Clinica Chimica Acta 411 (2010) 1606–1610

Contents lists available at ScienceDirect

Clinica Chimica Acta

j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / c l i n c h i m

Invited critical review

Biological variability of glycated hemoglobin

Federica Braga a,b,⁎, Alberto Dolci b, Andrea Mosca a, Mauro Panteghini a,b
a
Centro Interdipartimentale per la Riferibilità Metrologica in Medicina di Laboratorio (CIRME), Università degli Studi, Milano, Italy
b
Laboratorio Analisi Chimico-Cliniche, Azienda Ospedaliera ‘Luigi Sacco’, Milano, Italy

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

Article history: Background: The measurement of glycated hemoglobin (HbA1c) has a pivotal role in monitoring glycemic
Received 28 June 2010 state in diabetic patients. Furthermore, the American Diabetes Association has recently recommended the
Received in revised form 23 July 2010 use of HbA1c for diabetes diagnosis, but a clear definition of the clinically allowable measurement error is still
Accepted 23 July 2010 lacking. Information on biological variability of the analyte can be used to achieve this goal.
Available online 2 August 2010
Methods: We systematically reviewed the published studies on the biological variation of HbA1c to check
consistency of available data in order to accurately define analytical goals.
Keywords:
Results: The nine recruited studies were limited by choice of analytic methodology, population selection,
Biological variability
Glycated hemoglobin
protocol application and statistical analyses.
Diabetes mellitus Conclusions: There is an urgent need to determine biological variability of HbA1c using a specific and
traceable assay, appropriate protocol and appropriate statistical evaluation of data.
© 2010 Elsevier B.V. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1606
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607
3. Results and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607
3.1. Selected studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607
3.2. Definition of the measurand and influence of method specificity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607
3.3. Type of studied subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1607
3.4. Study duration and sampling frequency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608
3.5. Sample analysis and assessment of analytical variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1608
3.6. Statistical analysis of data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1609
Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1610
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1610

1. Introduction
Glycated hemoglobin or βN-1-deoxyfructosyl-hemoglobin
(HbA1c)1 is the product of a nonenzymatic reaction of glycation,
namely a condensation between the aldeidic group of glucose and the
amino group of the terminal valine in the β-chain of hemoglobin A0.
The amount of HbA1c is strictly related to blood glucose concentration.
⁎ Corresponding author. Laboratorio Analisi Chimico-Cliniche, Azienda Ospedaliera
‘Luigi Sacco’, Via GB Grassi 74, 20157 Milano, Italy. Fax: + 39 02 503 19835.
E-mail address: federica.braga@studenti.unimi.it (F. Braga).
1
Nonstandard abbreviations: HbA1c, glycated hemoglobin; ADA, American Diabetes
Association; NGSP, National Glycohemoglobin Standardization Programme; SI,
‘Sistème Internationale de Measure’; CVI, intra-individual biological variation; CVG,
inter-individual biological variation.
Considering the average life span of red cells, the HbA1c value should
mimic the mean glycemic value of the previous 2–3 months. The
American Diabetes Association (ADA) recommends HbA1c determi-
nation in patients with diabetes mellitus on therapy in order to
monitor the glyco-metabolic status in the medium–long term and
thus reduce the risk of vascular complications [1]. Studies performed
on non-diabetic subjects showed that even slight elevations of HbA1c
concentration in blood correlated with an increased cardiovascular
risk [2,3]. The “Standards of medical care in diabetes” recently
published by ADA, recommends the use of HbA1c as a diagnostic
criteria for diabetes, a finding which may further increase the clinical
relevance of HbA1c testing [1].
Many analytical methods are available for HbA1c determination
[4]. Techniques based on the difference in isoelectric point of HbA1c
and other hemoglobins, and immunochemical methods specifically

