HbA1c

(everything you wanted to know,

but were afraid to ask)
Graham Jones
Department of Chemical Pathology
St Vincent’s Hospital, Sydney
 HbA1c – An Update
• What is HbA1c
• Units / Standardisation
• Current Uses
• Diagnosis of DM
• Assays
• Conclusions
 Declarations
• Meeting travel support from BioRad
• Research and meeting travel support from
Roche
(Neither in the area of HbA1c)

Mention of any product does not imply

endorsement or criticism.
 Relevant Expertice
• Member: ADS, RCPA, AACB working
party on HbA1c for diagnosis
• Member: ADS, RCPA, ADEA, AACB
working party on HbA1c reporting
• Member: RCPA QAP HbA1c Whole Blood
program advisory committee

• Opinions expressed are my own

 Arthur Kornberg

“I never met a dull enzyme”

Nobel Prize in Physiology or Medicine 1959

"the mechanisms in the biological synthesis of DNA"
 Maria del Pilar Ospina, PhD

Sebia Electrophoresis Workshop

07-26-2011
AACC Annual Meeting

National Center for Environmental Health

Division of Laboratory Sciences
 Hb A > 96% Hb F < 1% Hb A2 < 3%
α1 β1 α1 γ1 α1 δ1

β2 α2 γ2 α2 δ2 α2
Hb A Hb A0
93-95%

Hb A1 = GHb
Fast hemoglobins
Glycated Hemoglobins
5-7%
Hb A1a1 Hb A1b Hb A1c
HbA1a2 pyruvate glucose
Fructose-1,6-diphosphate
Hb A2
Glucose-6-phosphate
Hb F
+ + +
 Definition of the Analyte
IFCC: HbA1c is defined as hemoglobin
molecules having a stable adduct of glucose
to the N-terminal valine of the hemoglobin β
chain
(βN-1-deoxyfructosyl-hemoglobin)
(nearly known as “DOF Haemoglobin”)
 G G
   
 
    N G  
N N
G G G
G  
N
  G
N
G

All of these are HbA1c

 G
  G
G G   N
G
 
    N
G
 
N   G
G   G
N

All of these are Glycated Haemoglobin

(but are not HbA1c)
 Definition of the Analyte
IFCC: HbA1c is defined as hemoglobin
molecules having a stable adduct of glucose
to the N-terminal valine of the hemoglobin β
chain
(βN-1-deoxyfructosyl-hemoglobin)
(nearly known as “DOF Haemoglobin”)

“HbA1c” is a ratio of HbA1c : (HbAo + HbA1c)

Both measurements are important

Units reflect ratio (mmol/mol)
 HbA1c
• Results = HbA1c / (HbAo + HbA1c)
• In the presence (normally) of:
– HbA1a+1b, HbA1a2, HbA1b
– Other glycated HbAs
– HbF, HbA2
– Other glycated HbF, HbA2
– Labile HbA1c, Carbamylated Hb
• Abnormal haemoglobins
– Glycated abnormal haemoglobins
Hb A Hb A0
93-95%

Hb A1 = GHb
Fast hemoglobins
Glycated Hemoglobins
5-7%
Hb A1a1 Hb A1b Hb A1c
HbA1a2 pyruvate glucose
Fructose-1,6-diphosphate
Hb A2
Glucose-6-phosphate
Hb F
+ + +
 What we actually measure:
Immunoassay:
HbA1c + HbA21c S1c + E1c etc / total Hb
Boronate affinity:
total glycated Hb / total Hb
HPLC:
HbA1c (+X) / (HbAo + Y + HbA1c + X)

• Methods highly correlated

• Calibration reduces differences
• Different interferences in different assays
2010 Consensus Statement on the Worldwide
Standardization of HbA1c
 2399 -2400

Diabetologica. Volume 50, Number 10, October 2007, 2042-

2043(2)
Collaborative project:
Clinicians, Educators, Pathologists, Scientists
Christchurch Diabetes Centre (Courtesy Chris Florkowski)
 HbA1c Language
• 40s – excellent diabetic control
• 50s – very good diabetic control
• 60s – seek possible improvement
• 70s – improvement needed
• 80s+ - very poor control

• Further promotion necessary

 HbA1c re-standardisation
• Most current assays measure ~ HbA1c
– Immunoassay, HPLC, CE
– Assay calibration (in NGSP %) adjusts result
to look like we are measuring Dowex 70
results: HbA1c, HbA1a1, HbA1a2, HbA1b,
other
• % units are providing a result for
something we used to call HbA1c
 Mathematical Oddity #1

