CERTIFICATION REPORT Certification of the catalytic...

CERTIFICATION REPORT

Certification of the catalytic activity concentration of aspartate transaminase

Certified Reference Material ERM®-AD457/IFCC

EU

R 23808

EN

-2009

The mission of the JRC-IRMM is to promote a common and reliable European measurement system in support of EU policies. European Commission Joint Research Centre Institute for Reference Materials and Measurements Contact information Reference materials sales Retieseweg 111 B-2440 Geel, Belgium E-mail: [email protected] Tel.: +32 (0)14 571 705 Fax: +32 (0)14 590 406 http://irmm.jrc.ec.europa.eu/ http://www.jrc.ec.europa.eu/ 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 this publication.

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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/ JRC 51414 EUR 23808 EN ISBN 978-92-79-12206-4 ISSN 1018-5593DOI 10.2787/10322 Luxembourg: Office for Official Publications of the European Communities © European Communities, 2009 Reproduction is authorised provided the source is acknowledged Printed in Belgium

CERTIFICATION REPORT

Certification of the catalytic activity concentration of aspartate transaminase

Certified Reference Material ERM®-AD457/IFCC

B. Toussaint(1), G. Schumann(2), R. Klauke(2), N. Meeus(1), R. Zeleny(1), S. Trapmann(1), H. Emons(1), H. Schimmel(1)

(1) Institute for Reference Materials and Measurements (IRMM), Joint Research Centre, European Commission, Geel (BE) (2) Institute for Clinical Chemistry, Medical School Hannover, Hannover (DE)

Disclaimer

Certain commercial equipment, instruments, and materials are identified in this report to specify adequately the experimental procedure. In no case does such identification imply recommendation or endorsement by the European Commission, nor does it imply that the

material or equipment is necessarily the best available for the purpose.

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Abstract The production and certification of ERM-AD457/IFCC, a new reference material for the enzyme aspartate transaminase (AST) [L-aspartate: 2-oxoglutarate-aminotransferase, EC 2.6.1.1], also called aspartate aminotransferase (ASAT), is described. The certified reference material is lyophilised and should be reconstituted by addition, gravimetrically controlled, of 2 g highly purified water comparable to bi-distilled water. The certified catalytic activity concentration and certified uncertainty of AST in the reconstituted material are (1.74 ± 0.05) µkat/L or (104.6 ± 2.7) U/L (k=2, obtained with the IFCC reference procedure at 37 °C).

The material was produced from a human type recombinant AST in E. coli and a buffer containing bovine serum albumin.

A first batch of the material was processed, filled into vials (1 mL per vial) and lyophilised. The vials were closed with a stopper and a metal cap. This batch was used as a trial batch to test the lyophilisation process, the homogeneity and the stability of AST in the material. A reconstitution protocol was also developed.

Following the positive results obtained on the trial batch, a second batch was produced in the same way. The homogeneity and the stability of this batch were assessed using an In Vitro Diagnostic assay. Then a feasibility study was carried out to characterise the material using a reference procedure from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Twelve expert laboratories participated in the study and familiarised themselves with the protocol. Finally, the characterisation of the material was performed. A good agreement between the results of the 12 laboratories allowed the value assignment of AST in the material in terms of catalytic activity concentration of AST (U/L or µkat/L) in the reconstituted material.

The material is aiming to control the IFCC reference procedure for AST at 37 °C. It can also be used to calibrate assays if the commutability of the material with patient samples is demonstrated for these assays.

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ABSTRACT ..............................................................................................................................1

GLOSSARY..............................................................................................................................5

1 INTRODUCTION AND DESIGN OF THE PROJECT ....................................................7 1.1 BACKGROUND: NEED FOR THE CRM.............................................................................................................. 7 1.2 CHOICE OF THE MATERIAL ............................................................................................................................. 8 1.3 DESIGN OF THE PROJECT ................................................................................................................................ 8

2 LIST OF PARTICIPANTS .................................................................................................10

3 PROCESSING ....................................................................................................................11 3.1 PROCESSING OF THE MATERIAL.................................................................................................................... 11

3.1.1. Filling ................................................................................................................................................ 11 3.1.2. Lyophilisation .................................................................................................................................. 11 3.1.3. Capping and labelling .................................................................................................................... 11

3.2 PROCESSING CONTROL................................................................................................................................. 11

4 HOMOGENEITY.................................................................................................................12 4.1 HOMOGENEITY ............................................................................................................................................ 12 4.2 MINIMUM SAMPLE INTAKE FOR ANALYSIS ................................................................................................... 13

5 STABILITY ..........................................................................................................................13 5.1 STABILITY OF THE NON-RECONSTITUTED CRM ........................................................................................... 13

5.1.1 Short-term stability .......................................................................................................................... 13 5.1.2 Long-term stability ........................................................................................................................... 14

5.2 STABILITY OF THE RECONSTITUTED CRM ................................................................................................... 16

6 CHARACTERISATION .....................................................................................................16 6.1 PREPARATIONS ............................................................................................................................................ 16 6.2 PERFORMANCE OF THE VALUE ASSIGNMENT MEASUREMENTS ..................................................................... 16 6.3 RECONSTITUTION OF ERM-AD457/IFCC ................................................................................................... 16 6.4 DATA ANALYSIS .......................................................................................................................................... 17 6.5 RESULTS ...................................................................................................................................................... 17

6.5.1 General considerations .................................................................................................................. 17 6.5.2 Control sample................................................................................................................................. 17 6.5.3 ERM-AD457/IFCC........................................................................................................................... 18 6.5.4 Value assignment............................................................................................................................ 19

7 UNCERTAINTY BUDGETS AND CERTIFIED VALUES ............................................20 7.1 ESTIMATION OF THE UNCERTAINTIES........................................................................................................... 20 7.2 CERTIFIED VALUES ...................................................................................................................................... 20

8 METROLOGICAL TRACEABILITY ................................................................................21

9 COMMUTABILITY .............................................................................................................21

10 INTENDED USE AND INSTRUCTIONS FOR USE ...................................................21

REFERENCES AND ACKNOWLEDGEMENTS ..............................................................23 REFERENCES...................................................................................................................................................... 23 ACKNOWLEDGEMENTS ...................................................................................................................................... 23

ANNEX 1 .................................................................................................................................24

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ANNEX 2 .................................................................................................................................25

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GLOSSARY ABTS 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid)