0009-8981/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.cca.2010.07.030
 F. Braga et al. / Clinica Chimica Acta 411 (2010) 1606–1610
measure HbA1c; other techniques, such as affinity chromatography,
determine all glycated hemoglobins, including molecules with
glucose bound to lysines β17 and β66 and to amino-terminal valines
of α-chain. The disagreement between results obtained with different
methods prompted to organize harmonization programmes on a
national basis, e.g. the National Glycohemoglobin Standardization
Programme (NGSP) [5]. Despite the improvement obtained by the
implementation of these programmes, results of HbA1c assayed in
different countries were, however, still significantly discordant [6].
Therefore, in 1995 the IFCC created a working group with the aim of
international standardization of HbA1c assays [7]. Following the
theory of metrological traceability, first it was unequivocally defined
the measurand ‘HbA1c’ as hemoglobin molecules having a special
hexapeptide in common, which is the stable adduct of glucose to the
amino-terminal valine of the hemoglobin β-chain (βN-1-deoxyfruc-
tosyl-hemoglobin). Then, a metrological reference system was
implemented, including specific reference methods for measuring
HbA1c as defined, reference materials and a network of laboratories
performing the reference measurement procedure in a strictly
controlled way [8]. Expression of HbA1c results in mmol βN-1-
deoxyfructosyl-hemoglobin/mol hemoglobin β-chain, in agreement
with the ‘Sistème Internationale de Measure’ (SI), was also recom-
mended [9,10].
Once the traceability of HbA1c results by the commercial assays to the
reference measurement system is obtained, it is important to define the
acceptable uncertainty of HbA1c measurements in view of their clinical
use [11]. Lacking outcome-related goals, there is a consensus for using
biological variation to formulate analytic variation limits [12]. In
particular, desirable imprecision performance (expressed as CV) should
be less than one-half of the intra-individual biological variability (CVI)
and desirable bias should be b0.25[(CVI)2 +(inter-individual biological
variation (CVG))2]1/2. The quality specification for desirable total error is
calculated as: bias goal +(k×imprecision goal), where k= 1.65 at
α=0.05 [13,14].
The accurate estimate of the biological variability of an analyte is,
therefore, a fundamental prerequisite for the definition of the
allowable measurement error, permitting reliable application of
laboratory measurements in clinical setting. In this study, we
systematically reviewed the published papers on biological variation
of HbA1c, with special focus on the suitability of data, their
concordance and possible causes of disagreement, with the aim to
ascertain whether such existing studies are valid and scientifically
sound enough to be used for attaining analytical goals for HbA1c.
2. Materials and methods
A PubMed search was performed, without any time limit, using as keywords:
“Biological variation & HbA1c”, “Intra-individual biological variation & HbA1c” and
“Inter-individual biological variation & HbA1c”. As unique inclusion/exclusion criterion,
recruited papers should have as explicit aim of the study the experimental assessment of
HbA1c biological variability components. We therefore ruled out papers reporting
biological variation data obtained from literature, even because quoted references in
such papers always referred to the already selected papers.
In evaluating published data, special attention was paid to the strategy of data
collection and assembly, by considering selection and type of enrolled subjects, study
duration, frequency and number of blood sampling, samples storage until analysis,
analytical specificity of employed methods and statistical evaluation of data. These
features were compared with the theory of biological variation as reported by Fraser
and Harris [15]. In particular, Table 1 lists the optimal features for an experimental
protocol for accurate estimate of biological variability and Table 2 summarizes the
recommended statistical approach for quantifying biological variability components.
