Laboratory performance in the World Federation of Hemophilia EQA programme, 2003-2008.

External quality assessment (EQA) has been shown to improve laboratory performance and diagnosis in haemostasis. We report here findings from the World Federation of Haemophilia (WFH) EQA programme during the period 2004-2007. Samples for PT, APTT, FVIII:C, FIX:C and VWF assays were distributed to centres in both established and emerging countries, and results were compared with results obtained by United Kingdom National External Quality Assessment Scheme (UK NEQAS) participants on the same samples. In general, good agreement was seen throughout between WFH and UK NEQAS for screening tests, and it was possible to identify an improvement in WFH centre agreement for results for VWF assays during the period of study. Agreement between emerging and established WFH centres was comparable for screening tests, possibly indicative of the relative simplicity of these tests and the degree of automation now employed in almost all haemostasis laboratories. However, CVs and performance compared with UK NEQAS participant results for factor assays amongst established centres was better than between emerging centres. Distribution of a questionnaire revealed different application of methodology for these assays, which may contribute to the observed difference in performance. Several centres participated in supplementary exercises, with comparable results obtained by emerging and established centres performing FVIII and fibrinogen measurement on cryoprecipitate, and all centres performing FVIII inhibitor assays correctly identifying the presence of an inhibitor. Participation in EQA programmes should continue to encourage improvement in laboratory performance and therefore improvements in the diagnosis and care of patients with haemophilia.

Risk of a first venous thrombotic event in carriers of a familial thrombophilic defect. The European Prospective Cohort on Thrombophilia (EPCOT).

BACKGROUND: Reliable risk estimates for venous thrombosis in families with inherited thrombophilia are scarce but necessary for determining optimal screening and treatment policies. OBJECTIVES: In the present analysis, we determined the risk of a first venous thrombotic event in carriers of a thrombophilic defect (i.e. antithrombin-, protein C- or protein S deficiency, or factor V Leiden). PATIENTS AND METHODS: The asymptomatic carriers had been tested prior to this study in nine European thrombosis centers because of a symptomatic carrier in the family, and were followed prospectively for 5.7 years on average between March 1994 and January 2001. Annually, data were recorded on the occurrence of risk situations for venous thrombosis and events (e.g. venous thrombosis, death). RESULTS: Twenty-six of the 575 asymptomatic carriers (4.5%) and seven of the 1118 controls (0.6%) experienced a first deep venous thrombosis or pulmonary embolism during follow-up. Of these events, 58% occurred spontaneously in the carriers compared with 43% in the controls. The incidence of first events was 0.8% per year (95% CI 0.5-1.2) in the carriers compared with 0.1% per year (95% CI 0.0-0.2) in the controls. The highest incidence was associated with antithrombin deficiency or combined defects, and the lowest incidence with factor V Leiden. CONCLUSIONS: The incidence of venous events in asymptomatic individuals from thrombophilic families does not exceed the risk of bleeding associated with long-term anticoagulant treatment in the literature (1-3%).

Stability of coagulation proteins in lyophilized plasma.

INTRODUCTION: External quality assessment (EQA) is an important component of quality assurance for laboratory tests of haemostasis. Lyophilization of plasma confers stability of labile clotting factors, allowing valid comparison of results between participating centres. However, elevated ambient temperatures in some geographical areas could affect the stability of lyophilized samples in transit. METHODS: The effect on lyophilized plasma samples of consistent elevated temperature with respect to haemostasis tests was determined in a single centre. The temperature to which packages were exposed during transit was also monitored. RESULTS: Survey packages were exposed to average temperatures up to 31.9 °C and maximum temperatures up to 39.7 °C over delivery periods between 1 and 8 weeks. In-house studies revealed samples to be stable over a 6-week period at a constant 30 °C, and only small changes were observed for samples exposed to 37 °C for 4 weeks. 6-week storage at 37 °C was associated with average changes of up to 15% in factor assay activity. CONCLUSION: Lyophilized EQA material employed in UK NEQAS surveys is stable under conditions encountered for the majority of participants, but in cases of delayed delivery of samples, the effect of temperature on sample integrity must be considered when assessing laboratory performance.

Chronic hepatitis in haemophilia.

Chronic hepatitis affects almost all haemophiliacs treated with non-virally inactivated clotting factor concentrates. The virus responsible is hepatitis C (HCV) and most patients have non-neutralising antibodies with circulating virus. Although the majority also have evidence of past infection with hepatitis B, less than 5% are chronic carriers of HBsAg. Chronic hepatitis C can be associated with severe and progressive liver disease but the development of complications is slow. Treatment with recombinant interferon alpha given subcutaneously normalises the liver function in 50% of patients, but 50% of responders relapse on stopping treatment. Liver transplantation is successful in patients with advanced liver disease and it offers the added advantage of phenotypic cure of the haemophilic state.