The case of the missing numbers!

mmol/mol NGSP
Measure in IFCC, convert to NGSP
40 5.8
41 5.9
42 6
% = (mmol/mol+23.5)/10.93
43 6.1
44 6.2
45 6.3
46 6.4
47
48
6.5
6.5
* 2 x 6.5%
49
* 6.6 (47 and 48 both
50 6.7 become 6.5)
51 6.8
52 6.9
53 7
54 7.1 Also occurs at: 5.4 (35 & 36)
55 7.2 7.5 (58,59), 8.6 (69,70)
56 7.3
% NGSP mmol/mol Measure in NGSP, convert to IFCC
6 42
6.1 43
6.2 44 mmol/mol = 10.93 x % - 23.5
6.3 45
6.4 46
6.5 48 “missing” 47 mmol/mol
6.6 49
6.7 50
6.8 51
6.9 52
7 53
7.1 54
7.2 55 Also missing:
7.3 56 35, 59, 72 and 83 mmol/mol
7.4 57
7.5 58
7.6 60
 RCPA QAP Whole Blood Program
NGSP 1-01 1-02
IFCC 1-01 1-02
Analyser Number Median Median
Abbott Architect c8000 1 5.9 10.6 Analyser Number Median Median
Arkray 7 5.7 10.4 Abbott Architect c8000 1 41.0 92.0
Axis-Shield Afinion 6 5.9 10.1
Arkray 1 39.0 90.0
Beckman Coulter DxC 600 1 5.9 10.5
Beckman Coulter AU400 1 6.0 10.2 Axis-Shield Afinion 3 39.9 84.0
BIO-RAD in2it 1 6.0 9.0 Beckman Coulter DxC 600 1 41.0 91.0
BIO-RAD Variant II 6 6.2 10.7
Beckman Coulter AU400 1 42.0 88.0
BIO-RAD D-10 8 6.1 10.5
BIO-RAD Variant II Turbo 12 6.2 10.6 BIO-RAD in2it 1 42.0 75.0
Primus CLC385 1 5.9 9.6 BIO-RAD Variant II 4 43.0 92.5
Primus PDQ 1 5.8 9.7
BIO-RAD D-10 6 43.0 90.2
Primus Ultra 2 1 5.9 9.8
Roche Cobas c501/c502 2 5.7 10.5 BIO-RAD Variant II Turbo 8 44.0 92.0
Roche Cobas Integra 400 3 6.0 10.3 Primus CLC385 1 41.0 81.0
Roche Cobas Integra 700/800 10 5.9 10.3
Primus Ultra 2 2 43.0 85.0
Sebia Capillary 1 6.1 10.3
Siemens Advia 1650/1800 2 5.8 9.9 Roche Cobas Integra 700/800 9 41.0 91.0
Siemens DCA 2000 18 5.9 10.1 Roche Cobas c501/c502 2 39.5 86.0
Siemens DCA Vantage 7 5.7 10.0
Sebia Capillary 1 43.0 89.0
Siemens Dimensions 2 5.9 10.4
Siemens Xpand 1 5.7 10.6 Siemens DCA 2000 4 40.0 85.5
Tosoh HLC-723GHb G7 1 6.3 10.9 Siemens DCA Vantage 6 39.0 85.5
Tosoh 1 6.1 10.8
Total and Overall Median 53 41.0 89.0
Total and Overall Median 111 5.9 10.3
Target SKML 5.7 10.3 Target SKML 39 89

RCPA Chemical Pathology Quality Assurance Programs – Used with permission

 +/- 6.4% (CV 3.2%)

RCPA QAP – 2012 – Used with permission

 Within-method Precision (CV%)

NGSP IFCC
(%) mmol/mol
Afinion 1.4% 2.5%
Integra 1.4% 2.9%
Variant II 1.9% 3.3%
D10 1.8% 5.5%

Overall 3.7% 5.4%

RCPA QAP – 2012 – Used with permission

 Mathematical Oddity #2

The case of the unequal CVs!

