The certification of anti-myeloperoxidase immunoglobulin in human serum ERM® - DA476/IFCC certification report.
Date: 2015-04-08
Institutions: Institute for Reference Materials and Measurements
Subjects: Community certification, analytical chemistry, medical research, research body, research method, research report

CERTIFICATION REPORT 

The certification of anti-myeloperoxidase immunoglobulin G 
in human serum ERM® - DA476/IFCC 

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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)  

Contact information 
Reference materials sales 
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All images © European Union 2015 

JRC94458 

EUR 27092 EN 

ISBN 978-92-79-45349-6(PDF) 
ISSN 1831-9424 (online) 
doi:10.2787/798324 

Luxembourg: Publications Office of the European Union, 2015 
© European Union, 2015 

Reproduction is authorised provided the source is acknowledged. 
Printed in Belgium 

Abstract 
This report describes the production and certification of ERM-DA476/IFCC, a new serum protein reference material intended for the standardisation of 
measurements of anti-myeloperoxidase immunoglobulin G (anti-MPO IgG) antibodies. The material was produced according to ISO Guide 34:2009 . 
The raw material used to prepare ERM-DA476/IFCC was a plasmapheresis material containing a high concentration of anti-MPO IgG. After a thorough 
commutability study lyophilised serum was selected as the format for the candidate reference material. Serum processing was performed based on 
the procedure used for the reference material ERM-DA470k/IFCC . The plasma was converted into serum which was then delipidated. After the 
addition of preservatives the processed serum was diluted with plasmapheresis solution containing albumin, prior to the transfer of 1 mL aliquots to 
glass vials. The serum was then lyophilised and the vials closed with rubber stoppers and screw caps under nitrogen atmosphere prior to storage at -
70 °C.  
The between unit-homogeneity was quantified and stability during dispatch and storage were assessed in accordance with ISO Guide 35:2006.  
The material was characterised by an inter-laboratory comparison exercise performed by laboratories of demonstrated competence and with 
adherence to ISO/IEC 17025, using a purified anti-MPO IgG preparation as calibrant. This was achieved using a value transfer protocol previously used 
in the characterisation of ERM-DA470k/IFCC [3]. Technically invalid results were removed. However no other outliers were eliminated on statistical 
grounds only.  
Uncertainties of the certified values were calculated in accordance to the Guide to the Expression of Uncertainty in Measurement (GUM) and include 
uncertainties relating to possible lack of homogeneity, instability and characterisation. 
The material is intended for the calibration of methods and quality control. As any reference material, it can also be used for control charts or 
validation studies. The CRM is available in glass vials containing the lyophilised residue of 1 g serum. The minimum amount of reconstituted sample 
to be used is 10 µL. 
The CRM was accepted as European Reference Material (ERM®) after peer evaluation by the partners of the European Reference Materials consortium.

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
CERTIFICATION REPORT 

The certification of anti-myeloperoxidase 
immunoglobulin G in human serum ERM® - DA476/IFCC 

Evanthia  Monogioudi1),  Dana  P.  Hutu1),  Gustavo  Martos1),  Emma  Tuddenham2), 
Joanna  Sheldon2),  Heinz  Schimmel1),  Stefanie  Trapmann1),  Pierluigi  Meroni3), 
Hendrik Emons1) and Ingrid Zegers1) 

1)European Commission, Joint Research Centre 
Institute for Reference Materials and Measurements (IRMM), 
B-2440 Geel (BE) 

2)Protein Reference Unit and Immunopathology Department, 
St Georges' Hospital, London (UK) 

3)Department of Clinical Sciences and Community Health & Istituto Auxologico Italiano, 
Milan (IT) 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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.  

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Summary 

This  report  describes  the  production  and  certification  of  ERM-DA476/IFCC,  a  new  serum 
protein  reference  material  intended  for  the  standardisation  of  measurements  of  anti-
myeloperoxidase immunoglobulin G (anti-MPO IgG) antibodies. The material was produced 
according to ISO Guide 34:2009 [1]. 

The  raw  material  used  to  prepare  ERM-DA476/IFCC  was  a  plasmapheresis  material 
containing  a  high  concentration  of  anti-MPO  IgG.  After  a  thorough  commutability  study 
lyophilised serum was selected as the format for the candidate reference material [2]. Serum 
processing  was  performed  based  on  the  procedure  used  for  the  reference  material  ERM-
DA470k/IFCC  [3].  The  plasma  was  converted  into  serum  which  was  then  delipidated.  After 
the addition of preservatives the processed serum was diluted with plasmapheresis solution 
containing albumin, prior to the transfer of 1 mL aliquots to glass vials. The serum was then 
lyophilised  and  the  vials  closed  with  rubber  stoppers  and  screw  caps  under  nitrogen 
atmosphere prior to storage at -70 °C.  

The between unit-homogeneity was quantified and stability during dispatch and storage were 
assessed in accordance with ISO Guide 35:2006 [4].  

The  material  was  characterised  by  an  inter-laboratory  comparison  exercise  performed  by 
laboratories of demonstrated competence and with adherence to ISO/IEC 17025 [5], using a 
purified  anti-MPO  IgG  preparation  as  calibrant.  This  was  achieved  using  a  value  transfer 
protocol previously used in the characterisation of ERM-DA470k/IFCC [3]. Technically invalid 
results were removed. However no other outliers were eliminated on statistical grounds only.  

Uncertainties  of  the  certified  values  were  calculated  in  accordance  to  the  Guide  to  the 
Expression  of  Uncertainty  in  Measurement  (GUM)  [6]  and  include  uncertainties  relating  to 
possible lack of homogeneity, instability and characterisation. 

The material is intended for the calibration of methods and quality control. As any reference 
material, it can also be used for control charts or validation studies. The CRM is available in 
glass  vials  containing  the  lyophilised  residue  of  1  g  serum.  The  minimum  amount  of 
reconstituted sample to be used is 10 µL. 

The  CRM  was  accepted  as  European  Reference  Material  (ERM®)  after peer  evaluation  by 
the partners of the European Reference Materials consortium. 

The following value was assigned: 

Certified value 2) 
[mg/L] 

anti-MPO IgG1) 

84 

1) Anti-myeloperoxidase immunoglobulin G as measured by immunoassays 

Uncertainty 3) 
[mg/L] 

9 

Mass Concentration  

2) Unweighted mean value of the means of accepted data sets each set obtained in a different laboratory and/or with 
a different method of determination. The certified mass concentration and its uncertainty are traceable to the stated 
value of the mass concentration in United States National Reference Preparation (USNRP) 12-0575C (Reimer et al., 
Am. J. Clin. Pathol. 77 (1982) 12-19) 

3)  The  certified  uncertainty  is  the  expanded  uncertainty  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 

The minimum sample intake is 10 µL 

1 

 
 
 
Table of contents 

Summary .............................................................................................................................. 1 
Table of contents ................................................................................................................. 3 
Glossary ............................................................................................................................... 5 
Introduction .............................................................................................................. 9 
1. 
Background ............................................................................................................... 9 
1.1 
Choice of the material ................................................................................................ 9 
1.2 
Design of the project .................................................................................................10 
1.3 
Participants .............................................................................................................12 
2. 
Provision of raw materials .........................................................................................12 
2.1 
Project management and evaluation .........................................................................12 
2.2 
Processing ................................................................................................................12 
2.3 
Homogeneity and stability studies .............................................................................12 
2.4 
Characterisation ........................................................................................................12 
2.5 
3. 
Material processing and process control .............................................................13 
3.1       Origin of the starting material ....................................................................................13 
3.2       Processing and processing control ...........................................................................13 
3.2.1.   Plasma conversion into serum ..................................................................................13 
3.2.2.   Serum processing .....................................................................................................13 
3.2.3.   Filling ........................................................................................................................13 
Homogeneity ...........................................................................................................14 
4. 
4.1 
Between-unit homogeneity........................................................................................14 
4.2  Within-unit homogeneity and minimum sample intake...............................................15 
Stability....................................................................................................................16 
5. 
Short-term stability study ..........................................................................................16 
5.1 
Long-term stability study ...........................................................................................17 
5.2 
Estimation of uncertainties ........................................................................................17 
5.3 
Characterisation .....................................................................................................18 
6. 
Selection of participants ............................................................................................18 
6.1 
General principles of the value assignment ...............................................................19 
6.2 
6.3 
Characterisation of the calibrant ...............................................................................21 
6.3.1.   Production and physico-chemical characterisation of the calibrant ...........................21 
6.3.2.   Value assignment of the calibrant .............................................................................22 
Characterisation of ERM-DA476/IFCC ......................................................................23 
6.4 
Uncertainty budget and certified values ...............................................................24 
7. 
Certified value ...........................................................................................................25 
7.1 
7.2 
Estimation of uncertainty ...........................................................................................25 
Metrological traceability and commutability .........................................................26 
8. 
8.1  Metrological traceability ............................................................................................26 

3 

 
Commutability ...........................................................................................................26 
8.2 
Instructions for use ................................................................................................27 
9. 
Safety information .....................................................................................................27 
9.1 
Storage conditions ....................................................................................................27 
9.2 
9.3 
Preparation and use of the material/Reconstitution ...................................................27 
9.4  Minimum sample intake ............................................................................................28 
Use of the certified value ..........................................................................................28 
9.5 
Acknowledgments ..............................................................................................................29 

References ..........................................................................................................................30 

