The UK National External Quality Assessment Scheme for heritable bleeding disorders.

Molecular genetic analysis of families with hemophilia and other heritable bleeding disorders is a frequently requested laboratory investigation. In the United Kingdom, laboratories undertaking genetic testing must participate in a recognized external quality assessment scheme for formal accreditation. The UK National External Quality Assessment Scheme (UK NEQAS) for heritable bleeding disorders was established in its current format in 2003, and currently has 27 registered participants in the United Kingdom, the European Union (EU), and the non-EU countries. Two exercises per annum are circulated to participants comprising either whole blood or DNA isolated from cell lines, and laboratories are allowed 6 weeks to analyze the samples and generate a report. Reports are assessed by a panel comprising clinicians and scientists with expertise in this area. Samples to date have involved analysis of the F8 gene (10 exercises), the F9 gene (4 exercises), and the VWF gene (3 exercises) and have comprised a wide spectrum of mutations representing the routine workload encountered in the molecular genetics laboratory. The majority of laboratories in each exercise passed, but a small number did not and reasons for failing included clerical errors, genotyping inaccuracies, and a failure to correctly interpret data. Overall we have seen an improvement in quality of reports submitted for assessment, with a more concise format that will be of value to referring clinicians and counsellors. Informal feedback from participants has been very positive.

Point of Care INR testing devices: performance of the Roche CoaguChek XS and XS Plus in the UK NEQAS BC external quality assessment programme for healthcare professionals: four years' experience.

BACKGROUND: Vitamin K antagonists have been used for many decades and have been traditionally monitored by the measurement of the International Normalised Ratio (INR) in the laboratory. Introduction of Point of Care (POC) testing devices to measure INR has resulted in many tests being undertaken in primary care. External Quality Assessment (EQA) of these POC devices is recommended to ensure accuracy and reliability of INR results outside a laboratory setting. AIM: To assess the quality of INR results for users of two POC devices (CoaguChek XS and CoaguChek XS Plus) over a four-year period. METHODS: Four surveys (two samples) were sent in each 12-month period. The median INR value of each sample was calculated and the percentage deviation from this median determined. Any results greater than 15% from the median were considered to be outside consensus which indicated a possible problem within the testing system. RESULTS: Variability of INR results in this UK National External Quality Assessment Scheme (NEQAS) programme was comparable to that in the UK NEQAS EQA programme for laboratory INR testing. Occurrence of persistent problems was lower in the POC programme than the laboratory programme. CONCLUSIONS: Utilisation of an EQA programme for POC devices in primary care is feasible and necessary. Our data suggest for those health professionals using EQA, the reliability and accuracy of INR testing matches the quality of laboratory testing.

Corn trypsin inhibitor in fluorogenic thrombin-generation measurements is only necessary at low tissue factor concentrations and influences the relationship between factor VIII coagulant activity and thrombogram parameters.

The fluorogenic calibrated automated thrombin-generation assay is influenced by contact pathway activation in platelet-rich and platelet-poor plasma. This influence lessens with increasing tissue factor (TF) concentrations and is inhibited by corn trypsin inhibitor (CTI). CTI is expensive and at what TF concentration its influence becomes irrelevant is unclear. Spiking of factor VIII (FVIII)-depleted plasma with FVIII, in samples without CTI, shows a plateau of thrombin generation at low normal FVIII levels. Given the association with thrombosis at high levels, a continuing increase in thrombin generation would be expected. We studied the effect of CTI on this relation by spiking experiments up to 4.8 IU/ml at 1 pmol/l TF and compared the influence of CTI at 1 and 5 pmol/l in platelet-poor plasma. CTI significantly influences thrombin generation in platelet-poor plasma at 1 pmol/l TF (difference of means for endogenous thrombin potential of 232.5 nmol/l per min, P<0.0001) and peak of 48 nmol/l (P<0.0001)) but not at 5 pmol/l. Spiking experiments without CTI confirm the hyperbolic relation between FVIII coagulant activity (FVIII:C) and endogenous thrombin potential with a plateau at 0.70-1.40 IU/ml. With CTI, a near-linear response up to 1.0 IU/ml was found with a plateau at 2.4-4.8 IU/ml. For peak thrombin, no plateau was reached with CTI. The present study confirms and extends previous data on CTI and the relationship between FVIII:C and thrombin generation. CTI is not necessary at 5 pmol/l TF, and thrombin generation remains dependent on FVIII:C up to 4.8 IU/ml at 1 pmol/l with CTI. Higher levels than previously thought may be needed to normalize thrombin generation.

Emerging technologies and quality assurance: the United Kingdom National External Quality Assessment Scheme perspective.

