Automated blood cell analysis is a routine function in the clinical hematology laboratory. We have all had simples taken for such a purpose, but very few realize that the flow cytometric instrumentation performing these tests may have fundamental limitations regarding measurement of red blood cell volume, red blood cell hemoglobin concentration, and accurate classification of white blood cell subpopulations. The `high-end' instruments have become very complex and expensive in order to `ensure' that an accurate and reliable result can be reported. Yet, there is no tool available to do these kinds of measurements any differently or better. In this proposal is described two methods for the clinical flow cytometry hematology laboratory that can overcome the problem associated with hemoglobin concentration and volume interferences when making red blood cell measurements and overcome the problem of refractive index and volume interference in the white blood cell differential measurement. This is done by simultaneously detearmining cell dry mass by measurements of phase using a two-frequency HeNe laser and cell volume by the principle of dye exclusion. This Phase I, research will characterize both techniques using particle and blood samples, on an experimental flow cytometer. The ultimate instrument will provide a unique capability as a low cost device for making accurate red blood cell and white blood cell dry mass and volume measurements.
