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HbA1c by

Capillary
Electrophoresis:
The Next
Generation of
Separation
Method
HbA1c & Diabetes Mellitus

HbA1c is a measure of hemoglobin glycation, and since


red blood cell have about a 120 day life span, HbA1c
reflects mean glycemia for the previous 4 months

Thus, HbA1c is a widely used biomarker in the


management of diabetes because it provides
information on the monitoring of long-term glycaemic
control and an assessment of the risk of developing
complications.
What is
HbA1c?
HbA1c: What are we looking for?

Hb A > 96,5% Hb F < 1% Hb A2 < 3,5%


α1 β1 α1 γ1 α1 δ1

β2 α2 γ2 α2 δ2 α2
HbA1c: What are we looking for?
Hb A
Hb A0
93-95%
Glycation at the
N-terminal Valin
of the β-globin chain
Hb A1 = GHb
Glycated Hbs
5-7%
Hb A1a Hb A1b Hb A1c
0,5% 0,5% 4-6%
Fructose-1,6-diphosphate pyruvate glucose
Hb A2Glucose-6-phosphate

Hb F
+ + +
1st Step: Unstable, reversible reaction between Glucose and the N-terminal valine of the β-chain (Schiff base)
2nd Step: During red blood cell circulation, some of the labile A1C is converted to form a stable HbA1c (Amadori
rearrangement)
HbA1c by
capillary
electrophoresis
Hb A0 Hb E, Hb D
HPLC
Long elution time
Just keep the essential

P4
Clear-cut separation
?
Hb A1c
A1a
A1b Unknown P3
? ?
? Hb F Unknown ?
? LA1c
? ?
%HbA1c =
100 x [HbA1c area / (HbA1c area + HbA0 area)]

CE uses the same calculation


formula as the IFCC Reference
Methods
Analytical
Performances of
the CE assay
• Precision

• Trueness

• Linearity
Poor trueness Poor trueness
Poor precision
Not accurate Good precision
Not accurate

Good trueness Good trueness


Poor precision Not accurate Good precision Accurate
For internal use only - Not for customers
distribution
« Only 1 method (labeled N) achieves the
minimum performance level of the BV model »

For internal use only - Not for customers


distribution
For internal use only - Not for customer
distribution
TOSOH

BIO-RAD

ROCHE

SEBIA

For internal use only - Not for customer


distribution
For internal use only-Not for customer
17
distribution
CAPILLARYS

For internal use only-Not for customer


distribution
Robustness of
the CE assay
• Labile A1c

• Carbamylated Hb

• Fœtal Hb
Analytical Interferences

Labile HbA1c

Incubation at 37°C with 0.5 mol/L Glucose (min)


0 10 20 30 60 90
Level 1
Labile HbA1c (%) * 0.8 3.1 4.5 5.7 9.3 10.4

HbA1c (mmol/mol) 34 34 33 33 33 34
Profile not modified
Level 2
Labile HbA1c (%) 1.5 3.1 4.7 6.2 9.1 10.5

HbA1c (mmol/mol) 60 59 60 59 58 58

Level 3
Labile HbA1c (%) 1.5 3.2 4.6 5.9 8.4 10.3

HbA1c (mmol/mol) 99 100 100 97 96 99


* Value estimated using HPLC method (Variant II - BioRad)

 No interference of labile HbA1c


HbA1c Labile study:
mix a normal sample with glucose solution

Variant II Tosoh G8
Journées Commerciales 4 - 8 Juillet 2011
Analytical Interferences

Carbamylated Hb

Incubation at 37°C for 3 h with KCNO (mmol/L)


0 0.12 0.25 0.5 0.75 1
Level 1
cHb (%) * 0.8 1.5 2.4 3.9 5.4 6.5

HbA1c (mmol/mol) 32 33 32 32 33 33

Level 2 Profile not modified


cHb (%) 0.9 1.6 2.0 3.8 4.7 5.3

HbA1c (mmol/mol) 53 53 51 53 51 52

Level 3
cHb (%) 2.0 2.6 3.4 5.4 6.5 8.1

HbA1c (mmol/mol) 87 86 85 85 85 87
* Value estimated using HPLC method (Variant II - BioRad)

 No interference of carbamylated Hb
Carbamylated Hb study:
mix a normal sample with KCNO solution

Variant II Tosoh G8
Journées Commerciales 4 - 8 Juillet 2011
*Values determined
by CAPILLARYS 2
Hb method (n=3)

No interference for the integration of HbA1c and HbA0


Behavior of the capillary
electrophoresis method
in the presence of Hb disorders
THALASSEMIA IN VIET NAM

Prof. Nguyen Anh Tri


Vietnam National Institute of Hematology and Blood Transfusion
Vietnam Thalassemia Association

26
Epidemiology of haemoglobinopathies in Vietnam

There hasn’t been a national epidemiology research yet.

