Clinical Biochemistry 50 (2017) 587–594

Contents lists available at ScienceDirect

Clinical Biochemistry

journal homepage: www.elsevier.com/locate/clinbiochem

Review

Pre-analytical and analytical aspects affecting clinical reliability of plasma

glucose results

Sara Pasqualetti ⁎, Federica Braga, Mauro Panteghini

Research Centre for Metrological Traceability in Laboratory Medicine (CIRME), University of Milan, Milano, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The measurement of plasma glucose (PG) plays a central role in recognizing disturbances in carbohydrate metab-
Received 6 February 2017 olism, with established decision limits that are globally accepted. This requires that PG results are reliable and un-
Received in revised form 1 March 2017 equivocally valid no matter where they are obtained. To control the pre-analytical variability of PG and prevent in
Accepted 10 March 2017
vitro glycolysis, the use of citrate as rapidly effective glycolysis inhibitor has been proposed. However, the com-
Available online 11 March 2017
mercial availability of several tubes with studies showing different performance has created confusion among
Keywords:
users. Moreover, and more importantly, studies have shown that tubes promptly inhibiting glycolysis give PG re-
Glucose sults that are significantly higher than tubes containing sodium fluoride only, used in the majority of studies gen-
Analytical performance specifications erating the current PG cut-points, with a different clinical classification of subjects. From the analytical point of
Standardization view, to be equivalent among different measuring systems, PG results should be traceable to a recognized
Uncertainty higher-order reference via the implementation of an unbroken metrological hierarchy. In doing this, it is impor-
EQAS tant that manufacturers of measuring systems consider the uncertainty accumulated through the different steps
of the selected traceability chain. In particular, PG results should fulfil analytical performance specifications de-
fined to fit the intended clinical application. Since PG has tight homeostatic control, its biological variability
may be used to define these limits. Alternatively, given the central diagnostic role of the analyte, an outcome
model showing the impact of analytical performance of test on clinical classifications of subjects can be used.
Using these specifications, performance assessment studies employing commutable control materials with
values assigned by reference procedure have shown that the quality of PG measurements is often far from desir-
able and that problems are exacerbated using point-of-care devices.
© 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
2. Pre-analytical sources of variation in PG testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 588
2.1. Not all citrate tubes are created equal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
2.2. Clinical impact of the optimization of pre-analytical phase in PG determination . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
3. Biological variation of PG . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 589
4. APS for PG testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
5. Analytical issues related to PG measurement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
5.1. Establishment of traceability of PG results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 590
5.2. Uncertainty of PG measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591

Abbreviations: WHO, World Health Organization; GDM, gestational diabetes mellitus; PG, plasma glucose; CVP, pre-analytical variation; CVI, within-subject biological variation; CVA,
analytical variation; ADA, American Diabetes Association; HAPO, Hyperglycaemia and Adverse Pregnancy Outcome Study; BV, biological variation; CVG, between-subject biological
variation; APS, analytical performance specifications; HbA1c, glycated haemoglobin; FP, false positives; FN, false negatives; IFG, impaired fasting glucose; TE, total error; EFLM,
European Federation of Clinical Chemistry and Laboratory Medicine; IVD, in vitro diagnostics; EQA, external quality assessment; POCT, point-of-care testing; CEG, consensus error grid;
CLSI, Clinical and Laboratory Standards Institute; FDA, Food and Drug Administration.
⁎ Corresponding author at: Clinical Pathology Unit, ASST Fatebenefratelli-Sacco, Via G.B. Grassi 74, 20157 Milano, Italy.
E-mail address: sara.pasqualetti@asst-fbf-sacco.it (S. Pasqualetti).

http://dx.doi.org/10.1016/j.clinbiochem.2017.03.009
0009-9120/© 2017 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.
588 S. Pasqualetti et al. / Clinical Biochemistry 50 (2017) 587–594

5.3. Performance of laboratories in terms of standardization of PG measurements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 591

5.4. Point-of-care testing (POCT) in hospitalized patients: a world apart? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 592

