Hemoglobin Electrophoresis

• Hemoglobin electrophoresis is used to separate

and identify the different hemoglobins by their

migration within an electric field.

• Hemoglobin variants separate at different rates

due to differences in their surface electrical

charge as determined by their amino acid

structure.

Why test is Performed?

• Hempoglobin electrophoresis is performed to find out

abnormal forms of hemoglobin (hemoglobinopathy).

• Many different types of hemoglobin (Hb) exist. The most

common ones are HbA, HbA2, HbF, HbS, HbC, Hgb H,

and Hgb M. Healthy adults only have significant levels of

HbA and HbA2.

• Some people may also have small amounts of HbF

(which is the main type of hemoglobin in an unborn

baby's body). Certain diseases are associated with high

HbF levels (when HbF is more than 2% of the total

hemoglobin).

Why test is Performed?

• HbS is an abnormal form of hemoglobin associated with sickle cell anemia. In people with this condition, the red blood cells have a crescent or sickle shape. These misformed cells then break down, or can block small blood vessels.

• HbC is an abnormal form of hemoglobin associated with hemolytic anemia. The symptoms are much milder than they are in sickle cell anemia.

• Other, less common, abnormal Hb molecules cause anemias.

Normal values

In adults, these hemoglobin molecules make up the following percentages of total hemoglobin: • Hgb A1: 95% to 98%

• Hgb A2: 2% to 3%

• Hgb F: 0.8% to 2%

• Hgb S: 0%

• Hgb C: 0%

In infants and children, these hemoglobin molecules make up the following percentages of total hemoglobin: • Hgb F (newborn): 50% to 80%

• Hgb F (6 months): 8%

• Hgb F (over 6 months): 1% to 2

Methods of electrophoresis

1. Cellulose Acetate At Alkaline pH

2. Citrate Agar Electrophoresis ( acid pH)

1-Cellulose Acetate At Alkaline pH

• Cellulose acetate Hb electrophoresis at

alkaline pH (at pH 8.4-8.6) is used as

routine procedure to detect variant

(abnormal) Hbs.

• This gives good separation of HbA, HbF,

HbS, and HbC. On alkaline electrophoresis

HbD and HbS have the same mobility and

HbC, HbE and HbO also co-migrate.

Principle of the test

• Electrophoresis is the movement charge particles in

an electric field. In an alkaline pH (8.2-8.6) Hb is a negatively charged molecule and will migrate toward the anode (+).

• The various Hbs move at different rates depending on their net negative charge, which in turn is controlled by the composition (amino acids) of the Hb molecule (globin chain).

• The red cell hemolysate (red blood cell membranes are destroyed to free the Hb molecules for testing) is placed in a cellulose acetate membrane, which is positioned in an electrophoresis tray with the inoculated hemolysate near the cathode (-).

Principle of the test

• One end of the cellulose acetate strip is immersed in the

buffer (pH 8.2-8.6) on the cathode side and the other end

is placed in the buffer on the anode (+) side.

• An electric current of specific voltage is allowed to run for

a timed period. During electrophoresis, the Hb molecules

migrate toward the anode because of their negative

charge.

Principle of the test

• The difference in the net charge of the Hb molecule

determines its mobility and manifests its self by the speed

with which it migrates to the positive pole. Example of the

fast Hbs are Hb Bart’s and the tow fastest variants Hb H

and I, while Hb C is the slowest common Hb.

• The cellulose acetate membrane is then stained in order

to color the proteins (Hbs). By noting the distance each

Hb has migrated and comparing this distance with the

migration distance of known controls, the types of

hemoglobins may be identified.

• Controls

Controls should include the following

haemolysates:

• HbAA: Normal adult blood

• HbAS: Blood from a person with sickle cell

trait

• HbAF: Blood from an infant below 3 m

• HbAC: Blood from a person with HbC trait

(when HbC is found locally)

2- Citrate Agar Electrophoresis ( acid pH)

• Citrate agar separates Hb fractions that migrate

together on cellulose acetate agar, all Hb

specimens that show an abnormal

electrophoretic pattern in alkaline media

(cellulose acetate agar) should undergo

electrophoresis on an acid citrate agar.

• In specialist laboratories agarose gel

electrophoresis at an acid pH (6.0) can be used

to separate these haemoglobins and also to

provide a clear separation of HbF from HbS and

HbC.

Citrate Agar Electrophoresis ( acid pH)

Reading of results