bluuebunny/crossref_metadata_2025 huggingface dataset:
Neutropenia: The accuracy and precision of the neutrophil count in leukopenic patients
Authors: Ross Dennis W. , McMaster Kitt
Published: August 2005

Neutropenia (absolute neutrophil count < 500/μl) is a life threatening clinical state commonly seen in cancer and leukemia patients on intensive chemotherapy. The purpose of this investigation is to determine the accuracy and precision of the absolute neutrophil count in these patients using two methods: (1) the percentage of neutrophils as determined by a 100 cell manual differential multiplied by the white blood cell count (NEUT‐M), and (2) the absolute neutrophil count determined directly by automated flow cytometry using the Hemalog‐D automated white cell differential counter (NEUT‐D) Technicon Instruments Corp., Tarrytown, NY. The coefficient of variation (cv) for repeated measurements was dependent on absolute neutrophil count. For manual determinations, the cv was 5% at 5000/μl, 18% at 500/μl, and 42% at 50/μl. For the cytometry method the cv was 2.5% at 5000/μl, 8% at 500/μl, and 17% at 50/μl. The accuracy of flow cytometric differential neutrophil counts using the Hemalog‐D system was found to be equivalent to the manual differential count. The precision, however, was more than twice that of the manual white cell differential count. In the neutropenic patient this analytical improvement was clinically useful as demonstrated by sequential observation of daily neutrophil counts.
Distinction between 5‐bromodeoxyuridine labeled and unlabeled mitotic cells by flow cytometry
Authors: Darzynkiewicz Zbigniew , Traganos Frank , Melamed Myron R.
Published: August 2005

Exponentially growing Friend leukemia cells are exposed to 5‐bromodeoxyuridine (BrdUrd) for the time period equivalent to one generation. After fixation and incubation with RNase, DNA in situ is partially denatured by acid and the cells are stained with acridine orange (AO). Staining with AO under these conditions reveals the extent of denatured versus double stranded DNA and enables one to distinguish mitotic from interphase cells. It is observed that all BrdUrd treated cells, regardless of cell cycle phase, have decreased both green and red fluorescence. This finding suggests that BrdUrd interacts with AO not only in the complexes of the dye with double stranded DNA, but also with the single stranded biopolymer. The BrdUrd‐attributed suppression of cell fluorescence is large enough to separate totally BrdUrd labeled from unlabeled mitotic populations. The combination of these two flow cytometric techniques, one which allows discrimination between interphase and metaphase cells and the other which allows further subdivision of metaphase cells into those which have incorporated BrdUrd and those which have not, provides an alternative to the autoradiographic procedure necessary for obtaining the fraction of labeled mitoses.
A video‐computer for the chronotropic and inotropic measurements of the beating of cultured heart cells
Authors: Harary Isaac , Wallace Garn , Bristol Greg
Published: August 2005

A video‐computer system has been developed to measure the chronotropy and inotropy of cultured heart cells. The motion of the cell is followed by recording of the changes of light intensity from the edge of a beating cell. With this system the velocityof contraction and relaxation, the time to peak contraction, relaxation time and the displacement of the cell in culture can be measured for the first time. Also, the rate of beating can be measured beyond the limits set by visual counting. With the application of this technique to the cultured heart cell system we have found that norepinephrine increases the velocity of relaxation thus reducing the ratio of contraction velocity to relaxation velocity, and reduces the twitch time. Increased external calcium, on the other hand, has little effect on either the velocity of contraction or relaxation but, like norepinephrine, decreases the twitch time.
Chromosome analysis by high illumination flow cytometry
Authors: Bartholdi Marty F. , Sinclair Douglas C. , Cram L. Scott
Published: August 2005

Fluorescence measurements from metaphase chromosomes of the Chinese hamster, stained with propidium iodide excited at high illumination irradiance, completely resolve each chromosome type. The measurements are performed in a specially designed flow cytometer that achieves high irradiance (4 MW/cm2) by using high power laser output (2 W at 488 nm) focused to small spot size (1% irradiance variation over 2 m̈m). The coefficient of variation of each chromosome peak is near 1.5%.

Saturation of the fluorescence transition and photobleaching, two consequences of high irradiance, are shown to occur. Even with a nonlinear dependence of fluorescence upon illumination irradiance, fluorescence retains a proportional response to chromosome type; each chromosome peak maintains a consistent ratio to the others at every irradiance. No perturbation of fluorescence by the optical or geometrical properties of the chromosomes is evident.

The advantages of high irradiance illumination are an increase in fluorescence sufficient to reduce the statistical error in photoelectron number to a low level and reduced influence of laser power fluctuations and variable chromosome flow trajectories on the precision. These benefits improve the resolution of chromosome analysis by flow cytometry, particularly the resolution of smaller chromosomes.
Differentiation of HL‐60 cells: Cell volume and cell cycle changes
Authors: Zucker Robert M. , Whittington Karen , Price Brandon J.
Published: August 2005

HL‐60 promyelocytic leukemic cells can differentiate into more mature myeloid cells with the addition of dimethylsulfoxide, butyric acid or retinoic acid and can differentiate into macrophages with the addition of phorbol ester 12‐0‐tetradecanoylphorbol‐13‐acetate (TPA). After the addition of an inducer, the HL‐60 cell volume shows a daily decrease while the cell number increases at a rate similar to the untreated control cells. Flow cytometry measurements show an increase in G1 cells and a decrease in S cells after day 1. Since the generation time is constant, the data suggest that the length of time spent in the different cell cycle stages has changed during differentiation.