 10.0

9.0
Mean = 7.64 SDs: 2.7 / 3 = 0.9
SD = 2.7
CVs: 3.6 / 5 = 0.72
HbA1c (NGSP Standardisation, %)

CV = 3.6%
8.0

7.0
Mean = 4.9
6.0 SD = 1.4
CV = 2.8%
5.0

4.0

3.0 SDs: 1.4 / 1.5 = 0.9 Mean = 60

SD = 3
CVs: 2.8 / 5 = 0.56 CV = 5%
2.0
Mean = 30
1.0 SD = 1.5
CV = 5%
0.0
0 10 20 30 40 50 60 70 80
HbA1c (IFCC standardisation, mmol/mol)
 2.4 120

2.2

2.0 100

1.8

HbA1c (IFCC, mmol/mol)

CV (IFCC) / CV (NGSP)

80
1.5

1.3
60
1.1

0.9
40
0.7

0.4 20
0.2

0.0 0
3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 12.0
HbA1c (NGSP, %)
 Current Role of HbA1c
• Managing patients with diagnosed
diabetes
Jones et al
Diagnosis

April 2011
 HbA1c for Diagnosis
• Current Diagnostic Criteria
• Why might HbA1c be better?
• (or worse)
• Possible implementation
• Making it happen
 Current Diagnostic Criteria
• Based on WHO/IDF 2006 Criteria
• Within setting of:
– Screening
– Testing
– Follow-up
• Plasma glucose is key parameter
NHMRC 2009
AUSDRisk ≥12  laboratory testing
 Medicare Schedule of Benefits
• 66551 Quantitation of glycosylated haemoglobin
performed in the management of established diabetes -

Fee: $16.90 Benefit: 75% = $12.70 85% = $14.40

• Glycosylated Haemoglobin - (Item 66551) The

requirement of "established diabetes" in this item may be
satisfied by:
(a) a statement of the diagnosis by the ordering practitioner
on the current request form or on a previous request form;
(b) two or more blood glucose levels that are in the diabetic
range; or
(c) an oral glucose tolerance test result that is in the
diabetic range.
 WHO

WHO 2011
WHO
WHO
 Less Assay Variation
• HbA1c assay improvement the product of
years of work
– NGSP
– IFCC
– CAP
– RCPA QAP
• Manufacturers
• Laboratories
 HbA1c Variability
• Same sample to different laboratories

• Results +/- 7% (NGSP)

• Eg “real value” 7.0% Mx 6.5 to 7.5

• Eg “real value” 53 mmol/mol Mx 43 to 53
(mostly closer than this)
 Compared to Glucose…
• Between lab CV for HbA1c: ~ 3.5%
(95% within +/- 7%)

• Between lab CV for glucose: ~ 3.5%

(95% within +/- 7%)

• No advantage for either

• Whole blood Quality Assurance Program
started 2012
 Pre-analytical variability
• Red cells are alive when collected
• Red cells are hungry
• Glucose goes down

• Approx 0.3 mmol/L

in 1st hour
(even in grey top tube)
• (adds +/- 0.15 mmol/L
to result uncertainty)

• HbA1c – very stable

 Variability in patient
• The concentration of all things in blood
changes over time.
• “within-subject biological variation”
• CVi (Coefficient of Variation in an individual)
 Within patient variation
• CVi Fasting Glucose: ~ 6%
(95% within +/- 12%, eg 7.0 mmol/L: 6.2 – 7.8)

• CVi 2 hour Glucose: >13%

(95% within +/- 26%, eg 11.0 mmol/L: 8.2 – 13.8)

• CVi HbA1c: 2 – 3%

• HbA1c lower variation in patients

 Total Result Variation
• Combining patient, sample and assay
variations:

• Fasting plasma glucose Result CV : 6.7%

• OGTT 2 hr Result CV: 13.4%
• HbA1c Result CV: 4.7%

HbA1c has lower total result variation

than fasting or 2 hour glucose
Total Result Variability and Diagnosis

Decision point

High CVi Low CVi

 Pathophysiology
• “Better marker of long term Glycaemic
exposure”

• “ … using HbA1c, a measure of chronic

glycaemia, for the diagnosis of diabetes, a
disease of chronic glycaemia.
– Shaw 2011
WHO 2011
HbA1c similar to Fasting and 2 hour glucose
for predicting diabetic complications
 Sampling
No need for fasting sample

