Calibration of the WHO 5th IS for Blood Coagulation Factor IX, Concentrate and Ph. Eur. Human Coagulation Factor IX Concentrate Biological Reference Preparation Batch 3 and investigation of the suitability of an IS as potency standard for purified full-length recombinant FIX.

A joint World Health Organization (WHO) - European Directorate for the Quality of Medicines & HealthCare (EDQM) study was run to calibrate the WHO 5th International Standard (IS) for Blood Coagulation Factor IX (FIX), Concentrate, and European Pharmacopoeia (Ph. Eur.) Human Coagulation Factor IX concentrate Biological Reference Preparation (BRP) Batch 3. The suitability of the 4th IS as a potency standard for purified full-length recombinant FIX (rFIX) was also investigated. Forty-nine laboratories contributed data for the calibration of 2 plasma-derived FIX candidates, relative to the 4th IS, from clotting and chromogenic assays. The intra-laboratory variability was reasonably low; the inter-laboratory variation was lower for sample B (14/148) than for sample C (14/162). Although there were no discrepancies between clotting and chromogenic assays, a significantly lower potency was obtained for sample C with clotting assays when buffer rather than FIX-deficient plasma was used as pre-diluent. A significant assay discrepancy was observed with estimates for the 4th IS for Blood Coagulation Factors FII, VII, IX, X, Plasma against the 4th IS, resulting in a clotting to chromogenic activity ratio of 1.11. The study also investigated the comparability of the plasma-derived concentrate standard with the rFIX products and considered the establishment of an IS for rFIX. The 3 rFIX products currently licensed were represented in this study. Data from 49 laboratories for 2 rFIX candidates were received, with additional results for another full-length rFIX test sample returned by 6 laboratories. The intra-laboratory variability when the rFIX samples were assayed against the 4th IS was acceptably low. Although the full-length rFIX could be assayed against the plasma-derived 4th IS and provided statistically valid results, there were large discrepancies among the clotting assays using different APTT reagents. The inter-laboratory variability of the chromogenic assays was similarly high. There were also significant clotting and chromogenic assay discrepancies. The data from the present study indicate that a recombinant standard for rFIX products will minimise assay discrepancies and improve inter-laboratory agreement. However, they also underline that the value assignment of the 1st rFIX IS needs careful consideration. The Expert Committee on Biological Standardization (ECBS) of WHO was therefore not requested to consider the establishment of an IS for rFIX. In order to ensure continued harmonised standards, sample B (14/148) was established as the WHO 5th IS for Blood Coagulation Factor IX, Concentrate, and as Ph. Eur. Human Coagulation Factor IX, concentrate BRP Batch 3 with the functional activity of 10.5 IU/ampoule.

Development and verification of an enzyme-linked immunosorbent assay for the quantification of toxoid A and toxoid B from Clostridioides difficile.

Clostridioides difficile (C. difficile) is the most common cause of nosocomial antibiotic associated diarrhoea. The incidence of C. difficile infection (CDI) has been rising worldwide over the last 20 years with consequent rises in morbidity, mortality and healthcare costs, although the incidence has fallen in the UK over the last few years. Confirmation of diagnosis and early intervention are critical to the management of CDI. The standard treatment for CDI is the administration of antibiotics. However, vaccination has been recognized as the most cost-effective treatment for the prevention and possible long-term protection against CDI episode. There are several promising vaccine candidates in various stages of development. Many of these vaccines have displayed good efficacy for CDI under laboratory conditions or in clinical trials. With the emergence of vaccines against C. difficile, here we describe the development and verification of an Enzyme Linked Immunosorbent Assay (ELISA) that can be used for the quality control testing of candidate vaccines against C. difficile through the measurement of vaccine antigen content. Verification of the assay was performed by assessment of specificity, sensitivity, intermediate precision and relative accuracy. The ELISAs were specific for the toxoids being detected and the detection limit of the assay for toxoid A was 4.88 ng/mL and 3.91 ng/mL for toxoid B. The geometric coefficients of variation for intermediate precision did not exceed 25% and relative accuracy was within 77-130%. We therefore conclude that the ELISA described here is sufficiently sensitive, specific, precise and accurate for use for the quality control testing of candidate C. difficile vaccines.

Cytokine release assays for the prediction of therapeutic mAb safety in first-in man trials--Whole blood cytokine release assays are poorly predictive for TGN1412 cytokine storm.

