Original Article Nepal Med Coll J 2014; 16(2-4): 103-108

Comparative study of glycated hemoglobin by ion exchange

chromatography and affinity binding nycocard reader in
type 2 diabetes mellitus
Gautam N,1 Dubey RK,1 Jayan A,1 Nepaune Y,1 Padmavathi P,1 Chaudhary S,2 Jha SK,3 Sinha AK1
Department of Biochemistry, 2Department of Internal Medicine, Universal College of Medical Sciences, Bhairahawa, Nepal,
1
3
Deuri Parbaha Primary Health Post, Dhanusha, Nepal.
Corresponding Author: Narayan Gautam, Associate Professor, Department of Biochemistry, Universal College of Medical Sciences,
PO: 53, Bhairahawa, Nepal, e-mail; [email protected]

ABSTRACT:
The aim of this study was to compare the level of glycated hemoglobin (HbA1c) in type 2 Diabetes Mellitus
(DM) patients by two different methods namely Ion Exchange Chromatography and Affinity Binding Nycocard
Reader. This is a cross-sectional study conducted on confirmed type 2 diabetes mellitus patients (n=100) who
visited Out Patients Department of the Universal College of Medical Sciences Teaching hospital, Bhairahawa,
Nepal from November 2012 to March 2013. The diagnosis of diabetes mellitus was done on the basis of their
fasting (164.46±45.33 mg/dl) and random (187.93±78.02 mg/dl) serum glucose level along with clinical
history highly suggestive of type 2 DM. The HbA1c values of (7.8±1.9 %) and (8.0±2.2 %) were found in
DM patients as estimated by those two different methods respectively. The highest frequency was observed
in HbA1c>8.0% indicating maximum cases were under very poor glycemic control. However, there were no
significant differences observed in HbA1c value showing both methods are comparable in nature and can be
used in lab for ease of estimation. The significant raised in HbA1c indicates complications associated with DM
and monitoring of therapy become hard for those patients. Despite having standard reference method for HbA1c
determination, the availability of report at the time of the patient visit can be made easy by using Nycocard
Reader and Ion Exchange Chromatography techniques without any delay in communicating glycemic control,
clinical decision-making and changes in treatment regimen.
Keywords: HbA1c, Ion exchange chromatrography, Nycocard reader, Type 2 Diabetes

