| | Re: Hemoglobin detection at 540 nm or 430 nm
The haemoglobins variants are separated electrophoretically on cellulose acetate plates in alkaline buffer medium. After separation, the fractions are fixed, stained and destained, in order to visualize the 2 haemoglobins variants: HbA, HbA2 and in case the pathological variants HbF and HbS, and finally the bands eluted in ammonia solution and Abs read at 546 nm
The red blood cells, from whole blood in EDTA-K3 routinely used as anticoagulant, are preliminary washed a few times with saline solution. To the last wash, in a tube add to 1 part of packed erythrocytes with 3 parts of lysing solution (hypotonic phosphate buffer 0.0132 M at pH 7.2, contained 1% Triton X-100 and 0.02% NaN3). Shake the tube on a vortex until the haemolysis is complete. In alternative, wash red cells 3 times in 150 mM NaCl and lyse mixing 50 µl of erythrocytes package with 300 µl complexone III hypotonic solution (4.83 mM) and freeze at -20 °C for 30 minutes. To obtain a better standardization of the method, measure the concentration of the Hb in the haemolysate obtained with a blood cell counter and dilute, with distilled water, in order to obtain a final concentration of Hb of 4 gr/dl. This can be obtained using the following formula : Final volume to achieve = Hb conc. g/dl x 0.5 ml blood volume used/4 g/dl Hb to achieve. Electrophoresis of hemolysate is carried out on cellulose acetate and running in hemoglobin buffer (0.17 M Tris, 0.45 mM EDTA, 40 mM glycine, 43 mM sucrose, pH 9.2), at 230 - 250 Volts, for 30-40 mins at room temperature. Bands are stained with 0.2% (w/v) Ponceau S in 3% (w/v) trichloroacetic acid, and after 3 - 5 min, rinsing in destaining solution (0.05 M [9.6 g/L] citric acid and 0.05 M [7.1 g/L] sodium phosphate dibasic and 0.01 g/L [0.16 mM] of NaN3). After drying, strips were cut into portions, HbA, HbA2, and blank (portion of the strip of about the same dimensions as the eluted bands), and in case the pathological variants HbF and HbS , and eluted with eluting solution (dilute 2 ml of 14.3 N ammonium hydroxide to 100 ml with dH2O) dispensing 5 ml in tube containing the HbA component, and 1 ml to those containing the A2 fraction and the blank portion. The tubes are agitated vigorously on a vortex mixer to complete elution; only dye is eluted, the hemoglobin pigment remaining in the membrane. The pieces of membrane are separated from eluition solution through a briefly centrifugation and as to sediment this remaining. The supernatant fluid is transferred to a spectrophotometer cell and the absorbance measured at 546 nm (540-550). The percentage of HbA2 hemoglobin is determined by the following equation:
---------------------------- x 100
(0.2*[x-b]) + (y - [b/5])
where : x = AbsHbA2; y = AbsHbA; and b = Abs blank.