bluuebunny/crossref_metadata_2025 huggingface dataset:
Improved BrdUrd‐Hoechst bivariate cell kinetic analysis by helium‐cadmium single laser excitation
Authors: Kubbies M. , Goller B. , Van Bockstaele D. R.
Published: August 2005

In laser based flow cytometers, UV excitation of Hoechst 33258 and propidium iodide (PI) or ethidium bromide (EB) is performed with 351/364 rum high power lines of UV‐capable argon ion lasers, which are expensive and short‐lived. In this paper we note for the first time that helium‐cadmium lasers emitting 10 to 30 mW at 325 nm are even more superior for cell kinetic bivariate bromodeoxyuridine (BrdUrd)/Hoechst PI or EB cell cycle analysis. HeCd single laser UV excitation gives comparable CVs for, cell cycle distributions, and almost normal G2D4/G1 ratios of 1.9 to 2.0 for all cell cycles. This is shown for synchronous and asynchronous cell populations on a FACStar+ and an Ortho Cytofluorograf. Therefore we recommend helium‐cadmium lasers as low‐power, cheap, and long‐lived UV excitation sources for the cytochemically simple but high resolution multiparameter BrdUrd‐Hoechst cell kinetic analysis. © 1992 Wiley‐Liss, Inc.
DNA signal splitting improves detection and analysis of tetraploid populations
Authors: Weaver Donald L. , Bagwell C. Bruce
Published: August 2005

Detection of DNA tetraploid populations requires a high index of suspicion at the time of data acquisition and frequently requires subsequent off‐line analysis for confirmation, including evaluation of the hypertetraploid region. To analyze these specimens, the flow cytometer operator must run all specimens with the G0G1 peak in low channels or rerun specimens in which tetraploidy is suspected with a lower photomultiplier tube (PMT) voltage or lower amplifier gain setting. Re‐analysis may not be possible in specimens with few cells. A simple modification to the cytometer allows PMT signal splitting with simultaneous processing of the signal by two different amplifiers. This allows simultaneous acquisition of histograms optimized for both the hypotetraploid and hypertetraploid regions. © 1992 Wiley‐Liss, Inc.
Noise, Sensitivity, and Resolution of Flow Cytometers
Authors: Steen Harald B.
Published: August 2005

The sensitivity and resolution of flow cytometers are functions of the signal produced by a given particle as well as by the noise in the presence of which the signal is detected. The noise is primarily due to the fact that emission of light as well as its detection by photoelectric devises are stochastic processes. This fact leads to equations describing how resolution and sensitivity are limited by the magnitude of the signal, the background, and the photoelectron quantum yield of the detector. The equations are pointing to a method by which the signal and noise of a flow cytometer can be measured in absolute terms, as well as a way to determine fluorescence sensitivity without having to extrapolate to the noise level. The equations appear to be validated when applied to measuring data obtained with two different flow cytometers. © 1992 Wiley‐Liss, Inc.
Reticulocyte quantification by flow cytometry, image analysis, and manual counting
Authors: Schimenti Kerry J. , Lacerna Kathleen , Wamble Alison , Maston Lisa , Iaffaldano Carol , Straight Margaret , Rabinovitch Albert , Lazarus Hillard M. , Jacobberger James W.
Published: August 2005

Reticulocyte counting by flow cytometry with thiazole orange was compared to manual or automated counting of new methylene blue stained blood smears. Forty‐nine samples were compared for manual counting from randomly chosen clinical samples. Two hundred and eighty‐nine samples from bone marrow transplant patients were compared during the period before and through chemo‐irradiation and engraftment. The slopes of correlation plots were less than 1 when flow cytometric data were the dependent variable, suggesting that thiazole orange is less sensitive than new methylene blue. In a third study, 407 samples from bone marrow transplant patients were compared after increasing the thiazole orange concentration. The reticulocyte fluorescence distribution was divided into four groups of the brightest (youngest) 40, 60, 80, and 100% of reticulocytes. The slopes from regression analysis were 0.25, 0.49, 0.78, and 1.14, respectively. This demonstrates that thiazole orange is more sensitive than new methyhene blue because the window of analysis includes an increased fraction of mature reticulocytes. In addition, the precision of each assay was measured. The rank order of precision from high to low was flow cytometry > image analysis ≫ manual counting. © 1992 Wiley‐Liss, Inc.
DNA quantification of squamous cell carcinoma of the oesophagus by flow cytometry and cytophotometric image analysis using formalin fixed paraffin embedded tissues
Authors: Dorman A. M. , Walsh T. N. , Droogan O. , Curran B. , Hourihane D. O'B. , Hennessy T. P. J. , Leader M.
Published: August 2005

