Factor VIII assay variability in postinfusion samples containing full length and B-domain deleted FVIII.

INTRODUCTION: Although the variability in factor VIII (FVIII):C measurement is well recognized, this has not been widely reported for post-FVIII infusion samples. AIM/METHODS: Three samples from haemophilia A patients were distributed in a UK National External Quality Assessment Scheme survey, each after treatment with either ReFacto AF, Kogenate FS or Advate. Fifty-two UK haemophilia centres performed FVIII assays using one-stage (n = 46) and chromogenic (n = 10) assays. Centres calibrated assays with the local plasma standard and with ReFacto AF laboratory standard for the ReFacto AF sample. RESULTS/CONCLUSIONS: Chromogenic assays gave significantly higher results than one-stage assays (P < 0.0001, 32% difference) in the post-Kogenate sample but not in the post-ReFacto AF (11% higher by chromogenic assay, ns) or post-Advate samples (3% lower by chromogenic, ns) when assays were calibrated with plasma standards. Twenty centres used all Instrumentation Laboratory (IL)-activated partial thromboplastin time reagents (Synthasil)/IL deficient plasma/reference plasma) in the one-stage assay and 15 used all Siemens reagents (Actin FS/Siemens deficient plasma/reference plasma); this made a significant difference to results post-ReFacto AF (41% higher by IL reagents, P < 0.0001) and Advate (39% higher by IL reagents, P < 0.0001), but not Kogenate (7% higher by IL, ns) when calibrated with plasma standards. Differences between results obtained with different one-stage assay reagents for monitoring Advate have implications for dosing patients. Furthermore, there was considerable inter-laboratory variation as indicated by CVs in the range 15-26% for chromogenic assay and 12-19% for one-stage assay results. This study suggests that external quality assessment schemes should offer participation in post-FVIII infusion schemes where haemophilic patients are monitored.

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.

Investigation of a prolonged APTT. Different approaches taken by laboratories to achieve the same diagnosis.

INTRODUCTION: The APTT is widely employed as part of a coagulation screening panel, used as a pre-operative assessment of bleeding risk, to detect hereditary and acquired haemostatic defects and to monitor anticoagulant therapy. External quality assessment (EQA) exercises assess laboratory performance of individual tests, but rarely assess the approach to investigation of an abnormal result. METHODS: A multicentre exercise was carried out to investigate the ability of laboratories to identify the cause of a prolonged APTT. A sample was distributed with a request to carry out whichever tests were considered necessary to achieve a probable diagnosis. RESULTS: One hundred and ten centres in the UK NEQAS programme took part, and all 104 centres providing an interpretation correctly identified deficiency of FVIII in the sample. However, of these, 10 centres reported additional defects, including lupus anticoagulant, FIX deficiency, FXII deficiency and a FVIII inhibitor. CONCLUSIONS: A markedly varied approach to investigation of a prolonged APTT was observed, although a lack of clinical information may have contributed to this finding.

Management of patients receiving oral anticoagulants using computer dosing software--does everyone agree? Data from a UK NEQAS (blood coagulation) exercise.

INTRODUCTION: The quality of anticoagulation management is not readily or frequently assessed, particularly between different centres. This study sought to evaluate agreement in oral anticoagulant management decisions between participating centres in UK NEQAS programmes. METHODS: Participants were asked to indicate whether they used computerized dosing support software (CDSS) and to complete a series of questions with respect to anticoagulant management provision. Four clinical scenarios were provided, together with past and current International Normalised Ratio (INR) results. Participants were asked to provide recommendations on the target INR they would assign to the patient, the dose of warfarin and a recall interval. RESULTS: Seven hundred and fifty-nine centres returned results, of which 28% were enrolled in the hospital-based EQA programme, and 72% were participants in the point-of-care testing programme. Six hundred (79%) reported use of CDSS. In one straightforward scenario, there was 99% agreement in dose recommendation. However, for three more complex scenarios, differences were apparent in target INRs employed and both dose and recall recommendations. In some cases, differences related to the software system employed. CONCLUSION: The study emphasizes large variation in the approach to managing these scenarios and warrants further investigation, together with education including promoting national guidelines for the assignment of target ranges.

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.

Platelet function testing: practice among UK National External Quality Assessment Scheme for Blood Coagulation participants, 2006.

