Protocol:F-cells by acid elution technique

Hb F is not uniformly distributed among red cells, except in the condition of deletional HPFH. Cells with detectable amounts of Hb F are called F-cells and they can be detected on blood smears by two techniques: the acid elution test of Kleihauer (13,14), and the immunofluorescence test using specific anti-Hb F monoclonal antibodies (15,16). Methods of staining red cells in suspension have been developed making possible the quantitation of erythrocytes by flow cytometry (17). Quantitative evaluation of F-cells may be useful to screen for HPFH, to monitor F cells in patients with sickle cell anaemia treated with hydroxyurea and to detect fetal cells in adult blood such as in case of fetal-maternal haemorrhage. The performance of different methods of flow cytometry for F cell counting has been recently reported (18).

Principle
The acid elution technique is based on the differential elution of fetal and adult haemoglobins from ethanol fixed red blood cells at acid pH (14).

Reagents

 * 1) Citric-acid phosphate buffer pH 3.3
 * Solution A: (citric acid 0.1 M): 21.01g of citric acid in 1 litre of distilled water.
 * Solution B: (NaH2PO4 0.2 M): 35.6 g NaH2PO4.2H2O in 1 litre of distilled water.
 * Mix 73.4 ml of solution A with 26.6 ml of solution B; check pH and adjust if necessary to pH 3.3.
 * 1) Ethanol: 80 % vol.
 * 2) Stains:
 * 3) 0.1% of erythrosine in water.
 * 4) Erlich's acid haematoxyline: dissolve 4.0 g of crystalline haematoxyline in 200 ml of ethanol 95 % vol and add 8 ml of 10% sodium iodate. Add 200 ml of water and boil the mixture. Cool and add 200ml of glycerin 6.0 g of aluminium ammonium sulphate and 200 ml of glacial acetic acid. Allow to stand the solution for at least 14 days.

Complete kits of reagents are commercially available.

Method

 * 1) Blood in any anticoagulant. Make thin smears and air dry for 10 to 60 minutes.
 * 2) Fix in ethanol 80 vol% for 5 minutes at 20-22oC.
 * 3) Rinse the smears with tap water and air dry.
 * 4) Stain with (a) for 3 minutes, then rinse with water and air dry.
 * 5) Counter-stain with (b) for 3 minutes.
 * 6) Rinse with tap water, air dry.
 * 7) Examine under light microscopy without oil immersion.

Interpretation
F cells are densely stained with erythrosine. Cells containing Hb A appear as ghost cells.

Normal values for adults are below 0.01%.

Comments
The method is not quite sensitive and gives too low values. Hb F at low concentration is eluted from red cells together with Hb A. Pink stained cells are of difficult interpretation.