Relationship between measured average glucose by continuous glucose
monitor and HbA 1c measured by three different routine laboratory methods
Tze Ping Loh, Sunil Kumar Sethi, Moh Sim Wong, E. Shyong Tai,
Shih Ling Kao
PII:
DOI:
Reference:

S0009-9120(15)00066-1
doi: 10.1016/j.clinbiochem.2015.02.012
CLB 8969

To appear in:

Clinical Biochemistry

Received date:
Revised date:
Accepted date:

26 December 2014
3 February 2015
23 February 2015

Please cite this article as: Loh Tze Ping, Sethi Sunil Kumar, Wong Moh Sim, Tai E.
Shyong, Kao Shih Ling, Relationship between measured average glucose by continuous
glucose monitor and HbA1c measured by three dierent routine laboratory methods,
Clinical Biochemistry (2015), doi: 10.1016/j.clinbiochem.2015.02.012

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Title: Relationship between measured average glucose by continuous glucose monitor

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and HbA1c measured by three different routine laboratory methods

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Category: Full Research Paper

Author: Tze Ping Loh,a* Sunil Kumar Sethi,a Moh Sim Wong,b E Shyong Tai,c Shih

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Department of Laboratory Medicine, National University Hospital, Singapore.

Department of Laboratory Medicine, Khoo Teck Puat Hospital, Singapore.

Department of Medicine, National University Hospital, Singapore.

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*Corresponding author

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Ling Kaoc

Tze Ping Loh

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National University Hospital, 5 Lower Kent Ridge Road, 119074 Singapore.

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Phone: +65-67724345

Email: tploh@hotmail.com

Running title: HbA1c and average glucose

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Abstract
Objectives

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The relationship between glycated hemoglobin A1c (HbA1c) and average glucose has
been described by the empirically derived estimated average glucose (eAG) equation

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in the A1c-derived Average Glucose (ADAG) study, with extensive calibration efforts
in four secondary reference HbA1c methods. It is not known if this relationship is
preserved when HbA1c is measured by routine laboratory methods under routine

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conditions.

Design and Methods

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We measured average glucose (mAG) by six days of continuous glucose monitoring

in 45 adults with stable HbA1c (<1% HbA1c change in the preceding two months).

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Subjects with medical conditions that may confound HbA1c measurement, including
anemia and hemoglobinopathy, were excluded. HbA1c were measured using Bio-Rad

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Variant II (cation-exchange HPLC), Bio-Rad in2it (boronate affinity HPLC) and

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Roche Tina-quant (immunoassay) methods.

Results

The average differences between eAG derived from the routine HbA1c methods and
mAG were 10.4% (Variant II), 6.0% (Tina-quant) and 1.0% (in2it). The regression
coefficients between the mAG and HbA1c are different between in2it (mAG, mmol/L
= 0.58%HbA1c + 2.3), Tina-quant and Variant II (both mAG, mmol/L =
0.66%HbA1c + 1.9). However, the 95% confidence intervals of the slope and bias of
these methods overlap. The correlation between mAG and HbA1c was greatest when

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measured using the Variant II (r2=0.84), followed by Tina-quant (r2=0.82) and in2it

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(r2=0.71).

Conclusions

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The relationship between HbA1c and measured average glucose is method-dependent

despite improved HbA1c standardization. The differences in relationship may reflect

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as discrepant eAG and home glucose monitoring results.

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Keywords: glycated hemoglobin A1c; glucose; continuous glucose monitoring;

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correlation; regression

Abbreviations

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HbA1c, Glycated hemoglobin A1c; NGSP, National Glycemic Standardization

Program; HPLC, high performance liquid chromatography; eAG, estimated average

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glucose; mAG, measured average glucose; CGM, continuous glucose monitoring;

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CVa, analytical coefficients of variation

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Introduction
Glycated hemoglobin A1c (HbA1c) is formed from non-enzymatic glycation of

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hemoglobin. HbA1c concentrations are highly correlated with average glycemia, and
reflect the glycemic control of a patient over the past two to three months. The

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relationship between average glucose and HbA1c has been described by the
empirically derived estimated average glucose (eAG) equation in the seminal A1c-

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derived Average Glucose (ADAG) study [1].

