bluuebunny/crossref_metadata_2025 huggingface dataset:
Parallel processing data acquisition system for multilaser flow cytometry and cell sorting
Authors: van den Engh Ger , Stokdijk Willem
Published: August 2005

This report describes the data acquisition electronics for a flow cytometer. The design differs from most instruments in that the signals from a large number of detectors are processed in parallel. Each of the input channels is capable of autonomously measuring and digitizing the fluorescence signals. The digitized values that belong to one particle are collected by digital circuitry and are presented as a compact data package on a special bus. In addition to the pulse values, the data package contains a time marker, information needed for sort decisions, and an error detection code. Specially designed electronic modules that read the information from the bus can take complex multiparameter sort decisions at a very high speed. All events can also be recorded as data lists by a computer. The lists can be used to reconstruct a sort or analysis run. The raw data lists can also be reduced to kinetic curves and/or (gated) multivariate histograms. As a result of the applied scheme of parallel pulse processing, the dead time of the system is independent of the number of parameters measured and the number and time separation of the excitation beams. The instrument has a cycle time of 5 m̈s, which corresponds to a throughput rate of 2 × 105 events/s. At this rate, the incidence of correlation errors is well below 1 in 108 analyzed particles. The system has proved to be reliable and convenient to use in a variety of experiments. Its high speed and low error rate make it well suited for high‐resolution measurements, rare‐event analysis, kinetic measurements, and high‐speed cell sorting.
Flow cytometric detection of mitotic cells using the bromodeoxyuridine/DNA technique in combination with 90° and forward scatter measurements
Authors: Nüsse Michael , Jülch Markus , Geido Elio , Bruno Silvia , Vinci Angela Di , Giaretti Walter , Ruoss Kurt
Published: August 2005

Mitotic cells could be well discriminated from the cells in the G1‐, S‐ and G2‐phases of the cell cycle using pulse labeling of S‐phase cells with bromodeoxyuridine (BrdUrd) and staining of the cells for incorporated BrdUrd and total DNA content. Unlabeled G2‐ and M‐phase cells could be measured as two separate peaks according to propidium iodide fluorescence. M‐phase cells showed lower propidium iodide fluorescence emission compared to G2‐phase cells. The fluorescence difference of M‐ and G2‐phase cells was caused by the different thermal denaturation of their DNA. Best separation of M‐and G2‐phase cells was obtained after 30–50 min heat treatment at 95°C. Mitotic index could be measured if no unlabeled S‐phase cells were present in the cell culture. With additional measurements of 90° scatter and/or forward scatter signals, mitotic cells could be clearly discriminated from both unlabeled G2‐ and S‐phase cells. The correct discrimination (about 99%) of mitotic cells from interphase cells was verified by visual analysis of the nuclear morphology after selective sorting. Unlabeled and labeled mitotic cells could be observed as pulselabeled cells progressed through the cell cycle. We conclude that this modified BrdUrd/DNA technique using prolonged thermal denaturation and the simultaneous measurement of scatter signals may offer additional information especially in the presence of BrdUrdunlabeled S‐phase cells.
Automated detection and recognition of live cells in tissue culture using image cytometry
Authors: Spadinger Ingrid , Poon Steven S. S. , Palcic Branko
Published: August 2005

An automated image cytometry device, the Cell Analyzer, was used to locate live V79 cells plated at low densities in a tissue culture flask. Cells and other objects were detected by moving the flask in steps across a linear solid‐state image sensor. The step size was selected to be small enough to allow detection of all the cells in the area being scanned but sufficiently large so that most cells would be detected on only one image line. To distinguish cells from other detected objects, a recognition algorithm utilizing 18 characteristic cell signal features was developed. The algorithm first tests whether a set of feature values falls within specified upper and lower bounds, and then applies a linear discriminant function to the remaining data to further discriminate cells from debris. False‐positive errors of 5% or less were achieved with this method, whereas 15–35% of cells were misclassified as debris.
Central values and variation of measured nuclear DNA content in imprints of normal tissues determined by image analysis
Authors: Taylor Suzanne R. , Titus‐Ernstoff Linda , Stitely Suzette
Published: August 2005

A total of 109 slides derived from 37 normal tissues were analyzed for nuclear DNA content using the Cell Analysis Systems (CAS) model 100 image analysis system with the Quantitative DNA Analysis (QDA) software module in order to determine the central values of DNA content and define normal limits. Analysis of the 109 slides revealed an overall mean measured DNA index of 0.997 with a standard deviation of 0.04. Analysis of replicate samples was essentially constant. There were no differences between samples obtained from autopsy and surgical specimens. Small variations were detected among stain batches, individual patients, and tissue types. These results indicate a high degree of accuracy and reproducibility of DNA content determinations using this system.
Resolving leukocytes using axial light loss
Authors: Stewart Carleton C. , Stewart Sigrid J. , Habbersett Robert C.
Published: August 2005

