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Hemoglobin A 2 Estimation

Hemoglobin A 2 Estimation. Prepared by Hadeel AL Sadoun. Normal Hemoglobin . Normal adult hemoglobin composed of: Hb A > 95 % Hb A2<3.5 % Hb F< 2 % Hb A: α 2 β 2 Hb A2: α 2 δ 2 Hb F: α 2 γ 2. In Thalssemia and haemoglobinpathies the Presence/absence of these Hgb varies.

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Hemoglobin A 2 Estimation

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  1. Hemoglobin A2 Estimation Prepared by Hadeel AL Sadoun

  2. Normal Hemoglobin • Normal adult hemoglobin composed of: • Hb A > 95 % • Hb A2<3.5 % • Hb F< 2 % • Hb A: α2β2 • Hb A2: α2δ 2 • Hb F: α2γ 2

  3. 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.

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

  5. Hemoglobin A2 quantitiation • Purpose: • Essential for the differential diagnosis of Thalssemia minor, IDA and other hypochromicmicrocytic 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.

  6. Β Thalassemia minor film Normal Film B Thal minor film

  7. 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

  8. 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

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