Suzanna Immanuel

Department of Clinical Pathology

FMUI / CMGH
Jakarta
 Clinical usefulness of HbA1c
 Variant Hb

 HbA1c methods

 The effect of variant Hb

 Comparison of HPLC Method with Imunoturbidimetric

Method for HbA1 determination
 Predict development of diabetic complications
(DCCT)
 HbA1c for diabetes diagnosis
 Gold standard in monitoring blood glucose control
 Used as treatment target in management of
diabetes
 Guide adjustment to therapy
 Macrovascular Microvascular
Brain
Cerebrovascular disease
Eye
• Transient ischemic attack
Retinopathy
• Cerebrovascular accident
Cataracts
• Cognitive impairment
Glaucoma

Heart
Coronary artery disease Kidney
• Coronary syndrome Nephropathy
• Myocardial infarction • Microalbuminuria
• Congestive heart • Gross albuminuria
failure • Kidney failure

Extremities
Peripheral vascular Nerves
disease Neuropathy
• Ulceration • Peripheral
• Gangrene • Autonomic
• Amputation
 15 Retinopathy
Nephropathy
13 Neuropathy
11 Microalbuminuria
Relative Risk

9
7
5
3
1
6 7 8 9 10 11 12
HbA1c (%)

Adapted from Skyler J. Endocrinol Metab Clin North Am. 1996;25:243

DCCT Research Group. N Engl J Med. 1993;329:977
 Risk reduction for each 1% reduction in
HbA1c in Type 2 diabetes
Amputation or
Any diabetes- death due to
related Micro vascular Myocardial Cataract Heart peripheral
endpoint complications infarction extraction failure
Risk reduction (%) associated with

vascular disease
0
-5
a 1% lower HbA1c

-10
14% 16%
-15 19%
21% *
-20
* *
-25 *
-30
-35 37%
43%
-40 *
*
*Epidemiological extrapolation showing benefit of a 1% reduction in mean HbA1c at 12 years

Stratton IM et al BMJ 2000; 321: 405–412.

 Clinical usefulness of HbA1c

 Variant Hb
 HbA1c methods

 The effect of variant Hb

 Comparison of HPLC Method with Imunoturbidimetric

Method for HbA1 determination
 Hb α β γ δ
F 2 2
A 2 2
Globin chain (%)

A2 2 2
H 4
Bart't 4

Hemoglobin is made of globin

chains such as α, β, γ and δ, and
they are called HbF, HbA, HbA2,
(Week)
HbH, HbBart’s, depending on the
Birth
Growth of solid and change in hemoglobin structure.
Also, as shown in the left figure,
expression volume of each
globin chain changes as it grows.
Especially in the fetus stage, HbF
has higher oxygen affinity than
HbA; therefore, oxygen can be
sent from the mother to the
fetus.
 Enzymatic condensation glucose processes in the N
terminal amino acid valine on the hemoglobin β chain

Formed Schiff base is unstable (aldimin, pre- HbA1c )

Transformed into a stable form ketoamine through

Amadori, formed HbA1c

Labile HbA1c

HbA1c
 HbA1c Molecule

COOH COOH

-chains

H2N H2N
COOH COOH

-chains

H2N H2N

HbA0 HbA1c
Hb A with glucose attached to the
N-terminal valine of the -chain
 About 50% of the HbA1c results from the blood glucose of
the preceding 30 days
 40% from the preceding 31 to 90 days
 Only 10% from the period between 91 and 120 days.

1-30 days
31 - 90 days
91-120 days
Hemoglobinopathy

Abnormalities synthesis or the

molecular structure of hemoglobin

Defects quantitative globin chains

Thalassemia characterized by decreased synthesis
of one or more globin chains

Hemoglobin Structural abnormalities of the

Variant globin chains
 Hb α β γ δ
F 2 2
A 2 2
A2 2 2
H 4
Bart't 4

β -chain Hemoglobin A is made of two α-chains and two β-

α -chain chains.
If this chain has some kind of genetic variance, it
will be called variant Hb. Theoretically, there are
more than 2,000 variant Hbs.
α-thalassemia is a status in which α-chain is difficult
to form, and β-thalassemia is a status in which β-
chain is difficult to form. These are both caused by
genetic consequences. When in a state of
thalassemia, normal red blood cells cannot be
formed. The major symptom will be anemia.
Typical variant Hb
HbS: Altered from β6Glu to Val (GAG-GTG)
HbC: Altered from β6Glu to Lys (GAG-AAG)
HbD: Altered from β121Glu to Gln (GAA-CAA)
 WORLD – Hb S is the most hemoglobin variant found,
followed by Hb E, Hb C, and Hb D.

