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CERTIFICATION REPORT
Certification of the amount-of-substance fraction of HbA1c
versus the sum of HbA0 and HbA1c in haemoglobin:
ERM®-AD500/IFCC
EU
R 2
75
74
- 20
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JRC-IRMM promotes a common and reliable European measurement provides reference measurements. European Commission
Joint Research Centre
Institute for Reference Materials and Measurements (IRMM)
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This publication is a Reference Materials Report by the Joint Research Centre, the European Commission’s in-house science service. It aims to provide
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JRC98713
EUR 27574 EN
ISBN 978-92-79-53878-0 (PDF)
ISSN 1831-9424 (online)
doi: 10.2787/7609
Luxembourg: Publications Office of the European Union, 2016
© European Union, 2016
Reproduction is authorised provided the source is acknowledged.
Printed in Belgium
Abstract
This report describes the production of ERM-AD500/IFCC, which is a set of 6 ampoules containing haemoglobin (with different levels of HbA1c) in a
buffer solution, certified for the amount-of-substance fraction of HbA1c versus the sum of HbA0 and HbA1c. HbA1c is defined as the stable adduct
from glucose and the N-terminal amino group of the β-chain of haemoglobin A0 that is beta-N-(1-deoxyfructose-1-yl) haemoglobin [ ]. The reference
material was produced in collaboration with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC).
This material was produced following ISO Guide 34:2009 and is certified in accordance with ISO Guide 35:2006.
The ampoules contain buffered solutions of haemoglobin with different amount of substance concentrations of HbA1c and HbA0. The base materials
(solutions of purified HbA1c and HbA0) were prepared from whole blood obtained from diabetic volunteers and provided by Roche Diagnostic GmbH,
Department for Biochemical Materials, Penzberg, Germany. Mixtures of these base materials were filled into the ampoules, with each ampoule
containing approximately 1 mg haemoglobin in 30 µL solution, sealed under an atmosphere of argon.
Between-unit homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006.
The certified value was obtained from the gravimetric preparation of mixtures, taking into account the amount of substance fraction of HbA1c in the
two base materials as well as the haemoglobin content determined using the ICSH reference method (Drabkin). The values were confirmed with two
datasets obtained using the reference method for HbA1c.
Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) and
include uncertainties related to possible inhomogeneity, instability and characterisation.
The material is intended for the calibration of the IFCC reference method or for the assessment of method performance. As with any reference
material, it can be used for establishing control charts or validation studies.
The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.
CERTIFICATION REPORT
Certification of the amount-of-substance fraction o f HbA1c versus the sum of HbA0 and HbA1c in
haemoglobin: ERM ®- AD500/IFCC
Amalia Muñoz a, Ingrid Zegers a, Jean Charoud-Got a, Cas Weykamp b, Renata Paleari c, Andrea Mosca c, Uwe Kobold d, Heinz Schimmel a, Hendrik
Emons a
aEuropean Commission, Joint Research Centre
Institute for Reference Materials and Measurements (IRMM)
Geel, Belgium
bStreekziekenhuis Koningin Beatrix (SKB)
Winterswijk, Netherlands
cDepartment of Physiopathology and Transplantation,
Università degli Studi di Milano (USM)
Milano, Italy
dRoche Diagnostic GmbH, Penzberg, Germany
Disclaimer Certain commercial equipment, instruments, and materials are identified in this paper 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.
1
Summary
This report describes the production of ERM-AD500/IFCC, which is a set of 6 ampoules containing haemoglobin (with different levels of HbA1c) in a buffer solution, certified for the amount-of-substance fraction of HbA1c versus the sum of HbA0 and HbA1c. HbA1c is defined as the stable adduct from glucose and the N-terminal amino group of the β-chain of haemoglobin A0 that is beta-N-(1-deoxyfructose-1-yl) haemoglobin [1]. The reference material was produced in collaboration with the International Federation for Clinical Chemistry and Laboratory Medicine (IFCC).
This material was produced following ISO Guide 34:2009 [2] and is certified in accordance with ISO Guide 35:2006 [3]
The ampoules contain buffered solutions of haemoglobin with different amount of substance concentrations of HbA1c and HbA0. The base materials (solutions of purified HbA1c and HbA0) were prepared from whole blood obtained from diabetic volunteers and provided by Roche Diagnostic GmbH, Department for Biochemical Materials, Penzberg, Germany. Mixtures of these base materials were filled into the ampoules, with each ampoule containing approximately 1 mg haemoglobin in 30 µL solution, sealed under an atmosphere of argon.
Between-unit homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006 [3].
The certified value was obtained from the gravimetric preparation of mixtures, taking into account the amount of substance fraction of HbA1c in the two base materials as well as the haemoglobin content determined using the ICSH reference method (Drabkin). The values were confirmed with two datasets obtained using the reference method for HbA1c [4, 5, 6, 8].
Uncertainties of the certified values were calculated in accordance with the Guide to the Expression of Uncertainty in Measurement (GUM) [7] and include uncertainties related to possible inhomogeneity, instability and characterisation.
The material is intended for the calibration of the IFCC reference method or for the assessment of method performance. As with any reference material, it can be used for establishing control charts or validation studies.
The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.
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The following values were assigned:
HAEMOGLOBIN IN BUFFER
HbA1c/(HbA1c + HbA0)1) Amount-of-substance fraction
Certified value 2) [mmol/mol]
Uncertainty
[mmol/mol]
blank 0.03) -0.0 ; +0.4 4)
level 1 28.6 0.95)
level 2 57.8 1.35)
level 3 86.7 2.25)
level 4 118.8 2.65)
level 5 153 55)
1) As defined by the procedure according to "Approved IFCC reference method for the measurement of HbA1c in human blood", Jeppsson et al., Clin. Chem. Lab. Med. 40 (2002) 78-89. The HbA1c amount of substance fraction is defined as the amount of substance of the glycated N-terminal hexapeptide of the β-chain versus the sum of the glycated and non-glycated N-terminal hexapeptide of the β-chain, after digestion of the haemoglobin with Glu-C.
2) The certified values and their uncertainties have been obtained from the gravimetric mixing of base materials characterised for their HbA1c amount of substance fraction. The values are traceable to the SI.
3) The HbA1c amount of substance fraction, as defined by the IFCC reference method, in the blank material is below the limit of detection (0.2 mmol/mol) for all individual characterisation measurements.
4) Expanded uncertainty with a confidence level of about 95 %. The standard uncertainty is 0.2 mmol/mol.
