CERTIFICATION REPORT Catalytic concentration of alanine aminotransferase (ALAT) determined by IFCC method at 37 C Certified Reference Material ERM-AD454/IFCC

Report EUR 19579 EN

The mission of IRMM is to promote a common and reliable European measurement system in support of EU policies.

European Commission Directorate-General Joint Research Centre Institute for Reference Materials and Measurements Contact information Hendrik Emons European Commission Directorate-General Joint Research Centre Institute for Reference Materials and Measurements Retieseweg 111 B-2440 Geel Belgium Email: hendrik.emons@cec.eu.int Tel.: +32 (0)14 571 722 Fax: +32 (0)14 590 406 http://www.erm-crm.org Legal Notice Neither the European Commission nor any person acting on behalf of the Commission is responsible for the use which might be made of the following information. A great deal of additional information on the European Union is available on the Internet. It can be accessed through the Europa server http://europa.eu.int EUR Report 19579 Luxembourg: Office for Official Publications of the European Communities ISBN 92-828-9685-4 European Communities, 2000 Reproduction is authorised provided the source is acknowledged

Printed in Belgium

CERTIFICATION REPORT Catalytic concentration of alanine aminotransferase (ALAT) determined by IFCC method at 37 C Certified Reference Material ERM-AD454/IFCC

T. Linsinger, N. Kristiansen, H. Schimmel, J. Pauwels

European Commission, Joint Research Centre Institute for Reference Materials and Measurements Geel, Belgium

L. Siekmann
Deutsche Gesellschaft fr Klinische Chemie e.V. Reference Institute of Bioanalysis Bonn, Germany

T. Linsinger, N. Kristiansen, H. Schimmel, J. Pauwels, L. Siekmann, F. Ceriotti, G. Ferard, F.H. Franck, J. Gella, W; Hlzel, P. Jrgensen, T. Kanno, A. Kessner, M.M. Mller*, M. Panteghini, F. Schiele, G. Schumann, G. Weidemann, K. Yoshida
International Federation of Clinical Chemistry Laboratory Medicine, on behalf of the working group for calibrators in clinical enymology *President of the IFCC

Report EUR 19579 EN

SUMMARY The Institute for Reference Materials and Measurements and the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) have characterised and certified a reference material (CRM) under the name and number ERM-AD454/IFCC. Reference Material ERM-AD454/IFCC was originally certified as IRMM/IFCC-454. This report describes the certification of the catalytic concentration of alanine aminotransferase (ALAT) in a purified lyophilised material of porcine origin (heart) when measured by the IFCC recommended method at 37 C. The catalytic concentration of ALAT in reconstituted material is certified to 186 4 U/L or 3.09 0.07 kat/L. It is the intention that the reference material should be used to control and optimise the performance of enzyme measurements, verify the comparability of results from different laboratories and be used as a reference material for manufacturers of reagents and diagnostic kits.

TABLE OF CONTENTS
SUMMARY...1 TABLE OF CONTENTS..3 GLOSSARY..4 1 INTRODUCTION ....................................................................................................................................5 2 PARTICIPATING LABORATORIES .....................................................................................................6 3 PREPARATION OF THE MATERIAL...................................................................................................7 3.1 3.2 3.3 3.4 3.5 CHOICE OF THE SOURCE OF THE ENZYME ...........................................................................7 EXTRACTION AND PURIFICATION OF THE ENZYME..........................................................7 CHOICE AND ADDITION OF THE MATRIX FOLLOWED BY LYOPHILISATION ..............7 PRODUCTION CONTROL ............................................................................................................7 COMMUTABILITY ........................................................................................................................8

