hemoglobin electrophoresis

Different isoelectric points of various hemoglobin have different positive and negative charges in a certain pH buffer, and the hemoglobin moves in different directions after electrophoresis. Basic Information Specialist classification: growth and development examination classification: blood examination Applicable gender: whether men and women apply fasting: fasting Tips: Before the examination, the diet is light and alcohol is prohibited. Normal value The electrophoresis method was HbA 95%, HbA 21.1% to 3.2%, and HbA 32% to 3%. The Singer method has a HbF < 4%. Clinical significance 1, the main component is HbS, and no HbB, can be seen in sickle cell hemoglobin disease. 2, HbS, HbF and HbA2 increased slightly, while HbA rarely or disappeared, combined with the investigation can be diagnosed as HbS-β-marine anemia, more common in the Mediterranean. 3, in addition to HbS, containing 10% -30% HbA and slightly increased HbF and HbA2, HbS-β-marine anemia can also be considered. 4. HbS accounts for 20%-30%, HbA accounts for 65%-75%, and contains normal HbF and HbA2, which can be diagnosed as HbS-α-marine anemia. 5, HbA disappears, HbC accounts for 28%-44% of total hemoglobin, can be diagnosed as hemoglobin disease, more common in black people, more target cells can be seen in blood. 6, there is HbS, HbD also appears, there are target cells in the blood film, can be diagnosed as hemoglobin D disease, more common in northern China. 7, HbE results in electrophoresis, can be diagnosed as hemoglobin E disease, mainly found in Southeast Asia, India, etc., China is more common in southern Guangdong. Precautions Waxy acid fiber membrane electrophoresis direct colorimetry Reagent: Electrophoresis with micro-hemoglobin of cellulose acetate membrane. operating: (1) The cellulose acetate membrane was cut into 1/3 (4 cm × 8 cm) in TEB buffer for 10 min or more. (2) Remove the cellulose acetate film and use a filter paper to remove excess water. 10 μl of 100 g/L dissolved blood was absorbed by a Hb pipette and uniformly applied to the lower edge of the glass pusher, and was placed on the matte side at a distance of 1.5 to 2.0 cm from the cathode end of the film. Make two copies of each specimen at the same time. (3) Electrophoresis: The buffer is electrophoresed with a trace amount of hemoglobin. The voltage is 180V and the time is 40min~1h. (The cells are clearly separated in each zone.) After the electrophoresis, the electrodes are exchanged, and the buffer can be used repeatedly. (4) Elution and quantification HbA, HbA2 and a control zone corresponding to the size of HbA2 were separately cut. If abnormal Hb zones (HbX) are also cut at the same time, they are placed in the corresponding tubes. Add 10 ml of TEB buffer to the HbA tube, add 2 ml of TEB buffer to each tube, soak for 30 min, shake constantly. After being completely eluted. The eluate was mixed and 721 spectrophotometer with a wavelength of 413 nm. The blank is zeroed and the absorbance of each tube is measured. Inspection process (1) Electrophoresis of trace hemoglobin: 1 Dip film: The cellulose acetate film is floated on the surface of TEB buffer, and it can be used for at least 20 minutes after it is evenly soaked and sunk. This prevents bubbles from being generated. 2 Dot: Remove the cellulose acetate film and use filter paper to remove excess water. The hemoglobin solution was aspirated in a micropipette and placed in a concave groove of a multi-sample applicator. The hemoglobin solution was taken by a multi-sample pipette and spotted on a matte side of the cellulose acetate film. 1.5 to 2 cm from the cathode end, the amount of spotting is about 3 to 5 μl, and the normal hemoglobin solution at the parallel position is used as a control. 3 Electrophoresis: Add an equal amount of BB buffer solution in the buffer tank on both sides of the micro-electrophoresis tank, and use a two-layer filter paper as a salt bridge on the cellulose acetate membrane support. The film was matte to the bottom, and the negative electrode was terminated by a spot and equilibrated for 5 min. Turn on the power. The voltage is 180V, the current amount is about 0.2mA/cm, and the electrophoresis time is 20-30min. The buffer in the tank can be used once more after switching the electrodes. 