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EA - 4/14  The Selection and Use of Reference Materials

Publication

Reference EA-4/14 INF:2003

The Selection

and Use of

Reference Materials

PURPOSE

The aim of this paper is to provide a short, simple and user-friendly guide for laboratories,
and accreditation and certification bodies. It employs mainly ISO and VIM definitions and
provides references to other more comprehensive and expert texts. It is intended to provide
help for the inexperienced rather than the expert and necessarily simplifies some topics. It
includes discussion and clarification of some misunderstood issues. Although based on the
requirements of chemical measurement, it is intended to be also of use in other areas of
measurement.

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EA - 4/14  The Selection and Use of Reference Materials

Authorship
The publication has been prepared by the EEE-RM working group

Official language
The text may be translated into other languages as required. The English language version
remains the definitive version.

Copyright
The copyright of this text is held by EA. The text may not be copied for resale.

Further information
For further information about this publication, contact your national member of EA or the EA
Secretariat at secretariat@european-accreditation.org

Please check our website for up-to-date information http://www.european-accreditation.org/

Category: Application documents and Technical Advisory documents for Conformity


Assessment Bodies

EA-4/14 is an informative document

Date of endorsement: 13 june 2002

Date of implementation: 13 June 2003

Tansitionnal period:

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EA - 4/14  The Selection and Use of Reference Materials

CONTENTS

ABSTRACT ............................................................................................................................. 4
INTRODUCTION .................................................................................................................... 4
1 - TYPES OF REFERENCE MATERIALS........................................................................ 5
2 - CLASSIFICATION OF REFERENCE MATERIALS .................................................... 5
3 - TRACEABILITY OF REFERENCE MATERIALS ........................................................ 6
4 - THE AVAILABILITY AND SELECTION OF REFERENCE MATERIALS ................ 6
5 - USES OF REFERENCE MATERIALS .......................................................................... 7
5.1 Method Validation and Measurement Uncertainty ................................................. 7
5.2 Verification of the Correct Use of a Method ............................................................ 7
5.3 Calibration ..................................................................................................................... 8
5.4 Quality Control and Quality Assurance (QC&QA) .................................................. 8
6 - ASSESSMENT OF THE SUITABILITY OF REFERENCE MATERIALS ................ 8
6.1 Certificates and Supporting Reports ......................................................................... 9
6.2 - Assessment of the Suitability of Reference Materials ......................................... 9
7 - IN-HOUSE PREPARATION OF REFERENCE MATERIALS ................................. 10
8 - DEFINITIONS.................................................................................................................. 10
9 - KEY REFERENCE MATERIALS PUBLICATIONS ................................................... 10
10 - REFERENCES ............................................................................................................. 11
FIGURE 1: Overlap between functions associated with Measurement Traceability
and Analytical Quality ...................................................................................................... 13
FIGURE 2: Assessment of the suitability of a Reference Material ........................... 13

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EA - 4/14  The Selection and Use of Reference Materials

THE SELECTION AND USE OF REFERENCE MATERIALS

A BASIC GUIDE FOR LABORATORIES AND ACCREDITATION BODIES

ABSTRACT
The aim of this paper is to provide a short, simple and user-friendly guide for laboratories,
and accreditation and certification bodies. It employs mainly ISO and VIM definitions and
provides references to other more comprehensive and expert texts. It is intended to provide
help for the inexperienced rather than the expert and necessarily simplifies some topics. It
includes discussion and clarification of some misunderstood issues. Although based on the
requirements of chemical measurement, it is intended to be also of use in other areas of
measurement.