• Convenience

• Compliance

• Not affected by intercurrent illness

Currently used for management
• If OK for management …

• Should be OK for diagnosis

• Also,

• Using for diagnosis starts management

 HbA1c for Diagnosis
• Current glucose-based criteria are from
WHO

• Using HbA1c for diagnosis keeps Australia

in line with WHO recommendations
• But
WHO 2011
 Cautions
• Limitations in HbA1c results
– Factors in the patient
– Factors in the assays
– Combination of the above
• Exclusions
– Not for pregnancy
– Not for rapid onset or children (type 1)
 Iron Deficiency (Patient)
• Non-diabetic iron deficient patients: HbA1c ave
7.4%
Reduced to 6.2% with 3 months iron replacement
Iron replete patients had ave HbA1c of 5.7%.
(Coban 2004).
• Reduction of HbA1c from 6.1 to 5.2% after
treatment of iron deficiency anaemia (microcytosis).
(El Agouza 2002)
• Note iron deficiency anaemia.
 Iron Deficiency (patient)
• Iron deficiency is important (Incr HbA1c)
• Not method dependent
• Mechanism unclear

• How hard do we look for it?

• How severe does it have to be?
 Kidney Disease (Patient)
• RBC half-life for normal subjects was 23.5 days,
haemodialysis ~ 15 days (Ly et al).
• Patients with stage 4 and 5 CKD (+/- dialysis)
showed a poorer relationship between HbA1c and
average glucose than in normal subjects (Vos
2012)
• Comment: HbA1c appears to be unsuitable in
dialysis patients and less useful in stage 4 and 5
patients. Fructosamine or glycated albumin may
be useful.
 Other (Patient)
Vitamin B12 deficiency and treatment
• Vitamin B12 deficiency increases HbA1c.
• Treatment causes a decrease in HbA1c by
increasing red cell production and thus lowering
the average red cell life-span at the time of
testing.
Anaemia of Chronic disease
• Associated with reduced red cell survival due to
actions of TNF alpha damaging red cells and
leading to phagocytosis.
 Other (Patient)
Aspirin, Vitamin C, Vitamin E
Appear on the WHO list
No interference at usual doses

Intracellular pH
Appears on the WHO list
?
 Assay Interferences
• Haemoglobinopathies
– Some assays not affected
– Some assays affected but can be seen
– Some assays affected and not seen
– Hb S, C, D E trait - use appropriate assays
• Eg Haemoglobin F and Immunoassay
– Less Beta chain (replaced by delta chain)
– Less HbA1c
– Same amount of Hb
– Lower HbA1c relative to total Hb
– SILENT in patient and assay!
 So...
Opinion:
• Most patients likely to be OK for HbA1c
measurement
• Interferences: few outliers in method
comparison studies
• RBC factors: range of patients in trials
likely to include common factors
• But: some patients affected significantly
 Implementation
• Need to turn current (limited) knowledge of
limitations into action plans

• Which patients to be cautious with

• Which assays
• Other protocols
 Implementation
• “HbA1c may be used for diagnosis”
• Glucose testing still valid
• Advice on conditions where glucose is
preferred:
• Eg: “HbA1c not recommended in iron
deficiency anaemia, stage 4 or 5 CKD,
severe intercurrent illness, B12 deficiency,
high likelihood of haemoglobinopathy
(type)…”
 Some concerns
• Errors found if glucose and HbA1c do not
“match”
• Diagnosis: no set of glucose results
what is “matching”

• Can we use fructosamine?

What are action limits
Correlation with HbA1c is poor

WHO 2011
Fructosamine
 WHO criteria
• HbA1c ≥6.4% (48 mmol/L) diagnostic
• Should be repeated
• No “pre-diabetic” state
(ADA has HbA1c 5.7 to 6.4% as a risk
factor)

• NZ: using 50 mmol/mol for diagnosis

40 – 50 mmol/mol as pre-diabetic
NZSSD Position Statement on the diagnosis of, and
screening for, Type 2 Diabetes Updated: September 2011
 In Australia
• Working Party of ADS, RCPA, AACB
– Jonathon Shaw
– Stephen Coliguri
– Michael D’Emden
– Wah Chung
– Peter Coleman
– Stephen Twigg
– Graham Jones (RCPA)
– Hans Schneider (RCPA)
– Ian Goodall (AACB)
 Hba1c for diagnosis - Planning
• Planning to recommend HbA1c for
diagnosis
• Based on WHO criteria
• Seeking Medicare Funding
• 6 Step process
• Up to step 3
• Will take at least until march 2013
 Conclusions
• HbA1c / (HbAo + HbA1c) is an important
and interesting test
• HbA1c is likely to come into routine use for
diagnosis of DM.
• Laboratories are important for:
– Quality of analysis and reporting
– Advice on requesting and interpreting
• A large learning and educational
component is required for correct use