Identification of Discrete Classes of Normal Human Peripheral Lymphocytes by Multiparameter Flow Analysis

Abstract

Summary

We have demonstrated that normal human peripheral leukocytes vitally stained with acridine orange can be separated into four groups, consisting of granulocytes, monocytes, and two types of lymphocytes, on the basis of green/red fluorescence ratio measurements which characterize nuclear and cytoplasmic staining properties. Evaluation of the two sorted lymphocyte populations has suggested two morphologically and functionally distinct subclasses which are normally classified as large and small lymphocytes. When considered by volume measurement alone, small and large lymphocytes were not discrete classes but had overlapping distributions. However, the fluorescence ratio distributions clearly indicated that these are discrete classes of cells rather than representing an arbitrary classification of a continuum.

Since green nuclear fluorescence is nearly uniform, the green/red fluorescence ratio measurement permits characterization of leukocytes as an inverse cytoplasmic granulation density function. Using these criteria, granulocytes characterized by coarse cytoplasmic granulation had lower green/red fluorescence ratio signal amplitudes than lymphocytes which have finer granulation. The red/green fluorescence ratio distributions also have been measured, but the degree of separation between the two lymphocyte types was less pronounced.