Haemoglobin Content of Individual Erythrocytes in Normal and Abnormal Blood

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Abstract
Summary. Procedures were developed for the measurement of the haemoglobin content of individual unstained human erythrocytes in blood smears, using a Vickers M85 scanning and integrating microdensitometer. Possible errors due to instrumental factors were investigated. Spatial distribution error was found to be unimportant when using a flying spot 1.8 μm diameter in the specimen plane. Measurements of an oxyhaemoglobin solution adhered to Bouguer's Law (i.e. absorbance was proportional to pathlength); microdensitometric results obtained at a given wavelength and with a given monochromator bandwidth could be corrected to correspond to λmax and minimal bandwidth by multiplication by an empirical constant.Seventy cells in each of nine control and 10 pathological blood samples were measured. Microdensitometric estimates of the MCH correlated well with those obtained by conventional methods, although the former were slightly higher.The mean coefficient of variation of the haemoglobin contents of the control samples was 14.1%, considerably higher than the instrumental uncertainty (under 4%). Seven of the nine control samples exhibited statistically normal distributions without significant skewness or kurtosis (skewness referring to an asymmetrical distribution around the mean, and kurtosis referring to an excess or deficit of observations in the flanks of the distribution curve); the remaining two samples may not have been completely normal in the biological sense. Seven of the 10 pathological samples had significant skewness and/or kurtosis, and only one pathological sample showed no abnormal parameter (MCH, coefficient of variation, skewness or kurtosis). Bimodal distributions appeared to be present in some cases.Microdensitometry of individual erythrocytes yields information not otherwise obtainable, and may prove valuable in the research investigation of a variety of blood conditions.