INTRODUCTION methods available for the routine measurement of

The prevalence of Type 2 Diabetes mellitus (T2DM) HbA1c based on different analytical principles, such
is increasing at alarming rates both in the developing as immunoassays, ion-exchange chromatography and
and the newly industrialized countries of the world.1 It affinity chromatography.3 The choice of diagnostic
is highly prevalent in the elderly and associated with method will depend on local considerations such as cost,
various co-morbities, such as obesity, hypertension, availability of equipment, population characteristics,
hyperlipidemia and cardiovascular disease. The glycated presence of a national quality assurance system etc.4
hemoglobin (HbA1c) is non-enzymatic condensation
The aim of this study was to compare the level of Glycated
of glucose to the valine residue of β-hemoglobin to
Hemoglobin (HbA1c) in T2DM patients by two different
form aldimine and then by amadori rearrangement
methods viz a viz Ion Exchange Chromatography and
to ketoamine. It has provided clinicians worldwide
Affinity Binding Nycocard Reader. We have designed
with a means whereby average blood glucose values
Diabetes patients into different categories like very good
can be quantified over the previous 8-10 weeks. The
glycemic control, good glycemic control, poor glycemic
true need for the international standardization of
control and very poor glycemic control to assess
HbA1c measurements became an important objective
association between the HbA1c value with Ion Exchange
for scientists and clinicians worldwide only after the
Chromatography and Affinity Binding Nycocard Reader.
results of the Diabetes Control and Complications
Trial (DCCT) were published in 1993. 2 Although
MATERIALS AND METHODS
high performance liquid chromatography is the gold
This is a comparative, cross-sectional study carried out
standard method, the disadvantage of it is that results
in Type 2 diabetes mellitus patients (n=100), visiting
are not available at the time of patient visit, delay in
Universal College of Medical Sciences Teaching hospital
reporting time, communicating health feedback, delay
(UCMSTH), Bhairahawa, south-western, Nepal, who
in clinical decision making, changes in the regimen
are confirmed by Fasting Blood Glucose (FBG) based
prescription may be missed. There are many commercial
on World Health Organization (WHO) criteria during
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period of November 2012 to March 2013. Fasting and diol configuration of glycated hemoglobin. An aliquot
Random blood Glucose concentration were determined of the reaction mixture is added to the rest device, and
for the patients on same day. The EDTA- blood samples, all the precipitate hemoglobin, conjugate bound and
stored at 4°C were assayed for HbA1c by using standard unbound, remains on top of the filters. Any excess of
protocols by following methods. colored conjugate is removed with washing solution.
The precipitate is evaluated by measuring the blue
ION EXCHANGE CHROMATOGRAPHY (glycated hemoglobin) and the red (total hemoglobin),
Principle: Whole blood is mixed with lysing reagent color intensity with the NycoCard reader II, the ratio
containing a detergent and borate ions. Elimination of the between them being proportional to the percentage of
labile Schiff’s base is thus achieved during the hemolysis. HbA1c in the sample.
The hemolysate is then mixed for 5 minutes with a weakly
Procedures: 5μL of whole blood was added to the cup
binding cation exchange resin. During this time, HbA0
with reagent 1 mixed well and incubated for 3 minutes.
binds to the resin. A special resin separator is used to
It was remixed to obtain a homogenous suspension.
remove the resin from the supernatant fluid which contains
Reaction mixtures (25 μL) were applied to a test devise
the HbA1. The glycohemoglobin percentage of total
by holding the pipette approximtely 0.5 cm above the
hemoglobin is determined by measuring the absorbance
test. The pipette was emptied quickly in the middle
of the glycohemoglobin and of the total hemoglobin
of the test. The reaction mixture was allowed to soak
fraction at 405 nm in ELISA reader (Erba LISA scan II) in
completely into the membrane for 10 seconds. Washing
comparison with a standard glycohemoglobin preparation
solution (25 μL) was added to the test device. The
carried through the test procedure.
washing solution was allowed to soak completely into
Procedure: 500 μl Lyse solution was added into cups the membrane for 10 seconds. The test result was read
and 100 μl Sample, Standard & Control were added into within 5 minutes using NycoCard reader II.
separate cups. The solution was mixed and incubated for
5 minutes at 15-25°C. The solution was than pipetted STATISTICAL ANALYSIS
into labeled Reagent (RGT) i.e, Ion Exchange Resin Data were processed with the statistical software; SPSS
prefilled in plastic tubes in Imidazole Borate buffer and version 16 and diagram were represented by Prism 6. The
Separator (SEP) was inserted so that rubber sleeve is 1 mean and SD were presented as continuous variables,
cm above surface of resin suspension. The mixture was and categories were expressed as percentages. For
than mixed in hematology mixer for 5 minutes. SEP comparison of the mean values, the one way ANOVA
was pushed down the resin so that to it firmly packed. was used, and for that of categories, the paired t-test
The solution was taken of the SEP to microtitre well and and Spearman correlation were selected. Regression
measure in ELISA plate reader at 405 nm. analysis of individual values obtained from the two
measuring methods was performed.
Total Hemoglobin estimation was done by taking 20
μl hemolysate from lyse sample. 5 ml of distil water
RESULTS
was added and mixed. The measurement was done in
ELISA plate reader at 405 nm and calculation is done
by using following formula:
OD HbA1c
% HbA1c= OD of Total Hb (std) X % HbA1c (std) X (sample)
sample OD of HbA1c (std) OD Total Hb
(sample)

AFFINITY BINDING NYCOCARD READER

Principle: HbA1c is a boronate affinity assay. The kit
contains test devices with a porous membrane filter,
test tubes prefilled with reagent and washing solution.
The reagent contains agents that lyse erythrocytes and
precipitate hemoglobin specifically, as well as a blue Fig.1: Frequency (%) among different age groups of Type 2
boronic acid conjugate that binds cis-diols of glycated DM patients (n=100)
hemoglobin. When the blood is added to the reagent,the The figure 1 shows the frequency of patients categorized
erythrocytes immediately lyses. All hemoglobin into different age groups where maximum percentage
precipitate the boronic acid conjugate binds to the cis- of patients affected with T2DM fall under age group

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Table 1: Age-wise distribution of basic characteristics in Type 2 DM patients

