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 • 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.
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 A2100 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