HbA1c

Glycated proteins

• Monitoring long term glucose control

• Retrospective index of the integrated plasma glucose

• Is not subject to the wide fluctuations

• Adjunct to blood glucose determination

• not for the diagnosis of diabetes mellitus

Hemoglobin

• Human adult hemoglobin – HbA 97%of the total, HbA2 2.5%, HbF 0.5%

• Minor hemoglobins – HbA1a HbA1b, HbA1c collectively referred to

as HbA1 – fast hemoglobins

• Glycation – Nonenzymatic addition of a sugar residue to amino groups – Neoglycoprotein, Glycation

• HbA1a1; – fructose 1, 6 diphosphate

• HbA1a2– glucose 6 phosphate

• HbA1b– pyruvic acid

• HbA1C– glucose– major fraction 80% of HbA1

• Hb A0 – Glycation at lysine residues , or α chain – measured by affinity chromatography

• Blood levels of Glycated hemoglobin – Depends

• on the life span of red cells• the blood glucose concentration

Glycated Hb

• Free of day to day fluctuations • Unaffected by exercise or recent food ingestion • Recent glucose values provide larger

contributions to glycated Hb than earlier values.• The plasma glucose in the preceding one month

makes up 50% of the HbA1c whereas days 60-120 determine only 25%.

• blood glucose over the preceding 6-8 week

Interpretation of Glycated hemoglobin

• sources of errors – Low Glycated hemoglobin

• hemolytic disease • shortened red blood cell survival • recent blood loss

• High Glycated hemoglobin– Iron deficiency anemia – the effect of hemoglobin variants such as Hb

F, S and C

• Carbamylated Hb • Labile intermediates pre Hb A1C, Schiff

base • depends on the specific method of

analysis • Labile fraction

– changes rapidly with acute changes in blood glucose

– spuriously alter Glycated Hb values

• Pre-Hb A1c – amounts to 5-8% of total Hb A1 in normal

people – 8-30 % in patients with diabetes

• Glycated Hb should be routinely monitored at least every 3 month in all insulin treated patients

Clinical utility of Glycated Hb

• For glycemic control to decrease long term complications of diabetes mellitus

• To reduce the risk of retinopathy, nephropathies, and neuropathy

• to delay the onset and to slow the progression of these complications

• Study; a 10% lower Hb A1c was assocated with a 45% lower risk of retinopathy

• An index of long term blood glucose concentration in patients with diabetes mellitus

• The goal is blood glucose control

Methods for the determination of glycated hemoglobin

• selection of method – Including sample volume, patient population,

and cost

• most widely used technique– affinity chromatography In the United States

• methods based on charge

• Total glycated hemoglobin (A1+A0), HbA1 (HbA1a1+A1a2+A1b+A1c).

• In Europe – HPLC and ion-exchange with less use of

affinity chromatography

Ion exchange chromatography

• Hemoglobin variants are separated based on charge difference

• Bed– cation exchange resin (negatively charged)

• Procedure– hemolysis of the patient sample, a buffer is applied

and the eluent collected. – Elution

• The ionic strength and pH of the eluent buffer are selected so that glycated hemoglobins are less positively charged

Ion exchange chromatography

• A second buffer of different ionic strength to elute the more positively charged main Hb fraction – this is read as total Hb

• glycated Hb is expressed as percentage of total Hb

Ion exchange chromatography

• Modifications – Flow rates are accelerated by centrifugation – Batch technique

• agitation of resin with hemolysate to adsorb Hb A

– Using two different buffers to separate HbA1a+b from A1c

Factors affecting Ion exchange chromatography

• The temperature of the reagents and columns – thermostatting the columns – applying a correction factor

• Control of pH and ionic strength

• Sample storage condition

• different minicolumns exhibit wide variability in performance

Factors affecting Ion exchange chromatography

• The labile pre-Hb A1 fraction– produce elevated results

• HbF– elutes with HbA1 produce falsely elevated results

• Alteration of charge on Hb – carbamylated Hb, alcoholism,lead poisoning and

acetylated Hb

• HbS,HbC and their glycated derivatives; misleading low values for HbA1

HPLC

• The principle – Cation exchange chromatography

• Procedure – Application of hemolysate – Elution

• stepped elution – phosphate buffer of increasing ionic strength

– Detection • absorbance at 415 and 690 nm

HPLC

• Good resolution of Hb A1 a+b from HbA1c – with sodium phosphate-cyanide buffer at different pH

values

• Elution as one peak (HbA1)– A rapid system – evaluation

• with a dual wavelength detector at 405 for HbA1 and 546 for HbA

– Interference • Hb variants Hb C Hb F carbamylated and acetylated forms

of Hb

HPLC

• Quantification – Integrating the area under the peaks

• An automated system – Step gradients

• using three phosphate buffers of increasing ionic strength

– Detection• at 415 and 690nm

– both Hb A1c and HbA1 is reported – Variant Hb are resolved (Hb F, S and C)

