Protocol:Hb F quantitation by alkali denaturation

Fetal haemoglobin is the prevalent haemoglobin type in the newborn (about 60-80%) then progressively decreases to very low levels, of less then 1%, by the second year of life. The decline is slower in &beta;-thalassaemia carriers.

Hb F levels may be increased in adults in a number of inherited (&delta;&beta;-thalassaemia, deletional and non-deletional HPFH, some &beta;-thalassaemia mutations) and acquired conditions, including pregnancy, recovery after bone marrow transplantation and aplastic anaemia, myelodisplastic syndromes, juvenile chronic myeloid leukaemia. The Hb F is restricted to a sub-population of erythrocytes termed “F-cells”.

Detection and quantitation of increased levels of Hb F are important for genetic counselling and prognosis in &beta;-thalassaemia and sickle cell anaemia. An increased level of Hb F may be associated with a milder phenotype.

Since the range of Hb F may be from 1% to 95%, no single method is accurate for its quantitation over the whole range. High performance liquid chromatography (HPLC), gives the best estimation of Hb F over the whole range (10,11). However, other methods are available for Hb F determination, such as alkali denaturation, immunological techniques (by immunodifusion, ELISA and immunoradiometria), IEF, column chromatography. All these techniques, except alkali denaturation, for several reasons are not readily available and therefore not widely used.

Principle
Hb F is relatively resistant to denaturation by alkalis as compared to Hb A. The method described (12) gives reliable results in the range of 0.8 to 25%, which are the levels most commonly found in carriers of &delta;&beta;-thalassaemia and of most of the forms of HPFH.

Sample

 * 1) Blood in any anticoagulant may be used.
 * 2) Wash the red cells x3 in 0.9% saline and then lyse by adding to the packed cells 2 volumes of water and 1 volume of toluene or carbon tetrachloride (CCl4).
 * Take Care Toxic reagents, use with care.
 * 1) After shaking in a mechanical agitator, centrifuge at 3000 rpm (1200 x g) for 30 minutes and pipette of the clear haemoglobin solution.

Reagent and solutions
Ready to use reagents are commercially available.
 * Drabkin’s solution:
 * NaOH 1.2 M
 * 4.8 g in 100 ml distilled H2O (fresh prepared)
 * (NH4)2SO4:saturated solution
 * 706 g in 1 Litre distilled H2O
 * heat to dissolve, then slowly at room temperature

Procedure

 * 1) Add 0.6 ml of haemolysate (Hb concentration 8-10 g/dl) to 10 ml Drabkin’s solution (Hb is converted to cyanmet form). 2.8 ml of the Hb solution is added to 0.2 ml of alkali solution and the mixture is well agitated.
 * 2) Exactly after 2 minutes add 2 ml of (NH4)2SO4 solution.
 * 3) After vigorous mixing the denaturated material is allowed to precipitate for 5-10 minutes.
 * 4) Remove the precipitate by filtration through a double layer of filter paper (Whatman no 6 or no 42) and read the optical density of the filtrate at 415 mm.
 * 5) A control solution is prepared by mixing 1.4 ml of Cyammet-Hb, 1.6 ml of distilled H2O and 2 ml of saturated ammonium sulphate. Dilute this solution 1:10 with distilled H2O to obtain a suitable optical density.

Calculation

 * OD415 test sample x 100

%of HbF= -
 * OD415 control x 20

Interpretation
The Hb F determination is one of the most difficult of the routine procedures for haemoglobinopathies study. The difficulty is not in the methodology but in obtaining accurate and reproducible results, which can be achieved only when the method is in frequent use and done by an experienced technician.

Percentage Hb F for normal adult is 0.2-1.0%. Values of Hb F higher than 20% are slightly underestimated with this method. Higher levels can be determined with Jonxis and Visser method or with HPLC.

Some variability has been found with different batches of filter paper. The concentration of the haemolysate is also important: more diluted samples may give higher results.