5) The uncertainty is the expanded uncertainty of the certified value with a coverage factor k = 2 corresponding to a level of confidence of about 95 % estimated in accordance with ISO/IEC Guide 98-3, Guide to the Expression of Uncertainty in Measurement (GUM:1995), ISO, 2008.
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Table of contents
Summary .............................................................................................................................. 1
Table of contents ................................................................................................................. 3
Glossary ............................................................................................................................... 5
1 Introduction .............................................................................................................. 9
1.1 Background ............................................................................................................... 9
1.2 Choice of the material ................................................................................................ 9
1.3 Design of the CRM project ........................................................................................10
2 Participants .............................................................................................................10
2.1 Project management and evaluation .........................................................................10
2.2 Processing ................................................................................................................10
2.3 Homogeneity study ...................................................................................................10
2.4 Stability study ...........................................................................................................11
2.5 Characterisation ........................................................................................................11
3 Material processing and process control .............................................................11
3.1 The starting material .................................................................................................11
3.2 Processing ................................................................................................................11
4 Homogeneity ...........................................................................................................11
4.1 Between-unit homogeneity........................................................................................12
4.2 Within-unit homogeneity and minimum sample intake...............................................14
5 Stability....................................................................................................................14
5.1 Short-term stability study ..........................................................................................14
5.2 Long-term stability study ...........................................................................................15
5.3 Estimation of uncertainties ........................................................................................15
6 Characterisation .....................................................................................................16
6.1 Selection of participants ............................................................................................16
6.2 Study setup ...............................................................................................................16
6.3 Characterisation of the base materials ......................................................................17
6.4 Amount-of-substance fractions and their uncertainties ..............................................18
6.5 Verification measurements........................................................................................18
7 Value Assignment ...................................................................................................19
7.1 Certified values and their uncertainties .....................................................................19
7.2 Additional material information ..................................................................................20
8 Metrological traceability and commutability .........................................................21
8.1 Metrological traceability ............................................................................................21
8.2 Commutability ...........................................................................................................21
9 Instructions for use ................................................................................................22
9.1 Safety information .....................................................................................................22
9.2 Storage conditions ....................................................................................................22
9.3 Use of the material ....................................................................................................22
9.4 Minimum sample intake ............................................................................................22
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9.5 Use of the certified value ..........................................................................................22
10 Acknowledgments ..................................................................................................24
11 References ..............................................................................................................25
Annexes ..............................................................................................................................26
5
Glossary
ANOVA Analysis of variance
b Slope in the equation of linear regression y = a + bx
c Mass concentration c = m / V (mass / volume)
CE/UV Capillary electrophoresis with ultraviolet detection
CLSI Clinical and Laboratory Standards Institute
CRM Certified reference material
EC European Commission
EDTA Ethylenediaminetetraacetic acid
ESI/MS Electrospray ionisation mass spectrometry
ERM® Trademark of European Reference Materials
EU European Union
GUM Guide to the Expression of Uncertainty in Measurements [ISO/IEC Guide 98-3:2008]
HbA0 Non-glycated haemoglobin
HbA1c Beta-N-(1-deoxyfructose-1-yl) haemoglobin
Hb Haemoglobin (total)
HPLC High performance liquid chromatography
IC Ion chromatography
ICSH International Council for Standardisation in Haematology
IEC International Electrotechnical Commission
IFCC International Federation of Clinical Chemistry and Laboratory Medicine
IRMM Institute for Reference Materials and Measurements of the JRC
ISO International Organization for Standardization
IVD In Vitro Diagnostics
JCTLM
JRC
Joint Committee for Traceability in Laboratory Medicine
Joint Research Centre of the European Commission
k Coverage factor
LC-MS Liquid chromatography-mass spectrometry
LOD Limit of detection
LOQ Limit of quantification
MES 2-Morpholinoethanesulfonic acid
MS Mass spectrometry
MSbetween Mean of squares between-unit from an ANOVA
MSwithin Mean of squares within-unit from an ANOVA
n Number of replicates per unit
6
N Number of samples (units) analysed
n.a. Not applicable
n.c. Not calculated
n.d. Not detectable
RSD Relative standard deviation
RSE Relative standard error (=RSD/√n)
s Standard deviation
sbb Between-unit standard deviation; an additional index "rel" is added when
appropriate
sbetween Standard deviation between groups as obtained from ANOVA; an additional index "rel" is added as appropriate
SD Standard deviation
SI International System of Units
swithin Standard deviation within groups as obtained from ANOVA; an additional index "rel" is added as appropriate
swb Within-unit standard deviation
T Temperature
t Time
ti Time point for each replicate
tα, df Critical t-value for a t-test, with a level of confidence of 1-α and df degrees of freedom
tsl Proposed shelf life
TRIS Tris(hydroxymethyl)aminomethane
u Standard uncertainty
U Expanded uncertainty
u*bb Standard uncertainty related to a maximum between-unit inhomogeneity
that could be hidden by method repeatability precision; an additional index "rel" is added as appropriate
ubb Standard uncertainty related to a possible between-unit inhomogeneity; an additional index "rel" is added as appropriate
uc Combined standard uncertainty; an additional index "rel" is added as appropriate
ucal Standard uncertainty of calibration
uchar Standard uncertainty of the material characterisation; an additional index "rel" is added as appropriate
uCRM Combined standard uncertainty of the certified value; an additional index "rel" is added as appropriate
UCRM Expanded uncertainty of the certified value; an additional index "rel" is added as appropriate
u∆ Combined standard uncertainty of measurement result and certified value
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ults Standard uncertainty of the long-term stability; an additional index "rel" is added as appropriate
umeas Standard measurement uncertainty
urec Standard uncertainty related to possible between-unit inhomogeneity modelled as rectangular distribution; an additional index "rel" is added as appropriate
usts Standard uncertainty of the short-term stability; an additional index "rel" is added as appropriate
x Arithmetic mean
nsx Arithmetic mean of all results of normal stock samples
refx Arithmetic mean of results of reference samples
α Significance level
∆meas Absolute difference between mean measured value and the certified value
νs,meas Degrees of freedom for the determination of the standard deviation smeas
MSwithinν Degrees of freedom of MSwithin
8
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1 Introduction
1.1 Background
Diabetes mellitus is a collection of disorders associated with hyperglycaemia, a chronic elevation of the concentration of glucose in the blood, as a result of insulin deficiency, insulin resistance or a combination of both. This condition affects more than 5 % of the world’s population. The major medical and social problems are the chronic diabetic complications, which in the end may lead to blindness, renal failure and the need for limb amputations. The risk for these complications strongly depends on the long-term glycaemic status of the patients. The closer the blood glucose concentrations are maintained within the normal intervals, the lower the risk of such complications. At present the diagnostic control for the estimation of this long-term glycaemic status is based on the determination of haemoglobin-glucose adducts (glycated haemoglobins) in blood, formed by non-enzymatic glycation of haemoglobin. The major form, about 80 %, of these glycated haemoglobins is haemoglobin A1c (HbA1c), resulting from the condensation of glucose with the N-terminal valine residue of each β-chain of haemoglobin and subsequent formation of a stable ketoamine. HbA1c has been defined as beta-N-(1-deoxyfructose-1-yl) haemoglobin, while HbA0 is the form of haemoglobin that is not glycated on the N-terminus. Formation of glycated haemoglobin is essentially irreversible, and the blood level depends on both the life span of the red blood cells and the blood glucose level. The important role played by the determination of HbA1c vs HbA0 levels to monitor the evolution of patients with diabetes mellitus under treatment implies that the analysis of HbA1c is very frequently requested in laboratory medicine.