INTRODUCTION
There are a number of authoritative and detailed texts on various aspects of reference
materials and these are listed at the end of this paper, together with some internationally
recognised (1-4) definitions. This paper aims to provide a simple guide on the use of
reference materials (RMs) within a wider quality programme. Reference materials are an
important tool in realising a number of aspects of measurement quality and are used for
method validation, calibration, estimation of measurement uncertainty, training and for
internal QC and external QA (proficiency testing) purposes.
In the wider sense, the validity of measurements can be assured when:
 work is carried out to a clearly defined customer requirement
 validated methods and equipment are used
 qualified and competent staff undertake the work
 comparability with measurements made in other laboratories is assured
(traceability and measurement uncertainty)
 independent evidence of performance is available (proficiency testing)
 well defined QC and QA procedures are employed, preferably involving third
party accreditation
Often a measurement operation serves more than one quality purpose and there can be
overlap of function as illustrated in Figure 1. Different types of reference materials are
required for different functions. For example, a certified reference material would be
desirable for method validation but a working level reference material would be adequate for
QC.
More detailed guidance on the QA of chemical measurements, including cover of reference
materials, calibration, QC and validation is provided in a joint CITAC Eurachem guide (5).

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1 - TYPES OF REFERENCE MATERIALS


RMs are used to support measurements concerned with chemical composition, biological,
clinical, physical, engineering properties and miscellaneous areas such as taste and odour.
They may be characterised for ‘identity’ (e.g. chemical structure, fibre type, microbiological
species etc.) or for ‘property values’ (e.g. amount of specified chemical entity, hardness
etc.). Some commonly encountered types of reference materials are as follows:
1. Pure substances characterised for chemical purity and/or trace impurities.
2. Standard solutions and gas mixtures, often prepared gravimetrically from pure
substances and used for calibration purposes.
3. Matrix reference materials, characterised for the composition of specified major, minor
or trace chemical constituents. Such materials may be prepared from matrices
containing the components of interest, or by preparing synthetic mixtures.
4. Physico-chemical reference materials characterised for properties such as melting
point, viscosity, and optical density.
5. Reference objects or artefacts characterised for functional properties such as taste,
odour, octane number, flash point and hardness. This type also includes microscopy
specimens characterised for properties ranging from fibre type to microbiological
specimens.

2 - CLASSIFICATION OF REFERENCE MATERIALS


Two classes of materials are recognised by ISO, namely ‘certified reference materials'
(CRMs) and ‘reference materials’ (RMs). CRMs must by definition be traceable to an
accurate realisation of the unit in which the property values are expressed. Each property
value must be accompanied by an uncertainty at a stated level of confidence. RMs are
materials whose property values are sufficiently homogeneous and well established to be
used for the calibration of an apparatus, the assessment of a measurement method, or for
assigning values to materials.
For most chemical reference materials produced before the late 1990s, the measurement
uncertainty values given by the producers are unlikely to have been estimated by the now
recommended ISO (6,7) procedure. The actual uncertainty can be expected to be larger
than stated by a factor of 2-3, where only within laboratory precision measurements are
used and by a smaller factor where certification included a range of validated methods and
a number of laboratories. Also some materials sold as CRMs have no stated traceability.
The following classes of reference materials are also encountered:
Primary reference material

Secondary reference materials Decreasing Uncertainty

In-house or working reference material

Other terminology, such as NIST Standard Reference Materials (SRMs) is also used and a
classification (class O - V) based on degree of traceability to SI has been proposed (8). Pan
(9) has also published a useful paper on this topic.

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An internationally recognised quality approval system to assure the competence of RM


producers (based on ISO Guide 34 and reference 10) is under consideration within the
International Laboratory Accreditation Co operation, but it will take some years for any
system to make a significant impact on the market place. Users must, therefore, be
cautious and seek clear evidence of the quality and traceability of the property values of
materials from suppliers as detailed below.

3 - TRACEABILITY OF REFERENCE MATERIALS


A short review of the concept and practice of traceability in chemical measurement is
available in reference 5. Reference materials are important tools for the transfer of
measurement accuracy between laboratories and their propert values should, where
feasible, be traceable to SI. Traceability is, however, a relatively new concept in the field of
chemical measurement and as a consequence very few chemical reference materials are
explicitly traceable to SI. A hierarchy of methods is, however, used for assigning property
values to materials and even if not stated, their traceability can be described as follows:

Measurement Method Traceability

Primary method SI
Method of known bias SI/International standard
Independent method(s) Results of specified methods
Interlaboratory comparison Results of specified methods