1. Introduction concentrations in whole blood stored at room temperature is affected

Diabetes mellitus is a group of metabolic diseases characterized by
chronic hyperglycaemia and disturbances of carbohydrate, fat and pro-
tein metabolism, resulting from defects in insulin production, action, or
both. Among public health problems, diabetes is one of the most fre-
quent: in 2014, the World Health Organization (WHO) globally estimat-
ed 422 million diabetic adults, with an age-standardized prevalence
that has doubled since 1980, rising from 4.7% to 8.5% [1], and, more re-
cently, the International Diabetes Federation has predicted that diabetes
will affect 642 million people by 2040 [2]. Diabetes can be distinguished
in two main etiopathogenetic categories: type 1, which results from the
progressive autoimmune destruction of the pancreatic β cells, and type
2, much more common, resulting from the progressive impairment of β
cells to adequately produce and secrete insulin, together with a cellular
resistance to insulin action. A third relevant form of glucose intolerance
may occur firstly during pregnancy, defining the framework of gesta-
tional diabetes mellitus (GDM) [3].
Early recognition of disturbances in glucose metabolism is relevant to
counteract the exposure of organs to high glucose concentrations and
avoid the long-term associated damage. To this regard, the measurement
of plasma glucose (PG) plays a central role for the diagnosis of diabetes
and the assessment of risk for the future development of diabetes and car-
diovascular disease [3]. It is therefore crucial that clinical laboratories pro-
vide accurate and reliable PG results to preserve the correctness of their
interpretation and thus the validity of the clinical information. Clinicians in-
deed expect test results at the highest degree of consistency with the clin-
ical status of patients. To comply with this demand, laboratories devote
energy by relocating the concept of reliability to the ability of providing
the highest level of result quality, counteracting analytical and extra-analyt-
ical sources of variation. The total variation of a laboratory result is made up
of three components, namely, pre-analytical variation (CVP), within-subject
biological variation (CVI) and analytical variation (CVA) [4]. CVP comprises
the variability derived from different steps occurring before the analysis
[5]. CVI expresses the physiological changes of analyte concentrations that
occur in a biological fluid in each individual [4]. Unlike CVP, this source of
variability is not reducible and is typically associated with a given analyte.
Finally, CVA is associated with the analytical performance in terms of mea-
surement error. Particularly, the result of a measurement may be affected
by a certain degree of deviation from the “true value” depending on two
main sources of error [6]. The first one occurs systematically and, once char-
by an average decrease of ~5–7% per hour [8]. Accordingly, it is impor-
tant to take precautions to avoid the loss of PG after blood drawing.
The prompt centrifugation of sample followed by the immediate sepa-
ration of plasma from corpuscular phase may guarantee the stability
of PG over time [9,10]. If this is not possible, the National Academy of
Clinical Biochemistry (NACB) guidelines recommend immediately plac-
ing blood tubes in an ice-water slurry and separate plasma from cells
within 30 min from drawing [10], even if immediate sample cooling
may not be ideal as suggested [11]. However, fulfilling either recom-
mendations is difficult in daily practice. Therefore, the use of a PG stabi-
lizer in tubes for blood collection represents the practical alternative. In
2002, NACB recommended the use of tube containing sodium fluoride
for effectively stabilizing PG [12]. Once entered in glycolytic cells, fluo-
ride inhibits the enolase, an enzyme acting downstream in the glycolytic
pathway. The complete stabilizing effect of fluoride takes, however, as
long as 4 h during which the loss of PG is virtually identical to that ob-
tained in absence of fluoride [13]. In 1988, Uchida et al. [14] proved
the ability of sample acidification by addition of citrate buffer to block
the activity of hexokinase and phosphofructokinase working upstream
in glycolysis, ensuring the stability of PG over a 10 h period after blood
drawing. Accordingly, a PG testing tube (Venosafe® Glycaemia, Terumo
Medical Corporation), containing a granular mixture of citrate buffer,
fluoride (useful to sustain the inhibitory effect of the additive over a lon-
ger time) and disodium EDTA as chelating agent, was marketed [15], but
no longer promoted for the unavailability of Venosafe tubes worldwide.
It took two decades for reconsidering this option, under the urgent need
to have a PG stabilizer more effective than fluoride [16]. In particular,
the efficacy of citrate to prevent glycolysis was proved by an indepen-
dent study tailored to evaluate the reliability of PG results obtained
using citric acid plasma [17]. Results for acidified samples kept
uncentrifuged for 24 h showed a negligible decrease of PG in compari-
son with results obtained by applying the NACB recommended proce-
dure for blood collection. This was more recently confirmed by
another study [18]. Following these results, experts definitively recom-
mended the use of citrate tubes in daily practice to prevent in vitro gly-
colysis in effective way and ensure reliable PG results [10,19].
Table 1
Published studies comparing citric/citrate buffer vs. sodium fluoride tubes.

