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.

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%).

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.

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.

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.

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.

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.

Patient self-management of oral anticoagulation and external quality assessment procedures.

The role of external quality assessment (EQA) is a contentious issue for patient self-management (PSM) of oral anticoagulation. Patients from general practices in the West Midlands undertaking PSM were recruited to compare efficacy of patients' and health professionals' EQA procedure using the UK National External Quality Assessment Scheme (NEQAS). Patients using Coaguchek (Roche Diagnostics) were trained to perform EQA as part of their PSM training. They undertook PSM for 26 weeks and were asked to perform EQA using material provided by the UK NEQAS twice at home without supervision and twice at the practice with supervision. Patients' results were compared with health care professional users of Coaguchek S. Twenty-three PSM patients were compared with 75 health care professional users of the NEQAS scheme. The PSM group international normalized ratio (INR) percentage time in range was 74%. There was no significant difference in the median results on NEQAS samples obtained by the patients and those obtained by professionals. Three patients were outwith consensus (results > 15% from the median INR) on more than one occasion. Patients were able to perform the EQA tests competently. The data show that good agreement can be achieved between patients analysing the same EQA samples, with coefficients of variation ranging from 22.3% to as low as 5.4%. Further study is required to determine how precision within these EQA schemes relates to the stability of treatment in patients' management of their own anticoagulation.

Rapid reversal of oral anticoagulation with warfarin by a prothrombin complex concentrate (Beriplex): efficacy and safety in 42 patients.

Beriplex, a prothrombin complex concentrate (PCC), was administered to 42 patients requiring immediate reversal of their oral anticoagulant therapy. The dose administered was determined using the pretreatment International Normalized Ratio (INR). Blood samples were obtained before treatment and at 20, 60 and 120 min after treatment. The following investigations were performed on all samples - INR, clotting factors II, VII, IX and X, coagulation inhibitors protein C (PC) and antithrombin (AT), and other markers of disseminated intravascular coagulation, plasma fibrinogen, D-dimer and platelet count. Immediate reversal of the INR, the vitamin K-dependent clotting factors and PC was achieved in virtually all patients. Reduced AT levels were present in 18 patients before treatment. Further slight AT reductions occurred in four patients, but other associated abnormalities of haemostasis were observed in only one of the four patients. One patient with severe peripheral vascular disease, sepsis and renal and cardiac failure died of a thrombotic stroke following leg amputation, 48 h after receiving Beriplex. No other arterial and no venous thromboembolic events occurred within 7 d of treatment. Beriplex is effective in rapidly reversing the anticoagulant effects of warfarin, including PC deficiency, without inducing coagulation activation. Caution should continue to be exercised in the use of these products in patients with disseminated intravascular coagulation, sepsis or liver disease.

A comparison of the efficacy and rate of response to oral and intravenous Vitamin K in reversal of over-anticoagulation with warfarin.

The role of oral Vitamin K administration in the reversal of anticoagulation is not yet clear because of a paucity of data on the early effects of treatment, apparent differences in efficacy between preparations and a lack of data comparing oral with intravenous administration. We have compared the effects on the International Normalized Ratio (INR) and activities of the Vitamin K-dependent clotting factors II, VII, IX and X at 4 h and 24 h after administration of three oral Vitamin K preparations and of intravenous Vitamin K in 64 anticoagulated patients who required non-urgent partial correction of anticoagulation. Our data confirm that correction of anticoagulation is more rapid after intravenous administration of Vitamin K than after oral administration of similar or larger doses. At 24 h, satisfactory correction of INR can be achieved using low-dose Vitamin K given by either the intravenous or oral route. Our data, and that from previous studies, suggest that there may be differences in efficacy between orally administered products. Administration of Vitamin K by either route was accompanied by changes in the activities of the Vitamin K-dependent clotting factors that reflected their respective biological half-lives. In the 24 h after treatment, the relationship between the INR and the individual Vitamin K-dependent clotting factors was similar to that described previously in stable anticoagulated patients. We conclude that the reversal of anticoagulation with warfarin is achieved more rapidly by intravenous administration of Vitamin K. Satisfactory, but slower, reversal of anticoagulation can be effected using oral Vitamin K, but there may be differences in efficacy between the products tested in our study.

The management of von Willebrand's disease-associated gastrointestinal angiodysplasia.

There is a recognized association between von Willebrand's disease and gastrointestinal angiodysplasia. Most previous publications have been reports of the association itself and there is little published on the management and long-term follow-up of affected patients. We report our experience and follow-up of six patients, and review the previous literature.