Axial light loss (ALL) is the measurement of the total light lost from the laser beam at 0 degrees when a particle passes through the beam. Used in combination with the monoclonal antibody CD45, ALL can effectively resolve lymphocytes, monocytes, granulocytes, and dead cells in viable or fixed preparations of human peripheral blood. A bivariate display of ALL vs. CD45 clearly resolves all granulocytes from lymphocytes; although degranulated granulocytes cannot be resolved with forward‐angle and right‐angle light scatter, they are clearly resolved in right‐angle scatter vs. CD45. A blood differential can be performed, with a single laser flow cytometer and three colors of fluorescence, when ALL is combined with fluoresceinated CD45 to resolve leukocytes, phycoerythrin‐labeled NKH1 to resolve natural killer cells, and biotinylated CD3 in combination with DuoCHROME, the phycoerythrin/Texas red conjugate fluorochrome from Becton Dickinson, to resolve T‐cells. B‐cells are the only cells negative for both phycoerythrin and Texas red. When PE CD4 is included, the CD3+ CD4+ T‐cell subset is resolved from the CD3+ CD4− subset comprising mainly the CD3+ CD8+ T‐cell subset. The addition of propidium iodide is unnecessary since ALL clearly resolves dead cells in a viable preparation of human peripheral blood. Furthermore, since ALL resolves these cells even after fixation in paraformal‐dehyde, all samples can be fixed prior to analysis, thereby minimizing the potential biohazard risk.
Flow cytometric analysis of mature adipocytes
Authors: Bernstein R. L. , Hyun William C. , Davis Jonathan H. , Fulwyler Mack J. , Pershadsingh Harrihar A.
Published: August 2005

Flow cytometry is an excellent method for studying the physiological function in adipocytes because their response to hormones, especially insulin, varies with cell maturity and therefore size. Adipocytes present a unique technical challenge. A freshly prepared adipocyte suspension contains cells and fat droplets ranging from 10 to >120 μm in diameter. Stored fat occupies 90–98% of the cell volume, making it difficult to distinguish cells from fat droplets. Other difficulties include buoyancy, large size, fragility, and tendency to aggregate and clog the sample tube and nozzle.

These obstacles were overcome by (1) maintaining the sample, sample line, sheath fluid, reservoir, and nozzle assembly at 37°C; (2) using a 200 μm diameter orifice; (3) using a short, 300 μm inside diameter Teflon sample delivery line; (4) injecting the sample at constant flow rate into the sheath fluid at low pressure; and (5) using the pH‐sensitive vital stain, biscarboxyethylcarboxyfluorescein (BCECF) to distinguish cells from fat droplets.

Stained cells are brightly fluorescent when excited at 488 nm. Because fat droplets do not fluoresce, they can be distinguished from fat cells by gating on the BCECF emission. The cytosolic pH of intact, viable, mature adipocytes was derived from the ratio of the fluorescent emission intensities at 520 and 620 nm and was estimated to be 7.2. Unlike BCECF, several other useful fluorescent probes of cell function, e.g., the intracellular calcium indicator, indo‐1, label both fat cells and fat droplets. Using a dual‐excitation‐laser flow cytometer and by gating on the BCECF emission in an adipocyte suspension labeled with both BCECF and indo‐1, one can exclusively monitor cell‐associated indo‐1 fluorescence in order to assess unambiguously intracellular calcium concentration in viable, mature adipocytes.
Optical plankton analyser: A flow cytometer for plankton analysis, I: Design considerations
Authors: Peeters J. C. H. , Dubelaar G. B. J. , Ringelberg J. , Visser J. W. M.
Published: August 2005

The design criteria for a flow cytometer (FCM) for the analysis of field samples of phytoplankton are described. The criteria are based on the occurrence of a wide variety of particle sizes in field samples, normally at low concentrations. The instrument should be able to analyse cells and colonies from 0.5 to 500 μm diameter and of over 2,000 μm length. A minimum flow rate of 4 μl · s−1 was calculated from natural plankton concentrations. Commercially available FCMs are not suited to measure this range of sizes at this rate. Further limitations of standard FCMs are uneven illumination or incomplete processing of long signals. In addition, long filamentous colonies can break into small fragments caused by too high acceleration in the standard flow cuvette. Recognition of these limitations is of importance for the flow cytometry of phytoplankton.

The new design was developed to avoid these limitations. A dynamic range 5 to 6 decades could be accomplished by a combination of logarithmic amplifiers, a slit‐shaped focal spot, and a pulse integration system that can process long pulses. Multilaser capability to identify different phytoplankton species, a low fluid shear cuvette, and a trigger gate‐extension for inhomogeneously fluorescent algal filaments were included in the design.
Optical plankton analyser: A flow cytometer for plankton analysis, II: Specifications
Authors: Dubelaar George B. J. , Groenewegen Ad C. , Stokdijk Willem , Van Den Engh G. J. , Visser Jan W. M.
Published: August 2005

An analysing flow cytometer, the optical plankton analyser (OPA), is presented. The instrument is designed for phytoplankton anaysis, having a sensitivity comparable with commercially available flow cytometers, but a significantly extended particle size range. Particles of 500 μm in width and over 1,000 μm in length can be analysed. Sample flow rates of up to 55 μl/s can be used. Also, the dynamic range of the instrument is significantly increased for particles larger than about 5 μm. The optics, hydraulics, and electronics of the instrument are described, including the best form for a low fluid shear cuvette. The new pulse quantification technique we call digital integration is presented. This technique is essential for the instrument to handle both short and very long particles with a large dynamic range. Test measurements demonstrating particle size range and dynamic range are presented. Dynamic ranges of 10,000 and 100,000 were typically observed, measuring field samples with Microcystis aeruginosa colonies, whereas one sample showed a dynamic range of 106. A simple method for interpretation of time of flight (TOF) data in terms of particle morphology is presented. The specifications of the instrument are given.
Use of a neural net computer system for analysis of flow cytometric data of phytoplankton populations
Authors: Frankel Donald S. , Olson Robert J. , Frankel Sheila L. , Chisholm Sallie W.
Published: August 2005

