CERTIFICATION REPORT: The certification of the catalytic activity concentration of alpha-amylase in ERM® AD456/IFCC

This report describes the production of ERM®-AD456/IFCC, which is a material certified for the catalytic activity concentration of alpha-amylase. This material was produced following ISO 17034:2016 and is certified in accordance with ISO Guide 35:2017. The starting material was pancreatic alpha-amylase purified from human tissue. The enzyme was diluted in a buffered solution which was filled into glass vials and lyophilised. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2017. The material was characterised by an interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025. Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity, instability and characterisation. The material is intended for the assessment of method performance of the primary reference measurement procedure (PRMP) for the catalytic activity concentration of alpha–amylase at 37 °C established by the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) [ , ]. In addition the material can also be used as trueness control or external quality control material for routine measurement systems if commutability has been proven for the assay concerned. As with any reference material, it can be used for establishing control charts or in validation studies. The CRM is available in glass vials containing lyophilised material from 1 mL of alpha-amylase solution which were sealed under an atmosphere of nitrogen. The minimum amount of sample to be used is 5 µL after reconstitution of the whole content of 1 vial in 1 mL.JRC.F.6-Reference Material

CERTIFICATION REPORT: The certification of the mass of lambda DNA in a solution Certified Reference Material: ERM®-AD442k

This report describes the processing and certification of genomic lambda deoxyribonucleic acid (DNA) in a solution. ERM®-AD442k is certified for its lambda DNA mass, expressed in ng/µL. The DNA copy number concentration in cp/µL is provided as an indicative value. The material was produced according to ISO Guide 34:2009. A volume of approximately 400 mL of lambda DNA at an approximate concentration of 450 ng/µL was purchased from Promega Corporation and Benelux BV (Madison, USA and Leiden, NL). After homogenising and diluting this solution with TE buffer, 3100 vials of ERM-AD442k were produced. Each ERM-AD442k vial contains a certified DNA mass concentration of 57.53 ng/µL with an expanded combined uncertainty of 1.07 ng/µL. Using two different next generation sequencing (NGS) techniques (i.e. an Illumina platform and the GS Junior platform from Roche), the nucleic acid sequence of the lambda DNA in ERM-AD442k was verified. Non-lambda DNA sequences were identified in this material. The relative proportion of those sequences was estimated by NGS and further quantified by quantitative polymerase chain reaction (qPCR). Traces of the contaminating DNA, mainly coming from the Escherichia coli (E. coli) host used by Promega Corporation and Benelux BV to produce the lambda DNA, were negligible. Between-vial homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Within-vial homogeneity was quantified to determine the minimum sample intake. The certified DNA mass concentration value was obtained by ultraviolet (UV) spectrophotometry and the indicative DNA copy number concentration value by digital PCR (dPCR). The DNA copy number concentration measured by dPCR is consistent with the DNA mass concentration determined by UV spectrophotometry. The measurements were performed according to the scope of accreditation to ISO/IEC 17025:2005. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence and with adherence to ISO/IEC 17025. Technically invalid results were removed; however no other outliers were eliminated on statistical grounds only. Uncertainties of the certified and indicative values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties relating to possible inhomogeneity and instability, and to characterisation. The material and its certified value are intended to be used for the calibration of DNA quantification methods, quality control and assessment of method performance. As any reference material, it can also be used to establish control charts or in validation studies. The indicative value of the material is, in contrast to the certified value, a value where the uncertainty was deemed too large to allow certification and is therefore less reliable than the certified value. The CRM is available in Axygen maximum recovery polypropylene vial containing a nominal volume of 1.1 mL lambda DNA in solution. The minimum amount of sample to be used is 50 µL for UV spectrophotometry and 68 µL for dPCR. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

Crystal structure of Bacillus subtilis TrmB, the tRNA (m(7)G46) methyltransferase

