bluuebunny/crossref_metadata_2025 huggingface dataset:
The multivariate prognostic index and nuclear DNA content are independent prognostic factors in primary breast cancer patients
Authors: Van Der Linden J. C. , Lindeman J. , Baak J. P. A. , Meijer C. J. L. M. , Herman C. J.
Published: August 2005

The predictive value of a previously described Multivariate Prognostic Index (which incorporates weighted values of the mitotic activity index, tumor size, and the axillary lymph node status), and the nuclear DNA content (DNA) was evaluated in 156 patients with primary invasive ductal breast cancer, diagnosed between 1980 and 1983. The results were analysed with respect to the occurrence of distant recurrence and survival of the patients after at least 3 yr of follow‐up (range 36–73 months; median 44 months).

Known prognostic factors such as lymph node status, tumor size, and the mitotic activity index correlated independently with distant recurrence. Furthermore, in respect to survival, the investigated prognostic factors (except DNA content) were significantly correlated.

The results indicate that the predictive value of the Multivariate Prognostic Index (MPI) is stronger (P < 0.001) than of the nuclear DNA content (P < 0.005) with respect to distant recurrence.

In a Cox multivariate regression analysis DNA ploidy turned out to be an independent prognostic factor once the MPI was selected. Furthermore, in Cox's analysis, DNA ploidy was the fourth selected variable after lymph node status, mitotic activity index, and tumor size in individual parameter analysis. The results of this study indicate that, with respect to breast cancer screening programs, it seems worthwhile to integrate morphometric features, the MPI, and DNA ploidy in a new prognostic model.
Characterizing aquatic bacteria according to population, cell size, and apparent DNA content by flow cytometry
Authors: Robertson B. R. , Button D. K.
Published: August 2005

Flow cytometry offers a rapid method for characterizing aquatic populations according to the properties of individual cells. This technology has been extended to aquatic bacteria by using high‐intensity UV excitation, condensing the laser beam onto a small area, using blemish‐free flow cells, optimizing organism staining protocol, segregating the optical signal produced with high‐transmittance optical filters collecting the signal with sensitive photomultipliers, and expanding the range of data displayed from individual samples with calibrated circuitry. Bacteria could be counted according to event frequency, and populations agreed with direct counts by epifluorescence microscopy. Forward scatter intensity was a linear function of volume for bacterial cells between 1.3 and 0.25 μm3 as calibrated by coulter impedance. Plastic spheres down to 0.014 μm3, 0.3 μm in diameter, were resolved. Aquatic bacteria 0.05 μm3 in volume were clearly resolved according to DNA content by staining with DAPI. The observed signal was DNA‐dependent because DNase treatment eliminated most fluorescence.

These procedures are suitable for direct analysis of the bacteria in marine and freshwater samples without interference from algae, sediment, or most DNA‐free organic particles. Cytograms indicated one or more clearly resolved subpopulations of bacteria of substantially smaller size and DNA content than the laboratory organisms typically classified.
Ternary representation of trivariate data
Authors: Sloot Peter M. A. , Figdor Carl G.
Published: August 2005

A new fast trivariate display technique based on the ratio of three measured variables and of relative cell number, obtained from flow cytometric measurements, is described.
Flow cytometric enumeration of colonies for in vitro chemotherapeutic drug evaluation
Authors: Thornthwaite Jerry T. , Loutzenhiser Kathy , Hornicek Francis J. , Donahue Roger , Allen Larry M.
Published: August 2005

The percentage of 50–100 μ colonies formed by LX‐T cells in medium containing agarose was determined microscopically, and this value was compared with the percentage determined by a flow cytometric method based on the forward and 90° light scatter of the colonies. As assessed by both in vitro methods, LX‐T cells exposed to chemotherapeutic agents formed fewer colonies as the drug concentration increased. However, flow cytometric analysis indicated that a change in the number of colonies formed was a consequence of changing chemotherapeutic drug concentration, whereas microscopic colony counting did not always detect the corresponding change in colony number. These experiments demonstrate that measurement of a drug's chemotherapeutic potential by flow cytometric counting of colonies is an alternative to the enumeration of colonies microscopically.
Analysis of bivariate flow karyotypes
Authors: Dean P. N. , Kolla S. , Van Dilla M. A.
Published: August 2005