Problems relating to the laboratory diagnosis of factor XIII deficiency: a UK NEQAS study.

Familial (F)XIII deficiency is an extremely rare bleeding disorder. In most laboratories the diagnosis is initially established through a clot-solubility screening test. We report here results from a series of UK NEQAS (Blood Coagulation). Proficiency Testing investigations, in which laboratories were provided with samples from normal individuals and from various subjects with FXIII deficiency with a request to perform their usual test for this disorder and to provide an interpretation of their results. Over 95% of centers were able to diagnose severe familial FXIII deficiency in previously untreated patients and to identify samples from normal subjects. However, both quantitative and qualitative methods produced widely variable results on samples obtained from previously treated individuals with FXIII deficiency but having measurable levels of FXIII. Data generated by UK NEQAS investigations suggested that solubility tests employing thrombin show greater sensitivity to FXIII deficiency, and this was confirmed in a subsequent single-center study. Our results lead us to recommend the use of thrombin and acetic acid in the clot-solubility screening test. Use of sensitive screening tests, and improvement in the accuracy and precision of quantitative FXIII assays will aid study of the clinical importance of moderate FXIII deficiency.

Lupus anticoagulant testing using plasma spiked with monoclonal antibodies: performance in the UK NEQAS proficiency testing programme.

We report here results from a United Kingdom National Quality Assessment Scheme (UK NEQAS) exercise in which both plasma spiked with monoclonal antibodies and plasma from a patient known to have lupus anticoagulant (LA) were distributed to 245 hemostasis laboratories with a request for them to test for possible LA using their routine screening procedure. In general, good agreement was seen in the diagnosis of samples spiked with monoclonal antibodies against beta2-glycoprotein 1 (beta2GP1) and prothrombin, the LA-positive patient sample, and a normal pooled plasma; over 87% of centers correctly identified each sample. However, methods employing platelet neutralizing procedures were associated with a higher proportion of false-negative responses with the antiprothrombin-spiked sample, and it is important to recognize that sensitivity and responsiveness of different methods may vary between artificial plasmas and different LA-positive patient plasmas.

SSC/ISTH classification of hemophilia A: can hemophilia center laboratories achieve the new criteria?

To assess the practicality of the recent Scientific and Standardization committee (SSC) of the International Society on Thrombosis and Haemostasis (ISTH) recommendations in respect of the classification of hemophilia we distributed samples from three untreated subjects with hemophilia A to 91 UK hemophilia centers (HCs), comprising 20 comprehensive care centers (CCCs) and 71 HCs. Laboratories were requested to perform their routine factor (F)VIII:C assays and to classify the severity of hemophilia. Median values of < 1 U dL-1 were obtained on two samples. However, for each of the two, approximately 30% of laboratories obtained results in the range 1-29 U dL-1 and 1-33 U dL-1 respectively. For one of these samples 17 laboratories diagnosed severe hemophilia despite obtaining FVIII:C levels in the range 1-5 U dL-1. The median FVIII:C for the third sample was 5.8 U dL-1 with a range of 1.5-36 U dL-1. For this sample eight centers diagnosed severe hemophilia. Fifty-four laboratories obtained a result > 5 U dL-1; 21 of these diagnosed mild hemophilia, 31 moderate hemophilia and two severe hemophilia. Results from CCCs were more accurate and more precise than those from HCs. Our results indicate a need for improved standardization of FVIII assays. In the UK there remains a lack of consensus in respect of the laboratory diagnostic criteria for the classification of hemophilia A.

Hereditary thrombophilia and fetal loss: a prospective follow-up study.

BACKGROUND: As the placental vessels are dependent on the normal balance of procoagulant and anticoagulant mechanisms, inherited thrombophilia may be associated with fetal loss. OBJECTIVES: We performed a prospective study to investigate the relation between inherited thrombophilia and fetal loss, and the influence of thromboprophylaxis on pregnancy outcome. PATIENTS AND METHODS: Women were enrolled in the European Prospective Cohort on Thrombophilia (EPCOT). These included women with factor (F)V Leiden or a deficiency of antithrombin, protein C or protein S. Controls were partners or acquaintances of thrombophilic individuals. A total of 191 women (131 with thrombophilia, 60 controls) had a pregnancy outcome during prospective follow-up. Risk of fetal loss and effect of thromboprophylaxis were estimated by frequency calculation and Cox regression modelling. RESULTS: The risk of fetal loss appeared slightly increased in women with thrombophilia without a previous history of fetal loss who did not use any anticoagulants during pregnancy (7/39 vs. 7/51; relative risk 1.4; 95% confidence interval 0.4, 4.7). Per type of defect the relative risk varied only minimally from 1.4 for FV Leiden to 1.6 for antithrombin deficiency compared with control women. Prophylactic anticoagulant treatment during pregnancy in 83 women with thrombophilia differed greatly in type, dose and duration, precluding solid conclusions on the effect of thromboprophylaxis on fetal loss. No clear benefit of anticoagulant prophylaxis was apparent. CONCLUSIONS: Women with thrombophilia appear to have an increased risk of fetal loss, although the likelihood of a positive outcome is high in both women with thrombophilia and in controls.