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Several other groups have examined also the relationship between average glucose by
continuous glucose monitoring (CGM) and HbA1c in other populations. The linear

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relationship between average glucose and HbA1c has been demonstrated in children
and adult with type 1 diabetes [2-4], and patients with concomitant advanced chronic

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kidney disease and type 2 diabetes [5], albeit with different correlation and regression

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characteristics. A selection of these publications are summarized in Table 1.

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The ADAG equation is the most widely used eAG equation in routine clinical practice
to translate HbA1c values into average glucose. The eAG equation in the ADAG study
was derived by correlating the mean of four HbA1c values, which were measured
using four different International Federation of Clinical Chemistry and Laboratory
Medicine (IFCC) secondary reference laboratory methods with rigorous calibration
efforts [6], and with average glucose obtained from capillary blood and CGM. Such
rigorous effort in HbA1c measurement is unlikely to be achieved in routine practice.

Therefore, it is important to understand the impact of using HbA1c measured by

routine laboratory methods under routine laboratory conditions on the relationship

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with average glucose. Such evidence is lacking in the literature. To fill this gap, we
compared HbA1c measured using three National Glycohemoglobin Standardization

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Program (NGSP)-certified methods that employed different analytical principles, and

estimated the discrepancy between measured average glucose (mAG) and eAG

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derived from the different HbA1c measurements. The relationship between HbA1c

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measured by the three routine laboratory methods and mAG was also examined.

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DirecNet
study [3]

48 subjects with type 1

diabetes, aged 4-<18 years

93% nonHispanic whites,

7% non-whites

ORiordan
et al. [4]
Lo et al.
[5]

234 subjects with type 1

diabetes, aged <19 years
43 subjects with stage 3-5
chronic kidney disease and
type 2 diabetes, aged 1880 years
45 subjects with (n = 36)
and without (n = 9) type 2
diabetes, aged 21-80 years

Not specified

JDRFCGM
study [2]

This study

94% whites, 6%
non-whites

Not specified

Chinese

Linear
correlation, r2
0.84

HbA1c assay

CGM

Mean of 4 assays
were used. Roche
Tina-quant, Primus
Ul- tra-2, Tosoh
G7, Roche A1c

Medtronic
Minimed

-0.90 (-1.83 to 0.03)

0.63

Tosoh HbA1c 2.2

Plus

+2.22

Not specified

Siemens DCA
2000+

Medtronic
MiniMed, Abbott
FreeStyle
Navigator
Abbott FreeStyle
Navigator

Subjects
encouraged
to performed
CGM as
often as
possible for
6 months
5 days

1.00 (0.78 to
1.22)

0.49 (0.35 to
0.62)
1.38 + [0.57
CKD status]

+5.95 (4.72 to 7.19)

0.20

-1.16

0.66 (CKD 3-5);

0.79 (CKD 3);
0.34 (CKD 4-5)

Siemens DCA
2000+
Tosoh A1c 2.2 and
Adams Arkray

Medtronic
Minimed Gold
Medtronic
Minimed

0.66 (0.58-0.72,
Variant II);
0.66 (0.56 to 0.76,
Tina-quant);
0.58 (0.46 to 0.70,
in2it)

1.9 (1.2 to 2.5,

Variant II);
1.9 (1.1 to 2.7, Tinaquant);
2.3 (1.5 to 3.1 in2it)

0.84 (Variant II);

0.82 (Tina-quant);
0.71 (in2it)

Bio-rad Variant II,

Roche Tina-quant,
Bio-rad in2it

Medtronic Enlite
sensor

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83% white, 8%
black, 8%
Hispanic, 2%
others