Within 3 hours after the addition of TPA to HL‐60 cells, selective adhesion of G1 cells occurs. Smaller sized cells are recovered from the flask bottom and larger sized cells are recovered from the supernate. Flow cytometric analysis reveals a G1 and S block in cells obtained from both the supernatant and from the flask bottom. After 1 day of TPA incubation, there is preferential adhesion of G1 and G2 cells with the nonadherent cells being primarily in the S and G2 cell cycle stages and undergoing a cell cycle traverse.
Multistation multiparameter flow cytometry: Some influences of instrumental factors on system performance
Authors: Shapiro Howard M. , Feinstein David M. , Kirsch Albert S. , Christenson Lisa
Published: August 2005

In an effort aimed at design of a flow cytometer/sorter system in which light scattering, fluorescence, and extinction measurements could be made in as many as five spatially separated illuminating beams, the performance of different mechanical components, light sources, illumination and collection optics, and flow chambers was compared by changing individual components while maintaining the same basic instrument configuration. A protocol was developed, and is here described, allowing rapid setup of a flow cytometer using a mercury arc lamp and two laser beams for illumination. When two or more illuminating beams are derived from a single laser, it is generally necessary to incorporate automatic gain control in the detector electronics to compensate for source intensity fluctuations and so to achieve acceptable measurement precision. It is, however, possible to use a single laser beam as a source for several flow cytometers without the need for such compensation. Mercury arc lamps provide adequate excitation in the near ultraviolet (uv) for DNA fluorochromes, such as Hoechst 33342, and may be preferable to ion lasers for this purpose. Reported differences in precision and sensitivity between stream‐in‐air and enclosed flow systems appear owing more to differences in the illumination and collection optics used in different instruments; five flow chamber designs gave equivalent performance when tested in the same instrument. Relatively inexpensive achromatic microscope objectives of numerical aperture (NA) approximately 0.5 may offer the best combination of depth of focus and light gathering power when used as fluorescence collection lenses for flow cytometers; image plane apertures improve system performance in fluorescence measurement.
Cell surface antigens reacting with antiretrovirus sera on normal mouse spleen cells: A flow cytometric study
Authors: Szabó Gábor , D‐tóth Ferenc , Réthy Antal
Published: August 2005

The development of erythroleukemia in Balb/c mice injected with Rauscher leukemia virus has been followed by indirect immunofluorescence technique and flow cytometry, using antiserum against disrupted virions. The progression of the disease was accompanied by a great increase in the number of large, immunofluorescence positive cells. A subpopulation of normal spleen cells was also highly positive. The expression of the antigens in normal cells was examined in relation to the cell cycle. The majority of the S‐G2/M phase cells were found in the antigen positive compartment of larger cells. A two‐color analysis of immunofluorescence and DNA content revealed that the distribution of antigen expression in G1 phase was broad, gradually decreasing from a low‐intensity mode. The cell with double DNA content distributed evenly around a modus of five‐fold higher intensity. In experiments with stimulated bonemarrow cells, superiority of S‐phase cells in anti‐Rauscher serum binding was found. Cell‐surface gp70 antigen is suggested to be involved in this cell‐cycle dependent binding of antibodies by normal cells.
A practical graphical method for estimating the fraction of cells in S in DNA histograms from clinical tumor samples containing aneuploid cell populations
Authors: Ritch Paul S. , Shackney Stanley E. , Schuette William H. , Talbot Thomas L. , Smith Charles A.
Published: August 2005

A graphical method for the analysis of unperturbed DNA histograms is presented in which the area of the normalized histogram subtended by the fraction of cells in S is represented by a trapezoid whose dimensions are dependent on features common to all such histograms. The technique takes measurement variability into account. This method was applied to a variety of synthetic DNA histograms. Overall, calculated values for the fraction of cells in S correlated well with actual values. This method was applied to 36 diploid cases of non‐Hodgkin lymphoma; results correlated well with those obtained by a computer‐based method. The results of the graphical‐method were also highly reproducible between different observers. The graphical method can be used in the presence of aneuploid cell populations. Techniques for calculating S fractions in the presence of aneuploidy in clinical samples are described. These techniques were applied to synthetic histograms of mixed diploid and aneuploid populations. Calculated values correlated well with actual values.
A differential amplifier circuit for reducing noise in axial light loss measurements
Authors: Steinkamp John A.
Published: August 2005

Simplified electronics have been developed for reducing laser excitation source light intensity fluctuations in flow cytometric axial light loss (extinction) measurements. By continuously monitoring the laser output with a photodiode detector and combining it with the axial light loss signal from a second detector using a high‐speed differential amplifier, background interference is reduced 10 to 40 dB. Oscilloscope waveforms and frequency distribution histograms recorded from uniform‐size polystyrene latex spheres and human mononuclear blood cells are used to illustrate the noise reduction capabilities.
Purifying human Y chromosomes by flow cytometry and sorting
Authors: Fantes Judith A. , Green Daryll K. , Cooke Howard J.
Published: August 2005

