Transcript Document

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 60120 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 Nterminal 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