Bivariate flow karyotype analysis is performed using data from chromosomes stained with two fluorescent dyes, typically chromomycin A3 and Hoechst‐33258, and measured in a flow cytometer or cell sorter (Carrano et al.: Proceedings of the National Academy of Sciences of the United States of America 76:1382–1384, 1979; Gray et al.: Proceedings of the National Academy of Sciences of the United States of America 72:1231–1234, 1975; Langlois et al.: Proceedings of the National Academy of Sciences of the United States of America 79:7876–7880, 1982). In the resulting bivariate histogram, most chromosome types appear as individual peaks. In sorting of chromosomes to purify a specific chromosomal type, its corresponding peak in the bivariate histogram is delineated by a rectangular region which surrounds it. All events (objects) that fall within this region trigger the sorting process. In most cases, peaks for different chromosomal types overlap to some extent, and in addition there is always an underlying background due to chromosome fragments and clumps. Thus the sorted population will not be pure; it may include more than one chromosome type and will include debris. To determine the purity of a sort, i.e., the percentage of the sorted material that is of the actual chromosomal type desired, two methods of mathematical analysis have been developed. In the more general method, the bivariate data within an analysis region that Includes the sort region, are fit with a series of bivariate Gaussian functions, one for each peak. In a simplified method, the data within the analysis region are transformed into a univariate distribution of either chromomycin A3 or Hoechst‐33258 fluorescence. The peaks in these univariate distributions are fit with univariate Gaussian functions. In both methods the purity is determined mathematically. The results of both methods agree well with independent methods of analysis.
Analysis of cytosolic ionized calcium variation in polymorphonuclear leukocytes using flow cytometry and Indo‐1 AM
Authors: Lopez Marc , Olive Daniel , Mannoni Patrice
Published: August 2005

The present report describes the increase in cytosolic‐free calcium levels (Ca2+i) induced by chemoattractants in polymorphonuclear leukocytes (PMN) using the new fluorescent Ca2+ chelator Indo‐1 AM. Increases in cytosolic Ca2+ were measured by flow cytometry. With this approach, 98% of PMN were found to respond to F‐Met‐Leu‐Phe (FMLP) and leukotriene B4 (LTB4) stimulation. Although both substances induced a rapid and relatively homogeneous rise in Ca2+i, only FMLP gave a sustained Ca2+i rise, whereas that induced by LTB4 appeared transient. In addition, the combined use of wide‐angle light scatter and electronic cell volume measurement allowed analysis of Ca2+i variations in the total peripheral blood cell population. The supernatant of a human fibrous histiocytoma cell line (GCT) was able to increase Ca2+ release in PMN. This activity may be ascribed to a new granulocytic activation factor, as neither human recombinant interleukin‐1α (IL‐1α), granulocyte‐colony stimulating factor (G‐CSF), nor granulocyte‐macrophage‐colony stimulating factor (GM‐CSF), which were present in this supernatant, were able to induce a Ca2+i rise in PMN.
Improvement of flow‐cytometric detection of multidrug‐resistant cells by cell‐volume normalization of intracellular daunorubicin content
Authors: Ross Douglas D. , Thompson Bruce W. , Ordóñez José V. , Joneckis Christopher C.
Published: August 2005

To improve the ability of flow cytometry to detect multidrug‐resistant cells, we studied the extent to which cell volume heterogeneity accounts for the variance of intracellular daunorubicin (DNR) content. For P388 murine or HL‐60 human leukemia cells exposed to DNR (1 m̈g/ml, 60 min), log intracellular DNR content varied in direct proportion to log cell volume measured by flow cytometry, with a correlation coefficient of .9. This relationship was confirmed by cell sorting based on intracellular DNR content with subsequent volume determination of the sorted cells. Normalization of intracellular DNR content for cell volume (thus obtaining intracellular DNR concentration) was accomplished by subtracting log cell volume from log intracellular DNR content for each cell. This resulted in a 34% decrease (range 23‐58%) in standard deviation compared to DNR content measurements without volume normalization for all cell types tested. Following exposure to DNR (as above), intracellular DNR content of drug‐sensitive P388 or HL‐60 cells measured by flow cytometry was 12‐ and 8‐fold greater than that of the multidrug‐resistant sublines P388/ADR and HL‐60/AR, respectively. However, because of the variance of intracellular DNR content, the predictive value of flow‐cytometric determination of intracellular DNR content as a discriminant assay for detecting the frequency of drug‐resistant cells in a mixed population was acceptable only when the frequency of resistant cells in the population exceeded 10%. In contrast, volume normalization of intracellular DNR content enhanced the ability of the flow‐cytometric assay to discriminate resistant cells by 10‐fold for P388 cells and 100‐fold for HL‐60 cells. Thus, by normalization of cell volume, the sensitivity and specificity of flowcytometric detection of multidrug‐resistant cells are improved.
1,3′‐diethy 1‐4,2′‐quinolylthiacyanine iodide as a “thiazole orange” analogue for nucleic acid staining
Authors: Van Bockstaele Dirk R. , Peetermans Marc E.
Published: August 2005

Flow cytometric evaluation of reticulocytes has become a valuable and more precise alternative for the microscopically determined reticulocyte count. Until now, the most promising results are obtained with the “thiazole orange” (TO) nucleic acid dye. We report on the use of a “thiazole orange” analogue, l,3′‐diethyl‐4,2′‐quinolylthiacyanine iodide (DEQTC), which gives comparable results and is commercially available at low cost.
Flow cytometry using paraffin‐embedded tissue: Five years on
Authors: Hedley David W.
Published: August 2005