Methods for measuring HbA1c are based on a variety of analytical principles such as the separation on the basis of charge (ion exchange chromatography in HPLC or mini-columns, electrophoresis), affinity binding of glycated haemoglobins (affinity chromatography) or immunoassays
. They rely on the use of a calibrator with values traceable to those provided by a reference system.
The EU Directive on In Vitro Diagnostic Medical Devices (IVD-MD) (Directive 98/79/EC) requires traceability of the assigned values of calibrants and control materials to reference measurement procedures and/or reference materials of higher order.
The IFCC Working Group on HbA1c Standardization has developed a reference measurement procedure. This method quantifies the cleaved N-terminal hexapeptide of the β-chain of haemoglobin. The glycated and non-glycated hexapeptides are first separated by HPLC and then quantified by mass spectrometry (HPLC-ESI/MS) or by UV detection after a second separation step by capillary electrophoresis (HPLC-CE/UV). The measurand is defined as HbA1c/(HbA1c + HbA0). Both detection systems give identical results when calibrated with mixtures of highly purified HbA0 and HbA1c. The performance of the reference measurement procedure was validated by a network of reference laboratories [5]. A modification of the procedure as described in [5] has been accepted by the IFCC network and is currently used by the IFCC network laboratories [6]. Several of the IFCC network laboratories are also listed as reference laboratories by the Joint Committee for Traceability in Laboratory Medicine (JCTLM).
ERM-AD500/IFCC consists of a set of 6 solutions with different amount-of-substance fractions of HbA1c. This material is intended to be used for the calibration of the IFCC reference method.
1.2 Choice of the material ERM-AD500/IFCC consists of a set of 6 ampoules each containing about 1 mg haemoglobin in approximately 30 µL buffer (50 mmol/L MES, 10 mmol/L KCN, 1 mmol/L EDTA, pH 6.2).
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The ampoules have different amount-of-substance fractions of HbA1c. The levels cover the clinically relevant measurement interval (25-110 mmol/mol HbA1c/(HbA1c + HbA0)) The different levels have been prepared by mixing solutions of purified HbA1c and HbA0 in different proportions. The ampoules have been closed after flushing them with argon.
The ampoule content of about 1 mg haemoglobin is also the amount of protein needed for the preparation of a sample for the reference measurement procedure. Therefore the first step of the procedure (addition of Glu-C enzyme solution and digestion buffer, which brings the solution to a final volume of about 500 µL), can be performed directly in the ampoules before transferring the solution to another vial for the incubation step.
1.3 Development of the CRM Two purified protein materials (HbA1c and HbA0) were prepared by Roche Diagnostic GmbH, Department for Biochemical Materials, Penzberg, Germany. For both materials the amount-of-substance fraction was determined using the IFCC reference method combined with standard addition experiments. The mass fraction of total haemoglobin was determined using the recommended reference method of the International Council for Standardisation in Haematology (ICSH; Drabkin method) [8]. It is measured at 540 nm in the form of cyanmethaemoglobin. This is formed from the reaction between the Drabkin’s reagent and the haemoglobin previously oxidised to methaemoglobin. The purified protein solutions were mixed gravimetrically to obtain the required levels of HbA1c.
The certified values were obtained from the gravimetric preparation of mixtures, taking into account the amount of substance fraction of HbA1c in the base materials. The values were confirmed with two datasets obtained using the reference measurement procedure for HbA1c [5, 6].
The values were confirmed using the IFCC reference measurement procedure for the measurement of the HbA1c amount of substance concentration in the fully processed material. The two variants of the IFCC measurement procedure were used, employing HPLC-ESI/MS and HPLC-CE/UV, respectively.
2 Participants
2.1 Project management and evaluation European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BE (accredited to ISO Guide 34, BELAC No. 268-RM)
2.2 Processing European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BE (accredited to ISO Guide 34, BELAC No. 268-RM)
2.3 Homogeneity study European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BE (accredited to ISO Guide 34)
Streekziekenhuis Koningin Beatrix (SKB), Winterswijk, NL
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2.4 Stability study European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BE (accredited to ISO Guide 34, BELAC No. 268-RM)
Streekziekenhuis Koningin Beatrix (SKB), Winterswijk, NL
Università degli Studi di Milano (USM), Milano, IT (accredited to ISO 15195:2004 and to ISO 17025:2005, Accredia LAT No. 217)
2.5 Characterisation European Commission, Joint Research Centre, Institute for Reference Materials and Measurements (IRMM), Geel, BE (accredited to ISO Guide 34, BELAC No. 268-RM)
Streekziekenhuis Koningin Beatrix (SKB), Winterswijk, NL
Università degli Studi di Milano (USM), Milano, IT (accredited to ISO 15195:2004 and to ISO 17025:2005, Accredia LAT No. 217)
3 Material processing and process control
3.1 The starting material
The starting materials were prepared by Roche Diagnostics GmbH from whole blood obtained from diabetic volunteers.
The purification of HbA1c was performed by multi-step liquid chromatography [9]. HbA0 and HbA1c were separated using cation exchange chromatography (SP Sephadex) and each of them was further purified by sequential affinity chromatography and cation exchange chromatography (SP Sephadex).
The amount-of-substance fraction in the starting materials was determined using the IFCC reference method [5, 6] combined with standard addition experiments [9]. On the basis of values for the starting materials six mixtures were produced with the following nominal HbA1c amount-of-substance fractions: 0, 30, 60, 90, 120, 150 mmol/mol (with acceptance limits of 10 %). These mixtures were transported to IRMM on cooling elements.
