Measurement of plasma free hemoglobin can be used to (1) approximate the value of red blood cells used. (2) Elevations and decreases in hemoglobin can be referenced to the clinical significance of erythrocyte elevations and decreases.

Operation method

Measurement of plasma free hemoglobin

Principle

Hemoglobin has peroxidase-like activity, which catalyzes the release of new ecological oxygen from H2O2, causing a color change in the oxidation of o-toluidine from colorless to eventually blue-violet. The content can be measured by comparing with the standard according to the depth of color development.

Materials and Instruments

Venous blood samples
o-Toluidine solution H2O2 Acetic acid solution Sodium citrate
Spectrophotometer Centrifuge

Move

1. 2-3ml of venous blood was withdrawn, anticoagulated with sodium citrate and centrifuged to separate the plasma.

2. Use spectrophotometer with a wavelength of 530nm, adjust the zero with a blank tube and determine the absorbance of the measuring tube.

3.Calculation formula

Plasma free Hb (mg/L) = absorbance of measuring tube / absorbance of standard tube × 100

Calculation of experimental results

Reference range: 0-40mg/L

Caveat

1. all containers to avoid hemoglobin contamination;2. blood collection should be smooth, vessels should be dry, test plasma should be fresh and separated in time to prevent artificial hemolysis;3. o-Benzidine solution is carcinogenic, be careful when testing, waste liquid should be properly disposed of to avoid contamination;4. Hydrogen peroxide should be freshly prepared and the concentration should be accurate.

Common Problems

Calculation of experimental results

Reference range: 0-40mg/L

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