Protocol:F-cells by immunofluorescence

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).

Materials

 * 1) Cleaned microscope slides: use pre-washed and pre-cleaned slides from BDH Superfrost (Cat.no. 406/0169/02).
 * Clean slides with acetone followed by ethanol, and allow to air dry for at least 1 hour.
 * Prepare 4-5 very thin smears, one cell thick, per individual using 1 &mu;l of fresh blood on the cleaned slides. Allow to dry for at least two days, for best results dry over one week.
 * 1) Fixative: acetone/ethanol/methanol (6:2:2 v/v).
 * 2) Phosphate buffered saline (PBS) Sigma P-4417.
 * 3) Trypsin solution: trypsin 0.1% in calcium chloride (CaCl2) 0.1% pH 7.8.
 * Prepare 10 ml solution [10 mg of trypsin (ICN Cat.No.150213) +10 mg CaCl2 +10 ml distilled water] and store at –20oC in 1 ml aliquots.
 * Alternatively, use trypsin tablets (Sigma; Cat.no. T-7168). Dissolve 1 tablet in 1 ml deionised water, store at –20°C in 10 &mu;l aliquots. Before use, make up to 500 &mu;l (i.e. add 490 &mu;l) with deionised water. Pre-warm to 37oC before use. NOTE: pH of CaCl2 very important.
 * 1) Anti-&gamma; monoclonal antibody (Sigma T-6653): undiluted supernatant.
 * 2) Tetramethylrodamine Isothiocyanate (TRITC) conjugate anti-mouse IgG (Sigma T-6653).
 * Store frozen in 10 &mu;l aliquots. Working solution: dilute 1:32 in PBS.
 * 1) Humidity chamber: moist tissue in plastic chamber.
 * 2) Glycerol: PBS (1:1 v/v) or anti-fade (Vectashield, Mounting Medium for Fluorescence; Vector Cat. No. H-1000).

Method

 * 1) Mark on the smear a small area of approximately 5 mm diameter using a diamond cutter.
 * 2) Fix smears in acetone: ethanol: methanol fixative for 20 minutes. Air dry for 2 minutes (do not overdry).
 * 3) Re-hydrate in PBS for 5 minutes (use plenty of PBS in a large glass container) and then rinse very briefly in distilled water. Air dry.
 * 4) Cover the circled area with 8 &mu;l pre-warmed (37oC) trypsin solution and incubate at 37 °C for 15 minutes in a humidity chamber. NOTE: time of trypsinization varies with age of slides but generally 15 minutes is sufficient.
 * 5) Wash in PBS for 5 minutes with gentle agitation and rinse in distilled water. Air dry.
 * 6) Cover trypsinized area with 5-10 &mu;l anti-&gamma; antibody, incubate at 37oC in humidity chamber for 30-40 minutes.
 * 7) Wash as before and air dry.
 * 8) Cover circled area with 5-10 &mu;l fluorescent anti-mouse IgG and incubate at 37oC for 20-30 minutes. In the humidity chamber
 * 9) Wash as before with PBS, rinse in distilled water, air dry.
 * 10) Mount in Glycerol: PBS or anti-fade.

Interpretation and comments
We suggest ~1,000 red cells are counted, which corresponds to 4-5 high power fields. Adult normal values are 0.3 to 4.4% F-cells. Females have a higher number of F-cells than males. The number of fields, of course, depends on the density of cells. Hence, it is very important to master the art of making thin blood smears. Whole blood can be stored up to a week prior to making smears, but it is preferable to use fresh blood. The air-dried smears can be stored wrapped in tissue paper at room temperature for years.