acterized, it can be in principle corrected. The second one relates to the abil- Authors (ref.) Plasma glucose Mean difference
ity of the analytical system to measure the same quantity over time. This concentrations (mmol/L) citrate vs. fluoride
can randomly fails, representing a source of variability that affects the mea- Del Pino IG et al. [20]a Mean: 6.43 vs. 5.98 +7.0%
surement in unpredictable way. Since the concept of “true value” is idealis- Szőke D et al. [21]a Range: 4.5–11.1 vs. 4.1–10.7 +6.7%
tic, as in practice it represents the best estimation of an unknown quantity, Van den Berg et al. [22]a Mean: 5.8 vs. 5.5 +5.5%
analytical results are always associated with a certain degree of uncertainty. Bonetti G et al. [23]a Median (range): 5.60 (5.47–5.73) +6.8%
vs. 5.21 (5.05–5.32)
Nevertheless, CVA should fulfil given specifications in order to assure the Carta M et al. [28]a Median (95% CI): 5.4 (5.1–5.7) vs. +5.9%
validity of the clinical information of test results [7]. 5.1 (4.8–5.3)
The characterization and management of any source of variability is im- Dimeski et al. [24]b Mean: 5.35 vs. 5.05 +5.9%
portant to assure that they do not invalidate the clinical usefulness of the Dimeski et al. [25]c Mean: 5.7 vs. 5.3 +7.5%
Juricic G et al. [26]c Mean (±SD): 6.2 (±1.1) +10.7%
test. The aim of this article is to explore and discuss the sources of variability
vs. 5.6 (±1.0)
potentially affecting the clinical reliability of PG measurements. Juricic G et al. [27]c Mean (±SD): 6.0 (±0.8) +8.5%
vs. 5.5 (±0.8)
Carta M et al. [28]c Median (95% CI): 5.6 (5.5–5.9) +9.8%
2. Pre-analytical sources of variation in PG testing vs. 5.1 (4.8–5.3)
a
Venosafe Terumo granular citric/citrate buffer tubes.
It is well known that in vitro PG concentrations are unstable due to b
Greiner Bio-One FC Mix Glucose dry citric/citrate buffer tubes.
c
ex-vivo glycolysis exerted by blood cells. Glucose at physiological Glucomedics Greiner Bio-One liquid citric/citrate buffer tubes.
 S. Pasqualetti et al. / Clinical Biochemistry 50 (2017) 587–594
2.1. Not all citrate tubes are created equal
After the release of the above-mentioned recommendation, several
groups have investigated the impact in PG results derived from the
shift from fluoride (the additive currently in use in the majority of the
healthcare facilities) to citrate tubes (Table 1) [20–28]. By the use of
citrate in granular form (Venosafe), Szőke et al. [21] experienced an av-
erage increase of 6.7%, in PG values, independent from the PG concen-
trations. The use of citrate in liquid form (Vacuette® Glucomedics,
Greiner Bio-One) also implied the obtainment of PG results significantly
higher in comparison to those obtained using fluoride only [25–28]. Im-
portantly, when studies compared head-to-head PG results obtained by
tubes using citrate in liquid (Glucomedics) vs. granular (Venosafe)
form, a significant bias between the two types of tubes (from 3.2% to
3.8%) was shown (Table 2) [28–31]. As Glucomedics tubes require the
use of a factor correcting the dilution effect of liquid additive, some au-
thors have suggested to modify the manufacturer's recommended fac-
tor to get results comparable to those obtained under controlled pre-
analytical conditions [25,32]. In our experience with Glucomedics
tubes, we speculated on some problems in tube manufacturing,
resulting in an imperfect vacuum action [30]. In fact, despite of blood
collection done by trained phlebotomists, we found several tubes not
perfectly filled. In this situation, even a theoretically accurate dilution
factor may become incorrect when tubes are not filled as intended.
In summary, although studies demonstrate that the use of citric acid
adds stability, literature data about the performance of different avail-
able citrate tubes are confused and selection of tubes containing citrate
should require great caution [30]. Recent events seems to corroborate
this caveat: at the end of 2015, Terumo decided to withdraw Venosafe
tubes from the market without a stated motivation, while few months
later an additional tube type (Vacuette® FC Mix Tube, Grainer Bio-
One) using a powder mixture of citrate/fluoride/EDTA has been
marketed. In this messy state of affairs, there is a strong and urgent
need for a well-designed study validating all the options using blood
acidification offered by the market against the reference approach for
blood collection recommended in guidelines for PG testing [10].
2.2. Clinical impact of the optimization of pre-analytical phase in PG
determination
As with the introduction of citrate tubes changes in PG values are
reasonably expected, population-based studies tailored to evaluate the
practical impact derived from these changes become advisable. In our
experience, the introduction of granular citrate tubes (Venosafe) deter-
mined a “shift to the right” in the PG distribution of outpatient popula-
tion [33]. Using the American Diabetes Association (ADA) cut-off for
desirable fasting PG (b5.60 mmol/L), the percentage of subjects with
undesirable fasting PG increased from ~26.8% to 45.2% when Venosafe
tubes replaced classical fluoride/oxalate tubes [34]. Moreover, by apply-
ing the diagnostic cut-point for diabetes (≥7.00 mmol/L) the prevalence
of subjects with abnormal fasting PG results increased from 17.8% to
23.3%, resulting in a 5.5% increase of diabetes diagnoses. Our data
Table 2
Published studies comparing plasma glucose results obtained by using tubes containing
citrate in liquid vs. granular form.
Authors (ref.) Plasma glucose Mean difference liquid
concentrations (mmol/L) vs. granular citrate
Bakliza A et al. [29] Mean (±SD): 5.8 (0.8) +3.2%
vs. 5.6 (0.7)
Pasqualetti S et al. [30] Range: 4.1–22.7 vs. 4.0–21.9 +3.8%
