Practical Hematology Lab LAB 13 Hemoglobin Electrophoresis Electrophoresis

Practical Hematology Lab - LAB 13 - Hemoglobin Electrophoresis

Electrophoresis ØElectrophoresis is a means of separating hemoglobin's. ØIt depends on the migration of the hemoglobin molecules dissolved in a buffer on, or in, a supporting medium when an electric current is passed through them. Hemoglobin electrophoresis ØIs a test that measures the different types of the oxygen -carrying substance (hemoglobin) in the blood. ØHemoglobin electrophoresis is performed to find out abnormal forms of hemoglobin (hemoglobinopathy).

Hemoglobin Electrophoresis ØMany different types of hemoglobin (Hb) exist. The most common ones are Hb. A, Hb. A 2, Hb. F, Hb. S, Hb. C, Hb H, and Hb M. ØHealthy adults only have significant levels of Hb. A and Hb. A 2. ØSome people may also have small amounts of Hb. F (which is the main type of hemoglobin in an unborn baby's body). Certain diseases are associated with high Hb. F levels (when Hb. F is more than 2% of the total hemoglobin).

Hemoglobin Electrophoresis ØHb S 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. ØHb C is an abnormal form of hemoglobin associated with hemolytic anemia. The symptoms are much milder than they are in sickle cell anemia.

Normal Values ØIn adults: • Hgb A 1 : 95% to 98% • Hgb A 2 : 2% to 3% • Hgb F : 0. 8% to 2% • Hgb S : 0% • Hgb C : 0% ØIn infants and children: • 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 p. H 2 - Citrate Agar Electrophoresis ( acid p. H)

1 -Cellulose Acetate At Alkaline p. H ØCellulose acetate Hb electrophoresis at alkaline p. H is the primary screening procedure used to detect variant (abnormal) Hbs, of which there are several hundreds. ØThe major portion of normal adult Hb is A. In addition, up to 3. 5% Hb A 2 is normally present, along with less than 2% Hb F. The more common mutant Hbs are S, C, E, D, G, and lepore.

1 -Cellulose Acetate At Alkaline p. H ØWhen an abnormal Hb is detected on cellulose acetate electrophoresis at an alkaline p. H (8. 2 -8. 6) further testing is frequently indicated: test for Hb S, quantitation of Hb A 2 and F, and citrate agar gel; acid/alkaline globin chain or neutral p. H electrophoresis may also be warranted.

Principle of Cellulose Acetate ØIn an alkaline p. H (8. 2 -8. 6) Hb is a negatively charged molecule and will migrate toward the anode (+). The various Hbs moves 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).

Principle of Cellulose Acetate ØThe 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 Cellulose Acetate ØOne end of the cellulose acetate strip is immersed in the buffer (p. H 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. ØDuring electrophoresis, the Hb molecules migrate toward the anode because of their negative charge. 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.

Principle of cellulose acetate ØThe 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. ØExample of the fast Hbs are Hb Bart’s and the two fastest variants Hb H and I, while Hb C is the slowest common Hb.

2 - Citrate Agar Electrophoresis ( acid p. H) ØCitrate agar separates Hb fractions that migrate together on cellulose acetate agar. ØAll Hb specimens that show an abnormal electrophoretic pattern in alkaline media (cellulose acetate agar) should undergo electrophoresis on an acid citrate agar.

2 - Citrate Agar Electrophoresis ( acid p. H) ØCitrate agar electrophoresis is used to confirm variant Hbs and further differentiates Hb S from Hb D and G, and Hb C from Hb E, O Arab, and CHarlem. . ØThe procedure should not be used as a screening procedure because many abnormal Hbs migrate with Hb A. However, this procedure is the method of choice when examining newborns (cord blood specimens) and infants under 3 months of age for some abnormal Hbs such as S and C because the test is able to detect quantities of Hb not easily seen by other techniques.

C s A D G A 2 E F