4 Staining: The electrophoresed film was taken out in the Ponceau red staining solution for about 10 minutes, and rinsed with 3% acetic acid several times until the background was white and dried. 5 Transparent: The cellulose acetate film is soaked in a transparent liquid, adhered to a clean glass plate, and the bubbles between the two are removed by a glass rod. A small amount of glacial acetic acid was poured into a chromatography cylinder, and the glass plate was reversely covered on the chromatography cylinder (with the film facing inward). Smoke with volatile glacial acetic acid for 10 to 20 minutes, and remove the film when it is transparent. (2) Cellulose acetate membrane electrophoresis staining colorimetric method: 1 Remove the 4 cm × 6 cm cellulose acetate membrane soaked in TEB buffer, and blot excess water with filter paper. At a distance of 1.5 cm from the cathode end of the matte surface, about 5 μl of a 50 g/L Hb solution was linearly and uniformly packed with a hemoglobin pipette. After the hemoglobin solution penetrated into the membrane, the membrane was inverted onto a filter paper bridge of the electrophoresis tank support plate. 2 Electrophoresis: voltage 180V, time 30~40min, subject to complete separation of Hb zones. The electrode is exchanged after electrophoresis, and the electrophoresis buffer can be used multiple times. 3 Dyeing: The film is immersed in the dyeing solution. It needs to be dyed for 2 hours in winter and 25~30°C in the summer. It can only be dyed for about 30 minutes. Note that if the staining is not transparent (such as the time is too short or the hemoglobin solution is too concentrated), or the voltage is too high, the HbA band is too concentrated, and the ribbon is easily peeled off, which affects the accuracy of the quantification. Wash with bleaching solution several times until the background is rinsed. 4 Quantification: The rinsed membrane was taken out, and each zone was cut, and a control zone corresponding to the size of HbA2 was placed in each corresponding test tube. 10 ml of the leachate was added to the HbA tube, and the remaining tubes were immersed in 2 ml, soaked for 20 minutes, and shaken from time to time. Make the ribbon completely elute (note that at higher temperatures, the elution time should not be too long, otherwise the eluent blue will decrease and gradually turn purple). The eluate was mixed, and the absorbance of each tube was measured by using a 721 spectrophotometer at a wavelength of 620 nm and zeroing with a blank. 5 calculation: the same direct colorimetry. Waxy acid fiber membrane electrophoresis direct colorimetry Reagent: Electrophoresis with micro-hemoglobin of cellulose acetate membrane. operating: (1) The cellulose acetate membrane was cut into 1/3 (4 cm × 8 cm) in TEB buffer for 10 min or more. (2) Remove the cellulose acetate film and use a filter paper to remove excess water. 10 μl of 100 g/L dissolved blood was absorbed by a Hb pipette and uniformly applied to the lower edge of the glass pusher, and was placed on the matte side at a distance of 1.5 to 2.0 cm from the cathode end of the film. Make two copies of each specimen at the same time. (3) Electrophoresis: electrophoresis of buffer and micro hemoglobin. The voltage is 180V and the time is 40min~1h. (The cells are clearly separated in each zone.) After the electrophoresis, the electrodes are exchanged, and the buffer can be used repeatedly. (4) Elution and quantification HbA, HbA2 and a control zone corresponding to the size of HbA2 were separately cut. If abnormal Hb zones (HbX) are also cut at the same time, they are placed in the corresponding tubes. Add 10 ml of TEB buffer to the HbA tube, add 2 ml of TEB buffer to each tube, soak for 30 min, shake constantly. After being completely eluted. The eluate was mixed and 721 spectrophotometer with a wavelength of 413 nm. The blank is zeroed and the absorbance of each tube is measured. Not suitable for the crowd No taboos. Adverse reactions and risks Risk of infection: If you use an unclean needle, you may be at risk of infection.