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.
The Power of Technology and Collaboration in Research - Rheumatology Research...
A1C Presentation-10Jun19MS (1).pptx
1. Created by Heidi Hanes, MT (ASCP) SH
Presented by Mark Swartz, MBA MT(ASCP)
Hemoglobin A1c-
The What…..
The Why…..
The How…..
1
2. •The What?
•What is HbA1c?
•The Why?
•Why do you need to test for HbA1c?
•The How?
•How to validate new instrument/assay?
2
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.
3
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.
4
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.
5
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.
7
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
8
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
9
11. Limitations HbA1c Testing
• Altered red blood cell lifespan
• The hemoglobin variants in patient population
• Chemically modified derivatives of hemoglobin
11
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)
13
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%
14
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%).
16
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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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.
21
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
25
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
26
Editor's Notes
The higher the A1c level the higher the average glucose level
Other names for it is glycohemoglobin and glycosylated hbg
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
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.
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
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.
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
I have included in your handout out standard templates for chemistry validation.
Measures range of random error in terms of
National Glycohemoglobin Standardization Program.
Should cover analytical measurement range of the test method
If using Error Index to be acceptable
Chose samples at random for accuracy – represent normal and abnormal population. Should cover analytical measurement range of the test method
Linearity and reference Range
covering reportable range of the method
Values should be ideally Looking for a straight line when plotted
Clinical reportable Range – allows for specimen dilutions.
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.