AIMS: Platelet function testing forms an important part of the laboratory investigation of a bleeding tendency; however, little standardisation and quality control is available for these tests. A UK National External Quality Assessment Scheme (UK NEQAS) for Blood Coagulation exercise sought to identify current practice among laboratories performing platelet function tests. METHODS: A questionnaire was circulated in March 2006 to establish the current status of platelet function testing practice among participants of UK NEQAS. Participants were asked specifically about practice in bleeding time testing, PFA-100 analyser use, platelet aggregometry methodology and additional tests of platelet function. RESULTS: 169 returned questionnaires revealed that 26 centres used bleeding time, the PFA-100 analyser and platelet aggregometry in their investigations; 13 used bleeding time and the PFA-100 only; 33 used bleeding time and platelet aggregometry; and 23 used the PFA-100 with platelet aggregometry. 58 centres reported that they performed only bleeding times in their investigations, 10 reported use of the PFA-100 only, and 6 reported use of aggregometry only. Marked variability was observed in methodology for each of these tests, and in many cases no form of quality control was employed. CONCLUSIONS: The data confirmed the lack of standardisation in methodology employed in different centres. Updated guidelines and standardisation of platelet function assessment are required to facilitate comparability between centres.

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.

Chlorination for degrading saxitoxins (paralytic shellfish poisons) in water.

Chlorination was investigated as a treatment option for degrading and thus removing saxitoxins (paralytic shellfish poisons, PSPs) produced by cyanobacteria (blue-green algae) from water. It was found to be effective with the order of ease of degradation of the saxitoxins being GTX5 (B1) approximately dcSTX > STX > GTX3 approximately C2 > C1 > GTX2. However the effectiveness of chlorine was pH dependent. Degradation as a function of pH was not linear with the degree of degradation increasing rapidly at around pH 7.5. At pH 9 > 90% removal was possible provided a residual of 0.5 mg l(-1) free chlorine was present after 30 min contact time. The more effective degradation at higher pH was unexpected as chlorine is known to be a weaker oxidant under these conditions. The more effective degradation, then, must be due to the toxins, which are ionisable molecules, being present in a form at higher pH which is more susceptible to oxidation. The feasibility of using chlorine to remove saxitoxins during water treatment will therefore depend strongly on the pH of the water being chlorinated. Degradation may be improved by pH adjustment but may not be a practical solution. Although saxitoxins were degraded in that the parent compounds were not detected by chemical analysis, there is no indication as to the nature of the degradation products. However, acute toxicity as determined by the mouse bioassay was eliminated.

Prescription safety eyewear: impact studies of lens and frame failure.

OBJECTIVES: To determine if a plano lens could be the test lens for all prescription (Rx) lenses and to investigate why Rx lenses pop out of safety eyewear. DESIGN: Plano and Rx polycarbonate lenses (n = 641) with varying thickness and edge geometry, mounted on steel lens holders, and Rx safety eyewear (n = 128) placed on headforms were impacted with test objects of varying diameter and hardness. Impacts were studied with 500 to 2,000 frames-per-second motion analysis. RESULTS: Plano lenses were at least, or more, prone to failure (dislodgment, perforation, shatter, or crack) than -3.00 or +3.00 lenses of the same minimum thickness. More than 40% of safety frames with removable lenses broke or had lenses pop out when impacted with energies expected in industry and sports. CONCLUSIONS: Plano lenses can be used as the test lenses for all Rx lenses made of the same material with the same minimal thickness. The ANSI Z87.1-1989 industrial standard for Rx eyewear is inadequate for sports or other activities with high-impact potential. The best lens-retention system has, as a component, a frame with a bevel perpendicular to a frontal impact force.

Lipid composition of seven APTT reagents in relation to heparin sensitivity.

The phospholipid content of different activated partial thromboplastin time (APTT) reagents was determined and compared to heparin sensitivity. The seven reagents included were those most widely used amongst participants of the U.K. National External Quality Assessment Scheme (NEQAS) at the time of study. Heparin sensitivity was assessed using the APTT ratios obtained by more than 300 NEQAS participants on five plasmas prepared from patients receiving unfractionated heparin. The concentrations of three neutral lipids and six phospholipids present in the seven APTT reagents were determined by high-performance thin-layer chromatography (HPTLC) and densitometry. Both the concentrations and the relative percentages of individual phospholipid components varied markedly between reagents. The total phospholipid concentration included a 12-fold range from 16 to 205 microgram/ml. Phosphatidylserine (PS) was completely lacking from one reagent prepared from vegetable material and ranged from 3 to 22 microgram/ml in the other six reagents containing extracts from animal tissue. The concentration of phosphatidylcholine ranged from 3 to 109 microgram/ml. There was no demonstrable relationship between the concentration of any individual lipid components and heparin sensitivity. However, the relative percentage phospholipid composition was important since a lower % of PS or phosphatidylinositol (PI) correlated with increasing heparin sensitivity.