Experimental principle
Hemoglobin has peroxidase-like activity, which can catalyze the release of new ecological oxygen by H2O2, and the color change of o-methylbenzidine is oxidized from colorless to blue-violet. According to the color depth and the standard, the content can be measured.
experimental method
material:
1.2g/L o-methylbenzidine solution
2.1% H2O2
3.10% acetic acid solution
4. Spectrophotometer
method:
1. Take 2-3 ml of venous blood, anticoagulate with sodium citrate, and centrifuge to separate plasma.
2. The operation was carried out according to the following table, and the absorbance of the measuring tube was measured by using a spectrophotometer at a wavelength of 530 nm and zeroing with a blank tube.
3. Calculation formula
Reagent (ml) | Measuring tube | Blank tube |
2 g/L o-methylbenzidine solution | 0.5 | 0.5 |
Patient plasma | 0.02 | / |
1% H 2 O 2 | 0.5 | 0.5 |
Mix and leave at room temperature for 10 min. | ||
10% acetic acid solution | 5.0 | 5.0 |
Leave at room temperature for 10 minutes | ||
3. Calculation formula
Plasma free Hb (mg / L) = measuring tube absorbance / standard tube absorbance × 100
Experimental result calculation
Reference range: 0-40mg/L
Precautions
1. All containers avoid hemoglobin contamination;
2. The blood collection should be smooth, the utensils should be dry, the plasma should be fresh, and separated in time to prevent artificial hemolysis;
3. The o-benzidine solution is carcinogenic, and care should be taken when testing. The waste liquid should be disposed of properly to avoid contamination;
4. The hydrogen peroxide should be freshly configured and the concentration should be accurate.
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