Indonesia – Currently, Hb E is the most hemoglobin variant

found

• Thailand
Triangle
• Cambodia
of HbE
• Laos

• Vichinsky E. Hemoglobin E syndromes. Hematology 2007;:79–83.

• Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring Harb Lab Press 2012;:a011734.
 Hb S homozygote incidence 0.3 - 1.3%

Hb S trait incidence 8 - 10%

Highest prevalence in Africa

Substitution of Glutamic acid (polar)  Valine (non

polar) in the 6th chain of β globin amino acid
(β6(A3) Glu  Val)

• McKenzie S, Williams J. Clinical laboratory hematology. 2nd ed. New York: Pearson; 2010.
• Hoffbrand A., Moss PAH. Genetic disorders of haemoglobin. In: Essential hematology. Blackwell Publishing Ltd.; 2011.
page 89–105.
Red blood cells with HbS have a tendency of becoming sickle-cell red
cells. Also, when other Hb variants such as homozygous type (HbSS),
HbC (HbSC), HbD (HbSD), etc. exist, such symptom worsens.
The main clinical symptoms are thrombosis and hemolysis.
 Substitution of GAGglu --> AAGlys at the 26th kodon of β globin chain.
Formation of βE chain synthesis

Heterozygote / Hemoglobin electroforesa :

• Hb E fraction commonly varies 25-30%
HB AE • Hb E fraction >39%, suspicion for thalassemia
β–Hb E
• Hb E fraction <25%, almost always have
Hb E thalassemia α trait or iron deficiency

Hemoglobin electroforesa :
Homozygote / • Hb E is the majority of hemoglobin fraction,
HB EE percentage of Hb E + Hb A2 : 85-99%.
• Hb F fraction < 15%
• Vichinsky E. Hemoglobin E syndromes. Hematology 2007;:79–83.
• Fucharoen S, Weatherall DJ. The hemoglobin E thalassemias. Cold Spring Harb Lab Press 2012;:a011734.
 HA-8180V
HbEβ26Glu→Lys

HbE
If β-thalassemia and HbE co-exist,
the symptom will be critical.
HbE was detected with HA-8180V.
 Substitution of glutamic amino acid with lysine at
the 6th position of A3 globin β helix chain
(β6[A3]GluLys )

Glutamic acid Lysine • Lowers the solubility of

(POLAR) (NON-POLAR) hemoglobin
• Changes in Hemoglobin
charge

Laudicina RJ. Hemoglobinopathies: qualitative defects. In: McKenzie SB, Williams JL, editors. Clinical Laboratory
Hematology. 2nd ed. New York: Pearson; 2010. p. 211-30.
 HA-8180V

HbC β6Glu→Lys

HbC
(*map from 1993)
Substitution of glutamic amino acid with glycine at the 121th
position of the GH4 globin β helix chain. (β121[GH4]GluGln)
 HbD homozygote
 HbD heterozygote

Clinical Symptoms:
 Heterozygote : Asymptomatic
 Homozygote : mild hemolytic anemia, target cells ↑ and
osmotic fragility ↓.
 Interaction between HbD – HbS molecule  deoxyHb
aggregation  mild sickle cell anemia
 HA-8180V

HbD β121Glu→Gln

HbD
 Clinical usefulness of HbA1c

 Variant Hb

 HbA1c methods
 The effect of variant Hb

 Comparison of HPLC Method with Imunoturbidimetric

Method for HbA1 determination
 HbA1c methodologies

Separation Chemical

Differences
charge

HPLC Ion Capillary Affinity Immuno Enzymatic

Exchange Electrophoresis Chromatography assays assays

IFCC Standardization
Ann Lab Med 2013;33:393-400. http://dx.doi.org/10.3343/alm.2013.33.6.393
 HbA1c Examination
HPLC – ION EXCHANGE
RBC lysate injected through
the negative charged column.

Positive charged hemoglobin

molecule moves slower
because of interaction with
the resine
HbA1c have more negative
charge compared to HbA0 
HbA1c is eluted first compared
to HbA0.
Hemoglobin molecule moves
out of the column, processed
by signal detector to become
chromatogram.
HbA1c Examination

HPLC – Boronic Affinity

RBC lysate was injected into the
column containing m-aminophenyl
boronic acid bound to the resin,
with a pH of 8.0

Glycated hemoglobin binds to the

resin, while not glycated hemoglobin
will pass through the column

Acid Buffer will break the bonds

of glycated hemoglobin in the
column.