A method of producing an enriched sample of human Y chromosomes from peripheral blood lymphocytes is described. Metaphase chromosomes were prepared from peripheral blood lymphocytes donated by 17 normal male individuals. A suspension of chromosomes in a polyamine buffer was produced from each sample, stained with the fluorescent dye Hoechst 33258, and passed through a flow cytometer and sorter. Following analysis of the 17 fluorescence distributions, a single donor was found giving a separate peak corresponding to the Y chromosome. Seventy percent of the chromosomes sorted from this peak were identified as Y chromosomes. Batches of a million Y chromosomes were produced from each of several 40 ml donations of peripheral blood. These were assessed for the amount of Y DNA present and used to construct a DNA library.
Flow cytometric classification of biopsy specimens from cervical intraepithelial neoplasia
Authors: Jakobsen A. , Kristensen P. Baek , Poulsen H. Kvist
Published: August 2005

The distribution of single‐cell DNA content was investigated in biopsy specimens from the human cervix of 121 women suspected of having intraepithelial neoplasia. Comparison of the results of the histopathological examination with the ploidy level showed that all normal specimens were diploid. Thus, no false‐positive results occurred. Most of the specimens classified as mild and moderate dysplasia were diploid as well. Aneuploid cell populations occurred in 78% of the lesions classified as severe dysplasia and carcinoma in situ. The ploidy level distribution permitted a natural division of the aneuploid cell populations into two groups with DNA indices either above or below 1.5. The importance of the aneuploidy in carcinogenesis is discussed.
Non‐small‐cell lung carcinoma: Tumor characterization on the basis of flow cytometrically determined cellular heterogeneity
Authors: Teodori Laura , Tirindelli‐Danesi Donatella , Mauro Francesco , De Vita Raffaele , Uccelli Raffaella , Botti Claudio , Modini Claudio , Nervi Carlo , Stipa Sergio , Teodori Laura
Published: August 2005

Some 150 tumor specimens from 49 patients with non‐small‐cell carcinoma of the lung (23 epidermoid, 14 adenocarcinoma, 12 large‐cell carcinoma) and three with nonneoplastic lung disease were analysed for cellular DNA content by flow cytometry. Monodispersed cells were stained with ethidium bromide and mithramycin. Normal specimens and samples from patients with nonneoplastic disease constantly yielded a single cell population with diploid DNA content. Twenty of 23 epidermoid carcinomas exhibited one or more than one aneuploid subpopulation. Ten of 12 large‐cell carcinomas were characterized by one aneuploid clone and 2/12 by two aneuploid clones. Adenocarcinoma exhibited multiclonal cell subpopulations (one to five aneuploid clones). Further information has been obtained on the differential presence of clones in various tumor areas and in infiltrated lymph nodes. These tumors appear characterized by a remarkable degree of cellular heterogeneity. The cytometric ploidy level(s) and the cell population multiclonal structure yield, in comparison with, and in addition to, pathology, indications of possible clinical interest. A correlation between the clonal DNA content and a prognostic parameter such as the tumor mass doubling time has been demonstrated.
Difference in chromatin sensitivity to heparin of chronic atrophic gastritis, of carcinoma‐free and carcinoma‐bearing stomach in comparison with normal gastric mucosa and gastric carcinoma as revealed by flow cytometry
Authors: Weiß H. , Heinz U. , Gütz H.‐J. , Schälicke W.
Published: August 2005

DNA distribution patterns from gastric mucosal cells corresponding to four groups defined by histological examination were measured by flow cytometry before and after treatment with heparin, a polyanion. Group I comprised normal gastric mucosal cells; group II, chronic atrophic gastric mucosal cells originating from a carcinoma free stomach; group III, chronic atrophic gastric mucosal cells originating from a carcinoma bearing stomach; and group IV, malignant gastric mucosal cells. The heparin concentrations used were 1.25, 1.5, and 5 U/ml cell suspension. Heparin caused increases in fluorescence intensity and in coefficients of variation, which are interpreted as a reflection of alterations in chromatin structure. For the four groups investigated, the heparin‐initiated changes were dependent, in varying degree, on concentration and time. Group I showed a much more extensive sensitivity to heparin than group IV. Group II and III reacted similarly to group I or group IV, depending on the source, i.e., either a carcinoma‐free stomach or a carcinoma‐bearing stomach. Further extension of this method might yield information concerning the real premalignant potential of a specific case of chronic atrophic gastritis.
Flow cytometric analysis of micronuclei found in cells after irradiation
Authors: Nüsse Michael , Kramer Johannes
Published: August 2005

Exposure of mammalian cells to either ionizing radiation or mutagenic and carcinogenetic substances can induce chromosome aberrations. These aberrations in turn may give rise to micronuclei which can be found in cells during the interphase after division. A two‐step method is presented that allows separation of micronuclei from cell nuclei. They can then be measured and analysed according to their DNA content in a flow cytometer. The method involves an initial detergent treatment of cells followed by a second treatment with sucrose and citric acid. Micronuclei with DNA content larger than 2% of the G1‐nuclei can be measured. The method is tested and compared with microscopic observations of micronucleated cells in irradiated, asynchronous, and synchronized Ehrlich ascites tumour cells growing in vitro. The agreement between the flow cytometric technique and microscopic observations is excellent when the dose‐dependent number of micronuclei per cell is taken into consideration.
An assay for pattern formation in dictyostelium discoideum using monoclonal antibodies, flow cytometry, and subsequent data analysis
Authors: Voet L. , Krefft M. , Mairhofer H. , Williams K. L.
Published: August 2005

An assay for determining the proportions of prespore cells in a simple multicellular organism, the slug stage of Dictyostelium discoideum, was established using a presporespecific monoclonal antibody and a fluorescence‐activated cell sorter. Appropriate techniques for data analysis were developed.