ANOVA analysis of variance

ASAT aspartate aminotransferase

AST aspartate transaminase

b slope in the equation of linear regression y = a + bx

bE catalytic activity concentration

CGPM General Conference on Weights and Measures

CI confidence interval

CRM Certified Reference Material

C-RSE Committee of Reference System for Enzymes

df degrees of freedom

dfwb degrees of freedom within bottle

ERM European Reference Material

f correction factor

F Snedecor F

Fcrit critical value of Snedecor F

IFCC International Federation of Clinical Chemistry and Laboratory Medicine

IRMM Institute for Reference Materials and Measurements

IUB International Union of Biochemistry and Molecular Biology

IUPAC International Federation of Pure and Applied Chemistry

IVD in vitro diagnostic

k coverage factor

m mass of water added in g

MDH malate dehydrogenase

MSbb mean sum of squares between bottles

MSq mean sum of squares

MSwb mean sum of squares within bottles

n number of subsamples analysed

NAD ß-nicotinamide adenine dinucleotide, oxidised form

NADH ß-nicotinamide adenine dinucleotide, reduced form

p number of data sets

r2 determination coefficient of the linear regression

RELA IFCC External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine

RSD relative standard deviation

sbb standard deviation between bottles

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sbb,rel relative standard deviation between bottles

swb standard deviation within bottles

SD standard deviation

SI International System of Units

SS sum of squares

t0.05,df t of Student test at 95 % confidence and df degree of freedom

ub standard uncertainty of the slope

ubb,rel relative standard uncertainty related to the between-bottle heterogeneity

u*bb standard uncertainty related to the between-bottle heterogeneity that can be hidden by the measurement repeatability

u*bb,rel relative standard uncertainty related to the between-bottle heterogeneity that can be hidden by the measurement repeatability

uc combined standard uncertainty

uc,rel relative combined standard uncertainty of the certified value

uchar,rel relative standard uncertainty related to the characterisation

uCRM combined standard uncertainty of the certified value ults,rel relative standard uncertainty related to the long-term stability of the material

um standard uncertainty of the measurement usts,rel relative standard uncertainty related to the short-term stability of the material

UCRM expanded uncertainty of the certified value

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1 Introduction and design of the project

1.1 Background: need for the CRM

Aspartate transaminase (AST) or aspartate aminotransferase (ASAT) is a vitamin B6 dependent enzyme (α2 dimer of about 2 x 400 amino acids) involved in amino acid metabolism. Together with its coenzyme vitamin B6 (pyridoxal 5-phosphate), it catalyzes the reversible transfer of an amino group replacing a keto group [1,2].

The reference interval for AST in serum is 13-37 U/L for women and 14-45 U/L for men, with a 90 % confidence interval [3]. In case of liver damage (cirrhosis, hepatitis), the catalytic activity concentration of AST may rise to 4000 U/L. In acute cases, AST catalytic activity concentration rises in 6 to 10 hours and may remain high for about 4 days. Therefore AST is a biomarker of the liver function.

The standardisation of catalytic activity concentration measurements in general is a very demanding task in clinical chemistry as the catalytic activity of an enzyme is a property measured by the catalyzed rate of a chemical reaction under specific experimental conditions. The measurement result is therefore procedure-dependent and the number of parameters having an influence on the enzyme activity is large and include temperature, pH, substrate nature and concentration, activators and inhibitors.

Therefore, the importance of a single and universally recognised routine procedure was raised in the seventies. In 1986, IFCC published a Primary Reference Measurement Procedure for the measurement of the catalytic activity concentration of AST at 30 °C [4].

However the idea of a unique procedure, based on the IFCC procedure, was not followed in practice due to the difficulty to reach a consensus between professionals on measurement and reaction conditions. Moreover the IFCC procedure for AST was not suitable for routine use because of long reactions times, limited linearity, need for a sample blank and for a temperature set at 30 °C whereas 37 °C was most commonly used in enzymatic reactions. As the use of a common procedure was not feasible, the calibration of routine procedures using validated calibrants, traceable to a reference measurement procedure, seemed to be the best approach [5,6]. Therefore, IFCC published another series of reference procedures for the measurement of catalytic activity concentrations of enzymes at 37 °C (alanine aminotransferase, creatine kinase, lactate dehydrogenase, γ-glutamyltransferase, α-amylase and aspartate aminotransferase). Part 5 describes the reference procedure for aspartate aminotransferase [7]. The reaction principle is illustrated in Figure 1. The procedure is based on the measurement of the temporal change of absorbance caused by NADH in the reaction mixture. The IFCC reference procedure foresees the addition of pyridoxal 5-phosphate, coenzyme of AST, to the reaction mixture.

L-aspartate + 2-oxoglutarate oxalacetate + L-glutamate

oxalacetate + NADH + H+ L-malate+ NAD+

Figure 1: Principle for the measurement of the catalytic activity concentration of AST using the IFCC reference procedure [7].

IRMM, the Institute for Reference Materials and Measurements, has now developed a certified reference material (CRM) which will be used as a quality control material for the IFCC reference procedure at 37 °C. The homogeneity and the stability of the reference material have been demonstrated and the certified value has been assigned using the IFCC

AST+coenzyme

MDH

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reference procedure at 37 °C. The material is certified for its catalytic activity concentration of AST in the reconstituted sample.

Together with the IFCC reference procedure, the new certified reference material is a key component of a Reference System for AST determination. Therefore the release of this material is an important step towards the standardisation of AST measurements and the ability to define common reference intervals for the clinical interpretation of patient data.

1.2 Choice of the material

A preparation from Asahi Kasei Corporation (Japan) was selected as a raw material. The enzyme is a cytosolic isoform and originates from human liver. The preparation contains a recombinant form of AST enzyme produced in E. coli. At Asahi Kasei Corporation, the enzyme was purified by hydrophobic and anion-exchange chromatography. It was then dissolved in a buffer at pH 7.5 containing bovine serum albumin, saccharose, pyridoxal phosphate and antibiotics. The liquid preparation was frozen.

In order to improve the long-term stability of the material, the material was planned to be lyophilised and a lyoprotectant was added by Asahi Kasei Corporation to the buffer.

The catalytic activity concentration was estimated at 205 U/L, lying within the measurement range of the reference measurement procedure.

The purity of the material, used by Asahi Kasei Corporation to make the liquid preparation, was guaranteed as containing no other enzyme with a relative catalytic activity concentration > 1.0 % of the total catalytic activity concentration of the preparation.

1.3 Design of the project

Asahi Kasei Corporation produced 2.5 L of liquid frozen material. This material was dispatched to IRMM which took care of the processing, storage and certification study. The liquid frozen material was stored at -70 °C.

