FETAL Hb and G6PD

Interpretation and principle of Fetal Hb and G6PD deficiency

In adults:

Hb A:90-97%- two alpha, 2 beta

Hb A2: 1-3.5%-two alpha, two delta

Hb F: 0-2%-two alpha, two gamma

Estimation of Fetal Hemoglobin

Method- ICSH, based on Betke method.

Principle:

•      Fetal hemoglobin is more resistant to denaturation by strong alkali than are other hemoglobins. For the test, alkali is added to a hemolysate containing a known amount of haemoglobin. After a specified time, the denaturation is stopped by adding saturated ammonium sulphate to lower the pH and precipitate the denatured hemoglobin. The amount of unaltered haemoglobin is measured and expressed as a proportion of alkali-resistant (i.e. fetal) haemoglobin.

•      The recommended

Reagents and equipment

•       ICSH (modified Drabkin) reagent. Stable for 2-3 weeks at room temperature if kept in a dark bottle, protected from light.

•       Sodium hydroxide 1.2 mol/l. Stable for four weeks at room temperature.

•       Saturated ammonium sulphate (NH4)SO4 500 g to one litre distilled or de-ionized water.

•       Spectrophotometer with wavelength setting at 413 nm; can also be used at 540 nm.

•       Stopwatch with a second hand.

•      Whatman No. 42 filter paper and funnel.

•      Blood in any anticoagulant can be used; a normal and a raised HbF control should be included with each batch of samples. The raised control can be made by mixing one volume of cord blood with nine volumes of normal adult blood of the same ABO group to obtain a Hb F of 10%

METHOD

1.  To prepare a lysate wash about 1-2 ml of the blood three times in isotonic saline. Add one volume of carbon tetrachloride and one volume of distilled or de-ionized water to two volumes of the washed cells. Shake vigorously for 4-5 minutes in a stoppered centrifuge tube. Centrifuge at about 1200g (3000 rev/min) for 30 minutes. Pipette the clear supernatant into a test-tube and adjust its hemoglobin concentration to about 100 g/l with water.

2.  Add 0.2 ml of the lysate to 4 ml reagent to make a hemoglobincyanide (HiCN) solution. Mix and stand for 10 minutes.

3.  Pipette 2.8 ml of HiCN solution into a test tube, add 0.2 ml of sodium hydroxide, mix well and start a stop-watch.

4.  At exactly two minutes add two ml of the saturated ammonium sulphate, mix well and allow to stand for five minutes to settle.

5.  Filter the solution through Whatman No 42 filter paper and collect the filtrate. Filter again if the filtrate is not absolutely clear.

6.   Add 3.5 ml distilled water to 0.1 ml of the HiCN solution as a standard.

7.  Read the absorbance of the filtrate and the standard in a spectrophotometer at 413 nm (alternatively 540 nm).

8. Calculate % Hb F = [A⁴¹³ test x 100] ¸ [A⁴¹³ standard x 20.16].

Interpretation:

Normal values for children at six months should be 2-3% or less, falling to less than 1% at one year; adults 0.2-1.0%.

  

•      Acid elution cytochemical method: sensitive procedure to identify individual cells containing Hb F even when present few.

•      Principle: Cells containing fetal hemoglobin are more resistant to acid elution than are normal cells. They appear as isolated, darkly red stained cells among a background of palely red stained ghost cells.

•      Carried out at pH 1.5

Interpretation:

•      Appear as isolated, darkly red stained cells among a background of palely red stained ghost cells in smear.

G6PD

•      G6PD-catalyses the oxidation of glucose-6-phosphogluconate with simultaneous reduction of dinucleotide phophate (NADP) to reduced NADP (NADPH):

      G6P+NADP                                 6PG+NADPH

                                                G6PD

G6PD deficiency leads to

a)     Mediterranean type-favism ( acute intravascular hemolysis following ingestion of broad beans)

b)    Type A in blacks-leads to primaquine sensitivity.    

1)    Fluorescent screening test for G6PD deficiency

Principle: NADPH generated by G6PD present in a lysate of blood cells, fluoresces under long wave ultraviolet (UV) light. In G6PD deficiency. There is inability to produce sufficient NADPH, this results in a lack of fluorenscene.

Interpretation: red cells with less than 20% of normal G6PD activity do not cause fluorescence.

2)Methemoglobin reduction test:

Principle: Sodium nitrite converts Hb to Hi(Methemoglobin). When no methylene blue is added, methemoglobin persists, but incubation of the samples with methylene blue allows stimulation of the pentose phosphate pathway in subjects with normal G6PD levels. The Hi is reduced during the incubation period. In G6PD-deficient subjects, the block in the pentose phosphate pathway prevets this reduction.

•      Interpretation:

1) Normal blood yields a color similar to that in normal reference( clear red).

2) Blood from deficient subject-brown color similar to that in deficient reference tube.

3) Cytochemical tests for demonstration of G6PD deficient cells

Red cells are treated with sodium nitrite to convert their oxyhaemoglobin (HbO₂) to methaemoglobin (Hi). In the presence of G6PD, Hi reconverts to HbO₂, but in G6PD deficiency, Hi persists. The blood is then incubated with a soluble tetrazolium compound (MTT), which will be reduced by HbO₂ (but not by Hi) to an insoluble formazan form.

Interpretation:

•      when G6PD activity is normal, all the red cells are stained.

•      In G6PD hemizygotes, majority of red cells are unstained.

•      In heterozygotes, usually 40-60% cells are unstained.