Hemoglobin molecules out of the

column, processed by signal
detector into a chromatogram
The Capillarys® system
uses the principle of
capillary electrophoresis
in free solution, charged
molecules are separated
by their electrophoretic This interaction leads to electro-
mobility in an alkaline Positives charges
from buffer solution osmotic flow (EOF) from the
buffer with a specific pH. anode to the cathode

Negatives charges Electro-osmotic flow carries all

from capillary wall proteins towards the cathodic side

 Separation occurs according to the electrolyte pH and electro-osmotic flow, from

cathode to anode.
 Hemoglobins were separated within 4 minutes, according to isoelectric point, by
migration in an electric field in the liquid phase. Direct detection of the
hemoglobin is made at an absorbance wavelength of 415 nm at their cathodic end
Immunoassay Method
 Common examination method
for HbA1c
 Antibody recognizes glycated
amino acid structures at the N
terminal of the β globin chain.
 4th through 10th amino acid of
the β globin chain.
 Other hemoglobin variants that
have mutation on the 4th
through 10th amino acid can
interferes with the examination.
 HbA1c Examination
ENZIMATIC

RBC lysate are subjected to proteolytic

process (A)
Releases amino acid, including N terminal
end of valine amino acid of the beta globin
chain (B)

Valine amino acid goes through the de-

glycated process, results H2O2, that reacts
and used in chromagenic reaction is used to
calculate HbA 1c (colours) (c)
Formed colours is equals to number of HbA1c
 Clinical usefulness of HbA1c

 Variant Hb

 HbA1c methods

 The effect of variant Hb

 Comparison of HPLC Method with Imunoturbidimetric
Method for HbA1 determination
Changes in molecule charge
In Variant Hb, there is substitution of amino acid that influenced
the Hb charge.
Substitution at the 26th kodon of the beta globin chain, GAG 
Hb E AAG, results in substitution of glutamic acid with lysine.
Substitution at the 121st codon of the beta globin chain, results
Hb D in substitution of glutamic acid with glutamine.

Changes in hemoglobin molecule charge

HB E and HB D co-eluted with HbA1c in

the ion exchange examination method False
Co-Elustion of Variant Hb

Area at the peak of Asymetric peak,

HbA1c curve cannot with shouldering
be measured, and the of the right side.
HbA1c percentage
cannot be measured.

CHROMATOGRAM OF DM PATIENT WITH HB AE

Substitution of amino acid at the glycated area
Substitution of glutamic acid by valine at the 6th
Hb S
amino acid of β globin chain.
Substitution of glutamic acid by glycine at the 6th
Hb D
amino acid of β globin chain.

Changes in amino acid composition of Hemoglobin

No Bonding of Anti-HbA1c (Immunoassay method)

False low value

 Hb F does not have valine amino
acid at the end of globin chain, so
glucose cannot bond with Hb F

False value of HbA1c in most examination

except in boronic affinity method.
 Effect IFCC Effect NGSP
Potential interferent
units, mmol/mol Units, %
Labile-HbA1c (13.8%) <1 < 0.1
Carbamylated
<1 < 0.1
haemoglobin (3%)
Hematocrit (3–14 mmol/L) <1 < 0.1
Icteric plasma (bilirubin
<1 < 0.1
268 mmol/L)

Clin Chem Lab Med 2011;49(4):647–651 2011 by Walter de Gruyter, Berlin. New York. DOI 10.1515/CCLM.2011.096
Journal of Clinical Laboratory Analysis 2014;28:428–434
Journal of Clinical Laboratory Analysis 2014;28:428–434
 HbA1c assay
HPLC method
HbS trait, HbC trait, HbE trait, and HbD trait

INTERFERENCE
Differences in retention time between HbA1c and hemoglobin variant

National Glycohemoglobin Standardization Program. HbA1c methods :effects of hemoglobin variants (HbC, HbS, HbE, and
HbD traits) and elevated fetal hemoglobin (HbF). [diakses: 26 Juli 2016]; diunduh dari: URL: http://www.ngsp.org/interf.asp
 Clinical usefulness of HbA1c

 Variant Hb

 HbA1c methods

 The effect of variant Hb

 Comparison of HPLC Method with
Imunoturbidimetric Method for HbA1
determination
Suzanna Immanuel, Novi Yanti
Department of Clinical Pathology
FMUI / CMGH
Jakarta
 Ion Exchange Chromatography
Immunoassay

Most

ADAMS TM A1cHA-8180V
(Arkray)
HPLC
Precision Cobas c501 Accuracy
(Roche)
Immunoturbidimetric