Flow cytometry has been used over the past 5 years to begin detailed exploration of the distribution and abundance of picoplankton in the oceans. Light scattering and fluorescence measurements on individual plankton cells in seawater samples allow construction of population signatures from size and pigment characteristics. The use of “list mode” data has made these studies possible, but on‐shore analysis of copious data does not permit on‐site reexamination of important or unexpected observations, and overall effort is greatly handicapped by data analysis time.

Here we describe the application of neural net computer technology to the analysis of flow cytometry data. Although the data used in this study are from oceanographic research, the results are general and should be directly applicable to flow cytometry data of any sort.

Neural net computers are ideally suited to perform the pattern recognition required for the quantitative analysis of flow cytometry data. Rather than being programmed to perform analysis, the neural net computer is “taught” how to analyze the cell populations by presenting examples of inputs and correct results. Once the system is “trained,” similar data sets can be analyzed rapidly and objectively, minimizing the need for laborious user interaction. The neural network described here offers the advantages of (1) adaptability to changing conditions and (2) potential real‐time analysis. High accuracy and processing speed near that required for real‐time classification have been achieved in a software simulation of the neural network on a Macintosh™ SE personal computer.
Algorithm to estimate cell biovolume using image analyzed microscopy
Authors: Sieracki Michael E. , Viles Charles L. , Webb Kenneth L.
Published: August 2005

This paper describes an algorithm for calculating the biovolume of cells with simple shapes, such as bacteria, flagellates, and simple ciliates, from a 2‐dimensional digital image. The method can be adapted to any image analysis system which allows access to the binary cell image—(i.e.), the pixels, or (x, y) points, composing the cell. The cell image is rotated to a standard orientation (horizontal), and a solid of revolution is calculated by digital integration. Verification and a critical assessment of the method are presented. The algorithm accounts for irregularities in cell shape that conventional methods based on length, width, and geometrical formulas do not.
Kinetics of bacterial processes in natural aquatic systems based on biomass as determined by high‐resolution flow cytometry
Authors: Button D. K. , Robertson B. R.
Published: August 2005

The two primary kinetic constants for describing the concentration dependency of nutrient uptake by microorganisms are shown to be maximal rate of substrate uptake and, rather than the Michaelis constant for transport, specific affinity. Of the two, the specific affinity is more important for describing natural aquatic microbial processes because it can be used independently at small substrate concentrations. Flow cytometry was used to evaluate specific affinities in natural populations of aquatic bacteria because it gives a convenient measure of biomass, which is an essential measurement in the specific‐affinity approach to microbial kinetics.

Total biomass, biomass in various filter fractions, and the specific affinity of the bacteria in each fraction were determined in samples from a near‐arctic lake. The partial growth rate of the pelagic bacteria from the 25 μg/liter of dissolved amino acids present (growth rate from the amino acid fraction alone) was determined to be 0.78 per day. By measuring activity in screened and whole‐system populations, the biomass of the bacteria associated with particles was computed to be 427 μg/liter.
Shipboard analytical flow cytometry of oceanic ultraphytoplankton
Authors: Li William K. W.
Published: August 2005

Shipboard analysis of marine ultraphytoplankton by flow cytometry is a powerful method to classify these cells according to in vivo fluorescence characteristics and size. At present, this ataxonomic‐allometric approach allows recognition of phycoerythrin‐containing cyanobacteria, cryptomonads, very small red‐fluorescing cells (presumably prochlorophytes), and eukaryotic algae of various sizes in many open ocean samples. The speed at which flow cytometric analysis can be performed on freshly collected samples permits a high degree of sampling resolution in both space and time. A flow cytometric view is presented of the vertical distribution of ultraphytoplankton at various sites in the north Atlantic and of experiments wherein phytoplankton were incubated in an artificial light gradient and under simulated in situ conditions.
Variance within homogeneous phytoplankton populations, I: Theoretical framework for interpreting histograms
Authors: Campbell J. W. , Yentsch C. M.
Published: August 2005

A framework is presented for interpreting frequency distributions of volume or fluorescence as measured by a flow cytometer on homogeneous phytoplankton populations. The framework, based on both laboratory experience and theoretical concepts, is illustrated with the use of a simulation model. Asynchronous, synchronous, and phased populations were simulated, with constant and variable growth patterns over the cell cycle. Though simulations produced a wide variety of histogram shapes, including multimodal distributions, the primary difference between asynchronous and synchronous/phased distributions lies in their temporal variation. Histograms that are constant in time indicate asynchronous populations; when populations are not asynchronous, their histogram shapes vary with a periodicity on the same time scale as the cell cycle. A probability density function for the case of asynchronous populations with a constant growth rate is derived. When fitted to simulated histograms this two‐parameter density function yields estimates of the two parameters: mean and variance of cell volume (or mass) at age 0.
Carbon dynamics of logarithmetic and stationary phase phytoplankton as determined by track autoradiography
Authors: Knoechel R. , Quinn E. M.
Published: August 2005

