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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 Delhi1

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?

NONot 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.22

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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Homozygous Variants

• How do we report A1c in presence of homozygous Hb variants.

• What are the common clinical queries?

• Methodology of choice?

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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 ChakrabortyPeerless Hospital, Kolkatadr_sutirtha@yahoo.com