The effects of slug size and age were determined. Small slugs have a lower percentage of prespore cells than large slugs. The percentage of prespore cells increases and then decreases in slugs aged between a few hours and 9 days. Pronounced effects were observed on the size of cells in aging slugs. In particular unlabelled (mostly prestalk) cells were larger than prespore cells in young slugs, but after 6 days migration they became considerably smaller than prespore cells. The fact that all unlabelled cells were coordinately shifted in size, suggests that these cells (which comprise prestalk, prestalklike, and predisc cells) are related to each other.
Cytometrically determined relative DNA content as an indicator of neoplasia in gastric lesionis,
Authors: Teodori Laura , Capurso Lucio , Cordelli Eugenia , De Vita Raffaele , Koch Maurizio , Tarquini Maurizio , Pallone Francesco , Mauro Francesco
Published: August 2005

Cellular samples from human gastric endoscopic biopsies were analysed in order to detect possible DNA content alterations as markers of cancerous and precancerous lesions of the digestive tract. Samples were derived from the stomach of normal donors (17 cases), and from patients clinically classified as affected by stomach adenocarcinoma (18 cases), chronic atrophic gastritis (20 cases), or other non‐neoplastic lesions (17 cases). Sample processing was performed by mechanical and enzymatic treatment to obtain monodispersed cells. Staining for flow cytometric analysis was achieved with ethidium bromide and mithramycin. Samples from normal donors constantly exhibited a single cell population with diploid DNA content. All but three neoplastic specimens exhibited both a diploid and an aneuploid cell subpopulation, with the DNA index of the aneuploid peak ranging from 1.10 to 1.85 (except a single instance with a value of 3. 13). The presence of a recognizable aneuploid subpopulation was also observed in 9 out of 20 chronic atrophic gastritis specimens. Such aneuploidy is similar to that observed for the adenocarcinoma, even if the fraction of aneuploid cells appears to be generally higher in the tumor than in the gastritis cases. All other cases of gastritis and of non‐neoplastic disease exhibited diploid cells only. The meaning of aneuploidy in some gastritis specimens is a phenomenon not yet fully explained. Still, aneuploidy appears to be a useful marker for recognizing the presence of suspect malignant cells in gastric lesions.
A follow‐up study of the development of Rauscher erythroleukemia,
Authors: Szabó Gábor , Mátyus László
Published: August 2005

Expression of cell membrane antigens recognized by antiserum raised against purified Friend murine leukemia virus (Fr‐MuLV) envelope glycoprotein (gp71) and by xenotropic MuLV‐coded cell surface antigen (XenCSA) specific antibodies was studied in the course of the development of Rauscher erythroleukemia in the spleen of Balb/c mice. DNA content vs immunofluorescence or light scattering of cells were simultaneously analyzed. At early stages of the disease (4–5 days after infection) the gp71 and XenCSA‐related antigen expression is enhanced mainly on S‐G2/ M‐phase cells as compared to the majority of G1‐phase cells or to the endogenous background of uninfected cells. Later (around 10 days after infection) an approximately tenfold increased gp'71‐related antigen density is reached in every phase of the cell cycle. These data show that the virus‐induced transition from resting to proliferating state is coupled to enhanced expression of both helper and defective viral env‐gene products in the cell membrane of mitotic and G1‐phase cells as well.
Analytic studies of foam cells from breast cancer precursors
Authors: King Eileen B. , Kromhout Lois K. , Chew Karen L. , Mayall Brian H. , Petrakis Nicholas L. , Jensen Ronald H. , Young Ian T.
Published: August 2005

A preliminary study of foam cells from nipple aspirate fluid demonstrated the ability of image analysis to discriminate categories of breast disease. Foam cell images numbering 471 were collected from nipple aspirate samples representing three to six cases of each of the four following disease categories based on breast tissue diagnosis: benign, nonproliferative; hyperplasia; atypical hyperplasia; and cancer. Twentytwo shape and density parameters were measured for each cell image. Using multivariate analysis, eight nuclear and three cytoplasmic parameters showed significant differences (P < 0.005) when tested among cell populations from the breast disease categories. Linear stepwise discriminant analysis enabled construction of a three‐parameter model that was optimal for distinguishing among cell populations from the four categories of breast disease. The means of all twenty‐three parameters were then evaluated on a per‐patient basis. A second three‐parameter model was constructed that distinguished, with 100% accuracy, patients with proliferative disease from those with nonproliferative disease. Grouping disease categories and comparing patients whose diagnosis was benign or hyperplasia versus atypical hyperplasia or malignant, the model placed patients in the correct group 83% of the time.
Use of flow cytometry in industrial microbiology for strain improvement programs
Authors: Betz J. W. , Aretz W. , Härtel W.
Published: August 2005