4 CERTIFICATION MEASUREMENTS...................................................................................................9 4.1 CERTIFICATION PROCEDURE ...................................................................................................9 4.2 QUALITY ASSURANCE................................................................................................................9 4.3 COMPONENTS OF CRM UNCERTAINTY..................................................................................9 5 RESULTS .................................................................................................................................................10 5.1 5.2 5.3 5.4 CERTIFICATION COLLABORATIVE STUDY ............................................................................10 STABILITY .....................................................................................................................................11 HOMOGENEITY.............................................................................................................................11 ESTIMATION OF THE COMBINED UNCERTAINTY ...............................................................11

6 CERTIFIED VALUE................................................................................................................................13 7 USE OF THE CRM ..................................................................................................................................14 7.1 DISPATCH AND INSTRUCTIONS FOR USE..............................................................................14 7.2 RECONSTITUTION OF THE MATERIAL ...................................................................................14 7.3 INTENDED USE .............................................................................................................................14 8 REFERENCES .........................................................................................................................................15

GLOSSARY ALAT ANOVA ASAT BCR CRM ERM GUM IFCC IRMM ISO LD MSB MSW NAD+ NADH RSD SI SOP WG-CCE UCRM uchar sbetw ubb swb sbb ults usts alanine aminotransferase analysis of variance aspartate aminotransferase Community Bureau of Reference certified reference material trademark European Reference Material Guide to the Expression of the Uncertainty in Measurement International Federation of Clinical Chemistry and Laboratory Medicine Institute for Reference Materials and Measurements International Organisation for Standardisation lactate dehydrogenase mean square between groups (ANOVA) mean square within groups (ANOVA) nicotinamide adenine dinucleotide, oxidised form nicotinamide adenine dinucleotide, reduced form relative standard deviation international system of units standard operating procedure working group for calibrators in clinical enzymology expanded (k=2) combined uncertainty of the CRM standard uncertainty of the characterisation standard deviation between certification laboratories (ANOVA) standard uncertainty of homogeneity standard deviation within groups from the hom. study (ANOVA) standard deviation between groups from the hom. study (ANOVA) standard uncertainty of stability during storage standard uncertainty of stability during transport

DEFINITIONS Catalytic activity (z) of an enzyme is a property quantified by the catalysed rate of conversion of a specified chemical reaction, produced in a specified assay system. Unit of catalytic activity: katal (kat) = mole per second (mol/s). For any measurement procedure 1 U = 1 mol/min = 16.67 nmol/s = 16.67 nkat. Catalytic (activity) concentration (b) is the catalytic activity of the component (enzyme) divided by the volume of the original system containing the enzyme (not the assay system). Unit of catalytic (activity) concentration: 1 kat/L = 103 mol . s-1 . m-3, 1 U/L = 16.67 nkat/L.