3.2 Processing The different mixtures were manually filled in 3 mL clear glass open ampoules. Each ampoule contains about 30 µL haemoglobin solution at a concentration of 34 mg/mL, i.e. about 1 mg haemoglobin per ampoule. The ampoules were flushed with argon before flame sealing. The reference material consists of a set of 6 ampoules with different levels of HbA1c (blank to level 5), assembled in a cardboard box. The material is stored at (-70 ± 5) °C.
4 Homogeneity
A key requirement for any reference material aliquotted into units is equivalence between those units. In this respect, the question is whether the variation between units is significant compared to the uncertainty of the certified value, and not whether the variation is statistically significant compared to the analytical variation. ISO Guide 34 [22] requires RM producers to quantify the between unit variation. This aspect is covered in between-unit homogeneity studies.
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For this material the minimum sample intake is the entire content of the ampoule.
4.1 Between-unit homogeneity The between-unit homogeneity was evaluated to ensure that the certified values of the CRM are valid for all units of the material, within the stated uncertainties.
The number of units selected corresponds to approximately the cube root of the total number of units produced. Ten units were selected using a random stratified sampling scheme covering the whole batch for the between-unit homogeneity test. For this, the batch was divided into ten groups (with a similar number of units) and one unit was selected randomly from each group. Three independent samples were taken from each selected unit, after its reconstitution, and analysed by the HPLC-CE/UV based reference measurement procedure. The measurements were performed under repeatability conditions, and in a randomised manner to be able to separate a potential analytical drift from a trend in the filling sequence. The results are shown in Annex A.
Regression analyses were performed to evaluate potential trends in the analytical sequence as well as trends in the filling sequence. For the blank level all values were below the calibration interval, so no uncertainty on the homogeneity could be determined. Seeing the high purity of the HbA0 base material, the absence of mixing, and the practical impossibility of forming HbA1c in the absence of glucose the uncertainty related to possible inhomogeneity for the blank level was considered to be negligible. For the level 5 a trend in the analytical sequence was observed at a 95 % confidence level, pointing at a changing parameter, e.g. a signal drift in the analytical system. No other trend was observed either in the filling or in the analytical sequence. The correction of biases, even if they are statistically not significant, was found to combine the smallest uncertainty with the highest probability to cover the true value [10]. Correction of trends is therefore expected to improve the sensitivity of the subsequent statistical analysis through a reduction in analytical variation without masking potential between-unit heterogeneities. As the analytical sequence and the unit numbers were not correlated, data for level 5 were corrected for the trend as shown below:
ibxx icorri ⋅−=_ Equation 1
b = slope of the linear regression
i = position of the result in the analytical sequence
The trend-corrected dataset was assessed for consistency using Grubbs outlier tests at a confidence level of 99 % on the individual results and on the unit means. No outlying individual results and outlying unit means were detected.
Quantification of between-unit inhomogeneity was undertaken by analysis of variance (ANOVA), which separates the between-unit variation (sbb) from the within-unit variation (swb). The latter is equivalent to the method repeatability if the individual samples were representative for the whole unit.
Evaluation by ANOVA requires mean values per unit, which follow at least a unimodal distribution and results for each unit that follow unimodal distributions with approximately the same standard deviations. Too few data are available for the unit means to make a clear statement of the distribution. Therefore, it was checked visually whether all individual data follow a unimodal distribution using histograms and normal probability plots. Minor deviations from unimodality of the individual values do not significantly affect the estimate of between-unit standard deviations.
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It should be noted that sbb,rel and swb,rel are estimates of the true standard deviations and are therefore subject to random fluctuations. Therefore, the mean square between groups (MSbetween) can be smaller than the mean squares within groups (MSwithin), resulting in negative arguments under the square root used for the estimation of the between-unit variation, whereas the true variation cannot be lower than zero. In this case, u*
bb, the maximum inhomogeneity that could be hidden by method repeatability, was calculated as described by Linsinger et al. [11]. u*
bb is comparable to the LOD of an analytical method, yielding the maximum inhomogeneity that might be undetected by the given study setup.
Method repeatability (swb,rel), between–unit standard deviation (sbb,rel) and u*bb,rel were
calculated as:
y within
rel,wb
MSs = Equation 2
yn
MSMS
s
withinbetween
rel,bb
−
= Equation 3
y
νn
MS
u MSwithin
within
*rel,bb
42
= Equation 4
MSwithin mean of squares within-unit from an ANOVA
MSbetween mean of squares between-unit from an ANOVA
y mean of all results of the homogeneity study
n mean number of replicates per unit
MSwithinν degrees of freedom of MSwithin
Table 1: Results of the homogeneity study
ERM-AD500/IFCC
HbA1c amount-of-substance fraction
swb,rel
[%]
sbb,rel
[%]
u*bb,rel
[%]
ubb,rel
[%]
Level 1 2.8 1.2 0.9 1.2
Level 2 1.7 0.3 0.5 0.5
Level 3 1.0 0.8 0.3 0.8
Level 4 0.8 0.5 0.3 0.5
Level 5 0.8 1.1 0.2 1.1
The homogeneity study showed no outlying unit means or trends in the filling sequence. Therefore the between-unit standard deviation can be used as estimate of ubb. As u*
bb sets the limits of the study to detect inhomogeneity, the larger value of sbb and u*
bb is adopted as uncertainty contribution to account for potential inhomogeneity.
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4.2 Within-unit homogeneity and minimum sample intake ERM-AD500/IFCC is intended to be used with the IFCC reference method. The entire content of the ampoule is used in the first samples preparation step. This was also the case for the stability, homogeneity and characterisation studies for this material. Therefore the minimum sample intake for the whole measurement procedure is the content of the ampoule (about 30 µL).
The reconstituted material is a true solution and is not expected to have any relevant inhomogeneity. Measures were taken to avoid agglomeration and to keep the molecules evenly distributed in the solution during processing.
5 Stability
Time, temperature, and light (including ultraviolet radiation) were regarded as the most relevant influences on the stability of the materials. The influence of ultraviolet or visible light was minimised by storing the material in cardboard boxes which reduces light exposure. In addition, materials are stored in the dark and dispatched in boxes, thus removing any possibility of degradation by light. Therefore, only the influences of time and temperature needed to be investigated.
Stability testing is necessary to establish the conditions for storage (long-term stability) as well as the conditions for dispatch of the materials to the customers (short-term stability).