Annexes…………………………………………………………………………...…………………31 

4 

Glossary 

ANCAs 

Anti-MPO 

ANOVA  

b 

c 

cij 

c0i 

Anti-neutrophil cytoplasmic antibodies 

Anti-myeloperoxidase 

Analysis of variance 

Slope in the equation of linear regression y = a + bx 

Mass concentration c = m / V (mass / volume) 

Concentration of the protein in dilution j of material i 

Concentration of the protein in material i 

canti-MPO IgG 

Concentration of the purified anti-MPO IgG 

cERM-DA476/IFCC 

Concentration of anti-MPO IgG in the ERM-DA476/IFCC 

CLSI 

CRM 

CR 

Cs 

CT 

df 

EC 

ELISA 

EQAS 
ERM® 

EU 

FR 

FT 

GUM 

HEPES 

HIV 

i 

IFCC 

IgG 

ISO 

IU 

IVD-MD 

JRC-IRMM 

j 

k 

Clinical and Laboratory Standards Institute 

Certified Reference Material 

Mass concentration of the protein in the calibrant 

Signals of unknown test samples 

Mass concentration of the protein in the target material 

Degrees of freedom 

European Commission 

Enzyme-linked immunosorbent assay 

External Quality Assurance Scheme 

Trademark of European Reference Materials 

European Union 

Dilution factor of the calibrant 

Dilution factor of TM 

Guide to the Expression of Uncertainty in Measurements  

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid 

Human immunodeficiency virus 

Different dilutions of TM 

International Federation of Clinical Chemistry and Laboratory Medicine 

Immunoglobulin G 

International Organization for Standardization  

International units 

In Vitro Diagnostic - Medical Devices 

Joint Research Centre – Institute of Reference Materials and 
Measurements 

Dilutions of the calibrant 

Coverage factor 

5 

 
M 

mintended 

mDD,j 

mDM,i 

mM,i 

mPD,i 

mPR,i 

MS 

MSbetween 

MSwithin  

n 

n.c. 

PES 

QC 

rel 

RM 

RSD 

RT 
R2 

s 

sbb

Molar mass 

Mass intended to be added 

Mass of diluent for dilution j 

Mass of the reconstituted material 

Measured mass of water added 

Mass of the diluent for the predilution 

Mass of the reconstituted material 

Mean of squares 

Mean of squares between-unit from an ANOVA 

Mean of squares within-unit from an ANOVA 

Number of replicates per unit 

Not calculated 

Polyethersulfone  

Quality Control 

Index denoting relative figures (uncertainties etc.) 

Reference Material 

Relative standard deviation 

Room Temperature 

Coefficient of determination of the linear regression 

Standard deviation 

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 

SEC 

sijk 

SS 

swithin 

swb 

T 

t 

ti 

t

ta

, df 

tsl 

ttt 

Size Exclusion Chromatography 
Signal of the kth measurement of material i within dilution j 

Sum of squares 

Standard deviation within groups as obtained from ANOVA; an additional 
index "rel" is added as appropriate 

Within-unit standard deviation 

Temperature 

Time 

Time elapse at time point i 

Mean of all ti 

Critical t-value for a t-test, with a level of confidence of 1-a
degrees of freedom 

 and df 

Chosen shelf life 

Chosen transport time 

6 

 
 
TIU 

TM 

TF 

TRIS 

u 

U 

ub 

ubal 

u*

bb  

ubb 

uc 

uchar,cal 

uchar  

uCRM 

UCRM  

Trypsin inhibitor unit 

Target material 

Transfer factor 

Tris(hydroxymethyl)aminomethane 

Standard uncertainty  

Expanded uncertainty 

Standard uncertainty of the slope 

Standard uncertainty related to the balance; an additional index "rel" is 
added as appropriate 

Standard uncertainty related to a maximum between-unit inhomogeneity 
that could be hidden by method repeatability; an additional index "rel" is 
added as appropriate 

Standard uncertainty related to a possible between-unit inhomogeneity;  
an additional index "rel" is added as appropriate 

Combined standard uncertainty; an additional index "rel" is added as 
appropriate 

Standard uncertainty of the calibrant characterisation; an additional index 
"rel" is added as appropriate 

Standard uncertainty of the material characterisation; an additional index 
"rel" is added as appropriate 

Combined standard uncertainty of the certified value; an additional index 
"rel" is added as appropriate 

Expanded uncertainty of the certified value; an additional index "rel" is 
added as appropriate 

uERM-DA470k/IFCC 

Standard uncertainty of the certified value for ERM-DA470k/IFCC 

u∆ 

ults 

upur 

usts 

Combined standard uncertainty of measurement result and certified 
value 

Standard uncertainty of the long-term stability; an additional index "rel" is 
added as appropriate 

Standard uncertainty for the purity of the calibrant; an additional index 
"rel" is added as appropriate 

Standard uncertainty of the short-term stability; an additional index "rel" 
is added as appropriate 

USNRP 

United States National Reference Preparation 

UV 

V 

y  

a  
D meas 

Ultraviolet 

Volume 

Mean of all results of the homogeneity study 

Significance level 

Absolute difference between mean measured value and the certified 
value 

7 

 
 
 
νMSwithin 
WG-HAT 

Degrees of freedom of MSwithin 

Working Group – Harmonisation of Autoantibody Tests 

8 

1. Introduction  

1.1 Background 

Autoimmune antibodies are important analytes in laboratory medicine. The measurement of 
their concentrations is used in routine medical evaluations and in a large number of specific 
indications, and can be performed repeatedly without (major) injury to the patient.  

In  2009  the  International  Federation  of  Clinical  Chemistry  and  Laboratory  Medicine  (IFCC) 
formed  a  new  working  group  with  a  mandate  for  the  Harmonisation  of  Autoantibody  Tests 
(known as WG-HAT). The detection and quantification of IgG antibodies to autoantigens are 
important for the diagnosis and monitoring of a number of autoimmune diseases. For every 
autoantibody  in  routine  use,  there  is  currently  marked  diversity  in  the  response  of  methods 
available  for  analysis  and  the  materials  used  for  assay  calibration.    There  are  materials 
designated  as  standards  for  some  of  these  methods,  however  they  are  not  fully 
characterised  and  often  used  inconsistently.  This  generates  large  variability  in  the  analysis 
results  (as  shown  in  EQAS  results)  and  a  potential  delay  in  the  diagnosis  and  subsequent 
follow-up of the disease.  

The  immunoassays  used  for  the  measurement  of  autoimmune  antibodies  can  be  sensitive 
and  specific,  and  are  convenient  in  a  clinical  setting  because  they  give  fast  results.  The 
signal  is  dependent  on  a  large  number  of  factors  such  as  antibody  specificity,  reaction 
kinetics and equilibria, multimeric state of the proteins, complex matrix effects, etc. Therefore 
the quantification with immunoassays requires the use of a proper calibrant.  

The  EU  Directive  on  In  Vitro  Diagnostic  Medical  Devices  (IVD-MD)  (Directive  98/79/EC) 
requires traceability of calibrants and control materials to reference measurement procedures 
and/or reference materials of higher order. 

A  calibrant  is  required  to  have  an  assigned  value  that  is  metrologically  traceable,  and 
accompanied by an uncertainty statement. The stability and homogeneity with respect to the 
certified property must be verified, and the calibrant must be commutable, i.e. resemble the 
patient samples [7]. These attributes are particularly challenging for serum protein calibrants, 
as they form a mixture of interacting proteins with different isoforms and complexes.  

1.2 Choice of the material 

One of the type of antibodies selected by IFCC for quantification and standardisation are the 
antibodies  against  the  neutrophil  enzyme  myeloperoxidase.  These  antibodies  are  detected 
as  perinuclear  anti-neutrophil  cytoplasmic  antibodies 
the 
cornerstones of the diagnosis of small vessel associated vasculitis. Microscopic polyangiitis 
and eosinophilic granulomatosis with polyangiitis are disorders mostly associated with ANCA 
antibodies directed against the naturally occurring myeloperoxidase [8].  

(ANCAs)  and 

represent 

The  requirements  for  a  material  to  be  used  as  a  reference  material  for  the  calibration  of 
immunoassay-based in vitro diagnostic devices or control products for anti-MPO IgG quality 
control are, next to requirements for homogeneity, stability, traceability and commutability: 

9 

 
 
(cid:1)  The concentration of the target protein in the final material should be high enough so 
that dilutions of the material can cover the relevant part of the measurement interval 
of the methods.  

(cid:1)  There must be continuity of the measurement results from methods calibrated against 
consecutive reference materials. This is an important issue in clinical chemistry, as 
the use of reference ranges and decision limits requires that measurement results are 
comparable over long time scales. 

According to the data received and analysed, the raw material selected for anti-MPO IgG is 
sufficiently  commutable  to  lead  to  a  considerable  reduction  of  inter-assay  variances. 
Therefore  it  was  decided  to  produce  the  new  material  according  to  procedures  described 
below.  

1.3 Design of the project 

After a thorough commutability study a plasmapheresis material with a high concentration of 
anti-MPO IgG autoantibodies was selected as the starting material [2]. It was converted into 
serum  and  processed  according  to  the  procedure  used  for  the  reference  material  ERM-
DA470k/IFCC.  The  procedure  consists  of  a  delipidation  step  followed  by  the  addition  of 
preservatives (Section 3.2). The processed serum was then transferred to vials (1 mL serum 
per  vial)  and  lyophilised.  The  vials  were  closed  under  nitrogen  with  rubber  stoppers  and 
screw caps and were then stored at -70 °C.  