1 INTRODUCTION Alanine aminotransferase (L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2., ALAT) is frequently determined in clinical laboratories. ALAT is mainly located in the liver and with lesser amounts in kidney, skeletal muscle and heart. Its predominance in the liver and its localisation in the cytoplasm account for its relative specificity in the diagnosis of hepatic disorders. The enzyme is normally present in human serum (or plasma), its catalytic concentration increasing in cases of hepatic injuries. During the symptomatic stage of acute viral hepatitis, ALAT is the first enzyme whose catalytic concentration is increased in plasma. In chronic hepatitis, serum ALAT catalytic concentration increases and is correlated with the progression of the disease. The catalytic concentration is in this case lower than when compared with acute hepatitis. ALAT may be elevated during the course of alcoholic and primary biliary cirrhosis and in cases of non-hepatobiliary diseases, e.g. infectious mononucleosis and parasitosis. In all these cases, the increase in enzyme catalytic concentration is moderate. ALAT is also very often determined together with aspartate aminotransferase (ASAT) to reveal toxic effects of drugs on the liver in clinical trials and/or during drug therapy. Determination of catalytic ALAT concentration is also used in blood banks to detect samples potentially infected with non A-, non B- and non C- hepatitis virus. The Community Bureau of Reference (BCR) of the European Commission has in the past co-ordinated the preparation and certification of enzyme reference materials namely gamma-glutamyltransferase (BCR 319), alkaline phosphatase (BCR 371), creatine kinase BB (BCR 299), alanine aminotransferase (ALAT, BCR 426), prostatic acid phosphatase (BCR 410) and lactate dehydrogenase isoenzyme 1 (LD1, BCR 404), creatine kinase MB (BCR 608) and pancreatic -amylase (BCR 476) [1-8]. With the exception of -amylase, these enzymes have been certified at 30 C. The continuous development of new methods by manufacturers based on different substrates, temperature, pH, cofactors etc. causes difficulties related to interpretation of interlaboratory results. On the 1998 IFCC General Conference in Seville, the working group for calibrators in clinical enzymology (WG-CCE) and the manufactures of diagnostic enzyme kits agreed to change the measurement temperature from 30 C to 37 C. This has resulted in a co-operation project between the Institute for Reference Materials and Measurements (IRMM), IFCC and diagnostic companies with the objective to prepare new standard operating procedures (SOPs) and recertify amongst others- the material for ALAT. The new IFCC recommended method for ALAT is optimised concerning both kinetic reactions and technical aspects of the measurements. Recent regulatory initiatives [9, 10] will have a major impact on the design of test systems, including those for enzyme measurements. European legislation foresees that manufacturers include in the technical documentation of their test systems adequate performance evaluation data showing the performances claimed by the manufacturer and supported by a reference measurement system with information on the reference methods, the reference materials, the known reference values, the accuracy and measurement units used [9]. Also, EN 45001 (to be substituted by the ISO/DIS 17025), used in several countries for accreditation of testing laboratories, requires demonstration of traceability of results: the overall programme of calibration of equipment shall be designed and operated so as to ensure that wherever applicable measurements made in the testing laboratory are traceable to national and international standards of measurement where available [10].

2 PARTICIPATING LABORATORIES Purification of the enzyme Centre du Mdecine Prventive, Vanduvre ls Nancy (FR) Lyophilisation and ampouling National Institute for Biological Standards and Control, Potters Bar (UK) Homogeneity and stability studies Centre du Mdecine Prventive, Vanduvre ls Nancy (FR) Certification exercise Ceriotti F, Laboratorio Centrale, Instituto Scientifico San Raffaele (IT) Ferard G, Centre Traumatologie et Orthopedie, Illkirch Grafenstaden (FR) Franck F.H, Department of Clinical Chemistry, Ziekenhuis Leyenburg, Den Haag (NL) Gella J, Biosystems S.A., Barcelona (ES) Hlzel W, Roche Diagnostics, Tutzing (DE) Jrgensen P, Department of Clinical Chemistry, Odense University Hospital, Odense (DK) Kanno T, Clinical Laboratories, Hamasatu University Hospital, Hamasatu City (JP) Kessner A, Beckman-Coulter, Inc., Brea, California (US) Panteghini M, Laboratory of Clinical Chemistry, Spedali Civili, Brescia (IT) Schiele F, Centre du Mdecine Prventive, Vanduvre ls Nancy (FR) Schumann G, Institut fr Klinische Chemie, Medizinische Hochschule, Hannover (DE) Weidemann G, Institut fr Klinische Chemie und Labormedizin, Klinikum der Stadt Nrnberg, Nrnberg (DE) Yoshida K, Health Care Business Administration, Asahi Chemical Industry Co., Tokyo (JP) Evaluation Reference Material Unit, Institute for Reference Material and Measurements (IRMM), Joint Research Centre, Geel (BE) Project management The ERM-AD454/IFCC 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 fr klinische Chemie, DGKC), Reference Institute of Bioanalysis (DE) and the IRMM (BE).