A trial batch was first processed. Glass vials suitable for lyophilisation and for further reconstitution of the material by injection through the septum were selected. A lyophilisation protocol was developed in order to reduce the water mass fraction of the material to less than 1 % mass fraction and the trial batch was lyophilised. This trial batch was used to investigate the homogeneity, the short-term stability and the catalytic activity concentration of the processed material. The trial batch was also tested for water mass fraction, hygroscopicity, glass temperature and accelerated degradation tests. Reconstitution of the lyophilised material was achieved by addition of 1 g of distilled water, gravimetrically controlled.

In parallel, a small batch of frozen material was processed but not lyophilised in order to to compare the catalytic activity of the lyophilised and non lyophilised material. At that stage, IRMM organised a meeting with the members of the IFCC Committee of Reference System for Enzymes (C-RSE) some manufacturers and experts in the field of enzymatic measurements to present the results of the trial batch and to agree on the best approach for the certification of the final batch.

As satisfactory results had been obtained on the trial batch regarding all investigated properties, IRMM processed in the same way a final batch, lyophilised and labelled as ERM-AD457/IFCC. The final batch was tested for homogeneity and short-term stability.

Stability tests were also performed on the material reconstituted with 1 g and 2 g of water and left at ambient temperature 2-6 hours before analysis. Both reconstitution protocols gave satisfactory results. However, according to clinicians, a catalytic activity concentration around 100 U/L is more suitable for the purpose of calibration. Therefore, a reconstitution protocol with 2 g of water was finally selected and was used for value assignment and long-term stability study.

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A feasibility study was first performed for the determination of AST catalytic activity concentration using the IFCC reference procedure at 37 °C [7] in 12 laboratories. The laboratories were provided with two samples from RELA, the IFCC External Quality Assessment Scheme for Reference Laboratories in Laboratory Medicine (trial 2007) as well as with a pool of human serum. The IFCC reference procedure is, like any procedure for enzymatic activity measurement, very sensitive to a series of influence quantities such as pH, temperature, reagents concentration as well as mixing. Any small deviation from the procedure might lead to a bias of the results. The IFCC reference procedure itself did not need to be calibrated as the enzyme activity is defined in terms of response with following this procedure (certified property as defined by the procedure).

Finally, the characterisation of the reference material was performed using the IFCC reference procedure. One control sample (pool of human serum), analysed during the feasibility study, was included in the sequence of analysis. The 12 participating laboratories either performed the measurements under an ISO/IEC 17025 and ISO 15195 accreditation, or within the scope of a quality system. Documented evidence of technical competence was obtained from all laboratories.

ERM-AD457/IFCC is certified for the catalytic activity concentration of AST in the reconstituted material. Therefore, the measurement results were corrected for exactly 2 g of water added for reconstitution (gravimetrically controlled).

Given the large amount of data which were necessary for the design of the project and in order to keep this report concise, the data concerning the trial batch, the development of the lyophilisation program and the feasibility study on the final batch are not shown. This report is focusing on the results from the certification of the final batch.

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2 List of participants Provision of the raw material Asahi Kasei Corporation, Shizuoka (JP) Provision of the pool of human serum Institute for Clinical Chemistry, Medical School Hannover, Hannover (DE)

Provision of the RELA material Deutsche Vereinte Gesellschaft für Klinische Chemie und Laboratoriumsmedizin e.V.

(DGKL), Bonn (DE) Processing of the raw material Institute for Reference Materials and Measurements (IRMM), Joint Research Centre,

European Commission, Geel (BE) Stability and homogeneity studies Institute for Clinical Chemistry, Medical School Hannover, Hannover (DE) (accred. ISO/IEC

17025, Deutscher Akkreditierungs Rat, Deutscher Kalibrierdienst DKD-K-20602) Universitat Autonoma de Barcelona, Departament de Bioquimica i Biologia Molecular, Unitat

de Bioquimica de Medicina, Barcelona (ES) Institute for Reference Materials and Measurements (IRMM), Joint Research Centre,

European Commission, Geel (BE) Characterisation Argentine Biochemical Foundation, Laboratory of Reference and Standardization in Clinical

Biochemistry, La Plata (AR) Asahi Kasei Pharma Corporation, Diagnostics Department, Shizuoka (JP) BioSystems S.A., Barcelona (ES) Centre de Médecine Préventive, Laboratoire de Biologie Clinique, Vandoeuvre Les Nancy

(FR) Diagnostica e Ricerca San Raffaele, Milano (IT) HagaZiekenhuis, Klinisch Chemisch en Hematologisch Laboratorium (KCHL), Den Haag

(NL) Institute for Clinical Chemistry, Medical School Hannover, Hannover (DE) (accred. ISO/IEC

17025 and ISO/IEC 15195, Deutscher Akkreditierungs Rat, Deutscher Kalibrierdienst DKD-K-20602)

Odense University Hospital, Department of Clinical Chemistry, Odense (DK) Roche Diagnostics, Department LR-HA2, Mannheim (DE) Università degli Studi di Milano, Laboratorio Analisi Chimico-Cliniche, Centro

Interdipartimentale di Ricerca sulla Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Milano (IT)

Universitat Autònoma de Barcelona, Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Medicina, Barcelona (ES)

Université Louis Pasteur, Institut Gilbert Laustriat, Ilkirch (FR) Data analysis Institute for Reference Materials and Measurements (IRMM), Joint Research Centre, European Commission, Geel (BE)

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3 Processing

3.1 Processing of the material

One mL of material was filled per vial, aiming at a catalytic activity concentration around 100 U/L after reconstitution by 2 g of distilled water.

3.1.1. Filling

Vials and stoppers were selected allowing lyophilisation and direct reconstitution of the material by injection through the septum.

The filling was achieved in a cleanroom, showing a particularly high quality of air corresponding to ISO level 5 in the classification according to ISO 14644-1 (no particle ≥ 5 µm). The incoming and outgoing air was filtered. Temperature, pressure and humidity were controlled. In the filling process, only sterile consumables and accessories which can be sterilised were used. Sterilisation of the accessories was carried out by autoclaving.

The material was allowed to thaw at 4 °C before it was placed at ambient temperature. The bottle was inverted several times and mixed by gentle swirling. The material was then filled into 3.5 mL glass vials using an automated filling device (Biomek 2000, Beckman Coulter). The labelling order followed the filling sequence.