The use of paraffin‐embedded tissue for flow cytometry is reviewed. A number of technical modifications of the original 1983 method have been described, aimed at improving the accuracy of DNA measurements by minimizing cell debris or reducing coefficients of variation, and at simplifying sample preparation. Over 100 clinical studies have now been reported, mainly assessing the effect of DNA index on prognosis, and those published up until mid‐1988 are summarized in an appendix. More recently there have been developments in the use of monoclonal antibodies to measure oncogene products or proliferation markers in addition to DNA content. Detailed clinical evaluation and standardization of these more sophisticated methods is still some way ahead, but as was the case with DNA index, the use of archival material from patients whose outcome is already known should speed this process.
A digital image microscopy system for rare‐event detection using fluorescent probes
Authors: Lee Benjamin R. , Haseman David B. , Reynolds C. Patrick
Published: August 2005

Instrumentation for rare‐event analysis should be capable of reliably detecting infrequent cells (< 1:10,000) while both excluding false‐positive signals and including true positive cells found in multicell clumps. We have developed a digital image microscopy (DIM) system in which a cytospin of 2 million cells is scanned with an intensified video camera (ISIT) using an IBM PC AT microcomputer‐controlled microscope stage. PASCAL software controls the stage and analyzes video input, storing the location of positive cells to magnetic disk. The user can then “replay” each positive cell under computer control for either visual confirmation or analysis using other fluorescent probes. The computer requires 24 min to scan a cytoprep of 2 million cells, while playback for visual confirmation by the user averages 5 min. Using Hoechst–33342 premarked cells seeded into bone marrow as a model system, we found that the DIM system reliably detects one target cell per million marrow cells. With appropriate immunological markers, this system will aid in evaluating bone marrow purged of tumor cells prior to transplantation and should also be useful for detection of minimal residual disease in blood or bone marrow from patients with leukemia or solid tumors.
Cell‐cycle studies by multiparametric automatic scanning of topographically preserved cells in culture
Authors: Colomb Evelyne , Kopp Francis , Spyratos Frédérique , Martin Pierre‐Marie
Published: August 2005

The authors have developed a new methodology for characterizing in situ the cell cycle of human mammary epithelial cell lines. Using a SAMBA 200 cell image processor (scanning cytometry), 15 densitometric and textural parameters were computed on each Feulgen‐stained nucleus. Parameters computed from the grey level cooccurrence and run‐length section matrices allowed assessment of the chromatin pattern. Multiparametric analysis of data defined: (1) the relative position of each cell; (2) the relative positions of groups of cells, each group corresponding to a definite phase of the cell cycle; and (3) the function of these parameters best separating these phases. Files then were constructed for each phase: G0/G1, S, G2/ and M. Using these three files as a reference to classify cells, it was possible to ascertain the phase of the cell cycle for each cell of a population.

The MDA AG human cell line synchronized by mitotic selection was used as a model to develop this method. The criteria used to assign cells to G0/G1, S, or G2 was DNA content. Classification in M phase was achieved by visual identification of mitotic cells. This method was checked on unsynchronized MDA AG and then applied to other human cell lines (MDA MB231, MCF‐7, T47D C111). Comparison of results obtained by scanning cytometry and flow cytometry showed the proportion of cells assigned to G0/G1, S, and G2/M by the two methods to be similar. This new method removes some of the limitations of flow cytometry by (1) allowing visual verification of each cell analyzed; (2) lowering the number of cells required for study; (3) discriminating between G2 and M; and (4) preserving cell topography.
Model system evaluating fluorescein‐labeled microbeads as internal standards to calibrate fluorescence intensity on flow cytometers
Authors: Vogt Robert F. , Cross G. David , Henderson L. Omar , Phillips Donald L.
Published: August 2005

Fluorescence intensity calibration was evaluated in a model system for flow cytometers using commercially available fluorescein‐labeled microbeads as internal standards and stabilized fluoresceinated thymus cell nuclei (Fluorotrol) as surrogates for stained mononuclear cells. Spectrophotometrically determined calibration values for the microbeads were used to generate a standard curve that converted green fluorescence histogram channels into molecular equivalents of soluble fluorescein (MESF). In 19 analyses repeated during a single run, the coefficients of variation (CVs) for the derived MESF values on both dimly and brightly stained Fluorotrol populations were less than 2%. In 26 separate determinations over 14 weeks, the CVs of the derived MESF values were less than 3%. The MESF values of the dim and bright Fluorotrol populations derived from the microbead standard curves were both about 50% lower than those determined by direct spectrophotometric analysis of Fluorotrol. The analytical imprecision of fluorescence intensity measurements in this idealized model system has a CV less than 3%, and the analytical inaccuracy shows that calibration in MESF units remains uncertain over about a two‐fold range.
New, simple flow cytometry technique to discriminate between internalized and membrane‐bound particles in phagocytosis
Authors: Fattorossi Andrea , Nisini Roberto , Pizzolo John G. , D'amelio Raffaele
Published: August 2005

