Screening hypochromism (sieve effect) in red blood cells: a quantitative analysis.

K Razi Naqvi

doi:10.1364/boe.5.001290

Abstract

Multiwavelength UV-visible spectroscopy, Kramers-Kronig analysis, and several other experimental and theoretical tools have been applied over the last several decades to fathom absorption and scattering of light by suspensions of micron-sized pigmented particles, including red blood cells, but a satisfactory quantitative analysis of the difference between the absorption spectra of suspension of intact and lysed red blood cells is still lacking. It is stressed that such a comparison is meaningful only if the pertinent spectra are free from, or have been corrected for, scattering losses, and it is shown that Duysens' theory can, whereas that of Vekshin cannot, account satisfactorily for the observed hypochromism of suspensions of red blood cells.

Highlights

When an absorbing-and-scattering specimen is investigated by using a standard absorption spectrophotometer, a measurement of the attenuance lg(P0/P) does not provide the true absorbance of the sample, and the measured attenuance depends on the acceptance angle of the instrument; here P0 denotes the output of the detector when it views the collimated incident beam leaving the reference cell and P is the output when the cell is filled by the sample under investigation
The purpose of the present paper is to point out that hypochromism, properly so called, should be used when one compares two spectra neither of which suffers from scattering losses, and to show that Duysens’ theory of screening hypochromism can be applied, in its original form [5] or in a trivially amended fashion [6], to understand the hypochromism of red blood cells
A different expression for the ration E /E was proposed by Vekshin [13, 14]; though his model can account for the observed hypochromism of red blood cells, it is physically unreasonable because the best fit between the model and the experimental data leads to the conclusion that the number of heme subunits per cluster is smaller than ten [14]

Summary

Introduction

When an absorbing-and-scattering specimen is investigated by using a standard absorption spectrophotometer, a measurement of the attenuance lg(P0/P) does not provide the true absorbance of the sample, and the measured attenuance depends on the acceptance angle of the instrument (or the sample-to-detector ratio); here P0 denotes the output of the detector when it views the collimated incident beam leaving the reference cell and P is the output when the cell is filled by the sample under investigation. An independent approach, utilizing similar ingredients, was presented some years later by Nonoyama and coauthors [4], who addressed an issue not treated in the earlier work, namely the lower absorbance (or hypochromism) of a suspension of pigmented cells as compared to the absorbance recorded by disrupting the cells and allowing the pigments to disperse throughout the sample holder. They commented that “this perceived hypochromism can be accounted for by considering two important issues: the acceptance angle of the instrument and the combined scattering and absorption effect of light on the particles”. The purpose of the present paper is to point out that hypochromism, properly so called, should be used when one compares two spectra neither of which suffers from scattering losses, and to show that Duysens’ theory of screening hypochromism can be applied, in its original form [5] or in a trivially amended fashion [6], to understand the hypochromism of red blood cells

Theoretical background

Flattening in the spectrum of red blood cells

Concluding remarks