This is the first comparative study of DNA quantification of oesophageal squamous cell carcinoma by flow cytometry (FCM) and image cytometry (ICM) using formalin fixed paraffin embedded tissue. The potential advantages of ICM include the identification of a reliable control cell population; avoidance of non‐tumour stromal and inflammatory cell nuclei, nuclear fragments, degenerate cell nuclei and doublets, triplets etc., which are not possible with FCM using archival tissue

Twenty‐eight cases, all of the same stage (stage 2a) and similar grade (well or moderately differentiated) were analysed. The cases were separated into two groups, those that had succumbed to tumour in less than 18 months (group A) and those that were tumour free at least 18 months post‐resection (group B)

Using ICM all 28 tumours yielded interpretable histograms by comparison to 25 of 28 using FCM. Aneuploidy was identified in 100% of cases in group A using ICM (in comparison to 73% by FCM) and in 73% of group B using ICM (in comparison to 44% by FCM). Any tumour aneuploid by FCM was also aneuploid byICM. Nine cases aneuploid by ICM were euploid by FCM. The mean 5C exceeding rate (% of cells whose nuclei contain a DNA mass equivalent to > 5 sets of 23 chromosomes) was 21% in group A and 14% in group B (P < 0.01). Euploidy was confined to tumours of those patients disease free for more than 18 months

The conclusions of this study are that: firstly, ICM is superior in its yield of interpretable histograms to FCM using formalin fixed paraffin embedded tissue; secondly, ICM is more sensitive in the identification of aneuploid stemlines than FCM; and thirdly, euploid tumours (as detected by ICM) appear to have a better prognosis than aneuploid tumours of similar stage and grade. © 1992 Wiley‐Liss, Inc.
Effect of random abnormal cell proportion on specimen classifier performance
Authors: Castleman Kenneth R. , Price Kenneth H. , White Benjamin S.
Published: August 2005

A series of papers had analyzed a simplified model of an automated cytology prescreening configuration consisting of a two‐class cell classifier followed by a two‐class specimen classifier. This has shown, among other things, that the proportion (p) of abnormal cells on an abnormal specimen dictates the number (N) of cells that must be classified before the specimen can be classified with specified accuracy (Anal Quant Cytol, 2:117–122, 1980). It has also shown that if a system designed assuming one fixed value, Po, encounters a specimen with a different fixed value, p, then the specimen classifier false negative rate will deviate significantly from the design value, increasing for p < po and vice versa (Cytometry, 2:155–158, 1981). Using a Gaussian approximation, Timmers and Gelsema (Cytometry, 6:22–25, 1985) extended this model to the case where p is a Beta‐distributed random variable. They showed that N increases dramatically with the width (coefficient of variation) of the distribution of p. They also concluded that the randomness of p imposes a fundamental lower limit on the specimen false negative rate below which it is impossible to go, even with an error‐free cell classifier.

In this paper we also extend the basic model to cover the case of random p, but by using an asymptotic expansion (rather than the Gaussian approximation), to develop an expression for N. We show that the limit cited by Timmers and Gelsema is not real, but is actually an artifact of the breakdown of the Gaussian approximation. This result means that any desired performance is, in fact, achievable, even with random p, (but potentially at the cost of very many cells examined). We also present a simple iterative design procedure using the basic model, and show that the cell classifier figure‐of‐merit from the earlier analysis remains valid for the case of random p. Using the Beta distribution, we examine numerically several situations to validate the extended model, and to illustrate the effects of the parameters. These results apply to all cell‐ and specimen‐classifier cascades, whether they are implemented by statistical techniques, neural networks, or other means. © 1993 Wiley‐Liss, Inc.
Detection of male cells in mixtures containing varying proportions of male and female cells by fluorescence in situ hybridization and G‐banding
Authors: White A. Denise , Sweeney Marion C.
Published: August 2005