The structure of Bacillus subtilis TrmB (BsTrmB), the tRNA (m(7)G46) methyltransferase, was determined at a resolution of 2.1 Å. This is the first structure of a member of the TrmB family to be determined by X-ray crystallography. It reveals a unique variant of the Rossmann-fold methyltransferase (RFM) structure, with the N-terminal helix folded on the opposite site of the catalytic domain. The architecture of the active site and a computational docking model of BsTrmB in complex with the methyl group donor S-adenosyl-l-methionine and the tRNA substrate provide an explanation for results from mutagenesis studies of an orthologous enzyme from Escherichia coli (EcTrmB). However, unlike EcTrmB, BsTrmB is shown here to be dimeric both in the crystal and in solution. The dimer interface has a hydrophobic core and buries a potassium ion and five water molecules. The evolutionary analysis of the putative interface residues in the TrmB family suggests that homodimerization may be a specific feature of TrmBs from Bacilli, which may represent an early stage of evolution to an obligatory dimer

Certification of Cystatin C in the Human Serum Reference Material ERM-DA471/IFCC - Certified Reference Material ERM®-DA471/IFCC

The production of ERM-DA471/IFCC, certified for the mass concentration of cystatin C, is described. Serum was produced from blood collected in 2 collection centres according to a procedure ensuring that it was obtained from healthy donors, and that the lipid content of the serum was low. The serum was processed, spiked with recombinant cystatin C, and lyophilised. It was verified that the material is homogenous and stable. The material was characterised using a pure protein primary reference preparation (PRP) as calibrant. The PRP was prepared from recombinant cystatin C, and its concentration determined by dry mass determination. The characterisation of ERM-DA471/IFCC was performed by particle enhanced immuno-nephelometry, particle enhanced immuno-turbidimetry and enzyme amplified single radial immuno-diffusion. The certified cystatin C mass concentration in ERM-DA471/IFCC, if reconstituted according to the specified procedure, is 5.48 mg/L, the expanded uncertainty (k = 2) is 0.15 mg/L.JRC.DG.D.2-Reference material

The certification of the mass concentration of immunoglobulin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA) in human serum: ERM® - DA483/IFCC

This report describes the production and certification of ERM-DA483/IFCC, a serum protein reference material intended for the standardisation of measurements of immunoglobulin G proteinase 3 anti-neutrophil cytoplasmic autoantibodies (IgG PR3 ANCA). The material was produced according to ISO Guide 34:2009 and is certified in accordance with ISO Guide 35:2006. The raw material used to prepare ERM-DA483/IFCC was a plasmapheresis material containing a high concentration of IgG PR3 ANCA. After a prior commutability study lyophilised serum was selected as the best format for the reference material. The processing of the serum was 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 buffer containing albumin, prior to the transfer of 1 mL aliquots to glass vials. The serum was then lyophilised and the vials were closed with rubber stoppers and screw caps under argon atmosphere prior to storage at -70 °C. The between unit-homogeneity was quantified and stability during dispatch and storage was assessed in accordance with ISO Guide 35:2006. The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence, using a purified IgG PR3 ANCA preparation as calibrant. This was achieved by applying a value transfer protocol previously used in the characterisation of ERM-DA470k/IFCC. Technically invalid results were removed, but no outliers were eliminated on statistical grounds alone. The uncertainty of the certified value was estimated in accordance to the Guide to the Expression of Uncertainty in Measurement (GUM) and included components relating to possible lack of homogeneity, stability and the property value measured during characterisation. The main purpose of this material is to be used for the calibration of immunoassay-based in vitro diagnostic devices or control products for IgG PR3 ANCA measurements. As any reference material, it can also be used for control charts or validation studies. The CRM is available in glass vials containing approximately 0.1 g of dried powder. The minimum sample intake to be used after reconstitution of the material is 5 μL. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium

Certification of C-reactive Protein in Reference Material ERM-DA472/IFCC

The production and certification of ERM-DA472/IFCC, a new reference material certified for C-reactive protein (CRP), is described. ERM-DA472/IFCC was characterised using the reference material ERM-DA470 as calibrant. This achieved using a value transfer protocol that can be considered as a reference procedure. the principles used to measure the CRP concentration were immunonephelometry and immunoturbidimetry. The measurements were performed with different platform/reagent combinations (Abbott, Beckmann Immage, BN II, different Hitachi instruments, and Olympus AU640). In total 8 laboratories participated in the value assignment. The certified CRP mass concentration is 41.8 mg/L, the expanded uncertainty (k = 2) 2.5 mg/L.JRC.D.2-Reference material