The therapeutic monoclonal antibody (mAb) TGN1412 (anti-CD28 superagonist) caused near-fatal cytokine release syndrome (CRS) in all six volunteers during a phase-I clinical trial. Several cytokine release assays (CRAs) with reported predictivity for TGN1412-induced CRS have since been developed for the preclinical safety testing of new therapeutic mAbs. The whole blood (WB) CRA is the most widely used, but its sensitivity for TGN1412-like cytokine release was recently criticized. In a comparative study, using group size required for 90% power with 5% significance as a measure of sensitivity, we found that WB and 10% (v/v) WB CRAs were the least sensitive for TGN1412 as these required the largest group sizes (n = 52 and 79, respectively). In contrast, the peripheral blood mononuclear cell (PBMC) solid phase (SP) CRA was the most sensitive for TGN1412 as it required the smallest group size (n = 4). Similarly, the PBMC SP CRA was more sensitive than the WB CRA for muromonab-CD3 (anti-CD3) which stimulates TGN1412-like cytokine release (n = 4 and 4519, respectively). Conversely, the WB CRA was far more sensitive than the PBMC SP CRA for alemtuzumab (anti-CD52) which stimulates FcγRI-mediated cytokine release (n = 8 and 180, respectively). Investigation of potential factors contributing to the different sensitivities revealed that removal of red blood cells (RBCs) from WB permitted PBMC-like TGN1412 responses in a SP CRA, which in turn could be inhibited by the addition of the RBC membrane protein glycophorin A (GYPA); this observation likely underlies, at least in part, the poor sensitivity of WB CRA for TGN1412. The use of PBMC SP CRA for the detection of TGN1412-like cytokine release is recommended in conjunction with adequately powered group sizes for dependable preclinical safety testing of new therapeutic mAbs.

Does climate change explain the decline of a trans-Saharan Afro-Palaearctic migrant?

There is an urgent need to understand how climate change will impact on demographic parameters of vulnerable species. Migrants are regarded as particularly vulnerable to climate change; phenological mismatch has resulted in the local decline of one passerine, whilst variations in the survival of others have been related to African weather conditions. However, there have been few demographic studies on trans-Saharan non-passerine migrants, despite these showing stronger declines across Europe than passerines. We therefore analyse the effects of climate on the survival and productivity of common sandpipers Actitis hypoleucos, a declining non-passerine long-distant migrant using 28 years' data from the Peak District, England. Adult survival rates were significantly negatively correlated with winter North Atlantic Oscillation (NAO), being lower when winters were warm and wet in western Europe and cool and dry in northwest Africa. Annual variation in the productivity of the population was positively correlated with June temperature, but not with an index of phenological mismatch. The 59% population decline appears largely to have been driven by reductions in adult survival, with local productivity poorly correlated with subsequent population change, suggesting a low degree of natal philopatry. Winter NAO was not significantly correlated with adult survival rates in a second, Scottish Borders population, studied for 12 years. Variation in climatic conditions alone does not therefore appear to be responsible for common sandpiper declines. Unlike some passerine migrants, there was no evidence for climate-driven reductions in productivity, although the apparent importance of immigration in determining local recruitment complicates the assessment of productivity effects. We suggest that further studies to diagnose common sandpiper declines should focus on changes in the condition of migratory stop-over or wintering locations. Where possible, these analyses should be repeated for other declining migrants.

Arthroplasty of the hip. Leg length is not important.

Discrepancy in leg length after total hip replacement has been associated with patient dissatisfaction. We prospectively studied 200 consecutive patients undergoing unilateral Charnley hip replacements to identify whether there is a demonstrable association between such disparity and postoperative function. Radiological measurements between defined points on the pelvis and femur of the operated hip were compared with the same points on the contralateral joint. A lengthening index was derived and statistical analysis used to compare this with validated functional outcome scores (Harris hip score and the SF36 Health Survey) and patient satisfaction. Our results showed no statistical association between leg-length discrepancy after hip arthroplasty and functional outcome or patient satisfaction.

The large subunit ribosomal RNA genes of Entrophospora infrequens comprise sequences related to two different glomalean families.

