Glycohemoglobin
Principle
Whole blood is mixed with a lysing reagent containing a
detergent and borate ions. Elimination of the labile Schiff’s
base is thus achieved during the hemolysis. The
hemolysate is the mixed
for 5 minutes with a weakly binding cation exchange resin.
During this time, HbA0 binds to the resin. After the mixing a
special resin separator is used toremove the resin from the
supernatant fluid which contains the HbA1. The
glycohemoglobin
percentage of total hemoglobin is determined by measuring
the absorbance of the glycohemoglobin and of the total
hemoglobin fraction at 415 nm. in comparison with a
standard
glycohemoglobin preparation carried through the test
procedure
Note: It’s possible measure the absorbances also at 405
nm but the sensitivity is lower of the 24%
Reagents composition
1. Ion exchange resin pH 7
Imidazole buffer
pH 7.6 ± 0.1
30 mmol/l
Borate
150 mmol/l
Thimerosal
0.1 g/l
2. Lysing reagent pH 7
Borate
1 mol/l
Detergents
0.25 %
Sodium Azide
0.065 %
3. Calibrator
Dried human hemoglobin* Concentration is stated on the
vial
4. Separator filters
20
According to the present laws the kit does not contain
substances classified as dangerous
Storage and Stability of Reagents
Store the Ion exchange resin and Lysing reagent at Room
temperature, Calibrator at 2 - 8° C
All the components are stable until the stated expiration
date if stored tighly closed and at correct temperature
Preparation and Stability of Working solution
Ion exchange resin liquid, ready to use and prefilled in
plastic tubes
Lysing reagent liquid and ready to use Calibrator
lyophilized
Reconstitute the Calibrator with 1 ml of distilled water and
allow to stand for 30 minutes
The reconstituted calibrator is stable for 30 days when
stored frozen at - 20° C
The Lysing reagent is stable 2 months after first opening
when stored at Room temperature
To guarantee a good riproducibility it is necessary, before
use, shake the Iron exchange resin
Bring reagents at Room Temperature before use
Precaution
BIOLOGICAL RISK
* Each unit of source material used in the preparation of the
Calibrator has been tested by a licensed method and found
non reactive for HbsAg and negative for Antibodies to HCV
and HIV 1/2.
However no known test can offer complete assurance that
products derived from human blood will not transmit
Hepatitis, AIDS or other infectious diseases.
This products, like all materials of human origin, should be
handled as potentially infectious biological material
Safety precautions
For in vitro diagnostic use
Do not pipette by mouth
Exercise the normal precautions required for handlig
laboratory reagents
Procedure
• Preparation of hemolysate •
Pipette into plastic tubes for Hemolysis:
Sample
Calibrator
Sample
100 µl
Calibrator
100 µl
Lysing reagent
500 µl
500 µl
Mix and incubate for 5 minutes at Room Temperature
Separation and determination of the Glycohemoglobin
Pipette into Ion exchange resin prefilled plastic tubes
Sample tube
Calibrator tube
Hemolysate (Sample)
100 µl
Hemolysate (Calibrator)
100 µl
Insert Resin separators so that the rubber sleeve is
approx.1 cm above the surface of the resin suspension. Mix
on a hematology mixer for 5 minutes. Push Separator filter
down until the resin is firmly packed. Transfer the
supernatant into cuvettes and read the Absorbance against
water
at Wavelength of 415 nm (A GLIC)
Absorbance should be read within 1 hour
Determination of Total Hemoglobin
Pipette into empty tubes
Sample tube
Calibrator tube
Hemolysate (Sample)
20 µl
Hemolysate (Calibrator)
20 µl
Distilled water
5ml
5ml
Mix carefully and read the Absorbance against water at
Wavelenght of 415 nm (A TOT)
Calculation
Determination of the Factor F by use of the Calibrator
A TOT Calibrator
F = ———————— x Calibrator Concentration (%)
A GLIC Calibrator
Calculation of Glycohemoglobin percentage in the sample:
A. GLIC Sample
GLYCOHEMOGLOBIN (HbA1) (%) = F x ———————
A. TOT Sample
Reference Values
Healty people and stabilised diabetics: 4.5 - 7%
Diabetics insufficiently controlled or with metabolic
imbalance: ≥ 8.5%
These values should only be used as a guideline.
Each laboratory should establish its Normal Reference
Range
Performance Characteristics
A. LINEARITY
T he reaction is linear between 4 and 24% of HbA1
B. INTRA - ASSAY PRECISION
Calibrator
Control
Sample
Mean (%)
6.9
13
7.2
SD
0.145
0.260
0.230
CV %
2.1
2.0
3.2
SD
0.076
0.403
0.446
CV %
1.1
3.1
6.2
C. INTER - ASSAY PRECISION
Calibrator
Control
Sample
Mean (%)
6.9
13
7.2
D. ACCURACY
Comparation between this method (y) and HPLC method
(x) gave the following results:
N = 26 r = 0.95 y = 0.870 x + 0.06
E. INTERFERENCE
High concentrations of Aldimin can influence the efficiency
of the Lysing reagent. In this case to extend the incubation
time up to 15 minutes ( See Procedure: Preparation of
Hemolysate)
Quality control
It is recommended to monitor the performance of assay
procedure with a commercial control
Bibliography
1. Gorka G.: Labor - Medizin 1, 30 - 31 (1985)
2. James T. M. et all: Clin. Biochem 14, 25 - 27 (1981)
3. Nuttall F. Q.: Diabetes Care 21, 1475 - 1480 (1998)
4. ISO 15223 Medical devices - Symbols to be used with medical
device labels, labelling and information to be supplied
For in vitro diagnostic use only.
The following symbols are used on labels
For in vitro diagnostic use
Use by (last day of the month)
Temperature limitation
Batch Code
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