The homogeneity, short term and long term stability of the material were assessed for anti-
MPO IgG.  

A  calibration  solution  was  prepared  and  characterised  to  facilitate  the  characterisation  of 
ERM-DA476/IFCC.  Anti-MPO 
IgG  was  purified  from  plasmapheresis  material  by  a 
combination  of  affinity  chromatography  and  size  exclusion  chromatography.  A  value  for the 
IgG  concentration  in  the  calibrant  was  assigned  using  5  routine  methods  selective  for  total 
IgG  (based  on  either  turbidimetry  or  nephelometry).  The  value  assignment  was  performed 
using  ERM-DA470k/IFCC  as  a  calibrant.  The  value  obtained  by  immunoassays  was 
consistent with that obtained by UV measurements. 

A value was assigned to the ERM-DA476/IFCC by using the purified anti-MPO IgG calibrant 
spiked  into  human  serum  and  routine  anti-MPO  IgG  procedures  (ELISA,  chemiluminescent 
and  fluoroenzyme  immunoassays).  This  was  achieved  using  a  value  transfer  protocol  that 
can be considered as reference procedure [3]. In the value transfer procedure 6 dilutions of 
the  target  material  were  measured  in  parallel  next  to  6  dilutions  of  the  calibrant.  The 
concentration of the target material was determined against the calibrant solutions (Figure 1). 
The procedure is described in detail in Chapter 6.2. 

The  characterisation  measurements  were  either  performed  under  an  ISO/IEC  17025 
accreditation or within the scope of an ISO 13485 quality system.  

10 

 
 
 
 
 
 
 
 
USNRP
12-0575C 

Total IgG methods

ERM-DA470/IFCC

Total IgG methods

ERM-DA470k/IFCC

Total IgG methods

Purified
anti-MPO IgG

anti-MPO IgG methods

ERM-DA476/IFCC

Figure 1: Traceability chain 

11 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
2. Participants 

2.1 Provision of raw materials  

       Statens Serum Institute, Amager, DK  

2.2 Project management and evaluation 

European Commission, Joint Research Centre, Institute for Reference Materials and 
Measurements  (IRMM),  Geel,  BE  (accredited  to  ISO  Guide  34  for  production  of 
certified reference materials, BELAC No. 268-RM) 

2.3 Processing  
•  European Commission, Joint Research Centre, Institute for Reference Materials and 
Measurements  (IRMM),  Geel,  BE  (accredited  to  ISO  Guide  34  for  production  of 
certified reference materials, BELAC No. 268-RM) 

•  Siemens Healthcare Diagnostics Products GmbH, Marburg, DE  

2.4 Homogeneity and stability studies 

INOVA Diagnostics, INC., San Diego, US  

2.5 Characterisation 

•  AESKU Diagnostics GmbH & Co., Wendelsheim, DE  
•  Bio-Rad Laboratories INC., California, US  
•  DAKO Denmark, Glostrup, DK 
•  EUROIMMUN Medizinische Labordiagnostika AG, Dassow, DE  
•  EuroDiagnostica AB, Malmö, SE  
•  European Commission, Joint Research Centre, Institute for Reference Materials and 
Measurements  (IRMM),  Geel,  BE  (accredited  to  ISO  Guide  34  for  production  of 
certified reference materials, BELAC No. 268-RM) 

IMMCO Diagnostics, Buffalo, US  

INOVA Diagnostics, INC., San Diego, US  

• 
• 
•  Lund University, Lund, SE  
•  Protein  Reference  Unit,  St.  Georges  Hospital,  London,  UK  (certified  to  UKAS  CPA 

1929) 

•  Phadia / Thermo Fisher Scientific, Freiburg, DE  
•  Roche Diagnostics GmbH, Penzberg, DE 
•  Siemens Healthcare Diagnostics Products GmbH, Marburg, DE  

12 

  
 
 
 
3. Material processing and process control 

3.1 Origin of the starting material 

The raw material was a  plasmapheresis material collected from patients diagnosed with an 
autoimmune, non-infectious disease, provided by Statens Serum Institute (DK). It was tested 
and found to be negative for Hepatitis B surface antigen, HIV 1&2, HIV antigen and Hepatitis 
C antibodies. 

3.2 Processing and processing control 

3.2.1. Plasma conversion into serum 

An  aliquot  (1  L)  of  the  plasmapheresis  material  was  thawed  and  warmed  up  to  37  °C.  A 
1/100 volume of 5 mg/mL protamine sulphate solution (Sigma-Aldrich, DE) was then added 
and the material was stirred for 10 minutes at 37 °C. After incubation for one hour at room  
temperature  (RT)  the  material  was  then  incubated  at  4  °C  for  40  hours  to  allow  the 
formation of fibrin. The fibrin was then removed by centrifugation at 10000 rpm for 10 min 
whereupon  the  supernatant  was  filter  sterilized  using  a  0.22  µm  PES  filter  (Corning 
Incorporated, USA). 

3.2.2. Serum processing 

The procedure for the processing of serum was based on that used for ERM-DA470k/IFCC 
[3].  Defibrinated  serum  was  treated  with  NaCl  (final  concentration  50  g/L,  Sigma-Aldrich, 
DE)  and  the  pH  adjusted  to  8.5  with  a  saturated  TRIS  solution  (Sigma-Aldrich,  DE).  The 
lipids present in the serum were removed by incubation for 1 h at 4 °C under slow stirring 
(100  rpm)  with  synthetic  amorphous  silica.  Protein  precipitates  and  silica  particles  were 
removed  by  centrifugation  at  10000  rpm  for  30  min.  The  clear  supernatant  was  dialysed 
against  nine  changes  of  isotonic  NaCl  solution  (0.9  %  w/v)  (Sigma-Aldrich,  DE)  over  24 
hours.  The  pH  was  then  adjusted  to  7.2  with  a  100  mmole/L  HEPES  solution  (Sigma-
Aldrich,  DE)  and  the  following  preservatives  added:  sodium  azide  (final  concentration  of 
0.95  g/L),  aprotinin  (final  concentration  of  61.5  TIU/mL)  and  benzamidine  hydrochloride 
monohydrate  (final  concentration  of  1  mmole/L).  All  three  preservatives  were  purchased 
from Sigma-Aldrich (DE). The serum was then filter sterilised through a 0.22 µm PES filter 
(Corning Incorporated, USA) prior to dilution 1:6 with Zenalb® 4.5 (Bio Products Laboratory, 
UK). 

3.2.3. Filling 

1 mL serum was transferred to each vial under clean-room conditions. Colourless threaded 
siliconised glass vials and white screw caps were used.  

Lyophilisation  was  started  within  one  hour  after  filling  the  vials.  Closing  the  bottles  using 
rubber  stoppers  was  performed  under  a  low-pressure  nitrogen  atmosphere.  Filling  and 
lyophilisation were carried out with the local defined processes in accordance with LP-00614 
(Filling and Labelling) and LP-00619 (Lyophilisation). 

13 

 
 
 
4. Homogeneity 

A key requirement for any certified reference material aliquoted into units is the equivalence 
between  those  units.  In  this  respect,  it  is  relevant  whether  the  variation  between  units  is 
significant  compared  to  the  uncertainty  of  the  certified  value.  Consequently,  ISO  Guide  34 
requires  RM  producers  to  quantify  the  between  unit  variation.  This  aspect  is  covered  in 
between-unit homogeneity studies. 

4.1 Between-unit homogeneity 

The between-unit homogeneity was evaluated to ensure that the certified value of the CRM 
was valid for all vials of the material, within the stated uncertainty. 

For the between unit homogeneity assessment, the number of units selected corresponds to 
approximately  the  cubic  root  of  the  total  number  of  units  produced  and  therefore  20  units 
were  selected  using  a  random  stratified  sampling  scheme,  covering  the  whole  batch.  For 
this, the batch was divided into twenty groups (with a similar number of units) and one unit 
was selected at random from each one. Three independent samples were taken from each of 
MPO IgG). The measurements were 
these units, and analysed by an ELISA (QUANTA Lite
performed  under  repeatability  conditions  and  in  a  randomised  manner  so  as  to  be  able  to 
separate a potential analytical drift from a trend in the filling sequence.  

TM 

Regression analyses were performed to evaluate potential trends in the analytical sequence 
as well as trends in the filling sequence. No trends were observed.  

The dataset was assessed for consistency using Grubbs outlier tests with a confidence level 
of 99 % on the individual results and the unit means. No outlying individual results or 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  are 
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.  The  distribution  of  the  mean  values  per  unit  was  visually 
assessed 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.  The  results  of  the  statistical  evaluation  of  the  homogeneity  studies  at  a  95  % 
confidence level show that the material is homogeneous. 

It should be noted that sbb,rel and swb,rel are estimates of the true standard deviations and are 
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. [9]. u*
bb is 
comparable  to  the  limit  of  detection  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*
calculated as:  

bb,rel  were 

s

,wb

rel

=

MS

within

y 

Equation 1 

14 

 
s

,bb

rel

=

u

*
,bb

rel

=

MS

between

MS

within

n
y

MS
within
n

4

ν

2

MSwithin

y

MSwithin 

mean square within a unit from an ANOVA  

MSbetween  mean squares between-unit from an ANOVA 

y  

n 

ν

MSwithin

mean of all results of the homogeneity study 

mean number of replicates per unit 

degrees of freedom of MSwithin  

Equation 2 

Equation 3 

The  results  from  the  homogeneity  studies  are  shown  in  Annex  A.  The  results  of  the 
evaluation  of  the  between-unit  variation  are  summarised  in  Table  1.  The  values  from  the 
equations  above  were  converted  into  relative  uncertainties.  The  uncertainty  contribution  for 
homogeneity was determined by the method repeatability. 

