Experiment (8):
Estimation of Glycosylated hemoglobin
The A1C test, also known as an HbA1C, hemoglobin A1C, glycated hemoglobin,
or glycosylated hemoglobin test (GHb), is a blood test that shows the average
blood sugar levels for the past two to three months. It is a broader test
than conventional glucose test, which measures the blood sugar at the time. The
A1C test is used to diagnose and monitor diabetes and the efficacy of therapy.
Hemoglobin A, a protein found inside red blood cells, carries oxygen throughout
the body. When there is glucose in the bloodstream, it can stick (glycate) to
hemoglobin A non-enzymatically. The more glucose that is in the blood, the
more it does this, creating a higher percentage of glycated hemoglobin proteins.
Once glucose sticks to a hemoglobin protein, it typically remains there for the
lifespan of the hemoglobin A protein (as long as 120 days).
Kit Contents:
- ion Exchange Resin (Predispensed Tubes)
- Lysing Reagent
- Control (10% GHb)
- Resin Separators
Reagent Preparation:
-The ion Exchange Resin tubes & the Lysing Reagent are ready to use.
-Reconstitute the Control with 1 ml of distilled water. Allow to stand for 10
mins. with occasional mixing. The reconstituted control is stable for at least 7
days when stored at 2-8 °C tightly sealed, and at least 4 weeks when stored at
-20°C. Do not thaw and refreeze.
Sample material:
Whole blood. Preferably fresh & collected in EDTA.
GHb in whole blood is reported to be stable for one week at 2-8°C
Principle:
(Colorimetric, Ion Exchange Resin method) for quantitative determination of Glycosylated
hemoglobin in blood.
After preparing the hemolysate of whole blood, hemoglobins are retained by a cationic
exchange resin leaving Hemoglobin A1C free in the supernatant. Hemoglobin A1C is
then specifically eluted after using a filter separator to remove the resin from the
supernatant, and is quantified by direct photometric reading at 415nm. The percent
glycosylated hemoglobin is determined by measuring absorbances of the glycosylated
hemoglobin (GHb) fraction & the total hemoglobin (THb) fraction. The ratio of the
absorbances of the Glycosylated hemoglobin & the Total hemoglobin fraction of the
Control and the Test is used to calculate the percent Glycosylated hemoglobin of the
sample.
Procedure:
A. Hemolysate Preparation
1. Dispense 0.5 ml Lysing Reagent into tubes labelled as Control (C) & Test (T).
2. Add 0.1 ml of the reconstituted control & well mixed blood sample into the appropriately
labelled tubes. Mix until complete lysis is evident.
3. Allow to stand for 5 minutes.
B. Glycosylated hemoglobin (GHb) Separation
1. Remove cap from the ion-Exchange Resin tubes and label as Control & Test.
2. Add 0.1 ml of the hemolysate from Step A into the appropriately labeled ion Exchange
Resin tubes.
3. Insert a resin Separator into each tube so that the rubber sleeve is approximately 1 cm
above the liquid level of the resin suspension.
4. Mix the tubes on a rocker, rotator or a vortex mixer continuously for 5 minutes.
5. Allow the resin to settle, and then push the resin separator into the tubes until the resin is
firmly packed.
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6. Pour or aspirate each supernatant directly into a cuvette and measure each absorbance A
(HbA1c ) against Distilled water at 415 nm against. The absorbance is stable for at least one
hour.
C. Total Hemoglobin (THb) fraction
1. Dispense 5.0 ml of Distilled water into tubes labelled as Control & Test.
2. Add to it 0.02 ml of hemolysate from Step A into the appropriately labelled
tube. Mix well.
3. Read each absorbance A (Hb TOTAL) against Distilled water at 415 nm. The
absorbance is stable for at least one hour.
The reference ranges for A1C results are:
No diabetes: below 5.7 percent
Borderline/prediabetes: 5.7 percent to 6.4 percent
Diabetes: 6.5 percent or higher
Calculations:
