ESTIMATION OF HAEMOGLOBIN F (HBF/ Foetal Hemoglobin) BY ALKALI DENATURATION METHOD

ESTIMATION OF HEMOGLOBIN F (HBF/Foetal Hemoglobin) BY ALKALI DENATURATION METHOD

Foetal hemoglobin (HbF) constitutes 70 to 90 % of the total hemoglobin at birth, but is replaced by the adult hemoglobin (HbA) during the first few months after birth. 

Hbf consists of two alpha (a) and two gamma (7) chains in its globin molecule, and each chain is linked with a haem group. When HbF is converted to HbA, an amino acid in the gamma chain is replaced, and the resulting chain is called a beta (P) chain. Thus

Globin chains in Hb A 2a and 2 B 

Globin chains in Hb F 2 a and, 27

In beta-thalassaemia, there is a decrease in the synthesis of beta chains, and therefore, an increase in HbF.

Principle of HBF

HbF is more resistant to denaturation by alkali than HbA. This property is used to estimate the amount of Hbf in a blood sample. A haemolysate is prepared. A measured quantity of the haemolysate is exposed to the action of an alkaline reagent for a defined period of time. 

Denaturation process is interrupted by adding a solution which simultaneously lowers the pH and precipitates the denatured hemoglobin. 

After filtration, the unaltered hemoglobin (HbF) is determined by reading the absorbance of the filtrate. This is expressed as a percentage of total initial amount of hemoglobin present in the specimen of blood. 

Specimen Blood anticoaguled with EDTA. 

Reagents of HBF

1. Sodium or potassium hydroxide (0.083 N): Dilute before use, I N NaOH or KOH 1:12. (8.3 ml of IN solution to 100 ml with distilled water).

2. Precipitating agent: Add 2 ml of concentrated hydrochloric acid to 500ml of half saturated ammonium sulphate solution. Prepare saturated ammonium sulphate solution by adding about 500g of the salt to 500ml water. Allow it to stand for 1-2 hours. Dilute the supernatant 1:2 to obtain a halfsaturated solution.

3. Sodium chloride 0.85 % solution. 

4. Chloroform

Technique of HBF

Step 1 Preparation of haemolysate

Centrifuge the specimen of blood.

Wash the cells three times with the sodium chloride solution.

For lysing the cells, shake each volume of packed cells (0.4 ml) vigorously with equal volume of distilled water (0.4 ml) and half volume of chloroform (0.2 ml) for two minutes.

Centrifuge at 3000 rpm for 20 minutes.

Remove the upper layer of haemoglobin. Filter to clear if necessary.

Determine the haemoglobin concentration of the haemolysate by one of the standard methods in use. e.g. cyanmethaemoglobin method.

Adjust the haemoglobin concentration of the haemolysate to approximately 10g %. For example, if haemoglobin content of the haemolysate is 13g%. add 0.3 ml of distilled water to 1.0 ml of the haemolysate to obtain the concentration equal to 10 g %. 

Step II Alkali denaturation

 Place 3.0 ml of 0.083 N hydroxide solution in a test tube.

Add 0.2 ml of the haemolysate quickly from a pipette and gently shake the tube for 10 seconds. Start a stop watch at the time of adding the haemolysate.

At the end of exactly 1 minute, add 6.8 ml precipitating agent.

Mix by inverting several times and filter through two layers of Whatman No. 1 filter paper. 

With normal adult haemoglobin, the filtrate is colourless whereas in the presence haemoglobin F. the filtrate may be light brown to deep red in colour.

Prepare a standard haemoglobin solution by diluting 0.2 ml of the haemolysate prepared in Step I to 200 ml with 0.04% ammonia solution. This corresponds to 5% alkali resistant haemoglobin.

Read the absorbance of the standard and test against a water blank using a blue filter (415 nm).

Calculations of HBF

(i) The amount of foetal haemoglobin (HbF) is expressed as a percentage of total haemoglobin (HbF + HbA).

(ii) During alkali denaturation, 0.2 ml of the haemolysate is diluted to 10 ml (1:50 dilution).

(iii) If all the haemoglobin is in the form of alkali resistant haemoglobin (HbF), the absorbance of 1:50 dilution should be equivalent to 100 % HbF.

(iv) Therefore, 0.2 ml of the haemolysate diluted to 200 ml (1:1000 dilution) is equivalent to 5 % HDF Alkali resistant hemoglobin (%) = Reading of test/Reading of standard

Normal range of HBF

in the normal adult blood, only 0.5 to 1.7 % of the total hemoglobin is alkali resistant (HbF).

Interpretation of HBF

Increased levels of upto 5 % HbF are found in various genetic and acquired disorders. In addition to beta thalassaemia, high HbF may be seen in leukaemias, aplastic anaemia and sickle cell anaemia.