This report describes a new flow cytometry technique to measure phagocytic activity and discriminate simultaneously between internalized and membranebound particles. Fluorescein‐conjugated heat‐killed Candida albicans (F‐Ca) are opsonized with purified antibodies or normal human serum and used as targets for human polymorphonuclear granulocytes (PMN). The procedure is based on the observation that F‐Ca lose their green fluorescence and acquire red fluorescence upon incubation with ethidium bromide (EB) through the resonance energy‐transfer phenomenon occurring between the two fluorochromes. PMN are incubated with opsonized F‐Ca particles for 20 min at 37°C or, as a control, at 4°C and in the presence of cytochalasin B, an inhibitor of the phagocytic process that does not affect membrane binding of F‐Ca. EB is added, and green and red fluorescence associated with PMN is evaluated using a mercury‐lamp‐powered instrument. Because EB does not penetrate intact cell membranes, internalized particles are not affected by EB and remain green, whereas membrane‐bound particles assume an intense red stain. By means of contour plot analysis, the number of PMN containing and/or binding F‐Ca particles can be readily assessed. The method described here allows precise quantitative analysis of the phagocytic process on the part of human PMN in a single, one‐step assay that does not require sophisticated instrumentation or reagents and should prove to become a test suitable for clinical purposes.
Cellular dna content parameters in untreated and recurrent squamous cell cancers of the head and neck
Authors: Ensley John F. , Maciorowski Zosia , Hassan Mohammad , Pietraszkiewicz Haline , Heilbrun Lance , Kish Julie A. , Tapazoglou Efstathios , Jacobs John R. , Al‐Sarraf Muhyi
Published: August 2005

The presence and degree of DNA aneuploidy as measured by the DNA index (DI) and the S phase fraction (SPF) were determined by flow cytometry in 294 specimens from 237 patients with untreated and recurrent squamous cell carcinomas of the head and neck (SCCHN). A descriptive analysis was performed in which the specimen DNA parameters were correlated with stage, size of primary, degree of lymph node involvement, morphological grade, and treatment status of the corresponding patients. Approximately 70% of the previously untreated specimens contained DNA aneuploid populations (DI > 1.10) and three quarters had SPF that were above 15%. There was a strong, direct as sociation between DI and SPF (P < 0.001). There was no correlation of the presence or degree of DNA aneuploidy with the stage of the tumor or the size of the primary or conventional morphological grade of the tumor. Specimens from patients with recurrent tumors and untreated patients with N3 lymph nodes had significantly lower rates of DNA aneuploidy and mean DI. Serial determinations of DNA aneuploidy in patients with SCCHN undergoing cytotoxic therapy are ongoing and may prove useful in the identification and understanding of resistance and response in this tumor.
A spectrophotometric method for determining the stream sample core diameter of a flow cytometer
Authors: Blume Philip
Published: August 2005

A simple, rapid, and accurate method is described for the determination of flow cytometer stream sample core diameters. A precisely formulated solution of potassium dichromate is introduced as a sample, the stream is collected, and its absorbance is measured using a spectrophotometer. This permits the calculation of the factor by which the original solution was diluted and, given the stream diameter, the sample core diameter.
Evaluation of algorithms for ratio imaging in fluorescence microscopy
Authors: Rogowska Jadwiga , Bright Gary R. , Preston Kendall , Taylor D. Lansing
Published: August 2005

Ratio imaging in fluorescence microscopy is used in measuring parameters such as pH, pCa, cytoplasmic porosity, and the relative concentration of fluorescent analogs within single cells. The fastest method for ratio imaging is to use lookup tables on special‐purpose image processors. Since lookup tables store integers in integer addresses, using a lookup table will generate rounding errors. The magnitude of the error will depend on the transformation performed and on the number of levels used in the lookup table. We examined ratio imaging by lookup table and computed the errors generated by both inversion and log subtraction methods. Both uniformly fluorescing fields and fluorescing cell images were employed to provide data for use in confirming our calculations and illustrating both the magnitude and spatial incidence of errors. It is shown that, through proper design of lookup tables, a significant reduction can be made in the errors generated in comparison with common methods available in most image processors.
Technique for cellular fluorescence distribution analysis
Authors: Robinson R. D. , Reeder J. E. , Wheeless L. L.
Published: August 2005

The usefulness of multidimensional slit‐scan flow cytometry in whole cell measurements is dependent on extracting relevant features from the cellular fluorescence distributions (slit‐scan contours). In addition, the extraction of these features must be rapid to allow for realtime data processing during acquisition. This paper describes two algorithms that have been used successfully to count the numbers of local maxima (peaks) and to find nuclear boundaries in a cellular fluorescence distribution. These routines are efficient, use only simple integer arithmetic, and have been implemented on several different microprocessors.
Autolysis is a potential source of false aneuploid peaks in flow cytometric DNA histograms
Authors: Alanen Kalle A. , Joensuu Heikki , Klemi Pekka J.
Published: August 2005

