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Laboratory Consultation in HbA1c Reporting:
Time to look beyond a number……
Dr Sutirtha Chakraborty, MD, FACB
Chief Consultant, Dept. of Biochemistry,
Peerless Hospital, Kolkata
Bio-Rad Hemoglobin Meet, 2016, New Delhi
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Hemoglobin
Definitions:
• Hb: hemoglobin
• HbA1: is a series of glycated variants resulting
from attachment of various carbohydrates to N
terminal valine of Hb
• Glycation results in increased negative charge and
hence runs fast on electrophoresis systems
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GHb: glycated hemoglobin
• HbA1a1: fructose 1,6 diphosphate N terminal
valine
• HbA1a2: glucose 6 phosphate N terminal valine
• HbA1b: unknown carbohydrate N terminal valine
• HbA1c: (60-80%): attachment of glucose to N
terminal amino acid valine of the beta chain of
hemoglobin
Beta N-1-deoxyfructosyl-hemoglobin
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Rahbar S. An abnormal Hemoglobin in
RBC’s of diabetics. Clin Chim Acta 1968
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DCCT & HbA1c
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UKPDS & HbA1c
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Major Principles of A1c assay
• Charge Difference:
- Ion Exchange HPLC, Capillary Electrophoresis
• Structural Difference:
- Affinity Chromatography, Immunoassay
• Chemical Difference:
- Enzymatic
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Standardization of HbA1c over 20 years
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Do all HPLC’s measure A1c equally well?
NO
Not in presence of common Hb Variants
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Impact of common Hb variants on A1c
Rohlfing et al. CCA 2016. 12
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Global Distribution of Hb Variants
• Hemoglobinopathies are common particularly in
countries with high prevalence of Diabetes.
• Hb S, C, E and D form the most common types of
hemoglobinopathy.
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Assumptions in clinical use of HbA1c
• Hemoglobin is present at a constant
concentration.
• RBC Life Span is Constant
• Microenvironment is constant.
Do we assume too much?
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• HbA1c is likely to be affected by iron deficiency
• IDA associated with a spurious increase in HbA1c
values; conversely, non-IDA may lead to a
decreased HbA1c.
• This might occasionally lead to confusion when
diagnosing diabetes using HbA1c.
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RBC Survival in Diabetic Vs Non Diabetics
Cohen RM et al. Blood 2008 16
RBC Lifespan of common Hb Variants
• Very minimal literature available on the impact of
Hb Variants on RBC lifespan.
• But there is evidence than RBC lifespan is shorten
in HbS, HbC and HbD.
• Are variant Hb exposed to the same amout of
glucose as a normal Hb?
Rhea JM et al, MLO 2012
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Impact on Microenvironment
Almost irreversible reaction
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Variation of A1c with Ethnicity
• A1c may differ between races despite similar blood glucose levels.
• The racial difference in A1c widens as the A1c increased.
• Diabetic complications also may differ between races with the
same A1c
• Need to determine whether A1c should be adjusted for ethnicity.
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Total influence of monthly glucose on A1c
MONTH Contribution
April 52%
March 26%
February 14.5%
January 6.5 %
If you were to do a HbA1c test on 30 th April this year ….?
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Conditions where A1c is unreliable
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Why A1c is not reliable in CKD
The relationship between CKD and A1c is complex:
Elevated A1c due to:
• -↓levels of erythropoietin
• -Potentially increased glycation
• -Higher levels of carbamylated Hb
• -Variable exposure to exogenous glucose through
dialysate
Lower A1c due to:
• -Shortened erythrocyte lifespan
• -Accelerated erythropoiesis upon erythropoietin
treatment.
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Hb E trait
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Where is the variant hemoglobin?
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Reporting A1c in heterozygous variants
• A1c mode or Dual Mode
• All variants need to be rerun in A2F mode.
• Hb D and Hb E variants need the Extended
programme on the D-10 for A1c estimation.
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• There is no standardized reporting format for
HbA1c that includes methodology, test
limitations or notification of variant
hemoglobins.
• Laboratories using IE-HPLC only 39% routinely
report the presence of hemoglobin variants,
and 10% report variants only if they cause
interference with the test.
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Interpretive Comments of HbA1c
Reports
Heterozygous Hb variants:
• Heterozygous Hb variant detected. The A1c
estimations is not analytically influenced in
presence of common variants like C, D, E, S
traits. Adviced Hb – HPLC for confirmation.
• If variant can be identified mention it.
• Note: A1c should not be used for diagnosis of
DM in presence of any variant Hb,
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What to do when A1c makes no sense
• The 2015 ADA Standards of Care state that
discrepancies between A1c and Lab Glucose
levels should warrant further exploration.
• Analytical & Clinical factors need considetation
• If A1c is unreliable, alternative strategies for
assessment of glycemic control should include
more frequent SMBG and CMG.
• Fructosamine, glycated albumin, and 1,5 AG.
• Limitations of Biomarkers: Lack of guidelines and
outcome data, poor standardization, No BRI/CDL.
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WHO : A1c in diagnosis of DM
• “HbA1c can be used as a diagnostic test for diabetes providing
that stringent quality assurance tests are in place and assays
are standardized to criteria aligned to the international
reference values, and there are no conditions present which
preclude its accurate measurement”.
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Should A1c assays which do not identify the presence of
heterozygous or homozygous Hb Variants be used to
diagnose Diabetes???????????????
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Contact:
Dr Sutirtha Chakraborty
Peerless Hospital, Kolkata
dr_sutirtha@yahoo.com
