Clinical Application

Contents
1 The Basics about HbA1c

Some different methodology in

2 Laboratory HbA1c Test

3 Theory of Ion-exchange HPLC

4 How to judge the chromatogram

The Basics about HbA1c
HbA1c-Glycated Material of Hemoglobin

Glucose moves into RBC, and react with hemoglobin inside

Erythrocyte
Glucose

HbA1c
 Biological Process of HbA1c Formation

intermediate material-
Hemoglobin Glucose LA1c HbA1c
Schiff Bases

Step 1 ： Fast, forming Liable A1c （ LA1c ）

Step 2 ： Slow, forming stable A1c （ HbA1c ）
 Hemoglobin constituent

Hb Hemoglobin

HbA HbA2 HbF

(ßß) () ()

95% 1-3.5% <2%

Non-glycated Hb
HbA0 HbA1 Glycated Hb

80-89% 6-8%

HbA1a HbA1b HbA1c

Fetal Hemoglobin is high in Fetus blood and newborn baby in 6 months. After 6 months, it decrease to
normal adult level
HbA1c is mainly applied for screening of gestational diabetes in Obstetrics & Gynecology Dept
 Separation Peaks by Ion Exchange
Chromatography （ normal sample ）

Hb % of Hb Combination of Hb and Glucide

HbA1a <1% HbA1 and 1,6 Fructose Diphosphate

HbA1b <1% Pyranic acid and amino acids at β chain terminal

HbF <1% Fetal Hemoglobin

LA1c <1% Intermediate material-Schiff Bases

HbA1c 4~6% Glucose and amino acids at β chain terminal

HbA0 <90% Non glycated hemoglobin

Some different methodology in Laboratory HbA1c
Test
HbA1c Assay Methods

3 Major Methods:

1. Ion Exchange Chromatography(HPLC)

Separate HbA1c

2. Borate Affinity Chromatography(HPLC)

Test total glycated hemoglobin

3. Immunoassay
Test HbA1c
Precision of Different Methods

Method Precision
Immunoassay/Borate affinity ++
LPLC +++
HPLC +++++
 Assay Objects of Different Methods

HbA (a2b2)

HbA1 HbA0
Non-glycated part
HbA1a HbA1c
HbA1b
Immunoassay and LPLC HPLC
Borate Affinity
 Ion Exchange Chromatography HPLC

Advantages ：
1. Fully Auto
2. Accurate, reliable
Analyte is HbA1c
High resolution to separate common Hb variants, providing
additional useful medical information
3. High Precision
CV<3%
Disadvantages ：
Require particular analyzer
High reagent cost
（ Operator should know how to distinguish abnormal
chromatographs ）
 Borate Affinity Chromatography

• Advantage ：
Separate glycated and non-glycated hemoglobin only, avoid
interference from variants

• Disadvantages ：
HbA1c is not tested directly.
Will be affected by high HbF
Homozygous Hb variant patient(like HbSS), it still just reports
HbA1c, will not provide relative information
 Immunoassay

• Antigen-antibody reaction
• Measuring turbidity or fluorescence signal, to calculate the ratio of
HbA1c

• It needs manual pretreatment steps

• Not good precision （ CV%>5% ）
• Only report a HbA1c number result, unable to provide
variant information
• Will be affected by high HbF
• Theory of Ion-exchange HPLC
 Ion Exchange Chromatography HPLC

• Golden Standard in HbA1c test

The only method used in DCCT Research
Reference method in NGSP standardization

• Principle ：
HbA1c carries different charges with other hemoglobins, it will
bind in varying degrees with ionogenic group on the surface
of chromatography column, in certain ionic strength buffer.
Therefore, HbA1c will be separated from other
elements(HbA1b, HbA1a, HbF and HbA0)
 Calculation in different model of H9
Calculated Formula ：

Variant
Mode:
 HPLC

HPLC:
High-Performance Liquid Chromatography
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Absorbing ability 　 Strong

HP Pump Absorbing ability 　 Weak

Sampling
Valve Ready to transfer mixture from
mixing cup to sample loop.
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
HP Pump

Valve

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
HP Pump
Sampling
Valve Inject mixture into column.

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Absorbing ability 　
HP Pump Strong
Absorbing ability 　 Weak

Valve Seperation
Elution of mixture inside column.
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Absorbing ability 　
HP Pump Strong
Absorbing ability 　 Weak

Valve

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Absorbing ability 　
HP Pump Strong
Absorbing ability 　 Weak

Valve

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Absorbing ability 　
HP Pump Strong
Absorbing ability 　 Weak

Valve

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Absorbing ability 　
HP Pump Strong
Absorbing ability 　 Weak

Valve

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
HP Pump

Valve

Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
HP Pump

Detector
Valve All the hemologbin will be
detected inside the test
Eluent module.
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Output

HP Pump Absorbance

Valve

Eluent Time
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Output

HP Pump Absorbance

Valve
Time
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Output
Absorbance
HP Pump

Valve
Time
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve Output
Absorbance
HP Pump

Valve
Time
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
HP Pump Absorbance