(n=100) results are expressed in (Mean ± SD)
Age-groups Age Ht Wt BMI BP(Systolic) BP(Diastolic)
(years) (m) (Kg) (Kg/sq.m.) (mm/Hg) (mm Hg)
21-30 26.5±2.12 1.69 ±0.02 54.5±6.36 19.08±2.71 145.0±21.21 90.0±14.14
31-40 37.0±3.46 1.63±0.03 60.75±3.77 22.83±0.84 147.5±15.0 97.5±17.08
41-50 46.6±3.34 1.62±0.06 62.7±9.79 24.18±4.55 145.5±19.50 103.5±19.44
51-60 55.68±3.54 1.63±0.05 61.35±8.64 23.34±4.28 144.71±12.61 97.06±13.38
61-70 66.67±2.94 1.63±0.03 61.59±6.25 23.25±2.3 154.44±20.06 105.56±15.28
71-80 75.08 ±3.88 1.65 ±0.06 63.74±4.56 23.45± 2.10 152.11±24.40 101.58±14.25
81-90 75.05±3.88 1.65±0.06 63.74±4.56 23.45±2.1 137.5±15.0 91.25±2.5
Total 60.13±13.26 1.66±0.08 65.5±6.66 23.71±1.09 147.55±16.96 100.20±14.23

51-60 years which was 33.0% followed by 61-70 years Table 1 shows the distribution of age-wise distribution
whose frequency was found to be 26%. of basic characteristics of T2DM patients. The mean age
(± SD) of study population was 60.13±13.26 years, the
mean Body Mass Index (BMI) calculated from mean
weight (63.74±4.56 kg) and mean height (1.65±0.06
m) was (23.45±2.1 Kg/m2), the mean systolic blood
pressure was 137.5±15.0 mm Hg and diastolic blood
pressure was 91.25±2.5 mm Hg respectively. The
maximum BMI (24.18±4.55 kg/sq.m.) was seen in the
age group 41-50 years, BP systolic (154.44±20.06 mm
Hg) and BP diastolic (105.56±15.28 mm Hg) were seen
in age group 61-70 years.
Table 2 shows the age-wise distribution of blood sugar
and HbA1c in T2DM. The mean (±SD) fasting blood
sugar and random blood sugar were 164.46±45.33 mg/dl
and 187.93±78.02 mg/dl respectively. The mean (±SD) of
HbA1c by Ion Exchange Chromatography was 7.8±1.9 %
Fig. 2: Frequency (%) of General characteristics of Type 2 and by Nycocard Reader was 8.0±2.2 %. The maximum
DM patients (n=100) FBS and RBS were seen in age group 41-50 years and
Figure 2 shows the frequency of general characteristics found to be 156.33±46.29 mg/dl and 215.0±113.14 mg/
of T2DM patients. According to Diet 72% were dl respectively. Moreover, the maximum HbA1c % was
Non-vegetarian and 28% were Vegetarian, 68% Non found in the age group 51-60 years whose value by Ion
smoker and 32% Smoker, 51% were Non-alcoholic Exchange Chromatography and Nycocard Reader were
and 49% alcoholic, 86% cases were Non-exercising found to be 8.2±2.3 % and 8.6±2.4 % respectively.
and 14% were exercising.
Table 2: Age-wise distributions of FBS, RBS and HbA1c in Type 2 DM patients (n=100) results are
expressed in (Mean ± SD)
FBS RBS
Age-groups HbA1c Ion Ex (%) HbA1c Nyco (%)
(mg/dl) (mg/dl)
21-30 145.22±21.34 167.33±65.76 8.0±0.3 6.3±0.8
31-40 174.0±14.14 173.0±56.80 6.8±1.6 7.1±1.7
41-50 156.33±46.29 215.0±113.14 7.6±1.9 7.2±1.1
51-60 131.67±2.52 194.33±24.62 8.2±2.3 8.6±2.5
61-70 170.2±37.76 196.0±39.7 8.2±2.1 8.2±2.4
71-80 163±25.46 155.2±62.67 7.4±1.6 7.2±1.8
81-90 135±10.11 177.0±60.23 7.7±1.2 8.2±1.6
Total 164.46±45.33 187.93±78.02 7.8±1.9 8.0 ±2.2

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Figure 4 shows mean ± SD of blood sugar level at

different level of HbA1c% by two different methods.
The maximum FBS level (155.0±28.27 mg/dl) and RBS
(200.40±94.72 mg/dl) were seen in HbA1c % level >8
% followed by 7.0-7.9 %, 6.0-6.9 % and 5.5-5.9 %
respectively.

