Hemoglobin A2 Estimation Prepared by Hadeel AL Sadoun

Normal Hemoglobin Normal adult hemoglobin composed of: Hb A: α2β2 Hb F< 2 % Hb A: α2β2 Hb A2: α2δ 2 Hb F: α2γ 2

In Thalssemia and haemoglobinpathies the Presence/absence of these Hgb varies. level of HbA, Hb F and Hb A2 can be affected. Thus both qualitative and quantitative method for Hgb measurement is needed. Hb Electrophoresis is the most common method used for the detection of Hgb.

Hgb Electrophoresis (+) Cellelouse acetate (-) (+) Citrate Agar (-) C F D S G A2 C E O F S A A2 D E G Methemoglobin Application point

Hemoglobin A2 quantitiation Purpose: Essential for the differential diagnosis of Thalssemia minor, IDA and other hypochromic microcytic anemia. HbA2 > 3.5% in B thal minor. Method used for HbA2 quantitiation: Hgb Electrophoresis using cellouse acetate followed by elution or densitometry. Anion Exchange Chromatography Immunochemical method using antisera specific to several Hemoglobins.

Β Thalassemia minor film Normal Film B Thal minor film

Anion Exchange Chromatography Principle: AEC contains 2 resin. Prepared from cellulose Positive resin coupled to Negative charged molecule Negative resin attract positively Charged molecule Buffer and PH level should be controlled To cause different Hg display net negative charges -ve charged Hg bind to +ve resin Helena Beta Thal Quick coloum

Principle Hgb then will removed selectively from the resin by altering PH or ionic strength of elution buffer. HbA2 fraction is eluted first and compared to the total Hgb by determining the absorbance Absorbance is detected using spectrophotometer. HbA2 is then calculated using the following formula: HbA2%= Hemoglobin A2 Absorbance 5 x Absorbance of total fraction of Hgb