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Haemophilia published by John Wiley & Sons Ltd Introduction: Advances in genomic sequencing have facilitated the sequencing of genes associated with disorders of haemostasis. The identification of variants within genes and access to curated data incorporating structural, functional, evolutionary as well as phenotypic data has become increasingly important in order to ascribe pathogenicity. Aim: The European Association for Haemophilia and Allied Disorders (EAHAD) Coagulation Factor Variant Database Project aims to provide a single port of entry to a web-accessible resource for variants in genes involved in clinical bleeding disorders. Results: New databases have evolved from previously developed single gene variant coagulation database projects, incorporating new data, new analysis tools and a new common database architecture with new interfaces and filters. These new databases currently present information about the genotype, phenotype (laboratory and clinical) and structural and functional effects of variants described in the genes of factor (F) VII (F7), FVIII (F8), FIX (F9) and von Willebrand factor (VWF). Conclusion: The project has improved the quality and quantity of information available to the haemostasis research and clinical communities, thereby enabling accurate classification of disease severity in order to make assessments of likely pathogenicity.

Introduction: Haemophilia treatment varies significantly between individuals, countries and regions and details of bleed rates, factor consumption and injection frequency are often not available. Aim: To provide an overview of the FVIII/FIX treatment practice and outcome for patients with haemophilia A (HA) or haemophilia B (HB) across Europe. Methods: Non-interventional, 12-month retrospective study where anonymized data were retrieved from haemophilia centres/registers in Belgium, France, Germany, Italy, Spain, Sweden and the United Kingdom. Male patients (all ages) receiving coagulation factor treatment 24 months prior to the study, with basal FVIII/FIX levels ≤5 IU dL-1, without inhibitors, were included. Data were summarized descriptively. Results: In total, 1346 patients with HA and 312 with HB were included in the analysis; 75% and 57% had severe disease (FVIII/FIX < 1 IU dL-1) respectively. Prophylaxis was most common for severe haemophilia, especially for children, whereas on-demand treatment was more common for moderate haemophilia in most countries. The mean (SD) prescribed prophylactic treatment ranged from 67.9 (30.4) to 108.4 (78.1) (HA) and 32.3 (10.2) to 97.7 (32.1) (HB) IU kg-1 per week, across countries. Most patients on prophylaxis were treated ≥3 times/week (HA) or two times/week (HB). The median annual bleeding rate (ABR) for patients on prophylaxis ranged from 1.0 to 4.0 for severe HA, and from 1.0 to 6.0 for severe HB, while those with moderate haemophilia generally had slightly higher ABRs. Median ABRs for on-demand-treated severe HA ranged from 4.5 to 18.0, and for HB, 1.5 to 14.0. Conclusion: Treatment practice varied greatly between centres and countries and patients treated on-demand and prophylactically both experienced bleeds, emphasizing the need for further optimization of care. (Less)

Patients with bleeding disorders previously frequently became infected with hepatitis C virus. We identified the number of patients infected in Scotland and assessed several aspects of the outcomes of HCV infection and its treatment comparing these with cohorts infected for other reasons. We calculated the number of individuals infected in Scotland (cohort A) starting with the total number of patients treated in Scottish haemophilia centres registered on the UKHCDO database between 1970 and 1989. Cases were then removed or added based on additional information from centre records. A second cohort B, consisted of 255 patients from cohort A and 47 patients HCV infected outside Scotland, but with follow-up data from Scottish centres around their HCV infection. We estimate that 455 patients with bleeding disorders became infected by coagulation factor provided by NHS Scotland. In 302 individuals with documented HCV infection, rates of natural clearance (17.4%), genotype spread (64% genotype 1) and responses to antiviral therapy (14.5% with monotherapy; 38.8% with combination therapy) were similar to those in other cohorts. Thirty-four liver biopsies were performed without adverse event and liver transplantation has been performed in 11 patients, seven for liver failure, four for hepatocellular carcinoma. Around 455 patients with bleeding disorders became HCV infected in Scotland before 1989. The natural history of HCV infection and responses to treatment are similar to those in other HCV-infected cohorts. Liver transplantation has been used successfully for the treatment of end-stage liver failure and hepatocellular carcinoma.

Microparticles (MPs) are considered to be important biological effectors of several different physiological and pathological processes. There is increasing evidence of their role in haemostasis and thrombosis, and also of their importance in cancer cell survival, invasiveness and metastasis. The level of circulating MPs has been assessed in many different disease states, and there are reports that patients with malignancy and patients with thrombosis have increased levels of circulating MPs and MP-dependent thrombogenic potential. Research into the function and effect of MPs is currently hampered by a lack of standardization in the methods used to identify and quantify them. As these methods improve it is likely that MP assays will be of use both diagnostically and therapeutically in the future.

Pharmacovigilance is an essential element of any drug treatment and considering the history of adverse events due to products used to treat inherited bleeding disorders, it should be an integral component of modern haemophilia treatment. Because inherited bleeding disorders and adverse events are rare, a multicentre, preferably multinational, adverse event reporting scheme for all clotting factor products is required. EUHASS is a European, prospective, multicentre adverse event reporting scheme in the field of inherited bleeding disorders.

