The Use of Reference Materials in international Reference Measurement Systems and for Comparison of Analytical Data

Transcription

1 The Use of Reference Materials in international Reference Measurement Systems and for Comparison of Analytical Data Hendrik Emons Institute for Reference Materials and Measurements (IRMM) Geel, Belgium

2 Reliable Measurement Results Approach 1: Standardisation harmonized methods PASC CEN EASC AIDMO COPANT ARSO ACCSQ PASC PASC Approach 2: Metrology ultimate analysis Traceability Measurement uncertainty MO/LDE9606B.CDR prescribed methods/procedures sampling, method-defined parameters Legislation prescribed method-performance characteristics SI-traceable parameters required quality assurance?

3 Sampling, Processing Analytical Process & QA Quality assurance Conservation Sample preparation Analyte identification & Quantitative measurement Evaluation Assessment Calibrants RM for qualitative analysis for calibration chemical identity often pure analyte Matrix CRMs Quality control as close as possible to real sample

4 Concepts & Terminology? Reference standard Measurement standard Standard material Calibration material Analytical standard Laboratory standard Reference material Laboratory reference material Proficiency testing material Reference substance Laboratory control material Quality control material

5 additional features: certificate certified value with uncertainty stated traceability The RM Family CRMs with uncertainties of property Calibrant values fit for calibration additional features: property value with uncertainty Certified Reference Material traceability CRMs CALs characteristics: QCMs homogeneity (fit for intended use) stability (fit for intended use) Quality Control Material H. Emons: ACQUAL 10 (2006) 690

6 Metrological Traceability or: traceable to a method 1 kg SI Standard International Standard 1 kg 1 kg National Standard Local Calibration Standard CRM representing a reference quantity or an artefact Local Calibration Standard Balance (Bio)Chemical Analysis Sample Sample

7 Challenges in modern Analysis Definition of the measurand & unit identity 'activity' Stability of 'reactive' molecules Stability of relevant material properties Certification of purity (analyt. characterisation & uncertainty calculation at c 100%) Shortage of reliable analytical methods Lack of common reference systems

8 Reference Measurement Systems Reference Method Reference Laboratories harmonized measurement target e.g., prothrombin time, Kjeldahl nitrogen, TOC Reference Material e.g., ERM-DA470 proteins in human plasma

9 CERTIFICATE OF ANALYSIS ERM - AD452 GAMMA-GLUTAMYLTRANSFERASE Certified value 1 Uncertainty 2 Catalytic concentration in reconstituted material U/L 1.90 µkat/l 2.4 U/L 0.04 µkat/l 1) This value is the unweighted mean of 12 sets of results, idependently obtained from 12 laboratories. It is traceable to the IFCC primary reference method at 37 C. The material must be reconstituted according to the specified procedure (see below). Values were converted from U/L into µkat/l by multiplication with ) Estimated expanded uncertainty U with a coverage factor k=2, corresponding to a level of confidence of about 95 %, as defined in the Guide to the Expression of Uncertainty in Measurement (GUM), ISO, Uncertainty contributions arising from characterisation as well as from homogeneity and stability assessment were taken into consideration. This certificate is valid until 5/2006; this validity may be extended as further evidence of stability becomes available. The minimum amount of sample to be used is 50 µl, after reconstitution according to the instruction the material can be regarded as a homogenous solution. NOTE European Reference Material ERM -AD452 was originally certified as IRMM/IFCC-452. It was produced and certified under the responsibility of the IRMM according to the principles laid down in the technical guidelines of the European Reference Materials co-operation agreement between BAM-IRMM-LGC. Information on these guidelines is available on the Internet ( Accepted as an ERM, Geel, May 2004 Signed: Prof. Dr. Hendrik Emons Unit for Reference Materials EC-DG JRC-IRMM Retieseweg Geel, Belgium