Carta M et al. [28] Median (95% CI): 5.6 (5.5–5.9) +3.7%
vs. 5.4 (5.1–5.7)
Juricic G et al. [31] Mean (±SD): 6.0 (1.0) vs. 5.8 (0.9) +3.4%
589
showing a significant change of clinical classification of patients with
the introduction of citrate tubes were corroborated by similar experi-
ences by others authors [35,36]. However, these reclassifications may
be misleading as current fasting PG cut-points [with the sole exception
of Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study-de-
rived limit for GDM diagnosis] were derived from studies using sodium
fluoride tubes and, thus, the decision limits are probably based on data
that were generated with approaches in collecting blood for PG mea-
surements that were negatively biased [37]. With regard to this aspect,
we previously claimed for an official position of diabetologist groups re-
garding the decision limits that should be applied to fasting PG results
using collection tubes minimizing pre-analytical variability: should
these be redefined or should they be maintained so that more subjects
at increased risk for diabetes will be identified earlier? [38].
In conclusion, since the optimization of pre-analytical phase of PG
measurement may lead to marked differences in clinical classification
of subjects, a second caveat, in addition to that related to the proper se-
lection of citrate tubes, refers to which decision limits should be applied
to PG after the introduction of citrate tubes. As an official position by
clinical associations is still lacking, it seems sensible for laboratories to
stay for the moment to tubes containing sodium fluoride only, as
these have been used in the majority of studies generating the current
PG cut-points for diabetes diagnosis, in order to not create confusion
among clinicians and adversely affect patient outcomes. We should,
however, emphasize that the use of tube containing fluoride alone re-
quires immediate centrifugation.
3. Biological variation of PG
In the assessment of clinical condition of an individual by measuring
specific constituents in his/her body fluids, it is essential to consider that
laboratory results vary due to the influence of biological variation (BV).
BV is characterized by the random fluctuation of the analyte concentra-
tions around the individual homeostatic set point, namely CVI, and by
the difference among homeostatic set points for the same analyte in dif-
ferent individuals in the same physical conditions, namely between-
subject variation (CVG) [4,39]. The knowledge of BV data for an analyte
permits the derivation of important information for the correct applica-
tion and clinical interpretation of its measurement [40]. In particular, BV
is a useful tool for establishing the reliability of test results through the
definition of analytical performance specifications (APS) of employed
assays [41].
Considering the importance of BV data in laboratory medicine, it is
essential to derive them in an accurate and reliable way. We recently
published an updated guideline for the production of high-quality BV
data to which readers are referred [42]. In general, published data are
of varying quality and quite heterogeneous and there is a need for appli-
cation of standards (i.e., a minimum set of attributes to enable the data
to be effectively transmitted and applied) [43]. The available literature
for BV of glycaemia consists of about twenty articles [44]. Published
CVI range from 3.7% [45] to 13.3% [46] and CVG from 2.7% [47] to 16.8%
(in a recent paper not yet enlisted in the Westgard's database [48]).
This high heterogeneity of data is certainly due to the lack of use of a
common experimental approach based on standardized protocol and
robust data analysis. For instance, the selection of subjects is a very crit-
ical point. Some studies have derived BV data for glycaemia on appar-
ently healthy subjects, while others on diseased patients. Even if some
author has supported the possibility to derive reliable BV data also by
enrolling diseased subjects, providing that they be metabolically stable
[49], the difficulty to evaluate the stability of a disease makes this ap-
proach poorly applicable in the clinical practice. Diabetes is a disorder
in glucose metabolism due to a progressive loss of insulin secretion in
a biologic environment of cellular insulin resistance. It is therefore rea-
sonable to consider this configuration unstable per se, since the expo-
sure to hyperglycaemia is chronic, but insulin deficiency as well as the
cellular resistance to its action is progressive. The implications of
590 S. Pasqualetti et al. / Clinical Biochemistry 50 (2017) 587–594
diabetes on day-to-day variability of fasting PG were first investigated
by Ollerton et al. [50], and definitively outlined in a more recent paper
showing a markedly different fluctuation amplitude of glycaemia in
healthy individuals and in diabetic patients, the latter as a result of the
combination of inherent BV with the variability dependent on the pres-
ence of disease and treatment [51]. This paper by Carlsen et al. nicely
complies with the previously mentioned standards for appraisal of BV
studies [43], so that the obtained results for CVI (5.4%) and CVG (5.6%)
of glycaemia can be assumed accurate.
According with the current statistical approach [42], the number of
specimens (n) that should be collected to ensure that the mean PG re-
sult is within ±5% of the individual's homeostatic set point can be ob-
tained according to the following statistics: 1.962 (CV2A + CV2I ) / 25.
Using an average CVA of 1.3% [52] and a CVI of 5.4% [51], for PG, it can
be estimated that each subject should theoretically undergo up to ap-
