Standardization of autoimmune testing - is it feasible?

Correct measurement of autoantibodies is essential for the diagnosis of autoimmune diseases. However, due to the variability of autoantibody results and the heterogeneity of testing, wrong diagnosis is a reality. For this and more reasons, harmonization of testing is of the outmost importance. In this review we have summarized the factors contributing to this variability. The ways with which the working group on harmonization of autoantibody testing of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has been trying to tackle the issue with the production and correct use of certified reference materials (CRMs), is discussed. Finally the advantages and the limitations of the use of CRMs are presented

Development of a Certified Reference Material for myeloperoxidase-anti-neutrophil cytoplasmic autoantibodies (MPO-ANCA).

A serum Certified Reference Material (CRM) for supporting reliable autoimmune diagnostics was recently released by the Institute for Reference Materials and Measurements (IRMM) of the Joint Research Centre of the European Commission. It was produced in collaboration with a Working Group on the Harmonisation of Autoimmune Tests of the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC WG-HAT). This material is aimed at facilitating the standardisation of measurements of anti-myeloperoxidase immunoglobulin G antibodies. The CRM could be used as a common calibrant by clinicians and manufacturers thereby significantly improving the comparability of results from commercial immunoassays used for IgG anti-MPO measurements. This paper provides information on the new CRM and its intended use

The certification of anti-myeloperoxidase immunoglobulin G in human serum ERM® - DA476/IFCC

This report describes the production and certification of ERM-DA476/IFCC, a new serum protein reference material intended for the standardisation of measurements of anti-myeloperoxidase immunoglobulin G (anti-MPO IgG) antibodies. The material was produced according to ISO Guide 34:2009. The raw material used to prepare ERM-DA476/IFCC was a plasmapheresis material containing a high concentration of anti-MPO IgG. After a thorough commutability study lyophilised serum was selected as the format for the candidate reference material. Serum processing was performed based on the procedure used for the reference material ERM-DA470k/IFCC. The plasma was converted into serum which was then delipidated. After the addition of preservatives the processed serum was diluted with plasmapheresis solution containing albumin, prior to the transfer of 1 mL aliquots to glass vials. The serum was then lyophilised and the vials closed with rubber stoppers and screw caps under nitrogen atmosphere prior to storage at -70 °C. The between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence and with adherence to ISO/IEC 17025 , using a purified anti-MPO IgG preparation as calibrant. This was achieved using a value transfer protocol previously used in the characterisation of ERM-DA470k/IFCC. Technically invalid results were removed. However no other outliers were eliminated on statistical grounds only. Uncertainties of the certified values were calculated in accordance to the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible lack of homogeneity, instability and characterisation. The material is intended for the calibration of methods and quality control. As any reference material, it can also be used for control charts or validation studies. The CRM is available in glass vials containing the lyophilised residue of 1 g serum. The minimum amount of reconstituted sample to be used is 10 μL. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium

Certified reference material against PR3 ANCA IgG autoantibodies. From development to certification.

Background The importance of the standardisation of immunoassays for autoantibodies has been widely discussed. The appropriate use of certified reference materials (CRM) could contribute to a more accurate diagnosis and follow-up of a series of diseases such as small vessel-associated vasculitis. This is a systemic autoimmune disorder during which two autoantibodies can be present, MPO ANCA IgG and PR3 ANCA IgG. Results from different commercially available immunoassays used for PR3 ANCA IgG measurement can vary significantly. Therefore the potential for improvement using a suitable certified reference material was assessed and led to the development of a CRM. Methods Thirty clinical samples were evaluated using 10 immunoassays. The correlation between results from these assays was assessed in a pairwise manner. Feasibility studies were conducted in order to find a reference material format most suitable for the preparation of a CRM. Results The evaluation of two sets of 30 clinical samples with 10 assays showed that differences between assays can result in different interpretations for individual clinical samples. Most of the samples had the same result classification in all assays. However, six of the samples tested led to inconsistent results. Conclusions The correlation between results from clinical samples was systematically good for combinations of eight of those assays. Therefore, it should be possible to improve the comparability of results using a commutable CRM for calibration. Based on these studies, a final format for the CRM was selected and eventually produced and certified for its PR3 ANCA IgG content

In Vivo Inflammation Does Not Impair ABCA1-Mediated Cholesterol Efflux Capacity of HDL

HDL provides atheroprotection by facilitating cholesterol efflex from lipid-laden macrophages in the vessel wall. In vitro studies have suggested impaired efflux capacity of HDL following inflammatory changes. We assessed the impact of acute severe sepsis and mild chronic inflammatory disease on the efflux capacity of HDL. We hypothesize that a more severe inflammatory state leads to stronger impaired cholesterol efflux capacity. Using lipid-laden THP1 cells and fibroblasts we were able to show that efflux capacity of HDL from both patients with severe sepsis or with Crohn's disease (active or in remission), either isolated using density gradient ultracentrifugation or using apoB precipitation, was not impaired. Yet plasma levels of HDL cholesterol and apoA-I were markedly lower in patients with sepsis. Based on the current observations we conclude that inflammatory disease does not interfere with the capacity of HDL to mediate cholesterol efflux. Our findings do not lend support to the biological relevance of HDL function changes in vitro

Clinical Studies into the Causes of Idiopathic Macular Telangiectasia Type 2: Sleep Apnoea and Macular Telangiectasia: The SAMTel Project

Purpose: To assess the prevalence of Obstructive Sleep Apnoea (OSA) in a population with Macular Telangiectasia Type 2 (MacTel) and how OSA impacts on MacTel progression. Methods: In this case-control study participants completed a questionnaire which incorporated the Berlin Questionnaire (BQ) and questions regarding anthropometric data and medical history. A subset was sequentially selected to undertake overnight sleep analysis using the ResMed ApneaLink™. Using data acquired from the Busselton Population and Medical Research Foundation participants were case-matched based on age, sex and body mass index (BMI) along with, where possible, the presence of hypertension and diabetes. Results: There were 57 (30 ApneaLink) MacTel and 183 controls, respectively. There was no difference in self-reported sleep disordered breathing outcomes between the cohorts using the BQ (p=0.95). Analysis of key indices from ApneaLink recordings found that those with an Apnoea – Hypopnoea Index (AHI) and Oxygen Desaturation Index (ODI) > 5 episodes per hour had a more advanced stage of MacTel (AHI p = 0.05, ODI p = 0.03). 2 year MacTel disease progression rates were unremarkable. Conclusion: Patients with MacTel have a high prevalence of OSA which appears to result in a more advanced form of the disease

Coherent combining of two high-brightness laser diodes phase-locked by a Michelson-type external cavity (Orale)

International audienceWe describe a new coherent beam combin- ing architecture based on the passive phase- locking of two laser diodes in a Michelson external cavity on their rear side, and their coherent combination on their front side

High-power operation of coherently coupled tapered laser diodes in an external cavity

We demonstrate a rear-side phase-locking architecture with two high-brightness diode lasers. This technique is based on the passive phase-locking of emitters in an external cavity on their rear facet, and their coherent combination on the front facet. Two high-brightness high-power tapered laser diodes are coherently combined using a Michelson-based cavity. The combining efficiency is above 80% and results in an output power of 6.7 W in a nearly diffraction-limited beam. The rear-side architecture is then used with a laser bar of 5 tapered emitters using an interferometric extended cavity, based on a diffractive optical element. We describe the experimental evaluation of the diffractive optical element, and the phase-locked operation of the laser bar