Accred Qual Assur (2006) 11: 474–480

DOI 10.1007/s00769-006-0170-4 DISCUSSION FORUM

Papers published in this section do not necessarily reflect the opinion of the Editors, the
Editorial Board and the Publisher.

Marina Patriarca
Ferdinando Chiodo
Estimates of uncertainty of measurement
Marco Castelli from proficiency testing data: a case study
Antonio Menditto

Received: 31 October 2005
Accepted: 26 April 2006
Published online: 27 June 2006


C Springer-Verlag 2006
Presented at the Eurachem PT Workshop
September 2005, Portorož, Slovenia.
M. Patriarca (
) · F. Chiodo ·
M. Castelli · A. Menditto
Department of Food Safety and
Veterinary Public Health, Istituto
Superiore di Sanità,
Viale Regina Elena 299,
00161 Rome, Italy
e-mail: marina.patriarca@iss.it
Tel.: + 390649902562
Fax: + 390649903686
Abstract The results obtained by a
laboratory over a number of
proficiency testing/external quality
assessment schemes (PT/EQAS)
rounds can give information on the
uncertainty of its measurements for a
given test, provided that conditions
such as full coverage of the routine
analytical range, traceability, and
small uncertainty of the assigned
values (compared to the spread of the
results) are met and provided that
systematic deviations and any other
sources of uncertainty are considered.
As organisers of the Italian EQAS
(ITEQAS) in occupational and
environmental laboratory medicine,
we tested this hypothesis using as
model data from well-performing
laboratories taking part in ITEQAS
for lead in blood over the last 2 years.
We also investigated how different
PT/EQAS features (frequency of
trials and number of samples) would
affect a laboratory estimate of its
uncertainty. Such information can be
helpful in improving PT/EQAS
organisation and define, for a given
test: (a) the state of the art of the
uncertainty of current measurement
procedures, (b) identify needs for
improvement of analytical
methodologies and (c) set targets for
acceptable uncertainty values.
Keywords Uncertainty estimate .
Lead in blood . External quality
assessment . Uncertainty of assigned
values

Introduction
The international standards on accreditation of calibration,
testing [1] and clinical [2] laboratories require the appli-
cants to have procedures for the estimate of the uncertainty
of the tests to be accredited. The theoretical principles and
methods for the expression of uncertainty in measurement
are laid down in the GUM [3]. Practical applications of
those principles to quantitative measurements in analyti-
cal chemistry are described in the EURACHEM/CITAC
Guide [4]. This last document, starting from measurement
models and identification of possible uncertainty sources,
exploits the concept of extracting information about un-
certainty of measurement from data already available in
laboratories operating under a quality management system,
such as data from validation studies, internal quality con-
trol and participation in proficiency testing (PT)/external
quality assessment schemes (EQAS). The rationale for the
use of PT/EQAS data to check or estimate a laboratory’s
uncertainty is based on the fact that the estimated uncer-
tainty for a given test should be compatible with the spread
of results obtained by that laboratory over a number of
PT/EQAS rounds [4]. This implies the PT/EQAS to offer
full coverage of the routine analytical range for real sam-
ples, traceability to stated references and small uncertainty
of the assigned values, compared to the spread of the re-
sults. Participants wishing to make such use of PT/EQAS
data should make sure that systematic deviations from the
assigned values and any other source of uncertainty not
covered by EQAS (e.g., sampling or storage of real sam-
ples) are taken into account by separate assessments.
However, PT/EQAS are organised in different ways, even
in the same field of analysis. In a survey of European EQAS
in occupational and environmental laboratory medicine
(OELM) [5], frequency of rounds, number of samples/trial
and methods to define assigned values varied widely. Few
schemes estimated the uncertainty of the assigned value
and/or took it into account in the evaluation of participants’
performance. As organisers of the Italian EQAS (ITEQAS)
in OELM, we wished to assess whether participants could