Familial thrombophilia and lifetime risk of venous thrombosis.

BACKGROUND: We started a large multicenter prospective follow-up study to provide reliable risk estimates of venous thrombosis in families with various thrombophilic defects. OBJECTIVES: This paper describes data collected at study entry on venous events experienced before study inclusion, i.e. the baseline data. PATIENTS/METHODS: All individuals (probands, relatives) registered in nine European thrombosis centers with the factor (F)V Leiden mutation, a deficiency of antithrombin, protein C or protein S, or a combination of these defects, were enrolled between March 1994 and September 1997. As control individuals, partners, friends or acquaintances of the thrombophilic participants were included. Incidence and relative risk of objectively confirmed venous thrombotic events (VTEs) prior to entry were calculated for the relatives with thrombophilia and the controls. RESULTS: Of the 846 relatives with thrombophilia (excluding probands), 139 (16%) had experienced a VTE with an incidence of 4.4 per 1000 person years. Of the controls, 15 of the 1212 (1%) controls had experienced a VTE with an incidence of 0.3 per 1000 person years. The risk of venous thrombosis associated with familial thrombophilia was 15.7 (95% CI 9.2-26.8) and remained similar after adjustment for regional and sex-effects (16.4; 95% CI 9.6-28.0). The highest incidence per 1000 person years was found in relatives with combined defects (8.4; 95% CI 5.6-12.2), and the lowest incidence was found in those with the FV Leiden mutation (1.5; 95% CI 0.8-2.6). CONCLUSIONS: Considerable differences in the lifetime risk of VTE were observed among individuals with different thrombophilia defects.

Treatment of the hypercalcaemia of malignancy with intravenous clodronate.

We studied the effects of intravenous clodronate (100-300 mg daily for 3-10 days) in 27 episodes of hypercalcaemia due to malignancy. Comparisons were also made between responses in patients with haematological malignancies and those with solid tumours. Following extracellular volume expansion, clodronate induced a significant decrease in serum calcium within 2 days of the start of treatment which was maximal at 7 days. This was associated with a decrease in bone resorption as judged by decreases in urinary excretion of hydroxyproline and calcium. Hypercalcaemia recurred 5-7 days after stopping treatment. Patients with solid tumours had higher pretreatment serum calcium values than those with haematological malignancies despite comparable values for fasting urinary excretion of calcium suggesting that renal tubular reabsorption of calcium was more markedly increased in patients with solid tumours. No difference was observed in the final calcium values between patients with solid tumours and haematological malignancies except in those patients in whom renal tubular reabsorption of calcium was markedly increased before treatment. We conclude that intravenous clodronate provides a safe and effective treatment of hypercalcaemia due to a wide range of tumour types provided that increased bone resorption contributes significantly to the hypercalcaemia. When increased renal reabsorption of calcium is the predominant mechanism for the maintenance of hypercalcaemia, the response to clodronate is incomplete.

Platelet aggregation in patients with peripheral vascular disease.

Platelet aggregation has been studied in 28 control subjects and 46 patients with peripheral vascular disease. The platelets from the patients with peripheral vascular disease were significantly more sensitive to the aggregating effects of adenosine diphosphate and adrenaline than those from the control group (P less than 0.001). In addition, spontaneous platelet aggregation was observed in vitro in several of the vascular patients; this response was not apparent in the control group. Increased platelet aggregation could add to the vascular impairment in the unoperated subject and might also play a part in premature graft failure. Platelet function studies should perhaps be included as a routine in the assessment of patients with peripheral vascular disease.

The effects of dietary omega-3 polyunsaturated fatty acids on erythrocyte membrane phospholipids, erythrocyte deformability and blood viscosity in healthy volunteers.