Table 1: Results of the homogeneity study 

CRM 

ERM-DA476/IFCC 

swb,rel 

[%] 

3.09 

sbb,rel 

[%] 
n.c.(1) 

u*

bb,rel 

[%] 

0.85 

1) n.c.: cannot be calculated as MSbetween < MSwithin 

bb sets the limits of the study to detect inhomogeneity it was adopted as the uncertainty 

As u*
contribution to account for potential inhomogeneity. 

4.2 Within-unit homogeneity and minimum sample intake 

The  minimum  sample  intake  is  the  minimum  amount  of  sample  which  is  shown  to  be 
representative  for  the  whole  unit  and  thus  can  be  used  in  an  analysis.  The  within-unit 
homogeneity  is  correlated  to  the  minimum  sample  intake  and  therefore  it  follows  that 
individual aliquots of a material below the minimum sample intake will not contain the same 
amount of analyte. Sample sizes equal or above the minimum sample intake guarantee the 
certified value within its stated uncertainty.  

The smallest sample intake tested was determined using the method information supplied by 
the  participants,  from  the  results  of  the  homogeneity/stability  experiments  and  from  the 
characterisation study. In all cases ELISA based methods were used. The smallest sample 
intake used was 10 µL, which was henceforth established as the minimum sample intake.  

The standard deviation within a bottle (swb) is lower than the expected method variability, so 
there is no indication of intrinsic heterogeneity or contamination at a sample intake of 10 µL. 

15 

 
-
 
 
 
 
 
 
 
 
5. Stability 

Stability  testing  is  necessary  to  establish  the  conditions  for  storage  (long-term  stability)  as 
well  as  the  conditions  for  dispatch  of  the  material  to  the  customers  (short-term  stability). 
During transport, especially in summer time, temperatures up to 60 °C can be reached and 
stability under these conditions must be demonstrated if the material is to be transported at 
ambient temperatures. 

The  stability  studies  were  performed  using  an  isochronous  design  [10].  In  this  approach, 
units were stored for a specified length of time at different temperature whereupon the units 
were  then  moved  to  conditions  where  further  degradation  was  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. The data were analysed by calculating the regression line for 
protein  concentration  in  relation  to  time,  and  determining  whether  it  is  significantly  different 
from zero. 

5.1 Short-term stability study 

During the short-term stability study, units were stored at -20 °C, 4 °C and 18 °C for 0, 1, 2 
and  4  weeks.  The  reference  temperature  was  set  at  -150  °C.  Two  units  per  storage  time 
were selected using a random stratified sampling scheme. Three samples, taken from each 
unit,  were  analysed  by  anti-MPO  BioFlash  ELISA  under  repeatability  conditions  in  a 
randomised  sequence,  to  be  able  to  separate  a  potential  analytical  drift  from  a  trend  over 
storage time. The values were corrected for the variable reconstitution volume.  

The  data  were  evaluated  for  each  temperature  individually.  The  results  were  screened  for 
outliers  using  the  single  and  double  Grubbs  test.  For  every  temperature  there  were  24 
measurements performed.  

Furthermore,  the  data  were  evaluated  against  storage  time  and  regression  lines  of  protein 
concentration  versus  time  were  calculated.  The  slope  of  the  regression  line  was  tested  for 
statistical  significance  (loss/increase  due  to  shipping  conditions)  and  it  was  found  to  be 
significantly different from 0 when the absolute value of the slope b divided by its uncertainty 
ub  (|b/ub|)  is  larger than t0.05,  22=  2.07.   There  were  no  indications  of  instability  at  any  of the 
temperatures for measurements performed with the BioFlash assay.  

Since  no  technical  reason  for  the  outliers  was  found,  all  data  were  retained  for  statistical 
analysis.  

The results of the short-term stability studies are shown in Annex B.  

No  technically  unexplained  outliers/Statistical  outliers  were  detected  and  thus  all  data  were 
retained for the estimation of usts. None of the trends were statistically significant at a 95 % 
confidence level for any of the temperatures. 

From  this  study  it  was  concluded  that  for  the  shipment  (up  to  2  weeks)  the  possible 
contribution of the transport to the uncertainty of the certified values is negligible compared to 
the overall uncertainty. For practical reasons and for retaining the uncertainty values as low 
as possible it is strongly recommended to ship the material on dry ice and within one week.  

16 

 
 
5.2 Long-term stability study 

During the  long-term  stability  study,  samples  were  stored  at -20  °C  and -70  °C for  0,  8,  16 
and 24 months. The reference temperature was set at -150 °C. Two units per storage time 
were  selected  using  a  random  stratified  sampling  scheme.  Three  samples  from  each  unit 
were  analysed  by  a  BioFlash  ELISA  immunoassay  under  repeatability  conditions,  in  a 
random  sequence  to  be  able  to  separate  any  potential  analytical  drift  from  a  trend  over 
storage time.  

The data were evaluated for each temperature individually. No outliers were found when the 
results were screened using the single and double Grubbs test. Furthermore, the data were 
plotted  against  storage  time  and  linear  regression  lines  of  protein  activity  (U/mL)  fraction 
versus  time  were  calculated.  The  slope  of  the  regression  lines  was  assessed  for  statistical 
significance  (loss/increase  due  to  storage  conditions).  The  slopes  of  the  regression  lines 
were not significantly different from zero (at a 95 % confidence level) for either temperature.  

No  technically  unexplained  outliers  were  observed  and  none  of  the  trends  was  statistically 
significant at a 95 % confidence level for any of the temperatures. The material can be stored 
at both -20°C and -70 °C. However, as it is envisaged to be stored for long periods of time, it 
should be preferably kept at -70 °C.  

5.3 Estimation of uncertainties 

Due  to  the  intrinsic  variation  of  measurement  results,  no  study  can  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 report that there was no detectable degradation during 
the timeframe studied.  

Uncertainties of stability during dispatch and storage were estimated as described in [11]. In 
this  approach,  the  uncertainty  of  the  linear  regression  line  with  a  slope  of  zero  was 
calculated.  The  uncertainty  contributions  usts  and  ults  are  calculated  as  the  product  of  the 
chosen transport time/shelf life and the uncertainty of the regression lines as: 

u

sts

,

rel

=

RSD

∑





t

i

2

t





t

tt

u

lts

,

rel

=

RSD

∑





t

i

2

t





t

sl

Equation 4 

Equation 5 

RSD  

relative standard deviation of all results of the stability study 

ti 

t  

ttt 

tsl 

time elapse at time point i 

mean of all ti  

chosen transport time (1 week at -20 ºC) 

chosen shelf life (24 months at -70 ºC) 

17 

 
 
(cid:215)
-
-
 
(cid:215)
-
-
 
 
 
 
The following uncertainties were estimated: 

-  usts,rel,  the  uncertainty  of  degradation  during  dispatch.  This  was  estimated  from  the  
-20 °C  studies.  The  uncertainty  describes  the  possible  change  during  a  dispatch  at  
-20 °C lasting for one week. 

-  ults,rel,  the  stability  during  storage.  This  uncertainty  contribution  was  estimated  from 
the  -70 °C  study.  The  uncertainty  contribution  describes  the  possible  degradation 
during 24 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 
storage at -20 °C for 1 week; ults,rel was calculated for storage at  -70 °C for 2 years. 

CRM 

ERM-DA476/IFCC 

usts ,rel 
[%] 
0.66 

ults,rel 
[%] 
1.95 

After  the  certification  study,  the  material  will  be  included  in  IRMM's  regular  stability 
monitoring programme. 

6. Characterisation  

The material characterisation is the process of determining the property value of a reference 
material. 

The  characterisation  of  both  the  anti-MPO  IgG  serving  as  calibrant  and  of  the  candidate 
CRM  was  based  on  an  inter-laboratory  comparison  of  expert  laboratories,  i.e.  the  protein 
concentration of the material was determined in different laboratories who applied their own 
methodology  and  instrumentation. This  approach  aims to  negate the  laboratory  bias,  which 
reduces the combined uncertainty.  

In  the  case  of  the  value  assignment  for  the  calibrant,  the  participant  laboratories  used 
turbidimetry and nephelometry for their measurements. For the characterisation of the ERM-
DA476/IFCC all participants used ELISA-type immunosorbent assays.  

6.1 Selection of participants  

Six laboratories were selected for the calibrant material and 10 for the target material based 
on  criteria  that  comprised  both  technical  competence  and  quality  management.  Each 
participant was required to operate a quality system and to deliver documented evidence of 
its laboratory proficiency in the field of in vitro diagnostics in relevant matrices by submitting 
results  for  intercomparison  exercises  or  method  validation  reports.  Having  a  formal 
accreditation was not mandatory, but meeting the requirements of ISO/IEC 17025 was. The 
accreditation  numbers  for  the  accredited  laboratory  are  stated  in  the  list  of  participants 
(Section 2). 

18 

 
 
 
 
 
 
6.2 General principles of the value assignment  

The laboratories were provided with detailed protocols and reporting sheets, as well as with 
vials  of  the  materials  to  be  analysed.  Laboratories  were  asked  to  specify  the  platform  and 
reagents used, and the order in which the measurements were performed. Both procedures 
required that the material was reconstituted the day before the measurements, according to 
the  procedure  described  in  the  certificate  of  ERM-DA470k/IFCC  and  in  the  present  report 
(Chapter 9.3). 