Intercept
(95% CI)
-2.59

1.35 (1.22 to
1.48)

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507 subjects that

comprised of 268 patients
with type 1 diabetes, 159
patients with diabetes and
80 subjects, aged 18-70
years
252 subjects with type 1
diabetes, aged 8-74 years

Slope
(95% CI)
1.59

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ADAG
study [1]

Duration of
CGM
At least 2
days
performed 4
times in 3
consecutive
motnhs
3 months

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Race

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Population

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Reference

2 days/
month for 3
consecutive
months
6 days

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Table 1. A selection of previously published studies that examined the relationship between HbA1c and average glucose. The regressions are expressed as

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average glucose (mmol/L) = slopeHbA1c (% unit) + intercept. CGM = continuous glucose monitor, CKD = chronic kidney disease.

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Materials and methods
This study was performed as part of a larger study examining glycemic and non-

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glycemic determinants of HbA1c in a Chinese population in Singapore. The inclusion

criteria were: age 21-80 years, Chinese ethnicity, and a screening HbA1c between 4.8-

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10%. Previous studies have shown that the relationship between average glucose and
HbA1c is different between types 1 and 2 diabetes [7,8]. In this study, we have chosen
to include examine patients with type 2 diabetes as Type 1 diabetes is uncommon in

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our population [9].

Patients with known factors that may alter the relationship between average glucose

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and HbA1c including pregnancy, use of erythropoietin, treatment with hemodialysis,

history of recent surgery, blood donation or transfusion in the past six months, were

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excluded. To ensure a stable metabolic state, patients with a history of recent

hospitalization or steroid use in the past six months as well as patients who had a

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excluded.

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weight change of greater than 3% in the month preceding recruitment were also

Forty-six consecutive subjects of the larger study were enrolled into this sub-study.
Participants with diabetes were on a stable dose of diabetic medications for the last
three months, and had stable glycemic control as evidenced by two sequential HbA1c
values in the last six months, which were within 1% of each other. The study was
approved by the ethics committee of National University Hospital, Singapore.
Informed consent was obtained from all participants.

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Data on demographics and medical history was obtained through an intervieweradministered questionnaire. Height and weight were measured. All participants

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underwent six days of CGM (Enlite sensor, Medtronic, Northridge, USA), in which
interstitial glucose concentrations were measured every five minutes. This CGM uses

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the glucose oxidase method to convert interstitial glucose into hydrogen peroxide that
reacts with the sensor surface to generate electrical signal that is proportional to the

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changes in interstitial glucose.

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The participants performed capillary blood glucose monitoring during CGM for
calibration purposes (One Touch Ultra, glucose oxidase biosensor method, Lifescan,

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Milipitas, USA). Capillary blood glucose readings were reported in plasma equivalent
units. Participants who had less than 72 hours of CGM readings were excluded from

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analysis. CGM data with a mean absolute difference compared to capillary blood
glucose of 18% or greater was also excluded, as recommended by the manufacturer.

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The measured average glucose (mAG) was the arithmetic mean of all interstitial

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glucose values obtained from the CGM.

Whole blood was collected into K2EDTA blood tubes for HbA1c measurement and
hemoglobin capillary zone electrophoresis (Sebia Capillarys, Norcross, GA)
examination at the end of the CGM period, immediately after removal of the sensor
from the patient. HbA1c was measured in an NGSP level I-certified clinical
laboratory, using the Variant II Dual Program in HbA1c mode (Bio-Rad Laboratories,
Hercules, CA), within four hours of sample collection. The sample was then
immediately stored at -70C. The archived samples were gradually thawed in iceslurry in a single cycle and kept chilled until measurement by the in2it (II) A1c (Bio-

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Rad Laboratories, Hercules, CA) and Tina-quant HbA1c Gen 3 (applied on c501
analyzer, Roche Diagnostics, Mannheim, Germany) methods within six months of

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sample collection. HbA1c concentrations in whole blood samples are stable for at least
twelve months when stored at -70C. The correlation coefficient (r) is >0.99 between

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fresh and freeze-thaw samples [10,11].