Serial flow cytometric nuclear DNA content analyses were performed from normal human spleen, thyroid, liver, and pancreas removed from ten patients at autopsy and stored for up to 8 d without any preservative to study the effect of autolysis on DNA histograms. Fine needle aspiration biopsy (FNAB) samples were taken in diluted ethanol and tissue biopsies from the same area in formalin for embedding into paraffin at the time of autopsy and serially thereafter. Histograms obtained from samples taken within 10 h after death had a symmetrical G1 peak with a small coefficient of variance (CV) except histograms produced from paraffin‐embedded pancreatic tissue, but bimodal distributions similar to those seen in aneuploid tumors were obtained from many samples stored longer than for 20 h. The DNA indices of the bimodal histograms were usually <1.3. The false peaks were more prominent in FNAB samples than in paraffin‐embedded samples. The time of appearance of the false aneuploid peaks varied individually, and they were usually first seen in samples taken from the pancreas, followed by the liver, the thyroid and the spleen. Because neoplasms may become necrotic in vivo and fixation of fresh surgical samples may be slow and incomplete, increased DNA staining caused by autolysis may be a source of false aneuploid peaks in DNA content analysis.
Accuracy of routine flow‐cytometric bitmap selection for three leukocyte populations
Authors: Sučić Mirna , Kolevska Tatjana , Kopjar Branko , Kosanović Mirjana , Drobnjak Marija , Žalud Ivica , Marušić Matko
Published: August 2005

A double‐blind study was performed with peripheral blood of 41 human subjects to check the accuracy of determination of lymphocyte, monocyte, and granulocyte windows with which every flow cytometric analysis of leukocyte markers starts. White blood cell suspensions were prepared according to the whole blood method and analyzed on an EPICS‐C flow cytometer using the two‐parameter 90° light scatter vs. forward angle light scatter (granularity vs. cell size) data distribution. Windows (bitmaps) for lymphocytes, monocytes, and granulocytes were drawn and numbers of cells determined in each. The proportions of lymphocytes, monocytes, and granulocytes were calculated in relation to total cell number, counted and in relation to the sum of cells in three bitmaps, and then compared with proportions determined by microscopic whole blood cell (WBC) differential and a WBC differential determined in an automated hematology analyzer. Average proportions of lymphocytes obtained by the flow cytometer were significantly lower than those obtained by either microscopic or automated differential, suggesting that some of the relevant cells were not included in the bitmaps. Granulocyte proportion related to total cell number was lower and that related to bitmap cell number higher than that obtained by microscopic and automatic differentials, suggesting that nongranulocytic cells were included in the granulocyte bitmaps. Proportions of lymphocytes and granulocytes obtained by the flow cytometer correlated well with those obtained by both microscopic and automatic differential. In contrast, the proportions of monocytes showed a poor correlation, which is probably due to their low number and delicate position in the distribution, and which makes them difficult to delineate.
Limitations in the measurement of c‐myc oncoprotein and other nuclear antigens by flow cytometry
Authors: Lincoln Sarah T. , Bauer Kenneth D.
Published: August 2005

Recent developments in cell fixation, quantitative immunocytochemistry, and flow cytometry allow for the quantification of a variety of oncoproteins and other proliferation‐associated antigens in both fresh and archival pathology material. These studies provide evidence that the standard tissue deparaffinization/dissociation technique significantly reduces the amount of c‐myc oncoprotein remaining for analysis. To examine the factor(s) responsible for this observation, individual variables of the deparaffinization/dissociation technique including type of fixative, pepsin concentration, pepsinization times, pH, and exposure to organic solvents were examined in HeLa‐S3 cells. The cells were stained with monoclonal antibodies either to the c‐myc oncoprotein or to p105, a prolifera‐tion‐associated nu' clear antigen. Protein‐levels were measured on the basis of anti‐c‐myc or anti‐p105 immunofluorescence by flow cytometry and were found not be affected significantly by type of fixative, exposure to organic solvents, acid pH solution, or mechanical disruption. Levels of c‐myc oncoprotein were reduced by over 50%, however, when cells were exposed to 0.5% pepsin, whereas p105 was more resilient with only an approximately 7% reduction following the same treatment. Thus, careful examination of aspects of the deparaffinization/dissociation technique appears to be a necessary prerequisite for quantification of specific nuclear proteins from dissociated tissue specimens.
Variance within homogeneous phytoplankton populations, II: Analysis of clonal cultures
Authors: Campbell J. W. , Yentsch C. M.
Published: August 2005

In part I of this series of articles, a framework was presented for interpreting histograms of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. In this paper, the analytical framework is applied to flow cytometric histograms from laboratory experiments involving clonal phytoplankton cultures. The density function derived in part I was modified to include a third parameter representing a linear shift of the origin. This modified density function was fitted to chlorophyll fluorescence histograms for populations believed to be asynchronous (grown in continuous light) and also to histograms from populations grown on a 14:10 (h:h) light/dark cycle. Near‐synchronous subpopulations sorted from an asynchronous population were also analyzed. In populations in which underlying assumptions (asynchronous divisions, constant growth) are valid, curve fits provide estimates of the inherent variability among cells at age 0. The implication of fitting the density function to populations in which these assumptions are not valid is discussed.
Variance within homogeneous phytoplankton populations, III: Analysis of natural populations
Authors: Campbell J. W. , Yentsch C. M. , Cucci T. L.
Published: August 2005

