Practical Hematology Lab
- LAB 6 -
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 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 – Anion Exchange Chromatography
• 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.
• Reagent 1 (Triton X-100) 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 100 µl from reagent 2 (Tris HCL) 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 5.0 ml
from reagent 2 (Tris HCL).
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:
% Hb A2 =
5 × Absorbance Hb A2 × 100%
12 × Absorbance Total Hb
Chromatographic Determination Of
Hemoglobin A2
Composition
Reagent A:
Resin : 25×2.5 ml
DEAE-cellulose, preweighted in tube
Reagent B:
Lysing solution : 1×20 ml
Triton × 100
Filters Separator : n. 25
Reagents Preparation
The reagents are ready to use
Storage And Stability
• 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.
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 separated by means of special
separating filters.
Hemolysate Preparation
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.
Hemoglobin A2 Preparation
1. 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.
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 (Abs HbA2).
Total Hemoglobin Fraction
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.
Calculations
Notes
1. This test must not be performed before six months age.
2. If the patient heterozygous for β- thalassemia also has
iron deficiency, the hemoglobin A2 may be within the
normal range.
3. If the patient has received a transfusion recently this test
should not be performed.
4. Some of the abnormal hemoglobin (Hb S, C, O, E, G, SG hybrid) are interfere with Hb A2 in this method. The
presence of the abnormal hemoglobin should be
confirmed by electrophoretic techniques. Hb F does not
interfere with this method.