Principle of the analysis 

The aim of the value transfer is to determine the transfer factor (TF) 

TF = CT/CR 

(1) 

where CT and CR are the mass concentration of the protein in the target material (TM, ERM-
DA476/IFCC) and in the calibrant (purified anti-MPO IgG), respectively.  

Six different dilutions of the calibrant are prepared. The mass concentration of the protein in 
each dilution will be: 

CR(i) = FR(i) x CR  

where FR is the dilution factor of the control and (i) denotes the different dilutions (i = 1 to 6). 

The concentration of the protein in the dilutions of the target material will be: 

CS(j) = FT(j) x CT 

(2) 

(3) 

Where FT is the dilution factor of TM and (j) denotes the different dilutions of TM (j = 1 to 6). 

For the transfer procedure a calibration run is made, and the calibration curve is constructed 
by  plotting  the  signals  against  the  different  dilutions  of  RM.  In  a  measurement  run  the 
different  dilutions  of TM  are  assayed. The  signals  of these  unknown  test  samples  CS(j)  are 
interpolated on the calibration curve, and are measured in relative concentrations of RM: 

CS(j) = FR(j) x CR 

A combination of (3) and (4) gives: 

CS(j) = FT(j) x CT = FR(j) x CR 

FR(j) = CT/CR x FT(j) 

(4) 

(5) 

which is the equation of a straight line through the origin (y = TF x). 

The  different  dilution  factors  FR(j)  obtained  by  interpolation  are  plotted  against  the  different 
dilution  factors  FT(j)  used  for  the  initial  dilution  of  TM.  Since  all  dilutions  are  controlled  by 
weighing, the uncertainty of FT(j) is negligible compared to that of the measurement results. 

Calculation of the dilutions 

The precision of the values for the dilutions were considerably improved by weighing all the 
liquids used for the reconstitution of the materials and for the preparation of the dilutions to a 
precision of at least 0.0001 g. 

The following mass corrections were applied: 

For the reconstitution: 

19 

 
 
 
 
=

f

iM,

m

intended
m

iM,

Where mintended is the mass intended to be added (1.0000 g), and mM,i is the measured mass 
of the water added to the vial 

For eventual predilutions: 

=

f

iP,

m
iPR,
+
m

iPR,

m

iPD,

where mPR,i and mPD,i are the masses of the of the reconstituted material and of the diluent for 
the predilution 

For the dilutions:  

=

f

ijD,

m

m

iDM,

iDM,
+
m

iDD,

for  the  dilution  j  of  the  material  i,  with  mDM,i  the  mass  of  the  reconstituted  (eventually 
prediluted) material, and mDD,i the mass of the diluent for dilution j. 

The concentration cij (with c = m / V (mass / volume) and the unit g/L) of the proteins in the 
dilutions is calculated via: 

cij = c0i x fM,i x fP,i x fD,ij  

with  c0i  of  the  protein  in  material  i,  and  cij  the  concentration  of  the  protein  in  dilution  j  of 
material i. 

Determination of the transfer factors (TFs) 

During  the  transfer  procedure  the  dilutions  of  the  reference  material  (either  ERM-
DA470k/IFCC in case of the characterisation of the calibrant or of anti-MPO IgG in the case 
of  the  characterisation  of  ERM-DA476/IFCC)  were  used  to  construct  the  calibration  curve. 
Based  on  this  curve  and  the  known  concentration  of  the  spiked  material,  the  values  of  the 
target material were calculated. The relative concentrations were corrected for the masses of 
the dilutions (and predilution).  

During  the  value  transfer  procedure  the  laboratories  measured  the  6  dilutions  of  the  target 
(Y1-Y6)  and  the  calibrant  (X1-X6)  in  triplicate  on  each  day.  For  both  materials,  single 
measurement results Sijk (signal of the kth measurement of material i within dilution j, in g/L) 
were  plotted  in  scatter  plots  Sijk  =  f(cij)  so  as  to  evaluate  outliers.  A  linear  regression  with 
intercept was performed on the means of the Sijk in function of the concentration cij. 

The following analyses were performed on all data: 

-   Evaluation of the studentised residuals for the identification of outliers  
-  Test  for  the  homoscedasticity  of  the  residuals  (whether  the  variances  are    
homogeneous) via a t-test 
-   Test for normality (visual inspection and normal probability plot) 
-   Test for linearity (visual inspection and evaluation of R2)  
-   Test for outliers of the regression model according to the method of Lund [12] (only 
in case of homoscedasticity)  
-   Verification that the intercept ± 4 times the s covers the origin.  

20 

 
 
 
 
 
 
The  TFs  were  calculated  as  the  ratio  of  the  slopes  of  the  linear  regression  lines  for  the 
candidate reference material and for the calibrant.  

The following general acceptance criteria were applied to the datasets: 

1.  R2 of the regression must be above 0.99 for data of all platforms  
2.  Data from at least 4 dilutions must be available 
3.  The dataset from any one day must contain at least 50 % of the data generated. 

on that day, otherwise all of the data generated on that day are declared non-valid 

4.  At least two daily value assignments must be valid 
5.  The day-to-day variation (RSD) of valid datasets for the protein and laboratory 

must be below 15 %   

Rationale for the acceptance criteria: 
- Overall R2 < 0.99 of regression indicates quality problems in the measurements (scattering, 
outliers, run-to-run variation).   

-  During  the  data  analysis,  the  results  where  the  linear  regression  went  through  0  (y  =  bx) 
were  compared  to  those  when  linear  regressions  were  performed  with  an  intercept  
(y = a+bx). The mean of means of the TFs between laboratories were comparable. However, 
since only measured data were used and no model other than the linearity within the actual 
measurement  interval  was  assumed  it  was  decided  to  process  the  data  allowing  for 
intercepts  in  the  linear  regression,  provided  that  the  intercept  ±  4  s  covers  the  origin.  As  a 
consequence,  the  criterion  for  the  acceptance  of  datasets  was  adapted,  and  a  (within 
laboratory) day-to-day RSD of 15 % was defined.  

6.3 Characterisation of the calibrant 

During the characterisation of the calibrant (anti-MPO IgG), each laboratory received one vial 
of purified anti-MPO IgG and five vials of its dilution prepared in JRC-IRMM as well as one 
vial  of  ERM-DA470k/IFCC  and  five  vials  of  its  dilution.  They  were  required  to  provide  18 
independent  results,  per  material  (three  per  dilution).  Participating  laboratories  were 
instructed  to  use  their  in-house  calibrant  to  determine  the  dose-response  function  of  the 
instrument.  The  units  for  material  characterisation  were  selected  using  a  random  stratified 
sampling scheme and covered the whole batch. The techniques used to measure the protein 
concentration  of  the  calibrant  were  nephelometry,  turbidimetry  and  spectrophotometry.  The 
measurements were performed with different platforms (Annex C, Table C1) and reagents. In 
total six laboratories participated in the characterisation study. 

6.3.1. Production and physico-chemical characterisation of the calibrant 

The anti-MPO IgG was purified from human serum (provided by Statens Serum Institute) in a 
three  step  purification  process.  Firstly,  total  IgG  was  separated  by  affinity  chromatography 
using a Protein A column. Secondly, the specific anti-MPO IgG was separated from all other 
IgGs eluting from the protein A column using an in-house prepared Hi-trap column prepared 
using commercially available purified human myeloperoxidase (Scipac, UK) according to the 
protocol  provided  by  the  manufacturer.  All  anti-MPO  IgG  containing  fractions  were  pooled, 
concentrated to a final volume of 500 µL using Amicon Ultra centricons with a 30 KDa cut-off 
(Milipore,  USA)  and  subsequently  applied  on  a  Superdex  200  10/300  GL  column  (GE 

21 

 
 
 
 
 
 
Healthcare, SE). The purity of the final material with respect to IgG selectivity for MPO was 
assessed  by  affinity  chromatography.  The  in-house  prepared  anti-MPO  Hi-Trap  column 
described above was used for this purpose. In the chromatograph only one peak was eluted 
under the acidic conditions of the eluting buffer (pH 2.7) and no peaks were observed during 
the  elution  with  the  binding  buffer  (pH  7.0).  The  background  noise  was  taken  into 
consideration for the ratio to the peak for anti-MPO IgG. The uncertainty of the purity of the 
material  was  set  as  three  times  this  ratio.  The  calculated  uncertainty  was  equal  to  0.5  % 
(Table 3). 

6.3.2. Value assignment of the calibrant 

The concentration of the calibrant was measured by nephelometry or turbidimetry (Annex C, 
Table C1). The participating laboratories provided us with concentration values for both the 
ERM-DA470k/IFCC and the purified anti-MPO IgG solution based on their in-house calibrant. 
Regression  lines  were  constructed  comparing  these  measured  concentrations  to  the 
gravimetrically  calculated  dilution  factors  of  these  samples.  The  slope  ratio  of  the  ERM-
DA470k/IFCC to the purified anti-MPO IgG was used to determine the value of the dilutions 
of the target material. 

The  data  were  assessed  for  both  their  compliance  with  the  analysis  protocol  and  for  their 
validity  based  on  technical  reasons.  The  following  criteria  were  considered  during  the 
evaluation:  

-  Compliance  with  the  analysis  protocol:  sample  preparations  and  measurements 

performed on a minimum of two days, and the analytical sequence. 

