagulant effects of dalteparin and rivaroxaban on TG 24 h after surgery. Haemostatic variables were assessed in plasma samples of 51 patients taken pre-operatively, peri-operatively, and 24 h post-operatively. Prophylaxis, once a day, with dalteparin or rivaroxaban, starting 6-8 h post-operatively, was administered in 25 (14 knee/11 hip) and 26 patients (13 knee/13 hip) respectively. TG, F1 + 2, TAT and D-dimer increased during surgery. Dalteparin patients showed a variable TG response 24 h after surgery: conversely, the effect of rivaroxaban on TG was consistent across individuals. Good correlation was seen between rivaroxaban levels and TG-lag-time (rs = 0 center dot 46, P = 0 center dot 01); TG-time-to-Peak (rs = 0 center dot 53, P = 0 center dot 005); TG-peak-thrombin (rs = -0 center dot 59, P = 0 center dot 001); and TG-velocity-index-rate (rs = -0 center dot 61, P = 0 center dot 0009). Patients who received rivaroxaban showed a greater decrease of TG, F1 + 2 and TAT (but not D-dimer) than those on dalteparin. TG increases during hip/knee replacement surgery. Rivaroxaban inhibits TG more than dalteparin at 24 h after surgery.

Point-of-care testing (POCT) in haematology has seen a significant increase in both the spectrum of tests available and the number of tests performed annually. POCT is frequently undertaken with the belief that this will reduce the turnaround time for results and so improve patient care. The most obvious example of POCT in haemostasis is the out-of-hospital monitoring of the International Normalized Ratio in patients receiving a vitamin K antagonist, such as warfarin. Other areas include the use of the Activated Clotting Time to monitor anticoagulation for patients on cardio-pulmonary bypass, platelet function testing to identify patients with apparent aspirin or clopidogrel resistance and thrombelastography to guide blood product replacement during cardiac and hepatic surgery. In contrast to laboratory testing, POCT is frequently undertaken by untrained or semi-trained individuals and in many cases is not subject to the same strict quality control programmes that exist in the central laboratory. Although external quality assessment programmes do exist for some POCT assays these are still relatively few. The use of POCT in haematology, particularly in the field of haemostasis, is likely to expand and it is important that systems are in place to ensure that the generated results are accurate and precise.

Platelet Factor 4 (PF4) prevents inhibition of blood coagulation proteases by heparin via formation of a putative enzyme-PF4 complex. To investigate the contribution of the latter, the activity of factor Xa (fXa) was determined in chromogenic assays measuring hydrolysis of a peptide substrate S2765 or cleavage of the macromolecular substrate prothrombin in the activating complex, prothrombinase. Upon pre-incubation with fXa and heparin, PF4 at about 250 nM decreased the k(cat), of S2765 hydrolysis about fivefold and that of prothrombin activation about 25-fold. In the presence of saturating fVa, inhibition of fXa by PF4 was abolished, while in the presence of limiting fVa, PF4 altered the interaction of fXa with fVa. Interestingly, high concentrations of PF4 restored fXa activity toward S2765 and prothrombin, indicating a dual effect of PF4 on fXa activities. These findings suggest that PF4 in the presence of heparin is an allosteric effector of the prothrombinase complex. (C) 2009 Elsevier Inc. All rights reserved.

This guideline provides a framework for the arrangement of point-of-care testing (POCT) services, previously known as near patient testing (patient self-testing not covered). POCT is defined as any analytical test performed outside the laboratory. Primary users are often non-laboratory healthcare workers. The guidance applies to units within hospitals as well as general practioner surgeries, community clinics and pharmacies. The head of the haematology laboratory or a point of care coordinator must take responsibility for all aspects of the POCT service, including quality and training. Depending on the size and nature of the POCT practice, a local POCT manager may also be required. Equipment selected should have received a successful independent performance evaluation. If an independent evaluation has not been performed the purchaser should assess the device according to the protocol in this document. POCT devices should generate results that are comparable to those of the local laboratory. An accredited external quality assessment programme and internal quality control system must be established. Manufacturers promoting POCT devices designed for non-laboratory sites, e.g. pharmacies, should undertake training and annual competency assessment, perhaps using a web-based system. A diagram to illustrate the stages for the implementation of a POCT service is illustrated.