use their results in EQAS to check or estimate the uncer-
tainty of their measurements for lead in blood. In addition,
we investigated how different PT/EQAS features would
affect a laboratory estimate of its uncertainty. Such infor-
mation can be helpful in improving PT/EQAS organisation,
to define the state of the art of the uncertainty of current
measurement procedures and to set targets for acceptable
uncertainty values for a given test.
Materials and methods
Scheme organization
The METOS Project (from “Metalli Tossici”, Toxic Met-
als) is an EQAS for laboratories performing specialised
analyses in OELM including lead in blood [6]. All con-
trol materials for this scheme are prepared in-house, from
animal blood spiked with known amounts of lead. The fre-
quency of trials is currently four times a year with three
unknown samples distributed in each trial. Unknown du-
plicates are included on a regular basis. Samples distributed
to laboratories are labelled with randomly generated con-
ventional codes in order to prevent the disclosure of the
sample identity to participants. Assigned values are defined
as consensus values (median of all participants’ results for
each sample). The report distributed to participants at the
end of each trial includes sample statistics and performance
indices for each analysed sample (compliance with stated
acceptability limits and z-score). Acceptability limits are
set by the organisers for each measurand at two concen-
trations and are wider at lower concentrations. Limits for
lead in blood are set as ± 20% at 100 µg l–1 and ± 10%
at 800 µg l–1 . Limits at any intermediate concentration
are obtained by interpolation. Z-scores are calculated in
the usual way [7], taking half of the acceptability limit as
the standard deviation for proficiency assessment at that
concentration [6].
Agreed acceptability limits for lead in blood
As part of collaborative work, the Network of Organisers of
EQAS in OELM has agreed common acceptability limits
for lead in blood as ± 4 µg dl–1 or ± 10% of the target
concentration, whichever is greater, with a view to reduce
them to ± 3 µg dl–1 or ± 10%, whichever is greater [8].
Uncertainty of assigned values
Since assigned values in ITEQAS are obtained as consen-
sus values (medians) of the participants’ results, it was
not possible to apply a rigorous procedure such as that de-
scribed in the GUM [3] for the estimate of their uncertainty.
However, a procedure to obtain an estimate of the uncer-
tainty of consensus values has been reported in ISO/FDIS
13528 [9] and this was applied to the values assigned to
the samples distributed in ITEQAS for lead in blood over
475
the last two years. According to [9], the uncertainty of the
assigned values was estimated as:
1.23 × s ∗
ux = √
p
where s ∗ is the robust standard deviation estimated from
the results provided by all participants using the Algorithm
A described elsewhere [9 , 10]. The number of participants
in each trial ranged from 55 to 64. All participants used ana-
lytical methods based on electrothermal atomic absorption
spectrometry. Expanded uncertainty was calculated using
a covering factor k = 2 for a confidence interval of 95%.
Estimate of uncertainty of participants’ results
from cumulative EQAS data
We used the results obtained by well-performing partici-
pants (laboratories, n = 14) in the ITEQAS for lead in blood
over the last 2 years. Laboratories were selected according
to (a) participation in all trials (n = 8) reporting results for
all samples (n = 24); (b) sum of |z-score| ≤ 48; (c) sum of
|z-score| in each trial ≤ 9; (d) average of z-scores not signif-
icantly different from zero, when compared to its standard
deviation. For each laboratory, the relative difference (Drel )
between the result provided (xi ) and the assigned value of
the respective EQA sample (Xref ) was calculated as:
xi − X ref
Drelx =
X ref
and the uncertainty of measurement was estimated as the
standard deviation of the relative differences. Expanded
uncertainty was calculated taking a covering factor k = 2
for a confidence level of 95%.
For each laboratory, the contribution of imprecision to to-
tal uncertainty was estimated as pooled standard deviation
of duplicates from the results provided in two different oc-
casions for each of seven samples distributed as unknown
duplicates, according to the formula:



 n
 (xi1 − xi2 )2
 i=1
SDpooled =
2n
A huge (compared to the others) difference (184 µg l–1 )
between one set of duplicates (Lab #1) was attributed to
occasional contamination of the sample and the set of data
removed.
Assessment of the effect of simulated variations of
scheme features on the estimate of laboratory
uncertainty
To test whether the frequency of trials (Tn ) or the number
of samples/trial affects the estimate of uncertainty, varia-
476
Table 1 Experimental design. Two sets of data (A bold line and B dashed line) were obtained from the whole database, by selecting trials
(T) or samples (S)
tions of scheme features (case A: fewer trials and case B:
fewer samples/trial) were simulated by selecting sub-sets
of data. As shown in Table 1, two groups of data were ex-
amined: (A) four sets each including two trials performed
1 year apart (set A1: all samples from T1 and T5 ; set A2:
all samples from T2 and T6 ; set A3: all samples from T3
and T7 ; set A4: all samples from T4 and T8 ); (B) three
sets each including only one sample from each of the eight
trials (set B1: all the S1 Tn ; set B2: all the S2 Tn ; set B3
all the S3 Tn ) where S1 , S2 , and S3 are the 1st, 2nd and
3rd samples (ordered by code), respectively. For each sub-
set, laboratories uncertainty estimates were obtained as de-
scribed above. Data were analysed by means of two-ways
ANOVA.
In addition, it was studied how the uncertainty estimates
varied if more data, from subsequent trials, were used for
the evaluation. To this aim, for each laboratory, uncer-
tainty of measurement was estimated, with the calcula-
tions described above, from the results of only the first
trial; then a second estimate was obtained from the results
of both the 1st and 2nd trial; a third from the results of
the 1st, 2nd and 3rd trial and so on, until all trials were
included.
Assessment of the reliability of the uncertainty
estimate
To assess the reliability of the uncertainty estimates ob-
tained by the method described above, the results obtained
in a new trial (immediately following those examined here)
were considered. Due to changes in the organization of the
programme (introduction of fees) only seven out of the 14
laboratories included in this study were still participating in
METOS. The results provided by these seven laboratories,
each accompanied by its estimated expanded uncertainty,
were compared with the assigned values (and their uncer-
tainty) of the three samples distributed in that trial: 105.8
(3.4) µg l–1 ; 205.9 (6.3) µg l–1 ; 401.1 (10.2) µg l–1 .
Results
Uncertainty of assigned values
The uncertainty of the assigned values ranged from to 3.9
to 1.5% within the concentration interval 51–718 µg l–1 ,
and was 7.0% for samples at lower concentration (22–
26 µg l–1 ). The relationship between relative uncertainty
(%) and sample concentration is shown in Fig. 1 and com-
pared to relative interlaboratory standard deviations (%)
50
40
Variation %
30
20
10
0
0 200 400 600 800
Assigned values, [Pb], µg l
-1
Fig. 1 Relative uncertainties % (
) of assigned values, estimated
from the participants’ data according to ISO/FDIS 13528, compared
to relative interlaboratory standard deviations %: a predicted by the
Horwitz equation (RSDR , dashed line) and b observed (robust stan-
dard deviations %,
) for the 24 samples distributed over a period of
2 years in ITEQAS
predicted by the Horwitz equation and to those observed
(robust standard deviations, %) in ITEQAS.
Estimate of uncertainty of participants’ results from
cumulative EQAS data
The estimated relative uncertainties over the concentration
range 22–718 µg l–1 ranged from 0.030 to 0.235 for the
14 laboratories included in this study (Fig. 2). The con-
tribution of imprecision to total uncertainty is also shown
in Fig. 2. The laboratories’ estimated expanded uncertain-
ties were compared, over the chosen concentration range,
with: (a) the expanded uncertainty of the assigned values,
(b) the acceptability limits defined in ITEQAS [6] (c) the
acceptability limits agreed by the Network of Organisers of
EQAS/PT in OELM [8]. Figure 3 shows this comparison
for two laboratories with either low or high uncertainty.
Assessment of the effect of simulated variations of
scheme features on the estimate of laboratory
uncertainty
The results of ANOVA analysis carried out on data set A
(four sets each including two trials performed 1 year apart)
and data set B (three sets each including only one sample
from each of the eight trials) were as follows:
– Set A, laboratories: F = 3.4142 (Fcrit = 1.9805),
p = 0.0015, df = 13; sets of trials: F = 2.4751
(Fcrit = 2.8451), p = 0.0758, df = 3;
 477