From separate study in last 5 years:


Alpha thalassemia: 1.7% - 28%
Βeta thalassemia : 0.3% - 15.9%
HbE gene: 0.4% - 35.6 %

Total prevalence: about 10% -> 10 million carriers

Estimate 2000 major thalassemia new born per year

; 27/10
Capillary Electrophoresis
Resolution matters! (9 min migration time)
Latest generation HPLC
(45 sec elution time)

HPLC HbA1c program


(1 min 30 sec elution time)

1’ 2’ 3’ 4’ 5’ 6’ 7’ 8’ 9’
Homozygous Hb E

No HbA = No HbA1c
HbA2 separated and
quantified

Alarm (!) if %HbA2 is above 3%


Why is it crucial to detect Hb disorders
when testing HbA1c?
Impact of hemoglobin disorders on HbA1c:
analytical or biological interference?

• Analytical interference:
HbA1c value obtained is not the correct value because the analytical
method failed to accurately measure HbA1c:
INCORRECT MEASUREMENT
• Biological interference:
HbA1c value obtained is the correct value but its interpretation is
misleading because of physiopathological conditions that alter the
RBC lifespan:
MISINTERPRETATION
Analytical Interference:
the result is wrong
Analytical interference:
the instrument is not measuring the right analyte,
it should measure HbA1c, and not the Hb Variant

Ion-exchange
Chromatography

Sample Heterozygous Hb Hope


Biological interference:
the result is correct but the
interpretation of this result is
misleading
HbA1c is a measure
of hemoglobin glycation,
and since red blood cell
has about a 120 day life span,
HbA1c reflects mean glycemia
for the previous 3 to 4 months

When clinicians interpret an HbA1c result,


they always assume
that the red cell lifespan is normal
The Pros and the Cons of using HbA1c for Diabetes Diagnosis
David B.Sacks; AACC Webinar April 10th 2012
Average lifespan of RBCs from hematologically normal or
Hb trait individuals

Impact of hemoglobin variants on Hb A1c interpretation: Do we assume too much?


Jeanne M. Rhea, PhD, Tiffany K. Roberts-Wilson, PhD, and Ross J. Molinaro, PhD, MT
MLO - June 2012
Red cell life span in heterozygotes for Beta-thalassemia ranges
from 81 to 93 days
Patients who are heterozygous for a
hemoglobin disorder are usually
asymptomatic

When these patients are tested for HbA1c


the clinicians are most of the time unaware
there is a physiopathological condition
that might lead to biological interference:

result can be mis-interpreted


Any method used for HbA1c testing should:

• be free of analytical interference : report an accurate


HbA1c value in the presence of common hemoglobin
disorders

• detect the biological interference: screening of the


common hemoglobin disorders, including both variants
and thalassemia
In the presence of Hemoglobin disorders

• The HbA1c target values usually chosen for diagnosis


purpose or for therapeutic decision may not be
applicable

HbA1c is not a reliable biomarker for the diagnosis of


diabetes mellitus
Immunoassay
All these
Enzymatic methods do not
detect the
variants
Boronate Affinity
• Immunoassays and
affinity chromatography
methods provided a
reportable Hb A1c value
without warning that
the patients do not have
any Hb A
• Red Cell Lifespan for
sickle cell patient is only
10 to 20 days: HbA1c
values obtained are
clinically misleading and
do not reflect glycemic
c
d
- Immunoassay using 1st generation antibodies
- Boronate chromatography
status of the patient
e – Immunoassay using 2nd generation antibodies
Boronate Ion-exchange Capillary
immunoassays
affinity HPLC Electrophoresis

Robustness Evolution of the Methods

Separation
Thanks for your attention