A combination of ‘certification procedures’ is sometimes employed, such as a consensus


value derived from an interlaboratory comparison where primary methods were used. In the
absence of formally stated traceability it will be necessary for the user to make judgements
about implicit traceability, based on the ‘certification’ data available in reports and the
technical literature. It is important to ensure that chemical interferences and matrix effects
are adequately addressed in arriving at both the certified value and its uncertainty. Unknown
levels of bias are not uncommon and contribute to lack of agreement of measurements.
The measurement uncertainty of the property value of a reference material employed in a
measurement process, will contribute to the uncertainty of the final measurement but should
contribute less than one third of the overall measurement uncertainty. Any underestimation
of the uncertainty of the RM property value will, of course, be carried through to
measurements where the RM is used.

4 - THE AVAILABILITY AND SELECTION OF REFERENCE MATERIALS


Generally the demand for reference materials exceeds supply in terms of the range of
materials and availability. It is rare to have a choice of alternative RMs and the user must
choose the most suitable material available. It is important therefore that users and
accreditation bodies understand any limitations of reference materials employed.
There are, however, several hundred organisations producing tens of thousands of
reference materials worldwide. Producers include internationally renowned institutions such
as NIST; collaborative government sponsored programs such as the EU BCR program,
semi-commercial sectoral or trade associations such as the American Oil Chemicals
Association and an increasing number of commercial organisations. The distinction between
government institutes and commercial businesses is disappearing with the privatisation of a
number of national laboratories.

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Not all materials that are used as reference materials are described as such. Commercially
available chemicals of varying purity, commercial matrix materials and products from
research programs are often used as standards or reference materials. In the absence of
‘certification’ data provided by the supplier it is the responsibility of the user to assess the
information available and undertake further characterisation as appropriate. Guidance on
the preparation of reference materials is given in ISO Guides 31, 34 and 35 and guides on
the preparation of working level reference materials are also available (12,13).
Information about reference materials is available from a number of sources. The COMAR
Database, contains information on more than 10,000 RMs/CRMS which can be accessed
directly or through institutes which provide an advisory service. Further information can be
obtained from the COMAR Central Secretariat (comar@BAM.de). Advisory services assist
users identify the type of material required for their task and identify a supplier. A database
covering reference materials currently under development has been prepared by CITAC and
ISO REMCO (14). A number of suppliers provide a comprehensive range of materials
including materials produced by other organisations and aim to provide a one-stop-shop for
users. A first internet database for a selection of RMs has recently appeared and is likely to
be followed by others (http://www.iaea.org/programmes/nahunet/e4/nmrm/index.htm)

5 - USES OF REFERENCE MATERIALS

A more detailed description of the use of RMs is available in ISO Guides 32 and 33

5.1 Method Validation and Measurement Uncertainty


Estimation of bias (the difference between the measured value and the true value) is one of
the most difficult elements of method validation, but appropriate RMs can provide valuable
information, within the limits of the uncertainty of the RMs certified value(s) and the
uncertainty of the method being validated. Although traceable certified values are highly
desirable, the estimation of bias differences between two or more methods can be
established by use of less rigorously certified RMs. Clearly the RMs must be within the
scope of the method in terms of matrix type, analyte concentration etc. and ideally a number
of RMs covering the full range of the method should be tested. Where minor modifications to
a well-established method are being evaluated then less rigorous bias studies can be
employed.
Replicate measurement of the RM, covering the full range of variables permitted by the
method being validated can be used to estimate the uncertainty associated with any bias,
which should normally be corrected for (15).
The uncertainty associated with an RM should be no greater than one third of that of the
sample measurement.

5.2 Verification of the Correct Use of a Method


The successful application of a valid method depends on its correct use, both with regard to
operator skill and suitability of equipment, reagents and standards. RMs can be used for
training, for checking infrequently used methods and for trouble shooting when unexpected
results are obtained.

