Training manual on GMO quantification: Proper calibration and estimation of measurement uncertainty

The content of this manual is based on the training course that was organised on the premises of the European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (Geel, BE) at the end of 2013. The training manual complements the training course that was intended to improve the quality of measurement results obtained when quantifying genetically modified organisms (GMO) in food and feed. Both, the training course and this manual, were developed in line with the current EU GMO legislation. The manual is addressed to laboratory managers and practitioners in analytical laboratories who perform GM quantification measurements and use reference materials for calibration, quality control and method validation including in-house verification. It is also intended for analysts who need to assess measurement uncertainties as required by (EC) No 1829/2003, (EC) No 619/2011 and ISO/IEC 17025:2005. This training document has been written by JRC-IRMM upon request of the European Union Reference Laboratory for Genetically Modified Food and Feed (EURL-GMFF) to further improve the reporting of National Reference Laboratories (NRLs) nominated under Regulation (EC) No 882/2004 and official GMO control laboratories within the EU. This manual is organised in four chapters covering the proper calibration of PCR methods, the estimation of measurement uncertainty, the establishment of metrological traceability of a measurement result and the way to prove the trueness of measurement results. The training manual is a didactic support of a previous guidance document that outlines issues related to the estimation of measurement uncertainty (MU) in the GMO sector. The training manual is also in line with the European technical guidance document for the flexible scope accreditation of laboratories quantifying GMOs, that is intended for laboratories that are acquiring or are holding a flexible scope of accreditation according to ISO/IEC 17025.JRC.D.2-Standards for Innovation and sustainable Developmen

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

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 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

Assessing the commutability of candidate reference materials for the harmonization of neurofilament light measurements in blood

OBJECTIVES: Neurofilament light chain (NfL) concentration in blood is a biomarker of neuro-axonal injury in the nervous system and there now exist several assays with high enough sensitivity to measure NfL in serum and plasma. There is a need for harmonization with the goal of creating a certified reference material (CRM) for NfL and an early step in such an effort is to determine the best matrix for the CRM. This is done in a commutability study and here the results of the first one for NfL in blood is presented. METHODS: Forty paired individual serum and plasma samples were analyzed for NfL on four different analytical platforms. Neat and differently spiked serum and plasma were evaluated for their suitability as a CRM using the difference in bias approach. RESULTS: The correlation between the different platforms with regards to measured NfL concentrations were very high (Spearman's ρ≥0.96). Samples spiked with cerebrospinal fluid (CSF) showed higher commutability compared to samples spiked with recombinant human NfL protein and serum seems to be a better choice than plasma as the matrix for a CRM. CONCLUSIONS: The results from this first commutability study on NfL in serum/plasma showed that it is feasible to create a CRM for NfL in blood and that spiking should be done using CSF rather than with recombinant human NfL protein

CERTIFICATION REPORT: The certification of electrophoretic mobility/zeta potential of silica particles in aqueous solution: ERM-FD306/SRM 1993

This report describes the production of ERM-FD306/SRM 1993, silica particles suspended in a borate buffer, certified for electrophoretic mobility and zeta potential by electrophoretic light scattering (ELS). This material was produced following ISO 17034:2016 and is certified in accordance with ISO Guide 35:2017. The certified reference material (CRM) was jointly produced by the Directorate F - Health, Consumers and Reference Materials of the European Commission’s Joint Research Centre (JRC) in Geel (Belgium) and the US National Institute of Standards and Technology (NIST), Gaithersburg (USA). The CRM was produced from a buffer-modified and diluted commercial colloidal silica slurry. Between-ampoule homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2017. The minimum sample intake for the ELS method was determined from the results and information provided by the laboratories that participated in the interlaboratory comparison (ILC) exercise. The material was characterised by an interlaboratory comparison between laboratories of demonstrated competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outlier was eliminated solely on statistical grounds. 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 quality control and assessment of method performance. The method-defined certified values are regarded as reliable estimates of the true values and ERM-FD306/SRM 1993 can therefore be used for calibration purposes. The CRM is available in 25 mL pre-scored amber glass ampoules each containing approximately 25 mL of suspension.JRC.F.6-Reference Material

CERTIFICATION REPORT The certification of electrophoretic mobility/zeta potential of silica particles in aqueous solution: ERM-FD305/SRM 1992

This report describes the production of ERM-FD305/SRM 1992, silica particles suspended in a borate buffer, certified for electrophoretic mobility and zeta potential by electrophoretic light scattering (ELS). This material was produced following ISO 17034:2016 and is certified in accordance with ISO Guide 35:2017. The certified reference material (CRM) was jointly produced by the Directorate F - Health, Consumers and Reference Materials of the European Commission’s Joint Research Centre (JRC) in Geel (Belgium) and the US National Institute of Standards and Technology (NIST), Gaithersburg (USA). The CRM was produced from a buffer-modified and diluted commercial colloidal silica slurry. Between-ampoule homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2017. The minimum sample intake for the ELS method was determined from the results and information provided by the laboratories that participated in the interlaboratory comparison (ILC) exercise. The material was characterised by an interlaboratory comparison between laboratories of demonstrated competence and adhering to ISO/IEC 17025. Technically invalid results were removed but no outlier was eliminated solely on statistical grounds. 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 quality control and assessment of method performance. The CRM is available in 5 mL pre-scored amber glass ampoules each containing approximately 5 mL of suspension.JRC.F.6-Reference Material

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

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles

Versailles project on advanced materials and standards (VAMAS) interlaboratory study on measuring the number concentration of colloidal gold nanoparticles

We describe the outcome of a large international interlaboratory study of the measurement of particle number concentration of colloidal nanoparticles, project 10 of the technical working area 34, "Nanoparticle Populations" of the Versailles Project on Advanced Materials and Standards (VAMAS). A total of 50 laboratories delivered results for the number concentration of 30 nm gold colloidal nanoparticles measured using particle tracking analysis (PTA), single particle inductively coupled plasma mass spectrometry (spICP-MS), ultraviolet-visible (UV-Vis) light spectroscopy, centrifugal liquid sedimentation (CLS) and small angle X-ray scattering (SAXS). The study provides quantitative data to evaluate the repeatability of these methods and their reproducibility in the measurement of number concentration of model nanoparticle systems following a common measurement protocol. We find that the population-averaging methods of SAXS, CLS and UV-Vis have high measurement repeatability and reproducibility, with between-labs variability of 2.6%, 11% and 1.4% respectively. However, results may be significantly biased for reasons including inaccurate material properties whose values are used to compute the number concentration. Particle-counting method results are less reproducibile than population-averaging methods, with measured between-labs variability of 68% and 46% for PTA and spICP-MS respectively. This study provides the stakeholder community with important comparative data to underpin measurement reproducibility and method validation for number concentration of nanoparticles