A flow cytometry (FCM) system was chosen to analyze and sort microbiological samples, e.g., bacteria, bacterial spores, yeasts, and fungal spores, without major changes in the commercially available state. The system was further improved by addition of a stepping motor‐driven scanning table that accepts standard petri dishes or microtiter plates. The electronics of the sorting system were changed to enable the sorter to deliver only one particle at a time, working in a “handshake” mode with the scanning table. Appropriate parameters, depending on the biological material and including all fluorescent stains that do not impair growth and productivity of cells were chosen to sort distinct bioparticles under aseptic conditions and to clone colonies or cultures out of them. A mutagenized sample of spores entering the germination cycle can be followed and thus provide a means to pick only viable growing cells despite the killing effect of the mutagen. One example of a typical strain improvement is illustrated. From a spore suspension of Rhizopus arrhizus, a subpopulation of morphologically different spores comprising about 5–10% of the whole population was cloned. From approximately 8,000 clones, 10 were isolated that produced approximately five‐ to six‐fold the amount of fungal lipase activity, compared to the original strain or to reisolated clones from the mean population of clones.
Three‐color immunofluorescence analysis of leu antigens on human peripheral blood using two lasers on a fluorescence‐activated cell sorter
Authors: Loken Michael R. , Lanier Lewis L.
Published: August 2005

A fluorescence‐activated cell sorter was modified to quantify simultaneously three immunofluorescence stains on a population of cells. A dye laser containing rhodamine 6G was used to obtain 600 nm light to excite Texas Red coupled avidin. An argon ion laser operating at 488 nm excited both fluorescein and phycoerythrin directly conjugated antibodies. The emission from these fluorophores could be independently quantified as demonstrated by the histograms generated by samples labeled separately with each of the three stains. This three‐color detection system was used to analyze human peripheral blood mononuclear cells for the expression of three antigens: Leu 2, Leu 7, and Leu 11. Upon reanalysis of the list mode data, several discrete subpopulations of lymphocytes could be identified based on the quantitative expression of these three antigens. Of particular interest in the normal sample studied was a population of dimly labeled Leu 2+ cells which were predominantly Leu 11+, a phenotype which is seen infrequently in normal individuals. This technique expands the combinations of antigens that can be studied at any one time and will facilitate the detection and functional analysis of cells in heterogeneous populations.
Fluorescence polarization of six membrane probes in embryonal carcinoma cells after differentiation as measured on a FACS II cell sorter
Authors: Schaap G. H. , De Josselin De Jong J. E. , Jongkind J. F.
Published: August 2005

Fluorescence polarization measurements on a FACS II cell sorter were compared with static measurements on a spectrofluorimeter using calibration solutions and Hoechst 33258‐labeled cells. For the flow cytometric measurements on the FACS we used a pseudodepolarizer for normalization of the output of the two photomultipliers. The results showed that fluorescein and fluoresceinated bovine serum albumin (BSA) solutions gave identical values on both instruments. The mean value for fluorescence polarization of Hoechst 33258‐labeled cells as measured on the FACS was the same as the value obtained with the spectrofluorimeter.

Subsequently the fluorescence polarization of six different membrane probes was determined using differentiating embryonal carcinoma cells as a model system. Differentiation was induced by treatment of the cells with retinoic acid together with cyclic AMP. With diphenylhexatriene (DPH) the fluorescence polarization increased from I‖/I = 1.55 to 1.74 upon differentiation. With a charged analog of DPH (TMA‐DPH) fluorescence polarization increased from I‖/I = 1.87 to 2.02. No appreciable changes in fluorescence polarization were observed in this cell system when anthroyloxysterate probes (12‐AS, 9‐AS, 6‐AS, 2‐AS) were used.
Flow cytometric analysis of the binding of eleven lectins to human T‐ and B‐cells and to human T‐ and B‐cell lines
Authors: Malin‐Berdel Jeannette , Valet Günther , Thiel Eckhart , Forrester John Anthony , Gürtler Lutz
Published: August 2005

The relative surface binding of 11 lectins to human peripheral blood T‐ and B‐lymphocytes, to Molt‐4 and JM T‐cell lines, and to 6410 and NC37 B‐cell lines was determined by flow cytometry. The lectins from Lens culinaris (LCA), Ricinus communis (RCA), Arachis hypogaea (PNA), Abrus precatorius (APA), Ulex europaeus (UEA‐F), Sarothamnus scoparius (SAS‐F), Helix pomatia (HPA), Phaseolus coccineus (L‐PHA), Glycine max (SBA), and Triticum vulgare (WGA) were fluoresceinated and incubated with living, formaldehyde‐fixed, or neuraminidase‐treated cells. Except LCA, which preferentially bound to the two B‐cell lines tested in this study, none of the other lectins exhibited selective binding to the undifferentiated cells of the cell lines. The T‐cell lines and, in part, the peripheral blood T‐cells bound less WGA, APA, LCA, and L‐PHA than the B‐cell lines and the peripheral blood B‐cells. Binding of PNA was found only after neuraminidase treatment of the cells; the binding of PNA, HPA, and UEA‐F after neuraminidase treatment was higher for the T‐cells than the B‐cells from peripheral blood. No significant differences were detected between both cell types for RCA, ConA, SBA, and SAS‐F.
Comparison of digital focus criteria for a TV microscope system
Authors: Harms H. , Aus H. M.
Published: August 2005