3 PREPARATION OF THE MATERIAL The preparation of the lyophilised material was performed in 1990 and is described in detail in the previous certification report [4]. However, a short summary of the main steps involved in the purification and characterisation of the enzyme that were performed in the beginning the nineties, is given below. 3.1 Choice of the source of the enzyme Use of material of animal origin can be equivalent to the material of human origin -provided that the source is carefully selected- and avoids ethical problems and minimises dangers of transmissible diseases. ALAT exists in two molecular forms in human and animal tissues, one cytoplasmic, the other mitochondrial. However, almost all ALAT present in human serum is of cytoplasmic origin. The mitochondrial isoenzyme is abundant in pig kidney while the cytoplasmic isoenzyme represents the major part of the enzyme in liver and heart [15]. In addition, pig heart ALAT is known to have catalytic properties very similar to those of the human serum ALAT [16]. The catalytic properties of the partially purified ALAT from pig heart were compared with those of ALAT present in human serum and were found to be close to those of the human serum enzyme with regard to catalytic characteristics such as Michaelis constant Km for each of its two substrates (L-alanine and 2-oxoglutarate) and for optimum pH. The degree of purity of the enzyme was selected to minimise contamination with possible interfering enzymes, and to meet the requirements of a good stability. 3.2 Extraction and purification of the enzyme The tissue homogenate was heated and centrifuged. The supernatant was precipitated with ammonium sulphate solution. The precipitate was dissolved and purified in a multi-step procedure comprising several chromatographic separations and dialysis steps. 3.3 Choice and addition of the matrix followed by lyophilisation A freeze-dried material was prepared to ensure good long-term stability. A relatively acidic pH value was shown to ensure a good storage stability of creatine kinase that also contains thiol groups [14]. Therefore, a matrix containing per litre: triethanolamine buffer 0.5 mol, pbovine serum albumin 30 g and saccharose 10 g at a pH of 6.4 was selected. 3.4 Production control Contaminating enzymes: The purified preparation was tested for the presence of possible contaminating enzymes. During the preparation, the material was in tested for various contaminating enzymes, namely ASAT, GGT, alkaline phosphatase, acid phosphatase, CK and LD. GGT, LD, alkaline phosphatase and acid phosphatase catalytic concentrations were not detectable. CK concentration was 0.01 of ALAT catalytic concentration. The ASAT catalytic concentration was 0.001 of ALAT catalytic concentration in the final preparation. Purity: The pig heart enzyme was homogeneous on electrophoresis in polyacrylamide gel of mass fraction 0.15. Only one protein band corresponding to the band of ALAT activity was revealed. Water content: Residual water content of the lyophilised material was measured by an automated Karl Fischer method in 10 ampoules. The results confirmed that adequate drying had taken place. 7

Testing of filling procedure: The mass of 34 ampoules of the solution taken at intervals throughout the filling procedure had a mean value of 1.0028 g with a range 1.0023 g to 1.0033 g. There was no evidence of any trend in the variation of mass through the filling procedure.

3.5 Commutability Commutability was tested analysing 49 serum samples and 8 samples of the pig-heart material. Measurements were performed with three different methods at measurements temperatures of 30 and 37 C respectively. From the evaluation of the data it appeared that the differences between the average ratios obtained for the sera and for the ALAT material, were not statistically significant, and would not introduce clinically significant errors if the material was used to transfer values between the methods.

CERTIFICATION MEASUREMENTS

4.1 Certification procedure In advance to the certification campaign, the laboratories volunteered to perform a training exercise by analysing 5 different commercial enzyme solutions. By this exercise it was possible to ensure the validity of the SOP for measuring enzyme as well as the performance of the individual laboratories. Certification was based on the agreement between the results obtained in the different laboratories, all of them using the same SOP to measure the catalytic concentration at 37 C [11]. The SOP contains information about instructions for calibration of pipettes, specifications and instructions for the calibration and verification of apparatus used in measurement, specifications for reagents and procedure for the preparation of solutions, specifications for the measurement conditions for the procedure and calculation of results. The measurement principle is as follows:
ALAT L Alanine + 2 Oxoglutarate Pyruvate + L Glutamate