We have examined, in normal subjects, the effects of a daily dietary supplement of fish oil concentrate ('maxEPA'), providing 3 g of omega-3 fatty acids, on erythrocyte membrane phospholipids, erythrocyte deformability and blood viscosity. After 3 weeks, incorporation of C20:5 omega 3 into erythrocyte phosphatidyl choline (PC) was greater compared to phosphatidyl ethanolamine (PE) and phosphatidyl serine (PS). After 6 weeks, there was no further increase in total erythrocyte C20:5 omega 3, but its distribution amongst phospholipid subclasses had changed. C20:5 omega 3 had increased further in PE and PS, but decreased in PC. Incorporation of C20:5 omega 3 also occurred into PC, PE and PS. omega-3 Fatty acids were incorporated almost entirely at the expense of C18:2 omega 6, but total unsaturation of phospholipids was increased. This is consistent with increased lipid fluidity, which may be an important determinant of erythrocyte deformability. The same dosage of maxEPA also resulted in a significant increase in erythrocyte deformability and a concomitant reduction in whole blood viscosity. Since plasma viscosity and haematocrit were unchanged it seems likely that the effects on blood rheology were mediated by changes in erythrocyte lipid fluidity. Modification of blood rheology by dietary omega-3 fatty acids is of potential value in the treatment of vascular disease.

A simple technique for studying immunoglobulin synthesis by normal and malignant plasma cells in vitro.

Plasma cells from human marrows are saturated with C-14 labelled amino acids, harvested and recultured in unlabelled growth medium. The appearance of radioactivity in the growth medium then provides a simple and rapid measure of protein synthesis. The secreted radio-labelled material is characterized by isoelectric focusing and autoradiography in acrylamide gels, a technique which has advantages over established serological methods.

Thromboxane and prostacyclin synthesis in experimental pancreas transplantation. Changes in parenchymal and vascular prostanoids.

The principal causes of failure of a pancreas transplant are rejection and vascular thrombosis. There is an unusually high attrition rate for pancreas transplants, but study models have been difficult to develop. In a rat model that allows study of acute rejection to the exclusion of nonspecific effects of transplant surgery on the pancreas, in vitro synthesis of prostacyclin (PGI2) and thromboxane A2 (TXA2) by transplanted pancreas and the blood vessels transplanted with it was measured using an RIA for their stable hydrolysis products 6-keto-prostaglandin F1 alpha and thromboxane B2 (TXB2). TXB2 synthesis was significantly greater in allotransplanted pancreas than isotransplanted pancreas from the 5th day after transplantation. Rejection was complete in the allografted group 7-9 days after transplantation. 6-Keto-prostaglandin F1 alpha synthesis was similar in the pancreas for both allografts and isografts. Similar changes were seen in aorta, celiac artery, superior mesenteric artery, and portal vein transplanted with the pancreas. In the transplanted aorta, TXB2 was significantly greater in the allograft group from the third posttransplant day. A group of CsA-treated allografts sampled after 9 days had transplanted pancreatic parenchymal and vascular prostanoid synthesis in the isograft range. The changes in PGI2 and TXA2 synthesis that accompany cellular rejection may mediate vascular failure in rejecting pancreas transplants, and changes in PGI2 and TXA2 synthesis in blood vessels transplanted with the pancreas could promote early vascular thrombosis.

Cortical and vascular prostaglandin synthesis during renal allograft rejection in the rat.

Alterations in local prostacyclin and thromboxane synthesis could mediate the changes in vascular perfusion and platelet deposition in acutely rejecting renal allografts and prostaglandin E2 (PGE2) has been implicated in the regulation of the immune response. 6-Keto-prostaglandin F1 alpha (6 KetoPGF1 alpha), thromboxane B2 (TxB2) (the stable degradation products of prostacyclin and thromboxane A2 [TxA2], respectively) and PGE2 were measured in incubates of cortical slices taken from rat renal allografts or isografts one to seven days after transplantation. 6 KetoPGF1 alpha and TxB2 synthesis was also measured in incubates of blood vessels supplying and transplanted with the kidney in these animals. During the phase of cellular rejection (3-5 days), TxB2 synthesis was selectively elevated in allografted renal cortex, renal artery, renal vein, and abdominal aorta in comparison with isografted tissues. There was also a small but significant rise in cortical PGE2 synthesis at this time, but vascular and cortical 6 KetoPGF1 alpha production remained unchanged. Renal infarction, occurring 7 days after transplantation, was accompanied by a nonspecific rise in the synthesis of all three prostaglandins by renal cortical slices. Increased tissue TxA2 synthesis may contribute to local thrombosis and decreased graft perfusion during acute rejection, thereby potentiating graft destruction.

Quality control and oral anticoagulation.