There have been four highly probable instances of variant Creutzfeldt-Jakob disease (vCJD) transmission by non-leucocyte depleted red cell concentrates and it is now clear that the infectious agent is transmissible by blood components. To date there in no reported evidence that the infectious agent has been transmitted by fractionated plasma products, e.g. factor VIII concentrate. This review outlines current and potential risk management strategies including donor deferral criteria, the potential for donor screening, blood component processing and prion reduction filters, plasma product manufacture and the difficulties in identification and notification of those considered 'at risk of vCJD for public health purposes'.

Summary. Objective: Pregnancy is associated with marked changes in vascular physiology and an increased risk of thrombosis. The aim of the study was to assess the effect of pregnancy on the acute release of tissue plasminogen activator (t-PA) from the endothelium. Methods and results: Ten primigravida pregnant women were recruited in the third trimester of pregnancy (week 36 ± 1) and compared with 20 age-matched non-pregnant women (day 9.8 ± 0.3 of menstrual cycle). Blood flow and plasma fibrinolytic factors were measured in both forearms by venous occlusion plethysmography and blood sampling, respectively, during unilateral brachial artery infusions of bradykinin (100–1000 pmol min−1). Pregnant women had higher plasma plasminogen activator inhibitor type 1 (PAI-1) antigen concentrations (77.1 ± 12.4 vs. 21.5 ± 9.8 ng mL−1; P = 0.004) that resulted in lower basal t-PA/PAI-1 ratios (0.2 ± 0.1 vs. 0.6 ± 0.1; P = 0.02) and plasma t-PA activity concentrations (0.17 ± 0.02 vs. 0.58 ± 0.06 IU mL−1; P

Since the 1970s, mortality in the hemophilia population has been dominated by human immunodeficiency virus (HIV) and few reports have described mortality in uninfected individuals. This study presents mortality in 6018 people with hemophilia A or B in the United Kingdom during 1977 to 1998 who were not infected with HIV, with follow-up until January 1, 2000. Given disease severity and factor inhibitor status, all-cause mortality did not differ significantly between hemophilia A and hemophilia B. In severe hemophilia, all-cause mortality did not change significantly during 1977 to 1999. During this period, it exceeded mortality in the general population by a factor of 2.69 (95% confidence interval [CI]: 2.37-3.05), and median life expectancy in severe hemophilia was 63 years. In moderate/mild hemophilia, all-cause mortality did not change significantly during 1985 to 1999, and median life expectancy was 75 years. Compared with mortality in the general population, mortality from bleeding and its consequences, and from liver diseases and Hodgkin disease, was increased, but for ischemic heart disease it was lower, at only 62% (95% CI: 51%-76%) of general population rates, and for 14 other specific causes it did not differ significantly from general population rates. There was no evidence of any death from variant Creutzfeldt-Jakob disease or from conditions that could be confused with it.

Thrombolytic therapy fails to achieve reperfusion in almost a third of patients with acute myocardial infarction. Thrombin activatable fibrinolysis inhibitor (TAFI) and soluble CD40 ligand (sCD40L) are novel endogenous fibrinolytic and atherothrombotic factors that determine clot stability. We investigated whether admission plasma thrombin activatable fibrinolysis inhibitor (TAFI) and soluble CD40 ligand (sCD40L) concentrations predicted reperfusion following thrombolytic therapy in patients with acute myocardial infarction.Prior to administration of thrombolytic therapy, venous blood was collected from 110 patients presenting with acute ST segment elevation myocardial infarction and plasma assayed for tissue plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor type-1 antigen (PAI-1), TAFI antigen and activity, C-reactive protein (CRP) and sCD40L concentrations. Reperfusion was determined using continuous ST segment monitoring.Reperfusion occurred in 77 (70%) patients with a mean treatment to reperfusion time of 83 +/- 46 min. Peak creatine kinase was significantly lower in patients who reperfused (1578 +/- 1199 versus 2200 +/- 1744 U/L; P0.05) and correlated with time to reperfusion (r = 0.44 [95% CI: 0.23 - 0.61], P = 0.0001). There was a modest correlation between plasma TAFI antigen and activity (r = 0.3 [95% CI: 0.04 - 0.53]; P0.05). There were no significant associations between coronary reperfusion and plasma concentrations of t-PA, PAI-1, TAFI, CRP or sCD40L.Systemic plasma TAFI, sCD40L and CRP concentrations do not predict reperfusion in patients receiving thrombolytic therapy for acute ST elevation myocardial infarction.

After surgery in haemophilia, haemostasis is difficult to maintain in the presence of an antifactor VIII antibody. This study assessed the pharmacokinetics of recombinant activated factor VII (rFVIIa) and its efficacy in securing post-operative haemostasis in haemophiliacs with inhibitors. Continuous infusion of rFVIIa was evaluated for elective major orthopaedic surgery in nine patients with neutralizing antibodies to FVIII and at high risk of bleeding. After an initial preoperative bolus of 90 micro g/kg, rFVIIa was infused at a fixed rate of 50 micro g/kg/h for a median of 20 d (range 7-20 d). The median plasma FVII coagulant activity (FVII:C) at 24 h, 72 h and 20 d after surgery was 38 IU/ml (range 22-169 IU/ml), 45 IU/ml (range 17-88 IU/ml) and 31 IU/ml (range 27-46 IU/ml) respectively. The median plasma FVIIa:C at the same time points was 51 (range 24-211), 63 (range 22-99) and 44 (range 28-76) IU/ml respectively. Median total rFVIIa clearance remained stable during the rFVIIa continuous infusion period and was 40 (range 9-70), 34 (range 17-86) and 48 (range 32-55)ml/kg/h at the end of 24 h, 72 h and 20 d infusion respectively. Post-operatively, there were bleeds in six patients, which settled readily after a single bolus of rFVIIa (60 micro g/kg). There was a good clinical outcome for all patients. These data indicate that rFVIIa infusion at 50 micro g/kg/h achieves continuous plasma FVII procoagulant activity in excess of 30 IU/ml (12-15 nmol/l) and provides adequate haemostatic control for inhibitor patients during major orthopaedic surgery.