Levels of circulating red blood cell (RBC)-derived vesicles are increased in sickle cell anaemia (SCA) and thalassaemia intermedia (TI) but the mechanisms, effects and controlling factors may differ. This study found that levels of vesicles and intravascular haemolysis were linked as shown by the correlation between levels of vesicles and plasma Hb. Vesicle levels were 6-fold greater in SCA and 4-fold greater in TI than in controls. The proportion of plasma Hb within vesicles was increased in SCA and TI with a significantly higher proportion in TI. We examined whether subpopulations of RBC expressing phosphatidylserine (PS) were a source of PS(+) vesicles and observed a significant association. Thrombin generation was promoted by the vesicles in which 40-50% expressed PS. In TI, markers of thrombin generation were significantly related to PS(+) RBC. Splenectomy in TI had significant effects including greater increases in vesicle levels, plasma Hb, PS(+) RBCs and thrombin generation markers than in unsplenectomised patients. In hydroxycarbamide (HC)-treated SCA patients these measures were decreased compared with untreated controls. The relationship between vesicle levels and plasma Hb suggests a mechanism linking vesiculation to haemolysis and consequently nitric oxide (NO) bioavailability and suggests a means by which HC treatment improves NO bioavailability.

The SmartCheck INR (Unipath, Bedford, England) is a point-of-care device for professional and patient self-monitoring of oral anticoagulant therapy. It measures the international normalized ratio (INR) and assesses test strip integrity, temperature, and sample quality. No significant differences were found in SmartCheck INR results from 3 different instruments or in 3 test-strip lots. Within run imprecision was 0.89% and 6.36% for low and high control samples, respectively (mean INR, 0.99 and 4.08, respectively). Comparability was assessed in 68 patients receiving warfarin by using PTHS Plus (Instrumentation Laboratory, Lexington, MA) and Innovin (Dade Behring, Marburg, Germany) thromboplastins. Good correlations were observed between the methods (r = 0.89 and r = 0.90, respectively) with no significant differences in means: SmartCheck INR, 2.82; Innovin, 2.75; PTHS Plus, 2.74. Clinical agreement with laboratory methods was 88% for Innovin and 97% for PTHS Plus. The SmartCheck INR was easy to use with no mechanical failures during the evaluation and a low test-strip failure rate of 4.7%. The SmartCheck INR provides accurate and reproducible results and is suitable for routine monitoring of oral anticoagulant therapy in the majority of patients.

Thrombotic thrombocytopenic purpura (TTP) has been linked to a severe deficiency in ADAMTS13 (a disintegrin and metalloprotease with thrombospondin type 1 motif, member 13) activity. Since the identification of ADAMTS13, and its target cleavage sequence in von Willebrand factor (VWF), several novel ADAMTS13 activity, antigen and autoantibody assays have been developed. Our aim was to evaluate the potential use of these novel assays. ADAMTS13 activity and inhibitors were measured by overnight incubation of patient plasma with pure VWF followed by multimer or collagen binding analysis. ADAMTS13 activity (Rapid peptide assay), antigen and immunoglobulin G anti-ADAMTS13 were measured by enzyme-linked immunosorbent assay. 118 samples from seven TTP patients (six adult idiopathic, one congenital) were studied longitudinally during episodes of TTP, their treatment and prophylaxis. ADAMTS13 antigen levels varied considerably between patients and sample times, but in new cases of acute TTP, rapid assays of ADAMTS13 antigen, on serial samples, maybe helpful in confirming the diagnosis. The rapid peptide ADAMTS13 activity assay showed good concordance of results with the older activity assay methods. The change in ADAMTS13 activity mirrored the autoantibody level and in 5/6 acquired TTP cases, a fall in antibody appeared to predict a rise in ADAMTS13 activity, potentially allowing modification of patient management based on autoantibody levels.

Idiopathic noncirrhotic intrahepatic portal hypertension (NCIPH), a chronic microangiopathy of the liver caused by arsenicosis from use of contaminated groundwater, was reported from Asia. This study aimed to see, if in the twenty-first century, arsenicosis was present in NCIPH patients at our hospital and, if present, to look for groundwater contamination by arsenic in their residential locality. Twenty-seven liver biopsy proven NCIPH patients, 25 portal hypertensive controls with hepatitis B or C related cirrhosis and 25 healthy controls, matched for residential locality, were studied. Eighty-four percent to 96 % of study subjects belonged to middle or lower socioeconomic category. Arsenicosis was looked for by estimation of arsenic levels in finger/toe nails and by skin examination. Arsenic levels in nails and in ground water (in NCIPH patients with arsenicosis) was estimated by mass spectrometry. Nail arsenic levels were raised in five (10 %) portal hypertensive study subjects [two NCIPH patients (both had skin arsenicosis) and three portal hypertensive controls]. All of these five patients were residents of West Bengal or Bangladesh. Skin arsenicosis was noted in three NCIPH patients (11 %) compared to none of disease/healthy controls. Ground water from residential locality of one NCIPH patient with arsenicosis (from Bangladesh) showed extremely high level of arsenic (79.5 μg/L). Arsenicosis and microangiopathy of liver, possibly caused by environmental contamination continues in parts of Asia. Further studies are needed to understand the mechanisms of such ‘poverty-linked thrombophilia’.

Introduction: Leucocyte filtration can reduce inflammation and end-organ damage. The aim of this study was to test the cardioprotective effect of systemic leucocyte filtration during cardiopulmonary bypass (CPB) for coronary revascularization. Methods: Sixty patients scheduled for elective coronary artery bypass grafting were prospectively randomised to receive either a test leucocyte-depleting (LD) filter or a control standard arterial line filter in the CPB circuit. Myocardial injury was determined by serum Troponin T concentration up to 72 h postoperatively. In addition, perioperative neutrophil counts, serum elastase and electrocardiograms (ECGs) were evaluated. Results: There was a peak of Troponin T release at 6 h in both groups. There was no difference between LD or control group Troponin T at any time point. No difference in neutrophil count was found. A greater rise in neutrophil elastase occurred in the LD group during CPB and 10 min post CPB (376 and 496 versus 108 and 228 mcg/L, p Conclusions: LD arterial line filters did not confer any cardioprotective effect as measured by Troponin T in elective coronary revascularization cases.

Comparative sensitivity of commonly used thromboplastins to ex vivo therapeutic rivaroxaban levels

Continuing aspirin up until surgery in cardiac surgical patients may increase peri-operative blood loss. It is possible that there is a subset of patients particularly sensitive to aspirin. The platelet function analyser (PFA-100(R)) can demonstrate the antiplatelet effect of aspirin. This study was designed to assess the effect of daily 75 mg aspirin on platelet function, as measured by the PFA-100(R), in 92 patients with ischaemic heart disease. Patients were classified into three groups according to their PFA-100(R) results; aspirin hyper-responders (16%), aspirin normal responders (33%) and aspirin non-responders (51%). The PFA-100(R) has potential as a screening tool to identify patients who are either hyper-responsive or resistant to aspirin. Pre-operative PFA-100(R) screening to isolate aspirin hyper-responders could enable the vast majority of patients to continue with aspirin therapy pre-operatively, avoiding the risks of stopping treatment.

Objective: The risk of stroke in patients with recently symptomatic carotid stenosis is considerably higher than in patients with asymptomatic stenosis. In the present study it was hypothesised that excessive platelet activation might partly contribute to this difference. Methods: A full blood count was done and whole blood flow cytometry used to measure platelet surface expression of CD62P, CD63, and PAC1 binding and the percentage of leucocyte–platelet complexes in patients with acute (0–21 days, n = 19) and convalescent (79–365 days) symptomatic (n = 16) and asymptomatic (n = 16) severe (⩾70%) carotid stenosis. Most patients were treated with aspirin (37.5–300 mg daily) although alternative antithrombotic regimens were more commonly used in the symptomatic group. Results: The mean platelet count was higher in patients with acute and convalescent symptomatic compared with asymptomatic carotid stenosis. There were no significant differences in the median percentage expression of CD62P and CD63, or PAC1 binding between the acute or convalescent symptomatic and asymptomatic patients. The median percentages of neutrophil–platelet (p = 0.004), monocyte–platelet (p = 0.046), and lymphocyte–platelet complexes (p = 0.02) were higher in acute symptomatic than in asymptomatic patients. In patients on aspirin monotherapy, the percentages of neutrophil–platelet and monocyte–platelet complexes (p = 0.03) were higher in acute symptomatic (n = 11) than asymptomatic patients (n = 14). In the convalescent phase, the median percentages of all leucocyte–platelet complexes in the symptomatic group dropped to levels similar to those found in the asymptomatic group. Conclusion: Increased platelet count and leucocyte–platelet complex formation may contribute to the early excess risk of stroke in patients with recently symptomatic carotid stenosis.