The Variant II method utilizes the cation-exchange principle to separate the

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hemoglobin species. The total analytical coefficients of variation (CVa) of this

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analyzer at HbA1c values of 5% and 10% are less than 2%. The Tina-quant method is
a latex-enhanced competitive turbidimetric immunoassay and has CVa of 2.1% at

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similar HbA1c concentrations. The in2it method uses the boronate affinity
chromatography principle to measure total glycated hemoglobin, which is then

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converted to HbA1c values. Its CVa is approximately 4% at HbA1c of 6.0%-10.4%

[12]. All the HbA1c methods are NGSP-certified. Hemoglobin concentration was

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measured using the XE-5000 analyzer (Sysmex Corp, Kobe, Japan). All HbA1c

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measurements were performed according to manufacturer's recommendation under

routine laboratory conditions.

Statistical analysis
The eAG were derived from the HbA1c results using the ADAG equation [eAG
(expressed in mmol/L) = 1.59 HbA1c (expressed in % unit) 2.59] [1]. BlandAltman plots were drawn to compare the difference between the eAG calculated from
the results of the three HbA1c methods and mAG. Simple linear regression was
performed with mAG as the dependent variable and HbA1c as the explanatory
variable.

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The statistical analyses were performed using Microsoft Office Excel version 2010

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(Microsoft Corporation, Redmond, WA) and SPSS version 19 (SPSS IBM Inc.,
Chicago, IL, USA). Data are shown as means (SD), and a p-value of <0.05 was

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considered statistically significant.

Results

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A total of 46 subjects were included in this study. One subject was regarded as an

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outlier by visual inspection of the scatter plot of mAG and HbA1c, and was excluded
from further analysis. The mean age was 56.8 years (SD: 9.8) and mean BMI was

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25.0 kg/m2 (4.3). Twenty-seven of these subjects (60%) were male. Thirty-six
subjects (80%) had type 2 diabetes. None of the subjects had anemia or

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hemoglobinopathy when examined by hemoglobin electrophoresis. The results of the

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laboratory tests are summarized in Table 2.

Mean (SD)

Reference interval

13.0 (0.8) (female)

10.9 15.1 (female)

14.6 (1.3) (male)

12.9 17.0 (male)

Mean corpuscular volume, fL

88.9 (4.0)

80.0 95.0

Mean corpuscular hemoglobin, pg

30.1 (1.6)

27.0 33.0

HbA1c, Variant II, %

7.0 (1.1)

4.5 5.6

HbA1c, in2it, %

6.8 (1.1)

4.5 5.6

HbA1c, Tina-quant, %

7.1 (1.1)

4.5 5.6

eAG, Variant II, %

8.5 (1.7)

eAG, in2it, mmol/L

8.2 (1.7)

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Laboratory tests, unit

Hemoglobin, g/dL

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8.6 (1.8)

Measured average glucose, mmol/L

7.7 (1.5)

Number of CGM readings per subject

1701 (57)

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eAG, Tina-quant, mmol/L

Table 2. Summary of laboratory results in this study. SD, standard deviation; HbA1c, glycated

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hemoglobin A1c; eAG, estimated average glucose; CGM, continuous glucose monitoring.

The eAG derived from the HbA1c measured by the three routine laboratory methods

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were higher than mAG (Table 2). These differences were reflected in the BlandAltman plots (Figure 1), which showed the percentage difference between the eAG

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and mAG (y-axis) as a function of the average of eAG and mAG (x-axis). There was
no proportional bias among all three HbA1c methods. The systematic bias, expressed

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as average percentage differences between eAG and mAG, was 10.4% (95%

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confidence interval: 7.7 to 13.2), 1.0% (-1.7 to 3.6) and 6.0% (4.3 to 7.7) for Variant

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II, in2it and Tina-quant methods respectively.

