Practical Hematology Lab LAB 6 Hemoglobin F Quantitation

Practical Hematology Lab - LAB 6 - Hemoglobin F Quantitation Fetal Hemoglobin

Definition: Is the main oxygen transport protein in the fetus during the last seven months of development in the uterus and in the newborn until roughly 6 months old. • Baby takes about 2 years to completely switch over to adult haemoglobin •

Function Fetal hemoglobin differs most from adult hemoglobin in that it is able to bind oxygen with greater affinity than the adult form, giving the developing fetus better access to oxygen from the mother's blood stream. • The transfer of oxygen is from the mother (less tightly bond) to the baby (more tightly bond). •

Characteristic: 1. Hemoglobin F is made up of 2 alpha chains and 2 gamma chains 2. Hemoglobin F does not turn into hemoglobin A. 3. Hb F and Hb A are completely different hemoglobin 4. Fetal hemoglobin is alkali-resistant hemoglobin 5. Newborn babies with sickle cell disease make hemoglobin F and hemoglobin S

Hb F increased in: 1. 2. 3. 4. 5. Hereditary persistence of fetal hemoglobin. Sickle cell anemia. Acquired a plastic anemia. Megaloblastic anemia. Paroxysmal nocturnal hemoglobin.

Principle • The test utilize the characteristics of Hb F to resist denaturation in an alkaline solution. • A red blood cell hemolysate is prepared to lyse the red blood cell and free the hemoglobin. • The hemolysate is added to cyanmethemoglobin reagent and then exposed to an alkaline reagent, sodium hydroxide, for specified period. • During this time normal Hb is destroyed, but the fetal Hb remains intact. • Ammonium sulfate is added to stop the denaturation process and to precipitate the denaturated hemoglobin. • The solution is filtered, cyanmethemoglobin solution measured spectrophotometerically to determine the percent of hemoglobin.

Procedure: 1. Prepare hemolysate (R 1) by add 0. 5 ml blood to 9. 5 ml drapkien then mixed. 2. Transfer 2. 8 ml from R 1 to new tube and add 200 µl Na. OH (2 N) mixed and incubation at R. T 2 min. 3. At the end of 2 min exactly add 2 ml saturation ammonium sulfate and mixed then incubation 5 -10 min at R. T. 4. Filter the solution by filter paper. 5. Read the filtrate at 540 nm. Total Hb: Add 0. 4 ml blood from (R 1) to 6. 75 ml D. W and mixed, Read it on 540 nm.

Calculation • %Hb F = • (Abs of Hb. F / Abs of total Hb*10) *100 • Normal range • 0 – 6 months : Up to 75% of Hb. F • 6 months - Adult: Up to 2% Hb. F

Discussion 1. Babies with sickle cell disease experience more problems as hemoglobin F is turned off. 2. In adults, fetal hemoglobin production can be reactivated pharmacologically, which is useful in the treatment of such diseases as sickle cell disease 3. Hydroxyurea, one of the new drugs used to treat sickle cell disease in adults works by turning hemoglobin F back on.