3. Results and discussion
3.1. Selected studies
Table 3 shows the main characteristics of selected studies on HbA1c
biological variability [16–24]. While all nine studies estimated HbA1c
CVI, only five did HbA1c CVG.
1607
Table 1
Main characteristics of an experimental protocol for accurate estimate of the biological
variability of an analyte. Adapted from ref. [15].
—Select apparently healthy subjects, using conditions that minimise pre-analytical
variablesa
—Specimens taken at set time intervals
—Specimens processed and stored frozen at − 80 °C
—When all specimens are available, analysis of all samples in a single run in
duplicate
a
Usual life styles, no drugs or alcohol, phlebotomy by the same person at the same
time of day, optimal protocol for sample transport, processing, and storage.
3.2. Definition of the measurand and influence of method specificity
According to the theory of metrological traceability, the standard-
ization of HbA1c measurement is based on the definition of the
‘measurand’. If the adopted methodologies have different analytical
specificities, assaying thus a different measurand rather than HbA1c
(like total glycated hemoglobins, also including hemoglobins glycated
on other sites differing from amino-terminal valine of the β-chain),
changes in biological variability, that strictly depends from the
characteristics of the measurand, can be expected [25]. Techniques
such as ionic exchange or affinity chromatography on microcolumns
as well as those based on electroendosmosis, used in the three oldest
papers [16–18], had specificity markedly different from the IFCC
reference measurement system. Godsland and Howey et al. correctly
defined the measurand as ‘HbA1’, meaning the whole set of glycated
hemoglobin fractions (HbA1a + 1b + 1c) [16,17]. On the other hand,
affinity chromatography methods used by Phillipou and Philips [18]
and, more recently, by Rohlfing et al. [23] are based on the reaction of
cis-1,2-diol groups of glucose (and other sugars)-hemoglobin adduct
with immobilized boronic acid, thus producing boronated esters. Such
methods, therefore, measure total glycated hemoglobins, including
the glucose bound to lysines and amino-terminal valines of α-chains.
Four reported studies used HPLC systems with ion-exchange
columns (BioRex 70, Pharmacia Mono S, or Tosoh 2.2) [19–21,24].
These methods show more specificity for HbA1c than the above-
mentioned techniques, even if correlations with the IFCC reference
measurement procedure still show a significant constant bias
witnessing some differences in terms of analytical specificity
[26,27]. The remaining study by Trapé et al. [22] used a competitive
immunoturbidimetric assay (Tina-quant HbA1c, Roche Diagnostics)
applied on an automatic analyzer of a different company (Synchron
CX7, Beckman Coulter). This method is known to display the same
specificity for HbA1c as the reference method and this study is indeed
the only one which is suitable from this point of view [28].
3.3. Type of studied subjects
In the enrollment protocol of the studies the limitation most
frequently found was the recruitment of diabetic patients. Indeed, to
select apparently healthy people is required by the theory of biological
variability, which aims to define the physiological fluctuation of analyte
Table 2
Recommended statistical approach for quantifying biological variability of an analyte.
Adapted from ref. [15].
Using all sample data, the components of variance should be dissected out as follows:
σ2total = σA2 + σ2I+ σG2
The analytical (σA2), intra-individual (σ2I), and inter-individual (σG2) variations
are calculated by analysis of variance:
—The analytical variation is estimated from the duplicate results for each specimen,
—The intra-individual variation is estimated from the serial results for each subject,
—The inter-individual variation is estimated from the total variance (σ2total) of data
minus the analytical and intra-individual components.
All the components of variation are then transformed to the relevant CVs using the
corresponding means.
1608 F. Braga et al. / Clinica Chimica Acta 411 (2010) 1606–1610