proximately five determinations to achieve a sufficiently accurate esti-
mate of the PG individual's homeostatic set point. Comparing these
data with those obtained for glycated haemoglobin (HbA1c), it is possi-
ble to note that for HbA1c no more than two samples are needed for a
reliable estimation of the homeostatic set point of an individual [53].
From the point of view of BV, HbA1c is therefore the elective test for
the diagnosis of diabetes, even if in certain frameworks the use of PG
could be the solely choice [3]. It is noticeable that the ADA standard
treats the two markers in equal way, suggesting repeating them in the
absence of unequivocal hyperglycaemia [3]. Simplistically, ADA argues
that if a PG result above the diagnostic cut-point is below the same
cut-point when repeated, this may be attributable to a laboratory
error [3], ignoring, however, the bigger impact of CVI on PG results.
4. APS for PG testing
To be clinically reliable and assure appropriate medical decisions,
laboratory results should fulfil an established degree of analytical qual-
ity expressed in term of APS. This provides an objective evidence that
the measuring system is appropriate for the intended clinical applica-
tion of the test. The approaches for deriving APS has recently been
revisited [54]. Particularly, three models to set APS have been identified:
model 1, based on the effect of analytical performance on clinical out-
comes obtained through direct (1a) or indirect outcome studies (1b);
model 2, based on BV data of the measurand, asserting that acceptable
analytical quality can be achieved when the analytical signal does not
obscure the biological one; and model 3, based on the state of the art
of the measurement [55]. Ceriotti et al. [56] recently reported the ratio-
nale for assigning measurands to one of those. In principle, model 1
should be used for measurands having a central role in the decision-
making of a specific disease, model 2 is suited for measurands with
high homeostatic control and model 3 should be used for measurands
that have neither central diagnostic role nor sufficient homeostatic
control.
Since PG plays a relevant role in diagnosis of diabetes and its values
are used to define glycaemic-related conditions, model 1 should be pre-
ferred to derive APS for PG. However, as studies investigating the direct
impact of performance of PG measurement on clinical outcome are not
available, model 1b has to be adopted. By the way, Horvath et al. [57]
stated that direct studies are not necessary when: a) the clinical deci-
sions associated with the test results are well defined, b) evidence
about the diagnostic accuracy of the test to classify patients for these
clinical decisions is available and is generalizable to the patient popula-
tion, and c) the consequences of correct/incorrect classification are
established, all conditions clearly fulfilled by PG testing. A pioneering
study approaching the APS issue using model 1b simulated the influ-
ence of analytical bias and imprecision of PG measurement on the mis-
classification of healthy subjects as diabetics [false positives (FP)] and of
diseased subject as healthy [false negatives (FN)]. Using a single PG de-
termination, a bias b0.1 mmol/L and an imprecision (as CV) b2% gave a
rate of FP and FN of 5% [58]. In a following study [59], the same authors
further investigated the impact of the two sources of analytical error on
clinical classification, setting a maximum APS for bias of 0.18 mmol/L
and for imprecision of 6% to allow a maximum rate of 5% in FP and FN
when two replicate blood samplings for PG testing were used (data de-
rived from Fig. 1D of ref. 59). A similar simulation based on the current
definition of impaired fasting glucose (IFG) status (i.e., fasting PG be-
tween 6.1 and 6.9 mmol/L) can be done. Particularly, it can be estimated
that to properly classify an individual with a PG of 6.5 mmol/L as IFG, the
measurement error should not exceed ±6.15% (0.4/6.5). Indeed, if the
measurement error is greater, a patient with a fasting PG of
6.5 mmol/L would be indifferently classified as healthy or diabetic and
this obviously would not be acceptable. We recently performed a simu-
lation analysis to investigate the impact of derived measurement error
(± 6.15%) on the clinical classification of the outpatient population
served by our institution. We retrospectively retrieved PG results from
outpatients for a 6-month period during which PG was measured by a
well-standardized and precise assay [52]. The clinical classification of
retrieved subjects (n = 6537) was 51.6% as healthy, 21.6% as IFG and
26.8% as diabetics. A + 6.15% error in PG measurement resulted in
7.7% of IFG subjects misdiagnosed as diabetes and 18.1% of healthy indi-
viduals classified as IFG. Conversely, a –6.15% error implied the shift of
6.2% subjects from diabetes to IFG category and of 12.6% IFG subjects
to healthy group. The IFG category represents a set of subjects at in-
creased risk to develop diabetes mellitus, for which the prevention of di-
abetes onset as well as of vascular hyperglycaemia-related
complications is accomplished with interventions lowering PG over
time [60,61]. FN, i.e., IFG subjects misclassified as normoglycaemic, are
therefore the most impacting results. In our outpatient population, mea-
suring PG with an error of −6.15% would imply that ~12% of individuals
would miss interventions necessary to stop the progression to diabetes
and the worsening of related outcomes. Of course, application of lower
FP/FN rates for diabetes diagnosis and/or IFG detection should require
more stringent APS.
As PG has high homeostatic control, it is also possible to use model 2
to derive APS for its measurement. To this regard, using Carlsen's BV