-  Method  performance,  i.e.  agreement  of  the  measurement  results  with  the  assigned 

value of their in-house QC sample. 

-  R2 of the regressions must be above 0.99 for data of all platforms.  

Based  on  the  criteria  above,  one  dataset  was  rejected  as  not  technically  valid.  The  results 
provided from this laboratory did not comply with the criteria for the correlation coefficients, 
showing  R2  values  <  0.99.  In  another  laboratory  (L1,  Annex  C,  Table  C1),  the  higher 
concentration analysed was not taken into account while plotting the results as it was outside 
the laboratory's working range of the method. 

The  5  datasets  accepted  based  on  technical  reasons  were  assessed  for  the  normal 
distribution of dataset means using normal probability plots and  were assessed for outlying 
means  using  the  Grubbs  test  and  using  the  Cochran  test  for  outlying  standard  deviations 
(both  at  a  99  %  confidence  level).  Standard  deviations  within  (swithin)  and  between  (sbetween) 
laboratories were calculated using one-way ANOVA.  

The results of the individual laboratories are given in Table C1 of the Annex C. 

The uncertainty relating to the characterisation of the calibrant (purified anti-MPO IgG) was 
estimated as the standard error of the mean of laboratory means (Table 3). The uncertainty 
of  the  reference  material  used  for  the  calibration  (ERM-DA470k/IFCC)  was  taken  into 
account  as  well  the  uncertainty  of  the  purification  of  the  material.  The  uncertainty  deriving 
from the gravimetric preparation of the dilutions was found to be very low and according to 
GUM  specifications  it  was  not  taken  into  account  for  the  final  uncertainty  of  the  calibrant 
(ubal,rel = 0.005 %).  

22 

 
 
 
 
 
Table 3: Uncertainty budget for purified anti-MPO IgG.  

Analyte 

anti-MPO 
IgG 

p 

5 

Mean concentration 
[mg/mL] 

s 
[mg/mL] 

uERM-DA470k,rel 
[%] 

uchar, cal rel 
[%] 

upur,rel 
[%] 

1.52 

0.03 

0.98 

0.88 

0.43 

These  different  contributions  were  combined  to  estimate  the  relative  uncertainty  of  the 
concentration value of the calibrant (uanti-MPO IgG, rel) as:  

u

IgG  MPO-anti

=

u

2
char

 cal,

rel

+

u

2
ERM
-
DA470k/IFC
C

+

u

2
 pur,

rel

Equation 6 

-  uchar,cal was estimated from the data in Annex C, Table C1.  

-  uERM-DA470k/IFCC was estimated in the report of the material [3] 

-  upur was estimated as described in Section 6.3.1. 

The  concentration  of  the  material  was  additionally  determined  by  UV  spectrophotometry  at 
280  nm  using  an  absorption  coefficient  of  1.36  (g/L)-1cm-1  (210000  M-1cm-1)  [13].  The 
concentration  which  was  measured  on  4  separate  days,  in  triplicate,  was  equal  to  1.43 
mg/mL.  This  value  was  not  used  for  the  calculation  of  the  certified  value  of  the  candidate 
reference material nor in the calculation of the uncertainties. 

6.4 Characterisation of ERM-DA476/IFCC 

During  the  target  material  characterisation  each  laboratory  received  four  vials  of  ERM-
DA476/IFCC  and  four  vials  of  anti-MPO  spiked  into  human  serum.  They  were  required  to 
perform  six  dilutions  from  these  vials  and  to  provide  72  independent  results,  three  per  vial 
per day, for each material. The vials for the materials characterisation were selected using a 
random stratified sampling scheme and covered the whole batch. The sample preparations 
and  measurements  had  to  be  spread  over  four  days  to  ensure  intermediate  precision 
conditions.  For  the  spiked  material  (calibrant),  participants  were  required  to  treat  them  as 
they  would  any  other  sample.  The  dilution  protocol  was  the  same  for  both  materials  (6.67, 
10, 15, 20, 50 and 100 (m/m) % of the reconstituted materials). 

The  techniques  used  to  measure  the  protein  concentration  were  traditional  ELISAs  and  in 
some cases its variations (chemiluminescent and fluoroenzyme immunoassays) specific for 
IgG anti-MPO. In total 10 laboratories participated in the value assignment, using 9 different 
kits (Annex C, Table C2). 

The  data  were  first  assessed  for  compliance  with  the  requested  analysis  protocol  and  for 
their  validity  based  on  technical  reasons.  The  following  criteria  were  considered  during  the 
evaluation:  

-  Compliance  with  the  analysis  protocol  (sample  preparations  and  measurements 

performed on a minimum of two days) and the analytical sequence. 

-  Method  performance,  i.e.  agreement  of  the  measurement  results  with  the  assigned 

value of their in-house QC sample 

23 

 
 
 
 
 
 
 
 
 
-  R2 of the regressions either linear or logarithmic must be above 0.99 for data of all 

platforms  

Linear regressions were used for the analyses of the data provided by laboratories 1, 3, 5-10 
(Annex C, Table C2). However, in the case of laboratories 2 and 4 the data provided  were 
following a logarithmic distribution and had to therefore be treated accordingly.  

Based on the above criteria, the results obtained from laboratory 10 were completely omitted 
and not used for the certification. In particular, the R2 values for all days  were much below 
the  set  limit  of  0.99  whilst  at  the  same  time  there  was  a  35  %  day-to-day  variation. 
Additionally  for  laboratory  3,  two  out  of  4  days  had  correlation  coefficients  below  0.99  and 
thus those two days were not taken into account for the final value assignment.  

The  valid  datasets  were  assessed  for  the  normal  distribution  of  the  dataset  means  using 
normal  probability  plots  and  were  assessed  for  outlying  means  using  the  Grubbs  test  and 
using  the  Cochran  test  for  outlying  standard  deviations  (both  at  a  99  %  confidence  level). 
Standard  deviations  within  (swithin)  and  between  (sbetween)  laboratories  were  calculated  using 
one-way ANOVA.  

The laboratory means follow normal distributions. None of the data contains outlying means 
and  variances.  The  datasets  were  therefore  consistent  and  the  mean  of  laboratory  means 
was  a  good  estimate  of  the  true  value.  The  value  assignment  data  of  the  individual 
laboratories are shown in Annex C, Table C3. 

7. Uncertainty budget and certified values 

Certified values are values that fulfil the highest standards of accuracy. Procedures at IRMM 
require  no  less  than  6  valid  datasets  to  assign  certified  values.  Full  uncertainty  budgets  in 
accordance  with  the  'Guide  to  the  Expression  of  Uncertainty  in  Measurement'  [6]  were 
established. A certified value was assigned to ERM-DA476/IFCC. 

The  assigned  uncertainty  consists  of  uncertainties  relating  to  the  characterisation  of  the 
target  material,  uchar  (Section  6),  to  the  combined  uncertainty  of  the  characterisation  of  the 
calibrant uchar,cal (Section 6), to the potential between-unit inhomogeneity, ubb (Section 4) and 
to the potential degradation during transport (usts) and long-term storage, ults (Section 5). The 
uncertainty relating to the use of analytical balance during the gravimetric preparation of the 
dilutions  of  both  the  calibrant  and  the  target  material  was  found  to  be  negligible  (equal  to 
0.005 and 0.006 % respectively) [4]. The uncertainty relating to possible degradation during 
transport and long-term storage is included in the results for usts and ults. The uncertainty of 
between-unit inhomogeneity was found to be too low to be calculated and thus the maximum 
hidden  heterogeneity  was  eventually  used  for  the  calculation  of  the  uncertainty  of  the 
certified  value.  These  different  contributions  were  combined  to  estimate  the  expanded, 
relative uncertainty of the certified value (UCRM, rel) with a coverage factor k as:  

U

CRM,

rel

(cid:215)=
k

u

2
char,

rel

+

u

2
char,

 cal,

rel

+

u

2
 bb,

rel

+

u

2
 sts,

rel

+

u

2
lts,

rel

Equation 7 

-  uchar was estimated as described in Section 7.1  

-  uchar,cal was estimated as described in Section 6.3.2 

-  ubb was estimated as described in Section 4.1. 

24 

 
 
 
 
 
 
 
 
 
-  usts was estimated as described in section 5.3 

-  ults was estimated as described in Section 5.3.  

7.1 Certified value 

The certified value was calculated from the average of the TF (Annex C, Table C3) and the 
value of the calibrant (Table 3, 3rd column) according to: 

cERM-DA476/IFCC = TFaverage * canti-MPO IgG 

Where canti-MPO  IgG is the concentration of anti-MPO IgG (calibrant) spiked gravimetrically into 
human serum. 

The uncertainty related to the characterisation of the ERM-DA476/IFCC is estimated as the 
standard error of the mean of laboratory means (Table 4). 

Table 4: Value assignment for ERM-DA476/IFCC 

CRM 

p  Mean [mg/L]  s [mg/L]  uchar, rel  [%] 

ERM-DA476/IFCC  9 

84.20 

10.69 

4.25 

7.2 Estimation of uncertainty 

The  relative  expanded  uncertainty  was  calculated  from  the  relative  combined  standard 
uncertainty  uCRM,rel  by  multiplication  with  a  coverage  factor  k  (UCRM=uCRM*k).  This  coverage 
factor was taken as 2 as there is a sufficient numbers of degrees of freedom of the different 
uncertainty  contributions.  The  relative  expanded  uncertainty  was  multiplied  by  the  mean  of 
dataset means to obtain the expanded uncertainty UCRM. 