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5.3 Calibration
Normally a pure substance RM is used for calibration of the measurement stage of a
method. Other components of the test method, such as sample digestion, separation and
derivatisation are, of course, not covered and loss of analyte, contamination and
interferences and their associated uncertainties must be addressed as part of the validation
of the method. The uncertainty associated with RM purity will contribute to the total
uncertainty of the measurement. For example, an RM certified as 99.9% pure, with an
expanded uncertainty U (k=2) of 0.1% will contribute an uncertainty component of 0.1% to
the overall measurement uncertainty budget. In the case of trace analysis, this level of
uncertainty will rarely be important but for assay work, it can be expected to be significant.
Some other methods, such as XRF analysis, use matrix RMs for calibration of the complete
analytical process. In addition to a close matrix match, the analyte form must be the same in
the samples and RMs, and the analytical concentrations of the RMs must span that of the
samples (12).
ISO Guide 32 and reference 7 provide additional useful information.

5.4 Quality Control and Quality Assurance (QC&QA)


RMs should be characterised with respect to homogeneity, stability, and the certified
property value(s). For in-house QC, however, the latter requirement can be relaxed, but
adequate homogeneity and stability are essential. Similar requirements apply to samples
used to establish how well or badly measurements made in different laboratories agree. In
the case of proficiency testing, homogeneity is essential and sample stability within the time-
scale of the exercise must be assessed and controlled. Although desirable, the cost of
certifying the property values of proficiency testing samples often prohibits this being done
and consensus mean values are often used instead. As a consequence, there often
remains some doubt concerning the reliability of assigned values used in proficiency testing
schemes. This is because, although the consensus mean of a set of data has value, ‘the
majority’ is not necessarily correct and as a consequence the values carry some
undisclosed element of uncertainty. The interpretation of proficiency testing data thus needs
to be carried out with caution.

6 - ASSESSMENT OF THE SUITABILITY OF REFERENCE MATERIALS


As previously indicated the key quality parameter is the uncertainty associated with
the certified value and the reliability of the uncertainty estimate. Uncertainty budgets should
be derived using the ISO approach (6,7). ‘Certification’ data should be stated together with
the expanded uncertainty, U, using a coverage factor k=2 which gives a level of confidence
of approximately 95%.
However, the full uncertainty data is often not available and it is necessary to consider other
quality criteria. Also, the non-expert may not be in a position to fully evaluate the
‘certification’ data and a quality check list, or a third party quality approval system, is
desirable. Such systems are under development but may take some time to become fully
established.
A protocol for assessing the suitability of RMs is detailed in Figure 2 and discussed below.
The user must assess the appropriateness and fitness for purpose of any RM based on the
customer and analytical requirements. Factors to be considered include the following:

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1. The suitability of a reference material depends on the details of the analytical


specification. Matrix effects and other factors such as concentration range can be more
important than the uncertainty of the certified value as detailed in (11). The factors to
consider include:
 Measurand including analyte
 Measurement range (concentration)
 Matrix match and potential interferences
 Sample size
 Homogeneity and stability
 Measurement uncertainty
 Value assignment procedures (measurement and statistical)
2. The validity of the ‘certification’ and uncertainty data, including conformance of key
procedures with ISO Guide 35 and other ISO requirements (6,7).
3. Track record of both the producer and the material. For example, when a RM in use has
been subjected to an interlaboratory comparison, cross-checked by use of different
methods, or there is experience of use in a number of laboratories over a period of
years.
4. Availability of a certificate and report conforming to ISO Guide 31.
5. Demonstrated conformance of the production of the reference materials with quality
standards such as ISO Guides 34 or ILAC requirements (10), or compliance of the
measurement of property values with ISO/IEC 17025 (16) requirements.
All or some of the requirements may be specified in the customer and analytical
specification, but often it will be necessary for the analyst to use professional judgement.
Finally, quality does not necessarily equate to small uncertainty and fitness for purpose
criteria need to be used.

6.1 Certificates and Supporting Reports


Ideally, a certificate complying with ISO Guide 31 and a report covering the characterisation,
certification and statistical analysis procedures, complying with ISO Guide 35, will be
available. However, many RMs, particularly older materials and materials not specifically
produced as RMs, may not fully comply with ISO Guides 31 and 35. Alternative, equivalent
information in whatever form it is available, that provides credible evidence of compliance
can be considered acceptable. Examples include the following: technical reports, trade
specifications, papers in journals or reports of scientific meetings and correspondence with
suppliers.