data reported above [51], minimum, desirable and optimum APS for im-
precision, bias, and total error (TE) can be derived according to Fraser et
al. [62]. Table 3 summarizes APS for PG measurements according to dif-
ferent models and different levels of quality. The similarity of APS de-
rived from both models 1 and 2 (i.e., outcome-based and BV-based)
confirms the equivalence of the two models advocated by the expert
group of the European Federation of Clinical Chemistry and Laboratory
Medicine (EFLM) when measurands, such as PG, with well-defined bio-
logical and clinical characteristics are considered. On the other hand, it is
important that the application of different levels of suitable quality is
considered in the context of the intended use of the PG measurement
[63].
5. Analytical issues related to PG measurement
5.1. Establishment of traceability of PG results
In laboratory medicine, the production of accurate and equivalent
results on patient samples requires the standardization of measure-
ments by the implementation of an unbroken metrological hierarchy
able to ensure traceability of results to the highest available level of ref-
erence [64]. In more depth, we recently described the “temple of labora-
tory standardization” including in its pillars the six requirements for the
correct implementation of traceability plans [65].
At the level of in vitro diagnostics (IVD) manufacturers, the traceabil-
ity should be promoted through the application of protocols enabling to
transfer the trueness from a suitable reference material (certified and
commutable) to commercial calibrators in a reliable way. Basics to the
measurement standardization and traceability is that a possible system-
atic measurement error, i.e., bias, in this phase is appropriately
corrected by realignment of measuring system by adjusting the value
 S. Pasqualetti et al. / Clinical Biochemistry 50 (2017) 587–594
Table 3
Analytical performance specifications for plasma glucose measurement.
Quality level Imprecision (as CV)
Outcome-based Biological variationa
Minimum b6.0%b,c b4.05%
Desirable b2.0%c b2.7%
Optimum – b1.35%
a
Calculated according to ref. 59, with biological variation data from Carlsen et al. [51].
b
Using mean of two replicate samplings.
c
According to a 5% rate of false positives and false negatives in diabetes diagnosis [58,59].
d
According to a 12.6% rate of false negatives in subjects with impaired plasma glucose [data from authors, unpublished].
assigned to the calibrator, providing unbiased (or “negligible biased”)
results on clinical samples [66]. In a previous paper [65], we described
the critical issues related to the available information related to the met-
rological traceability of IVD systems for measuring PG. In that study, the
strategies implemented by four major IVD companies for establishing
traceability of their commercial systems for PG determination were re-
ported. Briefly, in the case of PG, to assign traceable values to commer-
cial calibrators it is possible to use at least four different types of
metrological traceability chain, each with some specificities in trueness
transfer steps and differences in uncertainty accumulation [65]. Impor-
tantly, although it was possible to infer the different internal calibration
hierarchies applied by different IVD companies, it was evident that
there is quite incomplete information concerning the metrological
traceability of commercial assays for PG measurement.
5.2. Uncertainty of PG measurements
The uncertainty of measurement, defined as a parameter character-
izing the dispersion of the quantity values being reasonably attributed
to a measurand [67], occurs for each measurement step that is part of
the metrological traceability chain and its final amount on patient re-
sults depends on the contribution of different procedures that partici-
pate in transferring trueness. There are three main components of
uncertainty contributing to the measurement uncertainty budget: a)
the uncertainty of references (reference materials, reference proce-
dures), b) the uncertainty of commercial system calibrators (associated
to the manufacturer's calibrator values and depending on trueness
transfer process), and c) the uncertainty of random sources (measuring
system imprecision and individual laboratory performance) [68]. There-
by, in order to fulfil specifications for uncertainty associated to patient
results, it is essential to recognize the limits for uncertainty at each
level of the traceability chain [69]. We previously recommended an ap-
proach to set relative limits for uncertainties due to references (≤1/3 of
total uncertainty budget), commercial calibrators (≤50% of total budget)
and random sources (the remaining) as a defined proportion of the al-
lowable total uncertainty budget [70].
For measurement uncertainty, the goal that should be considered in
measuring patient samples is that related to the allowable CVA, consid-
ering that in a correct trueness transfer process the systematic measure-
ment error should be corrected. From data in Table 3 (CV column), we
may derive APS for combined standard uncertainty of PG measurement
at the level of patient results using, for instance, the BV model. By
expanding limits at the three quality levels by a coverage factor of 2
(95% level of confidence), minimum, desirable and optimum goals for
the expanded measurement uncertainty of PG results are 8.1%, 5.4%,
and 2.7%, respectively. According to these APS, references should display
in PG traceability chain an expanded uncertainty ≤1.8% (1/3 of 5.4%, de-
sirable quality level). From data in Table 1 (last column) of ref. 65, it is
evident that possibly only two of the current available references are