The introduction of the INR system for the monitoring of oral anticoagulant control represents a major advance, not only in terms of therapeutic efficacy but also in respect of patient safety. It is important to recognize that the system is only viable if careful attention is paid to the many important variables which contribute to its overall value. These include the choice of thromboplastin, assignation of ISI, determination of the mean normal prothrombin time and the method of end-point detection. Participation in independent external quality assessment schemes permits a unique opportunity for individual laboratories to identify, through laboratory performance analysis, problems relating to their own laboratory practice. Another major advantage, particularly of the larger schemes, is the identification of poor reagents and coagulometers and inconsistencies in their performance. One example of this is the recent identification of problems in respect of ISI assignment for thromboplastins used with coagulometers. This, in turn, provided the necessary stimulus to the potentially important development of calibrants for local ISI determination (21).

The therapeutic range for heparin therapy: relationship between six activated partial thromboplastin time reagents and two heparin assays.

The activated partial thromboplastin time (APTT) is the most commonly used test for laboratory monitoring of unfractionated heparin therapy. Since there are differences between APTT reagents in respect of responsiveness to heparin the widely used therapeutic range of 1.5-2.5 (APTT ratios) may not be appropriate for all reagents. The aim of this study was to assess the relationship between 6 different APTT reagents using a manual technique, 2 of these reagents used in combination with a coagulometer, a heparin assay by protamine titration and a chromogenic anti-Xa assay. Samples from 42 patients treated with unfractionated heparin for thromboembolic disease were studied, 12 of whom were receiving warfarin therapy with International Normalised Ratios (INR) of > 1.3. For normal subjects, APTT results were highly dependent on the method used and statistically significant differences were noted. The ratio of patient to mean normal APTT was calculated for each APTT method. When 30 samples from heparinised patients (with INRs of < 1.3) were analysed manually, the APTT ranges equivalent to 0.2-0.4 u/ml heparin by protamine titration (by regression analysis) were 1.6-1.9 for Boehringer reagent (the least responsive) up to 2.2-2.9 for Instrumentation Laboratory reagent (the most responsive). The concentration of heparin associated on average with APTT ratios of 1.5-2.5 varied approximately twofold to threefold between reagents.

A comparison of methods for the measurement of activated factor VII.

A number of different methods are available for the measurement of factor VIIa. Almost all of these employ ratios of two different measurements of factor VII. In order to determine which is the most sensitive to activated factor VII we have compared currently available methods in the following groups: two patients with haemophilia A following treatment with activated recombinant factor VII (rVIIa); 6 normal plasmas during cold promoted activation of factor VII; normal individuals (n = 23); and patients with unequivocal disseminated intravascular coagulation (DIC, n = 19). Factor VII was measured in an amidolytic assay (VII:Amid) and an antigen assay (VII:Ag). Clotting activity was measured using rabbit (VII:C Rab), human (VII:C Hum) and bovine (VII:C Bov) thromboplastin. Of the clotting assays the most sensitive to the presence of factor VIIa was that which utilised bovine thromboplastin. Amidolytic and immunological measurements were unaffected by the activity state of factor VII. The ratios VII:C Rab/VII:Ag and VII:C Rab/VII:Amid were insensitive to activated factor VII. The ratios most sensitive to the presence of factor VIIa were VII:C Bov/VII:Amid and VII:C Bov/VII:Ag. The ratios VII:C Bov/VII:C Rab and VII:C Bov/VII:C Hum are less sensitive but have the advantage for epidemiological studies of narrower reference ranges.

Changes in haematological indices, blood viscosity and inhibitors of coagulation during treatment of endometriosis with danazol.

The effects of treatment with danazol (600 mg daily) on haemostatic and haematological function were investigated in 18 pre-menopausal women with endometriosis. Blood samples were taken at 2 pre-treatment visits, at 6, 12 and 24 weeks on treatment, and at 6 weeks after discontinuation of the drug. Haemoglobin, red cell count, haematocrit and platelet count all rose significantly during treatment with danazol (p < 0.01 vs. baseline). Plasma fibrinogen levels fell significantly (p < 0.01), while whole blood viscosity increased during treatment and remained significantly elevated at follow-up. The prothrombin time shortened, but remained within normal limits, and there were no significant changes in factor VII:C, VIIa, or fibrinopeptide A. No significant changes were found in platelet function. Plasma B-beta 15-42 increased significantly. Functional levels of protein C, protein S, and antithrombin III, all rose significantly, above the normal range, while C4b-binding protein levels fell. We conclude that the observed changes in coagulation inhibitors and fibrinolytic activity may be considered beneficial in the context of venous thromboembolism. The rheological effects, however, indicate a degree of caution in the use of the drug in individuals considered to be at risk from arterial cardiovascular disease.