T Sharpless

Simultaneous staining of ribonucleic and deoxyribonucleic acids in unfixed cells using acridine orange in a flow cytofluorometric system.

Simultaneous staining of deoxyribonucleic (DNA) and ribonucleic acid (RNA) in nonfixed, but permeable, cells is described. Cells are made permeable by treatment with non-ionic detergent at low pH. RNA is denatured prior to, or during staining, by exposure of cells to chelating agents to ensure that DNA (native) and RNA (dentured) may be stained differentially with the metachromatic dye, acridine orange. The fluorescence of individual cells is measured in a flow cytofluorometer. A comparison between various staining procedures employing acridine orange or other intercalating dyes in unfixed cells is discussed in terms of staining specificity, cell permeability and preservation. Evidence is provided that acridine orange staining of unfixed cells may be used as a simple, fast means of obtaining information on cell ploidy levels and cell cycle status from DNA measurements (green fluorescence), and cell transcriptional activity from RNA staining (red fluorescence), in human and murine cells lines, peripheral blood and bone marrow specimens from patients with leukemia and mitogenically (phytohemagglutinin) or antigenically (mixed lymphocyte culture) stimulated human peripheral blood cultures. Exposure of cells to detergent at low pH as an alternative to cell fixation or hypotonic treatment is proposed as a fast, convenient method of making cells permeable to dyes.

Thermal denaturation of DNA in situ as studied by acridine orange staining and automated cytofluorometry

Abstract Thermal denaturation of nuclear DNA is studied in situ in individual cells or isolated cell nuclei by employing the property of the fluorochrome acridine orange (AO) to differentially stain native and denatured DNA and by using an automated flow-through cytofluorimeter for measurement of cell fluorescence. RNAse-treated cells, or cell nuclei, are heated, stained and measured while in suspension and AO-DNA interaction is studied under equilibrium conditions. Measurements are made rapidly (200 cells/sec); subpopulations of cells from a measured sample can be chosen on the basis of differences in their staining or light-scattering properties and analysed separately. DNA denaturation in situ is rapid; it approaches maximum during the first 5 min of cell heating. Divalent cations stabilize DNA against denaturation. At low pH the transition occurs at lower temperature and the width of the transition curves (‘melting profiles’) is increased. Decrease in ionic strength lowers the DNA melting temperature. This effect is much more pronounced in cells pretreated with acids under conditions known to remove histones. Histones thus appear to stabilize DNA in situ by providing counterions. At least four separate phases can be distinguished in melting profiles of DNA in situ; they are believed to indicate different melting points of DNA in complexes with particular histones. A decrease in cell (nuclear) ability to scatter light coincides with DNA melting in situ, possibly representing altered refractive and/or reflective properties of cell nuclei. Formaldehyde, commonly used to prevent DNA renaturation, is not used in the present method. The heat-induced alterations in nuclear chromatin are adequately stabilized after cell cooling in the absence of this agent. Cells heated at 60–85 °C exhibit increased total fluorescence after AO-staining, which is believed to be due to unmasking of new sites on DNA. This increase is neither correlated with DNA melting, nor with the presence of histones. Possibly, it reflects destruction of DNA superstructure maintained at lower temperatures by DNA associations with other than histone macromolecules (nuclear membrane).

Conformation of RNA in situ as studied by acridine orange staining and automated cytofluorometry

Abstract Secondary structure of RNA was studied in human leukemic SK-L7 cells by their staining with acridine orange (AO) and fluorescence measurements in a flow-through cytofluorimeter. Parallel measurements of RNAse-treated cells made it possible to distinguish and to measure separately those components of total cell fluorescence that are due to AO interaction with RNA. Like DNA, double helical and single-stranded RNA in situ stain differentially with AO. Hence, the extent of RNA in double helical conformation, interacting with AO by intercalation, can be measured. In 10 −3 M phosphate buffer, RNA in situ denatures at 40–60 °C. Mg ions stabilize RNA against thermal denaturation; EDTA markedly decreases stability of RNA in double helical conformation. A slow, progressive denaturation of RNA in situ is seen as a result of cell exposure to AO at low ionic strength at room temperature. It is presumed that the changes described reflect mostly alterations of secondary structure of rRNA. Results of our in situ studies conform with data of others on rRNA denaturation in intact isolated ribosomes. This suggests that the ribosome anchoring in cytoplasm is not associated with double helical rRNA regions. Cell staining with AO under conditions of full RNA denaturation and in the absence of DNA denaturation offers differential, simultaneous staining of RNA and DNA. This approach in cell staining provides a new parameter for automated cell classification based on both quantity and conformation of RNA in situ.