HbA1c% Mean±SD Mean±SD

Methods HbA1c Nyco HbA1c Icon Exchange

5.5-5.9% 5.7±0.1 5.1±0.2

6.0-6.9% 5.4±0.3 6.5±0.3

7.0=7.9% 7.5±0.3 7.4±0.3

>8.0% 10.1±1.8 9.7±1.6

Figure 3: Frequency (%) of General characteristics of Type

2 DM patients (n=100)
Figure 3 shows the frequency and Mean (±SD) of HbA1c
% by affinity binding Nycocard Reader and ion exchange
chromatography. The highest frequency of T2DM was
Figure 5: Comparision of HbAc % (Mean SD) by Nycocard
>8% (very poor glycemic control) by Nycocard Reader
Reader & Ion Exchange Chormatography
and Ion Exchange Chromatography whose mean (±SD)
HbA1c % were 10.1±1.8 and 9.7±1.6 respectively. It is Figure 5 shows the comparison between mean± SD of
followed by HbA1c level 6.0-6.9% (good glycemic control) HbA1c% by two methods. There was statistical non-
with 20% and 28% measured by Nycocard reader and Ion significant difference of HbA1c% by Nycocard Reader
Exchange Chromatography whose mean (±SD) HbA1c and Ion Exchange Chromatography (p>0.05)
% were 6.4±0.3 and 6.5±0.3 respectively. Then followed
by HbA1c level 7.0-7.9% (poor glycemic control) with
26% and 23% measured by Nycocard reader and Ion
Exchange Chromatography whose mean± SD HbA1c %
were 7.5±0.3 and 7.4±0.3 respectively. Lastly followed
by HbA1c level 5.5-5.9% (very good glycemic control)
with 10% and 4.0% measured by Nycocard reader and
Ion Exchange Chromatography whose mean± SD HbA1c
% were 5.7±0.1 and 5.1±0.2 respectively. The statistically
significant difference was observed between groups as
compared to very good glycemic control (p<0.0001).

Figure 6: Regression analysis between Nycocard

Reader (IFCC Standardized method) and Ion exchange
chromatography
The Regression analysis between Nycocard Reader
(IFCC Standardized method) and Ion exchange
chromatography yielded a coefficient of determination
FBS (mg/dl) FBS (mg/dl) RBS (mg/dl) RBS (mg/dl)
r2 = 0.24 (P < 0.0001) as shown in Figure 6. We also found
at different
HBA1c% by
at different
HBA1c% by
at different
HBA1c% by
at different
HBA1c% by
positive correlation between HbA1c% Nycocard Reader
Nycocard Ion Exchange Nycocard Ion Exchange
and Ion Exchange chromatography (p<0.0001), between
HbA1c% Nycocard Reader & FBS, RBS (p<0.001),
Figure 4: FBS &RBS (Mean±SD) Different level of
HbA1c% by Affinity Binding Nycocard Reader & Ion between HbA1c% Ion Exchange Chromatography &
Exchange Chromatography FBS, RBS (p<0.001) shown in table 3.