The  uncertainty  budget  after  taking  into  consideration  the  various  uncertainty  contributions 
and the relative combined uncertainty (uCRM,rel) is shown in Table 5. 

Table 5: Uncertainty budget for ERM-DA476/IFCC 

CRM  

uanti-MPO IgG rel  [%]  uchar, rel [%] 

ubb, rel 

usts, rel 

ults, rel 

uCRM,rel 

UCRM 

[%] 

[%] 

[%] 

[%] 

[mg/L] 

ERM-DA476/IFCC 

1.38 

4.25 

0.84 

0.66  1.95 

4.99 

8.40 

Finally  the  certified  value  for  the  ERM-DA476/IFCC  is  equal  to  84  mg/L  and  the  expanded 
uncertainty of this value is equal to 9 mg/L. 

25 

 
 
 
 
 
 
 
 
 
8. Metrological traceability and commutability 

8.1 Metrological traceability  

Identity 

The  mass  concentration  of  anti-MPO  IgG  in  ERM-DA476/IFCC  is  defined  by  the 
immunoassay  procedures  used  to  characterise  it.  The  assigned  value  is  therefore 
operationally defined by method. 

Quantity value 

The  measurements  of  anti-MPO  IgG  were  calibrated  with  ERM-DA470k/IFCC  by  applying 
the value transfer procedure described in this report. The value transfer measurements were 
strictly controlled with respect to adherence to the procedure and the adequate functioning of 
equipment and reagents was verified. Different combinations of reagents and platforms were 
used,  which  gave  consistent  results.  Therefore  the  certified  value  is  not  dependent  on  the 
individual methods.  

The certified values for anti-MPO IgG in ERM-DA476/IFCC were obtained by calibration with 
purified anti-MPO. 

The traceability chain is  based on the use of calibrated balances and a thorough control of 
the weighing procedure. The certified mass concentration and its uncertainty are traceable to 
the stated value of the mass concentration in USNRP 12-0575C [14] applying the procedures 
described in the certification report of ERM-DA470k/IFCC [3] and in the present report. 

8.2 Commutability 

Many  measurement  procedures  include  one  or  more  steps,  which  select  specific  analytes 
from the sample for the subsequent steps of the measurement process. It is difficult to mimic 
all  the  analytically  relevant  properties  of  real  samples  within  a  CRM.  The  degree  of 
equivalence  in  the  analytical  behaviour  of real  samples  and  a  CRM  with  respect  to  various 
measurement  procedures  (methods)  is  summarised  in  a  concept  called  'commutability  of  a 
reference material'. There are various definitions expressing this concept. For instance, the 
CSLI  Guideline  C-53A  [7]  recommends  the  use  of  the  following  definition  for  the  term 
commutability: 

"The  equivalence  of  the  mathematical  relationships  among  the  results  of  different 
measurement procedures for an RM and for representative samples of the type intended 
to be measured." 

The commutability of a CRM defines its fitness for use and, thus, is a crucial characteristic for 
the  application  of  different  measurement  methods.  When  commutability  of  a  CRM  is  not 
established  in  such  cases,  the  results  from  routinely  used  methods  cannot  be  legitimately 
compared with the certified value to determine whether a bias does not  exist in calibration, 
nor  can  the  CRM  be  used  as  a  calibrant.  For  instance,  CRMs  intended  to  be  used  to 
establish or verify metrological traceability of routine clinical measurement procedures must 
be commutable for the routine clinical measurement procedures for which they are intended 
to be used. 

Different  formats  of  candidate  reference  materials,  all  based  on  the  same  raw  material  as 
ERM-DA476/IFCC, have been tested and found to be commutable for combinations of seven 
methods  giving  correlating  results  (Annex  D,  Table  D1).  Therefore  ERM-DA476/IFCC  is 
expected  to  be  commutable  for  the  majority  of  IgG  anti-MPO  methods  [2].  However,  if 

26 

 
 
another  method  is  used  other  than  those  included  here  then  commutability  should  be 
verified. 

9. Instructions for use 

9.1 Safety information 

The usual laboratory safety measures apply. Do not discharge the waste into the drain. Each 
portion  of  donated  blood  used  in  the  production  of  the  material  has  been  tested  and found 
negative  for  Hepatitis  B  surface  antigen,  HIV  1&2,  HIV  antigen  and  Hepatitis  C  antibodies. 
However, the product must be handled with adequate care as any material of human origin. 
It is intended for in vitro analysis only. 

9.2 Storage conditions 

Unopened vials should be stored at both -20°C and -70 °C. However, as it is envisaged to be 
stored  for  long  periods  of  time,  it  should  be  preferably  kept  at  -70  °C.  If  microbial 
contamination has been excluded during the reconstitution procedure, the solution of ERM-
DA476/IFCC can be used for one week. It is advisable to cover the vial with the original seal 
after use and to store it at 2 to 8 °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. 

9.3 Preparation and use of the material/Reconstitution 

The material must be reconstituted according to the following procedure: 
•  Remove  the  vial  from  the  freezer  or  refrigerator  during  the  afternoon  of  the  day  before 

use and place the vial in the room where the balance is located for 1 hour. 

•  After 1 hour, tap the bottom of the vial gently on the surface of the table. Make sure that 

all the material has settled down on the bottom of the vial. Remove the screw cap. 

•  Weigh  the  vial  together  with  the  rubber  stopper.  Note  the  mass  or  press  the  “TARE” 
button on the balance. Lift the rubber stopper with care until air is allowed to enter the vial 
and the groove in the rubber stopper becomes accessible. 

•  Add 1.00 mL of water through the groove, and press the rubber stopper back into place. 
Weigh the vial and note the mass. If you have used the “TARE” function, the value can 
be  used  directly  for  the  mass  m.  Otherwise  the  first  mass  must  be  subtracted  from  the 
second to obtain m. 

•  The  concentration  of  a  particular  protein  in  the  solution,  corrected  for  the  reconstitution 
mass, can be obtained by multiplying the certified value for that protein with mintended / m, 
with mintended the mass intended to be added (1.000 g).  

•  Leave the vial at room temperature for one hour, then invert it carefully at least five times 

(do not shake it) during the next hour. 

•  Leave the vial at room temperature overnight. On the day of use invert the vial carefully 

five times during one hour before use. 

27 

 
 
 
 
9.4 Minimum sample intake 

The minimum representative sample intake is 10 µL. The entire content of the vial must be 
reconstituted. 

9.5 Use of the certified value 

The main purpose of this material is to be used for the calibration of immunoassay-based in 
vitro  diagnostic  devices  or  control  products  for  anti-MPO  IgG.  As  any  reference  material,  it 
can also be used for control charts or validation studies. 

When  the  material  is  used  as  a  calibrant  in  a  particular  assay  the  commutability  should  be 
verified for the assay concerned.  

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 [15].  

For  assessing  the  method  performance,  the  measured  values  of  the  CRMs  are  compared 
with the certified values. The procedure is described here in brief:  

-  Calculate  the  absolute  difference  between  mean  measured  value  and  the  certified 

value (D meas). 

-  Combine  measurement  uncertainty 

(umeas)  with 

the  uncertainty  of 

the  

certified value (uCRM): 

=D

u

u

+
2
meas u

2
CRM

-  Calculate the expanded uncertainty (UD ) from the combined uncertainty (uD

,) using an 
appropriate coverage factor, corresponding to a level of confidence of approximately 
95 % 
If  D meas  £   UD   no  significant  difference  between  the  measurement  result  and  the 
certified value, at a confidence level of about 95 % exists. 

- 

Use in quality control charts 

The materials can be used for quality control charts. Different CRM-units will give the same 
result as inhomogeneity was included in the uncertainties of the certified values.  

28 

 
 
 
 Acknowledgments 

The  authors  would  like  to  acknowledge  the  support  received  from  Håkan  Emteborg,  Jean 
Charoud-Got  and  Katharina  Teipel  from  IRMM  related  to  the  processing  of  this  CRM  and 
from Maria Concepción Contreras López concerning the set-up of the isochronous studies. 

Furthermore, the authors would like to thank Amalia Muñoz Piñeiro and Maria Karczmarczyk 
(IRMM)  for  reviewing  the  certification  report,  as  well  as  the  experts  of  the  Certification 
Advisory  Panel  "Biological  Macromolecules  and  Biological/Biochemical  Parameters",  Hez 
Hird  (Food  and  Environmental  Research  Agency,  UK),  Martin  Wagner  (University  for 
Veterinary  Medicine  Vienna,  AT)  and  Lothar  Siekmann  (University  of  Bonn,  DE),  for  their 
constructive  comments.  The  authors  are  grateful  for  the  active  involvement  of  the 
participating laboratories in the characterisation study.  

