1. Hemoglobin A1c- The What….. The Why….. The How…..
Created by Heidi Hanes, MT (ASCP) SH
Presented by Mark Swartz, MBA MT(ASCP)

2. The What? The Why? The How? What is HbA1c?
Why do you need to test for HbA1c?
The How?
How to validate new instrument/assay?

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.

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.
The higher the A1c level the higher the average glucose level
Other names for it is glycohemoglobin and glycosylated hbg

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.
Comparing effectiveness and safety of 26 weeks of Delamanid with the current prophylactic regime of Isoniazid
One of the new safety test for this protocol is HbA1c.
The addition of HbA1c is to see if participants over age 15 with high HbA1c are more susceptible to TB

6. HbA1c Methods

7. Molecular Charge HPLC and Electrophoresis Disadvantages
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 %
More common Hemoglobin variants usually detected.
Disadvantages
Implementing into laboratory can be demanding.
Financial burden on equipment purchase.
Ion-exchange high performance liquid chromatography
According to 2014 CAP survey.
It requires operator skilled enough to be able to detect variant HB that can’t be separated.

8. Molecular Structure Immunoassays Disadvantages
Uses structural variations of Hemoglobin
Antibodies are used that target N-terminal glycated amino acids on β chain
Commercially available
CVs between %
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
It quantifies the A1c
According to 2014 CAP survey
Patients with Hb F above 10% will have a falsely low A1c
Need to know patient population to see if this will be a factor

9. Molecular Structure Boronate Affinity Chromatography Disadvantages
Structural variations
M-aminophenylboronic acid reacts with bound glucose.
Commercial assays are available
CVs between %
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
Detect and quantify Hb with glucose
Selectively holds the glycated hemoglobin on the column.
2014 CAP survey
Most POC instrument use this or the immunoassays methods. While POC instruments are good for immediate testing such as in an in-office or home use the American Diabetes Association concluded that they should not be used for diagnostic purposes.

10. Examples of Instruments
Boronate Affinity Method
Trinity Biotech
HPLC
Sysmex BioRad Variant II Turbo
Tosoh Bioscience
Immunoassay
Roche
Abbott Architect

11. Limitations HbA1c Testing
Altered red blood cell lifespan
The hemoglobin variants in patient population
Chemically modified derivatives of hemoglobin
If quick turn over of RBC won’t have the 2-3 month average. Less time for glucose to adhere to RBC
Be aware if there are Hb variants in population such as Hb s, Hb E, elevated Hb F,
Seen in patients with renal failure can affect accuracy

12. Validation/Verification

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)
I have included in your handout out standard templates for chemistry validation.

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%
Measures range of random error in terms of
National Glycohemoglobin Standardization Program.

15. Precision

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 >
Error Index (Observed result – Expected Result)/Total Error) = 1 to -1 (95%).
Should cover analytical measurement range of the test method
If using Error Index to be acceptable

17. Accuracy / Correlation
Chose samples at random for accuracy – represent normal and abnormal population. Should cover analytical measurement range of the test method
Linearity and reference Range

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
covering reportable range of the method
Values should be ideally Looking for a straight line when plotted

19. Linearity
Clinical reportable Range – allows for specimen dilutions.

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.
high concentration samples run immediately before low concentration samples causes falsely elevated results. If carryover detected laboratory must determine the analyte concentration above which samples may be affected. On Resources under the SMILE validation section there is a guide and worksheet to perform carryover.

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.

22. Reference Range

23. Software/IT (If connected to LIS)
Specimen Stability Study (Time study)

24. Questions?

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

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 Clinical Laboratory News
College of American Pathologist