Evaluation and calibration of a microphotometric method for hematocrit determination in microvessels of transilluminated tissues is described. This method is based on the relation of the optical density (OD) of a microvessel to its hematocrit (Hct). The following requirements of the microphotometric system appear essential: narrow-band monochromatic light source with efficient false light suppression, high numerical aperture of the objective and low numerical aperture of the condensor. We used a video system to measure the intensities of incident (Io) and transmitted (I) light. For converting of Io and I into OD values, correcting procedures were evaluated to eliminate the influence of glare, shading, and fading. The calibration procedure was performed with glass tubes of inner diameter (ID) between 13 and 68 microns perfused with red cell suspensions. A function was fitted to the data, correlating OD to ID and Hct. The standard deviation of the original data from this function is +/- 0.02 units of fractional hematocrit. The presented method allows the continuous determination of the hematocrit in a microvessel as well as the off-line evaluation of the hematocrit distribution within a microvessel network.
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