29 

 
References 

1.  ISO  Guide  34,  General  requirements  for  the  competence  of  reference  materials 
producers, International Organization for Standardization, Geneva, Switzerland, 2009 

2.  D.P. Hutu, E. Connell, E. Monogioudi, P. Meroni, A. Wiik, H. Schimmel, J. Sheldon, I. 
Zegers  and the collaborating laboratories, First steps in the standardisation of MPO-
ANCA measurements, In Preparation 

3.  Certification report, Certification of proteins in the human serum. Certified Reference 
Material  ERM®  -  DA470k/IFCC,  European  Commission.  Joint  Research  Centre, 
Institute of Reference Materials and Measurements, EUR 23431 EN - 2008 

4.  ISO  Guide  35,  Reference  materials  –  General  and  statistical  principles  for 
certification,  International  Organization  for  Standardization,  Geneva,  Switzerland, 
2006 

5.  ISO/IEC  17025:2005,  General  requirements  for  the  competence  of  testing  and 
calibration  laboratories,  International  Organization  for  Standardization,  Geneva, 
Switzerland, 2005 

6.  ISO/IEC Guide 98-3, Guide to the Expression of Uncertainty in Measurement, (GUM 
1995), International Organization for Standardization, Geneva, Switzerland, 2008 

7.  H. Vesper, H. Emons, M. Gnezda, C. P. Jain, W. G. Miller, R. Rej, G. Schumann, J. 
Tate,  L.  Thienpont,  J.  E.  Vaks,  Characterization  and  Qualification  of  Commutable 
Reference  Materials  for  Laboratory  Medicine;  Approved  Guideline,  CLSI  document 
C53-A, Clinical and Laboratory Standards Institute, Wayne, PA, USA, 2010 

8.  R.J.  Falk,  J.C.  Jennette,  Anti-Neutrophil  Cytoplasmic  Autoantibodies  with  Specificity 
for  Myeloperoxidase  in  Patients  with  Systemic  Vasculitis  and  Idiopathic  Necrotizing 
and Crescentic Glomerulonephritis, N Engl J Med (1988) 318:1651-1657 

9.  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 

10. 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 

11. 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 

12. R.E.  Lund,  Tables  for  an  Approximate  Test  for  Outliers  in  Linear  Models. 

Technometrics 17 (1975) 473 - 476 

13. A.  Johnstone,  R.  Thorpe, 

Immunochemistry 

in  Practice,  Blackwell  Scientific 

Publications (1988), ISBN:0-632-01723-6 

14. C.B.  Reimer,  S.J.  Smith,  T.W.  Wells,  R.M.  Nakamura,  P.W.  Keitges,  R.F.  Ritchie, 
G.W.  Williams,  D.J.  Hanson,  D.B.  Dorsey.  Collaborative  calibration  of  the  U.S. 
national and the College of American Pathologists reference preparations for specific 
serum proteins. Am J Clin Pathol. (1982), 77:12–19 

15. T.P.J. Linsinger, ERM Application Note 1: Comparison of a measurement result with 

the certified value, www.erm-crm.org  

30 

 
 
 
Annexes 

Annex A: Results of the homogeneity measurements 

Source of 
Variation 

Between 
Units 

SS 

37.6 

Within Units 

118.3 

Total 

155.88 

df 

19 

40 

59 

MS 

s 

F 

F-crit 
95 % 

F-crit 
99 % 

1.98 

MSB<MSW  0.67 

1.9 

2.4 

2.96 

1.72 

Homogeneity 
Results 

Weight.Avg. 
[mg/L] 

Sbb [mg/L] 

Sbb  [%] 

Swb [mg/L] 

Swb  
[%] 

Ubb* 
[mg/L] 

ubb*[%] 

55.76 

MSB<MSW  MSB<MSW 

1.72 

3.08 

0.47 

0.84 

Table A1: Analysis of Variance of the homogeneity results 

31 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex B: Results of the short-term stability measurements 

)
L
m
U

/

(
s
e
u
l
a
V
d
e
r
u
s
a
e
M

300

280

260

240

220

200

180

0

1
2
Time (weeks)

3
Time (weeks)

4

0

300

280

260

240

220

200

180

)
L
m
U

/

(
s
e
u
l
a
V
d
e
r
u
s
a
e
M

/

)
)
L
L
m
m
/
U
U
(
(
s
s
e
u
lu
l
a
a
V
v
d
d
e
re
r
u
u
s
s
a
a
e
e
M
M

300

280

260

240

220

200

180

0

1

2

3
Time (weeks)

Time (weeks)

4

2

1
Time (weeks)

Time (weeks)

3

4

Fig.  B1:  Short-term  stability  data  (results  of  individual  replicates)  of  ERM-DA476/IFCC; 
Measured values by an ELISA immunoassay (U/mL), when stored for several weeks at -20 
°C  (a),  4 °C  (b)  and  18 °C  (c),  results  at  time  point  0  weeks  correspond  to  units  that  were 
stored at the reference temperature of -20 °C. 

CRM 

usts, 1 week [%] 

usts, 2 weeks [%] 

ERM-DA476/IFCC 

-20 °C  4 °C  18 °C 

-20 °C  4 °C  18 °C 

0.66 

0.54  0.55 

1.32 

1.07  1.09 

Table  B1:  Uncertainties  of  stability  during  storage  at  different  temperatures  for  1  and  2 
weeks 

32 

 
 
 
 
 
 
 
Annex C: Calibrant characterisation 

anti-MPO IgG 

Laboratory 

L1 

L2 

L3 

L4 

L5 

Platform 

Immage 

Method 

Nephelometry 

Cobas 6000/8000 

Turbidimetry 

BN Prospec 

Nephelometry 

Modular P 

Cobas c 501 

Turbidimetry 

Turbidimetry 

Concentration 
[mg/mL] 

1.47 

1.51 

1.53 

1.55 

1.52 

Table  C1:  All  laboratories  giving  acceptable  results,  together  with  the  platforms  they  used 
and the principles of the methods that these platforms employ are listed.  

ERM-DA476/IFCC 

Laboratory 

Method 

Principle 

Concentration 

[mg/L] 

L1 

L2 

L3 

L4 

L5 

L6 

L7 

L8 

L9 

L10 

Wieslab® Capture 
MPO-ANCA 

ImmuLisa ™ 

Autoimmune EIA 
Anti-
Myeloperoxidase 

Anti-
Myeloperoxidase 
ELISA (IgG) 
Wieslab® Capture 
MPO-ANCA 
Wieslab® MPO-
ANCA 

ELISA 

ELISA 

ELISA 

ELISA 

ELISA 

ELISA 

QUANTA Flash MPO 

EliA MPOS 

AESKULISA MPO 

TM 

QUANTA Lite
IgG 

MPO 

Chemiluminescent 
immunoassay 

Fluoro-enzyme 
immunoassay 

ELISA 

ELISA 

92 

68 

99 

75 

88 

73 

78 

93 

91 

- 

Table  C2:  All  laboratories  participating  in  the  value  assignment  of  the  target  material.  The 
methods  they  used  and  the  principles  that  these  methods  employ  are  listed.  On  the  last 
column the average concentrations of the ERM-DA476/IFCC are presented.  

33 

 
 
 
 
 
 
 
ERM-DA476/IFCC 

Laboratory  
code 

Day 1 
[slope 
ratio] 

Day 2 
[slope 
ratio] 

Day 3 
[slope 
ratio] 

Day 4 
[slope 
ratio] 

0.962 

0.860 

0.827 

0.809 

0.70 
1) 
0.751 
0.723 

0.674 

0.63 

0.918 

0.697 
0.866 

0.728 

0.61 
 1) 
0.660 
 1) 
0.712 

0.738 
0.889 
0.898 

0.61 

0.941 

0.721 
0.857 

0.589 

0.785 
0.866 
0.925 

0.622 
0.812 
0.771 
1): results excluded for technical reasons 

0.720 
no data 
0.747 

L1 

L2 

L3 
L4 
L5 
L6 
L7 
L8 
L9 

TF 
[mean 
slope 
ratio] 
0.864 

0.639 

0.930 

0.707 
0.815 

0.676 

0.717 
0.856 
0.835 

Day-to-
Day 
variation 
[%] 
7.91 

6.37 

1.75 

5.46 
9.82 
9.19 

9.60 
10.69 
10.69 

Table  C3:  Individual  results  for  the  value  assignment  for  the  characterisation  of  the  target 
material,  ERM-DA476/IFCC.  The  relative  uncertainties  per  laboratory  and  their  day-to-day 
variation are presented.  

34 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Annex D: Commutability 

Laboratory 

Method 

L1 

L2 

L3 

L4 

L5 

L6 

L7 

Varelisa™ MPO ANCA 

EliA MPOS 

Wieslab® Capture MPO-
ANCA 

Wieslab® Anti-MPO ELISA 

Anti-MPO ELISA 

QUANTA Lite MPO IgG 
ELISA 

Anti-MPO ORG 519 

Principle 

ELISA 

Fluoro-enzyme 
immunoassay 

ELISA 

ELISA 

ELISA 

ELISA 

ELISA 

Table  D1:  Laboratories that  participated  in the  commutability  studies  and  the methods  they 
used 

35 

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
36 

 
 
European Commission 

EUR 27092 EN – Joint Research Centre – Institute for Reference Materials and Measurements 
Title:  
The certification of anti-myeloperoxidase immunoglobulin G in human serum ERM® - DA476/IFCC 
Author(s): Evanthia Monogioudi, Dana P. Hutu, Gustavo Martos, Emma Tuddenham, Joanna Sheldon, Heinz Schimmel,  

  Stefanie Trapmann, Pierluigi Meroni, Hendrik Emons and Ingrid Zegers 

Luxembourg: Publications Office of the European Union 
2015 –36 pp. – 21.0 x 29.7 cm 
EUR – Scientific and Technical Research series – ISSN 1831-9424 
ISBN 978-92-79-45349-6 
doi:10.2787/798324 

 
 
 
 
 
 
 
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. 

L
A
-
N
A
-
2
7
0
9
2
-
E
N
-
N

doi:10.2787/798324 

ISBN: 978-92-79-45349-6