3.1.2. Lyophilisation

The freeze-drier, also located inside the cleanroom, was cleaned and sterilised before use. Its temperature was set at -20 °C for storage of the vials immediately after filling. Stoppers were entered half way into the vials and the shelves of the freeze-drier were loaded equally.

The lyophilisation program was developed at IRMM and aimed at a residual water mass fraction < 1 % in the lyophilised material in order to avoid microbial growth. The glass temperature of AST in solution was determined and taken into account in the program in order to guarantee the long-term stability of the enzyme after lyophilisation.

After lyophilisation, the vials were filled with argon and closed with a slight underpressure (800 mbar) to avoid any loss of material during reconstitution. The mass fraction of residual water in the lyophilised material was 0.6 %. The catalytic activity concentration of AST after lyophilisation showed a relative decrease of 10.9 % compared to the frozen material. However, the resulting catalytic activity of the material was fitting within the requirements of the clinicians, around 100 U/L after reconstitution with 2 g of water.

3.1.3. Capping and labelling

Vials were sealed with a metal cap with a central hole making the septum accessible for reconstitution. This was achieved on a semi-automatic capping machine. Finally, the vials were labelled on an automated system, in the same order as the filling sequence.

3.2 Processing control The repeatability and the trueness of the filling using Biomek were optimised before processing with water and with a solution of 10 % sucrose (mass fraction), close to the viscosity of the reference material. Repeatability and trueness of filling were then verified during processing on 8 vials of the AST material. The repeatability showed a RSD of 0.3 % and there was no evidence of any trend in the variation of mass trough the filling procedure. The trueness was estimated at 98 % (volume fraction), determined using the density of a solution of sucrose 10 % (mass fraction) for calculation. This result is very satisfactory as it corresponds to the expected trueness on such an automated system for non-viscous liquids such as water.

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The program of the freeze-drier was adapted to freeze the material at a temperature lower than the glass temperature of the material. Probes were positioned in the material at different places of the freeze-drier to monitor temperature and electrical resistance.

The water mass fraction of the material in the vials after lyophilisation was determined by Karl-Fischer Titration in 3 vials. The result of 0.6 % (mass fraction) confirmed that an adequate drying had taken place.

4 Homogeneity 4.1 Homogeneity The homogeneity of ERM-AD457/IFCC was verified by measuring the catalytic activity concentration of AST in duplicate in 20 vials taken randomly over the whole batch. The measurements were performed using a Modular P analyser (Roche Diagnostics) with a measurement interval of 4-800 U/L. Reconstitution was achieved, in this study, with 1 g of water, gravimetrically controlled. The AST catalytic activity concentration, around 200 U/L, was corrected for the mass of the water used to reconstitute each sample.

Grubbs tests were performed to detect outlying individual results as well as averages measured for each vial. Two outlier samples were found at the 95 % confidence level with the Double Grubbs test, considering averages. No technical explanation could be given for these outliers. No outlier was detected considering individual results. Therefore, all results were taken into account for analysis.

Regression analyses were used to evaluate drifts in results related to the analysis sequence or to the filling sequence. No significant trend (at the 99 % confidence level) was observed.

Using normal probability plots and histograms respectively, it was verified that the individual data and the vial averages were normally distributed and unimodal.

A one-way ANOVA was carried out grouping the data by sample number. The results show that a significant difference was observed between the samples (F > Fcrit). However, the same statistics carried out on the data without the ouliers show a non-significant difference between the samples (F < Fcrit). The total RSD of the results including the outliers was 0.8 % whereas the repeatability of the method, in the same laboratory using a control sample (target concentration: 237 U/L, 22 samples) was 1.1 %. Therefore, these outliers were not considered as a sign of heterogeneity.

A one-way ANOVA was used to calculate the between bottle standard deviation (sbb) and the maximum standard uncertainty related to the inhomogeneity that can be hidden by the method repeatability (u*bb), with the formulas:

(MSbb = mean sum of squares between bottles; MSwb = mean sum of squares within bottles; n = number of replicates; dfwb = degrees of freedom within bottles)

Both values were converted into relative uncertainties. The relative between bottle heterogeneity (sbb,rel) was 0.72 %. The relative maximum hidden heterogeneity (u*bb, rel) was 0.24 %. The largest of these values, sbb,rel, was included into the calculation of the overall uncertainty of the certified values (Section 7.1). These results were obtained for a CRM reconstituted with 1 g of water. However, as mentioned in the design of the project (Section 1.3), for clinical reasons, the CRM was finally certified for a reconstitution with 2 g of water. In order to extend the validity of the homogeneity study to the material reconstituted with 2 g of water, thus 2-fold diluted, the repeatability of the method at lower concentration was taken into account. As the limit of quantification of the method (4 U/L) is far below the concentration of the 2-fold diluted sample

nMSMS

s wbbbbb

−= 4

wb*bb

2

wbdfnMS

u ⋅=

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(100 U/L), the precision of the method is very unlikely to decrease significantly for the analysis of this material. As the method repeatability (relative maximum hidden heterogeneity) for the material reconstituted with 1 g of water is around three fold lower than the relative between bottle heterogeneity, it is also very unlikely that after two fold dilution of the material, the method repeatability becomes higher than the relative between bottle heterogeneity. The material can thus be considered as homogeneous after reconstitution with 2 g of water.

4.2 Minimum sample intake for analysis The reconstituted material forms a clear solution, and a true solution is not expected to have any relevant heterogeneity in protein concentration at sample intakes even of a few nL volume. The sample intake of homogeneity studies, on the material reconstituted with 1 g of water and analysed with a Modular P analyser, was 7 μL. The RSD within a bottle (swb,rel) was 0.6 % which is lower than the measurement variability (RSD = 1.1 % for control samples at 237 U/L, n = 22), so there is no indication of intrinsic heterogeneity at a sample intake of 7 μL. When the material is reconstituted with 2 g of water, the sample intake for which there is no indication of heterogeneity, according to the results described above, must logically be increased to 14 µL.

5 Stability Short and long-term stability studies were carried out using an isochronous set-up [9] that consists of the simultaneous analysis of reference and test samples. For each study a defined set of samples was exposed for different periods of time to elevated temperatures and then brought back to the reference temperature (-70 °C). At the end of the study all samples were analysed for the catalytic activity concentration of AST within one analytical run under repeatability conditions. The data were analysed by determining the regression line for the enzyme concentration in function of time [10].

