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Updated: Jul 28, 2026
A Rapid and Chemical-free Hemoglobin Assay with Photothermal Angular Light Scattering
Published on: December 7, 2016
Mechanism of interference of a polymerized hemoglobin blood substitute in an alkaline phosphatase method
J J Chance1, E J Norris, M H Kroll
1Division of Clinical Chemistry, Department of Pathology, Johns Hopkins University School of Medicine, 600 N. Wolfe St., Baltimore, MD 21287-7065, USA.
Background:
Hemoglobin-based oxygen carriers can cause profound interferences in many analytical procedures. We determined the mechanism of interference in the assay of alkaline phosphatase activity and identified approaches that might be used to correct for this interference.
Methods:
Interference of a polymerized hemoglobin blood substitute with the assay of alkaline phosphatase was examined with a Hitachi 917 analyzer and ultraviolet-visible spectrophotometry.
Results:
Hemoglobin-based oxygen carrier solutions had substantial absorbance at 415 nm, the wavelength of analysis used to measure the formation of 4-nitrophenol. In addition to offsetting the initial absorbance at the analytical wavelength, polymerized hemoglobin gave rise to a strong negative interference plot because of alkali denaturation of the substitute. The same interference mechanism was also observed for native hemoglobin (hemolysate), indicating that the interference was not derived from the polymerization process. The interference can be corrected by implementing a rate-correction procedure, or the interference can be avoided by measurement at 450 nm.
Conclusions:
The interference of polymerized hemoglobin in the alkaline phosphatase assay is a result of an absorbance offset caused by alkali denaturation of hemoglobin. The interference can be corrected or avoided by modifying the calculation or the analytical wavelength. The correction strategy may also be applicable to improving the hemolysis index for this method.