We have developed a novel assay for quantitation of human von Willebrand factor (vWF) messenger RNA (mRNA) using the ABI 7700™ quantitative PCR system (Applied Biosystems, Warrington, Cheshire, UK). To allow accurate measurement of the vWF mRNA concentration in samples, we have used 18S ribosomal RNA as an internal control. The specificity of the assay was demonstrated by polyacrylamide gel electrophoresis and ABI Prism® BigDye™ terminator cycle sequencing of the PCR product. We have also assessed the assay by using RNA from both a hepatocyte cell line which does not express vWF mRNA, and an endothelial cell line which does express vWF mRNA. By mixing varying amounts of these RNAs together, we were able to adjust the vWF quantity in known increments while keeping the 18S ribosomal RNA internal control constant. The quantitation of vWF mRNA correlated with the proportion of endothelial cell RNA (r2=0.912, p

The European Study on the Clinical Outcomes and Resource Utilization associated with Haemophilia Care was designed to compare various health outcomes associated with on-demand and prophylactic factor substitution methods in European haemophilia patients. While the primary objective of this research is to conduct an economic analysis, an important component of this study is to evaluate quality-of-life differences that may exist between patients who utilize these two styles of therapy. Quality-of-life research has emerged as a primary measure of health outcomes because it allows the augmentation of traditional clinical indicators of health with data gathered from the patient's perspective. A total of 1033 haemophilia patients from 16 European haemophilia treatment centres were enrolled in this study. The SF-36, a multidimensional quality-of-life instrument, was administered to all participants. This instrument measures eight health-related quality-of-life dimensions: physical functioning, physical role limitations, bodily pain, general health, vitality, social functioning, emotional role limitations, and mental health. All haemophilia subjects enrolled in the study scored significantly lower than the population normative means in the three physical dimensions and in the general health dimension. HIV-negative haemophiliac subjects differed significantly by factor substitution type in a multivariate analysis examining all eight health dimensions. Univariate analyses testing each dimension separately indicated that patients treated prophylactically reported significantly less bodily pain, better general health, and scored significantly higher in the physical functioning, mental health, and social functioning dimensions. While these results suggest that health-related quality-of-life may be better for haemophilia patients treated prophylactically, future prospective studies that gather periodic quality-of-life data over time should be conducted.

Over the past 10 years considerable use has been made of recombinant factor VIIa (rFVIIa) for the treatment of patients with inhibitors to coagulation factors. During this time, its place in the management of acute bleeds and surgery has become better defined. Although pharmacokinetic studies report the half-life of rFVIIa as 2.7 h, there is considerable intersubject variability. Moreover, rFVIIa is cleared more rapidly in children than in adults. Assays for the measurement of rFVIIa plasma levels are not readily available in clinical diagnostic laboratories, although there is evidence that plasma FVII:C levels, measured by a one-stage prothombin-based assay, reflect the plasma concentration of rFVIIa:C. The level of FVII:C required to achieve haemostasis in different clinical circumstances remains uncertain. In order to overcome the logistic difficulties of repeated frequent bolus injections, and potentially to minimise usage, administration of rFVIIa by continuous infusion has been reported. However, there is some uncertainty as to whether continuous infusion of rFVIIa has similar therapeutic efficacy to an equivalent total dose administered by bolus injections. The extensive clinical experience with rFVIIa in haemophilic patients with inhibitors has been recorded in descriptive accounts of the Compassionate Use Programme and the Emergency Use Study. On the basis of the apparent clinical efficacy and safety reported in these studies, prospective randomised trials of different dose regimens have been undertaken for the treatment of acute bleeds and surgery. These have helped to define the minimum dose needed to achieve haemostasis. There remains considerable uncertainty about the minimal effective dose and appropriate duration of therapy in different clinical circumstances. There is therefore a need for the development of evidence-based guidelines for the use of rFVIIa in bolus and continuous infusion regimens in different settings, and for the therapeutic value of measuring plasma concentrations of rFVIIa, to facilitate the optimal use of this product. Furthermore, additional randomised clinical trials will help ensure that rFVIIa is used in the most clinically and cost effective way.

We conducted a multicentre, cross- sectional study of 1042 haemophilia subjects across Europe to compare various health outcomes associated with on-demand vs. prophylactic factor-substitution therapy. Demographic, medical history, and healthcare resource utilization data were analysed along with the number of bleeding events over the past 6 months. Treatment-cost data were also examined to provide preliminary information for future economic studies. A logistic regression analysis, controlling for other statistically significant covariates, showed that patients treated on demand were 3.4 times more likely to have had a joint bleed over the previous 6 months than those treated with prophylaxis. Multiple regression analyses further confirmed these findings, because on-demand subjects had, on average, 5.15 more joint bleeds over the reporting period than patients treated with prophylaxis. Notably, these findings were even more dramatic for younger haemophilia patients when our study sample was stratified by age. Due to the high cost of factor replacement, healthcare costs were significantly higher for subjects treated prophylactically. While hospital costs for prophylaxis subjects were, on average, lower, statistically significant cost savings for prophylactic subjects were not noted. These results suggest that clinicians and health policy decision-makers should consider the advantages of prophylactic therapy for haemophilia patients in formulating treatment protocols and allocating health resources.

