Journal of Histochemistry & Cytochemistry

Herman, C J; Bunnag, B

doi: 10.1177/24.1.943438pmid: 943438

The immediate goal of the Cytology Automation Program of the National Cancer Institute is the development of an automated system which will screen appropriate specimens from asymptomatic women for squamous cell carcinoma of the cervix and its precursor lesions, dysplasia and carcinoma in situ. This system should make one of three decisions on each specimen: normal, abnormal or inadequate with an acceptably low false negative rate. It is expected that the next step in evaluation of patients whose specimens have been identified as abnormal by the automated system would be a manual evaluation of a traditional cytologic specimen.

Tolles, W E

doi: 10.1177/24.1.1254937pmid: 1254937

Rate-zonal sedimentation of cells from the female genital tract has been employed as a means of separating many of the different cellular components from each other. Ultra-slow streak photography of forward scattered light from the sedimentation spectrum has been used as a monitor of the success of the individual sedimentation maneuver. The presence of convention currents vitiates the separation potential of the technique, and this is readily detected by the photographic monitor. In addition, these photographs clearly indicate the positions of bands of cellular entities of different sedimentation velocities. Further study of the quantitative aspects of the light scattered from each part of the spectrum reveals that it possesses a quantitative representation of the concentration of cells at each level of the spectrum. The photographs and densitometric scans support these expectations. To avoid the limitations of film as a data-logging medium and to obtain real-time system utilization, a direct photoelectric scanner has been assembled. Preliminary tests indicate ready feasibility.

Wolley, R C; Dembitzer, H M; Herz, F; Schreiber, K; Koss, L G

doi: 10.1177/24.1.1254908pmid: 1254908

A simple and reliable method of determining the degree of dispersion of a cell suspension has been developed using the Perkin-Elmer Uni-Smear Spinner. Optimum conditions regarding rate and duration of spin, etc., were first ascertained using dispersed cell cultures including human cervical cancer cells as well as gynecologic samples. After spinning, single cells in suspension appeared as isolated cells on the slides. Cell aggregates, on the other hand, remained together. Therefore, the distribution of cells in various sized aggregates could be easily quantitated and the slides retained for future review. This method was used to evaluate the dispersing effects of trypsin, ethylenediaminetetraacetate and and syringing human on human gynecology samples obtained by routine cervical scrapes. None of the dispersion methods has, so far, produced an adequate monodispersed cell suspension without unacceptable cell loss.

Boltz, R C; Todd, P; Gaines, R A; Milito, R P; Docherty, J J; Thompson, C J; Notter, M F; Richardson, L S; Mortel, R

doi: 10.1177/24.1.176264pmid: 176264

The application of cell electrophoresis to cytodiagnosis requires that a scientifically established basis exists for identifying abnormal cells electrophoretically, that research to detect such differences in the cytodiagnostic setting is possible and that a rapid and simple method of cell electrophoresis is adaptable to the clinical setting. Data are presented indicating modifications of electrophoretic mobility due to herpes simplex virus type 1 infection and Rous sarcoma virus transformation of culture cells. A simple apparatus for electrophoretically separating cells on a density gradient and collecting them for subsequent analysis is described, and results of experiments with this apparatus are consistent with those obtained by microscopic electrophoresis. Laser-doppler spectroscopic electrophoresis is suggested as a rapid method adaptable to clinical application.

Latt, S A; Stetten, G

doi: 10.1177/24.1.943439pmid: 943439

Absorption, fluroescence and circular dichroism measrements on 33258 Hoechst-deoxyribonucleic acid (DNA) complexes are consistent with the existence of two types of dye-binding interactions. One type, which persists at elevated solution ionic strength, is highly specific for adenine-thymine-rich DNA. Dye bound under this condition exhibits efficient fluorescence and strong optical activity. A less specific, largely electrostatic interaction is associated with less intense fluorescence and weaker optical activity. The fluorescence of 33258 Hoechst and several other bisbenzimidazole dyes is less when bound to poly(deoxyadenylate-5-bromodeoxyuridylate) than when bound to poly(deoxyadenlyate-deoxythymidylate). Quenching of 33258 Hoechst fluorescence can also be used to detect biosynthetic incorporation of 5-bromodeoxyuridine into the DNA of living cells. This property of 33258 Hoechst should allow fluorescence-activated cell and chromosome sorting according to the extent of DNA synthesis, providing a bridge between biochemical and cytologic analyses of processes related to DNA replication.

