Cell classification by laser light scattering: identification and separation of unstained leukocytes

We have used a flow-system cell sorter to separate unfixed, unstained human leukocyte cells into morphologically distinct populations based only on the intensity of 488-nm wavelength laser light simultaneously scattered by each cell at two different angles. Three populations were observed as distinct peaks in a two-parameter pulse-height distribution and were then physically sorted into separate classes and stained for cytological examination. The three groups consisted of lymphocytes, monocytes, and neutrophils. Each group contained between 77 and 98 per cent of a single cell type. Blood from an irradiated monkey was also sorted and showed the presence of a fourth peak which consisted of 61 per cent eosinophils. Thus, multiangle light-scattering information from unfixed, unstained cells may be a promising technique for rapid morphologic analysis and may have application, for example, as a highspeed automated leukocyte differential. We anticipate that this method may be useful in other clinical applications where morphologic differences are diagnostically important. One of the principal advantages of the method is elimination of fixation and staining of the samples; this is a nondestructive testing technique.