Track autoradiographic analysis of photosynthetic radiocarbon incorporation at the cellular level indicated that the carbon uptake rate and carbon pool size of exponentially growing (log phase) Scenedesmus cells was threefold that of stationary phase cells, while carbon turnover rates were similar. Carbon fixation was uncoupled from growth and cell division in the stationary phase cells, which were larger and contained less chlorophyll per unit volume than log phase cells. Changes in the temporal pattern of isotope incorporation were evident at the cell level prior to the cessation of division and transition to stationary phase, while bulk carbon fixation responded only the second day after cell division ceased. The carbon uptake patterns of a marine nanoplankter from a nutrient‐enriched natural sample resembled that of log phase cells while the control population pattern resembled that of stationary cells. The physical, biochemical, and metabolic differences between log and stationary phase cells are potentially measurable by flow cytometry procedures currently in use and under development. The use of flow cytometry to sort cell types for analysis by track auto‐radiography and subsequent correlation of metabolic characteristics with flow cytometry signatures is a feasible means of investigating the heterogeneity of phytoplankton metabolic state in the marine environment.
Discrimination of eukaryotic phytoplankton cell types from light scatter and autofluorescence properties measured by flow cytometry
Authors: Olson R. J. , Zettler E. R. , Anderson O. K.
Published: August 2005

Flow cytometric methods for recognizing several groups of eukaryotic marine phytoplankton were tested using 26 laboratory cultures. Each culture was divided into three aliquots, and these samples were analyzed for (1) Coulter volume; (2) light scatter (magnitude and polarization properties of forward scattered light and magnitude of right‐angle scattered light) and autofluorescence emission (phycoerythrin and chlorophyll); and (3) autofluorescence excitation (by 488 nm and 515 nm light).

Three kinds of cells could be easily distinguished from others in the culture collection: (1) The two cryptophytes and the rhodophyte had high phycoerythrin/chlorophyll ratios; (2) the two coccolithophores depolarized forward scattered light; and (3) the two pennate diatoms scattered only a relatively small amount of light in the forward direction compared with that at right angles. Mean chlorophyll fluorescence excited by blue light relative to that excited by green light was highest in the four chlorophytes, but there was overlap between some of these and some other kinds of cells. Unresolved cell types included centric diatoms, dinoflagellates, and naked coccolithophores. Forward light scatter and Coulter volume were closely related (except for the pennate diatoms) over a range of about 0.01 to 30 pL (equivalent spherical diameter about 3 to 40 μm), according to a logarithmic function.
Using phytoplankton and flow cytometry to analyze grazing by marine organisms
Authors: Cucci Terry L. , Shumway Sandra E. , Brown Wendy S. , Newell Carter R.
Published: August 2005

Phytoplankton can, through their auto‐fluorescent characteristics, be thought of as tracer particles in much the same way as fluorescent microspheres when used in particle uptake experiments. Flow cytometric techniques can be used to differentiate phytoplankton from other suspended particles by the two primary autofluorescing photosynthetic pigments, chlorophyll and phycoerythrin. Based on these characteristics, phytoplankton assemblages have been used to assess grazing rates, particle selectivity, and endocytotic abilities in various marine species, from single‐celled organisms to higher invertebrates.
Reference standards for flow cytometry and application in comparative studies of nuclear DNA content
Authors: Tiersch Terrence R. , Chandler Robert W. , Wachtel Stephen S. , Elias Sherman
Published: August 2005

Nuclear DNA mass in cells from a reference species can be used to obtain high‐resolution estimates of DNA mass from a target species. In our study of DNA mass in cells from 45 selected species, representing each of the major vertebrate classes, we have obtained values of from 1.5 to 110.0 pg of DNA. Because values in or near this range would be expected in the study of nuclear DNA mass in vertebrates and other organisms, the species in this report can provide a useful catalogue of references for comparative studies of DNA.
Different opinions on classification of DNA histograms produced from paraffin‐embedded tissue
Authors: Joensuu Heikki , Kallioniemi Olli‐Pekka
Published: August 2005

Flow cytometric DNA ploidy determination has been regarded as an objective prognostic parameter in several types of human cancer. To test whether DNA histograms are similarly interpreted, a series of flow cytometric DNA histograms was posted to six investigators working in the field for independent classification. The histograms were produced from paraffin‐embedded adrenal adenomas or non‐neoplastic tissue and had several different patterns. Only 44% of the histograms were similarly classified by all investigators, and 85% by five of the six participants, when DNA ploidy was evaluated. Different criteria for tetraploidy existed, and also some uncertainty in classifying peridiploid and small aneuploid peaks. It is concluded that lack of consensus on histogram classification may result in widely varying percentages of DNA aneuploid tumors found even if the data are similar. Until general agreement is reached on the definition of DNA aneuploidy and its subclasses, classification of DNA histograms is variable and subjective.
Thermal denaturation of DNA for immunochemical staining of incorporated bromodeoxyuridine (BrdUrd): Critical factors that affect the amount of fluorescence and the shape of BrdUrd/DNA histogram
Authors: Hoy C. A. , Seamer L. C. , Schimke R. T.
Published: August 2005

