Microspectrophotometric studies of Romanowsky stained blood cells

Abstract

SUMMARY This paper describes a comparison of the performance of two standardized Romanowsky blood stains, namely those of Marshall et al. and Wittekind et al., both containing azure B and eosin alone. Stain performance is assessed objectively by the use of three complementary techniques, all based on the visible absorbance spectra of stained cellular substrates. The first of these techniques is a simple comparison of the shapes and heights of the absorbance spectra. The second technique uses the CIE Colorimetric System, and thus permits the quantitation of colour in a manner that agrees with human observation. CIE co‐ordinates (chromaticity points, luminance) are calculated directly from absorbance spectra. The third technique is that of spectral subtraction, which yields a set of factors which describe the quantities of component dyes which are bound by the object. This technique, unlike the other two, requires a priori knowledge of the dyes used in the stains, and their spectra when bound to cellular substrates. Although the differences between the two methods are subtle, and hard for the subjective observer to define, the objective methods described here do show statistically significant differences. Wittekind's stain produces less intense staining, except for lymphocyte and monocyte cytoplasms. To the human eye, the differential coloration of these two substrates is more pronounced, but the difference between all nuclei and cytoplasms is less marked. The major difference in the uptake of dye components is in the small quantities of eosin dimer that are bound in this technique.