Hemoglobin F QuantitationFetal Hemoglobin

Practical Hematology Lab

- LAB 8 -

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. Hereditary persistence of fetal hemoglobin.

2. Sickle cell anemia.

3. Acquired a plastic anemia.

4. Megaloblastic anemia.

5. Paroxysmal nocturnal hemoglobin.

Principle• Hemoglobin F, unlike other physiological hemoglobin's, is

not denatured in a highly alkaline medium and then can be separated by filtration after selective precipitation of denatured hemoglobin's.

• The remaining concentration of the Hb F in the solution is photo metrically determined and related to the total Hemoglobin's.

Procedure:

Hemolysate Preparation :1. wash RBCs 3 times with saline

2. Add 1 ml RBCs to 1.4 ml distilled water, and mix for 30 sec.

3. Add 0.4 ml of reagent D (Toluol) for 1 ml hemolysate.

4. Mix and centrifuge for 10 min.

Hb F Preparation:5. Add 100 µl from the supernatant to 1.5 ml of reagent A ( NaOH).

6. Mix for 2 min exactly, then add 3.5 ml of reagent B (ammonium sulfate + HCL)

7. Turn the tube ubside down for 5 min and let to settle for 5 min, then filtrate.

8. Read the absorbance at 410 nm against a blank of reagent C (ammonium hydroxide).

Total Hemoglobin Preparation:

1. Add 100 µl from hemolysate to 5 ml of reagent C (ammonium hydroxide) and mix.

2. Take 100 µl from the previous tube to 1900 µl D.W.

3. Read the absorbance at 410 nm against a blank from reagent C (ammonium hydroxide).

Calculation:

% Hb F = Absorbance Hb F × 5

Absorbance Total Hb

Normal Range:

Hb F ≤ 2

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.