5.1 Stability of the non-reconstituted CRM

5.1.1 Short-term stability A short-term stability study was performed in order to assess the possible effect of transport at different temperatures on the stability of the material. The reference temperature was -70 °C. Test samples were kept for 0, 2, 4 and 8 weeks at -20, 4, 18 and 60 °C before being brought back to the reference temperature. For each combination of time and temperature 2 samples were analysed in triplicate. The samples were analysed using the Modular P analyser. The values were corrected for the variable mass of water added (target mass: 1 g). The results are shown in Table 1.

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Table 1: Short-term stability study, test for significance of the slope: slope (b), standard uncertainty (ub) and relative standard uncertainty (usts,rel) after two weeks storage at the specified temperature.

Temperature [°C]

b [(U/L)/week]

ub [(U/L)/week] |b/ub| t0.05,df usts,rel

[%]

-20 0.02 0.31 0.065 2.07 0.3 4 0.07 0.18 0.389 2.07 0.2 18 0.03 0.34 0.088 2.07 0.3 60 -8.11 0.45 18.022 2.07 5.1

For each temperature there were 24 measurements, i.e. 22 degrees of freedom for the linear regression. The slope of the protein concentration as a function of time is significantly different from 0 if the absolute value of slope b divided by its uncertainty ub (|b/ub|) is larger than t0.05, 22 = 2.07. When samples were kept at -20, 4 and 18 °C, none of the slopes was significantly different from 0. However at 60 °C, a significant (at a 95 % confidence level) negative slope was found. It was concluded from this study that the uncertainty due to degradation during dispatch is negligible, if the material is shipped with cooling elements.

5.1.2 Long-term stability

A one-year stability study was performed in order to confirm the stability of the material upon storage at -20 and 4 °C. The reference temperature was -70 °C. The test samples were kept for 0, 4, 8 and 12 months at -20 °C and 4 °C. For each combination of time and temperature 2 samples were analysed in triplicate. The samples were analysed using the Modular P analyser. The values were corrected for the variable mass of water added in the vials (target mass: 2 g). An ANOVA and a trend analysis were performed on the results. The ANOVA of the data, comparing the results grouped by temperature, is presented in Table 2. There was a significant difference between the catalytic activity concentration of AST in the samples stored at the reference temperature (-70 °C), the test temperatures -20 °C and 4 °C (F > Fcrit). However, no significant difference was observed when considering the results from samples stored at -70 °C and -20 °C only. No significant difference was observed either between the samples stored at -70 °C and 4 °C (F < Fcrit). However this is probably due to the low number of data taken into account and the value of F remains relatively high. The test for significance of the slope was performed. For each temperature, there were 24 measurements, or 22 degrees of freedom for each linear regression at each temperature. None of the slopes (AST catalytic activity concentration versus time) was significantly different from 0 at a 95 % confidence level.

In addition, a 2 years-stability study was performed. Test samples were kept for 0, 12, 18 and 24 months at -20 °C and 4 °C. In the same ways as for the 1-year study, for each combination of time and temperature 2 samples were analysed in triplicate. The samples were analysed using the Modular P analyser. The values were corrected for the variable mass of water added in the vials (target mass: 2 g). The ANOVA of the data, presented in Table 2, showed no significant difference between the catalytic activity concentration of AST in the samples stored at the reference temperature and the test temperatures -20 °C and 4 °C (F < Fcrit). The test for significance of the slope indicated that none of the slopes (AST catalytic activity concentration versus time) was significantly different from 0 at a 95 % confidence level.

These results indicate that it is safe to store the material for two years at -20 °C and at 4 °C. Therefore, until further stability monitoring data are available, the CRM shall be stored at -20 °C.

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The uncertainty contribution for the long-term stability corresponds to the maximum uncertainty due to instability that could be hidden by the measurement variation after a period of 24 months at -20 °C.

Table 2: Long-term stability study, One-way ANOVA.

1 year storage

Groups (=storage

temperature) [°C]

Number of

replicates

Average catalytic activity

concentration [U/L]

Variance of the catalytic activity concentration

[U²/L²]

-70 6 113.9 1.0 -20 18 113.7 0.6 4 18 112.9 1.4

ANOVA for 1 year storage at -70 °C, -20 °C and 4 °C Source of Variation SS df MSq F Fcrit Between Groups 8.56 2 4.28 4.35 3,23 Within Groups 38.32 39 0.98 Total 46.88 41 ANOVA for 1 year storage at -70 °C and -20 °C Source of Variation SS df MSq F Fcrit Between Groups 0.05 1 0.05 0.07 4.30 Within Groups 15.04 22 0.68 Total 15.09 23 ANOVA for 1 year storage at -70 °C and 4 °C Source of Variation SS df MSq F Fcrit Between Groups 4.40 1 4.40 3.42 4.30 Within Groups 28.30 22 1.29 Total 32.70 12 2 years storage

Groups (=storage

temperature) [°C]

Number of

replicates

Average catalytic activity

concentration [U/L]

Variance of the catalytic activity concentration

[U²/L²]

-70 6 112.2 2.9 -20 18 112.6 1.3 4 18 112.8 4.1

ANOVA for 2 years storage at -70 °C, -20 °C and 4 °C Source of Variation SS df MSq F Fcrit Between Groups 1.76 2 0.88 0.32 3.24

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Within Groups 106.59 39 2.73 Total 108.35 41

5.2 Stability of the reconstituted CRM Three samples of ERM-AD457/IFCC were reconstituted with 1 g of water and were analysed immediately and after 1, 2, 4 and 6 hours at ambient temperature. Three other samples of the CRM were reconstituted with 2 g of water and were analysed in the same way. The measurements were performed in duplicate, using the Modular P analyser. The trend analysis, using the t of Student test, showed no significant slope (|b/ub| < t0.05,4) which indicates that the CRM may be stored at ambient temperature for 6 hours after reconstitution with 1 g or 2 g of water.

6 Characterisation

6.1 Preparations A feasibility study was organised using the IFCC reference procedure for AST at 37 °C [7] to determine the catalytic activity concentration of AST in the RELA material and in a human serum pool. Twelve laboratories participated in the exercise. It was checked if the procedure was applied properly and it turned out that there was no outlier.

Note: The publication for the primary reference measurement procedure has an error on page 730 and on page 731. The participants of the study were informed that the correct factor for the LDHstock and for the MDHstock was 1905 instead of 2302. The measurements were performed by use of correctly adjusted preparations of LDHstock and MDHstock.