The stability studies were carried out using an isochronous design [12]. In this approach, samples were stored for a particular period of time at different temperature conditions. Afterwards, the samples were moved to conditions where further degradation can be assumed to be negligible (reference conditions). At the end of the isochronous storage, the samples were analysed simultaneously under repeatability conditions. Analysis of the material (after various exposure times and temperatures) under repeatability conditions greatly improves the sensitivity of the stability tests.
5.1 Short-term stability study For the short-term stability study, samples from level 3 were stored at 4 °C and -40 °C for 0, 1, 3 and 4 weeks (at each temperature). The reference samples were stored at temperatures below -140 °C (stored above liquid nitrogen). One unit per storage time was selected using a random stratified sampling scheme. From each unit, two samples were measured by ion exchange HPLC (Biorad Variant II, Dual Kit). The measurements were performed under repeatability conditions.
The data were evaluated individually for each temperature. The results were screened for outliers using the single and double Grubbs test on a confidence level of 99 %. The two results for the unit stored at the reference temperature (t = 0 weeks) are outliers for the study at 4 °C.
In addition, the data were evaluated against storage time, and regression lines of amount-of-substance fraction versus time were calculated, to test for potential increases/decrease of the measurand due to shipping conditions. The slopes of the regression lines were tested for statistical significance. The slope was significantly different from 0 at a 95 % confidence level for both temperatures, but not at 99 % confidence level.
The results of the measurements are shown in Annex B.
However, due to the very limited number of units used in the study it cannot be excluded that the trend is a statistical artefact.
Therefore the data from the long-term stability study performed for level 2 at -70 °C were evaluated (see below). In case of shipment at temperatures at -70 °C or below the
15
uncertainty associated with the short term stability for a period of one month is 0.05 %, which is negligible with respect to other uncertainties. Therefore the material can be safely shipped on dry ice.
5.2 Long-term stability study For the long-term stability study, samples were stored at -70 °C for 0, 3, 6 and 12 months. The reference temperature was set to below -140 °C. Two units per storage time were selected using a random stratified sampling scheme. From each unit, three samples were measured by HPLC-ESI/MS (IFCC reference measurement procedure). The measurements were performed under repeatability conditions, in a random sequence to be able to separate any potential analytical drift from a trend over storage time.
The long-term stability data were evaluated individually for each temperature. The results were screened for outliers using the single and double Grubbs test at a confidence level of 99 %. No outlying individual results were found.
In addition, the data were plotted against storage time and linear regression lines of amount-of-substance fraction versus time were calculated. The slopes of the regression lines were tested for statistical significance (loss/increase due to storage). The slopes were not significantly different from 0 at a 95 % confidence level.
The results of the long-term stability measurements are shown in Annex C.
As no technically unexplained outliers were observed and the trend was not statistically significant on a 95 % confidence level the material can be stored at –70 °C.
5.3 Estimation of uncertainties Due to the intrinsic variation of measurement results, no study can entirely rule out degradation of materials, even in the absence of statistically significant trends. It is therefore necessary to quantify the potential degradation that could be hidden by the method repeatability, i.e. to estimate the uncertainty of stability. This means that, even under ideal conditions, the outcome of a stability study can only be that there is no detectable degradation within an uncertainty to be estimated.
The uncertainties of stability during dispatch and storage were estimated, as described in [13]. In this approach, the uncertainty of the linear regression line with a slope of zero was calculated. The uncertainty contributions usts and ults were calculated as the product of the chosen transport time/shelf life and the uncertainty of the regression lines as:
( ) tt
i
relrelsts t
tt
su ⋅−
=∑
2, Equation 5
( ) sl
i
relrellts t
tt
su ⋅−
=∑
2, Equation 6
srel relative standard deviation of all results of the stability study
ti time elapsed at time point i
t mean of all ti
ttt chosen transport time (1 week at below -70 ºC)
tsl chosen shelf life (12 months at -70 ºC)
16
The following uncertainties were estimated:
- usts,rel, the uncertainty of degradation during dispatch. This was estimated from the -70 °C study. The uncertainty describes the possible change during a dispatch on dry ice lasting for one month.
- ults,rel, the stability during storage. This uncertainty contribution was estimated from the -70 °C study. The uncertainty contribution describes the possible degradation during 12 months storage at -70 °C.
The results of these evaluations are summarised in Table 2.
Table 2: Uncertainties of stability during dispatch and storage. usts,rel was calculated for a temperature of below -70 °C and 1 month; ults,rel was calculated for a storage temperature of -70 °C and 12 months
usts ,rel
[%] ults,rel
[%] HbA1c amount of substances fraction 0.05 % 0.65 %
No significant degradation during dispatch even at -70 °C was observed. Therefore, the material can be transported on dry ice.
After the certification study, the material will be included in the IRMM's regular stability monitoring programme, to control its further stability.
6 Characterisation
The material characterisation is the process of determining the property value(s) of a reference material.
The characterisation of the material was performed using the IFCC reference measurement procedure [5,6]. After cleavage of haemoglobin into peptides, the N-terminal hexapeptide corresponding to the β-chain of haemoglobin is selected as final and unique target for the determination of both forms HbA0 and HbA1c, the latter defined as beta-N-(1-deoxyfructose-1-yl).
HPLC-ESI/MS and HPLC-CE/UV were used to determine the identity and amount-of-substance fraction of the isoform of HbA1c forming the glycated N-terminal hexapeptide of the β-chain versus HbA1c plus HbA0 and other isoforms forming the non-glycated hexapeptide of the β-chain. This quantity is referred to as the amount-of-substance fraction of HbA1c versus HbA1c plus HbA0.
6.1 Selection of participants Two laboratories were selected based on criteria that comprised both technical competence and quality management aspects. Each participant was required to operate a quality system and to deliver documented evidence of its laboratory proficiency in the use of the IFCC reference measurement procedure for HbA1c. Having a formal accreditation was not mandatory, but meeting the requirements of ISO/IEC 17025 was obligatory. Additionally, one of the participating laboratories was accredited for ISO 15195:2004 for laboratory medicine - Requirements for reference measurement laboratories.
6.2 Study setup
Solutions containing purified HbA1c and HbA0, respectively, were produced by Roche Diagnostic GmbH (DE) and provided to laboratory 1 (L01). L01 assessed the amount of
17
substance fraction of HvA1c in the bulk materials using the IFCC reference measurement procedure employing HPLC-ESI/MS with a standard addition protocol according to Finke et al. [9]. A second dataset for the characterisation of the base materials was obtained by HPLC-CE/UV by laboratory 2 (L02). The mass fraction of total haemoglobin was determined using the recommended ICSH reference method (Drabkin method) [8]. The solutions were mixed gravimetrically in order to produce 6 solutions with different amount-of-substance fractions of HbA1c, and approximately equal total haemoglobin concentrations. The amount-of-substance fraction of HbA1c in the 6 levels was verified by L01 and L02 using the two variants of the reference measurement procedure, employing HPLC-ESI/MS and HPLC-CE/UV, respectively, calibrated with sets of calibrators that were independent from the present material.