Significant inter‐ and intraexperimental variations of the relative antibromodeoxyuridine fluorescence were found during measurement of DNA synthesis rates using flow cytometric analysis of 5‐bromodeoxyuridine (BrdUrd)‐labeled cells with an anti‐BrdUrd antibody. Fluctuations in other endpoints associated with levels of denaturation (integrity of DNA and cell size) were also observed to vary widely among samples that were otherwise thought to have been treated identically. Therefore, the denaturation step has been carefully re‐examined, and several critical factors were identified that influence the denaturation and subsequent binding of the anti‐BrdUrd to the labeled DNA. These factors include cell density, volume of water, and pH of the sample during heating. Appropriate adjustments are now included in the protocol, resulting in more consistent anti‐BrdUrd measurements in the face of routine (and sometimes necessary) experimental variations.
Flow cytometric evaluation of DNA stainability with propidium iodide after histone H1 extraction
Authors: Giangarè Maria Chiara , Prosperi Ennio , Pedrali‐Noy Guido , Bottiroli Giovanni
Published: August 2005

A flow cytometric evaluation of the effect of the histone H1 extraction on DNA stainability with propidium iodide was performed on isolated HeLa nuclei. Selective removal of the lysine‐rich protein was attained by using two established techniques involving treatment with 0.7 M NaCl or low pH. DNA stainability was monitored at different dye/DNA‐P ratios, varying from low to high saturating concentrations. Depletion of the histone H1from nuclei results in the transition from low to high affinity of a portion of binding sites, as shown by (1) the increase in fluorescence intensity after staining with the dye at low saturating concentrations and (2) the higher value of the fluorescence intensity ratio (FI5FI50) exhib ited by H1‐depleted nuclei stained with a low (5 μg/ml) vs. a high (50 μg/ml) concentration, as compared with control samples.
Simultaneous flow cytometric analysis of surface markers and nuclear Ki‐67 antigen in leukemia and lymphoma
Authors: Drach Johannes , Gattringer Claus , Glassl Herta , Schwarting Roland , Stein Harald , Huber Heinz
Published: August 2005

The monoclonal antibody Ki‐67 identifies an antigen present during the late G1, S, G2, and M phases of the cell cycle, whereas resting cells do not express this antigen. Immunostaining with Ki‐67 provides a simple method with which to determine the growth fraction of a malignant cell population without requiring a laborious procedure or use of radioactive materials. Thus far, detection of Ki‐67–positive cells by flow cytometry was limited because of nuclear location of the antigen. In this study, periodate‐lysine‐paraformaldhyde (PLP) fixation of cells in suspension, labeling with Ki‐67, and the subsequent flow cytometric analysis of the tumor growth fraction is described. Fixation with PLP at −10°C for 15 min rendered the plasma membrane permeable without destroying cell surface antigens. Thus double immunofluorescence studies using both a surface marker and Ki‐67 could be performed. This offers the additional advantage of being able to define the phenotype of proliferating cells. This method was applied to determine the growth fraction in peripheral blood and bone marrow samples of patients with leukemia and non‐Hodgkin's lymphoma. The results of Ki‐67 studies in 91 patients are shown. A wide variability of individual Ki‐67 values was observed within each entity. Use of this flow cytometric procedure substantially facilitates the quantification of proliferating cells in pathological blood and bone marrow samples.
Use of xantham gum to suspend large particles during flow cytometric analysis and sorting
Authors: Freyer James P. , Fillak David , Jett James H.
Published: August 2005

In this report we describe the use of xantham gum as a biologically inert material for increasing the viscosity of a suspension of cells or particles during flow cytometric analysis and sorting. A 0.1% concentration of xantham gum in culture medium or saline will increase the viscosity ∼︁9‐fold. For suspensions of multicellular spheroids 100–400 μm in diameter the measured sedimentation velocity was ∼︁9 times slower than that in medium alone. Thus, spheroids of 100 μm diameter remain in suspension in 0.1% xantham gum for 66 min, compared to 7.5 min in culture medium. This allows extended periods of sorting without stirring or agitating the sample suspension. The xantham gum solution is noncytotoxic for periods up to 8 h as measured by clonogenicity assay. Xantham gum has the added advantage that the viscosity is significantly reduced when the solution is subjected to shear stress, such as during flow. This technique should be applicable to extended sorting of suspensions of spheroids, plant cells, and other large particles, as well as for analyzing and sorting single cells for extended periods.
Modified box‐cox transform for modulating the dynamic range of flow cytometry data
Authors: Dvorak James A. , Banks Steven M.
Published: August 2005

We describe an algorithm,

based upon Box‐Cox transformations as an alternative to nonlinear electronic amplifiers to expand or compress high‐ or low‐amplitude flow cytometer‐derived signals. If the indexing parameter λ < 1, input channels in the high‐amplitude input range are compressed in the output range as occurs when an electronic logarithmic amplifier is used. However, if λ > 1, input channels in the low‐amplitude input range are compressed in the output range as occurs when an electronic power amplifier is used. Our modified Box‐Cox transform can be implemented either during data collection or off‐line for the transformation of previously collected raw data. The transform is the equivalent of an infinite class of nonlinear amplifiers. As the transform is implemented in software, it does not suffer from many of the disadvantages of nonlinear electronic amplifiers.
A unified model of eukaryotic chromosomes
Authors: Manuelidis Laura , Chen Terence L.
Published: August 2005