A theoretical framework for interpreting flow cytometric histograms from homogeneous phytoplankton populations was developed in part I of this series of articles and applied to chlorophyll fluorescence histograms from clonal cultures in part II. In this paper, we demonstrate the application of this framework to the analysis of cell volume distributions found in a natural assemblage of phytoplankton from the Gulf of California. Flow cytometric analyses of a surface water sample incubated for a period of 61 h revealed the sequential growth and decline of three distinct subpopulations. Cell volume distributions for each subpopulation measured at different times were analyzed, and the theoretical density function described in parts I and II was fitted to these distributions. The range of cell volumes within each subpopulation was similar to that predicted for asynchronous populations.
Rapid analytical technique for the assessment of cell metabolic activity in marine microalgae
Authors: Dorsey Judy , Yentsch Clarice M. , Mayo Sara , McKenna Colleen
Published: August 2005

A standard method for the assessment of cell viability has been developed for marine phytoplankton using an inexpensive stain, fluorescein diacetate (FDA), at. 75 μM for 10 min. A flow cytometer was used as the fluorescence detector, providing an assessment of viability for each individual particle. Cell size and chlorophyll fluorescence per cell were assessed simultaneously, permitting an assignment of viability to specific subpopulations, thus increasing the power of the technique. A reasonable correspondence between FDA mean fluorescence intensity per cell and an independent metabolic indicator, photosynthetic capacity measured by 14C, was found. Both FDA mean fluorescence intensity and photosynthetic capacity vary as a function of cell volume. Recovery after extended periods of darkness indicate that cells that are FDA negative may not be dead, but merely quiescent or inactive.
A simple method to preserve oceanic phytoplankton for flow cytometric analyses
Authors: Vaulot D. , Courties C. , Partensky F.
Published: August 2005

A simple method was developed to preserve marine phytoplankton populations so that delayed flow cytometric analyses could be performed. The method consisted of immediate fixation with 1% glutaraldehyde (final concentration) followed by storage in liquid nitrogen. The method was tested on individual algal species and on natural samples from both coastal and pelagic waters. In most cases, it caused little cell loss and preserved well both forward angle light scatter and chlorophyll fluorescence, but phycoerythrin fluorescence sometimes was significantly increased. The technique performed best for the small‐sized picoplankton (below 2 μm) such as Syne‐chococcus cyanobacteria or the newly discovered oceanic prochlorophytes. For larger‐sized cells it had to be applied on a case by case basis as some fragile species, particularly dinoflagellates and cryptophytes, were poorly preserved.
A flow cytometric approach to assessing the environmental and physiological status of phytoplankton
Authors: Demers Serge , Davis Kimberly , Cucci Terry L.
Published: August 2005

Individual particle analysis using a flow cytometer (FCM) was made on natural phytoplankton communities in oligotrophic waters. Our objective was to develop an assay to yield information on the nutrient history of individual cells using FCM. Results from nutrient assays showed that both biovolume and total red fluorescence are affected by the nutrient conditions in the incubator. The light effect was measured by changes in the chlorophyll content of the cells, and after the 12 h incubation the cells seemed well adapted to the light conditions. The estimated kinetic constant for the chlorophyll synthesis averaged 1.5 × 1.0−2 h−1, whereas the growth rate calculated from the changes in the cell numbers changed from 0.14 to > 2.5 doubling per day. The smallest size fraction presented the highest growth rate ( > 2.5 doublings per day). The relationship between the total red fluorescence as estimated with the FCM and the biovolume revealed that the cells from the 2 m samples at the beginning of the experiment were probably nutrient limited. Important changes in the size of the cells under nutrient limitation were also observed. The FCM data suggest that the FCM is a valuable tool for estimating the relative growth response and nutritional state of natural phytoplankton populations.
Origin and biology of cancer metastasis
Authors: Fidler Isaiah J.
Published: August 2005

Metastasis, the spread of cells from a primary neoplasm to distant sites where they grow, contributes to the death of most cancer patients. The process of metastasis is not random. Rather, the process consists of a series of linked, sequential steps that must be completed by tumor cells if a metastasis is to develop. Thus, metastatic cells must succeed in invasion and embolization, survive in the circulation, arrest in a distant capillary bed, and extravasate into and multiply in organ parenchyma. Although some of the steps in this process contain stochastic elements, as a whole metastasis favors the survival and growth of a few subpopulations of cells that preexist within the parent neoplasm. Moreover, metastases can have a clonal origin, and different metastases can originate from the proliferation of single cells. The outcome of metastasis depends on the interaction of metastatic cells with different organ environments. Organ‐specific metastases have been demonstrated in a variety of experimental tumor systems, and even within one organ, site‐specific tumor growth can be found.

