Practical
Clinical Hematology
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 HBA2 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:
 Blood 50 µl.
 Distilled water 200 µl.
 Good mixing and leaved it for some time
at R.T.
Separation and reading HbA2
1. 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.
2. Take 50 µl from hemolysate and put it on the upper
disc and let the column drain to waste.
3. Add 200 µl from reagent (1) buffer and put it on the
upper disc and let the column drain to waste.
4. Place the micro column over a test tube and add 3.0
ml from buffer.
5. Collect the elute ( Hb A2 fraction)
6. 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
Today Lab:
 The reagents are stable up to the expiry date stated on
the labels, if stored at 15-25 °C.
 Strong temperature variations may alter resin
equilibrium and consequently its functionality; if
erroneously stored at 2-8 °C the resin has to remain at
room temperature for at least three days before using.
 Tubes containing yellowish-white resin indicate
chemical degradation and cannot be used.
 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.
1. Dispense into tube 300 μl Lysing Solution (Reagent B).
2. Place 50 μl of the well-mixed blood sample.
3. Mix well and allow to stand for 5 minutes.
Add 100 μl of the hemolysate in the resin tube (RA).
2. Position the Filter Separators in the tubes so that the
rubber sleeve is approximately 1 cm above the liquid
level.
3. Place the tubes on the rocker or rotator and gently
mix continuously for 5 minutes (alternatively turn
upside down at least six times at intervals of one
minute).
4. Remove the tubes from the rocker or rotator. Push
the Filter Separator into the tubes proceeding slowly
until the resin is firmly packed.
1.
5. The supernatant may be poured into another tube or
directly into a cuvette for absorbance measurement.
6. Read the absorbance values at 415 nm against a reagent
blank made of liquid phase obtained from a test tube
without hemolysate (A HbA2).
1. Pipette in empty test tube (Hb Total) 20 µl from
hemolisate and 10 ml from distilled water.
2. Mix and read the absorbance against a reagent blank
made of distilled water at 415 nm.
Calculation