Pyruvate +

NADH

LD H + Lactate +

NAD +

The change in NADH-concentration is measured photometrically. Each participant received 7 vials of the lyophilised ALAT material together with the samples to be used for internal quality control, the SOP, the reconstitution procedure and data sheets for reporting results and the requested information. Three vials were reconstituted on each of two days, and one measurement of catalytic concentration of ALAT on each vial was performed on the day of reconstitution. The remaining vial was a spare sample. All vials were shipped on dry ice to the participants on March 1999 and were delivered in less than 2 days. 4.2 Quality assurance Traceability of gravimetry, volumetry and thermometry was demonstrated and documented. Traceability of spectrophotometry was assured through potassium dichromate solutions certified by the German Metrology Institute (Physikalisch-Technische Bundesanstalt). 4.3 Components of CRM uncertainty Uncertainty of the CRM was estimated according to the Guide to the Expression of Uncertainty in Measurement (GUM) [12]. For this, estimates of the uncertainty of the characterisation (uchar), homogeneity (ubb) and stability (ults) are required as described by Pauwels et al. [17]. Uncertainty of the characterisation was derived from the certification measurements. An estimation for the inhomogeneity was derived from the homogeneity study of the original material. For assessment of stability, data from the original stability study and results from stability monitoring afterwards were available. The method is described in greater detail elsewhere [13].

RESULTS

5.1 Certification collaborative study On beforehand, it had been decided that an intralaboratory RSD above 2.5 % could not be accepted for the certification measurements. One of the 13 laboratories that participated in the certification exercise did not match his criterion. It was decided to exclude the set of results from this laboratory prior to evaluation. The individual results together with laboratory averages of 6 measurements, standard deviations and relative standard deviation (RSD) from 12 laboratories are given in Table 1 (results rounded to the same digit). The results were obtained from single measurements of each of six vials that were performed in March/April 1999.
Laboratory lab 01 lab 03 lab 04 lab 05 lab 07 lab 08 lab 10 lab 11 lab 13 lab 14 lab 15 lab 16 187.9 182.2 187.7 185.2 181.8 183.8 187.7 184.8 186.1 186.7 189.3 191.1 Experimental results [U/L] 187.29 187.2 184.29 188.3 187.5 183.2 182.2 179.4 180.3 181.0 188.6 190.5 186.7 185.8 187.7 180.7 183.5 186.2 183.9 181.0 183.3 183.4 180.9 182.0 181.9 184.0 182.5 182.0 177.9 179.9 187.2 184.4 185.5 187.8 186.1 185.0 185.4 183.5 184.5 186.0 187.6 186.2 186.7 183.1 183.8 186.6 187.5 184.1 185.3 185.3 188.8 190.7 187.8 190.3 190.8 191.6 189.5 192.8 192.4 191.8 Table 1. Individual results accepted for the certification. Mean [U/L] 187.1 181.4 187.8 183.4 182.2 181.6 186.5 184.9 185.6 185.9 189.6 191.5 Stdev [U/L] 1.5 1.4 1.6 2.2 1.0 2.4 1.4 0.8 1.7 1.2 1.2 1.2 RSD [%] 0.8 0.8 0.9 1.2 0.5 1.3 0.7 0.5 0.9 0.7 0.6 0.6

Estimation of individual uncertainties of the catalytic concentration of ALAT in the lyophilised material was done in compliance with the GUM. The uncertainty from the interlaboratory study is referred to as uchar and consists of the uncertainties obtained from the between-lab reproducibility and within-lab repeatability. The standard error of the mean s ( ) was used as the best estimation of the combined effect of these influences. The total n uchar amounts to 0.49 % (0.91 U/L). An analysis of variance (ANOVA) was performed, the results of which are shown in Table 2. The results show that the variation of laboratory averages can be mainly contributed to differences between the averages while variation within the laboratories is only a minor contribution.
mean of means 185.63 U/L standard deviation between means s 3.15 U/L number of laboratories n 12 standard error of the mean of means 0.49 % ANOVA s within labs 1.53 U/L s between labs 3.09 U/L Table 2. Results of the statistical analysis of accepted results.

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5.2 Stability Only an accelerated stability study had been performed for the previous certification. However, data from stability monitoring organised by the IRMM allowed an assessment of the stability of the CRM. These data are shown in Table 3. source original certified value monitoring after 40 months monitoring after 50 months monitoring after 62 months result [U/L] 129 127 127 127

Table 3. Results of the original certification and later stability checks

As can be seen in Table 3, three times the same result was obtained in the stability monitoring. These results were very close to the certified value. However, calculation the regression line through the four data would result in a predicted loss of 0.027 % per month and an ults of 2.5 % at 6 years. It was felt that this loss was unrealistic and solely based on the bias of the laboratory performing the measurements. On the other hand, omitting the certified value would result in unlimited calculated stability, which is also not realistic. The standard deviation between the four values (= 1 U/L = 0.78 % ) was therefore used as ults. Because no degradation was visible during the shared cost action, only the differences between certified value and the results of the shared cost action remained as uncertainty. 5.3 Homogeneity Data for the assessment of homogeneity were taken from the original certification. 20 ampoules had been analysed in duplicate, both with and without incubation with pyridoxal phosphate. The two data sets were pooled to get a more accurate estimation of ubb. Because of the different averages of the two sets, data had to be normalised to their group average before the ANOVA over the complete set could be performed.

average MSB: MSW: swb: sbb:

1 0.000168 0.000139 1.18 % 0.27 %

Table 4. Results of the ANOVA over the homogeneity study with MSB and MSW as the mean squares between and within groups of the ANOVA, respectively. swb and sbb are the within bottle and between bottle variation, respectively.

This uncertainty was larger then the minimum between bottle uncertainty of 0.25 %, which was estimated according to Linsinger et al. [18] 5.4 Estimation of the combined uncertainty The uncertainty of the CRM can be estimated by summation of the contributions of characterisation, homogeneity and stability [15]. The individual uncertainty components for characterisation, inhomogeneity and instability described in the equation below are added, and multiplied by a coverage factor of 2 to give a combined expanded uncertainty. As uncertainties have the format of standard deviations, addition is done quadratically.
2 2 2 U CRM = k u char + u bb + u lts + u sts

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UCRM ........................... expanded uncertainty of the CRM k ....................................... coverage factor uchar................................ uncertainty of the certified property of the batch ubb................................... between-bottle inhomogeneity ults ................................... uncertainty of long-term stability (storage) usts ...................... uncertainty of stability during transports Transport conditions will be chosen that make the uncertainty of stability during transport negligible. uchar of the batch amounts to 0.49 %. ubb and ults were estimated 0.27 % and 0.78 % respectively. Thus, expanded uncertainty UCRM amounts to

U CRM = 2 *

0.49

+ 0.27

+ 0.78

= 1.92% = 3.57 U/L

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6 CERTIFIED VALUE The certified value and the corresponding expanded uncertainty are given below. Catalytic concentration of ALAT as determined at 37 C as determined by the 37 C IFCC reference method: 186 4 U/L or 3.09 0.07 kat/L The catalytic activity in kat/L was calculated by multiplication of the results in U/L by 0.01667. The certified value is valid until 3/2005. The validity can be extended if further evidence of the stability of the material is obtained.The results of the laboratory averages and the certified value with its expanded uncertainty estimated according to the GUM are given in Figure 2. The figure was obtained by estimating expanded uncertainties for the individual laboratories using the between laboratory standard deviation (sbetw) of the certification exercise from the ANOVA as the best estimate of laboratory bias. Thus,
2 slab ), with slab being the standard 6 deviation of the individual laboratories for the 6 measurements performed. 2 laboratory uncertainty was calculated using ( 2 * sbetw +

200 195 190 conc. [U/l] 185 180 175 170 lab 01 lab 03 lab 04 lab 05 lab 07 lab 08 lab 10 lab 11 lab 13 lab 14 lab 15 lab 16 mean

Figure 1. Laboratory averages, their expanded uncertainty (k=2), the certified value and its expanded uncertainty (k=2). The shaded area corresponds to the certified range. In contrast to previously certified materials for which the 95 % confidence interval of the mean of laboratory means was used as uncertainty, an expanded combined uncertainty according to the GUM was calculated this time. This included also influences of homogeneity and stability. The certified uncertainty of the present certification is therefore not readily comparable with the certified uncertainty of the certification at 30 C.