SummaryWe examined recombinant activated factor VII (rVIIa) administered by continuous infusion to eight patients with inhibitors to factor VIII, undergoing elective surgery. rVIIa was infused at a fixed rate of 16.5 μg/kg/h for a median of 13.5 days (range 1-26). There was effective haemostasis at this infusion rate in only one of two minor procedures and two of six major operations. Three patients experienced excessive bleeding despite plasma factor VII activity around 10 IU/ml. Serious bleeding occurred in two other patients caused by procedural errors unrelated to rVIIa and required re-operation. The median rVIIa clearance on day 1 was 57 ml/h/kg (range 18-100) and on day 3 was 100 ml/h/kg (range 61-200). Clearance on the final infusion day was not significantly different from day 3. The infusion did not induce pathological activation of the coagulation mechanism. The only thrombotic adverse events were two episodes of superficial thrombophlebitis of the infused vein in one subject. In conclusion, the 16.5 μg/kg/h infusion rate reliably achieves plasma factor VII activity levels of 10 IU/ml, but this level does not provide reliable haemostasis.

We present two cases in which the occurrence of acquired haemophilia is associated with the use of depot preparations of the thioxanthenes zuclopenthixol and flupenthixol. These drugs have not previously been implicated in the aetiology of acquired haemophilia.

To estimate the resources required to manage patients with haemophilia in Scotland, we studied the demographic features, hospital admissions and causes of deaths for individuals with haemophilia A and B and von Willebrand disease, treated with blood products, during the period 1980-94 living in central Scotland. Data were obtained from 413 adults and children (93% ascertainment). The age distribution in 1980 revealed a paucity of individuals over 60 years but the number in this age group increased over the study period. Of those with haemophilia A and B, 63 and two respectively, became HIV positive. Hospital admissions rose from 103 to 168 per annum; the number of annual bed days utilized also increased, but there was marked annual fluctuation (790-1832). The rate of admission was greater for those with severe haemophilia A and this increased during the 15-year period mainly due to the clinical consequences of human immunodeficiency virus (HIV) and hepatitis C virus (HCV). The admission rate for haemophilia B was significantly lower than that for haemophilia A, and was similar for all degrees of severity of the disorder. Throughout the 15-year period the incidence of admissions for acute bleeds was constant, as was the average duration in hospital. For those with a factor VIII inhibitor, the rate of admission was about double the rate of those without an inhibitor, although the duration of hospital stay was similar for both groups. There were 61 deaths; the death rate increased during the study period principally due to HIV and HCV, and 12 patients died from haemorrhage. We conclude that: (i) the life expectancy for haemophiliacs in Scotland was generally increasing, although HIV and HCV caused increasing mortality and morbidity (as shown by the increase in hospital admissions); (ii) hospital bed usage for the treatment of acute bleeds continued to be required, but fluctuated greatly; and (iii) the clinical impression that haemophilia B is less clinically severe than haemophilia A is confirmed by objective data. The planning implications for haemophilia care in Scotland and similar countries are discussed.

Coagulation factor concentrates are known to inhibit a variety of immune reactions when assessed in vitro. This study assessed the immunomodulatory activity of a wide range of coagulation factor concentrates by measuring their inhibition of PHA-stimulated lymphocyte proliferation and reduction in IL-2 secretion. The hypothesis that TGF-β is responsible for most of these effects was tested by measuring biologically active TGF-β and immunoreactive TGF-β1 in the concentrates and comparing the levels recorded with immunosuppressive activity. In addition, the coagulation factors were compared directly with a standard preparation of TGF-β in a TGF-β-specific bioassay and in lymphocyte proliferation assays. Although there was a broad correlation between levels of total or active TGF-β and immunosuppressive activity across all of the coagulation factors tested, individual data sets showed clear discrepancies. Implying that TGF-β probably serves as a surrogate marker for other immunomodulatory contaminants and that neither TGF-β nor any other single substance could account for all of the immunosuppressive activity observed. Furthermore, there was a difference of more than 100-fold in the relative potencies of coagulation factors and pure TGF-β, when compared in immunosuppression assays, indicating that the different assays did not measure the same substance. Whereas anti-TGF-β antibody almost completely blocked the activity of coagulation factor concentrates (TGF-β-specific bioassay) and abrogated the effect of authentic TGF-β (immunosuppression assays) at high concentrations it achieved