6.2 Performance of the value assignment measurements The twelve laboratories participated in the value assignment of ERM-AD457/IFCC using the same method. As described in Section 1.3, the IFCC reference procedure does not need to be calibrated. However, several influence quantities were checked before analysis in order to ensure a high accuracy of the results. Six samples of ERM-AD457/IFCC were analysed by each laboratory over two days (3 samples per day, one measurement). Two samples of human serum pool were as well analysed (one sample per day, one measurement). Raw data were reported and the calculations were made at IRMM.

The laboratories were provided with detailed protocols and reporting sheets, as well as with vials of ERM-AD457/IFCC and human pool serum. The laboratories were asked to specify the instrument and reagents used. The order in which the measurements were performed was predefined.

The wavelength of the spectrometer was adjusted at 339 nm. The linearity of the detector was checked in each laboratory following a common protocol (Annex 1).

Pipettes, balances and pH-meters were calibrated. pH-meters were also checked immediately before the adjustment of the pH in the solution 1 and the solution 2 of the reference procedure.

The control of the temperature in the cuvettes is also of utmost importance as a variation of 0.1 °C might lead to a relative change of catalytic activity concentration measured of 0.5 %. The temperature in the cuvettes was set at 37.0 °C.

6.3 Reconstitution of ERM-AD457/IFCC The material was reconstituted by addition of 2.00 g ± 0.02 g of distilled water through the septum of the vial, according to the procedure described in Annex 2. The reconstituted material had to be analysed within 6 hours following reconstitution.

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6.4 Data analysis Correction factor As the material is certified in terms of concentration, the mass of water added for reconstitution needs to be taken into consideration to determine the catalytic activity concentration of AST in the reconstituted material. The reconstitution process was controlled by gravimetry. Therefore, the catalytic activity concentration (bE) of AST in U/L was corrected for the mass of water added in g (m) using the correction factor (f):

f=(bE x m)/2.00 The symbol bE was used in this report to express the catalytic activity concentration instead of the symbol b [11,12], which is already used for the slope of the linear regression.

Note: The material is reconstituted in terms of mass of water added although other CRMs for enzyme (ERM-AD452 to 455, IRMM/IFCC-456) were reconstituted in terms of volume of water added. The use of a gravimetrically controlled reconstitution for ERM-AD457 allows the simple correction of the catalytic activity concentration by the mass of water added without taking into consideration the temperature of the water used for reconstitution.

6.5 Results

6.5.1 General considerations Twelve laboratories participated in the characterisation of the material. The experimental conditions reported by each laboratory were compared to the IFCC reference procedure and checked for deviations. No relevant deviation was observed. In addition, the pool of human serum, which had been analysed by all laboratories during the feasibility study, was again analysed as a control sample during the characterisation study of ERM-AD457/IFCC, five months later.

6.5.2 Control sample Table 3 presents the mean result obtained in each laboratory for the control sample analysed over the two days of the characterisation study. Laboratories are represented by codes from L0 to L 11.

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Table 3: Results obtained in each laboratory for the control sample (human serum pool) using the IFCC reference procedure (n=2)

Control sample

Laboratory Laboratory mean (U/L)

RSD (%)

L0 112.20 1.13

L1 107.65 0.99

L2 111.25 0.83

L3 109.83 0.66

L4 110.00 0.00

L5 111.50 0.63

L6 113.20 0.89

L7 111.35 0.70

L8 94.00 6.02

L9 107.94 2.13

L10 107.39 0.49

L11 110.09 1.91

L8 was clearly identified as an outlier, visually and with the Dixon, Nalimov and Grubbs tests. No explanation could be found to these results. Moreover, L8 showed a larger standard deviation than the other laboratories.

6.5.3 ERM-AD457/IFCC In the characterisation study of ERM-AD457/IFCC, some laboratories showed results systematically above or below the mean or with a larger SD but no outlier was detected (Figure 2). L8 gave the results with the lowest mean but was not an outlier. The results showed a normal and unimodal distribution.

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Figure 2: ERM-AD457/IFCC: Laboratory means and their standard deviation (CI = Confidence Interval of the mean of the means)

6.5.4 Value assignment

In order to assign a value to ERM-AD457/IFCC, all individual values reported by the laboratories were corrected for the mass of water added during reconstitution and for the temperature of the laboratory, as described in section 6.4. Then the mean of the laboratory means was calculated as presented in Table 4. After rounding, the value assigned to ERM-AD457/IFCC was 104.6 U/L.

Table 4: Value assignment of ERM-AD457/IFCC: mean and RSD of the single values (n = 6) in each laboratory and mean of the means.

Laboratory Mean

[U/L]

RSD

[%]

L0 107.02 0.64

L1 104.66 1.01

L2 108.29 1.32

L3 105.26 1.83

L4 102.93 0.59

L5 102.13 2.37

L6 108.74 1.37

L7 108.43 0.90

L8 99.44 2.37

L9 101.31 1.30

L10 102.81 0.67

L11 104.42 1.68

Mean of the means [U/L]

104.62

RSD of the mean of the means [%]

2.90

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7 Uncertainty budgets and certified values

7.1 Estimation of the uncertainties The certified uncertainties consist of relative standard uncertainties related to characterisation (uchar,rel), between-bottle heterogeneity (ubb,rel), and degradation during long-term storage (ults,rel) [10].

• uchar,rel was estimated as the relative standard uncertainty of the mean of laboratory means, i.e. RSD/√p with RSD the relative standard deviation of the mean of means and p the number of datasets.

• ubb,rel was estimated as the relative standard deviation between-units (sbb,rel) or the maximum heterogeneity potentially hidden by method repeatability (u*

bb,rel) as defined in Section 4.1. The higher of these two values was taken as a conservative estimate of heterogeneity.

• ults,rel was estimated from stability tests and were taken from the 24 months stability study, -20 °C (section 5.1.2) .

The relative combined standard uncertainty was calculated as the square root of the sum of squares of the individual contributions, according to:

2rellts,

2relbb,

2relchar,relc, uuuu ++=

The various uncertainty contributions and the relative combined standard uncertainty (uc,rel) are shown in Table 5. Table 5: Uncertainty budget for ERM-AD457/IFCC

uchar,rel [%]

ubb,rel [%]

ults,rel [%]

uc,rel [%]

AST 0.84 0.72 0.62 1.17 The relative combined standard uncertainty uc,rel was multiplied by a coverage factor of 2 to obtain the relative expanded uncertainty 2.54 %, with a level of confidence of 95 %.