Valve
Time
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
HP Pump Absorbance

Valve
Time
Eluent
 HPLC Simulated Diagram

Column Test Module

Sample

6-Way
Valve
Absorbance
HP Pump

Valve
Time
Eluent
How to judge the chromatogram
 chromatogram
The signal-time curve of the response signal generated by the chromatographic column
effluent through the detector, the longitudinal coordinates of which are the signal strength,
and the horizontal coordinates are the time

Sample separation diagram

06/16/2023
Calculate
Method:
1.Base line no drift
2. peak sharp
3. Normal
distribution curve
4. Accurately fit
the HbA1c peak
5.Separation
thoroughly
Chromatograph

• A0

• HbA1c

• LA1c (Schiff Bases ) and

Aminomethyl- acetylization Hb

• HbA1a & HbA1b

•Total Area 4-20

• A1c Result
Normal Graph
 04 Chromatogram standard

Parameter Point
1 Pressure Refer the information of label

2 ADC In range or not (415nm 24000-26000;

500nm 16000-18000 forH9)
3 Peak Retention time
4 Total area IN range or not
5 Calibrator Factor(K) offset(b) (K:0.8~1.3 ， B:-
2~+3 ）

6 QC In 1 SD range In range or not

06/16/2023
 05 Relation
Chromatogram

Test Stepping Liquid flow

module RT(S)
value rate(mL/min)
Tem
RT Press
perat Variant 115 1.56±0.02 55±2
ure
ure

Environ
Column Liquid
ment principle ：
temperat flow
tempera Determine the flow rate, do not
ure rate change, you can adjust the temperature,
ture simply said is the fixed flow rate,
adjustment temperature

25℃ 25- Step

approximately 40℃ value

06/16/2023
 Non Method-specific Interferences

Physiological Conditions that lead to change of RBC

lifespan
False Low
Sickle Cell Anemia
Hemolytic Anemia
Vitamin C and E high(inhibit Glycation progress)
False High
Iron Deficiency Anemia
Post Splenectomy Surgery
Polycythemia Vera, Plateau Region Patient
Gestation Period
Hb is lower, so HbA1c is lower than normal adult
 Method-specific Interferences

– Hb Variants(HbS, HbC, HbD, HbE)

– Hb Derivatives(acetylization, formylation)
Patients taking Aspirin in long term, have high
acetylization-Hb, results will be higher
Renal failure patient

– Schiff Bases High

Fast-develop Type 1 DM patients
– HbF
– Severe Jaundice, Hyperlipemia
 Hb Variant Prevalence

There finds more than 1000 kinds of Hb variants all over

the world
Latest data bout Hb variant incidence rate is 7% globally*

• African American ： HbS 7.8% ； HbC 2.3%

• Prevalence of HbE may be 30% in Southeast Asia
• About one in 100 Hispanic Americans/Latinos has sickle
cell trait * *

*Kohne E. Hemoglobinopathies: clinical manifestations, diagnosis, and treatment. 1. Dtsch Arztebl Int. 2011;108(31-32):532-540.
** Bry L, Chen PC, Sacks DB. Effects of hemoglobin variants and chemically modified derivatives on assays
for glycohemoglobin. Clinical Chemistry. 2001;47(2):153–163.
 Hb Variant Prevalence

The four types of variation of hemoglobin in the world are:

HbE 、 HbS 、 HbC 、 HbD.
 Hb Variants Affect A1c Result

Lynn Bry, Philip C. Chen, and David B. Sacks. Effects of Hemoglobin Variants and Chemically Modified Derivatives on Assays
for
Glycohemoglobin Clinical Chemistry,, 153–163 , 2001
 Hb Variants Affect A1c Result

Example ：
• Variant peak after
A0 peak
• HbF normal
• G8 Fast Mode was
1% lower

G8 Fast Mode
G8 Variant Mode
 Hb Variant and Blood Sugar Monitoring

• Hb variants affect RBC lifespan, then affect the

glycation progress furtherly

• These patients might have different clinical index to

evaluate blood sugar

Kohne E. Hemoglobinopathies: clinical manifestations, diagnosis, and

treatment. 1. Dtsch Arztebl Int. 2011;108(31-32):532-540
 Suggestion on Methodology

1. Method should be traceable to IFCC reference

method, or NGSP certified(required to renew every
year)

2. Laboratory should know all possible interferences of

the method
 HbA1c
HbA1
Total
GHB

Before DCCT and UKPD trial, there were 800 laboratories testing HbA1c in
United States, CAP survey indicated that there was huge difference in
different laboratories, using varying methods
 Standardization of HbA1c Test

• Since 1996, the United States began National Glycohemoglobin

Standardization Program(NGSP)

• the Central Primary Reference Laboratory uses Ion Exchange

Chromatography HPLC

• The NGSP consists of a Steering Committee and a network of

reference laboratories, including the Central Primary Reference
Laboratory(CPRL), Primary Reference Laboratory(PRL) and Secondary
Reference Laboratory(SRL)
 www.ngsp.org

Purpose:

1. Standardize HbA1c test results to DCCT

2. DCCT established the direct relationships between HbA1c levels

and outcome risks in patients with diabetes
 NGSP Flow Chart

NGSP Network

1 2 3

Calibration Certification Proficiency Testing

(Precision & Bias)
Fresh Blood Fresh Blood
Fresh Blood

Manufacturer

Routine Clinical Lab

 NGSP Contribution to HbA1c Test
Chaos to Order
CAP Survey: Mean +/- 2sd
22.0
21.0 1993 1999 2001 2008
20.0
HbA1c

19.0 HbA1
18.0
Total
17.0
GHB
16.0
%GHB

15.0
DCCT
Target
14.0
13.0
12.0
11.0
10.0
9.0
8.0
7.0
NGSP Certified Not Certified NGSP Certified Not Certified NGSP Certified Not Certified

According to survey in 2008, CV among 2000 CAP laboratories which were using different
methods was in 1.3~7.1%. It indicated that HbA1c results had been standardized effectively
 IFCC Standardization of HbA1c

the IFCC established a working group on HbA1c

standardization in 1995

 Reference methods: HPLC-MS(mass spectroscopy ) or

HPLC-CE(capillary electrophoresis)
 Purified HbA1c and HbA0 as calibrators, 6 levels
 Internal CV0.47 ～ 2.07% ； external CV1.35 ～ 2.27%
Difference between NGSP/IFCC Reference Methods

NGSP IFCC Difference

%HbA1c %HbA1c

4 2.1 1.9
5 3.2 1.8 Non-Diabetes
6 4.3 1.7
7 5.4 1.6 Control
8 6.4 1.6
9 7.5 1.5
10 8.6 1.4 Treatment needed
11 9.7 1.3
12 10.7 1.3
 IFCC and Other Reference Systems

JDS/JSCC% = 0.927*IFCC% + 1.73

NGSP% = 0.915 * IFCC% + 2.15
Sweden% = 0.989 * IFCC% + 0.88

IFCC NGSP JDS

%HbA1c %HbA1c %HbA1c
2.0 4 3.6
4.3 6 5.6
6.4 8 7.6
8.6 10 9.6
10.8 12 11.6
 2010 Consensus Statement on the Worldwide
Standardization of HbA1c

1. HbA1c test results should be standardized worldwide, including the reference system and
results reporting.
2. The IFCC reference system for HbA1c represents the only valid anchor to implement
standardization of the measurement.
3. HbA1c results are to be reported by clinical laboratories worldwide in SI (Système
International) units (mmol/mol) and derived NGSP units (%), using the IFCC-NGSP
master equation (DCCT units).
4. HbA1c conversion tables including both SI (IFCC) and NGSP units should be easily
accessible to the diabetes community
5. Editors of journals and other printed material are strongly recommended to require that
submitted manuscripts report HbA1c in both SI (IFCC) and NGSP/DCCT units.
6. The reportable term for glycated hemoglobin is HbA1c, although other abbreviations may
be used in guidelines and educational material (A1C).

2010 in Annals of Clinical Biochemistry, Clinical Chemistry, Clinical Chemistry and Laboratory Medicine,
Diabetes Care, Diabetes Research and Clinical Practice, Diabetologia, Diabetic Medicine and Pediatric
Diabetes.
 NGSP/IFCC Conversion Table
NGSP = [0.09148 * IFCC] + 2.152
HbA1c Clinical Use and the Standardization

HbA1c
1993
Blood Sugar Monitoring

Standardization

Diabetes Screening/Diagnosis
2010
 Requirements for Assaying System

1. Report: Report HbA1c

2. Precision: Internal CV<3%, better less than 2% ；

3. Accuracy: Reference range  0.5%HbA1c

4. Accuracy Verification:
1 ） Participate in external quality assessment
2 ） Test traceable standard material ；
3 ） Compare results to certified laboratory

Note: No direct evidences proves that other parameters(e.g. Total

Hemoglobin, Fructosamine, Glycosylated Serum Protein) relate to
outcome risks in patients with diabetes
 Inaccurate HbA1c affect clinical judgment

Different Internal CV
（ CI 95% ）
Why HbA1c should
be accurate? CV % 6.0% A1C 6.5% A1C 7.0% A1C
2 5.76-6.24 6.24 - 6.76 6.72 - 7.28
Normal level is closed
to Medical Decision 3 5.64-6.36 6.11 - 6.89 6.58 - 7.42
Level
5 5.4-6.6 5.85 – 7.15 6.3 – 7.7
HbA1c is a diagnosis
index of DM
（ only internal CV is No difference No difference
<3%, can external CV between normal between good
be <5% ） and diabetes blood sugar
control and bad
 Abnormal Result Handling

 HbA1c sample, results less than 4% or above

than 15%, should be retested, and operator
need communicating with the clinicians
Lifotronic H9/H8 Linearity Range 3.0%-18%

 Further tests are needed when results were

not matched with clinical symptoms
 Result Report

1. HbA1c results are to be reported by clinical

laboratories in NGSP units (%), providing IFCC
unit at same time(mmol/mol)
2. If the clinicians need, the method should be
mentioned on report