Clinical Chemistry, Vol 21, 362-369, Copyright © 1975 by the American Association for Clinical Chemistry

Use of Leuco-Dyes in the Quantitative Colorimetric Microdetermination of Hemoglobin and Other Heme Compounds

¹ Department of Medicine, College of Medical Sciences, University of Minnesota, Minneapolis, Minn. 55455.

S. S., at the Department of Medicine, University of Minnesota Medical School, Mayo Memorial Bldg., Box 291, Minneapolis, Minn. 55455.

Sensitivity, stability, and specificity of the color-producing reaction of hydrogen peroxide with benzidine, leucomalachite green, or o-dianisidine were tested in numerous systems containing hemoglobin and other hemoproteins. Use of urea or low temperature (to -12 °C), or both, was highly beneficial, especially with leuco-malachite green, for which the color reaction was stable, after about 15 min, for longer than 24 h, with a colorless blank. Absorbance was 0.3 at a final hemoglobin concentration of 0.27 mg/liter. Nonspecific color produced by substances such as FeCl₃ and ascorbic acid was completely eliminated. Of the three leuco-dyes studied, only benzidine yielded a completely linear calibration curve, but its relative instability and reported carcinogenicity are serious disadvantages. No system tested eliminated completely the known inhibition by plasma of the peroxidase activity of these leuco dyes.

Submitted on October 22, 1974
Accepted on December 13, 1974