Existing focusing methods, such as standard deviation of gray values, gradients, information content of the image and the lateral inhibition function, were compared, and a new combination criterion was developed. This combined method consists of using the standard deviation of the image for coarse focusing followed by a modified form of the lateral inhibition for the fine focus. The lateral inhibitor model of the eye is a recursive filter that can be simplified to a nonrecursive filtering algorithm, as postulated by Rechenberg. The reported focus method operates over a focusing range (z‐axis) of ± 300 μm and is accurate within ±0.1 μm in the transmission, dark‐field, and phase contrast microscope.
“Cytomic” data system modules—modern electronic devices for flow cytometric data handling and presentation
Authors: Kachel V. , Schedler K. , Schneider H. , Haack L.
Published: August 2005

Flow cytometric investigations can be very complex when several parameters of cells are to be measured and cells must be sorted. On the other hand, many questions may be answered by a single‐parameter analysis. The Cytomic modular data system can easily be adapted to such different needs of flow cytometry. It consists of four types of modules which can be used as stand‐alone elements or can be combined into a larger data system: CYTOMIC 12, a one‐and two‐parameter analyzer; CYTOMIC 123, a module mainly provided for on‐line three‐parameter data uptake and processing; CYTOMIC SORT, a fully software‐controlled sort module, where sort windows may be selected by a built‐in light pen; and the CYTOMIC LIST list mode module, which is able to collect up to eight cell parameters unclassified on a 1.2‐MB floppy disk. Each module built from Z80 microprocessor Eurocards has its own keyboard, display screen, power supply, analog‐to‐digital pulse height conversion equipment, and an intermodule data communication bus. Each module has a preprogrammed set of functions, which is specially tailored for flow cytometric applications. This kind of data system allows the user to start with a single module for one‐and two‐parameter analysis. When required, the system may successively be enlarged without losing the stand‐alone feature of each module.
A simple device for the collection of cells sorted by flow cytometry
Authors: Patrick Catherine W. , Keller Robert H.
Published: August 2005

A simple device for the collection of cells sorted by flow cytometry under sterile and nonsterile conditions is described. Cells collected in this manner reflect a high recovery rate with superior preservation of viability and cytomorphology.
Instruments for rapid cytophotometric analysis of morphologically identified clinical cell material
Authors: Carlson Leon , Auer Gert , Kudynowski Jan , Lindblom Roland , Zetterberg Anders
Published: August 2005

Two instruments for cytophotometric analysis are described. The first is a generally applicable object stage scanning microspectrophotometer for the wavelength region 240–700 nm. The microscopic image is shown on a monitor screen and delineation of the analyzed objects is made with the aid of a light pen. The second instrument is a high speed microscope photometer specially adapted for the measurement of DNA, nuclear protein, and projected nuclear area following combined Feulgen and Naphthol Yellow S staining. A two‐dimensional CCD sensor measures the light intensity in the microscopic image with a resolution of 0.3 μm. The objects are selected by the operator pointing the light pen at the image screen. The integrated absorbance at two different wavelengths, together with the projected area, is automatically measured for each object and presented in histogram form. Cells in clusters or sections can be measured under operator control with the help of the light pen. The time required for measuring 100 cells is 5–10 min in an ordinary preparation.

The application of these instruments to the grading of cellular atypia and in the prognostic evaluation of malignant tumors is demonstrated on material from developing squamous bronchial carcinoma and in invasive mammary and prostatic adenocarcinoma.
Rotational and translational swimming of human spermatozoa: A dynamic laser light scattering study
Authors: Rigler Rudolf , Thyberg Per
Published: August 2005

The rotational swimming motion of human spermatozoa is evaluated from measurements of depolarized dynamic laser light scattering at zero angle. The analysis is based on a Maxwellian angular velocity distribution and yields a rotational frequency of about 4 Hz that is ascribed to the rotation of the sperm head. From comparison with the translational swimming motion, a propelling efficiency of about 10 μm per turn is deduced. This parameter describes the linkage between the rotational and translational swimming motion and is likely to be discriminatory in the analysis of physiological and pathological sperm motions.
7‐amino‐actinomycin D as a specific fluorophore for DNA content analysis by laser flow cytometry
Authors: Zelenin Alexander V. , Poletaev Andrey I. , Stepanova Natalija G. , Barsky Viktor E. , Kolesnikov Viktor A. , Nikitin Sergei M. , Zhuze Alexey L. , Gnutchev Nikolai V.
Published: August 2005

A technique for DNA amount determination by flow cytometry based on the use of 7‐amino‐actinomycin D (7‐amino‐AMD), a fluorescent analogue of antibiotic actinomycin has been investigated, and a particular staining procedure has been developed. The procedure includes short fixation in 70% ethanol and staining for 20 min in 10‐5M solution of 7‐amino‐AMD at pH7. The results of DNA content measurements are very reproducible. The histograms obtained have a coefficient of variation less than 3%.