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USE OF THE CRM

7.1 Dispatch and instructions for use Dispatch to the customer will be done under cooled conditions. Upon receipt by the customer, it is advisable to keep the material at 20 C for long-term storage. Upon arrival, the material can be stored refrigerated (2-8 C) for not longer than 3 months until used. 7.2 1. 2. Reconstitution of the material Take the ampoule out of the freezer and allow reaching room temperature. Tap the vertically positioned ampoule gently to ensure that the lyophilised material is at the bottom of the ampoule. Score the ampoule at the constriction with a sharp file and open, by applying a redhot glass rod to the score for about 1 s, while holding the ampoule almost horizontally to prevent glass from entering the ampoule. Weigh the ampoule with its contents to the nearest 0.1 mg. Reconstitute by slow addition to the sides of the ampoule of (1.00 0.01) ml distilled water (20-22 C) with calibrated volumetric equipment. Note the temperature. Weigh the ampoule after adding the water Seal the ampoule with an inert plastic film, invert several times and mix contents by gentle swirling. Allow to stand at room temperature for 1 hour. During this time, swirl ampoule every 20 minutes Calculate the volume of water at 20 C from the weight of the volume taking into account the temperature-dependent density. The catalytic concentration of ALAT must be measured within 4 hours following the reconstitution.

3.

4. 5.

6. 7.

8. 9.

7.3 Intended use The material is intended to provide, when reconstituted, a solution with a known catalytic concentration of human ALAT 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 ALAT 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 ALAT 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.

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REFERENCES

1. Schiele F, Siest G, Moss DW, Colinet E. The certification of the catalytic concentration of gamma-glutamyltransferase in a reconstituted lyophilized material (CRM 319), 1986; CEC Report EUR 10628 EN. 2. Schiele F, Siest G, Moss DW, Colinet E. The certification of the catalytic concentration of alkaline phosphatase in a reconstituted lyophilized material (CRM 371), 1988; CEC Report EUR 11774 EN. 3. Steghens JP, Mathieu M, Horder M, Moss DW, Colinet E, Profilis C. The certification of the catalytic concentration of creatine kinase BB in a reconstituted lyophilized material (CRM 299),1992; CEC Report EUR 13886 EN. 4. Schiele F, Siest G, Colinet E, Profilis C. The certification of the catalytic concentration of alanine aminotransferase from pig heart in a reconstituted lyophilized material (CRM 426), 1992; CEC Report EUR 14475 EN. 5. Moss DW, Francis JM, Colinet E, Profilis C. The certification of the catalytic concentration of human prostatic acid phosphatase in a reconstituted lyophilized material (CRM 410), 1992; CEC Report EUR 14476 EN. 6. Maire I, Flandrois C, Lahet C, Mathieu M, Colinet E, Profilis C. The certification of the catalytic concentration of lactate dehydrogenase isoenzyme 1 (LD-1) in a reconstituted lyophilized material (CRM 404), 1994; CEC Report EUR 15404 EN. 7. Gella FJ, Frey E, Canalas F, Dirscherl C, Moss DW. The certification of the catalytic concentration of human creatine kinase-2 (CK-MB) in a reconstituted lyophilised material CRM 608, 1999; CEC Report EUR (in press). 8. Gella FJ, Gubern G, Canalas F, Profilis C, Colinet E. The certification of the catalytic concentration of human pancreatic -amylase in a reconstituted lyophilised material (CRM 476), 1995; CEC Report EUR 16476 EN. 9. Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on in vitro diagnostic medical devices. Official Journal of the European Communities 1998 (Dec 7): L 331/1-L 331/37. 10. The joint European Standards Institution. EN 45001. General criteria for the operation of testing laboratories, 1989; Brussels: CEN/CENELEC. 11. IFCC, WG-CCE, Standard operating procedure for the measurement of catalytic concentration of enzymes; alanine aminotransferase [L-alanine: 2-oxoglutarate aminotransferase, EC 2.6.1.2.], Clin Chem Lab Med (in preparation) 12. Guide to the Expression of Uncertainty in Measurement, 1995; ISO, ISBN 92-6710188-9, Geneva, Switzerland. 13. Linsinger T, Pauwels J Schimmel H, Lamberty A, van der Veen A, Schumann G, Siekmann L (2001). A pragmatic approach for the estimation of the uncertainty of CRMs in accordance with the GUM: Application to the Certification of four Enzyme CRMs, Fres. J. Anal. Chem (in preparation) 14. Nealon DA, Pettit SM, Henderson AR. Effect of serum pH on storage stability and reaction lag phase of human creatine kinase isoenzymes. Clin Chem 1980;26:1165-9