200
a 0.25

160
0.20
Relative u or SDpooled

120

-1
[Pb], µg l
0.15

80
0.10

40
0.05

0
0.00 0 100 200 300 400 500 600 700 800
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #14
Median, [Pb], µg l-1
Laboratora code
Fig. 3 Expanded uncertainty of measurement of two laboratories
b 100
(heavy and light solid line) compared over the concentration range
90 with: a the expanded uncertainty of the assigned values (dotted line);
80
b the acceptability limits defined in ITEQAS (dashed/dotted line);
c the acceptability limits agreed by the Network of Organisers of
(relative DSpooled/u relative)%

70 EQAS/PT in OELM (dashed line)

60

50
0 .1 2
40 Variation of uncertainty estimate 0 .1 0
30
0 .0 8
20
0 .0 6
10

0 .0 4
0
#1 #2 #3 #4 #5 #6 #7 #8 #9 #10 #11 #12 #13 #1 4
Laboratory code
0 .0 2

Fig. 2 a Relative uncertainties estimated from all EQAS results 0 .0 0

(closed bars) and intermediate precision estimated as relative pooled -0 .0 2
SD from seven unknown duplicates (open bars) for the 14 laborato-
ries included in the study (pooled SD for lab#1 calculated from six -0 .0 4
duplicates). b Contribution of imprecision to total uncertainty shown
-0 .0 6
as the ratio between relative uncertainty and relative pooled SD of
duplicates for the 14 laboratories included in the study 1 -2 1 -3 1 -4 1 -5 1 -6 1 -7 1 -8
Cumulated trials

– Set B, laboratories: F = 3.1286 (Fcrit = 2.1192), Fig. 4 Variations of the uncertainty estimates, for each laboratory
(—— lab#1, — ◦ — lab#2, —— lab#3, —  — lab#4, —
—
p = 0.064, df = 13; sets of samples F = 6.5591 lab#5, —•— lab #6, —— lab#7, —— lab#8, ––lab#9, – ◦ –
(Fcrit = 3.3690), p = 0.049, df = 2. lab#10, ––lab#11, –  – lab#12, –
– lab #13, –•– lab#14) observed
when data from subsequent exercises (trials: 1 and − 2; 1, 2 and 3;
For each laboratory, the changes of the uncertainty esti- and so on, until all trials from 1–8) were cumulated. Changes are
mates, observed when data from subsequent exercises (tri- shown as the difference to the estimate of the first trial
als: 1 and 2; 1, 2 and 3; and so on, till all trials from 1 to
8) were cumulated, are shown in Fig. 4, as the differences
from the uncertainty value estimated from the data of the Discussion
first trial only.
According to the EURACHEM/CITAC Guide [4], pre-
requisites for the use of PT/EQAS data to estimate the
Assessment of the reliability of the uncertainty uncertainty of measurement are full coverage of the rou-
estimate based on previous trials tine analytical range for real samples, traceability to stated
references and small uncertainty of the assigned values,
The comparison of laboratories’ results accompanied by compared to the spread of the results. In ITEQAS, the
their estimated expanded uncertainty and the assigned concentrations of samples distributed to participants cov-
values showed that, in all cases, the expanded uncer- ered the range of blood Pb levels that may occur following
tainty intervals around the data provided by the labo- environmental or occupational exposure. Robust statistics
ratory overlapped the interval assigned value ± its ex- (consensus median of the participants’ results) are used as
panded uncertainty. An example for the sample at con- the assigned values, checked against sample formulation
centration 401 µg l–1 is given in Fig. 5. Similar plots and the results of homogeneity tests. Consensus values are
were obtained for the samples at concentrations 105.8 and not traceable to SI, and, in some cases, there may be no
205.9 µg l–1 . real consensus amongst the participants or the consensus
may be biased by the general use of faulty methodology.
478

