Hemoglobin A2
Introduction



HBA2 is a protein which in humans is encoded by
the HBA2 gene.
Hemoglobin A2 is a normal variant of hemoglobin
A that consists of two alpha and two delta chains
and is found in small quantity in normal human
blood.
Normal value of Hb A2 in the adult is
( 1.8% – 3.5%).

HbA2 elevated up to 8% indicates:
 β.
Thalassemia trait
 Homozygous Thalassemia

Decreased level may be found in:
 iron
deficiency anemia.
 Hb H disease.
 hereditary persistence of Hb F.
 fibroblastic anemia.
 carriers of α Thalassemia.
HbA2 test


The anion exchange micro chromatography procedure is
an accurate and easily performed method for HbA2
Quantization.
Principle:
– A hemolysate is prepared from the patients red blood
cells.
– A specific amount of hemolysate is then added to the
top of the resin column.
– The diethyl amino ethyl DEDE resin is a preparation of
cellulose attached to positively charged molecules,
thus giving the cellulose appositive charge.
Principle



When the hemolysate is added to the column, the
PH of the buffer present determines the net
negative charge of the Hb, which then binds to the
positively charged cellulose resin.
The Hb are selectively removed from cellulose
according to the PH of the developer.
In this procedure the HbA2 (originally bound to
the resin) is released from the resin and eluted by
the developer as it passes through the column.

Most other normal and abnormal HbS
remain bound to the resin in the column.

The eluted HbA2 is then measured
spectrophotometrically and compared with
the amount of total Hb in the specimen to
calculate the percent of HbA2 present.
Procedure
 Hemolysate
–
–
–
preparation:
1- Blood 50 µl.
2- Distilled water 200 µl.
3- Good mixing and leaved it
for
some time at R.T.
Separation and reading HbA2
1.
2.
3.
Remove the upper of the Micro column and then
snap the tip off the bottom. Then, using the
rounded end of a pipette, push the upper disc
down to resin surface taking care not to
compress it. Let the micro column drain
completely to waste.
Take 50 µl from hemolysate and put it on the
upper disc and let the column drain to waste.
Add 200 µl from reagent (1) buffer and put it on
the upper disc and let the column drain to waste.
4.
5.
6.
Place the micro column over a test tube
and add 3.0 ml from buffer.
Collect the elute ( Hb A2 fraction)
Shake thoroughly and read absorbance
(Abs) of the HbA2 fraction at 415 nm
against distilled water (Abs HbA2)
Reading of total hemoglobin

Put in test tube 12.0 ml distilled water and
50 µl hemolysate and then read at 415nm
against distilled water.
Calculation

% Hb A2 = (Abs HbA2 / Abs Hb total) X 25
Determination of A2 hemoglobin (Hb A2) in blood

Principle
The hemolisate is directly placed in the test
tubes containing DEAE-cellulose resin.
Hemoglobin A2 unlike all remaining
hemoglobin is not linked by the resin and
then can be separed by means of special
separating filters.
Procedure

Hemolisate preparation
1- Blood 50 µl
2- hemolysis reagent 300 µl, Wait 5 minutes.
3- Good mixing and leaved it for 5 min at R.T.
Separation and reading HbA2

Pipet in test tube 1 ( resin) Hb A2 100 µl from
hemolisate.

Turn upside down test tube 1 till complete
resuspension of the resin. Continue to shake
gently the test tubes for 5 minutes using a stirrer
or turn upside down al least 6 times at intervals of
1 minute.
Separate the liquid phase by gently pressing
the separating filter in the test tube.
 Determine at 415 nm the absorbance of the
liquid phase of the test tube (A HbA2)
against a reagent blank made of liquid
phase from a test tube without hemolisate.

Reading of total hemoglobin


Pipet in Test tube 2 (empty) Hb tot 20 µl from
hemolisate and 10 ml from distilled water.
read the absorbance of total hemoglobin (A Hb tot)
against a reagent blank made of distilled water.
Calculation

% Hb A2 = (Abs HbA2 / Abs Hb total X 22) X 100