Vascular events commonly recur in stroke patients on aspirin, and may reflect incomplete inhibition of platelet function with aspirin therapy. The platelet function analyser (PFA-100) activates platelets by aspirating a blood sample at a moderately high shear rate through a capillary to a biologically active membrane with a central aperture. The membrane is coated with collagen, and either ADP (C-ADP) or epinephrine (C-EPI). The time taken for activated platelets to adhere, aggregate, and occlude the aperture is called the closure time. Previous studies have shown that aspirin prolongs the C-EPI closure time, without prolongation of the C-ADP closure time, in the majority of control subjects. We hypothesised that the PFA-100 would provide a sensitive assay for the detection of early and convalescent phase cerebrovascular disease (CVD) patients who had incomplete inhibition of platelet function with aspirin. We investigated potential cyclooxygenase-dependent and -independent mechanisms that might influence the responsiveness to aspirin using the PFA-100. Patients were studied during the early (< or = 4 weeks, n=57) and convalescent phases ((< or = 3 months, n=46) after ischaemic stroke or TIA. To investigate potential mechanisms that could contribute to aspirin responsiveness on the PFA-100, we measured von Willebrand factor antigen levels, and carried out platelet aggregometry experiments in platelet-rich plasma in response to sodium arachidonate (1 mM) and ADP (5 microM). Sixty percent of patients in the early phase and 43% of patients in the convalescent phase did not have prolonged C-EPI closure times on 75-300 mg of aspirin daily, and were defined as aspirin non-responders. Median C-ADP closure times were significantly shorter in aspirin non-responders than aspirin-responders in both the early and convalescent phases after symptom onset (P=0.008), suggesting platelet hyper-reactivity to collagen or ADP in the aspirin non-responder subgroup. There was a significant inverse relationship between plasma von Willebrand factor antigen levels and C-EPI closure times in both early and convalescent phase CVD patients (P=0.008). Mean von Willebrand factor antigen levels were significantly higher in aspirin non-responders than aspirin responsive patients in the early (P=0.001), but not convalescent phase (P=0.2) after stroke and TIA. None of the patients studied were defined as being aspirin-resistant using sodium arachidonate- or ADP-induced platelet aggregometry. A large proportion of ischaemic CVD patients have incomplete inhibition of platelet function with low to medium dose aspirin using the PFA-100. The results suggest that cyclooxygenase-independent mechanisms, including elevated von Willebrand factor antigen levels, play an important role in mediating aspirin non-responsiveness on the PFA-100.

BACKGROUND: The aim of this study was to assess whether the quality of FFP produced from whole blood stored at 4 degrees C overnight is adequate for its intended purpose.STUDY DESIGN AND METHODS: Fresh-frozen plasma (FFP) separated from whole blood (n = 60) leukodepleted (LD) after storage at 4 degrees C overnight (18-24 hr from donation, Day 1 FFP) was compared with that LD within 8 hours of donation (Day 0 FFP, the current standard method).RESULTS: In more than 95 percent of Day 1 FFP units, levels of factor (F) II, FV, FVII, FVIII, F IX, FX, FXI, and FXII were greater than 0.50 U per mL except for von Willebrand factor (VWF) antigen and FVIII, where 92 and 87 percent of units, respectively, contained greater than 0.50 IU per mL. Compared with historical data on FFP stored for 8 hours, fibrinogen, FV, FVIII, and FXI were reduced by 12, 15, 23, and 7 percent, respectively, but other factors were not significantly reduced. Levels of VWF-cleaving protease activity were not different between FFP prepared from paired units of blood (n = 3) held for 8 or 24 hours, but were below the reference range in an additional 2 of 6 units held for 24 hours. The activities of protein S, protein C, antithrombin III, and alpha(2)-antiplasmin were reduced by less than 10 percent in Day 1 FFP (n = 20), but with final levels above the lower limit of the normal range in greater than 95 percent of units. Activated FXII antigen was not significantly raised in plasma stored for 18 to 24 hours, but levels of prothrombin fragment 1 + 2 were slightly increased (0.88 ng/mL, 18-24 hr; 0.65 ng/mL, < 8 hr).CONCLUSION: These data suggest that there is good retention of relevant coagulation factor activity in plasma produced from whole blood stored at 4 degrees C for 18 to 24 hours and that this would be an acceptable product for most patients requiring FFP.