700 Table 2 Ratio between uncertainty of the assigned value (uref ) and
ITEQAS standard deviation for proficiency testing (σ) in comparison
600 with the recommended ISO criteria (uref /σ ≤ 0.3)
σ
[Pb], µg l -1

500 Assigned values, [Pb] (µg/l) uref a (µg/l) b

(µg/l) uref /σ
400 22.0 1.57 6.57 0.24
300 25.9 1.79 6.79 0.26
200 51.0 1.99 7.86 0.25
#4 #5 #8 #9 #10 #11 #12 62.8 2.32 8.37 0.28
100
Laboratories 76.8 2.48 9.10 0.27
79.9 2.16 9.12 0.24
Fig. 5 Results (each accompanied by its expanded uncertainty)
obtained, for the sample at 401.1 µg l–1 , by the seven laboratories that 100.6 2.58 10.00 0.26
took part in a new trial following those used to estimate laboratory 102.5 2.86 10.11 0.28
uncertainty, compared to the assigned value (and its uncertainty), 197.6 4.73 14.20 0.33
401.1 (10.2) µg l–1
201.8 4.13 14.28 0.29
203.1 3.49 14.41 0.24
However, it is recognised that, for tests where sufficient 205.9 4.71 14.50 0.32
experience exists, as in the case presented here for lead 285.4 6.95 17.97 0.39
in blood, consensus values are usually very close to those 345.5 7.49 20.80 0.36
provided by formulation, consensus of expert laboratories 379.2 8.22 22.04 0.37
or reference values obtained from reference laboratories or 391.0 7.35 22.53 0.33
certified reference materials [11]. The uncertainty of the 391.2 5.77 22.47 0.26
assigned values (Fig. 1) was from 5 to 8-fold smaller than 394.9 7.01 22.75 0.31
typical values observed over the course of the years for the 429.2 8.84 23.90 0.37
dispersion of participants’ results at three concentrations 435.3 9.22 24.63 0.37
levels (21%, 14% and 11% at 100, 400 and 700 µg l–1 , 584.5 10.48 31.00 0.34
respectively) [8] and much smaller than the corresponding 695.9 19.03 35.77 0.53
reproducibility RSDs calculated from the Horwitz equa-
709.1 15.15 36.18 0.42
tion [12] (modified according to Thompson [13]), which
718.5 17.97 36.87 0.49
range from 17 to 22% for concentrations between 22 and
718 µg l–1 ). However, this exercise also showed that the a
Uncertainty of the assigned values for ITEQAS samples, estimated
criteria set by ISO FDIS 13528 [9] with respect to the according to ISO/FDIS 13528
standard deviation for proficiency testing (u ≤ 0.3σ ) were
b
Standard deviation for proficiency testing used in ITEQAS to cal-
culate acceptability limits (2σ )
not fully met (Table 2). The uncertainty of five out of 24
samples was less than 0.4σ and for the three samples at
concentrations >700 µg l–1 the uncertainty reached about (Fig. 3, low uncertainty). Taking the concentration level
half of the chosen standard deviation for proficiency testing. of 400 µg l–1 as an example, laboratories’ uncertainties
Although this observation may call for a revision of current were from 2.6 to 12.1-fold greater than those of the as-
procedures for the determination of assigned values or the signed values. The comparison of the estimated standard
evaluation of laboratory performance in ITEQAS, for the uncertainties from participants’ data with the acceptability
purpose of this study the uncertainty of the assigned values limits set in ITEQAS and by the Network reveals that in six
was considered acceptable. and eight cases, respectively, the laboratory uncertainty is
The estimated uncertainties of the participants ranged close or wider than the stated limits (ratio uncertainty/limit
from 3.0 to 23.5% (Fig. 2A) with the majority of values ≥ 1.0). As a consequence, these laboratories may experi-
(n = 10) laying between 6.2 and 13.9%. This is consistent ence a higher percentage of non compliant results.
with reported estimates of uncertainty for lead in blood Analysis of variance showed that sampling of trial and
from single laboratory studies of methods based on elec- samples in order to simulate participation of laboratories
trothermal atomic absorption spectrometry [14, 15]. The to one trial per year (set A) or the distribution of only one
contribution of imprecision to total uncertainty (Fig. 2A, sample per trial (set B) significantly influenced the esti-
B) ranged from 21.3 to 91%. It is worth noting that im- mate of laboratory uncertainty from PT/EQAS data. This
precision accounted for more than two-thirds of the total observation prompted us to assess the minimum number of
uncertainty in seven cases, but less than 50% in the other results necessary to give a reliable estimate of uncertainty.
seven. This points out that sources other than imprecision To this aim, for each laboratory, separate estimates of its
contributed to the uncertainty of measurement in some lab- uncertainty were obtained using data only from the first
oratories and that, in general, uncertainty values based only trial and, subsequently, from the first and second trials and
on within-laboratory intermediate precision are likely to be so on, until data from all trials were included. When, for
underestimated. each laboratory, the variations of the uncertainty estimates
The uncertainty of the assigned values contributed little based on data from one, two or more trials were studied
to the estimate of uncertainty for all but one laboratory (Fig. 4), consistent estimates of uncertainty were gener-
 479

