Letter to the editor

Abstract

Fluorescent location of rat leukaemia cells in resin sectionsCells which have the ability to migrate are associated with a recently described proteolytic enzyme (Steven and Al-Ahmad, 1983) referred to as guanidinobenzoatase (GBase).GBase degrades fibronectin and in particular the peptide GlyArgGlyAsp, which is thought to link fibronectin to cell surfaces (Pierschbacher and Ruoslahti, 1984). This activity may explain why migratory cells possess this unusual enzyme. GBase is inhibited by 9-aminoacridine which stacks at the active centre leading to intense fluorescence of cells possessing this enzyme (Steven et al., 1985). We have employed 9-aminoacridine to locate invading leukaemia T-cells in LKB 2218-500 historesin sections of formalin-Jined rat kidney tissue obtained from animals with T-cell lymphoblastic leukemia (Dibley et al., 1975; Jackson et al., 1984). Historesin sections I pm thick were stained with 9-aminoacridine ( I O v 3~) for 5 min, then washed with isotonic saline for 30 sec prior to fluorescent microscopy in a Leitz Orthoplan microscope employing UV illumination. The leukemia cells exhibited yellow surface fluorescence (Fig. I ) whilst the surrounding host tissue was very weakly stained. The lumen of the tubules appeared blue and did not bind 9-aminoacridine. Individual leukaemia cells can be located by this technique. Much of the yellow fluorescence of 9-aminoacridine staining was lost during photography; this problem was overcome by co-stacking propidium iodide on the previously stacked 9-aminoacridine, which resulted in cells with pink sulface fluorescence and better photographic contrast . For this putpose, the 9-aminoacridine-stained slides were dipped in propidium iodide (6 x 1 0 -5~) for I min and washed with water for I0 sec prior to microscopic examination using W illumination. Figure 2 shows clusters of leukemia cells surrounding kidney tubules whilst Figure 3 Figure 4 Figure 5, whilst Figure 6 shows a mass of leukaemia cells and a partially destroyed kidney parenchyma. shows individual leukaemia cells in the intertubular spaces. The staining of circulating leukaemia cells in the kidney vasculature is demonstrated in together with heavy infiltration by leukaemia cells in the intertubular tissue. Accumulation of leukemia cells within and around a glomerulus is shown inAlthough propidium iodide is conventionally used as a nuclear stain, we have utilized the prior stacking of 9aminoacridine on the cell surface of leukemia cells to co-stack propidium iodide, obtaining cells with pink fluorescent surfaces (see arrows in Figs. 2, 5 and 6). Historesin sections were employed to preserve the cell structure by preventing shrinking; this is best illustrated in Figures I , 2, 4 and 5, where the irregular surface of the leukemia cells is clearly defined by the yellow or pink surface fluorescence. We believe that chemofluorescent probes can be of value in locating small clusters of leukemia cells and even isolated cells. These cells might otherwise escape identification by conventional staining techni...