Clinical Chemistry, Vol 29, 2022-2025, Copyright © 1983 by American Association for Clinical Chemistry

Spectrophotometry of occult blood in feces

CL Welch and DS Young

This method for measuring fecal occult blood is based on the heme- catalyzed oxidation of tetramethylbenzidine by H2O2. An aliquot of heated stool homogenate is mixed with acetic acid to chemically separate heme from globin. The heme is extracted into ethyl acetate and reacted with the reagent and H2O2 to produce a green oxidation product. The reaction is followed kinetically for 30 to 60 s at 660 nm. A660 is linearly related to the amount of hemoglobin. The lower limit of detection is 1 to 2 mg of hemoglobin per gram (wet weight) of feces. Within-day precision (CV) of the analysis for hemoglobin added to stool specimens (4 to 30 mg/g) ranged from 2.3 to 7.6%, between-day CV from 2.1 to 8.1%. Analytical recovery of hemoglobin added to fecal specimens (4 to 30 mg/g) ranged from 86.7 to 106.2%. Of the substances known to interfere with conventional dye-oxidation tests for fecal occult blood, only myoglobin and ascorbic acid interfere with hemoglobin quantification by our procedure. The test is fast, inexpensive, and easy to perform, and involves equipment available in hospital laboratories.