6.2 - Assessment of the Suitability of Reference Materials


Laboratories must be able to explain and justify the basis of selection of all RMs and of
course any decision not to use a RM. In the absence of specific information it is not possible
to assess the quality of an RM. The rigour with which an assessment needs to be conducted
depends on the criticality of the measurement, the level of the technical requirement and the
expected influence of the particular RM on the validity of the measurement. Only where the
choice of RM can be expected to significantly affect measurement results is a formal
suitability assessment required.

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7 - IN-HOUSE PREPARATION OF REFERENCE MATERIALS


High quality RMs are demanding and costly to produce and if materials are available from
other sources it is not normally cost effective for laboratories to make their own. However
should this be necessary, there are guides available (12,13) to help the non-specialist
laboratory prepare their own RMs. Some of the key issues that need to be considered are:
selection of materials (appropriateness, native material versus spikes, material preparation
etc.), homogeneity testing, preparation and packaging (homogeneity, contamination,
stability etc.), stability testing, certification studies, uncertainty estimation, documentation
and QA, certification approval, storage, distribution and relifing.

8 - DEFINITIONS
Calibration (1): Set of operations that establish, under specified conditions, the relationship
between the values of quantities indicated by a measurement instrument or measuring
system or values represented by a material measure or a reference material, and the
corresponding values realised by standards.
Certified Reference Material (1): Reference material, accompanied by a certificate, one or
more of whose property values are certified by a procedure which establishes traceability to
an accurate realisation of the unit in which the property values are expressed, and for which
each certified value is accompanied by an uncertainty at a stated level of confidence.
Primary method (2): A primary method is a method having the highest metrological
qualities, whose operation can be completely described, and understood and for which a
complete uncertainty statement can be written down in terms of SI units. A primary direct
method measures the value of an unknown without reference to a standard of the same
quantity. A primary ratio method measures the ratio of an unknown to a standard of the
same quantity; its operation must be completely described by a measurement equation. The
methods identified as having the potential to be primary methods are: isotope dilution mass
spectrometry: gravimetry, covering gravimetric mixtures and “gravimetric analysis”;
titrimetry; coulometry; determination of freezing point depression; differential scanning
calorimetry and nuclear magnetic resonance spectroscopy. Other methods such as
chromatography, which has extensive applications in organic chemical analysis, have also
been proposed.
Reference material (RM) (1): Material or substance one or more of whose property values
are sufficiently homogeneous and well established to be used for the calibration of an
apparatus, the assessment of a measurement method, or for assigning values to materials.
Traceability (1): Property of a result of a measurement or the value of a standard whereby
it can be related to stated references, usually national or international standards, through an
unbroken chain of comparisons all having stated uncertainties.
Uncertainty of measurement (1): Parameter associated with the result of a measurement
that characterises the dispersion of the values that could reasonably be attributed to the
measurand.
Validation (3): Confirmation by examination and provision of objective evidence that the
particular requirements of a specified intended use are fulfilled. Method validation has not
been formally defined but a Guide on the topic is available (4)

9 - KEY REFERENCE MATERIALS PUBLICATIONS


The following guides have been prepared by ISO REMCO, the international
committee that leads on reference material matters:
 ISO Guide 30:1992 Terms and definitions used in connection with reference
materials

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EA - 4/14  The Selection and Use of Reference Materials

 ISO Guide 31:2000 Contents of certificates of reference materials


 ISO Guide 32:1997 Calibration of chemical analysis and use of certified
reference materials
 ISO Guide 33:2000 Uses of certified reference materials
 ISO Guide 34:2000 General requirements for the competence of reference
material producers
 ISO Guide 35:1989 Certification of reference materials-General and statistical
principles
 ISO/REMCO Document N 330 List of producers of certified reference materials,
Information by Task Group 3 “Promotion”
Other guides include:
 European Commission Document, BCR/48/93 (Dec 1994): Guidelines for the
production and certification of BCR reference materials
 NIST Publication 260-100 (1993): Standard Reference Materials - Handbook for
SRM Users
 IUPAC ‘Orange Book’: Recommended Reference Materials for the Realisation of
Physico chemical Properties, Edited K N Marsh, Blackwell Scientific
Publications, 1987
 World Health Organisation (WHO) Guidelines for the Preparation and
Characterisation and Establishment of International and other Standards and
Reference Reagents for Biological Substances, Technical Report Series No 800
(1990)