Fibrinogen is the major plasma protein coagulation factor. Low plasma fibrinogen concentrations are therefore associated with an increased risk of bleeding due to impaired primary and secondary haemostasis. Fibrinogen is a classical positive acute-phase reactant protein and is an independent predictor of coronary heart disease events. This review considers available methods for measurement of fibrinogen and makes recommendations as to their appropriate use. Total clottable fibrinogen assays are the definitive and reference method for plasma fibrinogen measurement. However, they are time-consuming and are rarely required in clinical practice. Clotting rate assays remain the routine method of choice for investigation, monitoring and treatment of bleeding disorders associated with low plasma fibrinogen concentrations. They are appropriately sited in haematology or haemostasis laboratories, with facilities for further relevant investigations, and expert advice from consultant haematologists on appropriate management. They have also been used in the majority of studies investigating increasing fibrinogen concentrations as a cardiovascular risk factor. Prothrombin-time derived assays are widely used, because they are less expensive and come at no extra cost with prothrombin time assays. However, their results vary widely with analysers and reagents, show discrepancies with clotting rate assays for both low and normal plasma fibrinogen samples, and at present they are not recommended by routine clinical use. Immunoassays (radial immunodiffusion, enzyme-linked immunosorbent assay or nephelometric) are useful in (a) differentiating hypofibrinogenaemia from dysfibrinogenaemia, and (b) assessing cardiovascular risk and acute-phase reactions. Unlike clottable fibrinogen assays, immunoassays can be performed in dipotassium edetate anticoagulated samples.

Flow cytometric studies suggest that platelets are activated in ischaemic stroke or transient ischaemic attack (TIA). However, few studies have measured circulating leucocyte-platelet complexes in this patient population. Whole blood flow cytometry was used to quantify the expression of CD62P-, CD63-, and PAC1-binding, and the percentages of leucocyte-platelet complexes in acute (1-27 d, n = 79) and convalescent (79-725 d, n = 70) ischaemic cerebrovascular disease (CVD) patients compared with controls without CVD (n = 27). We performed a full blood count, and measured plasma levels of soluble P-selectin, soluble E-selectin, and von Willebrand factor antigen (VWF:Ag) as additional markers of platelet and/or endothelial cell activation. The median percentage CD62P expression and the median percentage monocyte-platelet complexes were higher in both acute and convalescent CVD patients than controls (P

Summary. Using a diluted whole blood method of flowcytometric analysis, we have shown that platelets could beactivated in vitro in the presence of high concentrations(100 nmol/l) of recombinant factor (F) VIIa (rFVIIa; Novo-Seven ) and 2AE5 mmol/l calcium chloride. This was dem-onstrated by a significant increase in the mean percentageof platelets expressing CD62P and their mean fluorescentintensity (MFI) after 30 min versus platelets incubated withcalcium or rFVIIa alone or diluted blood alone. The presenceof rFVIIa and calcium increased the exposure of the PAC-1activation epitope of glycoprotein (Gp) IIb/IIIa. This effectwas equally influenced by the presence of calcium alone butnot by rFVIIa. The effect of rFVIIa was time and concen-tration dependent. Thrombin generation was also neces-sary, as the effect of rFVIIa was completely abrogated by theadditional presence of hirudin. Furthermore, soy beantrypsin inhibitor (SBTI) but not corn trypsin inhibitor (CTI)abrogated CD62P exposure, suggesting that thrombin wasderived via FX but not FXII activation. Exposure of CD62Pdemonstrated a significant lag phase, sometimes of the orderof > 30 min, as well as large intersubject variation. Signi-ficant platelet activation was observed at a concentration aslow as 25 nmol/l rFVIIa. Platelet–leucocyte aggregationwas also increased in the presence of 25 nmol/l rFVIIa andcalcium. No significant difference was observed betweenlevels of CD62P in diluted whole blood and platelet-richplasma adjusted to an identical platelet count after theirexposure to rFVIIa and calcium for 30 min.Keywords: factor VIIa, blood platelets, platelet membraneglycoproteins, flow cytometry.Factor (F) VII circulates in blood as a single chain zymogenat a concentration of 10 nmol/l. It is unusual amongstcoagulation factor zymogens in that it appears to have asmall degree of proteolytic activity before the liberation ofan activation peptide and conversion to the activetwo chain form (FVIIa). However, minute amounts(10–100 pmol/l, i.e. approximately 1%) of FVIIa aredetectable in plasma. When associated with tissue factor(TF), the activity of FVIIa is increased by a factor of 10