10 DESCRIPTION OF THE SAMPLE Each sample is in lyophilised form and equivalent to about 1 ml of a solution of a partially purified pig kidney gamma-glutamyltransferase. The preparation has been stabilised by incorporation in a matrix of bovine serum albumin at a mass concentration of 60 g/l. No contamination, as assessed by measurements of their catalytic activity, has been detected for the following enzymes: aspartate and alanine aminotransferases, lactate dehydrogenase, leucine arylaminase and alkaline phosphatase. The material is kept under nitrogen gas in sealed glass ampoules. The mass of lyophilised material contained in an ampoule is about 0.06 g with a residual water mass fraction of the sample of below ANALYTICAL METHOD USED FOR CERTIFICATION Catalytic concentration in reconstituted material as determined by the IFCC method at 37 C described in Clinical Chemistry and Laboratory Medicine 40: PARTICIPANTS Centre du Médecine Préventive, Vandœuvre lès Nancy (FR) National Institute for Biological Standards and Control, London (UK) St. Radeboud Ziekenhuis, Nijmegen (NL) Royal Postgraduate Medical School, London (UK) Laboratorio Centrale, Instituto Scientifico San Raffaele (IT) Centre Traumatologie et Orthopedie, Illkirch Grafenstaden (FR) Department of Clinical Chemistry, Ziekenhuis Leyenburg, Den Haag (NL) Biosystems S.A., Barcelona (ES) Roche Diagnostics, Tutzing (DE) Department of Clinical Chemistry, Odense University Hospital, Odense (DK) Clinical Laboratories, Hamasatu University Hospital, Hamasatu City (JP) Beckman-Coulter Inc., Brea, California (US) Laboratory of Clinical Chemistry, Spedali Civili, Brescia (IT) Centre du Médecine Préventive, Vandœuvre lès Nancy (FR) Institut für Klinische Chemie, Medizinische Hochschule, Hannover (DE) Institut für Klinische Chemie und Labormedizin, Klinikum der Stadt Nürnberg, Nürnberg (DE) Health Care Business Administration, Asahi Chemical Industry Co., Tokyo (JP) Institute for Reference Material and Measurements (IRMM), Joint Research Centre, Geel (BE) Project management ERM AD452 was produced and certified in a close co-operation between the above mentioned institutions on behalf of IFCC in the frame of a project on standardisation of enzyme measurement initiated by WG-CCE. The work of certification was co-ordinated by the German Society of Clinical Chemistry (Deutsche Gesellschaft für klinische Chemie, DGKC), Reference Institute of Bioanalysis (DE) and the IRMM (BE). SAFETY INFORMATION Avoid swallowing as well as prolongated and repeated contact with skin. Do not discharge the waste into the drain. INSTRUCTIONS FOR USE The material is intended to provide, when reconstituted, a solution with a known catalytic concentration of human GGT that can be used for intra-laboratory quality control of the measurement procedure and to verify comparability of results from laboratories using this measurement procedure. The certified reference material can also be used for evaluation of in vitro test systems for GGT measurements by method comparison with the 37 C IFCC reference measurement procedure. The material can also be used for the calibration of lower order procedures for measuring GGT activities provided they have the same or similar analytical specificities as the reference measurement procedure used for the certification. Commutability of the results from the undiluted material to dilutions has to be checked thoroughly before using the material at several concentration levels for calibration. Reconstitution of the material 1. Take the ampoule out of the freezer and allow reaching room temperature. 2. Tap the vertically positioned ampoule gently to ensure that the lyophilised material is at the bottom of the ampoule. 3. Score the ampoule at the constriction with a sharp file and open, by applying a red-hot glass rod to the score for about 1 second, while holding the ampoule almost horizontally to prevent glass from entering the ampoule. 4. Weigh the ampoule with its contents to the nearest 0.1 mg 5. Reconstitute by slow addition to the sides of the ampoule of (1.00 ± 0.01) ml distilled water (20-22 ºC) with calibrated volumetric equipment. Note the temperature. 6. Weigh the ampoule after adding the water 7. Seal the ampoule with an inert plastic film, invert several times and mix contents by gentle swirling. Allow to stand at room temperature for 20 min. Swirl ampoule again and let stand for 10 min. Total reconstitution time is approximately 30 min. 8. Calculate the volume of water at 20 ºC from the mass of the volume taking into account the temperaturedependent density. 9. The catalytic concentration of Gamma-glutamyltransferase must be measured within 4 hours following the reconstitution. STORAGE Unopened ampoules should be stored at 20 C. LEGAL NOTICE Neither IRMM, its contractors nor any person acting on their behalf: (a) make any warranty or representation, express or implied, that the use of any information, material, apparatus, method or process disclosed in this document does not infringe any privately owned intellectual property rights; or (b) assume any liability with respect to, or for damages resulting from, the use of any information, material, apparatus, method or process disclosed in this document save for loss or damage arising solely and directly from the negligence of IRMM. NOTE A detailed technical report is available on A paper copy can be obtained from IRMM on explicit request. European Commission Directorate General Joint Research Centre Institute for Reference Materials and Measurements (IRMM) Retieseweg 111, B Geel (Belgium) Telephone: Telefax:

11 Uncertainty of the certified value CRM 2 lts 2 bb U = k u + u + u 2 char Contributions to uncertainty: Long term stability u lts Homogeneity u bb Characterisation u char several measurements needed!!! Check uncertainty information for your CRM!!!

12 Uses of Reference Materials Method development and validation evaluation of trueness uncertainty estimation Calibration Proof of method performance statistical quality control establishing traceable results equipment qualification Proficiency testing training and verification of competence

13 Method Validation / Verification Comparison of result and CRM data 1. Determine difference ( m ) measured vs. certified value m = c m c CRM 2. Determine uncertainty of the cert. value (u CRM ) 3. Determine uncertainty of the measurements (u m ) 4. Combine u CRM and u m to u u = u m u CRM 5. Compare m with 2 * u if m < 2 u no difference

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16 Selecting your required RM metrological level? (A) I matrix k? K eq? Real-world sample (A) II sol, g analytical problem commutability? Analytical procedure what is first? Reference Materials

17 Thank You!!! RM Unit (February 2006)