Table 3 tions and the study duration. Such duration should be neither too
Studies on biological variability of HbA1c. short (a few days) nor too long (years), so that the variability study
Study Author, No. and No. of Analytical CVI, CVG, may not be related to periods different from those used in clinical
no. year type of samples method % % practice for the measurements of specific analyte nor influenced by
subjects (frequency) additional causes (e.g. seasonal variation) [21,30]. It seems, therefore,
1 Godsland, 10 healthy 11–27 Ion-exchange 1.8 – reasonable to adjust the duration of studies evaluating biological
1985 [16] volunteers (7 days) chromatography variability of HbA1c between 1 and 6 months, which corresponds to
(6 M, 4 F)
the advised frequency of the assay in different clinical situations [1]. If
2 Howey et 8 diabetics 6 (3–4 days) Electroendosmosis 7.3 10.8
al., 1989 the majority of studies apply this criterion, the choice by the other
[17] authors to extend the duration beyond 6 months appears question-
3 Phillipou 73 4 (1 month) Affinity 4.2a; – able [18,19,22,24]. It is indeed likely that an evaluation lasting for
et al., diabeticsa,b chromatography 5.1b more than 6 months would include variability sources beside the
1993 [18] 4 7.1a; –
biological one. For instance, Kolatkar et al. [19] attributed to an
(3 months) 9.8b
4 Kolatkar 29 NS HPLC (ion- 2.4 – intensive treatment regimen the decrease in variability of HbA1c
et al., diabetics (3 months) exchange) concentrations in diabetic patients found along the wide study period
1994 [19] NS 4.1 – (6 years), wrongly interpreting this result as a reduction of the HbA1c
(12 months)
CVI. The variation observed in these subjects may undoubtedly be
5 Kilpatrick 12 healthy 10 (15 days) HPLC (ion- 1.9 6.8
et al., volunteers exchange) utilized for clinical decision making, but certainly not to estimate the
1998 [20] (7 M, 5 F) biological variability and the reference change value of HbA1c, which
6 Garde et 11 healthy 5 (7 days) HPLC (ion- b 0.7 3.3 must be assessed on a healthy population for a suitable time period to
al., 2000 volunteers exchange) be adopted as the basic criterion for interpretation of HbA1c results
[21] (F only)
and evaluation of glycemic control in diabetic patients over time.
7 Trapé et 47 4–7 Immunoturbidimetry 7.9c; –
al., 2000 diabeticsc,d,e (6 months) 5.4d;
[22] 3.9e 3.5. Sample analysis and assessment of analytical variability
8 Rohlfing 45 healthy 12 (7 days) HPLC (affinity) 1.7f 4.0
et al., volunteers
Apart from the method employed, in the retrieved studies two
2002 (M only)
[23]
further limitations can be found when compared to the ideal protocol
9 Desmeules 38 5 (1 year) HPLC (ion- 4.8 12.8 mentioned in Table 1. First, HbA1c measurements were often not
et al., pediatric exchange) performed in duplicate. Duplicate measurement permits a direct
2010 [24] non- evaluation of the within-run analytical variability, and that is better
diabetics,
than deriving it from the results of the laboratory internal quality
but with
cystic control programme, sometimes obtained on non-commutable mate-
fibrosis rials displaying a different behaviour from patient samples. Second,
(24 M, 14 F) HbA1c measurements along the study were often performed on fresh
CVI, intra-individual variability; CVG, inter-individual variability; M, males; F, females; samples the same day of blood collection, spreading them on several
NS, not specified. analytical runs, instead of performing measurements for all appro-
a
39 on stable metabolic control (16 on short-term, 23 on long-term), b34 on variable priately stored samples only in a single batch and, consequently,
metabolic control (13 on short-term, 21 on long-term); c13 on stable glycemic control,
d exclude from the total variability calculation the effect of between-run
10 on acceptable diabetic control, e24 on poor glycemic control; fanalytical variability
included. analytical variation, so difficult to be correctly assessed.
To perform all the determinations in duplicate on the same analytical
concentrations in a biological fluid around its homeostatic set point [15]. run represents the ideal experimental model for a biological variability
The presence of disease, mainly if unstable and not well controlled, may study, limiting to within-run variability the influence of the analytical
markedly modify the obtained information by amplifying such fluctua- variability on total variability of results, with less chance of error in the
tion. Fig. 1 clearly shows the difference between CVI values obtained in subsequent estimate of biological variability, obtained by subtraction
healthy volunteers (from b0.7% to 1.9%) and in diabetic patients (from 2.4 (Table 2). Unfortunately, the only study which correctly included sample
to 9.8%) with no overlap between the two groups. The same is also freezing in order to perform all HbA1c determinations on the same batch
evident for CVG in Fig. 2. Some authors [29] have argued that the at the end of blood collection phase, did not assay samples in duplicate
biological variability of HbA1c could be more efficiently evaluated in and got the information on analytical variability from the results obtained
diabetic patients, because the reference change value in clinical practice is on control materials [21]. This did not allow an accurate definition of CVI,
used to monitor HbA1c concentrations in such patients rather than in as total variability of experimental results was less than the analytical
non-diabetic subjects. This approach, however, does not take into account variability alone obtained from quality control data, that was obviously
that the disease itself may act as an extra-component of the variability overestimated [21].
around the HbA1c set point in the individual. This pathophysiologic In some studies, blood samples were not frozen and were,
change should actually be highlighted by the reference change value, not therefore, assayed in different runs, estimating analytical variability
used to derive it. For similar reasons, it is also questionable to carry out a from results obtained on control materials [18,20,24]. Other studies
biological variability study on a pediatric cohort affected by cystic fibrosis obtained the analytical variability from literature data [19]. The
instead of on ostensibly healthy children [24]. extreme case of such approximation can be found in the study by
Four studies correctly performed the evaluation on healthy Rohlfing et al. [23], in which, in addition to the lack of mentioning
volunteers, but two of those enrolled only subjects of one gender, sample storage conditions, singlicate measurements of samples made
females [21] or males [23], possibly because the samples did not it necessary to subtract to total experimental variability of results the
belong to specifically enrolled population. “within-day” analytical variability, obtained from quality control data,
so overestimated that only an approximate and possibly under-
3.4. Study duration and sampling frequency estimated CVI was reported as “probably b1%”.
Homogeneity of collection and in the number of samples drawn
It is well known that a biological variability estimate is strictly from each individual is another critical procedural point; the availability
dependent on the time interval between consecutive sample collec- of too many samples from the same subject may imply the technical
 F. Braga et al. / Clinica Chimica Acta 411 (2010) 1606–1610 1609