A revised model of DNA packaging into chromosomes is presented. Its features are consistent with observed structural dimensions and the molecular periodicities related to transcription, replication and matrix attachment domains. Thetransitions between euchromatic, heterochromatic and metaphase states are explained simply. Molecular and physical properties of chromosomal bands, and their correlation with specific DNA sequence motifs are discussed.
Direct nonradioactive in situ hybridization of somatic cell hybrid DNA to human lymphocyte chromosomes
Authors: Kievits T. , Devilee P. , Wiegant J. , Wapenaar M. C. , Cornelisse C. J. , van Ommen G. J. B. , Pearson P. L.
Published: August 2005

Biotinylated DNA from various human–rodent hybrids was hybridized to human lymphocyte spreads after preannealing of the repeated sequences with sonicated total human DNA. Fluorescent labeling was achieved by successive treatments with fluorescein‐labeled avidin and biotinylated antiavidin antibody. The use of labeled total DNA from hybrids with known chromosome composition permits the fluorescent staining‐(“painting”) of specific chromosomes, or parts thereof, in human lymphocyte metaphases. Alternatively, the human chromosome content of cell hybrids with unknown chromosome composition is directly assessed from the labeling pattern of human lymphocyte spreads using the total hybrid DNA as probe.
High resolution mapping of Xenopus laevis 5S and ribosomal RNA genes by EM in situ hybridization
Authors: Narayanswami Sandya , Hamkalo Barbara A.
Published: August 2005

We have developed a modification of in situ hybridization at the electron microscope level that permits simultaneous detection of at least two sequences. Probes are labelled with either biotin or AAF and detected with two distinct sizes of colloidal gold. This protocol has been applied to map the positions of Xenopus laevis oocyte‐type 5S genes relative to ribosomal precursor genes in several independently derived cell lines. The results for the line TRXO, which expresses some oocyte 5S RNA, indicate that this inappropriate expression is not due to translocation from telomeric sites into the nucleolus organizer, as previously hypothesized (7). In addition we found that four other Xenopus cell lines, none of which express these genes, also contain distinct, 5S oocyte translocations. These results suggest that an alteration in chromosome position is insufficient to result in gene activation and that sequences which are telomeric‐proximal are exceptionally prone to translocation.
Flow cytometric quantification of human chromosome specific repetitive DNA sequences by single and bicolor fluorescent in situ hybridization to lymphocyte interphase nuclei
Authors: van Dekken H. , Arkesteijn G. J. A. , Visser J. W. M. , Bauman J. G. J.
Published: August 2005

Fluorescent in situ hybridization allows for rapid and precise detection of specific nucleic acid sequences in interphase and metaphase cells. We applied fluorescent in situ hybridization to human lymphocyte interphase nuclei in suspension to determine differences in amounts of chromosome specific target sequences amongst individuals by dual beam flow cytometry. Biotinylated chromosome 1 and Y specific repetitive satellite DNA probes were used to measure chromosome 1 and Y polymorphism amongst eight healthy volunteers. The Y probe fluorescence was found to vary considerably in male volunteers (mean fluorescence 169, S.D. 35.6). It was also detectable in female volunteers (mean fluorescence 81, S.D. 10.7), because 5–10% of this repetitive sequence is located on autosomes. The Y probe fluorescence in males was correlated with the position of the Y chromosome cluster in bivariate flow karyotypes. When chromosome 1 polymorphism was studied, one person out of the group of eight appeared to be highly polymorphic, with a probe fluorescence 26% below the average. By means of fluorescent in situ hybridization on a glass slide and bivariate flow karyotyping, this 26% difference was found to be caused by a reduction of the centromere associated satellite DNA on one of the homologues of chromosome 1.

The simultaneous hybridization to human lymphocyte interphase nuclei of biotinylated chromosome 1 specific repetitive DNA plus AAF‐modified chromosome Y specific DNA was detected by triple beam flow cytometry. The bicolor double hybridized nuclei could be easily distinguished from the controls. When the sensitivity of this bicolor hybridization is improved, this approach could be useful for automatic detection of numerical chromosome aberrations, using one of the two probes as an internal control.
A computer program for analyzing bivariate flow karyotypes
Authors: van den Engh Ger , Hanson Don , Trask Barb
Published: August 2005

This article describes a computer program for analyzing bivariate flow karyotypes of human chromosomes stained with Hoechst 33258 (HO) and chromomycin A3 (CA). The karyotype first is divided into regions that contain chromosome peaks. The chromosomes that are associated with those areas are identified. The distributions in these areas then are fitted with mathematical functions of increasing complexity. The process starts by fitting a specified number of univariate Gauss functions to projections of the HO and CA distributions of each area. The final fit can include multiple bivariate Gauss functions, including a background function for debris subtraction. The results of one stage in the fitting process serve as seed values for the next, more complex step. Since the program autonomously estimates the starting values for the iterative fitting procedures, the fit results are insensitive to operator bias and the program will consistently converge to the same solutions. The resulting table of parameter values can be used to compare flow karyotypes to a reference data set.
Clinical applications of flow karyotyping in myelocytic leukemia by stimulation of different subpopulations of cells in blood or bone marrow samples
Authors: Arkesteijn G. J. A. , van Dekken H. , Martens A. C. M. , Hagenbeek A.
Published: August 2005