able to fulfil this recommendation. IVD manufacturers may spend differ-
ent amounts of the total uncertainty budget to allow traceability of their
analytical system to higher-order references. In some cases, given that
too much of the total uncertainty budget is spent by the selected
591
Bias Total error
Outcome-based Biological variationa Outcome-based Biological variationa
±0.18 mmol/Lb,c ±3.0% – ±9.6%
±0.10 mmol/Lc ±1.95% ±6.15%d ±6.4%
– ±1.0% – ±3.2%
references, it becomes difficult, if not impossible, to achieve the accept-
able limits of measurement uncertainty on clinical samples.
Our analysis highlights how strongly the measurement uncertainty
of PG may be dependent on the type of chain adopted by the IVD com-
panies to implement the traceability of their calibrators. On the other
hand, the PG example shows that, in a standardization program, the def-
inition of the uncertainty budget limits across the entire metrological
traceability chain represents an effective tool that may objectively
drive reference providers and IDV manufactures during their activities
of traceability implementation, helping correctly allocating different re-
sponsibilities [71].
5.3. Performance of laboratories in terms of standardization of PG
measurements
As previously discussed, laboratory results require an unequivocal
interpretation independently of the measuring system performing the
analysis. To assure the interchangeability of patient results among labo-
ratories the unique tool is the implementation of metrological-based
approach for calibrating different marketed measuring systems [64,
68]. However, even though IVD manufacturers are requested to assure
traceability of their calibrators to the highest available references, it is
up to our profession to verify the effective accomplishment to this
duty through a post-market surveillance targeted to verify the analytical
validity of measurements [65,72]. Basics to the verification of result
equivalence among laboratories and of standardization of measure-
ments is the implementation of external quality assessment (EQA) pro-
grams properly structured to judge the quality of the commercial assays
in terms of traceability, accuracy and fitness for intended clinical appli-
cation [7,73]. Requirements characterizing appropriate EQA programs
are: a) the target value of EQA materials assigned with a reference pro-
cedure performed by an accredited laboratory, b) their proved
commutability to allow the transferability of laboratory performance
checked on EQA materials to clinical samples, and 3) the use of objective
APS to verify the suitability of laboratory measurements in clinical
setting.
EQA efficacy in showing the status of measurement standardization
strongly relies on the establishment of limits within which a measure-
ment error is allowable [74]. Previously, we have proposed the addition
of APS derived from models established during the EFLM conference to
the Miller's categorization of EQA programs [75] as criteria to evaluate
the performance of laboratories participating to EQA [76]. In particular,
Miller's categories 1 and 2, which fulfil the metrological requirements
highlighted above, should be each split in two sub-categories: 1/2A
EQA category, in which high-order models 1 and 2 for APS are applied,
and 1/2B EQA category, in which other low-order models are employed
[76].
Although the implementation of EQA programs fulfilling above-
mentioned requirements is difficult, their unique benefits have been in-
controvertibly proved [77–79]. Since 2005, the Dutch EQA scheme has
introduced a scoring system based on BV (data derived from Westgard
website [44], not checked for their robustness), together with commut-
able EQA materials value-assigned by reference procedures, to monitor
standardization of analytical system in The Netherlands [80]. In a pilot
592 S. Pasqualetti et al. / Clinical Biochemistry 50 (2017) 587–594
study, the model was extended to other three European countries (10
participating laboratories each) to evaluate the level of equivalence of
results for different biochemical analytes, including glycaemia [81]. Lab-
oratory performance was considered acceptable if the results were
within the desirable TE area with a probability of 95%. With a TE limit
for PG of ± 7.2% (higher than the ± 6.4% reported in Table 3), the PG
measurements did not met the acceptable performance criterion in a
significant number of exercises performed by participating laboratories.
Another recent study used commutable materials (pooled sera) with
value targeted by the reference procedure for surveying traceability of
different PG measuring systems marketed by four major IVD companies
[82]. In this study, each of the five materials was assayed in triplicates to
minimize the contribution of random error. By applying APS for bias re-
ported in Table 3, most, but not all, of the measuring systems met the
minimum quality specification (i.e., ±3.0%), but only one system was
able to achieve the desirable bias goal of ±2.0%.
Quite recently, the INPUtS project, aiming to evaluate the perfor-
mance of laboratories across five European countries, has reported an
update on the quality of PG measurement [83]. The obtained results
have shown that the overall performance of PG measurement in each
country, expressed in term of TE, was unable to meet the desirable
APS for TE of ±6.4%. The average TE in different countries were substan-
tially close to each other, all between desirable and minimum APS (from
6.7% to 8.3%; all countries TE, 7.5%).
In summary, using robustly derived APS, the assessment of stan-
dardization by employing commutable control materials with values
