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Clin Chem Lab Med 2014; aop
Letter to the Editor
Sutirtha Chakraborty*, Sidhartha Sankar Ghosh, Anupam Das, Prasad Sawant
and Anders Kallner
Can EDTA, EDTA-fluoride, and buffered citrate
tubes be used for measurement of HbA1c on the
Bio-Rad D10?
Keywords: analysis of variance components; diabetes
monitoring; different tubes; emergency co-requests; error
grid.
DOI 10.1515/cclm-2014-0644
Received June 19, 2014; accepted July 6, 2014
To the Editor,
The measurement of HbA1c in blood has become the gold
standard for long-term control of the glycemic state of diabetic patients. The concentration of blood glucose indicates the real-time glycemic status at the time of sampling,
whereas the HbA1c concentration indicates the glycemic
status over the preceding 8–12 weeks. In clinical practice,
both glucose and HbA1c concentrations are important in
diabetes monitoring because they provide complementary
information. Recently, HbA1c has also been recommended
in the diagnosis of diabetes [1].
Simultaneous requests of HbA1c and glucose are
common, and alone or together may trigger a revision of
the therapeutic regime. Post-prandial glucose concentrations significantly contribute to HbA1c concentration.
Therefore, post-prandial glucose requests are required if
the therapeutic targets of HbA1c are not met [2]. Because
*Corresponding author: Dr. Sutirtha Chakraborty, Department of
Biochemistry, Peerless Hospital and B. K. Roy Research Centre,
Kolkata 700094, India, Phone: +91 33 24622462 (extension 2204),
E-mail: [email protected]; [email protected]
Sidhartha Sankar Ghosh: Department of Biochemistry, Peerless
Hospital and B. K. Roy Research Centre, Kolkata, India
Anupam Das: Department of Quality Assurance, Peerless Hospital
and B. K. Roy Research Centre, Kolkata, India
Prasad Sawant: Asian Institute of Oncology Pvt Ltd, Department of
Laboratory Medicine, Mumbai, India
Anders Kallner: Department of Clinical Chemistry, Karolinska
University Hospital, Stockholm, Sweden
HbA1c does not require a fasting patient, co-request
becomes a convenient and efficient procedure in rational
diabetes monitoring.
Glucose concentration is measured in blood, serum,
or plasma, whereas for HbA1c concentration, EDTA blood
is recommended [3]. To minimize glycolysis, in vitro fluoride is added to samples for glucose concentration measurements. This addition gives full effect after about 2 h,
whereas buffering the EDTA-fluoride to pH of approximately 5.5 [4, 5] has been shown to instantaneously inhibit
glycolysis.
We have only found two studies that validate the use
of anticoagulants other than EDTA in the measurements of
HbA1c [6, 7]. These were limited to 6 and 30 patient samples,
respectively. Although claiming equivalence of results
using EDTA and EDTA-fluoride tubes, no statistics were
presented. We investigated EDTA anticoagulated blood
with added fluoride and buffered fluoride for measuring
HbA1c concentration. This is a methodological change and
thus requires [8] validating if the “in-house measurement
procedure” is fit for purpose, i.e., to describe the degree
of agreement among results obtained from EDTA, EDTAfluoride, and buffered EDTA-fluoride tubes.
The study cohort included in-patients and out-patients
who were either diabetic or screened for glycemic impairment. This ensures that the cohort includes samples from
the entire measuring interval although with an expected
predominance of increased values.
Samples were collected in vacuum tubes; Vacutainers® were Potassium EDTA and Sodium Fluoride-Sodium
EDTA (Becton Dickinson, Franklin Lakes, NJ, USA) and
Terumo Venosafe® – Glycemia Buffered Fluoride Sodium
EDTA tubes (Terumo, Belgium). The latter were buffered
by citrate and thus contain two potent calcium complex
binders.
HbA1c concentrations were measured using a Bio-Rad
D10 cation exchange high-performance liquid chromatography (HPLC) platform (Bio-Rad Laboratories, Hercules,
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2 Chakraborty et al.: Can glucose tubes be used for measurement of HbA1c?
140
120
100
HbA1c mmol/mol
CA, USA) using the Bio-Rad calibrators with assigned
values traceable to the IFCC Reference Measurement Procedure. The results were reported in relative substance
concentration units, mmol/mol.
Venous blood, 2 mL, was collected from 104 patients.
Owing to limited amounts of material, the study was made
in two sets. In the first 104 patients, results were compared
after collection in EDTA and EDTA-fluoride tubes (set I).
In the second, another set of results from 104 patient
were obtained in EDTA blood and were compared with
those from buffered EDTA-fluoride tubes (set II). All pairs
of samples were processed on the same day. The validation was focused on estimating any bias between results
obtained in the different tubes and the imprecision of
measurement procedures. The bias was estimated using
regression analysis [9] and absolute and relative difference graphs. Excel spreadsheet programs were used for
regression and difference analyses. Nonparametric tests
were performed, when appropriate, using Sigma Plot (v.
12.5 Systat, Germany). A detailed imprecision evaluation
was completed using “analysis of variance components”
(ANOVA) as described in CLSI EP15 [10] and Kallner [11].
The clinical importance of the differences was evaluated
using error grids comprising A-, B-, and C-zones [12]. Significance was considered at p < 0.05.