Examples are presented in which normal as well as abnormal chromosome distributions could be obtained from the same individual by means of bivariate flow karyotyping. Selective stimulation of T‐lymphocytes obtained by E‐rosetting from the blood of a patient with acute myelocytic leukemia resulted in a normal flow karyogram. The specific stimulation of myelocytic leukemia cells with granulocyte‐macrophage colony stimulating factor (GM‐CSF) and interleukin 3 (IL‐3) yielded flow karyograms displaying the leukemia‐associated chromosome abnormalities. The resulting flow karyograms could be used to discriminate between homolog differences, which appear normally in virtually every individual, and leukemia‐associated chromosomal aberrations. In the case of a female chronic myelocytic leukemia patient who received bone marrow form an HLA‐identical male donor, specific stimulation of various subsets of cells enabled to discriminate between leukemic host cells and non‐leukemic donor cells. Both the leukemia‐specific translocations and sex chromosomes were used as markers to analyse the flow karyograms obtained from the same sample.
High gradient magnetic cell separation with MACS
Authors: Miltenyi Stefan , Müller Werner , Weichel Walter , Radbruch Andreas
Published: August 2005

A flexible, fast and simple magnetic cell sorting system for separation of large numbers of cells according to specific cell surface markers was developed and tested. Cells stained sequentially with biotinylated antibodies, fluorochrome‐conjugated avidin, and superparamagnetic biotinylated‐microparticles (about 100 nm diameter) are separated on high gradient magnetic (HGM) columns. Unlabelled cells pass through the column, while labelled cells are retained. The retained cells can be easily eluted. More than 109 cells can be processed in about 15 min. Enrichment rates of more than 100‐fold and depletion rates of several 1,000‐fold can be achieved. The simultaneous tagging of cells with fluorochromes and very small, invisible magnetic beads makes this system an ideal complement to flow cytometry. Light scatter and fluorescent parameters of the cells are not changed by the bound particles. Magnetically separated cells can be analysed by fluorescence microscopy or flow cytometry or sorted by fluorescence‐activated cell sorting without further treatment. Magnetic tagging and separation does not affect cell viability and proliferation.
Multiparametric analysis of cell membrane permeability by two colour flow cytometry with complementary fluorescent probes
Authors: Dive Caroline , Watson James V. , Workman Paul
Published: August 2005

We describe an improved twin‐probe multiparameter flow cytometric technique to examine cell membrane permeability. Ability to retain preloaded intracellular bis‐carboxyethyl carboxy fluorescein (BCECF, green fluorescence) and to exclude extracellular propidium (red fluorescence) is measured, simultaneously with forward and right‐angle scatter. This has significant advantages over an earlier method using fluorescein together with ethidium. In addition to the two expected cell populations which were stained green positive, red negative (by convention membrane “intact” and “viable,” Region 1) and green negative, red positive (“membrane‐damaged” and “non‐viable,” Region 3), a third population was seen which fluoresced neither green nor red and displayed intermediate light scatter characteristics (Region 2). This was true for each of 9 cell types in vitro. For EMT6 mouse mammary tumour cells held under sub‐optimal conditions or treated with membrane‐active drugs, progression from Region 1 to Region 2 was observed, followed by further progression from Region 2 to Region 3. Cells eventually accumulated in Region 3. These results suggest that sequential changes in membrane structure lead to increased permeability, first with respect to intracellular BCECF and in turn to extracellular propidium.
Characteristic immunophenotyping artefact seen in patients with anti‐mouse immunoglobulin antibodies
Authors: Wilks D. , Byrom N. , Walker L. , Habeshaw J. , Dalgleish A.
Published: August 2005

Patients treated with murine monoclonal antibodies develop anti‐mouse immunoglobulin antibodies which can interfere with flow cytometry of lymphocytes, giving rise to high levels of non‐specific staining. This artefact can be avoided by using separated, washed lymphocytes for immunophenotyping.
Chlorophyll α fluorescence measurements of isolated spinach thylakoids obtained by using single‐laser‐based flow cytometry
Authors: Xu Chunhe , Auger Julie , Govindjee
Published: August 2005

Flow cytometry data of spinach thylakoid membrane preparations indicate the presence of a homogeneous thylakoid population. Fluorescence data from a flow cytometer and comparison with data from two other fluorometers show that cholorophyll α fluorescence detected with a flow cytometer has the character of maximum fluorescence (Fmax), not of the constant component (Fo). This conclusion is important since Fo measures fluorescence that is affected mostly by changes in excitation energy transfer and Fmax‐Fo (the variable fluorescence) by changes in photochemistry. This was demonstrated by: (1) The light intensity as well as diffusion rate dependence of the quenching effect of various quinones (p‐benzoquinone, phenyl‐benzoquinone, and 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐p‐benzoquinone, DBMIB) on fluorescence yield; quenching for the same concentration of these quinones was lower at the higher than at the lower light intensities. (2) Temperature dependence of the fluorescence yield; increasing the temperature from 20 to 70°C did not show an increase in fluorescence yield using a flow cytometer in contrast to measurements with weak excitation light, but similar to those obtained for Fmax. (3) Addition of an inhibitor diuron up to 100 μM did not change the fluorescence intensity. A comparison of quenching of fluorescence by various quinones obtained by flow cytometry with those by other fluorometers suggests that the high intensity used in the cytometry produces unique results: the rate of reduction of quinones in much larger than the rate of equilibration with the bulk quinones.
Flow cytometric recognition of clastogen induced chromatin damage in G0/G1 lymphocytes by non‐stoichiometric Hoechst fluorochrome binding
Authors: Kubbies M.
Published: August 2005

