Oxygen saturation determination in frozen blood

A spectroscopic method for O₂ saturation determination in frozen blood has been developed. In the 500-575 nm range, frozen oxygenated and deoxygenated blood had essentially the same spectral absorption character as liquid blood, although apparent absorbance was much higher than liquid blood. The standard two-wavelength method for spectroscopic determination of O₂ saturation of blood was inadequate to correct for the nonspecific light loss through the ice crystals. A method for O₂ saturation determination using three wavelength (506, 523, and 560 nm) measurements is reported here. Compared with blood O₂ saturation determined by the Van Slyke technique, this three-wavelength method demonstrated an accuracy of 2-5% within 95% confidence limits. It also compared favorably with a previously reported four-wavelength geometric computational technique. The rate of freezing did not influence the absorption spectra nor the percentage saturation determination. Samples could be stored at -25° up to 49 days with no change in O₂ saturation. Our calculations reveal that the upper limit of blood O₂ saturation increase in a closed system due to freezing is 2.3%. The upper limits of variation in blood O₂ saturation due to freezing blood with various hemoglobin concentrations or at various initial pH was calculated to be about 1% or less. This method may be adapted for microspectrophotometric determination of regional arteriovenous blood O₂ saturation difference in quick frozen tissue.