The relative expanded uncertainty was then multiplied with the mean of dataset means (104.62 U/L) to obtain an expanded uncertainty UCRM of 2.65 U/L. 7.2 Certified values The certified catalytic activity concentration and certified uncertainty of AST in the ERM-AD457/IFCC reconstituted with 2 g of water are:

(1.74 ± 0.05) µkat/L or

(104.6 ± 2.7) U/L

The International Union of Pure and Applied Chemistry and the International Union of Biochemistry (IUB) recommended that enzyme concentration is expressed in terms of katals per liter (kat/L) [13]. This is a special name and symbol approved by CGPM (General Conference on Weights and Measures) and is consistent with the International System of Units (SI) as kat = mol/s. Historically, different units have been introduced. Therefore the Commission on Enzymes of the IUB proposed the term international unit (U) as the quantity of enzyme that catalyzes the reaction of 1 µmol of substrate per minute. Catalytic activity concentration is then to be expressed in terms of U/L [14]. Enzyme units (U) are still more commonly used than the katal in practice, especially in biochemistry. Therefore the certified values for ERM-AD457/IFCC are expressed both in µkat/L and in U/L in this certification

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report as well as on the certificate. The catalytic activity concentration in µkat/L can easily be converted in U/L by multiplying with the factor f = 60.

1 µkat/L = 60 U/L

1 U = 10-6 mol/60 s = 16.7 x 10-9 mol/s

8 Metrological traceability

The measurement of the catalytic activity concentration of AST in ERM-AD457/IFCC was performed using the IFCC reference procedure for AST at 37 °C [7]. This procedure does not require calibration as the enzyme activity is defined in terms of response to this procedure.

As the different steps of the reaction mixture preparation, following the reference procedure, are gravimetrically controlled and as the linearity of response of the spectrophotometer was verified using standard solutions also prepared by gravimetry, the certified values are traceable to the SI, applying the IFCC reference procedure [7].

9 Commutability

Before the start of the certification campaign of ERM-AD457/IFCC, the IFCC Committee for Reference Systems for Enzymes (C-RSE) performed preliminary commutability studies on the raw material. They declared the results convincing enough to process the material and to certify its AST catalytic activity concentration. However, if ERM-AD457/IFCC would be used to calibrate routine procedures, expanded commutability studies should be performed with these procedures.

10 Intended use and instructions for use

The material is primarily intended to be used as a control for the performances of the IFCC reference procedure for the determination of AST at 37 °C [7].

When the material is used as a calibrant in a particular measurement procedure the commutability should be verified for the assay concerned. Reconstitution of the material To prepare the material for use, the entire content of the vial must be reconstituted with 2 g of distilled water according to the procedure described in Annex 2. Storage

Unopened vials should be stored at -20 °C ± 5 °C. After reconstitution, the material should be used within 6 hours, as it was verified that changes to the certified concentration observed during that period at ambient temperature are not significant. It is nevertheless advisable to cover the vial with the original seal and to store it at 2 to 8 °C, for maximum 6 hours, if the reconstituted material must be stored before use.

However, the European Commission cannot be held responsible for changes that happen during storage of the material at the customer's premises, especially of opened samples. Use of the certified values

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The certified combined standard uncertainty of the certified value should be included in the uncertainty of the measurement result obtained with the IFCC reference procedure.

1. Calculate the combined standard uncertainty (uCRM) from the certified value. This is obtained by dividing the expanded uncertainty (UCRM) given in the abstract and in Section 7.2, as well as on the certificate, by the coverage factor k = 2.

2. Estimate the standard measurement uncertainty (um) of the measured quantity value. As a very rough approximation, the repeatability standard deviation of the mean can be used.

3. Combine the two uncertainties: 2CRM

2mc uuu +=

4. Check whether 2*uc is larger than the difference between the certified and the measured value. If this is the case, the measurement result lies within the limits of the respective uncertainties of the certified values.

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References and acknowledgements References

[1] E. E. Snell, S. J. Di Mari in The Enzymes, ed. Boyer, P.D. (Academic, New York), 3rd Ed., Vol. 2 (1970) 335-370.

[2] A. E. Braunstein in The Enzymes, ed. Boyer, P.D. (Academic, New York), 3rd Ed., Vol. 9 (1970) 379-481.

[3] P. Rustad, Klinisk Biokemi i Norden 15:2 (2003) 10-17.

[4] H. U. Bergmeyer, M. Hørder, R. Rej, International Federation of Clinical Chemistry (IFCC). Approved recommendation (1985) on IFCC methods for the measurement of catalytic activity concentration of enzymes. Part 2. IFCC method for aspartate aminotransferase. J Clin Chem Clin Biochem 24 (1986) 497-510.

[5] M. Panteghini, F. Ceriotti, G. Schumann, L. Siekmann, Clin Chem Lab Med 39 (2001) 795-800.

[6] K. Lorentz, Clin Chem Lab Med 40 (2002) 549.

[7] G. Schumann, R. Bonora, F. Ceriotti, G. Férard, C. A. Ferrero, P. F. H. Franck, F.-Javier Gella, W. Hoelzel, P. J. Jørgensen, T. Kanno, A. Kessner, R. Klauke, N. Kristiansen, J.-M. Lessinger, T. P. J. Linsinger, H. Misaki, M. Panteghini, J. Pauwels, F. Schiele, H. Schimmel, G. Weidemann, L. Siekmann, Clin Chem Lab Med 40 (2002) 725-733.

[8] A. Lamberty, H. Schimmel, and J. Pauwels, Fresenius J. Anal. Chem. 360 (1998) 359–361.

[9] T. P. Linsinger , J. Pauwels, A. Lamberty , H. G. Schimmel , A. M. van der Veen , L. Siekmann, Fresenius J Anal Chem 370 (2001) 183-188 A.

[10] J. Pauwels, H. Schimmel, and A. Lamberty, Clin Biochem 31 (1998) 437-439.

[11] R. Dybkaer, Metrologia 37 (2000) 671-676.

[12] R. Dybkaer, Pure Appl Chem 73 (2001) 927-931.

[13] Comptes Rendus de la 21e Conférence Générale des Poids et Mesures (1999), 2001, 334.

[14] C. A. Burtis, E. R. Ashwood and D. E. Bruns, Tietz textbook of Clinical Chemistry and Molecular Diagnostics, 4rd Edition, 2006, 644-645.