The absorption maximum of the complex of 7‐amino‐AMD with DNA is situated in the green spectrum region, making this stain particularly suitable for argon laser flow cytometry.
Studies on characterization of mosquito cell lines
Authors: Wagh Ulhas V. , Chiplonkar Jayant M. , Tyagi Nirmala S. , Dighe Rajani P.
Published: August 2005

Results of studies on characterization of mosquito cell lines are described; these include chromosome analysis, image cytometric, and flow cytometric estimation of DNA, tumorogenicity and angiogenicity, concanavalin‐A‐induced agglutination, and proton spin‐lattice relaxation time (T1). The established mosquito cell lines are not diploid lines, although their stem‐lines are diploid. Feulgen cytometry and flow cytometry reveal the inherent heterogeneity in DNA contents and support the observations on chromosome frequency distribution. Two cell lines exhibit angiogenic property and cell infiltration inchorio‐allantoic membrane of chick embryos; however, none of the cell lines tested by inoculating hamsters, mice, or adult mosquitoes exhibit tumorogenicity. The agglutinability patterns of these cells in response to concanavalin‐A treatment reveal the coexistence of agglutinable and unagglutinable cells. The mosquito cells differ from mammalian and chick cells in respect of their proton spinlattice relaxation time.
Progression of mammary adenocarcinomas as reflected by nuclear DNA content
Authors: Auer Gert U. , Fallenius Anders G. , Erhardt Kerstin Y. , Sundelin Birgitta S. B.
Published: August 2005

In 18 breast cancer patients the DNA histograms observed in the primary tumor at the date of diagnosis were compared with those found in the corresponding local and distant metastases at autopsy up to more than 12 yr later. All patients, except one, exhibited the same type of DNA histogram in both the primary tumor and its metastases. In one patient the DNA histogram changed from an euploid type in the primary breast carcinoma to an aneuploid type in the metastases.

The results are interpreted as indicating that mammary adenocarcinoma in general exhibit a high degree of stability of the nuclear DNA content during the history of the disease. It is suggested that in breast cancer progression of the tumor disease is more likely due to a net increase arid/or dissemination of tumor cells exhibiting similar genetic properties and malignancy potential than to a progressive dedifferentiation and increase of malignancy of the tumor cells.
The prognostic significance of DNA measurements in endometrial carcinoma
Authors: Moberger B. , Auer G. , Forsslund G. , Moberger G.
Published: August 2005

DNA analysis was performed in 71 cases of endometrial carcinoma, selected from a retrospective series of 445 cases registered at the Department of Gynecology, Radiumhemmet, Karolinska Hospital, Stockholm, during 1973–1975. The histological material from 37 patients surviving more than eight years was compared with that from those who died from cancer within two years.

The prognostic value of the DNA distribution pattern of the tumors in relation to the clinical stage and the histological grade of the tumors was evaluated. Patients with neardiploid or ‐tetraploid tumors were found to have a significantly lower death rate than those with aneuploid tumors. The DNA distribution pattern was also found to correlate better with the survival rate than the clinical stage or the histological grade of the tumors.
A morphometric study of vascularization in uterine cervix cancers
Authors: Révész L. , Siracka Eva
Published: August 2005

The stromal, parenchymal, and vascular cellular constituents were identified and their relative proportion determined in biopsy specimens derived from untreated uterine cervix carcinomas. In the stroma of the tumors that eventually responded well to radiation treatment resulting in a prolonged survival of the patients, the proportion of vascular cells was increased in comparison to the cases that were associated with a poor patient survival. The observations were interpreted in terms of the radiobiological oxygen effect reflecting the radiosensitivity variation of cells in tumor areas vascularized to varying extent. It was concluded that the determination of a tumor vascularity index as a routine parameter would be of a great prognostic value and would contribute to the characterization of individual tumors, leading to improved therapeutic efficiency.
Convention on nomenclature for DNA cytometry
Authors: Hiddemann Wolfgang , Schumann Johannes , Andreeff Michael , Barlogie Barthel , Herman Chester J. , Leif Robert C. , Mayall Brian H. , Murphy Robert F. , Sandberg Avery A.
Published: August 2005

The Committee on Nomenclature of the Society for Analytical Cytology presents guidelines for the analysis of DNA content by cytometry. These guidelines cover: staining of DNA; cytogenetic and cytometric terminology; DNA index; resolution of measurements; and cytometric standards.
Morphologic changes in rat urothelial cells during carcinogenesis: I. Histologic and cytologic changes
Authors: King E. B. , Vanderlaan M. , Jensen R. H. , Kromhout L. K. , Hoffman J. W.
Published: August 2005

The methods of either image or flow cytometry applied to developing bladder tumors in rats requires satisfactory cell samples and a system for cell classification that is related to the lesions from which the cells are derived. Seven‐ to eight‐week‐old male Fisher 344 rats were fed 0.05% of the carcinogen N‐butyl–4‐N‐hydroxybutylnitrosamine (BBN) in drinking water for 10 wk and then returned to tap water. Animals were killed at 14, 26, 34, 45 and 62 wk after the start of carcinogen feeding. Age‐matched untreated animals were controls. Bladders were fixed, embedded, sectioned, and histologically evaluated, or were dissociated with a trypsin/EDTA solution into single cells that were Papanicolaou stained and evaluated for cytopathologic changes. Overnight urines were collected before killing; urine sediments were Papanicolaou stained and evaluated. Histologic features were hyperplasia at 14 wk, followed by slowly progressing papillary transitional cell tumors that eventually led to invasive carcinoma and were similar to those reported for this animal model. Treated animals had cytologic features of repair at 14 and 26 wk and neoplastic features at 45 and 62 wk. Both reparative and neoplastic changes were found at 34 wk. Cells were much more numerous in urines from treated rats (> 1,000 per sample) than in urines from controls (< 1,000 per sample). Although exfoliated cells in urine samples were generally of poor quality, as many as 11% of cells were adequately preserved. Dissociated bladder cells provided an optimal sample representative of the lesions from which they were derived; cells were of excellent quality for detailed morphologic and quantitative studies.
The use of correspondence analysis in the assessment of morphologic changes during carcinogenesis
Authors: Gelsema E. S. , Hunink M. , Queiros C. E. , Timmers T.
Published: August 2005