Changes in chromatin structure were induced in human peripheral blood lymphocytes. Resting G0/G1 cells were exposed to either X‐rays, mitomycin C, or bleomycin and stimulated with PHA. Exposure to such agents provokes an increase in the non‐cycling cell fraction; and a distinctive, non‐cycling G‐/G1 subpopulation appears which is characterized by a 23% reduced Hoechst fluorescence intensity. This novel subpopulation was found as early as 24 h after PHA stimulation; it was still present in 72 h cultures. Bromodeoxyuridine (BrdUrd/Hoechst) 33258‐ethidium bromide (EB) flow cytometric analysis revealed increments of this subpopulation from 2% of the non‐cycling cell fraction in the control culture to 29% (X‐rays), 15% (mitomycin C), and 24% (bleomycin) after clastogen exposure. In the presence of the ligase inhibitor 3‐aminobenzamide, this aberrant cell population increased significantly after X‐ray treatment. With the aid of a viable BrdUrd/Hoechst staining assay, the newly identified non‐cycling subpopulation with decreased Hoechst 33258 binding was identified as a distinctive signal cluster. Other than the regular non‐cycling and cycling cell fractions, this subpopulation with non‐stoichiometric Hoechst dye binding showed progressive uptake of ethidium bromide; however, by such criteria 44% of the subpopulation was still viable. It is concluded that the clastogen induced subpopulation of noncycling cells represents damaged cells with altered dye binding properties.
Bromodeoxyuridine incorporation into DNA of human leukemia cells is not concentration dependent
Authors: Powell Bayard L. , Gregory Betsy W. , Kute Timothy E. , Morgan Timothy M. , Lyerly E. Susan , Capizzi Robert L.
Published: August 2005

Leukemia blasts isolated from bone marrow aspirates of 44 adults with acute leukemia were incubated for 1 h with 0.008–32 μM bromodeoxyuridine (Brd‐Urd). After dual labeling with monoclonal anti‐BrdUrd antibodies and propidium iodide, the cells were analyzed by flow cytometry. Percent labeled cells and intensity of labeling were similar over concentrations of BrdUrd ranging from 0.8–32 μM—a 40‐fold range. Therefore, despite potential interpatient variability in nucleoside pharmacokinetics, commonly used doses of BrdUrd which are intended to achieve steady‐state plasma concentrations in the 8.0 μM range can be expected to provide a reliable estimate of the S‐phase fraction.
Detection of S cells and evaluation of DNA denaturation protocols by image cytometry of fluorescent BrdUrd labelling
Authors: Humbert Catherine , Giroud Françoise , Brugal Gérard
Published: August 2005

This paper addresses the problem of detecting weak incorporation of BrdUrd and the related efficiency of the denaturation protocols used to unmask this thymidine analog. Evidence is presented that measuring the distribution of BrdUrd‐tagged fluorescence intensities by image cytometry generates a standard deviation threshold that discriminates between positive and negative MRC5 cells in vitro. A comparison of the thresholding by standard deviation (SDT) with the usual thresholding by the nuclear total fluorescence intensity (FIT) demonstrated that SDT has a significantly higher sensitivity (99.4–100%, depending on the denaturation protocols) than FIT (94.7 and 74.3%, respectively), although both tests have a high specificity (93% and 100%, respectively) for detecting S cells. Since detecting the S cells is not only dependent on the test used, but also on the denaturation protocols, a quality index (QI) was derived from the standard deviation and the mean value of the nonspecific fluorescence of negative cell population versus BrdUrd fluorescence of positive cell population. The following DNA denaturation protocols have been assessed according to QI: acidic denaturation, thermal denaturation in formamide, and thermal denaturation in distilled water. Each denaturation procedure was preceded or not by incubation in either proteinase K or Triton X‐100. The results showed that thermal denaturation in formamide, especially when preceded by proteinase K incubation, revealed the largest difference between negative and positive cells. This work also demonstrated that image cytometry of BrdUrd‐labelled cells can be suitable for clinical application because of the high sensitivity provided and the small samples needed.
Evaluation of immunocytochemical detection methods of incorporated bromodeoxyuridine in hybridomas
Authors: Williams Lance S. , Nordberg Judith A. , Chau Pao C.
Published: August 2005

Bivariate distributions obtained fron nominal acid hydrolysis or thermal treatment methods used in the cell cycle analysis of incorporated bromodeoxyuridine were shown to be unacceptable with hybridomas. Four different cell treatment and staining methods were compared. These methods are acid hydrolysis, thermal denaturation, nuclei extraction with pepsin digestion, and simultaneous pepsin digestion and acid hydrolysis. The nuclei extraction method was determined to be the most appropriate for the immunocytochemical staining of incorporated bromodeoxyuridine in hybridomas. The resulting bivariate distribution provides a clear distinction between labelled and unlabelled cell fractions. The method based on nuclei extraction with pepsin digestion was optimized for a hybridoma line used in this study.