6.3 Characterisation of the base materials
The batch characterisation of the base materials (purified protein solutions for HbA1c and HbA0) was performed according to the IFCC reference measurement procedure using HPLC-ESI/MS and HPLC-CE/UV [9]. With this approach the purity is determined for Hba1c (as defined by the reference method, haemoglobin glycated on the N-terminal fragment generated by cleavage of haemoglobin by Glu-C) or HbA0 (not glycated on the N-terminus) with respect to the total haemoglobin content (sum of HbA1c and HbA0). The values are summarised in Table 3.
Table 3: Results for the characterisation of HbA1c and HbA0 solutions
Replicate
HbA1c amount of substances fraction in the HbA1c solution
[%]
HbA0 amount of substances fraction in
the HbA0 solution (L01) [%]
L01 L02 1 91.1 90.47 >99.98 2 91.7 90.41 >99.98 3 91.8 90.49 >99.98 4 90.8 90.54 >99.98 Mean 91.35 90.48 >99.98 SD 0.48 0.05 --
Results for the measurement of the total haemoglobin concentration in the base materials by the method of Drabkin [8] are summarised in Table 4. The uncertainty associated with the repeatability is the relevant contribution to the uncertainty, as other uncertainty contributions cancel out in the calculation of the amount-of-substance fraction.
Table 4: Total haemoglobin content in HbA1c and HbA0 base materials
Replicate Total haemoglobin in the HbA1c solution
[mg/g]
Total haemoglobin in the HbA0 solution
[mg/g] 1 30.91 159.92 2 30.91 161.34 3 31.19 160.99 4 31.31 160.36 5 31.07 160.29 6 31.16 161.27 Mean 31.09 160.70 SD 0.16 0.58
18
6.4 Amount-of-substance fractions and their uncertainties The certified amount-of-substance fractions are based on the gravimetry for the mixing of HbA1c and HbA0 solutions, taking into account the amount-of-substance fractions in the HbA1c and HbA0 base solutions and their total haemoglobin concentration. The estimated uncertainties related to the characterisation were derived by combining the uncertainty related to the repeatability for the determination of total haemoglobin in the HbA1c and HbA0 solutions (0.21 and 0.13 %, respectively) with the uncertainty for the purity estimation. For the HbA1c base material the uncertainty for the purity estimation was derived assuming a rectangular distribution for the two datasets (urec = 0.56 %). This urec was combined with the uncertainties related to the repeatability of the datasets from L01 and L02 (0.26 % and 0.029 %, respectively). The uncertainty related to the weighings was negligible. For the HbA0 base the uncertainty was estimated on the basis of the limit of detection (LOD) of the IFCC reference procedure. The certified value for the blank level is within the interval [0,0.4] mmol/mol with a level of confidence of about 95 %.
Table 5: Amount-of-substance fractions of HbA1c based on gravimetry
Mass HbA1c solution
Mass HbA0 solution
[HbA1c] amount-of-substance fraction uchar, rel
ERM-AD500/IFCC
[g] [g] mmol/mol %
Blank 0.00000 6.29064 0.0 -
Level 1 1.06404 6.33584 28.61 0.66
Level 2 2.12765 6.05850 57.84 0.66
Level 3 3.17217 5.81923 86.73 0.66
Level 4 4.24155 5.45774 118.83 0.66
Level 5 5.28833 5.04118 153.38 0.66
6.5 Verification measurements
The values were confirmed using the IFCC reference measurement procedure for the measurement of the HbA1c amount of substance concentration in the fully processed materials. Two variants of the IFCC measurement procedure were used, employing HPLC-ESI/MS and HPLC-CE/UV, respectively (measurements performed in laboratories L01 and L02, respectively).
19
Table 6: Results from verification measurements of the amount-of-substance fractions of HbA1c based on the IFCC reference method
HbA1c amount-of-substance fraction
L01 Average (SD)1)
L02 Average (SD) 1)
Average between L01
and LO2 Gravimetry
Difference between
gravimetry and confirmation
measurements
ERM-AD500/IFCC
mmol/mol mmol/mol mmol/mol mmol/mol mmol/mol
Blank <LOQ <LOQ <LOQ 0.0 -
Level 1 28.7 (0.2) 29.2 (0.8) 29.0 28.6 0.3
Level 2 58.6 (0.8) 58.7 (0.9) 58.7 57.8 0.8
Level 3 87.4 (1.0) 87.4 (0.8) 87.4 86.7 0.7
Level 4 119.5 (0.8)
119.1 (0.9) 119.3 118.8 0.5
Level 5 154.6 (0.8)
153.3 (0.9) 153.9 153.4 0.5
1) Standard deviation over triplicate measurements
The results of the verification measurements from the two independent laboratories agree with each other and agree with the values derived from gravimetric mixing of base materials within the uncertainties.
7 Value Assignment
Certified values were assigned.
Certified values are values that fulfil the highest standards of accuracy. Full uncertainty budgets in accordance with the 'Guide to the Expression of Uncertainty in Measurement' [7] were established.
7.1 Certified values and their uncertainties The certified values are based on the masses and HbA1c amount-of-substance fractions of the HbA1c and HbA0 base materials used in the gravimetrical preparation.
The assigned uncertainty consists of uncertainties relating to characterisation, uchar (Section 6), potential between-unit inhomogeneity, ubb (Section 4.1), and potential degradation during transport, usts, and long-term storage, ults (Section 5). These different contributions were combined to estimate the relative expanded uncertainty of the certified value (UCRM, rel) with a coverage factor k given as:
2rel char,
2rel lts,
2rel sts,
2rel bb,rel CRM, uuuukU +++⋅= Equation 7
- uchar was estimated as described in Section 6.4
- ubb was estimated as described in Section 4.1.
20
- usts and ults were estimated as described in section 5.3
The uncertainty related to degradation during transport was found to be negligible.
For the blank level, the LOD of the IFCC reference method using the standard addition approach was used to describe the 95 % confidence interval of the certified mass fraction ([0.0, 0.4] mmol/mol). This was supported by the high purity of the HbA0 base material and the absence of any mixing step.
A coverage factor k of 2 was applied to obtain the expanded uncertainties. The certified values and their uncertainties are summarised in Table 7.