Summary Background A newly discovered DNA virus, transfusion- transmitted virus (TTV), has been implicated as a cause of post-transfusion hepatitis. We investigated the frequency of TTV viraemia in UK blood donors, and the extent to which TTV contaminates blood products such as factor VIII and IX clotting factors. We also investigated the possible aetiological role of TTV in cryptogenic fulminant hepatic failure (FHF). Methods We extracted DNA from plasma of blood donors and patients with FHF, and from blood products (factor VIII and IX clotting-factor concentrates, immunoglobulin preparations). We detected TTV by PCR using primers from a conserved region in the TTV genome. Findings TTV viraemia was detected in 19 (1·9%) of 1000 non-remunerated regular blood donors. Infection occurred more frequently in older donors (mean age 53 years), compared with the age prolife of donors infected with hepatitis C virus and other parenterally-transmitted viruses. TTV contamination was found in ten (56%) of 18 batches of factor VIII and IX concentrate manufactured from such non- remunerated donors, and in seven (44%) of 16 batches of commercially available products. Whereas solvent or detergent treatment had little effect on the detection of TTV in factor VIII and IX by PCR, this virucidal step seemed to inactivate TTV infectivity. TTV infection was detected in four (19%) of 21 patients with FHF; in three cases, infection was detected at the onset of disease and could thus not be excluded from its aetiology. Interpretation TTV viraemia is frequent in the blood-donor population, and transmission of TTV through transfusion of blood components may have occurred extensively. Clinical assessment of infected donors and recipients of blood and blood products, and assessment of TTV's aetiological role in hepatic and extra-hepatic disease, are urgently needed.

The development of auto-antibodies to factor VIII results in a severe bleeding disorder, which has a mortality of up to 22%. It is predominantly a disease of the elderly, and probably results from a failure of the mechanisms which normally maintain immunological unresponsiveness to FVIII. An increasingly large therapeutic armamentarium often enables bleeds to be treated effectively with coagulation factor concentrates (or occasionally, desmopressin) while an attempt is made to lower the concentration of the antibody by immunomodulation with intravenous immunoglobulin and immunosuppressive drugs. A consequence of the high cost of the coagulation factor concentrates and the not infrequent need to provide treatment over long periods of time is that such treatment often has serious resource implications.

SummaryCirculating factor VIII (fVIII) levels increase during inflammation suggesting that fVIII synthesis or secretion is stimulated during acute inflammation. To examine the mechanisms underlying this increase in circulating factor VIII, we have developed a sensitive and reliable semi-quantitative assay for fVIII mRNA utilising competitive reverse transcriptase-polymerase chain reaction (RT-PCR, and used this to study two human liver cell lines, Hep-G2 and Chang Liver cells. These cells were cultured under basal conditions or following treatment with interleukin-1, -2 and 6 (IL-1, -2, and -6). Following 18h culture with IL-6 (maximum concentration 40U/ml), these levels had risen 6 and 9 fold respectively, with no concomitant rise in control RNA levels. The dose responses for both cell types were similar, with an ED50 of 11 U/ml. The time course of this response was also similar in both cell lines with the increase in fVIII mRNA first reaching significance by 3 h, and reaching maximum levels by 12 h. IL-1 and IL-2 had no effect at any of the doses studied. This study provides the first evidence for regulated expression of fVIII in human cell lines, and suggests that increased plasma fVIII levels during the acute phase response may be due to increased expression of fVIII mRNA.

SummaryHepatitis G virus (HGV), a novel flavivirus, has been implicated as a cause of posttransfusion hepatitis. We have performed a longitudinal study in a cohort of haemophiliacs (n = 68) who previously received non-virus inactivated coagulation factor concentrates to assess both patterns of HGV viraemia and any associated liver disease. Hepatitis C virus (HCV) RNA was present in 58/68 and co-infection with human immunodeficiency virus (HIV) was present in 15/68.HGV RNA was detected in 17/68 (25%) samples from the mid-1980s. There was no association between either HIV infection (p = 0.74) or co-infection with a particular HCV genotype (p = 0.62). However, there was a relationship between HGV viraemia and the severity of haemophilia (p = 0.0004) with HGV RNA detected in 5/19, 9/16 and 3/32 patients with mild, moderate and severe haemophilia respectively.A longitudinal study was performed in 15/17 haemophiliacs with HGV viraemia using stored serum samples from the 1980s and 1990s. HGV viraemia persisted in 8/15 and cleared in 7/15 over a variable period of time. A Weibull model was constructed to estimate the duration of HGV viraemia in the study group. The 75th and 90th percentiles for the duration of HGV were estimated to be 8.7 years (95%, confidence interval 4.8-15.7) and 23.6 years (95% confidence interval 11.8-47.1) respectively. Laparoscopic liver inspection/biopsy was performed in 25/68. There was no association between severity of liver disease and HGV viraemia (p = 0.43). This study demonstrates considerable variation in patterns of HGV viraemia in haemophiliacs. We found little evidence to implicate HGV as a major cause of chronic liver disease in haemophiliacs.

