Method for preparing a sample in a scan capillary for immunofluorescent

Abstract

An assay and sample mixture for the enumeration of fluorescently stained target components of a whole blood sample by an imaging instrument. The sample preparation method ensures that the amount of target components per unit of volume of the whole blood sample is preserved by elimination of certain non-quantitative preparation steps while producing an even hematocrit layer within a scan capillary. Typical target components include white blood cells that express certain surface antigens, such as CD-4 and CD-8 proteins. To inhibit aggregation of the red blood cells, a reagent is added to an aliquot of whole blood sample. The aliquot of whole blood is mixed and with a preselected amount of a fluorescent dye and ligand complex which tags the target components. The sample and fluorescent complex are allowed to incubate a sufficient amount of time to bind enough of the fluorescent complex to the target components to provide a fluorescent signal from the target components which will be distinguishable from the fluorescent signal from the unbound fluorescent complex in the sample. The resulting mixture allows the imaging instrument to detect peak intensities of fluorescence from the target components, thereby allowing the target components to be volumetrically enumerated with an improved level of accuracy and efficiency.