10 - REFERENCES
1. International vocabulary of basic and general terms in metrology (VIM), second
edition, 1993, ISO/BIPM/IEC/IFCC/IUPAC/IUPAP/IOML, Published by ISO
2. Minutes of 4th CCQM meeting, Feb 1998, Paris

3. ISO 9000, Quality management systems - Fundamentals and vocabulary (ISO


9000:2000);
4. Eurachem Guide: The fitness for Purpose of Analytical Methods – A Laboratory
Guide to Method Validation and Related Topics, 1998, Published by LGC UK
(see www.eurachem.org)
5. CITAC Eurachem Guide , Guide to Quality in Analytical Chemistry, An Aid to
Accreditation, 2001, In press – see Eurachem or CITAC web site.
6. Guide to the expression of uncertainty in measurement, first edition, 1995,
ISO/BIPM/IEC/IFCC/IUPAC/IUPAP/IOML. (Published by ISO)
7. Eurachem/CITAC Guide, Quantifying Uncertainty in Analytical Measurement, 2.
edition 2000, Published by LGC, UK (see www.eurachem.org)
8. P De Bievre et al, Accred Qual Assur, 1996, 1, 3-13
9. X R Pan, Metrologia, 1997, 34, 35-39,

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10. ILAC Guidelines for the Competence of Reference Material Producers, ILAC
G12, 2000 (see www.ILAC.org)
11. A Marschal, Traceability and Calibration in Analytical Chemistry - Principles and
Applications to Real Life in Connection with ISO 9000, EN 45000 And ISO Guide
25, Eurolab Symposium, Firenze, April 1994
12. B Brookman and R Walker, Guidelines for the In-House Production of Reference
Materials, March 1997, LGC Report, UK
13. J M Christensen, Guidelines for Preparation and Certification of Reference
Materials for Chemical Analysis in Occupational Health, NORDREF, 1998 (ISBN:
87-7904-010-1)
14. S D Rasberry and C L Monti, Worldwide Production of CRMs: 1996 Status
Report, NIST Report Feb 1996
15. S L R Ellison and A Williams, Measurement Uncertainty: The Key to the Use of
Recovery Factors, pp 30-37, The Use of Recovery Factors in Trace Analysis, Ed
M Parkany, RSC, 1996
16. ISO/IEC 17025 General requirements for the competence of testing and
calibration laboratories (ISO/IEC 17025:1999)

Prepared by Bernard King on behalf of and with assistance from the EEE-RM
working group.

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EA - 4/14  The Selection and Use of Reference Materials

FIGURE 1: Overlap between functions associated with Measurement


Traceability and Analytical Quality

Validation Traceable
calibration

Measurement Uncertainty/
Traceability
QC/QA

Valid measurement

FIGURE 2: Assessment of the suitability of a Reference Material

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Define analytical Select candidate RM and


requirement (Note 1) obtain supporting
information (Note 2)

No
Reported RM charac-teristics fully match analytical
requirement Limitations but RM is
best available and
meets minimum
requirements
Yes
Yes
No

Supporting evidence concerning quality No Limitations but RM is


is satisfactory (Note 3) best available and
meets minimum
requirements
Yes No
Yes

REFERENCE MATERIAL SUITABLE RM NOT SUITABLE, SEEK ALTERNATIVE


OR DOWN GRADE REQUIREMENT

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Notes to Fig. 2:

Note 1: The analytical requirements specification should include details concerning the
measurand, concentration, traceability, measurement uncertainty, etc

Note 2: Key characterists should be available in the RM Certificate. Additional information,


for example details of the method(s) used for value assignment and the full measurement
uncertainty budget should also be available in the Certificate or in a supporting report.

Note 3: See p4 and p5

June 2002

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