The efficiency and sensitivity of fluorescence in situ hybridization (FISH) and G‐banding for the detection of male cells in mixtures of male and female cells derived from peripheral blood (PB) and bone marrow (BM) are compared. Zero false positive and negative rates were obtained with G‐banding in all studies. False positive rates obtained with FISH were low (0–2.5%) in both PB and BM. False negative rates varied from 0–13.3%. Variations due to differences in fluorescent counterstain or scorers were not significant and in some cases the percentage of Y‐positive cells as determined by FISH agreed more closely with the estimated frequency of male cells than that obtained by G‐banding. The minimum limits of detection for FISH were 5% male cells on counting 500 PB or BM interphases; 10% male cells on counting 100 PB metaphases; and 20% male cells on counting 50 BM metaphases. This is in agreement with the theoretical limits of detection [95% confidence limit (CL)]. However, minimum levels of detection for female cells were below the 95% CL. It was not possible to reliably distinguish 95% female cells from 100% on counting 500 PB or BM interphases; 90% from 100% on counting 100 PB metaphases; or 80% from 100% on counting 50 BM metaphases. © 1993 Wiley‐Liss, Inc.
Microscopic analysis of interactions between HIV particles and living leukocytes
Authors: Wang Tao , Petty Howard R.
Published: August 2005

Video‐enhanced/‐intensified optical microscopy has been used to study the interaction of non‐infectious HIV8E5 particles with living cells. The purified particles retained gp120 antigenic sites. Fluorescent lipids were intercalated into the particles' envelopes. When incubated with CD4+ cell lines, roughly 90% of the cells bound HIV8E5 particles. The extent of fusion and endocytosis varied among the cell lines tested. CD4− control cells did not significantly bind, fuse, or internalize particles. To control for non‐specific exchange of the fluorescent label, HIV8E5 were bound to CD4− murine WEHI cells using concanavalin A; no apparent fusion or internalization took place. We suggest that both fusion and internalization are important mechanisms of virion‐cell interaction. Adherent human peripheral blood mononuclear cells were much less efficient in binding HIV8E5 than non‐adherent mononuclear cells. Both endocytosis and apparent fusion were observed for lymphocytes. Our results indicate that cells interact with HIV8E5 by multiple pathways and that these pathways are strongly affected by cell type (lymphocyte or monocyte) and origin (normal or transformed). These methods may be useful in characterizing viral entry and in anti‐viral drug screening. © 1993 Wiley‐Liss, Inc.
Cytofluorometric assay for the determination of thymidine uptake and phosphorylation in living cells
Authors: Hengstschläuger Markus , Wawra Edgar
Published: August 2005

Thymidine kinase is a key enzyme for the application of drugs in chemotherapy and for diagnosis. Although of great interest, its regulation during cell cycle and differentiation is difficult to study, as current techniques for isolation of cells in different phases of growth are unsatisfactory. An assay that allows the determination of enzymatic activity in situ in single cells would be much faster than present methods and would elegantly avoid synchronization procedures.

We synthesized different analogues of thymidine with the 5‐methyl group substituted by a fluorochrome. At least three of these compounds were phosphorylated by thymidine kinase in cell free extracts and were taken up and phos‐phorylated by cells in culture. The cyto‐fluorometric signal of the accumulated fluorochrome in any given cell reflected the thymidine kinase activity of this cell. Simultaneous measurement of cell‐cycle dependent parameters allowed the correlation of thymidine kinase activity with the phase of growth in mixed cell populations. © 1993 Wiley‐Liss, Inc.
Expression of silver‐stained nucleolar organizer regions is coupled to cell cycle in rat thymic cells
Authors: Carbajo Santiago , Orfao Alberto , Vicente‐Villardón José L. , Carbajo‐Pérez Eduardo
Published: August 2005

To directly analyze the relationship between the expression of silver‐stained nucleolar organizer regions (AgNORs) and cell proliferation, thymic cells from newborn rats were separately sorted at the G0–G1‐phase and early‐mid and iate‐mid S‐phase of the cell cycle according to their DNA content. Different AgNOR‐derived parameters (mean area and numbers of AgNORs per cell and mean AgNOR‐particle area) were evaluated after silver staining of cytospins. A linear correlation was observed between the mean area and numbers of AgNORs per cell, both parameters increasing progressively from G0–G1‐phase to early‐mid and late‐mid S‐phase. An increase of the mean AgNOR‐particle area was also seen between G0–G1 and S‐phase but this was not significant along the S‐phase. A bias on the selection of S‐phase cells linked to intrathymic maturation can be ruled out as S‐phase cells labeled with bromodeoxyuridine were found throughout the thymus, and, moreover, the analysis of the frequency distribution of nuclear area did not show a bimodal pattern. It is concluded that the expression of AgNORs—evaluated as AgNOR area/cell or AgNOR numbers/cell—is causally or indirectly coupled to DNA synthesis and, thus, AgNORs can be considered as a cell proliferation marker. © 1993 Wiley‐Liss, Inc.
Cytometric analysis of blood cells from malaria‐infected patients and in vitro infected blood
Authors: Bunyaratvej Ahnond , Butthep Punnee , Bunyaratvej Porntip
Published: August 2005

