Quantitative, corrected fluorescence emission spectra from the nuclei of acridine orange-stained bladder epithelial cells were obtained with a corrected spectrum microspectrofluorophotometer (MSF)5 which is calibrated with a phosphor particle fluorescence standard. The spectra demonstrated that the green fluorescence band (1,,,2530 nm) is stoichiometrically related to the amount of nucleic acids (NA) present in a given nucleus, provided this spectroscopic species has reached saturation. The shape of this spectral band is constant. On the basis of this information, we used a simple microfluorometer, (SFM). calibrated with the phosphor particle standard, to routinely measure the amount of green fluorescence emission from the nuclei of AO-stained bladder epithelial cells. The amount of green fluorescence per normal nucleus never exceeded 0.

This study was undertaken to provide further information concerning the chemical heterogeneity of heparins and the relationships between the anticoagulant activity (USP assay) and the anionic density of the heparin. A sample of commercial heparin was fractionated into 13 fractions by sequential extraction in a two-phase system of 1-butanol-aqueous NaCl containing excess hexadecylpyridinium chloride. The anionic density distribution was characterized by the fractional distribution of uronate among the fractions. The fractions were characterized by several molar ratios of constituents, molecular weight, charge density, and anticoagulant activity in recalcified sheep plasma. The heparin was broadly distributed among the last 10 fractions; the first three contained impurities which were completely separated from the heparin fractions. The heparin fractions differ systematically in anionic density but are of substantially the same molecular weight. Anticoagulant activity increased markedly with anionic density, ranging from 81 units/mg for the heparin fraction with the lowest anionic density up to a high of 243 units/mg. The relationship between anticoagulant activity and either anionic density or its square is nonlinear. However, in the latter case an initial linear relationship was observed for anticoagulant activities of less than 200 units/mg.

SynopsisBinding isotherms for acridine orange (AD)-heparin systems can be evaluated solely on the basis of quantitative fluorescence spectroscopic measurements. The evaluation of thermodynamic parameters indicates that the interactions of A 0 with heparins from several animal sources are similar to each other in magnitude. Binding is highly exothermic (AH = -6 kcal mol-') and is stabilized by dye-polymer and dye-dye (cooperative) interactions, as well as by entropic factors ( I S = +7 e.u.). The predominant stabilizing factor appears to he t,he electrostatic attraction between the A 0 cation and the heparin polyanion, although the other factors are important as well. At 24°C the value of the cooperative binding constants for the various heparins range from 8.8 to 11.3 X 105M-', corresponding to a free energy of -8 kcal mol-'. The degree of cooperativity, which is a direct measure of dye-dye interaction, varies with po1ymer:dye ratio; the theoretical basis for this variation remains to he elucidated. Electrophoretic data indicate that each heparin sample consists of a mixture of species, each with its own charge density. This precludes definitive interpretation of observed small differences in the values of the thermodynamic parameters among the various samples until each sample can be resolved into its components.

The irreversible photochemical fading of fluorescence of acridine orange-heparin complexes indicates that fading involves the reaction of a "reactive" excited bound dye dimer with one in the ground state. A kinetic parameter, r", is derived, which has a constant value over a considerable range of conditions, and which is directly related to rate constants for photophysical and photochemical processes. This parameter appears to be characteristic for heparin and may serve to identify it in the presence of other glycosaminoglycans.

A system for screening cervical cytological preparations is described which employs the Leitz Texture Analyzer System (E. Leitz, Rockleigh, N. J.) quantitative staining with acridine orange, and a fluorescence standard. The instrumentation scans cells on microscope slides and detects objects which it interprets to be nuclei with excess total nuclear green fluorescence intensity (Previous results employing manual measurements have indicated that normal nuclei do not produce total nuclear green fluorescence greater than a specific absolute intensity level). Detected objects are identified by visual observation. Cells (102,000) from 65 patients (29 normal, 36 abnormal) have been examined. In each abnormal sample, at least one abnormal cell was detected. In over half of the samples, three or fewer other objects (e.g. clumps of polymorphonuclear leukocytes) were detected. These are easily distinguishable from single nuclei, and could be discarded by someone with minimal cytological training.