SummaryThe effects on blood flow and plasma fibrinolytic and coagulation parameters of intraarterial substance P, an endothelium dependent vasodilator, and sodium nitroprusside, a control endothelium independent vasodilator, were studied in the human forearm circulation. At subsystemic locally active doses, both substance P (2-8 pmol/min) and sodium nitroprusside (2-8 μg/min) caused dose-dependent vasodilatation (p

To assess the effect of HIV infection and the introduction of virus-inactivated concentrates, we conducted a retrospective 20-year longitudinal study of hepatitis B virus (HBV) serology and look for HBV DNA in recent serum samples of 63 multiply transfused haemophiliacs. Of 63 haemophiliacs, 51 had evidence of previous HBV infection and 12 vaccinees had anti-HBs only. Of 40 HIV-negative, two had persistent HBsAg but all were HBV DNA negative. All 23 HIV-positive were HBsAg-negative. Loss of anti-HBc(46% vs. 17.5%) and anti-HBs (32% vs. 14%) was more commonly seen in HIV-infected compared with noninfected individuals. One HIV-positive individual had HBV DNA detectable by PCR. Restrospective testing demonstrated that re-emergence was associated with loss of anti-HBs and advanced HIV infection (CD4

Mean platelet volume (MPV), a measure of platelet size, has previously been noted to change in a time dependent manner in ethylene-diaminetetraacetic acid (EDTA) and citrate anticoagulants. We measured MPV in healthy volunteers using several different anticoagulants and at different temperatures. In ten volunteers MPV was measured (Sysmex NE 8000 autoanalyser) serially over a 29 h period in 15 different anticoagulant/antiplatelet combinations kept at room temperature and on ice-water mixture. We evaluated the following mixtures: K3EDTA, K3EDTA/prostaglandin E1 (PGE1), K3EDTA/PGE1/theophylline, Na2EDTA, Na2EDTA/PGE1, citrate 1:9, citrate 1:4, citrate 1:9/PGE1/theophylline, citrate 1:4/PGE1/theophylline, citrate 1:9/PGE1, citrate 1:9/dextrose, citrate 1:4/dextrose, acid-citrate-dextrose (ACD) 15%, ACD 11.9%, ACD 11.9%/Na2EDTA. In a second experiment we evaluated MPV, at room temperature only, in a further 20 volunteers in K3EDTA, citrate 1:9, ACD 11.9%/Na2EDTA, ACD 11.9% (total = 30) and citrate-phosphate-dextrose-adenine (CPDA; total = 20). Over the 29 h period there was a significant positive trend of increasing MPV in K3EDTA, CPDA and ACD 11.9%/Na2EDTA and a negative trend in citrate 1:9. ACD 11.9% showed no significant change in MPV with time at room temperature. This methodology should enable the wider application of MPV measurement for routine clinical and research studies.

To assess the relative infectivity and pathogenicity of variants of hepatitis C virus (HCV) genotypes, the distribution of genotypes in hemophilic patients who had been treated with nonvirally inactivated factor concentrates or cryoprecipitates prepared from local blood donors was compared with those found in the respective blood donor populations. Genotype frequencies differed markedly in the four countries investigated (Scotland, Hungary, South Africa, and Thailand) but in each, the HCV genotype distributions in hemophiliacs and blood donors were similar. In addition, HCV genotypes in recipients of commercially manufactured concentrates were similar to those found in the US general population. These findings provide no evidence that HCV genotypes differ significantly from each other in replication rate, transmissibility, or infectivity.

A strategy is described for the initial detection, management and elimination of factor VIII inhibitors arising in patients with congenital and acquired haemophilia A. It is suggested that children with severe haemophilia A should be screened every 3 months up to the age of 10 years for inhibitors using the Bethesda method. Factor VIII inhibitors arising in these patients should be abolished using immune-tolerance induction wherever possible. Such regimes should be started as early as possible, preferably when the inhibitor titre is < 10 Bethesda Units (BU)/ml, and should not be interrupted. High-intensity regimes are recommended for patients whose inhibitors exceed 10 BU. Autoantibodies to factor VIII giving rise to acquired haemophilia should be abolished using high-dose immunoglobulin or conventional immunosuppression. The choice of haemostatic agent for the treatment of severe bleeding should be based upon the clinical circumstances and the current inhibitor value, measured using both human and porcine factor VIII in the Bethesda assay. The past anamnestic response should also be considered when choosing treatment for minor bleeding episodes.

In this study, we assessed the effectiveness of interferon treatment in 31 hemophiliacs with chronic hepatitis C virus (HCV) infection. Interferon alfa-2a (3 MU three times weekly) was administered for 6 months. Response was assessed by both serial alanine transaminase (ALT) and HCV RNA levels measured by a sensitive semiquantitative polymerase chain reaction (PCR) method. HCV genotype was determined by restriction fragment length polymorphism (RFLP), and evidence of changing genotypes during interferon therapy was sought. Severity of liver disease was assessed by both noninvasive and invasive methods, including laparoscopic liver inspection and biopsy. Sustained normalization of ALT levels occurred in eight patients (28%), and seven (24%) became nonviremic as assessed by PCR (

SummaryRecent studies using assays for surrogate markers of thrombogenic-ity in man have demonstrated that activation of the coagulation system occurs following infusion of clinical doses of prothrombin complex concentrates (PCC) but not after the same doses of high-purity factor IX concentrates (HP-FIX) in patients with haemophilia B. Here we have investigated the mechanism of such thrombogenesis by applying assays that detect early-through to late-events in coagulation system activation in a pharmacokinetic cross-over study of 50 IU/kg PCC and a new HP-FIX product in haemophilia B patients. Satisfactory recoveries and half-lives were observed for both concentrates.HP-FIX caused no increases in thrombin-antithrombin III complex (TAT), prothrombin activation peptide fragment F1+2 (F1+2), factor X activation peptide (FXAP) or factor Vila (FVIIa). In contrast the same dose of factor IX in the form of PCC was followed by significant increases over pre-infusion levels of TAT, F1+2 and FXAP, but not FVIIa. Elevations of FIXAP occurred after both HP-FIX and PCC but did not reach normal levels and were attributed to normalisation of the FIX concentration in those patients whose levels of FIXAP were initially low. We conclude that the thrombogenic trigger associated with PCC infusion occurs at the level of factor X activation. In the absence of any increase in FVIIa, we would attribute this to the likely presence of FIXa in the PCC.