HPLC

• HPLC methods – have excellent precision – recommended as reference method

• interference – Carbamylated and acetylated Hb and possibly

other derivatives• slightly higher results

Electrophoresis

• Agar gel at pH 6.3 resolution of Hb A and HbA1• The gel contains negatively charged moieties• Quantification performed by scanning densitometry at

415 nm• HbA1c is also commercially available • Results agree with that of HPLC or column but are less

precise• Minor variations in pH, ionic strength or temperature

have little effect on results • HbF migrates the same as HbA1and causes falsely

elevated value • Hb C and S do not • The labile form should be removed

Isoelectric focusing

• Principle; migration in gel containing a pH gradient

• Matrix; acrylamide gel • pH range of 6-8 • On completion the gels are fixed and then

scanned by a microdensitometer • Hb A1c resolved from HbA1a, A1b, S and F • Results showed close agreement with other

methods • The equipment is expensive

Immunoassay

• Anti serum raised against purified human HbA1c

• Available methods

• RIA format

• Enzyme immunoassay format

• Agglutination inhibition

Immunoassay

• Antibodies raised against the Amadori product of glucose (ketoamine linkage) plus the first few amino acids at the N-terminal of β-chain

• Agglutinator; a synthetic polymer containing multiple copies of the immunoreactive portion of HbA1c, light scattering

Immunoassay

• Excellent precision • The antibodies do not recognize labile

intermediates or other glycated hemoglobins • Other Hb variants such as HbF, A2, S,

carbamylated Hb are not detected.• Correlate well with HPLC but exhibit lower

values • Due to different calibration, detection by HPLC

of substances other than HbA1c

Affinity chromatography

• Principle– m-aminophenyl boronic acid is immobilized by cross

linking to beaded agarose or other matrix (e.g., glass fiber)

– The boronic acid react with the cis-diol groups of glucose

– Dissociation • By Sorbitol

– Detection • Absorbance of bound and non bound fractions measured at

415 nm

Affinity chromatography

• Advantage – No interference non glycated Hb – Negligible interference from the labile

intermediate form – Unaffected by variations in temperature – Reasonably good precision – Hemoglobin variants

• Hb F, S, and C produce little effect

Affinity chromatography

• Report – Affinity methods measure total glycated Hb – Commercially available systems are

calibrated to also report a HbA1c standardized value

• Specimen– Patients need not be fasting – Venous blood containing EDTA, oxalate, or

fluoride – Whole blood my be stored at 4°C for up to 1

week– storage at -20°C or 80°C is not

recommended

• heparinized samples– should be assayed within 2 days and may not

be suitable for other methods (electrophoresis)

• Preparation of hemolysate– Packed cell

• Centrifuge – remove the plasma and buffy coat

• Wash with saline

– Removal of labile glycated Hb • Incubation of RBC in saline • in buffer solutions at pH 5 to 6 • by dialysis or ultrafiltration of hemolysate

• Preparation of column– Bring the column to room temperature – Remove the caps – Pour off upper buffer – Add equilibration buffer let drain and discard

the eluate

Assay standardization

• The absence of a reference method and a single glycated Hb standard has generated confusion

• Interlaboratory comparisons are not possible

• calibration – significantly improves precision and facilitates

direct comparison of results obtained by different methods

Assay standardization

• Calibrator– lyophilized hemolysate assayed by a precise

HPLC method for Hb A1c

• adoption of a universal standard will enhance the clinical utility of glycated Hb

Reference interval

• Values for glycated Hb are expressed as a percentage of total Hb

• Three major glyacted Hb species – HbA1, HbA1c, or total glycated Hb

• Reference intervals vary depending on – method – the glycated Hb component – whether the labile fraction is included

Reference intervals

• Reference intervals show some increase with age

• poorly controlled diabetes – values may extent to twice the upper limit of the

reference interval

• Values grater than 20% should prompt further studies

• There is no specific level of HbA1c below which the risk of diabetic complications is eliminated completely

Reference intervals

• Each laboratory should establish its own nondiabetic reference interval

• Assay precision is important; each 1% change = 25-35 mg/dl change