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The relationships between the mAG and HbA1c measured by the three laboratory
methods are shown in Figure 2 together with the regression and correlation
coefficients. While numerically different, the slope and intercept of the three
laboratory methods have overlapping standard errors. The correlation between mAG
and HbA1c was greatest when measured using the Variant II (r2=0.84), followed by
Tina-quant (r2=0.82) and in2it (r2=0.71).

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Figure 1. The Bland-Altman plots showing the difference between measured average glucose
and estimated average glucose (eAG) derived from (panel A) in2it, (panel B) Tina-quant and
(panel C) Variant II. The average percentage difference (solid line), its 95% confidence

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intervals (dotted lines) and 1.96 standard deviations (dashed lines) are also displayed.

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Figure 2. The relationships between the measured average glucose and HbA1c measured by
the three routine laboratory methods. B = slope, SE = standard error, C = intercept, Adj r2 =
adjusted r2.

Discussion
The reporting of average glucose is thought to be more intuitive for a patient to
understand his glycemic control. The eAG equation derived from the ADAG study
provides a convenient tool for converting HbA1c results to estimated average glucose.
In certain regions, clinical laboratories are encouraged to report eAG together with
HbA1c to help patients better understand and manage their glycemic control [13,14].

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Nevertheless, there are several limitations of the ADAG equation that have been well

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argued [15]. Firstly, the eAG assumes that relationship between HbA1c and average
glucose is mathematically consistent between subjects. However, the extent to which

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hemoglobin is glycated at a given concentration of glucose also depends on factors

such as red blood cell lifespan and turnover, rate of glucose uptake into the blood
cells, rate of glycation and deglycation. These intricate factors are different between

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individuals and contribute to discordant eAG and self-monitored mean blood glucose

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seen in routine practice [16].

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Secondly, subjects included in the ADAG study were not sufficiently representative
to allow reliable expression of HbA1c as eAG in all patients. The subjects included in

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the ADAG study were mostly non-pregnant white adults without renal failure or
hemoglobin variants [1]. The narrow inclusion criteria meant that this equation should

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not be used beyond these populations without exercising caution or additional studies

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(Table 1). There are racial and ethnic differences in the relationship between HbA1c
and blood glucose [17]. It would be of interest to evaluate the relationship between
mean glucose and HbA1c in other ethnic populations.

Finally, the wide scatter of mAG around the regression line meant that a high
proportion of patients are likely to have significantly different true average glucose
when compared to the eAG. By some estimates, up to 100% difference may be seen
in some patients when the analytical bias in HbA1c assay is considered [15].

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In this study, the application of the ADAG-derived eAG equation was associated with
an over-estimation of average glucose compared to mAG. The degree of over-

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estimation of eAG varied with the laboratory method used to measure HbA1c. The
over-estimation was greatest for Variant II (10%), followed by Tina-quant (6%) and

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in2it (1%). The overestimation of average glucose implied that for the same degree of
glycemia, the HbA1c is higher in our cohort compared to those in the ADAG study.

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One possible explanation for this may be the ethnicity of our study population. Asian

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and black populations are known to have higher HbA1c values compared with
Caucasians at the same level of fasting glycemia [17]. The subjects included in the

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ADAG study are predominantly Caucasians and people of African descent, whereas

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our cohort is entirely Chinese.

The eAG equations (regression coefficients shown in Figure 2) derived from this

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study for each method were different from the ADAG study, which could also explain

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the reason for the over-estimation of average glucose seen in our study subjects. This
may be related to between-method biases in the HbA1c instruments, which remain
present despite the manufacturer receiving NGSP certification
(http://www.ngsp.org/CAPdata.asp). The NGSP has significantly reduced the bias and
imprecision of most routine laboratory methods since its inception [18] and continued
tightening of the NGSP performance criteria may reduce these differences. Of note,
the ADAG equation has a residual HbA1c of 1.63% when eAG is 0 mmol/L. This
suggests that the equation includes some non-glycemic factors. A recent in-silico
semi-mechanistic modeling study has found that red blood cell lifespan and nonspecificity of the routine (NGSP) HbA1c assays were able to explain the non-

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proportional relationship between HbA1c and average glucose [19]. Alternatively, the
discordance may be related to the difference in the calibration characteristics, and

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hence bias, of the CGM and glucometers used between the studies.