Gratzner, H G; Pollack, A; Ingram, D J; Leif, R C

doi: 10.1177/24.1.815428pmid: 815428

Antibodies to 5-bromodeoxyuridine (BrdU) or iododeoxyuridine may be used to identify cells or regions of chromosomes in which de novo deoxyribonucleic acid synthesis has occurred. The antibodies to BrdU were produced in rabbits by injection of the antigen, a conjugate between bovine serum albumin and bromouridine (BrU), or iodouridine. Specific antibodies were produced by affinity chromatography on AH-Sepharose 4B to which had been coupled BrU. Anti-BrU cross-reacts with iodeodeoxyuridine. Indirect antibody techniques have been used to monitor deoxyribonucleic acid synthesis in nuclei; anti-BrdU treatment was followed by goat anti-rabbit immunoglobulin G labeled with either fluorescein or horseradish peroxidase. By use of these techniques, labeling indices were determined in cell cultures which had been pulsed with 3H-BrdU. The immunologic technique compared favorably with the autoradiographic methods performed concurrently on the same cultures. Metaphase chromosomes from synchronous CHO cell which had been pulse labled with BrdU at different time intervals during S phase were subjected to these immunologic procedures. Chromosome banding was observed with both the fluoresence and peroxidase methods. Chromosomes from cells not containing BrdU did not exhibit banding.

Traganos, F; Darzynkiewicz, Z; Sharpless, T; Melamed, M R

doi: 10.1177/24.1.1254934pmid: 1254934

Binding of the fluorochrome acridine orange (AO) to nucleic acids in situ is studied by automated cytofluorometry in two differentiating cell systems: Friend virus-transformed murine erythroleukemia induced to differentiate by dimethyl sulfoxide, and phytohemagglutinin-stimulated human lymphocytes. The specificity of the stain for deoxyribonucleic acid is discussed on the basis of data obtained by cell treatment with nucleases. Evidence is presented that in the case of Friend leukemia cells, but not phytohemagglutinin-stimulated lymphocytes, a significant change in the number of AO-intercalating sites in DNA occurrs during differentiation. These results suggest that changes in nuclear chromatin occurring during cell differentiation may be correlated, in some but not all systems, with changes in accessibility of DNA in situ to intercalating dyes. The role of divalent cations, especially Mg2+, in the conformation of nuclear chromatin and in modulation of the accessibility of nucleic acids to AO is discussed. The method provides a tool for the study of nucleic acid-protein interaction in situ, and in some cell systems it may be applicable as a marker for recognition of cell transformation, differentiation or neoplasia.

Darzynkiewicz, Z; Traganos, F; Arlin, Z A; Sharpless, A T; Melamed, M R

doi: 10.1177/24.1.1254935pmid: 1254935

Thermal denaturation of deoxyribonucleic acid (DNA) in situ in individual unbroken cells is studied by a cytofluorometric method. This method allows us to investigate DNA denaturation in the presence of divalent cations at concentrations reported to be necessary to maintain native structure of nuclear chromatin. Under these conditions the pattern of DNA denaturation is very different than when studied in the presence of ethylenediaminetetraacetate or citrate. The results suggest that with divalent cations present, the histone basic charges are more uniformly distributed along whole nuclear DNA. Various cell types exhibit great differences in sensitivity to DNA denaturation when assayed in the presence of 1 mM MgCl2. Human lymphocytes, monocytes and certain kinds of human leukemic cells show differences large enough to be used as a parameter for their recognition in mixed samples. Possible applications of the method in basic research on chromatin conformation and as a tool for cell recognition in diagnostic cytology or in the classification of human leukemia are proposed.

West, S S; Golden, J F; Menter, J M; Love, L D

doi: 10.1177/24.1.1254936pmid: 1254936

The fading behavior of the 670 nm fluorescence emission band produced by unfixed rat mast cells stained with acridine orange (AO) has been found to be in excellent agreement with the behavior predicted by second order chemical kinetics. The reciprocal of fluorescence intensity plotted against time yields a straight line. When due account is taken of dye/cell ratio and the intensity of fluorescence-exciting radiation, Io (measured with the standard phosphor particle), the slope of this straight line is a constant, k'', which is independent of dye/cell ratio and Io. k'' differs from the second order photochemical rate constant by a constant factor. The fading of a given AO-biopolymer complex is described by a particular value of k''. Two values of k'' have been found for rat mast cell granules, indicating the presence of two different AO-biopolymer complexes. Fading of fluorescence may serve to identify particular intracellular biopolymers in individual cells even when present in a heterogeneous population.

Crissman, H A; Oka, M S; Steinkamp, J A

doi: 10.1177/24.1.56392pmid: 56392

Quantitative fluorescent staining and analysis of cellular deoxyribonucleic acid (DNA) were accomplished using three groups of reagents having different mechanisms of action for DNA binding. These reagents included (a) the fluorescent antitumor antibiotics mithramycin, chromomycin A3 and olivomycin; (b) the Feulgen reagents acriflavine and flavophosphine N and (c) the intercalating dyes ethidium bromide and propidium iodide. Propidium iodide (PI) was used in combination with fluorescein isothiocyanate (FITC) to stain both cellular DNA and protein, respectively. Multiparameter analysis of PI/FITC-stained cells provided a direct correlation of DNA and protein for cells in all stages of the cell cycle. Nuclear-to-cytoplasmic ratio determinations were also performed on PI/FITC-stained cells by analysis of the time duration of the red (DNA) and green (protein) fluorescence signals from each cell. These staining and analysis techniques provide alternative methods for directly determining the quantitative relationship between cellular DNA and protein and will be extremely useful in investigations where fluctuations of these parameters are of importance for assessing experimental results.