Blood samples from malaria‐infected patients and from in vitro culture were analyzed using the H*1 hematology analyzer. An attempt to find a hematologic parameter for detecting the malaria infection and to characterize the pathophysiological changes of red cells was made. The study included 18 malaria‐infected patients (10 with Plasmodium falciparum and 8 with Plasmodium vivax) and 52 normal, healthy volunteers. Increased young large lymphocyte or large unstained cell count (LUC over 3%) in the peripheral blood of malaria‐infected patients was evidence for malaria infection. Increased population dispersions of red cell volume (red cell distribution width: RDW) and intraerythrocytic hemoglobin concentration (hemoglobin distribution width: HDW) were demonstrated both in clinical samples and cultured blood. The increased RDW correlated with an increased percentage of macrocytes (r = 0.64, P = 0.004). Comparison of HDW and percentage of hypochromic red cells between the clinical specimen and the cultured blood supports the finding that changes in red cell hemoglobin concentration were mainly due to the response of the patient to malaria infection and partly due to the effect of malaria parasites on the red cells. © 1993 Wiley‐Liss, Inc.
Quantification of fluorescence properties of lymphocytes in peripheral blood mononuclear cell suspensions using a latent class model
Authors: van Putten W. L. J. , de Vries W. , Reinders P. , Levering W. , van der Linden R. , Tanke H. J. , Bolhuis R. L. H. , Gratama J. W.
Published: August 2005

Lymphocytes, monocytes, granulocytes, and other blood cells can be distinguished on the basis of their forward (FSC) and sideward (SSC) light scatter properties and their expression of CD45 and CD14. A FSC,SSC gate can be set to include > 95% of the lymphocytes using a “back gating” procedure on the CD45+, CD14− cells. However, nonlymphoid cells such as monocytes have light scattering properties similar to lymphocytes. This problem occurs particularly in patient populations where the light scattering properties of lymphocyte subsets have changed (e.g., due to activation) and are similar to those of the monocytes. Thus, immunophenotyping using antibodies specific for other markers than CD45 and CD14 does not allow a direct assessment of the percentage of all lymphocytes positive for those markers. In order to optimize immunophenotyping we have developed an analytic model in which the FSC,SSC dot plot is partitioned into six nonoverlapping light scatter regions. Each light scatter region contains a mixture population of different cell types, i.e., lymphocytes, monocytes, granulocytes, and other cells. The proportions of each cell type are known from the CD45,CD14 expression within each light scatter region. Under the assumption of independence of fluorescence and scatter properties conditional on cell type, the expression of markers other than CD45 or CD14 are derived from the cell type composition and the fluorescence properties on the other markers of each light scatter region. The underlying statistical model is a latent class model, and maximum likelihood estimates are computed using the expectation‐maximization (EM) algorithm. The application of the model for immunophenotyping of lymphocytes of healthy individuals and cancer patients receiving immunotherapy is shown. © 1993 Wiley‐Liss, Inc.
Chromatid contamination can impair the purity of flow‐sorted metaphase chromosomes
Authors: Telenius Håkan , de Vos Diana , Blennow Elisabeth , Willat Lionel R. , Ponder Bruce A. J. , Carter Nigel P.
Published: August 2005

We describe a phenomenon where a second flow karyotype is superimposed on the normal metaphase flow karyotype of human lymphoblastoid chromosomes. The events of this second flow karyotype contain half the DNA content of corresponding events in the normal flow karyotype and, when sorted, show the morphology of single chromatids. DNA amplified by degenerate oligonucleotideprimed PCR of 300 chomatids sorted from a single peak and hybridized onto normal metaphase spreads painted the entire length of the corresponding chromosome type. In retrospect, we could observe this phenomenon at a low level in 37% of randomly selected, previously analyzed flow karyotypes and in several published flow karyotypes from other laboratories. Experimentally, chromatid frequency was shown to vary with the colcemid blocking conditions as well as with the cell line used. At the extreme, we obtained a 4:10 ratio of chromatids to metaphase chromosomes, whereas this could be reduced tenfold by altering the colcemid blocking conditions in the same cell line. The presence of chromatids in chromosome preparations has important implications for the purity of isolated fractions used for generating libraries and in PCR analysis. © 1993 Wiley‐Liss, Inc.
Three‐marker phenotypic analysis of lymphocytes based on two‐color immunofluorescence using a multinomial model for flow cytometric counts and maximum likelihood estimation
Authors: van Putten W. L. J. , Kortboyer J. , Bolhuis R. L. H. , Gratama J. W.
Published: August 2005