The conclusion that metastasis is a highly selective process that is influenced by both the intrinsic properties of tumor cells and by host factors is optimistic. A selective process is regulated and therefore can be studied and then manipulated.
Flow cytometry chamber with 4π light collection suitable for epifluorescence microscopes
Authors: Watson James V.
Published: August 2005

A compact, solid, spherico‐ellipsoidal chamber (SEC), which has approaching 4π (“all around”) light collection, has been developed for flow cytometry. This was mounted onto the stage of a standard fluorescence photomicroscope, and the camera was replaced by a photomultiplier. Both components can be added or removed in minutes. The increased light collection efficiency of the SEC (about 85%) compared with about 4% from standard chambers enabled a fluorescence microscope with a 50 W mercury vapour lamp to “double” as a flow cytometer. The system was tested with microbeads and cells stained for DNA with ethidium bromide, and results were comparable to those obtained with our laser‐based instrument.
Measurement variability in DNA flow cytometry of replicate samples
Authors: Wheeless Leon L. , Coon John S. , Cox Christopher , Deitch Arline D. , Devere White Ralph W. , Koss Leopold G. , Melamed Myron R. , O'Connell Mary J. , Reeder Jay E. , Weinstein Ronald S. , Wersto Robert P.
Published: August 2005

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter‐ and intralaboratory variability. Replicate “cocktail” samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and‐the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set‐up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory databases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.
Flow cytometric analysis of protein thiol groups in relation to the cell cycle and the intracellular content of glutathione in rat hepatocytes
Authors: Principe P. , Wilson G. D. , Riley P. A. , Slater T. F.
Published: August 2005

The relationship between protein thiols (PSH) and cell proliferation was examined in ethanol‐fixed rat hepatocytes. A new protocol was developed for simultaneous measurement of protein thiol vs. DNA content by flow cytometry. The fluorescent dye o‐phthalaldehyde (OPT) was used for flow cytometric measurements of protein thiol groups. The influence of nonprotein thiols was examined by monitoring the cell cycle of cells in which the glutathione content (GSH) was modified by treatment with buthionine sulphoximine (BSO). Three rat liver cell lines (IAR 20, IAR 6.1, IAR 6.1RT7) were used: these cell lines possess different growth characteristics and degrees of tumorigenicity, which made it possible toanalyse changes in PSH during normal and deranged cell proliferation. The effects on the cell cycle of the changes in PSH due to the depletion of GSH were measured by 5‐bromo‐2'‐deoxyuridine (BrdUrd) incorporation and flow cytometry. The data obtained can be summarised as follows: (a) OPT fluorescence increases with increasing DNA content in all rat liver cell lines examined; (b) the greatest variation in PSH content occurs in G1. There is a smaller variation in G2 + M, and PSH levels are relatively invariant throughout S‐phase; (c) a higher content of PSH is found in the tumorigenic cell lines; (d) the amount and distribution of PSH is not affected by BSO treatment; (e) kinetic studies indicate that BSO treatment has no effect on the ability of the IAR rat liver cell lines to progress through the cycle.
Dual‐parameter flow cytometric analysis of an early lymphocyte activation antigen (CK226) and DNA content
Authors: Zocchi M. Raffaella , Heltai Silvia , Poggi Alessandro
Published: August 2005

This study provides a direct correlation, via dual‐parameter flow cytometric analysis (simultaneous assessement of surface immunofluorescence and DNA content), between activated T‐cell entry into the S/G2/M phases of the cell cycle and the kinetics of expression of a novel T‐cell activation antigen, termed CK226. This molecule was identified by the specific monoclonal antibody on the leukaemic T‐cell line CEM/K, and it was expressed by 8–30% of resting peripheral blood lymphocytes and the majority of monocytes and granulocytes. A large fraction of activated lymphocytes acquired the CK226 antigen before DNA synthesis. Moreover, this molecule was expressed on virtually all G0/G1 and S/G2/M phase cells by day 2 after phytohaemoagglutinin (PHA) activation and at day 6 after stimulation in a mixed lymphocyte culture. The time course of expression of other known activation antigens, such as Tac and transferrin receptor, was comparable to that of CK226. Based on the relationships between CK226 expression on cycling cells and the stimulatory effects of the specific monoclonal antibody, we conclude that CK226 should be considered an early activation antigen, which defines a new pathway of T‐cell activation.
Separation of hepatocytes of different acinar zones by flow cytometry
Authors: Thalhammer T. , Gessl A. , Braakman I. , Graf J.
Published: August 2005

Hepatocytes in the proximal (zone 1) and distal (zone 3) regions of the liver acinus are selectively stained by perfusion of the isolated rat liver with 0.2–20 μM acridine orange (AO). After 10–60 min of anterograde perfusion, AO fluorescence is visible in zone 1 cells, whereas retrograde perfusion stains cells of zone 3. In this paper, we describe a technique to isolate a mixed population of fluorescent and nonfluorescent hepatocytes (cells from all acinar zones, which do not loose the zone specific AO labeling) and to separate these cells according to their zonal origin by fluorescence activated cell sorting. The zonal populations obtained were either fluorescent or non‐fluorescent (purity >95%). Separated cell fractions differed in their enzyme content (5' nucleotidase, succinate‐dehydrogenase, β‐glucuronidase). An unidentified AO metabolite, which is not found in bile after retrograde perfusion (not formed in zone 3 cells), is also absent after retrograde perfusion in sorted fluorescent cells (zone 3 cells), indicating zonal purity of sorted cells.
Flow cytometric analysis of the kinetic effects of cisplatin on lung cancer cells
Authors: Fujikane Toshiaki , Shimizu Tetsuo , Tsuji Tadakatsu , Ishida Sakae , Ohsaki Yoshinobu , Onodera Sokichi
Published: August 2005