The certification of mass concentration of Beta-2-microglobulin in human serum: ERM-DA470k/IFCC

This report describes the additional certification of the mass concentration of Beta-2-microglobulin (B2M) in ERM-DA470k/IFCC, a human serum material. The material was certified following ISO Guide 34:2009. The material was released in 2008 and was certified for the mass concentration of 12 proteins in human serum. A full description of the processing steps can be found in the original report. Between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006. Within-unit homogeneity was estimated to determine the minimum sample intake. The material was characterised by an intercomparison among laboratories of demonstrated competence and adhering to ISO/IEC 17025. Uncertainties of the certified values were calculated in compliance with the Guide to the Expression of Uncertainty in Measurement (GUM) and include uncertainties related to possible inhomogeneity, to instability and to characterisation. The material is intended for the calibration of immunoassay-based in-vitro diagnostic devices or control products for the proteins certified. As for any calibrator it should be verified that it is commutable. The material is produced in a similar manner as ERM-DA470, the use of which has led to a significant reduction in the between-method and between-laboratory variation for the proteins certified (B2M was not certified in this material) [ , ]. It was verified during the value assignment procedure that there were no significant matrix effects, and that different methods produced consistent results. However, when the material is used as a calibrator, the commutability should be verified for the particular assay concerned. The Certified Reference Material (CRM) is available in the lyophilised form of a 1.0 mL portion of serum with additives (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES), sodium azide, benzamidine hydrochloride, sodium chloride and aprotinin). The material is kept under nitrogen gas in threaded glass bottles with rubber stoppers and polypropylene screw caps. The water mass fraction of the sample is (4.3 ± 0.6) mg/g. The lyophilised powder has to be reconstituted with (1.00 ± 0.01) g of distilled water. The minimum amount of reconstituted sample to be used is 2 µL. The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.JRC.D.2-Standards for Innovation and sustainable Developmen

CERTIFICATION REPORT: The certification of Amyloid β1-42 in CSF in ERM®-DA480/IFCC, ERM®-DA481/IFCC and ERM®-DA482/IFCC

This report describes the production of ERM®-DA480/IFCC, ERM®-DA481/IFCC and ERM®-DA482/IFCC, which are human cerebrospinal fluid (CSF) materials certified for the mass concentration of amyloid β1-42 peptide (Aβ1-42). These materials were produced by the European Commission, Joint Research Centre (EC-JRC) in collaboration with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) following ISO Guide 34:2009 and are certified in accordance with ISO Guide 35:2006. The starting material used to prepare ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC was human CSF collected from normal pressure hydrocephalus patients by continuous lumbar drainage. After collection, the CSF was aliquoted and frozen at -80 °C. For the preparation of each certified reference material (CRM) a selected number of CSF donations were thawed, pooled, mixed, filled in microvials and stored at (-70 ± 10) °C immediately thereafter. 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 interlaboratory comparison of laboratories of demonstrated competence and adhering to ISO/IEC 17025 [ ]. Technically invalid results were removed but no outlier was eliminated on statistical grounds only. Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) [ ] and include uncertainties related to possible inhomogeneity, instability and characterisation. The materials are intended for the calibration of methods, quality control and/or the assessment of method performance. As with any reference material, they can be used for establishing control charts or validation studies. The CRMs are available in microvials containing at least 0.5 mL of frozen liquid. The minimum amount of sample to be used is 15 µL.JRC.F.6-Reference Material

To harmonize and standardize: making measurement results comparable

Clinical chemistry is a field where the reliability of measurement results has a major impact on decisions affecting human beings directly. The conditions under which the measurements are performed and the way their results are used bring about very stringent requirements on the methods. Measurements are expected to give reliable results after a single measurement performed directly in complex matrixes like serum or urine, and data should preferably be available within minutes or a few hours. The results need to be reliable (i.e. accurate) enough to be able to compare them within a laboratory with reference ranges and cut-off points. The main criterion however is that the measurement target is a clinically significant parameter. These requirements can currently met for hundreds of analytes.JRC.D.2-Standards for Innovation and sustainable Developmen