Table7: Certified values and their uncertainties for ERM-AD500/IFCC
ERM-AD500/IFCC
Certified value1) [mmol/mol]
uchar
[mmol/mol]
ubb
[mmol/mol]
usts
[mmol/mol]
ults,
[mmol/mol]
UCRM
[mmol/mol]
Blank 0.0 -0.0 ; +0.2 n.a.4) n.a.4) n.a.4) -0.0 ; +0.42)
Level 1 28.6 0.19 0.34 <0.1 0.19 0.93)
Level 2 57.8 0.38 0.29 <0.1 0.38 1.33)
Level 3 86.7 0.58 0.69 <0.1 0.56 2.23)
Level 4 118.8 0.79 0.59 <0.1 0.77 2.63)
Level 5 153 1.02 1.69 <0.1 1.00 53) 1) Reported on the basis of gravimetric mixing (Section 6.4) 2) Expanded uncertainty with a confidence level of about 95 %. 3) Expanded (k = 2) and rounded uncertainty. 4) n.a.: not applicable
7.2 Additional material information The data provided in this section should be regarded as informative only on the general composition of the material and cannot be, in any case, used as certified or indicative value. The mass fraction of total Hb in solution was determined by using the ICSH reference method (Drabkin method) [8]. This colorimetric method was applied by two different laboratories. The results are presented in Table 7.
21
Table7: Total Haemoglobin (Hb) mass concentration in ERM-AD500/IFCC as determined with the Drabkin method.
Haemoglobin mass concentration
L01 L02 Mean
ERM-AD500/IFCC
[g/L] [g/L] [g/L]
Blank 33.6 34.3 34.0
Level 1 34.7 34.1 34.4
Level 2 34.4 34.9 34.7
Level 3 34.2 34.7 34.5
Level 4 33.6 34.0 33.8
Level 5 32.6 33.4 33.0
8 Metrological traceability and commutability
8.1 Metrological traceability
The measurand is the amount-of-substance fraction of the isoforms of HbA1c forming the glycated N-terminal hexapeptide of the β-chain versus HbA1c plus HbA0 and other isoforms forming the non-glycated hexapeptide of the β-chain, as determined by the IFCC reference measurement procedure [5,6]. This quantity is referred to as the amount-of-substance fraction of HbA1c versus HbA1c plus HbA0. The measurand is method-defined and can only be obtained by following the specified procedure. The measurand is therefore operationally defined by the IFCC reference measurement procedure.
Quantity value
Traceability of the obtained results is based on the traceability of all relevant input factors, including the balances used for the gravimetric mixing. Instruments in individual laboratories were verified and calibrated with tools ensuring traceability to the International System of units (SI). The values are therefore traceable to the SI.
8.2 Commutability
The intended use of this reference material is the calibration of the IFCC reference measurement procedure and analogous methods evaluating the stable glycated N-terminal hexapeptide of the β-chain of Hb.
Commutability of the material with routine in vitro diagnostic devices has not been assessed. The user would need to assess the commutability if ERM-AD500/IFCC is used as calibrant for routine in vitro diagnostic devices.
22
9 Instructions for use
9.1 Safety information The usual laboratory safety measures apply.
The material is for in-vitro use only.
9.2 Storage conditions
Shipment will be carried out on dry ice. On receipt, the materials should be stored in the dark at -70 °C.
Please note that the European Commission cannot be held responsible for changes that happen during storage of the material at the customer's premises, especially for opened ampoules.
9.3 Use of the material The intended use of this reference material is the calibration of the IFCC reference measurement procedure as implemented by IFCC Network for HbA1c (http://www.ifcchba1c.net/), evaluating the stable glycated N-terminal hexapeptide of the β-chain of Hb. As with any reference material, it can be used for establishing control charts or validation studies.
The ampoules contain about 1 mg haemoglobin, which is also the amount of protein needed for the preparation of a sample for the reference measurement procedure. Therefore the first step of the procedure (addition of Glu-C enzyme solution and digestion buffer, bringing it to a final volume of about 500 µL) can be performed directly in the ampoules before transferring the solution to another vial for the incubation step.
To make them ready for use the ampoules have to be brought to room temperature. They are opened by breaking off the upper part. First gently tap with your finger to get all the liquid to the bottom part of the ampoule. Protect your hands from broken glass by using a paper towel, light cloth or piece of gauze when opening the ampoule. To open the ampoule, hold it with both hands, with one thumb against the narrow top section. The black spot should be turned away from the thumb. Hold the bottom of the ampoule firmly while pulling the top section toward you with easy, even pressure. A light pressure should cleanly snap the ampoule open.
For the calibration of measurements according to the IFCC reference method the certified values of ERM-AD500/IFCC should be used. The calibration curve should not be forced through the origin.
9.4 Minimum sample intake The minimum sample intake is the whole content of the ampoule.
9.5 Use of the certified value The purpose of this material is the calibration of the IFCC reference measurement procedure for the amount-of-substance fraction of HbA1c versus HbA1c plus HbA0.
Comparing an analytical result with the certified value
A result is unbiased if the combined standard uncertainty of measurement and certified value covers the difference between the certified value and the measurement result (see also ERM Application Note 1, www.erm-crm.org [14].
23
When assessing the method performance, the measured values of the CRMs are compared with the certified values. The procedure is summarised here:
- Calculate the absolute difference between mean measured value and the certified value (∆meas).
- Combine the measurement uncertainty (umeas) with the uncertainty of the certified value (uCRM): 22
CRMmeas uuu +=∆
- Calculate the expanded uncertainty (U∆) from the combined uncertainty (u∆,) using an appropriate coverage factor, corresponding to a level of confidence of approximately 95 %
- If ∆meas ≤ U∆ then no significant difference exists between the measurement result and the certified value, at a confidence level of approximately 95 %.
24
10 Acknowledgments
The authors would like to acknowledge the support received from Albert Oostra, John Seghers, Paul De Vos, Håkan Emteborg, Virginie Tregoat, Sébastien Boulo, Guy Auclair, Brigitte Toussaint, Elena Scaravelli, Jenny Chung, Dana Hutu, and Katja Hanisch from IRMM relating to the processing of this CRM and from Maria Concepcion Contreras concerning the set-up of the required isochronous studies. Authors would also like to thank Janine Slootstra and Robbert Slingerland (Isala, Zwolle, NL) and Carla Siebelder (Streekziekenhuis Koningin Beatrix, Winterswijk,NL) for their contribution to the production of the material.
