Screening for abnormal hemoglobin

Summary

This test is primarily used to screen for abnormal hemoglobin.

Operation method

Acetate film electrophoresis

Principle

Hemoglobin is a binding protein that consists of hemoglobin and bead protein. Normal human beings contain six peptide chains of hemoglobin: a, b, d, e, g and z. The genes for these six peptide chains are expressed at different stages of human development. The genes for these six peptide chains are expressed differently at different stages of human development. There are three types of hemoglobin in normal adult erythrocytes, namely, HbA, HbA2, and HbF. HbA (a2b2) is the most dominant hemoglobin in normal adult erythrocytes, accounting for more than 96% of the total hemoglobin in erythrocytes, with a PI=6.7. HbA2 (a2d2) is the second largest component of normal adult erythrocytes, accounting for about 2-3% of the total hemoglobin, with a PI=6.7. HbF ( a2g2) is the second largest component of normal adult erythrocytes, accounting for about 2-3% of the total hemoglobin. HbF(a2g2) is fetal hemoglobin, which decreases sharply and continuously to less than 1% of total hemoglobin six months after birth, and its PI is close to 6.7. All kinds of hemoglobins are negatively charged in buffer solution at pH 8.5, and all of them are moving toward the anode in the electric field, but they have different speeds due to the difference in the number of charges at different iso-electric points. HbA has the fastest swimming speed towards the anode because it has the most charges, and the color of the zone with the most content is the darkest. HbA has the darkest color and is followed by a lighter zone called HbA2. HbF is close to the isoelectric point of HbA and is usually inseparable from HbA. Anomalous hemoglobins are red proteins that are structurally abnormal due to genetic mutations. Anomalous hemoglobins have electrophoretic behaviors that are different from those of normal hemoglobins, so they can be detected by electrophoresis. For example, HbS is an abnormal hemoglobin formed by the replacement of glutamic acid by valine at position 6 of the b-chain. This variant has two fewer negative charges than HbA, and therefore moves toward the anode more slowly than HbA, appearing between HbA and HbA2. There are many electrophoretic methods for analyzing hemoglobin, such as filter paper electrophoresis, starch gel electrophoresis and polyacrylamide gel electrophoresis. However, the most commonly used method for screening abnormal hemoglobin is acetate film electrophoresis. Because the acetate fiber film to do support body electrophoresis operation is simple, fast electrophoresis speed, strong resolution zone band concentration and the dye is not adsorbed, easy to quantify. The detection rate of abnormal hemoglobin screening with cellulose acetate film electrophoresis is 1~2‰.

Materials and Instruments

Blood Samples
TBE buffer night Boric acid buffer Lichtenstein staining solution Rinsing solution
Electrophoresis apparatus Electrophoresis tank Cellulose acetate film

Move

1. Membrane immersion: Place the cellulose acetate film membrane side down into the immersion solution, soak it through and then press it into the bottom of the liquid with ophthalmic forceps.

2. Blood collection: Disinfect the earlobe area with iodine vapour, collect blood with a disposable blood collection needle and suck the blood on the tip of the twice-folded filter paper.

3. Sample addition: place the soaked cellulose acetate film membrane face up on the filter paper, use the filter paper to absorb the excess liquid, and use the blood-soaked filter paper to paint dots (about 0.2cm in diameter) or lines at 1.5cm from one side.

4. Electrophoresis: put the cellulose acetate film membrane with added samples face downward on the buffer gauze in the electrophoresis tank, put the added sample end on the negative pole, and turn on the power supply after 2 minutes of resting, adjust the voltage to 200 volts, and electrophoresis for 40 minutes.

5. After the end of electrophoresis, turn off the power, observe the electrophoresis pattern, and record the red zone (a few) and distribution (position).

6. Staining: Place the cellulose acetate film into the staining solution for 2 minutes, then transfer it to the rinsing solution and wash it until the back bottom is white, then observe the results.

Caveat

1. The cellulose acetate membrane must be soaked through (no white spots), before adding samples from the soaking solution, both to absorb the excess liquid can not make the membrane too dry, otherwise it will affect the electrophoresis results.

2. When adding samples, pay attention to be added to the membrane surface of cellulose acetate, electrophoresis should also be placed on the buffer gauze in the electrophoresis tank with the membrane face down.

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