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15. Morino Y, Tanase S. Special Aspects of various transaminases. C. Aminotransferases utilizing pyruvate. In: Christen P, Metzler DE eds. Transaminases. New York: J Wiley and Sons,, 1985, pp 384-389 16. Gruber W, Mllering H, Perras L. Isolation, pH optima and apparent Michaelis constants of highly purified enzymes from human and animal sources. J Clin Chem Clin Biochem 15 (1977), 576-573 17. Pauwels J, van der Veen A M H , Lamberty A, Schimmel H. Evaluation of uncertainty of reference materials, Accred Qual Assur 5:95-99 (2000) 18. Linsinger TPJ, Pauwels J, van der Veen AMH, Schimmel H, Lamberty A (2001). Homogeneity and stability of reference materials, Accred Qual Assur (submitted)

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EUR 19579 DG Joint Research Centre, Institute for Reference Materials and Measurements Catalytic concentration of alanine aminotransferase (ALAT) determined by IFCC method at 37 C Certified Reference Material ERM AD454/IFCC Authors: T. Linsinger, N. Kristiansen, H. Schimmel, J. Pauwels, L. Siekmann, F. Ceriotti, G. Ferard, F.H. Franck, J. Gella, W; Hlzel, P. Jrgensen, T. Kanno, A. Kessner, M.M. Mller, M. Panteghini, F. Schiele, G. Schumann, G. Weidemann, K. Yoshida Luxembourg: Office for Official Publications of the European Communities 2005 16 pp. 21.0 x 29.7 cm Scientific and Technical Research series ISBN 92-828-9685-4

Abstract The Institute for Reference Materials and Measurements and the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC) have characterised and certified a reference material (CRM) under the name and number ERMAD454/IFCC. Reference Material ERM-AD454/IFCC was originally certified as IRMM/IFCC-454. This report describes the certification of the catalytic concentration of alanine aminotransferase (ALAT) in a purified lyophilised material of porcine origin (heart) when measured by the IFCC recommended method at 37 C. The catalytic concentration of ALAT in reconstituted material is certified to 186 4 U/L or 3.09 0.07 kat/L. It is the intention that the reference material should be used to control and optimise the performance of enzyme measurements, verify the comparability of results from different laboratories and be used as a reference material for manufacturers of reagents and diagnostic kits.

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The mission of the Joint Research Centre is to provide customer-driven scientific and technical support for the conception, development, implementation and monitoring of European Union policies. As a service of the European Commission, the JRC functions as a reference centre of science and technology for the Community. Close to the policy-making process, it serves the common interest of the Member States, while being independent of special interests, whether private or national.

LA-NA-19579-EN-C