The effects of cisplatin on the cell cycle and DNA synthesis of human lung adenocarcinoma cell line PC‐9 were examined by flow cytometry. The cellular DNA content and the bromodeoxyuridine (BrdUrd) incorporation rate were measured simultaneously using a monoclonal anti‐BrdUrd antibody. Following exposure to cisplatin (1.0 μg/ml) for 1 and 24 hr, the bivariate DNA/BrdUrd distributions revealed a delayed S‐phase transit and an accumulation of cells in the G2M phase. The BrdUrd‐linked green fluorescence intensity continued to decrease with the lapse of time. However, early‐and mid‐S‐phase cells soon recovered DNA synthesis activity, and the former showed higher activity than the control cells. These findings suggested the vigorous DNA synthesis of cells in early S phase. However, for quantitative analysis of chemotherapeutic effects, some problems remained to be resolved regarding the condition for DNA denaturation and its alteration by the agents.
Cytokinetic analysis of lung cancer by bromodeoxyuridine labeling of cytology specimens
Authors: Ten Velde G. P. M. , Schutte B. , Reijnders M. M. J. , Bosman F. T. , Blijham G. H.
Published: August 2005

Recent flow cytometric (FCM) studies have indicated the prognostic value of S‐phase cells (SPF) in lung cancer. More refined cytokinetic analysis can be obtained by dual‐parameter FCM, labeling S‐phase cells with 5‐bromodeoxyuridine (BrdUrd), which can be detected using a monoclonal antibody (MoAb) to BrdUrd. Tumor cells obtained through bronchoscopic brush were incubated for 1 hr in RPMI 1640 medium with 10% fetal calf serum and 10 m̈M BrdUrd. After fixation in ethanol, pepsin treatment, and DNA denaturation, the nuclei were stained with anti‐BrdUrd MoAb and propidium iodide. From 14 of 20 patients, sufficient material was obtained (three adenocarcinoma and seven squamous cell, one giant cell, and three small cell carcinoma). The measured SPF ranged from 5.2% to 26%. The labeling index (LI), calculated as the ratio of the number of BrdUrd‐la‐beled cells to the total number of aneuploid cells, or diploid cells in the case of a diploid tumor, ranged from 1.2% to 16.7%; LI and SPF correlated significantly (r = 0.69). In this study, we have demonstrated the feasibility of determining the actively DNA‐synthesizing cells on brush material from lung cancer cells. In addition, some extra information can be obtained about the SPF population, including the fraction of unlabeled SPF, which could be of prognostic significance.
INCA: Software for consort 30 analysis of flow cytometric calcium determinations
Authors: Keij Jan F. , Griffioen Arjan W. , The T. H. , Rijkers Ger T.
Published: August 2005

The INCA program converts Consort 30‐generated fluorescence list mode data collected from Indo‐1‐stained cells to absolute intracellular calcium concentrations (nM Ca2+i). The calcium data are plotted vs. time, allowing the user to analyze the fractions of cells responding to a given stimulus. Converted files can be restored to disk after replacing FL1 and FL2 with time and calcium, respectively, for future analysis.
Automatically inferred markov network models for classification of chromosomal band pattern structures
Authors: Granum Erik , Thomason Michael G.
Published: August 2005

A structural pattern recognition approach to the analysis and classification of metaphase chromosome band patterns is presented.

An operational method of representing band pattern profiles as sharp edgedidealized profilesis outlined. These profiles are nonlinearly scaled to a few, but fixed number of “density” levels. Previous experience has shown that profiles of six levels are appropriate and that the differences between successive bands in these profiles are suitable for classification. String representations, which focuses on the sequences of transitions between local band pattern levels, are derived from such “difference profiles.”

A method of syntactic analysis of the band transition sequences by dynamic programming for optimal (maximal probability) string‐to‐network alignments is described. It develops automatic data‐driven inference of band pattern models (Markov networks) per class, and uses these models for classification. The method does not use centromere information, but assumes the p‐q‐orientation of the band pattern profiles to be known a priori.

It is experimentally established that the method can build Markov network models, which, when used for classification, show a recognition rate of about 92% on test data. The experiments used 200 samples (chromosome profiles) for each of the 22 autosome chromosome types and are designed to also investigate various classifier design problems. It is found that the use of a priori knowledge of Denver Group assignment only improved classification by 1 or 2%. A scheme for typewise normalization of the class relationship measures prove useful, partly through improvements on average results and partly through a more evenly distributed error pattern. The choice of reference of the p‐q‐orientation of the band patterns is found to be unimportant, and results of timing of the execution time of the analysis show that recent and efficient implementations can process one cell in less than 1 min on current standard hardware. A measure of divergence between data sets and Markov network models is shown to provide usable estimates of experimental classification performance.