ally obtained after four consecutive trials. When treating
data according to this model, a significant variation of the
estimated uncertainty suggests a corresponding change in
laboratory performance or an analytical problem. In this
study, the variations observed for four laboratories when
data from trial no. 8 were cumulated with the others are
due to the greater analytical difficulties experienced with
a sample at the lowest extreme of the concentration range
(22 µg l–1 ) included in that trial. The reliability of these
uncertainty estimates was independently confirmed by the
analysis of the data reported by some of the laboratories in
a new trial (Fig. 5).
Conclusions
A model for the estimate of uncertainty from PT/EQAS
data was applied to data from well-performing laboratories
taking part in ITEQAS for lead in blood. The effect of
frequency of trials and number of samples analysed was
simulated by sampling of datasets. Reliable estimates were
obtained when data from at least four subsequent trials were
combined.
The approach of using PT/EQAS data to estimate the
uncertainty of routine analysis is particularly valuable as
it provides independent information on the performance of
the laboratory over the entire analytical range. Establishing
traceability and evaluating uncertainty are the most difficult
issues to be implemented in routine analytical laboratories.
Surveys of participants in ITEQAS have shown that only
30% of them estimate the uncertainty of their measure-
ment [16]. Similar situations may occur in other countries
or fields of clinical analysis. Although a number of in-
ternational activities are in place to provide the means to
improve traceability in chemistry and laboratory medicine,
at present in many situations PT/EQAS may still represent
the only way to demonstrate equivalence of measurements
and the only means available to a routine laboratory to trace
its results to an independently agreed value.
This study also provides some information on the uncer-
tainty of measurement of lead in blood in routine labora-
tories, in relation with stated acceptability limits for such
tests at national and international level. Such information
is valuable in order to identify needs for improvement of
analytical methodologies and to set targets for acceptable
uncertainty values for a given test, to the benefit of all
parties, i.e., the laboratory itself, accreditation bodies and
end-users of the data.
Provision of information that can be used for a labora-
tory’s estimate of its uncertainty and reduction of the uncer-
tainty of the assigned values could substantially increase
the value of participation in PT/EQAS schemes. This last
issue is of particular value for laboratories with the best
performances, in order to obtain full benefit from their par-
ticipation in PT/EQAS, but the additional costs may result
in reduced effectiveness of the scheme (fewer participants).
In the field of OELM, extensive collaboration is already in
place among scheme organisers [17, 18] and one of the main
issues is to develop common approaches to traceability and
reduced uncertainty of the assigned values at acceptable
cost [17].
Acknowledgements The authors acknowledge the financial sup-
port from the Italian Ministry of Health, Project “Analysis of risk
connected with the presence of residues in food of animal origin -
SARA”, 2003–2006.

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