Different sensitivity of chromatin to acid denaturation in quiescent and cycling cells as revealed by flow cytometry

The properties of DNA in situ as reflected by its staining with acridine orange are different in quiescent nonstimulated lymphocytes as compared with interphase lymphocytes that have entered the cell cycle after stimulation by mitogens. The difference is seen after cell treatment with buffers at pH 1.5 (1.3-1.9 range) followed by staining with acridine orange at pH 2.6 (2.3-2.9). Under these conditions the red metachromatic fluorescence of the acridine orange-DNA complex is higher in quiescent cells than in the cycling lymphocytes while the orthochromatic green fluorescence is higher in the cycling, interphase cells. The results suggest that DNA in condensed chromatin of quiescent lymphocytes (as in metaphase chromosomes) is more sensitive to acid-denaturation than DNA in dispersed chromatin of the cycling interphase cells. The phenomenon is used for flow cytometric differentiation between G0 and G1 cells and between G2 and M cells. In contrast to normal lymphocytes the method applied to neoplastic cells indicates the presence of cell subpopulations with condensed chromatin but with DNA content characteristic not only of G1 but also of S and G2 cells. The possibility that these cells represent quiescent (resting) subpopulations, arrested in G1, S and/or G2, is discussed.

Bladder cancer diagnosis by flow cytometry. Correlation between cell samples from biopsy and bladder irrigation fluid

Results of flow cytometry (FCM) examinations of bladder irrigation specimens were compared with those of FCM examinations of cell suspensions from bladder biopsies of 44 urologic patients. The fluorescent dye, acridine orange (AO), was used to stain DNA and RNA differentially and abnormal urothelial cells were identified by their relative content of nucleic acids. Granulocytes and squamous cells could be distinguished from transitional cells in this procedure, and did not interfere with the analyses. Of 28 patients with papillary carcinoma, carcinoma in situ, and invasive carcinoma, 27 were identified through FCM examination of irrigation cytology specimens; the one false‐negative result was from a low‐grade papillary carcinoma. Of 7 patients with papilloma, FCM examinations of irrigation specimens were positive in 4 and negative in 3. Results of FCM studies of biopsy specimens were in good but not complete agreement with those of irrigation specimens. In several cases, irrigation FCM disclosed tumor stemlines that were not identified in biopsy specimens. Discrepancies of this kind seemed most likely due to differences in sampling. Irrigation FCM seems to be a sensitive method for assessing multiple‐site bladder tumors, and may be a useful technique for monitoring the course of conservatively managed bladder tumors.

Flow Cytometry in Bladder Cancer Detection and Evaluation Using Acridine Orange Metachromatic Nucleic Acid Staining of Irrigation Cytology Specimens

A new technique for simultaneous multiparameter deoxyribonucleic acid, ribonucleic acid and nuclear size measurements by flow cytometry was applied to the examination of bladder irrigation cytology specimens from 107 urologic patients. The cell samples from patients with bladder carcinoma could be distinguished from normal by 2 features: 1) an increase in the proportion of bladder epithelial cells with more than diploid deoxyribonucleic acid and 2) aneuploid cell peaks. These criteria identified 12 of 13 cases of invasive carcinoma, 24 of 28 cases of carcinoma in situ and 11 of 13 cases of papillary carcinoma. An increased proportion of cells with more than diploid deoxyribonucleic acid or aneuploidy was found in 9 of 14 patients with papilloma and 6 of 19 patients with a history of bladder tumors but no evident disease at present--these were believed owing to increased epithelial proliferative rates or nuclear chromatin abnormalities not visible by light microscopy. None of the 20 patients who had never had bladder tumors was abnormal. While the results in this small clinical trial have been most encouraging an additional descriptor of nuclear chromatin structure is believed necessary to discriminate benign, reactive proliferative epithelium from neoplasm when the latter is near diploid or shedding few cells. Studies to develop such a parameter presently are under way.

Discrimination of cycling and non-cycling lymphocytes by BUdR-suppressed acridine orange fluorescence in a flow cytometric system

Abstract Stimulated lymphocytes which pass through the cell cycle may be distinguished from dormant G0 lymphocytes rapidly by flow cytometry. The method is based on cell incubation with 5-bromodeoxyuridine (BUdR) and their subsequent staining with acridine orange under conditions in which cellular DNA and RNA stain differentially. The DNA-specific green fluorescence of stimulated, cycling cells is suppressed while RNA-specific red fluorescence is affected only minimally. It is possible, therefore, to distinguish cycling vs non-cycling cells based on two entirely different parameters, i.e. BUdR incorporation and RNA content.

