An accurate method of plasma volume measurement by direct analysis of Evans blue spectra in plasma without dye extraction: origins of albumin-space variations during maximal exercise.

The Evans blue dye (EBD) dilution method, including a dye extraction step, is a standard way of measuring plasma volume. This report describes a new direct spectrophotometric method, which is simple and specific and avoids the dye extraction step. To begin with, all the contaminants which may appear during a plasma volume study were added to plasma samples, prior to the absorbance measurements. In this way we calculated correction factors for the haemolysis and the turbidity of the plasma samples, without dye. The study of the visible spectra showed that the correction factors could be obtained by measuring absorbances at only four visible wavelengths: 780, 720, 619 and 578 nm. The addition of various amounts of contaminants to dye-containing plasma samples allowed us to obtain precise values for the absorbance errors. The previously defined correction factors were then applied, and the residual absorbance errors were found to become nil. This spectrophotometric method can be used to check the efficiency of a dye-extraction procedure, as well as to study other biological fluids containing EBD. When used to analyse a chronological series of blood samples this method appeared to provide an effective way of simultaneously studying the processes of plasma volume concentration and albumin extravasation induced by maximal exercise.