• The D2 region of the large subunit (LSU) ribosomal RNA gene of isolates of Entrophospora infrequens from trap cultures, the type fungus for the genus Entrophospora, was investigated for sequence variation. • LSU rRNA genes were amplified using PCR from multiple (50 spores) and six single spore DNA extractions. Recombinant clones (261) from these amplifications were analysed for sequence differences using a combination of PCR-single strand conformational polymorphism (PCR-SSCP) and sequencing. • Single spores of glomalean fungi have been previously shown to contain high levels of ribosomal RNA gene sequence diversity. From single and multiple spore extractions, 64 glomalean sequences were obtained, of which 61 were unique. These were related to two glomalean families: the Glomaceae (41/61) and the Gigasporaceae (20/61). No evidence of Acaulosporaceae-like sequences was found. • Sequences related to both families were found within three single spores. Sequences related only to the Gigasporaceae were found in two single spores. The remaining spore only contained sequences related to the Glomaceae. The multiple spore PCR contained sequences related to both families. • The implications of these results for the current taxonomy of this unculturable species are discussed.

Nutritional differences in patients with proximal femoral fractures.

BACKGROUND: The reason why elderly human hips tend to break in one of two anatomical regions is uncertain. Nutritional factors may affect the site of fracture. OBJECTIVE: To assess possible nutritional differences in patients with proximal femoral fractures. DESIGN: Prospective observational cohort study. SETTING: University teaching hospital. SUBJECTS: 119 consecutive patients over the age of 65 with a hip fracture admitted to the trauma wards in a single centre. METHODS: One researcher measured triceps, biceps and supra-iliac skinfold thickness, and mid upper arm circumference on admission and on the fifth post-operative day. Body mass index was calculated for each patient, and used to classify patients as severely, moderately or mildly malnourished, normal, overweight or obese. Logistic regression was used to determine the influence of various factors on fracture site. RESULTS: According to their body mass index, 31% of patients were classified as malnourished and 11% as severely malnourished. Patients with intracapsular fractures were significantly more malnourished than patients with trochanteric fractures (P < 0.008). Nutritional status was not related to post-operative complications. Ability to weigh a patient on the fifth post-operative day was the single most important prognostic indicator for complications. CONCLUSIONS: Patients with intracapsular fractures are more malnourished. Those with trochanteric fractures tend to be overweight.

Coping with the epidemic. The impact of proximal femoral fractures upon the acute orthopaedic services in northeast Scotland.

Increasing health care costs and an aging population have made hip fractures one of the most pressing concerns for developed nations in the latter half of the twentieth century. Using data collected in 1970 and in 1993, the extent of the hip fracture problem in the northeast of Scotland is assessed. The results demonstrate a 101% increase in the number of patients admitted with hip fractures, and an increase in the proportion of patients aged 75 years and over by 158%. Although there has been no change in the number of acute orthopaedic beds in the region, the increase in patient numbers have been managed by approximately halving the in-patient stay through the provision of rural convalescent/rehabilitation beds. Through this approach considerable savings can be made by the health service without reducing the standard of care that is required by the elderly patient later in the post-operative period. In addition, this approach will help focus local community services upon the long-term placement of the patient, in keeping with current government policy.

Bilateral, asymmetrical congenital dislocation of the radial heads in trisomy 8 syndrome.

Congenital radial head dislocation (CRHD) can occur as an isolated abnormality, as part of an upper-limb anomaly or as a feature of at least fourteen syndromes. The dislocation may be unilateral or bilateral, and rarely can be bilaterally asymmetrical. CRHD is often asymptomatic, and may go undiagnosed and remain undetected until after a radiography has been obtained for an incidental injury. It is therefore important to be able to differentiate congenital from traumatic dislocation of the radial head. We report a mentally retarded female, known to have trisomy 8, who presented with stiffness of her elbow joints and no history of preceding trauma. Radiographs confirmed bilateral asymmetrical radial head dislocation. This combination of anterior and posterior CRHD co-existing in the same patient has not been described previously with trisomy 8 syndrome.

Antibiotic prophylaxis: theory and reality in orthopaedics.

Marked differences were found when prophylactic antibiotic regimens and accuracy of administration were compared in the orthopaedic trauma and elective surgery units of one city. Consultants carrying out elective, primary joint replacements advocated similar antibiotic policies. There was 100% compliance in correct prescription and over 99% in administration of antibiotic doses. An audit of eight surgeons who undertook emergency surgery on adult hip fractures found three different prophylactic antibiotic regimens and that half of the consultants wished no antibiotics to be given. Only 33% of the patients were prescribed the schedule that their consultant wished and there were frequent inaccuracies in dose administration. Of 48 doses prescribed, five were omitted and in addition, four were recorded as having been given although they were unprescribed. Whilst antibiotic prophylaxis is universally accepted and the drugs are prescribed and administered accurately in cases of elective joint arthroplasty, the converse is true for emergency hip surgery.