Diabetes Flashcards
Forms of glycated HbA1
- HbA1a1 - fructose-diphosphate to N-terminal valine of beta-chains
- HbA1a2 - glucose-6-phosphate to N-terminal valine of beta-chains
- HbA1b - pyruvic acid linked to N-terminal valine of beta-chain
- HbA1c - glucose to N-terminal valine of beta chains, and stabilised by Amadori reaction
What is the major fraction of HbA1?
HbA1c (80%)
Advantages of HbA1c over glucose to diagnose diabetes
Nonfasting
Very low biological variability
Stable sample
Prognostic - microvascular cx
Advantages and disadvantages of HbA1c measurement by immunoassay and HPLC
Immunoassay misses HbF, is slower, may be artefactually lowered by hypertriglyceridaemia. HPLC was used in the DCCT trial and therefore HbA1c from HPLC measurements has prognostic implications. Immunoassay is more specific and therefore not likely to be interefered with by labile HbA1c.
methods for HbA1c
Separate glycated from non-glycated Hb by charge (cation exchange, electrophoresis) or structure (affinity, IA).
- Cation exchange chromatography
- Affinity chromatography
- Immunoassay
- Capillary electrophoresis
- Enzymatic
Your lab’s method for HbA1c
Cation exchange chromatography
Anticoagulated blood diluted with a haemolysis reagent containing borate.
Hb a cation, attracted to solid phase (negatively charged resin)
3 phosphate buffers of increasing ionic strength
Absorbance measured at 415nm and 690nm
Quantification by integrating area under peaks
Advantages and disadvantages of HbA1c by cation exchange chromatography
No intereference from labile fraction or carbamylated Hb but may be prone to interference from Hb variants that co-elute with peaks of interest.
The method of HbA1c measurement in the DCCT trial
Affinity chromatography for HbA1c
m-aminophenyl boronic acid
Specific interaction between glucose on glycated Hb and immobilised boronic acid
haemolysate applied to affinity colum and GHb containing coplanar cis-diol groups interacts strongly with boronic acid immobilised on agarose gel
Non-glycated Hb elutes directly off column with first buffer
After elution of non-glycated fraction, bound Hb is dissociated by use of counter-ligand eg sorbitol (competes with glycated Hb for boronic acid sites)
Absorbance measured at 414nm and ratio determined
Detects all GHbs but calibrated to report a standardised HbA1c value.
Immunoassay method for HbA1c
Antibody targetd against Amadori product + first 4-8 aas at N terminal end of beta chain of Hb.
Variable assay design - immunoturbidimetry to latex-enhanced competittive immunoturbidimetry and enzymatic detection.
Latex agglutination process:
agglutinator (synthetic polymer containing multiple copies of immunoreactive portion of HbA1c binds anti-HbA1c mAb attached to latex beads. HbA1c in pt sample competes for antibody on latex, inhibiting agglutination and decreasing light scattering.
Advantages and disadvantages of immunoassay for HbA1c
Not affected by problems related to electrical change
Good for automation
But, non-linear calibration requires multilevel calibration, limited stability of reagent total Hb measured separately on a different method, introducing further analytical uncertainty
Capillary electrophoresis
liquid-flow capillary electrophoresis in free solution
charged mcules separated by electrophoretic mobility in alkaline buffer with specific pH 9.4
separation also dependent on electrolyte pH and electroosmotic flow
Optical detector made of deuterium lamp and optical fibres at cathodic end at wavelength of 414nm. (Absorption spectroscopy)
Advantages of capillary electrophoresis for HbA1c
high resolving ability (due to high voltage that can be applied)
small sample volume
Interferences in HBA1c measurement
method dependent.
1. variant Hb in HPLC methods
2. icterus increases HbA1c in charge separation techniques (bili migrates with fast Hb and absorbs at detecting wavelength)
3. hyperlipidaemia causes false increase as lipids elute in first HbA1c fraction and absorb at 415nm - method specific
4. Acetylation by aspirin on both alph ana beta chains of HbA. Acetylation confers a negative charge resulting in modified electrophoretic and cation exchange chromatographic properties. degree of in vivo interference unclear
5. Carbamylation by urea in renal failure - method specific
When should HbA1c NOT be used for the diagnosis of diabetes
- Type 1 diabetes
- short duration diabetes symptoms
- Child/young person < 18 yo
- high risk of diabetes but acutely ill
- taking meds that may cause rapid glucose rise eg corticosteroids, anti-psychotics
- acute pancreatic damage/pancreatic surgery
HbA1c assay standardisation
NGSP formed by AACC to calibrate all method results to DCCT-equivalent values
Improved harmonisation of results and reduced imprecision
Results obtained using NGSP-certified assays can be compared directly with results of DCCT and UKPDS, allowing alignment with clinical outcomes data.
Different approach adopted by IFCC working group developed a primary reference material and two candidate reference methods. HbA1c results obtained using IFCC reference methods are 1.5-2% lower than NGSP results, possibly becasue of the detection of glycated components other than HBA1c by HPLC (DCCT method)
