Fetal Hemoglobin (HbF) (Alkali resistant Hemoglobin)

Fetal Hemoglobin (HbF)

Sample

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

Purpose of the test (Indications)

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 roughly up to 2 to 4 months.
3. Fetal Hb has more affinity than the adult Hb and it helps to give more oxygen to the fetus from the mother 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.
   

   Hb F structure

3. HbF has a greater affinity to bind with oxygen than the 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.
4. HbF makes 50 to 90% of Hb in the newborn.
   1. The rest of the Hb in the newborn is HbA1 and HbA2.
      

      Newborn Hb pattern

5. HbF is replaced by adult Hb (HbA) by 6 to 12 months of age. This will be less than 1% of the adult.
6. In the normal situation in the first year of an infant, this HbF is replaced by HbA1 and HbA2.
   1. If HbF persists more than 5% after 6 months, then that is a sign of abnormality.
7. HbF is resistant to alkali denaturation, so this technique is useful for the screening of the patient.
8. Electrophoresis is more reliable for the confirmation that the 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. HbF can be diagnosed by the following methods:
   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 based on the fact that fetal hemoglobin is more resistant to strong alkali than the other hemoglobins.
   1. Mix blood with a small amount of distal water which will lead 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 Hb alkali denaturation

Normal Fetal hemoglobin (HbF)

Source 1

Age	%Hb F
one day	77.0 to ± 7.3
5 days	76.8 ± 5.8
3 weeks	70.±0  7.3
6 to 9 weeks	52.9 ± 11.0
3 to 4 months	23.2 ± 16.0
6 month	4.7 ± 2.2
8 months to 11 months	1.6 ± 1.0
Adult	<2.0

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)

Age  in days of the fetus/newborn	HbF %
0 to 10	56 to 87
11 to 20	55 to 83
21 to 30	51 to 76
31 to 40	46 to 70
41 to 50	38 to 62
51 to 60	31 to 54
61 to 70	24 to 44
71 to 80	17 to 34
81 to 90	12 to 28
91 to 100	8 to 24
101 to 110	7 to 18
111 to 120	5 to 15
121 to 130	4 to 10
131 to 140	<6.1
141 to 364	<4.1
One year and above	<2.1

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 15-35 %).
   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 manifestation.
   3. Thalassemia with other hemoglobinopathies.
4. The continuous production of HbF leads to severe anemia and death.

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