The aim of this study was to investigate whether abnormalities in the fibrinolytic system and in the naturally occurring anticoagulant proteins could contribute to the thrombotic risk in essential thrombocythemia. Euglobulin lysis time, fibrin plate lysis area, tissue plasminogen activator antigen, and activity and plasminogen activator inhibitor antigen were measured before and after venous occlusion in a group of 16 patients with essential thrombocythemia and in 16 healthy age and sex matched controls. In addition, resting levels of antithrombin III, D-dimer, prothrombin fragment 1 + 2, and protein C and S were assessed. The results were related to the presence or absence of a thrombotic history. The results demonstrated that the patients had a significantly elevated fibrin plate lysis area and significantly decreased plasminogen activator antigen, both at baseline and after venous occlusion. They also had significantly decreased levels of plasma protein C and total protein S. There was a modest, non-significant elevation in the plasma concentration of D-Dimer and F 1 + 2. Those patients with a history of thrombosis had significantly lower protein C levels compared with individuals without a thrombotic history. We conclude that patients with essential thrombocythemia have evidence of activated fibrinolysis in the resting state and after stimulation. This, and the decreased levels of protein C and total protein S, may be secondary to chronic clinically occult thrombosis occurring in myeloproliferative disorders.

Guy’s and St Thomas’ NHS Foundation Trust, London, Russells Hall Hospital, Dudley, West Midlands, Arrowe Park Hospital Arrowe Park Road Upton Wirral, Wellcome Trust Sanger Institute, Hinxton, Cambridge, St. James Hospital, James Street, Dublin, Gartnavel General Hospital 21 Shelley Road Glasgow, Addenbrooke’s Hospital, Cambridge, Salisbury Healthcare NHS Trust, Salisbury, Wiltshire, Cambridge Institute for Medical Research, Hills Road, Cambridge, John Radcliffe Hospital, Headley Way, Headington, Oxford, Royal Infirmary, Little France Crescent, Edinburgh, Sandwell and West Birmingham Hospitals, Dudley Road, Birmingham, Royal Hallamshire Hospital, Glossop Road, Sheffield, and Belfast City Hospital, Lisburn Road Belfast, UK

This study was designed to assess whether factors other than high haemoglobin, thrombocytosis and abnormal platelet function predispose to thrombosis in polycythaemia rubra vera (PRV). Components of the fibrinolytic system and concentrations of the naturally occurring anticoagulants were measured in patients and controls in the resting state; the fibrinolytic capacity was reassessed after venous occlusion. The results were related to presence or absence of a history of thromboembolism. Under resting conditions, patients with PRV had reduced plasminogen activator inhibitor antigen levels and higher fibrin plate lysis area and tissue plasminogen activator activity. Protein C, protein S and factor V levels were reduced. Those patients with a history of thromboembolism had decreased tissue plasminogen activator activity after venous occlusion compared to those who had not experienced a thrombosis. We conclude that reduced fibrinolytic capacity may predispose to thrombosis in PRV. Despite treatment to normalize haemoglobin levels, the patients have persistent activation of their fibrinolytic systems. This, and reduced levels of proteins C and S, may be secondary to a chronic, clinically occult, disseminated intravascular coagulation.

The effect of factor VIII concentrate, from commercial and National Health Services manufacturers, on in vitro lymphocyte proliferative response to allogeneic stimulator cells was investigated. Factor VIII preparations 'purified' by ion exchange or by monoclonal antibody affinity had no effect in this assay but lymphocyte proliferation in response to allogeneic cells was markedly and consistently enhanced by some intermediate purity factor VIII concentrates and, to a lesser extent, by a factor IX preparation. These preparations did not stimulate lymphocyte proliferation in the absence of other mitogens. The co-mitogenic factor(s) present in intermediate purity factor VIII was not identified. However, enhanced proliferation was not due to factor VIII itself, nor to albumin or fibronectin. The clinical relevance of the immunomodulatory activity of intermediate purity factor VIII concentrates in vitro is discussed.

Immunological studies were performed on a group of 44 haemophilia A and 15 haemophilia B patients who were treated exclusively with blood products manufactured by the Scottish National Blood Transfusion Service (SNBTS). All patients were HIV seronegative throughout the study. Of the haemophilia A patients 14 (32%) had CD4+ lymphocyte subset counts less than or equal to 0.5 x 10(9)/l, compared with one (6%) haemophilia B patient and four (8%) controls. The percentage of activated T cells was greater than 5% in 19/33 (57%) with haemophilia A, 5/9 (55%) haemophilia B and 14/50 (28%) of control subjects. beta 2 microglobulin values greater than or equal to 2.0 mg/l were observed in 19 (43%) haemophilia A and four (26%) haemophilia B patients, compared with one (2%) control. No significant increases in serum interleukin-2 receptor concentrations were observed in 15 haemophilia A and one haemophilia B patients. Significantly elevated levels of IgG, IgM and IgA were observed in the haemophilia A group, but elevation of immunoglobulins was restricted to the IgG class in the haemophilia B group. Of the haemophilia A patients 16/30 (53%) and 6/11 (54%) haemophilia B patients had depression of cell-mediated immunity (CMI) as assessed by delayed-type hypersensitivity responses to intradermally injected recall antigens. There was no correlation between factor VIII or factor IX usage and changes in lymphocyte subsets, beta 2 microglobulin, and immunoglobulin levels. There was, however, a strong correlation between annual factor VIII usage and the degree of depression of CMI for those with haemophilia A but not for those with haemophilia B. No correlation between alterations in the immune parameters and disturbance of liver function tests was observed in either haemophilia A or haemophilia B patients. We conclude that alloantigen or non-HIV viral exposure due to repeated administration of factor concentrates brings about alterations in the immune response, and that these changes are more marked following exposure to intermediate purity factor VIII compared with factor IX concentrate.

