section

Haematological procedures Haemoglobin analysis

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PROTOCOL Cellulose acetate electrophoresis

Cellulose acetate is an acetate salt of cellulose produced by treating cotton with acetic acid using sulphuric acid as a catalyst. Migration takes place on the buffer film on the surface of the cellulose acetate plate or membrane. Separation of the proteins is primarily by charge. Cellulose acetate electrophoresis may be used for qualitative identification of variants, but also with elution for quantitation of the haemoglobins, A2, A, S, D, Lepore, -chain variants, Hb H and Hb Bart's.

Reagents and materials a) Tris-EDTA Boric Acid (TEB) buffer, pH 8.4. Tris hydroxymethyl amino methane (TRIS): 10.2 g Ethylene diamine tetracetic acid (EDTA): 0.6 g Boric Acid: 3.2 g Make up to 1 litre with distilled water b) Whatman No. 3 chromatography paper. c) Cellulose acetate membranes supplied Schleicher and Schuell, 40x300 mm. Alternatives are made by Sartorius and by Shandon d) HbA2 control, as supplied by the National Institute for Biological Standards The control has been produced by freeze drying a solution of haemoglobin prepared from human cells and made stable by the addition of sucrose (200 mM), potassium cyanide (6mM) and chloramphenicol (1 mg/dl). This control was established by the World Health Organization in 1994 as the international and Control NIBSC.

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Reference Reagent with an assigned value of 5.3% of total haemoglobins present.

Equipment (i) (ii) (iii) (iv)

Power supply capable of delivering a constant current, 0-80 mA and up to 400 volts. An horizontal electrophoresis tank with adjustable bridge gaps and a polarity indicator (eg. Shandon). Roller mixer. A single beam SP6-200 - Spectrophotometer Pye Unicam.

Method 1. Haemolysate is prepared from whole blood (in K2EDTA) as previously described. 2. The electrophoresis tank is prepared by filling the tank with 900ml approximately of TEB buffer wicks are cut from Grade No. 3 chromatography paper and were placed along the 22 cm long bridges in the tank. 3. The cellulose acetate membranes are cut in 40xl00 mm each and soaked (shiny side down) in TEB buffer for 5 minutes. Five strips are plotted and placed on the electrophoresis tanks. 4. Voltage current is applied at 250 V for 5 minutes to the membranes to equilibrate the membranes with the buffer. 5. The current is turned off and 8-10 l haemolysate (10 g/l) is applied on each membrane at the cathodal end using a capillary tube. 6. Then the voltage is set at 250-300 V working at constant current of 2 mA for 7. The electrophoresis is run for approximately 45 minutes to one hour until there is a clear area between the bands. 8. The current is then turned off and the separated HbA2 on the cellulose acetate membrane is cut and immersed in a tube containing 4 ml of distilled

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each strip.

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water and the HbA in a tube containing 16ml distilled water.

If a

haemoglobin variant is present then this is cut separately into 4 or 16 ml distilled water depending on the quantity of the variant present. Note that a blank is prepared from the same run by cutting a piece of clear cellulose acetate strip that was immersed in 4ml distilled water. 9. The tubes are then placed on a roller mixer for 30 minutes for the haemoglobin elution. 10. The strips are removed and the tubes are then centrifuged for 10 minutes at 3.000 rpm. 11. The absorbance of each haemoglobin is read at 413 nm against the blank on a spectrophotometer.

Interpretation The results are calculated as follows: Absorbance of HbA2 x 100 % of HbA2 = -------------------------------------------------Abs. of Total (HbA x 4) + absorbance of HbA2 The haemoglobins migrate on the cellulose acetate membrane from cathode to anode in the following order: HbA2, Hb E, Hb C, Hb D, Hb S, Hb Lepore, Hb F, Hb A and the fast moving haemoglobins Bart's and Hb H (Fig 3.2). The normal range for HbA2 is 2.4 to 3.2% Factors affecting the result are: correct pH, correct concentration of the buffer and the temperature of the buffer, which may be influenced by the voltage or the electrophoretic tanks with buffer refrigerated at 4 oC. Ideally the method should be conducted at an environmental temperature below 23 oC. It is necessary, therefore, in warm climates to have air conditioning in the laboratory. The quality of the carrier membrane must be good and poor quality should be recognised and discarded. The membrane should be kept moist. environmental temperature. It is advisable, especially in hot climates to keep

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References
1. 2. Lafferty, J. (1999) College of America Pathologists hemoglobinopathy survey HG-B.Chicago, IL: College of American Pathologists. Cotton, F., Lin, C., Fontaine, B., Gulbis, B., Janssens, J., Varteongen, F. (1999) Evaluation of a capillary electrophoresis method for routine determination of hemoglobins A2 and F. Clinical Chemistry, 45,237-243. Mario, N., Baudin, B., Aussel, C., Giboudeau, J.(1997) Capillary isoelectric focusing and high performance cation-exchange chromatography compared for the qualitative and quantitative analysis of hemoglobin variants. Clinical Chemistry, 43, 2137-2142. Efremov, G.D., et al. (1974) Microchromatography of Hemoglobins.II. A Rapid Microchromatographic method for the determination of hemoglobin A2. J.Lab. Clin., 83, 657. Galanello, R.,Melis, M.A., Muroni, P., Cao, A., (1977) Quantitation of Hb A2 with DE-52 microchromatography in whole blood as screening test for beta-thalassemia heterozygotes. Acta Haematologica, 57(1),32-36. Galanello, R., Barella, S., Gasperini, D., Perseu, L., Paglietti, E., Sollaino, C., Paderi, L., Pirroni, M.G., Maccioni, L., Mosca, A. (1995) Evalution of an automatic HPLC analyser for thalassemia and haemoglobin variants screening. Journal of Automatic Chemistry, 17(2), 7376.

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7. Clarke, M.C., Higgins, T.N. (2000) Laboratory investigation of hemoglobinopathies and thalassemias: review and update. Clinical Chemistry, 46(8), 1284-1290.

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8. Clarke GM, Higgins TN. Laboratory Investigation of Hemoglobinopathies and Thalassemias: review and update. Clinical Chemistry 46:1284-1290; 2000. 9. Lorey F, Cunningham G, Vichinsky EP, Lubin BH, Witkowska HE, Matsunaga A, Azimi M, Sherwin J, Eastman J, Farina F, Waye JS, Chui DH. Universal newborn screening for Hb H disease in California. Genet. Test; 5:93-100, 2001.

10. Galanello R, Barella S, Gasperini D, Perseu L, Paglietti E, Sollaino C, Paderi L, Pirroni MG, Maccioni L, Mosca A. Evaluation of an automatic HPLC analyser for thalassemia and haemoglobin variants screening. Journal of Automatic Chemistry, 17:73-76, 1995.

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