Size and refractive index dependence of simple forward angle scattering measurements in a flow system using sharply-focused illumination.

We have investigated the properties of three forward angle light scattering signals in a flow cytophotometer (Bio/Physics FC-200) using a line focused illuminating beam of about 5 ,z half-intensity width: (a) extinction of the illuminating beam; (b) scattering in the direction parallel to the focal line, integrated between 2 and 20#{176}; (c) scattering perpendicular to the focal line, integrated between 5 and 25#{176}. Signals were compared qualitatively in terms of pulse shape, and quantitatively in terms of total intensity (time integral) and pulse width (integral rise time) measurements, with respect to variations in particle diameter and refractive index at two wavelengths (488 or 633 nm). Test particles were transparent, spherical dextran gel (Sephadex) beads, having diameters continuously distributed between about 10 and 40 �z of several distinct refractive indices in the range 1.01-1.04, relative to water- comparable to fixed (hydrated) and living cells. Parallel and perpendicular scatter signals exhibit different, characteristic pulse shapes which remain distinctive over the whole range of diameter, refractive index and wavelength studied. These shapes evidently depend on geometrical factors which are different for the two scattering directions. Parallel scatter pulses rise and fall rapidly at the edges, but their amplitudes change relatively little while the center of the particle traverses the beam. Perpendicular scattering arises primarily from the edges of the particle, each of which produces a sharp pulse as it passes through the beam. Leading and trailing edge pulses are detected unequally and mirror-symmetrically on opposite sides of the beam. Perpendicular scatter pulsewidth (i.e. , the separation in time of these two pulses) is accurately proportional to particle diameter (at constant flow velocity) and independent of refractive index and wavelength within experimental error. Integrated extinction intensity is comparable to extinction under uniform, parallel illumination; its dependence on diameter, refractive index and wavelength is in good agreement with the predictions of anomalous diffraction theory. This permits a simple absolute calibration ofthe pulsewidth scale. Perpendicular scatter intensity increases smoothly with bead diameter, and also depends strongly on refractive index in the range studied. Parallel scatter intensity shows a more complex dependence on these parameters, but is relatively much less sensitive to refractive index. These results are discussed in terms of an approximate separation of refractive from diffractive effects due to the undirectionally focused illumination. We conclude that unambiguous and fairly precise estimates of both size and refractive index for cell-like particles can be obtained from a single (perpendicular) scatter signal in a flow system using line-focused illumination.

RECOGNITION OF CELLS IN MITOSIS BY FLOW CYTOFLUORMETRY

Cells in mitosis may be distinguished from interphase cells based on difference in chromatin structure as revealed by two different methods of staining with acridine orange. In the first method, cells are heated and then stained at neutral pH; the difference in stainability between mitotic and interphase cells reflects the difference in the extent of deoxyribonucleic acid denatured by heat in these cells. At a given temperature the deoxyribonucleic acid of the mitotic cell appears to be more extensively denatured than that of the interphase cell. In the second method, cells are treated with buffer at pH 1.5 (1.3 to 1.9) and then stained at pH 2.6 (2.3 to 2.9). The mechanisms involved in the differential stainability of interphase versus mitotic cells at that low pH are currently under investigation. In both methods, in addition to enumerating cells in mitosis, it is possible to quantitate cells in G1, S and G2 phases of the cell cycle.

Interphase and metaphase chromatin: Different stainability of DNA with acridine orange after treatment at low pH☆

Abstract The stainability of DNA in situ with acridine orange is different in interphase and mitotic cells. The difference is evident after pretreatment of cells with buffers at low pH (1.3–1.9). When acid treatment is followed by cell staining at neutral pH, the DNA of cells in mitosis, in comparison with DNA of interphase cells, exhibits lower green fluorescence and similar red fluorescence. Apparently partial extraction of acid-soluble constituents of chromatin at pH 1.3–1.9 increases the accessibility of DNA for AO intercalation and this increase is more extensive in interphase than in mitotic cells. When acid-treated cells are stained at pH 2.3–3.2, the DNA of mitotic cells in comparison with DNA of cells in interphase shows lower green fluorescence and markedly higher red fluorescence. It is presumed that staining at low pH potentiates acid denaturation of DNA in situ (and/or precludes the fast reassociation of the denatured DNA) and that the acid denaturation of DNA is more extensive in metaphase chromatin. Due to differences in the stainability of DNA in interphase vs mitotic cells, it is possible to discriminate between cells in G1, S, G2 and M phases, respectively. Consequently, the method may be used to automatically analyse the cell cycle including measurements of mitotic indices.