Hemoglobin determined in 15 microL of whole blood by "front-face" fluorometry.

Abstract

We describe the rapid, inexpensive fluorometry of hemoglobin in undiluted whole blood. The procedure consists only of adding a standard quantity of a fluorescent dye to a measured volume (approximately 15 microL) of blood, together with some solubilizing detergent. The assay is based on the attenuation of the dye's fluorescence (excited within the region 400--440 nm) that results from the competitive absorption of exciting light by the hemoglobin present--the "inner filter effect." The wavelength that one can use is optional and will determine which dyes can be used. Measurements are made with the hematofluorometer, a "front-face" filter fluorometer (Blumberg et al., J. Lab. Clin. Med. 89: 712--723, 1977). We demonstrate the validity of the method for two dyes, rhodamine B and fluorescein dibutyrate, which we used with hematofluorometers that were designed to determine blood zinc protoporphyrin and bilirubin, respectively. Our method exhibited a standard error of about 4 g of hemoglobin per liter vs the comparison method (Coulter Counter method, for which the CV is 1.2%). The CV is about 3%. The method seems appropriate for "field" use (i.e., use outside the laboratory) in anemia-screening programs.