Furthermore, the authors would like to thank Katrien Busschots, Tsvetelina Gerganova and Robert Koeber (IRMM) for reviewing the certification report, as well as the experts of the Certification Advisory Panel "Biological Macromolecules and Biological/Biochemical Parameters", H. Hird (Food and Environmental Research Agency, UK), M. Wagner (University for Veterinary Medicine Vienna, AT) and L. Siekmann (University of Bonn, DE) for their constructive comments.
25
11 References
1 K. Miedema, Standardization of HbA1c and optimal range of monitoring. Scand. J. Clin. Lab. Invest. Suppl., 240 (2005) 61-72
2 ISO Guide 34:2009, General requirements for the competence of reference materials producers, International Organization for Standardization, Geneva, Switzerland
3 ISO Guide 35:2006, Reference materials – General and statistical principles for certification, International Organization for Standardization, Geneva, Switzerland
4 J. Büttner, Proceeding of the IFCC Meeting on Reference Materials and Reference Systems co-sponsored by WHO. Eur. J. Clin. Chem. Clin. Biochem., 33 (1995) 975-1022
5 J.-O. Jeppsson, U. Kobold, J. Barr, A. Finke, W. Hoelzel, T. Hoshino, K. Miedema, A. Mosca, P. Mauri, R. Paroni, L. Thienpont, M. Umemoto, C. W. Weykamp, Approved IFCC reference method for the measurement of HbA1c in human blood. Clin. Chem. Lab. Med. 40 (2002) 78-89
6 P. Kaiser, T. Akerboom, P. Molnar, H. Reinauer, Modified HPLC-Electrospray Ionization/Mass Spectrometry Method for HbA1c Based on IFCC Reference Measurement Procedure. Clin. Chem. 54 (2008) 1018-1022
7 ISO/IEC Guide 98-3:2008, Guide to the Expression of Uncertainty in Measurement, (GUM 1995), International Organization for Standardization, Geneva, Switzerland
8 International Committee for Standardization in Haematology (ICSH), Recommendations for reference method for haemoglobinometry in human blood (ICSH Standard EP 6/2 1977) and specifications for international haemoglobincyanide reference preparation (ICSH Standard EP 6/3 1977). J. Clin. Pathol. 31 (1978) 139-143
9 A. Finke, U. Kobold, W. Hoelzel, C. W. Weykamp, K. Miedema, J.-O. Jeppsson, Preparation of a candidate primary reference material for the international standardisation of HbA1c determinations. Clin. Chem. Lab. Med., 36 (1998) 299-308
10 G.E. O’Donnell, D.B: Hibbert, Treatment of bias in estimating measurement uncertainty, Analyst 130 (2005) 721-729
11 T.P.J. Linsinger, J. Pauwels, A.M.H. van der Veen, H. Schimmel, A. Lamberty, Homogeneity and stability of reference materials, Accred. Qual. Assur. 6 (2001) 20-25
12 A. Lamberty, H. Schimmel, J. Pauwels, The study of the stability of reference materials by isochronous measurements, Fres. J. Anal. Chem. 360 (1998) 359-361
13 T.P.J. Linsinger, J. Pauwels, A. Lamberty, H. Schimmel, A.M.H. van der Veen, L. Siekmann, Estimating the uncertainty of stability for matrix CRMs, Fres. J. Anal. Chem. 370 (2001) 183-188
14 T.P.J. Linsinger, ERM Application Note 1: Comparison of a measurement result with the certified value, www.erm-crm.org (last accessed on 18/11/2015)
26
Annexes
Annex A: Results of the homogeneity measurements
Level 1
The triangles, crosses and stars correspond to the first, second and third replicate, respectively.
Level 2
The triangles, crosses and stars correspond to the first, second and third replicate, respectively.
0
5
10
15
20
25
30
35
0 200 400 600 800 1000
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Unit number
0
10
20
30
40
50
60
70
0 200 400 600 800 1000
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Unit number
27
Level 3
The triangles, crosses and stars correspond to the first, second and third replicate, respectively.
Level 4
The triangles, crosses and stars correspond to the first, second and third replicate, respectively.
0
10
20
30
40
50
60
70
80
90
100
0 200 400 600 800 1000
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Unit number
0
20
40
60
80
100
120
140
0 200 400 600 800 1000
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Unit number
28
Level 5
The triangles, crosses and stars correspond to the first, second and third replicate, respectively.
0
20
40
60
80
100
120
140
160
180
0 200 400 600 800 1000
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Unit number
29
Annex B: Results of the short-term stability measurements
4 °C
The diamonds and the squares correspond to the first and second replicate, respectively
-40 °C
The diamonds and the squares correspond to the first and second replicate, respectively
0
20
40
60
80
100
120
0 1 2 3 4
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Time in week
0
20
40
60
80
100
120
0 1 2 3 4
Hb
A1
c a
mo
un
t-o
f-su
bst
an
ce f
ract
ion
[mm
ol/
mo
l]
Time in week
30
Annex C: Results of the long-term stability measurements
0.00
10.00
20.00
30.00
40.00
50.00
60.00
0 3 6 9 12
Hb
A1
c a
mo
un
t-o
f-su
bst
an
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ract
ion
[mm
ol/
mo
l]
Months
European Commission
EUR 27574 EN – Joint Research Centre – Institute for Reference Materials and Measurements
Title: CERTIFICATION REPORT Certification of the amount-of-substance fraction of HbA1c versus the sum of
HbA0 and HbA1c in haemoglobin: ERM®- AD500/IFCC
Author(s): Amalia Muñoz, Ingrid Zegers, Jean Charoud-Got, Cas Weykamp, Renata Paleari, Andrea Mosca, Uwe Kobold, Heinz
Schimmel, Hendrik Emons
Luxembourg: Publications Office of the European Union
2016 – 30 pp. – 21.0 x 29.7 cm
EUR – Scientific and Technical Research series – ISSN 1831-9424
ISBN 978-92-79-53878-0
doi: 10.2787/7609
As the Commission's in-house science service, the Joint Research Centre's mission is to provide EU policies
with independent, evidence-based scientific and technical support throughout the whole policy cycle.
Working in close cooperation with policy Directorates-General, the JRC addresses key societal challenges
while stimulating innovation through developing new methods, tools and standards, and sharing its know-
how with the Member States, the scientific community and international partners.
Key policy areas include: environment and climate change; energy and transport; agriculture and food
security; health and consumer protection; information society and digital agenda; safety and security,
including nuclear; all supported through a cross-cutting and multi-disciplinary approach.
LA
-NA
-27
57
4-E
N-N
doi: 10.2787/7609
ISBN: 978-92-79-53878-0