Acknowledgements The authors thank O. Couteau, J. Charoud-Got and I. Zegers from IRMM for the internal review of this report, and the experts of the Certification Advisory Panel ‘Biological Macromolecules and Biological/Biochemical Parameters’, R. Dybkaer (Frederiksberg Hospital, DK), A. Heissenberger (Umweltbundesamt, AT) and U. Örnemark (LGC Standards, SE) for reviewing of the certification documents. We also thank R. Koeber (IRMM) for comments on the certification study. The authors are grateful for the active involvement of the participating laboratories in the characterisation study, and have appreciated the thoughtful discussions with G. Schumann, director of the accredited reference laboratory of the German Society for Clinical Chemistry (DGKL), and Rainer Klauke, deputy of the reference laboratory. The contribution of the Committee for Reference Systems of Enzymes of the IFCC throughout the certification project is gratefully acknowledged.

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ANNEX 1 Check of the linearity of a spectrometer with ABTS (2,2'-azino-bis(3-ethylbenzthiazoline-6-sulphonic acid) Guidelines by G. Schumann and R. Klauke Measurement procedure: 1. Prepare about 100 mL of an aqueous ABTS stock solution with an absorbance of ≈ 2.5 at

340 nm (mass of ABTS for 100 ml water: ≈ 3.7 mg). 2. Make the following dilutions of the stock solutions using distilled water as solvent. Use a

balance for the exact determination masses of the volumes of stock solution and water. 3. Report the masses.

Dilution ABTS stock solution

Water Approximate absorbance

1 10 mL 0 mL 2.50

2 9 mL 1 mL 2.25

3 8 mL 2 mL 2.00

4 7 mL 3 mL 1.75

5 6 mL 4 mL 1.50

Blank 0 mL 10 mL 0.00

4. Measure the absorbance of each dilution at 339 nm. Use water for the blank. The

measurement for the blank and the sample shall be performed in the same cuvette. 5. Report the absorbances. 6. Check the linearity of the relationship between the absorbance and the volume fraction of

ABTS stock solution. The slope of the linear regression is reflecting the change of absorbance due to the change of the volume fraction of the ABTS stock solution. Calculate the slope of the linear regression in each interval, between two successive dilutions. In case of linearity, the slopes are constant.

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ANNEX 2 Reconstitution protocol with 2 g of water Each vial contains 1 mL of AST material freeze-dried in a glass vial with stopper and cap suitable for injection (aluminium cap with a hole in its centre). Therefore the reconstitution must be performed by injection through the rubber stopper without removing the aluminium cap. The whole content of the vial is reconstituted at once. Procedure: 1. Check that the room temperature stays between 20 °C and 25 °C. 2. Take the vial out of the freezer and allow to reach room temperature. 3. Tap the vertically positioned vial gently to ensure that the lyophilised material is at the

bottom of the vial. 4. Weigh the vial with its contents to the nearest 0.1 mg. Record the mass m1. 5. Take an injection needle (e.g. 0.55 mm x 25 mm, purple). Insert it through the septum

and leave it in to create a venting of the vial (see Figure 1). Leave some space to introduce a second injection needle in the same stopper.

6. Take syringe of 2 mL (e.g. single-use insulin syringe), with another injection needle (e.g. 0.9 mm x 40 mm, yellow).

7. Using the syringe, reconstitute by slow injection of (2.00 ± 0.02) mL distilled water through the stopper.

8. Remove syringe and needles. 9. Weigh the vial after adding the water. Record the mass m2. 10. Calculate the mass of water added in g (m3) :

m3= m2- m1 and calculate the correction factor (f) which will be applied to the catalytic activity

concentration of AST (bE) determined in the reconstituted material : f=(bE x m3)/2.00

11. Invert the vial several times and mix content by gentle swirling. Allow to stand at room temperature for 20 min. Swirl the vial again and then allow to stand for 10 min. The total reconstitution time is approximately 30 min.

The reconstituted vials must be kept between 2 °C and 8 °C and the catalytic activity concentration of AST must be measured within 6 hours following reconstitution. The procedure for sampling is left to the discretion of the laboratory (either open the vial with a crimp and use calibrated pipettes or aspirate the reconstituted material through the stopper using a clean syringe and injection needle).

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Figure 1 by G. Schumann Material:

Injection needle 0.9 mm x 40 mm (yellow): Injection of water, aspiration of reconstituted reference material

Injection needle 0.55 mm x 25 mm (purple): Venting of the vial

2 ml syringe („single-use insulin syringe“)

European Commission EUR 23808 EN – Joint Research Centre – Institute for Reference Materials and Measurements Title: Certification of the catalytic activity concentration of aspartate transaminase, ERM®-AD457/IFCC Author(s): B. Toussaint, G. Schumann, R. Klauke, N.Meeus, R. Zeleny, S. Trapmann, H. Emons, H. Schimmel Luxembourg: Office for Official Publications of the European Communities 2009 – 26 pp. – 21.0 x 29.7 cm EUR – Scientific and Technical Research series – ISSN 1018-5593 ISBN 978-92-79-12206-4 Abstract The production and certification of ERM-AD457/IFCC, a new reference material for the enzyme aspartate transaminase (AST) [L-aspartate: 2-oxoglutarate-aminotransferase, EC 2.6.1.1], also called aspartate aminotransferase (ASAT), is described. The certified reference material is lyophilised and should be reconstituted by addition, gravimetrically controlled, of 2 g highly purified water comparable to bi-distilled water. The certified catalytic activity concentration and certified uncertainty of AST in the reconstituted material are (1.74 ± 0.05) µkat/L or (104.6 ± 2.7) U/L (k=2, obtained with the IFCC reference procedure at 37 °C).

The material was produced from a human type recombinant AST in E. Coli and a buffer containing bovine serum albumin.

A first batch of the material was processed, filled into vials (1 mL per vial) and lyophilised. The vials were closed with a stopper and a metal cap. This batch was used as a trial batch to test the lyophilisation process, the homogeneity and the stability of AST in the material. A reconstitution protocol was also developed.

Following the positive results obtained on the trial batch, a second batch was produced in the same way. The homogeneity and the stability of this batch were assessed using an In Vitro Diagnostic assay. Then a feasibility study was carried out to characterise the material using a reference procedure from the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC). Twelve expert laboratories participated in the study and familiarised themselves with the protocol. Finally, the characterisation of the material was performed. A good agreement between the results of the 12 laboratories allowed the value assignment of AST in the material in terms of catalytic activity concentration of AST (U/L or µkat/L) in the reconstituted material. The material is aiming to control the IFCC reference procedure for AST at 37 °C. It can also be used to calibrate assays if the commutability of the material with patient samples is demonstrated for these assays

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