Fetal Hemoglobin (HbF) (Alkali resistant Hemoglobin)
Fetal Hemoglobin (HbF)
- Hemolysate is prepared from whole blood (EDTA, citrated, or heparinized).
- Avoid hemolysis.
Purpose of the test (Indications)
- Fetal Hb is done to rule out Thalassemia.
- Fetal Hb may be done on pregnant women to evaluate the fetal-maternal hemorrhage.
- HbF may be done in hemolytic anemia, hereditary persistence of fetal hemoglobin, and other hemoglobinopathies.
Hemoglobin formation process:
- Normal Hemoglobin synthesis is dependent on three processes.
- There is a need for an adequate iron supply.
- Adequate synthesis of protoporphyrins which is the precursor for heme.
- Adequate synthesis of globins.
- Hemoglobin seen on electrophoresis shows a very small band of HbF.
- HbF is the normal hemoglobin in fetuses and infants.
Definition of Fetal Hemoglobin (HbF):
- It is the main fetal protein carrying oxygen in the fetus during the last 7 months of the fetal uterine life.
- It will persist in the newborn for roughly up to 2 to 4 months.
- Fetal Hb has more affinity than adult Hb, and it helps to give more oxygen to the fetus from the mother’s circulation.
- Fetal Hb will disappear by the 6th month and only persists in thalassemia.
Fetal hemoglobin facts:
- Adult hemoglobin A1 (HbA1) consists of α2 β2 chains is 95 to 97%.
- Adult hemoglobin A2 (HbA2) consists of α2 δ2 chains is 2 to 3%.
- Fetal hemoglobin (HbF) consists of α2 γ2 chains are 1 to 2%.
- In adults, Hb A2 and HbF are present in trace amounts.
- 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.
- HbF is the major Hb present during gestation.
- HbF makes 50 to 90% of Hb in the newborn.
- The rest of the Hb in the newborn is HbA1 and HbA2.
- HbF is replaced by adult Hb (HbA) at 6 to 12 months. This will be less than 1% of the adult.
- In the normal situation in an infant’s first year, this HbF is replaced by HbA1 and HbA2.
- If HbF persists over 5% after 6 months, then that is a sign of abnormality.
- HbF is resistant to alkali denaturation, so this technique is useful for screening the patient.
- Electrophoresis is more reliable for confirming the alkali denaturation test and more helpful at a higher level of HbF.
Feto-maternal hemorrhage and fetal Hemoglobin (HbF):
- Fetal Hb may be present in mother circulation because of fetal-maternal hemorrhage, which causes leakage of the cells into the maternal circulation.
- Increased Feto-maternal hemorrhage may be seen in:
- Trauma to the mother.
- Placental abruption.
- Fetal Hydrops.
- Placental tumor.
- Amniocentesis in the third trimester.
- Massive fetal-maternal hemorrhage may be the cause of 1 out of 50 stillbirths.
- Leakage of fetal RBC may start after the mid-first trimester.
- Near the final term, 50% of the mother show fetal RBC.
- Total blood loss in this way is 2 ml or less in 96 to 98 %.
- Increased Risk of fetal-maternal hemorrhage is due to the integrity of placental circulation.
- Risk factors for Feto-maternal hemorrhage are:
- Maternal trauma.
- Placental abortion.
- Placental tumors.
- Third-trimester amniocentesis.
- Hydrops fetalis.
- Pale fetal organs (thin pale, loose, and dry skin).
Lab diagnosis of fetal hemoglobin (HbF):
- HbF can be diagnosed by the following methods:
- Acid elution stain.
- Flow cytometry.
- Alkali denaturation method.
Procedure for alkali denaturation method for Fetal hemoglobin (HbF):
- This is based on the fact that fetal hemoglobin is more resistant to strong alkali than the other hemoglobins.
- Mix blood with a small amount of distal water, leading to hemolysis.
- Due to hemolysis, there will be the release of free hemoglobin.
- Now centrifuge the sample for several minutes.
- Mix 5 mL of supernatant (pink in color) with 1mL of 1% NaOH.
- Check the color after 2 minutes:
- Fetal hemoglobin (HbF) will be pink.
- Adult hemoglobin (HbA) will be yellow-brown because it is less stable and will convert into hematin.
- Check the color after 2 minutes:
Normal Fetal hemoglobin (HbF)
|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|
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:
- Hereditary causes.
- Homozygous beta-thalassemia (20 to 100% HbF).
- Heterozygous beta-thalassemia (up to 5 % HbF).
- Hereditary persistence of HbF (Homozygous 100 % and in heterozygous is 15-35 %).
- Sickle cell anemia (≤ 30 % HbF).
- Acquired causes (up to 10 % HbF).
- Pernicious anemia.
- Refractory normoblastic anemia.
- Pure red aplasia.
- Aplastic anemia.
- Sideroblastic anemia.
- Pregnancy and molar pregnancy.
- Juvenile chronic myeloid leukemia.
- Chronic renal diseases.
- Leakage of fetal RBC into maternal circulation ( FMH ).
Thalassemia and Fetal Hemoglobin (HbF)
- There is no Thalassemia hemoglobin, actually, this is a complex group of genetic abnormalities of globin chain synthesis.
- Thalassemia is divided into two main groups.
- There are three major clinical groups:
- Thalassemia major shows HbF 40% to 90%. It is associated with severe and often life-threatening clinical manifestations.
- Thalassemia minor shows HbF 5 to 10 %. It has mild clinical manifestations.
- Thalassemia with other hemoglobinopathies.
- The continuous production of HbF leads to severe anemia and death.