Prepared byHadeel AL Sadoun

Hemoglobin A2 Estimation

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

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

Methemoglobin Application

point

SA A2 D E G

FA F DSG

A2CEO

Hemoglobin A2 quantitiationPurpose:

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 ChromatographyImmunochemical 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 cellulosePositive resin coupled to Negative charged moleculeNegative resin attract

positively Charged moleculeBuffer and PH level should be controlledTo 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