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The correlation between the mAG and HbA1c in this study was good, with r2 values
ranging from 0.71 to 0.84. The degree of correlation is inversely related to the CVa of
the HbA1c laboratory method. The high CVa of the in2it instrument may increase the

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variability of each HbA1c measurement, leading to poorer correlation between in2it

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and mAG. The correlation coefficients in this study are comparable to the ADAG

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study, where an r2 value of 0.84 was achieved [1].

The high degree of correlation was achieved in this study despite the relatively short

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period of intensive glucose monitoring of six days compared to three months in the
ADAG study. This may be attributable to the use of precise laboratory methods

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(Variant II and Tina-quant), the inclusion of patients with stable glycemia (<1% unit

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change in HbA1c in the preceding two months before enrollment), and the high
number of CGM readings achieved in this study (Table 2).

One potential limitation of this study is the use of samples that have been stored at 70C and subjected to a single freeze-thaw cycle. Under this condition, a high degree
of correlation (r2 = 0.82) was obtained for the Tina-quant immunoassay method,
while the in2it boronate affinity method has poorer correlation (r2 = 0.71). Besides the
difference in CVa between the methods, the difference may be caused by degradation
of HbA1c molecule during the freeze-thaw cycle in a manner that affects certain
method more than others. We have sought to minimize any potential degradation by

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slowly thawing the samples in ice slurry and keeping the samples chilled until testing.
Of note, the ADAG study also subjected their samples to a single freeze-thaw cycle

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on freshly collected samples would address this limitation.

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prior to HbA1c measurement [1]. A direct comparison between the different methods

In summary, the relationship between HbA1c and measured average glucose is

method-dependent despite improved standardization of HbA1c. The differences in

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relationship may reflect as discrepant eAG and readings on glucometers. Patients who

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routinely perform self-monitoring of blood glucose may observe a significant

discordance of their results with the eAG reported by the laboratory using the ADAG

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equation, which may confuse as well as undermine their confidence in the ability of
the laboratory to produce accurate results. Laboratory and clinical practitioners should

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thus be aware of such differences when considering the use of the ADAG equation in
their reports. Method-specific eAG equations may reconcile some of these differences

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but require further study.

Conflict of interest
The authors have no competing interest or disclosure to declare.

Acknowledgement
We would like to thank the staff of the Department of Laboratory Medicine, National
University Hospital and Khoo Teck Puat Hospital for the kind technical assistance.
We also gratefully acknowledge the funding from the National Medical Research
Council (Singapore) NMRC//NIG/1050/2011, awarded to Dr Shih Ling Kao.

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[18] Little RR, Rohlfing CL. The long and winding road to optimal HbA1c

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measurement. Clin Chim Acta. 2013;418:63-71.

[19] Lled-Garca R, Mazer NA, Karlsson MO. A semi-mechanistic model of the

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relationship between average glucose and HbA1c in healthy and diabetic subjects. J

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Pharmacokinet Pharmacodyn 2013;40(2):129-42.

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Highlights

We measured average glucose (AG) by continuous glucose monitor in 46

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subjects.
Their HbA1c was measured by 3 routine methods (in2it, Tina-quant, Variant

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II).

Measured AG was higher than those estimated using ADAG equation by 1.0%
to 10.4%.

Estimated AG (eAG) equations derived from routine methods were different

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HbA1c-AG relationship is method-dependent; eAG may contradict home

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glucose monitor.

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from ADAG.

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