Detection of Infrequent Cells in Blood and Bone Marrow by Flow Cytometry
I. INTRODUCTION Peripheral blood and bone marrow are composed of heterogeneous cell mixtures. Traditionally, the composition of blood cells has been performed by microscopic examination of cytochemically stained cell smears. This technique, however, is subjective and nucleated cells which appear at a frequency below 5% may be overlooked. The introduction of flow cytometry to discriminate between cell populations has significantly improved the ability to identify accurately and enumerate cell populations which cannot be distinguished by morphological features (1). A further improvement of the sensitivity of flow cytometric examination of heterogeneous cell mixtures has been obtained by multidimensional analysis of the data (2). Cell populations are identified by the simultaneous assessment of lightscattering and fluorescence parameters. Light-scattering parameters measure cell size and cell granularity. Fluorescence parameters can be used to assess cell surface antigens, intracellular antigens, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and protein content. By choosing an unique set of lineage-specific and or lineage-associated monoclonal antibodies, this technique has enabled the assignment of clusters of cells in bone marrow to a specific lineage and a maturational stage (3-6). Although cells can be identified in frequencies as low as 1/105 to 1/106, the practical detection limit of multidimensional flow cytometry to identify cells is 1/104 (7,8). The ability reliably to detect infrequent cell populations such as hematopoietic progenitor cells, hematopoietic stem cells, and micrometastases in peripheral blood is becoming increasingly significant in the management of patients with cancer (9-14). In this chapter, various approaches are described which enable the enumeration of infrequent cell types by flow cytometry.