The distribution of the HbA1c concentrations in the
cohorts was slightly skewed to higher values as expected
and failed the Shapiro-Wilks normality test. The medians
of the results of the two sets, collected in EDTA, were
not statistically different using Mann-Whitney rank sum
test. The differences between the paired results also did
not pass the normality test, and thus, the significance of
pairwise differences was tested using Wilcoxon’s signed
rank test. The results and comparisons are shown as
box-plots in Figure 1, which also shows the skewness of
the data. There were no suspected outliers visible in the
scatter plots, and the coefficients of determination (r2)
were high ( > 0.99). The average difference between the
observations in set I was 0.5 mmol/mol, and in set II, –0.5
mmol/mol, whereas the median difference between the
measurements was 0.0 mmol/mol in both sets. Although
the numerical differences were small, a statistical significant difference was shown by Wilcoxon’s signed rank test
between the EDTA tubes and those with additives.
Regression functions were calculated using Deming
regression with a relative variance of independent and
dependent variables (λ) set to 1. The slope was 1.00 and
0.99, and the intercept, 0.14 and –0.04 in set I and set II,
respectively.
We described the clinical performance in terms of
error grids [12]. In this approach, the widths of various
80
60
40
20
0
Set 1
A
EDT
o
+Flu
Set 1
Set 2
A
EDT
o
f Flu
+buf
Set 2
Sampling tube
Figure 1 Box-plots of the results in the investigated tubes.
Boxes represent the interquartile interval, the whiskers the 5th and
95th percentiles. Outside the whiskers, each observation is shown.
The solid line in the box represents the median, and the dotted, the
average. The difference between these central measures illustrates
the skewness of the data distribution.
zones of accepted results are defined and the number
of observations in each zone is counted. The A-zone is
defined symmetrically around the equal line as a ± 5%
deviation. It should ideally contain 95% of the observation. For set I, 92% of the observations were within the
A-zone, and for set II, 97%. The remaining observations
were in the B-zone, defined as ± 10%, symmetrical around
the equal line and thus including the A-zone. There were
none outside its limits, i.e., in the C-zone.
The imprecision study comprised measuring five
samples each day for 5 days using a large enough sample,
appropriately aliquoted from one and the same patient for
each set. The evaluation was done using ANOVA components. This has the advantage of offering within- and pure
between-series variances [10, 11]. Their sum (combined
uncertainty) corresponds to within-laboratory variance.
The detailed results of the analysis of the variance components are shown in Table 1. The imprecision was of the
same order of magnitude independent of the tube and
within the target total imprecision of 3%. The minimal
difference [13] between two successive measurements at
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Chakraborty et al.: Can glucose tubes be used for measurement of HbA1c? 3
Table 1 Imprecision of measurements.
Sample set I
EDTA
Within
Pure between Laboratory
Concentration mmol/mol
%
0.47
0.19
0.51
0.9
0.4
1.0
53.0
Sample set II
EDTA-fluoride
mmol/mol
%
0.45
0.23
0.50
0.8
0.4
0.9
54.0
a level of probability of 95% is thus about 1.2 mmol/mol
(2.2% at the tested concentration).
The data analysis shows that there is a statistically
significant difference between the results attributable to
the tubes. This difference, estimated as a mean difference
by regression analysis or at specified concentration, is of
the same order of magnitude, or less, than the minimal
significant detectable difference [13] and will therefore
not have any clinical implications. Sacks et al. [14] discussed the analytical goals for HbA1c measurements. The
presentation of data is not fully comparable, but the recommendation suggests an intra-laboratory coefficient of
variations of < 2%; our corresponding value was < 1.5% for
both types of tubes in our study (Table 1).
Expressing the relative performance of the methods
as the deviation from the equal line in terms of different
zones makes the performance understandable outside
the analytical laboratory and metrology, e.g., among clinicians. The high correlation coefficient and proportion
within the A-zone and lack of observations in the C-zone
are measures of equality of individual results.
The statistical power of the EP15 may not be sufficient
to create an imprecision profile, but the degrees of freedom
allow a comparison of the combined uncertainty of the
methods. The ANOVA components allows the isolation
of within (repeatability) and between (reproducibility)
variation. The uncertainty contribution in our study was
mainly from the repeatability component, i.e., the reproducibility was high. It is not unusual to observe that the
between-series variance is smaller than that within series
in measurement procedures where the measuring system
is stable, i.e., calibration is reproducible and the reagents
stable. Using ANOVA components is therefore desirable
in validating and verifying measurement procedures. The
present study therefore recommends that improvements
of the performance should focus on the repeatability of
the measurements.
Simultaneous requests for HbA1c and glucose concentration measurements are common. It is demonstrated that
HbA1c can be measured equally well using hemolysates from
EDTA
mmol/mol
%
Buffered EDTA-fluoride
mmol/mol
%
0.58
0.45
0.74
1.0
0.8
1.3
57.5
0.58
0.16
0.60
1.0
0.3
1.1
56.9
EDTA tubes, EDTA-fluoride, and buffered EDTA-fluoride
tubes on the Bio-Rad D10. This will contribute to rational
treatment of patients, savings of materials, and reduction
of inconveniences for patients and staff. Subsequent measurement of HbA1c concentration helps distinguish between
reactive and diabetic hyperglycemia and can safely be
requested from the original glucose tube. The present study
is limited to one measurement procedure, and the compatibility of different glucose tubes for HbA1c measurements
using non-HPLC methods needs further verification.
Author contributions: All the authors have accepted
responsibility for the entire content of this submitted
manuscript and approved submission.
Financial support: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
Competing interests: The funding organization(s) played
no role in the study design; in the collection, analysis, and
interpretation of data; in the writing of the report; or in the
decision to submit the report for publication.
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