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Zegers I.,European Commission | Beetham R.,Frenchay Hospital | Keller T.,ACOMED Statistik | Sheldon J.,Protein Reference Unit | And 5 more authors.
Clinical Chemistry | Year: 2013

BACKGROUND: Different methods for ceruloplasmin tend to give different results in external quality assessment schemes. During the production of the certified reference material ERM-DA470k/IFCC discrepant measurement results were also found for ceruloplasmin measured with different methods, and consequently the protein could not be certified in the material. METHODS: We performed a commutability study with 30 serum samples and the reference materials ERMDA470, ERM-DA470k/IFCC, and ERM-DA472/ IFCC, using 6 different methods. Data were analyzed according to the CLSI Guideline C53-A to assess whether the reference materials had the same behavior as the serum samples with respect to measurement results obtained with combinations of the methods used. RESULTS: Measurement results from different methods showed a good linear correlation for the serum samples. ERM-DA470 showed marked noncommutability for certain combinations of methods. ERMDA470k/ IFCC and ERM-DA472/IFCC were commutable for more combinations of methods. The lack of commutability of ERM-DA470 for certain combinations of methods correlates with results from the UK National External Quality Assessment Service showing discrepancies between results from these methods. For serum stored in the presence of sodium azide the results from different methods are essentially equivalent. CONCLUSIONS: Ceruloplasmin in ERM-DA470 is a fully documented example of a situation in which, due to lack of commutability, the use of a common material for calibration did not lead to harmonization. © 2013 American Association for Clinical Chemistry. Source

Meroni P.L.,University of Milan | Biggioggero M.,University of Milan | Pierangeli S.S.,University of Texas Medical Branch | Sheldon J.,Protein Reference Unit | And 2 more authors.
Nature Reviews Rheumatology | Year: 2014

Autoantibody measurement is an excellent tool to confirm the diagnosis of rheumatic autoimmune diseases. Hence, reliability and harmonization of autoantibody testing are essential, but these issues are still a matter of debate. Intrinsic variability in analytes and reagents as well as heterogeneity of the techniques are the main reasons for discrepancies in inter-laboratory variations and reporting of test results. This lack of reliability might be responsible for wrong or missed diagnoses, as well as additional costs due to assay repetition, unnecessary use of confirmatory tests and/or consequent diagnostic investigations. To overcome such issues, the standardization of autoantibody testing requires efforts on all aspects of the assays, including the definition of the analyte, the pre-analytical stages, the calibration method and the reporting of results. As part of such efforts, the availability of suitable reference materials for calibration and quality control would enable the development of a reliable reference system. Strong-positive sera from patients have been used as reference materials in most of the autoantibody assays for rheumatic diseases; however, antigen-affinity-purified immunoglobulin fractions or in some cases reliable monoclonal antibody preparations offer more adequate tools for standardization. Systematic assessments of reference materials are currently underway, and preliminary results appear to be encouraging. © 2014 Macmillan Publishers Limited. All rights reserved. Source

Willis R.,University of Texas Medical Branch | Grossi C.,Experimental Laboratory of Immunological and Rheumatologic Researches | Orietta Borghi M.,Experimental Laboratory of Immunological and Rheumatologic Researches | Orietta Borghi M.,University of Milan | And 5 more authors.
Lupus | Year: 2014

International standards for anti-beta2 glycoprotein I (anti-β2GPI) testing are needed. We evaluated the suitability of polyclonal/monoclonal candidate reference materials (RM) for the assay. IgG/IgM anti-β2GPI were affinity-purified (AP) from high-positive antiphospholipid syndrome sera and IgG from HCAL clone supernatant. Igs were tested for purity by SDS-PAGE, pooled, concentrated, sterile-filtered and the protein concentration determined. One unit was defined as the binding activity of 1-μg/ml of AP anti-β2GPI Ig. IgG/IgM RM were each assigned a unit value using the respective AP material as a calibrator. Polyclonal/monoclonal RM and 30 samples were evaluated for linearity, unit equivalency and commutability. Polyclonal AP material was assigned a value of 100 U IgG and 15-U IgM anti-β2GPI, respectively. IgG-RM had a value of 270 IgG and the IgM-RM of 220.3 IgM anti-β2GPI U. The linearity (R2) of each RM curve for the various assays ranged from 0.96 to 0.99. Commutability samples fit very well within 95% prediction intervals and had excellent correlation when comparing assays. IgG and IgM polyclonal and IgG monoclonal RM displayed excellent linearity and commutability, being good candidates for better standardization of anti-β2GPI immunoassays. © The Author(s) 2014 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav. Source

Ladhani S.,Public Health England | Ladhani S.,St Georges, University of London | Oeser C.,St Georges, University of London | Sheldon J.,Protein Reference Unit | And 3 more authors.
Vaccine | Year: 2011

Immunoglobulin deficiency has been reported in 21% of UK children with Hib vaccine failure but its clinical significance and long-term consequences are not known. This study aimed to estimate the prevalence of immunoglobulin deficiency in children with Hib vaccine failure several years after infection and to determine their risk of recurrent infections. The families of children who developed invasive Hib disease after prior immunisation were identified through national surveillance. A completed questionnaire and blood sample was provided by 170 children at a median of 4 years after infection, equivalent to 1035 child-years of follow-up. Nineteen (11.2%) children had immunoglobulin deficiency, including IgA (n=12), IgM (n=5) and all three immunoglobulin classes (n=2). Immunoglobulin deficiency was associated with younger age (<2 years) at initial Hib disease (12/19 [63.2%] vs. 60/151 [39.7%], P=0.05) and parental reporting of their child receiving >2 antibiotic courses annually in early childhood (11/19 [57.9%] vs. 39/151 [25.8%], P=0.004].). In a logistic regression model, Hib vaccine failure cases that had received multiple antibiotic courses in early childhood were 3.8 times (95% CI, 1.4-10.6; P=0.01) more likely to be immunoglobulin deficient at follow-up than those with fewer or no antibiotic courses. Thus, the prevalence of immunoglobulin deficiency in children with Hib vaccine failure at a median of four years after infection is half that reported at the time of the original infection. A proportion of children with Hib vaccine failure, especially where it occurs at a young age, appear to have a maturational delay in development of normal immunoglobulin concentrations. © 2011 Elsevier Ltd. Source

Sheldon J.,Protein Reference Unit | Dellavance A.,Research and Development Division
Frontiers in Immunology | Year: 2015

Producing robust, certified, traceable reference material for autoantibody testing is a vital element in maintaining the validity of results that are generated in the daily clinical laboratory routine. This is a huge challenge because of the high number of variables involved in the detection and measurement of the autoantibodies. The production of such materials is time consuming and needs rigorous attention to detail; this is best achieved by an overarching independent body who will oversee the process in a "not for profit" manner. Much effort has been made to build international standards for quantitative and qualitative assays based on monoclonal antibodies, obtained from affinity purification and plasmapheresis. The big challenge is to respect individual differences in immune response to the same antigen. A promising ongoing initiative is the construction of pools with monospecific samples from different individuals. © 2015 Sheldon and Dellavance. Source

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