Fetal Hemoglobin (HbF)

Sample for Fetal Hemoglobin (HbF)

1. Hemolysate is prepared from whole blood (EDTA, citrated, or heparinized).
2. Avoid hemolysis.

Purpose of the test (Indications) for Fetal Hemoglobin (HbF)

1. Fetal Hb is done to rule out Thalassemia.
2. Fetal Hb may be done on pregnant women to evaluate the fetal-maternal hemorrhage.
3. HbF may be done in hemolytic anemia, hereditary persistence of fetal hemoglobin, and other hemoglobinopathies.

Hemoglobin formation process:

1. Normal Hemoglobin synthesis is dependent on three processes.
   1. There is a need for an adequate iron supply.
   2. Adequate synthesis of protoporphyrins which is the precursor for heme.
   3. Adequate synthesis of globins.
2. Hemoglobin seen on electrophoresis shows a very small band of HbF.

Hb electrophoresis normal pattern

3. HbF is the normal hemoglobin in fetuses and infants.

Definition of Fetal Hemoglobin (HbF): 

1. It is the main fetal protein carrying oxygen in the fetus during the last 7 months of the fetal uterine life.
2. It will persist in the newborn for roughly 2 to 4 months.
3. Fetal Hb has more affinity than adult Hb, and it helps to give more oxygen to the fetus from the mother’s circulation.
4. Fetal Hb will disappear by the 6th month and only persists in thalassemia.

Fetal hemoglobin facts:

1. Adult hemoglobin A1 (HbA1) consists of α2 β2 chains is 95% to 97%.
2. Adult hemoglobin A2 (HbA2) consists of α2 δ2 chains is 2% to 3%.
3. Fetal hemoglobin (HbF) consists of α2 γ2 chains are 1% to 2%.
4. In adults, Hb A2 and HbF are present in trace amounts.

Fetal Hemoglobin (HbF): Hb F structure

5. HbF has a greater affinity to bind with oxygen than adult hemoglobin and gives better oxygen to the developing fetus from the mother’s blood circulation.
   1. HbF is the major Hb present during gestation.
6. HbF makes up 50% to 90% of Hb in newborns.
   1. The rest of the Hb in the newborn is HbA1 and HbA2.

Newborn Hb pattern

7. HbF is replaced by adult Hb (HbA) at 6 to 12 months. This will be less than 1% of the adult.
8. In the normal situation in an infant’s first year, this HbF is replaced by HbA1 and HbA2.
   1. If HbF persists over 5% after 6 months, then that is a sign of abnormality.
9. HbF is resistant to alkali denaturation, so this technique is useful for screening the patient.
10. Electrophoresis is more reliable for confirming the HbF by alkali denaturation test and more helpful at a higher level of HbF.

Fetal Hb characteristic features

Feto-maternal hemorrhage and fetal Hemoglobin (HbF):

1. Fetal Hb may be present in mother circulation because of fetal-maternal hemorrhage, which causes leakage of the cells into the maternal circulation.
2. Increased Feto-maternal hemorrhage may be seen in:
   1. Trauma to the mother.
   2. Placental abruption.
   3. Fetal Hydrops.
   4. Placental tumor.
   5. Amniocentesis in the third trimester.
3. Massive fetal-maternal hemorrhage may be the cause of 1 out of 50 stillbirths.
4. Leakage of fetal RBC may start after the mid-first trimester.
5. Near the final term, 50% of the mother show fetal RBC.
6. Total blood loss in this way is 2 ml or less in 96 to 98 %.
7. Increased Risk of fetal-maternal hemorrhage is due to the integrity of placental circulation.
8. Risk factors for Feto-maternal hemorrhage are:
   1. Maternal trauma.
   2. Placental abortion.
   3. Placental tumors.
   4. Third-trimester amniocentesis.
   5. Hydrops fetalis.
   6. Twin.
   7. Pale fetal organs (thin pale, loose, and dry skin).

Lab diagnosis of fetal hemoglobin (HbF):

1. The following methods can diagnose HbF:
   1. Acid elution stain.
   2. Flow cytometry.
   3. Chromatography.
   4. Electrophoresis.
   5. Alkali denaturation method.

Procedure for alkali denaturation method for Fetal hemoglobin (HbF):

1. This is because fetal hemoglobin is more resistant to strong alkalis than other hemoglobins.
   1. Mix blood with a small amount of distal water, leading to hemolysis.
   2. Due to hemolysis, there will be the release of free hemoglobin.
2. Now centrifuge the sample for several minutes.
3. Mix 5 mL of supernatant (pink in color) with 1mL of 1% NaOH.
4. Result:
   1. Check the color after 2 minutes:
      1. Fetal hemoglobin (HbF) will be pink.
      2. Adult hemoglobin (HbA) will be yellow-brown because it is less stable and will convert into hematin.

Fetal Hemoglobin (HbF): Fetal Hb alkali denaturation

Normal Fetal hemoglobin (HbF)

Source 1

Source 2 Fetal HbF

• <1% of RBCs

Source 4 Fetal HbF

• Newborn = 60% to 90%.
• By 6 months = 2%.
• Adult = 0 to 2%.

Another source of Fetal Hemoglobin (Hb F)

Increased Fetal hemoglobin (HbF) has been seen in:

1. Hereditary causes.
   1. Homozygous beta-thalassemia (20% to 100% HbF).
   2. Heterozygous beta-thalassemia (up to 5% HbF).
   3. Hereditary persistence of HbF (Homozygous 100 % and in heterozygous is 15% to 35 %).
   4. Sickle cell anemia (≤ 30 % HbF).
2. Acquired causes (up to 10% HbF).
   1. Pernicious anemia.
   2. Refractory normoblastic anemia.
   3. Pure red aplasia.
   4. Aplastic anemia.
   5. Sideroblastic anemia.
   6. Pregnancy and molar pregnancy.
   7. Juvenile chronic myeloid leukemia.
   8. Hyperthyroidism.
   9. Erythroleukemia.
   10. Chronic renal diseases.
   11. Leakage of fetal RBC into maternal circulation ( FMH ).

Thalassemia and Fetal Hemoglobin (HbF)

1. There is no Thalassemia hemoglobin. Actually, this is a complex group of genetic abnormalities of globin chain synthesis.
2. Thalassemia is divided into two main groups.
   1. α-thalassemia.
   2. β-thalassemia.
3. There are three major clinical groups:
   1. Thalassemia major shows HbF 40% to 90%. It is associated with severe and often life-threatening clinical manifestations.
   2. Thalassemia minor shows HbF 5% to 10 %. It has mild clinical manifestations.
   3. Thalassemia with other hemoglobinopathies.
4. The continuous production of HbF leads to severe anemia and death.

Hemoglobin electrophoresis showing Thalassemia

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