Fig. 1. Comparison between intra-individual biological variability (expressed as CVI) obtained in studies enrolling apparently healthy individuals and in studies evaluating diabetic
subjects. Study numbers as in Table 3. Dashed line shows CVI value as reported by Ricòs et al. (www.westgard.com/biodatabase1.htm).

impossibility to perform all measurements in the same run. A range
from a minimum of 11 to a maximum of 27 weekly blood samples
obtained in subjects during the same study means to estimate the
biological variability in a period from b3 to N6 months, making
incorrect to approximate individual data to a mean CVI value [16].
3.6. Statistical analysis of data
The statistical method used to assess variability components was
not specified (totally or partially) in three papers [16,19,22]; the
remaining studies correctly employed analysis of variance (ANOVA).
CV G estimate was added to CV I estimate in five studies
[17,20,21,23,24], three of them carried out in groups of apparently
healthy subjects (CVG values from 3.3% to 6.8%) [20,21,23].
4. Conclusions
A clear message from this systematic review of available literature
is the obvious lack of robust data on biological variability of HbA1c
concentrations in blood (Table 4). Despite all limitations and remarks
reported in the present paper, it should, however, be noted that the
information currently available on HbA1c biological variability is
generally taken as definitive in most scientific and professional circles.
The most popular informations on biological variability of biochemical
and hematological analytes, employed in daily practice worldwide,
are those compiled by Ricòs et al., freely available at www.westgard.
com/biodatabase1.htm. Values of biological variability components
for HbA1c listed in this database (CVI 3.4%, CVG 5.1%) are apparently
obtained from the (weighed?) mean of results available in the
literature, taken as valid by the authors of the compilation. It is,
however, impossible to understand the criterion (if one) of study
evaluation and selection of data, so that some doubts appear justified.
The observation reported in the present study and, particularly, the
finding of a substantial difference of variability results obtained in
healthy individuals and diabetic patients (Figs. 1 and 2) show the
fallacy of determining a single value of CVI and CVG simply using the
mean of available results. Despite all reported limitations, keeping
into account the available data obtained in healthy subjects [16,20],
the CVI of HbA1c should approximately be 1.8–1.9%, much lower than
that reported by Ricòs et al. in the mentioned database. It is, however,

Fig. 2. Comparison between inter-individual biological variability (expressed as CVG) obtained in studies enrolling apparently healthy individuals and in studies evaluating diseased
subjects. Study numbers as in Table 3. Dashed line shows CVG value as reported by Ricòs et al. (www.westgard.com/biodatabase1.htm).
1610 F. Braga et al. / Clinica Chimica Acta 411 (2010) 1606–1610

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