Correspondence analysis is a statistical technique originally developed for the analysis of frequency and contingency tables. It may also be successfully applied to the problem of the assessment of the characteristics of samples of cytological material. The present article describes the use of this formalism on feature data measured on urothelial cells in populations obtained from rats at different time points after exposition to a carcinogenic agent. After the application of correspondence analysis, the projections of the samples corresponding to the different time points onto the plane spanned by the first two factorial axes constitute a trajectory, preserving the time ordering. The locations of the projections of cell samples of untreated animals (controls) with respect to this trajectory suggest that two different processes inducing different morphological changes are at work: Early abnormal samples are characterized by a lower‐than‐normal value of nuclear stain content, whereas late abnormals are mainly characterized by a higher‐than‐normal nuclear stain value. It is shown that populations of selected atypical cells show less variation throughout the carcinogenic process than populations of randomly selected cells.
Quantitation of cell kinetic responses using simultaneous flow cytometric measurements of DNA and nuclear protein
Authors: Pollack A. , Moulis H. , Block N. L. , Irvin G. L.
Published: August 2005

A rapid procedure was developed for the simultaneous flow cytometric analysis of nuclear protein using fluorescein isothiocyanate, and DNA using propidium iodide in isolated nuclei. The staining procedure did not involve centrifugation and was easily adapted to the staining of human peripheral blood lymphocytes stimulated with phytohemagglutinin, EL4 murine lymphoid tumor cells in suspension culture, and R3327‐G rat prostatic adenocarcinoma solid tumor specimens. Histograms of unstimulated and PHA‐stimulated HPBL perturbed by actinomycin D, hydroxyurea, 3H‐TdR, colcemid, or hydroxyurea + colcemid showed that (1) resting, noncycling G1 (G1Q) cells are distinguished from late G1 (G1AB) cells, (2) early G2 (G2A) cells are distinguished from late G2 (G2B) cells, and (3) mitotic cells are distinguished from G2 cells. Treatment with hydroxyurea resulted in a build‐up of cells having high nuclear protein content and 2C DNA content (G1AB), while incubation with 3H‐TdR caused an increase in the number of cells with high nuclear protein content and 4C DNA content (G2B). Colcemid‐blocked mitotic cells were identified as having low nuclear protein content (lower than G2A nuclei) and 4C DNA content.

The nuclear DNA/protein histograms of untreated and colcemid‐treated log‐phase EL4 cells provided information concerning G1A, G1B, S, G2A, G2B, and M. The method was also used to quantitate the response of androgen‐sensitive rat prostatic R3327‐G tumors to androgen deprivation following castration. Sample preparation and staining for correlated nuclear DNA/protein measurements takes approximately the same amount of time as for single parameter nuclear DNA measurements.
Concentration‐dependent effects of potassium dichromate on the cell cycle
Authors: Bakke Oddmund , Jakobsen Karin , Eik‐Nes Kristen B.
Published: August 2005

Hexavalent chromium is found to be a strong mutagen, and it also is a potential carcinogen in man. DNA flow cytometry, growth measurements, and determinations of mitotic index show that 1–2 μM K2Cr2O7 produces a prolongation of the G2 phase of the cell cycle in NHIK 3025 cells. By increasing the chromate concentrations (>2 μM K2Cr2O7) the cells are also arrested in G2 phase. We have found, using synchronized cells and measuring cell cycle time, that the most chromate‐sensitive part of the cell cycle is S phase. This phase is also somewhat prolonged, and the cells became arrested in early S phase at high toxic K2Cr2O7 concentrations (8 μM). Our results thus indicate that K2Cr2O7 has an effect within S phase—maybe on DNA/RNA synthesis—and also interferes with processes necessary for progression through the G2 phase.
Calculation of S‐phase numbers of four major cell categories in human bone marrow from DNA‐flow cytometry and counterflow centrifugation data
Authors: Beumer Tom , Crissman Harry , Wessels Hans , Haanen Clemens
Published: August 2005

Fractionation of heterogeneous cell populations into a number of fractions differing in cell size and composition, followed by measurement of DNA profiles and differential morphology of the samples allows the calculation, using a mathematical deconvolution procedure, of the cell proliferation of the individual categories. The method was applied to human bone marrow, fractionated by counterflow centrifugation into 10 to 15 fractions. Calculated percentage of S‐phase cells in four major categories, including myeloid, erythroid, monocytic, and lymphoid cells, were in good agreement with data obtained by tritiated thymidine autoradiography.