HbA1c lecture The number of people with type 2 diabetes is likely to double during the next 20 years (1), leading to an increased burden of cardiovascular disease (2), renal failure (2), blindness (2), and risk of colon, breast, and other cancers (3). Diabetes profoundly alters macronutrient metabolism; the roles of diet and especially carbohydrate type and quality are therefore of considerable interest. Since 1970, the total availability of sugars has increased by ;20% (4), and high-fructose corn syrup now represents nearly 50% of caloric sweetener use in the United States (4,5). Increased total fructose consumption (from both sucrose and highfructose corn syrup) has been implicated in the development of the obesity epidemic in the United States (6) and has been singled out in diabetes guidelines because Diabetes associations (2,7,8) have taken a harm-reduction approach to fructose recommendations, setting an upper threshold for intake that is based on putative adverse effects on serum lipids. The American Diabetes Association guidelines, however, acknowledge that fructose produces a lower glycemic response in people with diabetes when it replaces sucrose and starch in the diet (7). Fructose has also been shown to improve glycemia without adversely affecting lipids when exchanged for other carbohydrate in controlled feeding trials in people with type 2 diabetes (9–15). In the absence of clear guidance on the role of fructose in glycemic control, we conducted a systematic review and meta-analysis of controlled feeding trials to assess the effects of isocaloric, oral fructose exchange for carbohydrates on fasting glucose, fasting insulin, and glycated blood proteins (glycated hemoglobin [HbA1c], glycated albumin, and fructosamine) in individuals with diabetes.

1. Created by Heidi Hanes, MT (ASCP) SH
Presented by Mark Swartz, MBA MT(ASCP)
Hemoglobin A1c-
The What…..
The Why…..
The How…..
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2. •The What?
•What is HbA1c?
•The Why?
•Why do you need to test for HbA1c?
•The How?
•How to validate new instrument/assay?
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3. Objectives
•Define HbA1c and usage in protocol testing.
•Distinguish between the different commonly used
HbA1c methods.
•Explain testing required for validation of new
instrument/assay.
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4. Hemoglobin A1c
• It is a specific glycated hemoglobin.
• Hemoglobin is modified at the N-terminal valine residue of each β
chain of Hb A.
• Essential component to both the diagnosis and management of
diabetes mellitus.
• Reflects average blood glucose concentration during the previous 8 to
12 weeks.
• HbA1c is directly related to risk for diabetic complications.
• Integral component of the standard of care for diabetic patients.
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5. PHOENIx is Rising
• The A5300/I2003/PHOENIx
• It is an international, multisite clinical trial.
• Protecting households on exposure to newly diagnosed MDR TB
patients.
• Comparing Delamanid instead Isoniazid for preventing confirmed or
probable active TB.
• Does high HbA1c make participants over age 15 more susceptible to
TB.
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6. HbA1c Methods
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7. Molecular Charge
• HPLC and Electrophoresis
• Separates glycated from non-glycated Hemoglobin
• Differences in charge – glycated has a decreased positive charge.
• Several fully automated systems
• Analysis time typically < 5 minutes
• CVs was 1.6-2.7%
• More common Hemoglobin variants usually detected.
• Disadvantages
• Implementing into laboratory can be demanding.
• Financial burden on equipment purchase.
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8. Molecular Structure
• Immunoassays
• Uses structural variations of Hemoglobin
• Antibodies are used that target N-terminal glycated amino acids on β chain
• Commercially available
• CVs between 1.6-6.1%
• Affordable
• Easy to operate
• Can be added to existing instruments.
• Disadvantages
• Interferences from variants Hemoglobin depends
• Where the antibody is targeted
• Whether a patient’s mutation is in the first few amino acids of the β-Chain
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9. Molecular Structure
• Boronate Affinity Chromatography
• Structural variations
• M-aminophenylboronic acid reacts with bound glucose.
• Commercial assays are available
• CVs between 2.1-3.1%
• Less interference from common Hemoglobin variants
• Disadvantages
• Cannot detect the presence of variants
• Affected by elevated Hemoglobin F above 10-15%
• Requires additional equipment
• Need for technical staff
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10. Examples of Instruments
• Boronate Affinity Method
• Trinity Biotech
• HPLC
• Sysmex BioRad Variant II Turbo
• Tosoh Bioscience
• Immunoassay
• Roche
• Abbott Architect
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11. Limitations HbA1c Testing
• Altered red blood cell lifespan
• The hemoglobin variants in patient population
• Chemically modified derivatives of hemoglobin
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12. Validation/Verification
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13. Validation Process
• Calibration
• Precision
• Accuracy/Correlation (Old vs New)
• Linearity
• Carryover (Should be provided by manufacturer)
• Software/IT (If connected to LIS)
• Specimen Stability Study (Time study)
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14. Precision
• The agreement of the measurements of replicate runs of the same
sample.
• 20 samples two levels
• Run between day and within day.
• Coefficient of variation (CV).
• CV =/< manufacturer’s specification or less than the TEa
• Total allowable error for HbA1c is 6%
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15. Precision
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16. Accuracy/Correlation
• True value of a substance being measured
• 20 samples, tested in duplicate
• Normal and abnormal population.
• Reference instrument/ known values
• Use patients, EQA or control samples
• Correlation coefficient should be > 0.975.
• Error Index (Observed result – Expected Result)/Total Error) = 1 to -1
(95%).
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17. 17
Accuracy / Correlation

18. Linearity
• Relationship between measured results and known concentration of
an analyte for a stated range.
• minimum of 5 samples
• Run in duplicate
• Equal distant from each other.
• Quality Control, Calibrators or Commercial Linearity
• Plotted immediately to identify and correct any outliers
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19. 19
Linearity

20. Carryover Studies
• Used to ensure no carryover from a high sample to next.
• Known high patient samples followed by known low patient samples
• If carryover detected must determine the analyte concentration
above which require a dilution.
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21. Reference Range
• Set of values occurring in a healthy non-diseased population.
• If adopting reference range must validate to study population
• 20 healthy, non-diseased individuals
• Acceptable if agreement is >90%.
• If outside performed another 20 samples.
• If outside need to analyze 120 samples to establish a new reference
range.
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22. 22
Reference Range

23. •Software/IT (If connected to LIS)
•Specimen Stability Study (Time
study)
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24. Questions?
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25. Acknowledgement
The presenter would like to thank the following:
DAIDS -Daniella Livnat and Joe Fitzgibbon
Johns Hopkins University - SMILE
Dr. Lori Sokoll - Principal Investigator
Anne Leach- Project Manager
Staff- Peggy Coulter, Mark Swartz, Orlinda Maforo, Amy Rada,
Afton Dorasamy, Mandana Godard, Arden Bongco, Eunhee Rim,
and Bre’Shey Jones
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26. References
• Sun Diagnostics Total allowable Error Limit Table
• Considerations in Choosing Hemoglobin A1C Methods by
Timothy Hanley, MD PhD and Healther Signorelli, DO, April 1
2015 Clinical Laboratory News
• College of American Pathologist
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