Udgivelsesdato: 2009-Mar Patients with haemophilia complicated by inhibitors have a significant burden of joint disease, which is associated with a negative impact on their quality of life. Successful elective orthopaedic surgery can result in decreased bleed frequency into a new joint, less time spent in hospital, increased mobility and improved well being. This paper describes a new protocol for use of recombinant activated factor VII (rFVIIa) in elective orthopaedic surgery, based on a review of published data as well as the personal experience of a group of expert physicians. The protocol offers guidance on the planning of the surgery and preoperative testing as well as the bolus schedule for rFVIIa and advice on the concomitant use of antifibrinolytic agents and fibrin sealants. A total of 10 operations involving 13 procedures in eight patients in five comprehensive care centres have been undertaken until now using the protocol, which employs an initial bolus dose of rFVIIa in the range of 120-180 microg kg(-1) to cover surgery. The clinical experience reported here encompasses all cases of elective orthopaedic surgery using rFVIIa as initial treatment carried out in the UK and Republic of Ireland over the last 2 years. In all cases, there was good control of haemostasis during surgery and the final outcome was rated as 'excellent' or 'extremely satisfactory' by the reporting clinicians. Although the initial cost of product to cover surgery such as arthroplasty is high, it needs to be borne in mind that this may be offset in subsequent years by savings resulting from avoidance of bleeding episodes in the affected joint.

Over activity of the fibrinolytic system (hyperfibrinolysis) occurs in cirrhosis and has been shown to correlate with the risk of variceal hemorrhage. We have developed a model for assessing acute tissue plasminogen activator (t-PA) release in vivo in man. The aims of the study were to assess the contribution of basal and stimulated t-PA release to hyperfibrinolysis in patients with alcoholic cirrhosis.Bilateral forearm blood flow and plasma fibrinolytic variables were measured in 8 patients with biopsy proven alcohol induced cirrhosis, ascites and portal hypertension, and 8 age and sex matched healthy controls during infusion of bradykinin (100-900 pmol/min; endothelium-dependent vasodilator that releases t-PA) followed by sodium nitroprusside (SNP 2-8 microg/min; a control endothelium-independent vasodilator).Baseline plasma t-PA antigen concentrations were higher in patients (14+/-2 vs 9+/-1 ng/mL; p0.05) whereas plasma plasminogen activator inhibitor type-1 (PAI-1) antigen concentrations were similar (59+/-16 vs 55+/-11 ng/mL; p=NS). This resulted in an increased t-PA activity (3+/-1 vs 0+/-0 IU/mL; p0.05) and reduced PAI-1 activity (9+/-2 vs 21+/-2 AU/mL; p0.05) indicating a relative deficiency of PAI-1 in patients with cirrhosis. Bradykinin and SNP caused dose-dependent vasodilatation (p0.001 for both) that did not differ between the two groups. Bradykinin caused a similar release of t-PA antigen (p0.05 for both) in both patients and controls (24+/-17 vs 23+/-7 ng/100 mL/min; p=ns) without affecting PAI-1 concentrations. Local t-PA activity was increased in patients following acute stimulated t-PA release (5+/-1 vs 3+/-1 IU/mL; p0.05). SNP caused no significant change in fibrinolytic parameters.Patients with alcoholic cirrhosis have a higher basal plasma t-PA activity because of a failure to increase plasma concentrations of its inhibitor, PAI-1. Furthermore, despite releasing normal amounts of t-PA acutely, higher t-PA activity remained due to the relative deficiency of PAI-1. This suggests that the pathogenesis of hyperfibrinolysis in alcoholic cirrhosis is the result of a relative PAI-1 deficiency and enhanced basal t-PA release.

Microparticles are circulating, phospholipid rich, submicron particles released from the membranes of endothelial cells, platelets, leucocytes and erythrocytes. Investigation into their biological activity has revealed diverse actions in coagulation, cell signalling and cellular interactions. These actions are mediated through their phospholipid rich surfaces and the expression of cell surface molecules which reflect their cell of origin and its state of activation. Microparticle numbers are reported to be elevated in a number of conditions where vascular dysfunction and inflammation are important pathophysiological mechanisms, for example coronary artery disease or thrombotic microangiopathies. Currently, there are a variety of different methods used for the quantitation of circulating microparticles; however with standardisation their assessment may prove to be of clinical value, reflecting the state of the vasculature. Knowledge of the functional properties of microparticles will contribute to our understanding of the mechanisms underlying vascular dysfunction and prothrombotic states.