assigned by reference procedure have shown that the current quality
of PG measurements is often far from desirable, confirming the need
of some improvements in order to assure the clinical reliability of
results.
5.4. Point-of-care testing (POCT) in hospitalized patients: a world apart?
There are some critical situations, in which disglycaemic conditions
should be quickly recognized and appropriately treated, where POCT
glucometers are employed [84]. Given that those devices directly pro-
vide glucose results usable by clinicians to make therapeutic decisions,
the accuracy of their measurements is essential to ensure safe and reli-
able information to the end-users. The current situation about APS for
glucometers within the hospital scenario lacks, however, an interna-
tional consensus. The ISO 15197:2013 standard, drafted for glucose
self-testing in managing diabetes, outlines as goal for accuracy that at
least 95% of results provided by POCT must fall within ±0.83 mmol/L
of the comparison laboratory results at glucose concentrations
b5.56 mmol/L and within ±15% from the reference at glucose concen-
trations ≥ 5.56 mmol/L [85]. Moreover, the standard requires that at
least 99% of individual results fall within consensus error grid (CEG)
zone A or B (when accuracy is evaluated with three strip lots) [85]. Clin-
ical and Laboratory Standards Institute (CLSI) POCT12-A3 criteria states
that a maximum of 5% of results should be N±0.67 mmol/L, when refer-
ence results are b 5.56 mmol/L, or within ±12.5% for reference results
≥ 5.56 mmol/L [86]. Finally, the Food and Drug Administration (FDA)
draft guidance 1755 suggest that no N1% of POCT results should be N±
0.40 mmol/L from reference, when PG concentrations are
b3.9 mmol/L, or N±10% from reference results, when ≥ 3.9 mmol/L
[87]. Alternatively, 100% of POCT glucose results should be within
±0.83 mmol/L when PG b3.9 mmol/L or within ±20% from reference
when PG is ≥3.9 mmol/L [87]. By taking into account the tight relation-
ship between glucometer results and patient outcome in diabetic sub-
jects when results are interpreted to set therapy, ADA also
recommended the use of CEG criteria to evaluate glucometers, provid-
ing an estimation of the clinical risk in association to the inaccuracy of
POCT results, based on clinical judgment of experts [88]. Overall, accept-
ability criteria currently recommended for glucose POCT are larger than
those proposed for the PG measurements provided by central laborato-
ries (Table 3). Moreover, excepting for FDA guidance, available
standards for POCT accuracy always refers to a percentage of provided
POCT measurements instead of the 100% of results. Theoretically, the
use of tolerance limits b100% leaves IVD manufacturers free to market
POCT devices providing a low, but still substantial, percentage of results
out of tolerance limits, not valid for clinical application and unsafe for
patients [89].
Recently, we evaluated the performance of three POCT glucometers
for use in the hospital setting by comparing their results to those by an
automated assay shown traceable to higher-order references [52]. All
the three evaluated glucometers gave an average bias that was unable
to achieve the minimum quality goal reported in Table 3. We speculated
for a poor standardization of these devices to justify the observed inac-
curacy. However, all results, except two borderline values for one
glucometer, were within the low-risk zone according to CEG analysis.
Therefore, the relevant inaccuracy, when judged by APS based on [55],
would not have any significant impact on patients' outcome, according
to the experts' opinion. The question is, therefore, how accurate must
the POCT devices be? In our opinion, the answer comes on the heels
of the debate about the benefits and safety of their use in hospitals.
For instance, if the intended application relates to healthcare profes-
sionals maintaining tight glycaemic control protocols in clinical settings,
it seems sensible that the principles applied to a PG measurement from
central laboratory must be similarly applied to POCT glucometers to
permit their applicability in hospital frameworks.
6. Conclusions
10 years after the Gambino's editorial, defining glucose as “a simple
molecule that is not simple to quantify” [90], some issues still persist in
its measurement. We have discussed the importance to stabilize glucose
concentration after blood drawing as well as the role of citrate, for long
neglected, as effective stabilizer, highlighting, however, the poor com-
parability of results obtained in published studies using different
marketed tubes, which has created confusion among users. This claims
for the need of a well-designed study tailored to evaluate their effective
reliability. Moreover, since the introduction of citrate affects PG results,
we have express the urgent need of a position by diabetologists regard-
ing the diagnostic cut-points. Our work also emphasizes the central role
of traceability implementation as a unique tool effectively standardizing
PG results regardless the employed measuring systems. A poor process
of standardization may affect, however, the reliability of the PG mea-
surement by increasing its uncertainty. The availability of many options
useful as higher-order references can make the effective implementa-
tion of a calibration hierarchy arduous, pointing out on the need of un-
equivocally defined APS to judge the quality of achieved results. Finally,
the presented approach of defining APS, based on the EFLM criteria, can
be considered a good exemplification to be translated to other
measurands on which important clinical decisions are based.
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