Bland-Altman
Sacks DB. Diabetes Mellitus. In: Burtis CA, Ashwood ER, Bruns DE, editors. Tietz textbook of clinical chemistry and molecular diagnostics. 5th ed.
Missouri: Elsevier Saunders; 2012.p. 1415-56
 Cross Sectional

120 K3EDTA whole blood for HbA1c assay

at Clinical Pathology Laboratory from June-July 2016

HbA1c Sample
<6,5 60
6,5 – 8,0 30
>8,0 30

Sample is measured using two methods

• Perkeni. Konsensus pengelolaan dan pencegahan diabetes melitus tipe 2 di Indonesia. 2011.
• NCCLS. Method comparison and bias estimation using patient samples; approved guidelines. 2nd edition. NCCLS document EP9-A2 (ISBN 1-
56238-472-4). NCCLS, Pennsylvania. 2002.
 HPLC Method
Spreading the hemoglobin fraction based on interaction from ionic
strength of hemoglobin fraction with material in the column

High pressure  Buffer with programing Hemoglobin separation

through sample gradient  increase the based on ionic strength
in column ionic strength in column (retention time)

Absorbance is measured by λ 420 nm Flow cell

Chromatogram
Service training HA-8180V. Arkray infinity Inc.2011
Leaflet Automatic Glycohemoglobin Analyzer ADAMSTM A1c HA-8180V. Arkray Inc. 2011
 Y : Scale

S–A1c

S–A1c
X : time retention

(a) (b)

Chomatogram on normal HbA1c (a) and diabetic HbA1c (b)

 NGSP CV within-run : < 2%
CV between day :< 3.5%
WITHIN RUN
CV ADAMSTM A1c-8180V Cobas c501
Normal control 0% 1,15 %
Abnormal control 0% 0,41 %
BETWEEN DAY
CV ADAMSTM A1c-8180V Cobas c501
Normal control 0,93 % 1,34 %
Abnormal control 1,11 % 1,02 %
•NCCLS. Evaluation of Precision Performance of Quantitative Measurement Methods; Approved Guideline;Second Edition.
NCCLS document EP5-A2 (ISBN 1-56238-542-9). NCCLS. Pennsylvania. 2004.
•Miedema K. Standardization of HbA1c and optimal range of monitoring. Scand J Clin Lab Invest 2005; 65 (Suppl 240):61-72.
 ADAMSTM A1c HA-8180 and Cobas c501
meet the manufactory ranges

Normal control Abnormal control

ADAMSTM : 5,1 – 6,1 % ADAMSTM : 9,5 – 11,3 %
c501 : 4,73 – 6,83 % C501 : 8,7 – 12,3 %

•Leaflet A1c-3. Roche Diagnostics. Manheim;2014. p1-8

•Leaflet ExtendSURETM Haemoglobin A1c Lyophilised controls. Canterbury Scientific. New Zealand; 2014
 Mean difference of HbA1c assay between
ADAMSTM A1c HA-8180V and Cobas c501

abnormal distribution

ADAMSTM A1c HA-8180V (HPLC)

median 6,40 % (4,10 % - 14,00 %) No difference
Cobas c501 (Imunoturbidimetri)
median 6,45 % (4,10 % - 13,90 %)
 Kolmogorov
-Smirnov Uji Wilcoxon

0,000 0,057

No difference
Abnormal
distribution
• Alcala HG, Arguelles AR, Carvallo BC. Effect of the method to measure level of
glycated hemoglobin on individual clinical decisions; comparison of an immunoassay
with high performance liquid chromatography. Am J Clin Pathol 2009;132:332-335.
• Cully M, Burns G, Engel WD, dkk. Homogenous immunoturbidimetric determination
of hemoglobin A1c. Recent Prog Clin Chem 1992
 r= 0,995 p<0,0000

Very
strong
correlation

Spearman correlation of HbA1c assay between HPLC

method and immunoturbidimetry method
 96,7% data is in limits of agreement

Mean

Limits of
agrrement
-3,6 until
3,9

HbA1c assay HPLC method and imunotubidimetric

method in Bland-Altman Plot
 This may have an effect of variant Hb on HbA1c
measurement depending on the method that is
performed
 HbA1C method must be standardized to NGSP or IFCC
method
 HbA1c is related to good glycemic control & decrease
risks of complications DM.
 ADAMSTM HA-8180V & Cobas C501 for HbA1c
determination:
 Showed good precision & accuracy
 Showed a very strong correlation
 Can be used to diagnose & monitoring therapy for DM