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Table 3: Correlation between Glucose concentration glucose binds to m-aminophenylboronic acid and has
and HbA1c minimal interference from haemoglobinopathies, HbF
and carbamylated Hb. It measures not only glycation of
HbA1c HbA1c (Ion
Variables N-terminal valine on β chain, but also β chains glycated
(Nyco) Exchange)
at other sites and glycated α chains.5 The coefficient of
HbA1c (Nyco) 1 0.49**
variation (CV) <5% is usually obtained. There is no
HbA1c (Ion Exchange) 0.49** 1 biochemical interference from hemoglobin variant for
FBS 0.16* 0.11* the affinity and immunochemical methods but liable
RBS 0.23* 0.18* to interference with RBC turnover in blood is high.2
**p<0.0001,*p<0.001 Adopting the new IFCC standardization procedure
will result in HbA1c percentage values being lowered
DISCUSSION because of the higher specificity on the reference
The significant rise in HbA1c level in the present study method.3 The IFCC reference is unaffected by interfering
indicates the complication associated with Diabetes substances that result in the non-specificity in certain
mellitus and monitoring of therapy become harder for methodologies. The conversion formulae of IFCC into
those patients. Moreover, there were significant rise in DCCT/NGSP unit (%) is equal to 0.09148XIFCC units
systolic and diastolic BP in study population indicating (mmol/mol) +2.152 and vice versa DCCT/NGSP unit
risk of hypertension and associated cardiovascular (%) is equal to10.93XDCCT/NSGP unit (%) - 23.50.4
diseases. Hypertension is common in patients with type
2 diabetes, with prevalence rate of 40-60% over the age Although HPLC is the standard method for assay of
range of 45-75 years.5 Hypertension multiplies the risk HbA1c the disadvantage is that the results are not
of cardiovascular and renal disease already present in available at the time of the patient visit. In present study,
diabetic patients. The major reasons for the increasing the average time consumption by those two methods
number of people with T2DM are population growth, are less (5 min by Nycocard reader and 15 min by Ion
aging, urbanization and increasing prevalence of obesity exchange chromatography), blood glucose concentration
and physical inactivity. Long term complications of correlate with HbA1c % as well as statistical non-
T2DM include retinopathy, nephropathy, peripheral and significant difference show the comparability nature
autonomic neuropathy and cardiovascular diseases cause of these two methods with ease of performance. In
huge medical and socioeconomic burden on the society one of the randomized study comprising 201 patients
and impose enormous strains on health care systems.6 (both type 1 and type 2 diabetic patients with insulin
Medical burden of diabetic patients increases long before treatment), one group received immediate feedback,
actual diagnosis for the disease is established.7 while the other group received delayed feedback via
phone call or letter. Patients were followed for 1 year.
The maximum value of HbA1c was observed for age Results indicated that immediate A1C feedback helped
groups 51-60 years are 8.6 ± 2.5 % and 8.2 ± 2.1 % by to improve subsequent glycemic control at 6 and 12
Ion Exchange Chromatography and Affinity Binding months.9 The availability of report at the time of the
Nycocard Reader respectively. Amongst the DM patient visit can be made easy with the use of Nycocard
patients, the frequency was found to be highest for Reader and Ion Exchange Chromatography techniques
the HbA1c value >8.0 % (very poor glycemic control) by without any delay in communicating glycemic control,
these two methods. This indicates the maximum patients opportunities for clinical decision-making and changes
are at risk for developing complication related with in treatment regimen. Implementing inexpensive, easy-
Diabetes mellitus. This also reflects poor management to-use interventions can reduce the huge economic
of diabetic patients in this region because of ignorance, burden of diabetes. Many of these interventions are
poverty, poor health education, unawareness about cost effective and/or cost saving; even in developing
Diabetes control programme etc. A positive correlation countries.10 The measurement of glycated hemoglobin is
between fasting glucose, random glucose and glycated central to good-quality diabetes care. This is a measure
hemoglobin in cases indicates the exposure of glucose by which healthcare providers can relate blood glucose
correlate with the glycation which corroborate with the control to the risk of complications. The working
study done by Baral et al.8 group by International Diabetes Federation (IDF) was
Ion Exchange Chromatography HbA1c has lower established to develop a standard and harmonise HbA1c
isoelectric point and migrates faster than other reporting.11
Hemoglobin (Hb) components. The current ion exchange The conventional methods, reference ranges, diagnostic
assays correct for HbF and carbamylated Hb and does criteria, risk assessment pattern are updated regularly for
not interfere by them. In a Boronate Affinity HbA1c, increasing its quality and reliability for the diagnosis and
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management of patients with Diabetes mellitus. Better control and risk of macrovascular and microvascular
management plans based on the current evidence can complications in type 2 diabetes. UKPDS 38. BMJ 1998;
317: 703-13.
help significantly to reduce the long-term complications
6. Wei M, Gaskill SP, Haffner SM, Stern MP. Effects of diabetes
associated with type 2 diabetes. and level of glycemia on all-cause and cardiovascular
mortality: The San Antonio Heart Study. Diabetes Care
ACKNOWELEDGEMENT 1998; 21:1167–1172.
Our sincere thanks to all participants of this study and also
7. Duckworth W, Abraira C, Moritz T, Reda D, Emanuele
to Dr V.K. Pahwa, CEO and Dr. Anand Kumar, Principal, N, Reaven PD, et al. Glucose Control and Vascular
UCMS for their constant bolster to write this manuscript. Complications in Veterans with Type 2 Diabetes. N Engl J
The financial support for purchasing kit of Ion Exchange Med 2009; 360:129-39.
Chromatography was made by Universal College of Medical 8. Baral N, Koner BC, Karki P, Ramaprasad C, Lamsal M,
Sciences, Bhairahawa, Nepal. Koirala S. Evaluation of New WHO Diagnostic Criteria for
Disclosure: None Declared. Diabetes on the Prevalence of Abnormal Glucose Tolerance
in a Heterogeneous Nepali Population – The Implications of
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