The pattern of tests employed and technologies developed within hemostasis laboratories has changed considerably within the last 10 years. These changes have presented challenges to external quality assessment (EQA) providers, including the United Kingdom National External Quality Assessment Scheme (NEQAS). EQA for point-of-care devices used for monitoring oral anticoagulant therapy has focused on provision of suitable material to assess performance of devices designed for capillary blood testing, and on education of a user group not usually trained in laboratory quality control procedures. Development of novel therapeutic agents for hemophilia has presented challenges regarding standardization of assays for monitoring treatment, whereas advances related to laboratory testing and automation have not always been accompanied by improved accuracy and precision. EQA provision has also been shown to be of value in molecular genetic screening tests for thrombophilia, and in highlighting standardization issues related to D-dimer measurement in the exclusion of deep vein thrombosis. The increasing prevalence of screening tests of global hemostasis, such as thrombin generation tests and thromboelastography, presents additional challenges to EQA providers in the attempt to standardize these new and potentially beneficial technologies.

Multilaboratory testing in thrombophilia through the United Kingdom National External Quality Assessment Scheme (Blood Coagulation) Quality Assurance Program.

We describe here results from the United Kingdom National External Quality Assessment Scheme (UK NEQAS) Thrombophilia Screening Program, in which an average of 21% of 280 centers reported an incorrect diagnosis for a series of plasma samples. Three case studies are described, showing causes of error in individual laboratories, related to the source of reference plasma or reagents. Methodological bias is also described. For protein C (PC) assays 18% of centers reported PC deficiency in a patient homozygous for factor V Leiden. Studies in the NEQAS laboratory confirmed the effect of activated protein C resistance (APCR) on clot-based PC activity assays. Differences in results obtained for PS-deficient subjects with different protein S (PS) activity kits are reported; several subjects would be misdiagnosed as normal with one kit if the manufacturer's reported reference range was adopted instead of a locally determined reference range. Antithrombin (AT) assays were shown to vary in their sensitivity to different molecular defects in the antithrombin gene; 77% of centers employing human thrombin-based activity assays reported a normal AT level in a patient with antithrombin Cambridge II. Sensitivity of the APC resistance test in the absence of factor V-deficient plasma was shown to be improved through normalization of results, and errors in the genetic diagnosis of factor V Leiden and the P20210A prothrombin gene mutation are described. Errors in the diagnosis of thrombophilic defects can therefore be identified through participation in EQA programs, and following dissemination of information, improvements in diagnosis can be demonstrated.

Quasi-achromatic laser Doppler anemometry systems based on a diffractive beam splitter.

We propose a new beam-splitter system that makes it possible to use nonstabilized laser diodes for laser Doppler anemometry (LDA) systems by making the system wavelength independent. The beam splitter consists of two linear diffraction gratings that produce two parallel beams with a beam spacing that is wavelength dependent. This ensures passive wavelength compensation for the fringe spacing in the measurement volume. One can choose the distance between the two parallel beams by changing the distance between the two gratings, whereas the distance to the measurement volume can be designed by choice of a condensing lens with the proper focal length. This means that the system can be designed to have a desired fringe spacing in the measurement volume. The gratings are implemented as surface-relief holograms in photoresist, which makes it possible to mass produce the beam-splitter system at low cost through replication of the structure. The method for passive wavelength compensation for the fringe spacing is demonstrated both theoretically and experimentally.

Evaluation of the Roche LightCycler: a simple and rapid method for direct detection of factor V Leiden and prothrombin G20210A genotypes from blood samples without the need for DNA extraction.

The Roche LightCycler is a micro-volume thermocycler that combines extremely rapid polymerase chain reaction with fluorescence resonance energy transfer analysis of amplified products. We have evaluated the use of minimally processed blood samples for detection of two point mutations known to increase the risk of venous thromboembolism. Results from the LightCycler using the supernatant of diluted heated blood were compared with those gained by traditional methods based on polymerase chain reaction and restriction enzyme digestion. For factor V Leiden mutation, there was complete agreement between both methods in detection of wild-type (n = 82), heterozygous (n = 100) and homozygous (n = 18) genotypes. Similarly, the prothrombin G20210A mutation showed complete agreement for wild-type (n = 135), heterozygous (n = 63) and homozygous (n = 2) subjects.

Holographic common-path interferometer for angular displacement measurements with spatial phase stepping and extended measurement range.

A novel technique for extending the unambiguous measurement range for differential measurements of angular deflections is presented. The technique utilizes a common-path interferometer that simultaneously probes the out-of-plane displacement of three points on the object surface. The system is based on a single laser diode, and all the optical functions of the system are implemented in a dedicated holographic optical element (HOE). The HOE automatically provides spatially phase-stepped interference signals for real-time phase measurement. It is therefore not necessary to employ any polarizing optics or active elements to introduce the phase stepping. The common-path scheme combined with the HOE provides a system that is inherently stable, since the HOE operates as both transmitter and receiver in the system. The system is compact, is robust, and has the potential for being mass-produced at a low cost and is thus well suited for industrial use, such as in commercial vibrometers. The technique is demonstrated in a system for measuring angular deflections of a plane mirror. The technique, however, is not restricted to this use alone and can easily be configured to probe other types of surface displacements, e.g., the deflection of a diaphragm. In the present configuration, the system can measure angular deflections with a sensitivity of 2.5 x 10(-7) rad over a measurement range that is approximately 3.5 x 10(-3) rad, i.e., a dynamic range of approximately 1:14,000. Furthermore, the system can easily be reconfigured for a desired angular sensitivity and measurement range.