The simultaneous flow cytometric study of multiple (≥ 3) markers on individual cells is restricted by technical reasons, e.g., the type of flow cytometer, and the availability of (monoclonal) antibodies (mAb) conjugated with the appropriate fluorochromes. However, a n‐way classification (n ≥ 3) may be derived from multiple 2‐way classifications. The 2‐way classifications are obtained by the use of only two fluorochromes, but each fluorochrome may be used for the simultaneous labelling of 2 or more mAb. We present a formal statistical model, based on an underlying multinomial distribution for the observed quadrant counts, by which data from such multiple 2‐way classifications can be analyzed. The model is restricted to 3‐marker phenotypic analyses, but can be extended to n‐way classifications (n > 3). Maximum likelihood estimates are obtained by the application of the EM algorithm. The model was tested on 20 samples of peripheral blood mononuclear cells to study the coexpression of CD45RA, CD45RO, and Leu 8 by lymphocyte subsets defined by the CD56 (MHC‐unrestricted cytotoxic cells) and CD3 (T cells) markers. Application of the model gave an excellent fit in all but one cases. In addition, the model reduced the effect of inter‐assay variation on the estimates and it provided an analysis of consistency over the data, which allows the detection of outliers due to staining errors. © 1993 Wiley‐Liss, Inc.
Method for counting mitoses by image processing in feulgen stained breast cancer sections
Authors: ten Kate T. K. , Beliën J. A. M. , Smeulders A. W. M. , Baak J. P. A.
Published: August 2005

This study describes an image processing method for the assessment of the mitotic count in Feulgen‐stained breast cancer sections. The segmentation procedure was optimized to eliminate 95–98% of the nonmitoses, whereas 11% of the mitoses did not survive the segmentation procedure. Contour features and optical density measurements of the remaining objects were computed to allow for classification. Twelve specimens were analyzed, nine used to serve as a training set, and three put aside for later use as independent test set. The fully automatic image processing method correctly classified 81% of the mitoses at the specimen level while inserting 30% false positives. The automatic procedure strongly correlated with the interactive counting procedure (r = 0.98). Although the fully automatic method provided satisfactory results, it is not yet suited for clinical practice. The automated method with an interactive evaluation step gave an accurate reflection of the mitotic count showing an almost perfect correlation with the results of the interactive morphometry (r = 0.998). Therefore this semiautomated method may be useful as prescreening device. © 1993 Wiley‐Liss, Inc.
Carotenoids located in human lymphocyte subpopulations and natural killer cells by Raman microspectroscopy
Authors: Puppels G. J. , Garritsen H. S. P. , Kummer J. A. , Greve J.
Published: August 2005

The presence and subcellular location of carotenoids in human lymphocyte sub‐populations (CD4+, CD8+, T‐cell receptor‐γδ+, and CD19+ ) and natural killer cells (CD16+ ) were studied by means of Raman microspectroscopy. In CD4+ lymphocytes a high concentration (∼10−3M) of carotenoids was found in the Gall body. In CD8+ lymphocytes, T‐cell‐receptor‐γδ+ lymphocytes and in natural killer cells carotenoids appeared to be concentrated (∼10−4M) in the Golgi complex. The concentration of carotenoids in CD19+ lymphocytes was found to be below the present detection limit, which is ∼10−6 to 10−5 M. The results provide new possibilities to investigate the mechanism(s) behind the suggested protective role of carotenoids against the development of cancers. © 1993 Wiley‐Liss, Inc.
Characterization of hypoxia‐dependent peroxide production in cultures of Saccharomyces cerevisiae using flow cytometry: A model for ischemic tissue destruction
Authors: Yurkow Edward J. , McKenzie Maureen A.
Published: August 2005

Peroxide production in cultures of Saccharomyces cerevisiae was measured using the H2O2‐sensitive fluorescent probe 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) and flow cytometry. Aeration of cultures of S. cerevisiae exposed to a period of hypoxia was found to induce elevated levels of peroxide that were 100‐fold higher than the levels observed in cultures maintained under exclusively aerated or hypoxic conditions. Simultaneous viability analysis, using the fluorescent DNA‐intercalating dye propidium iodide, indicated that the increase in peroxide generation preceded cell damage and death. Various agents were found to influence the effect of peroxides on cell viability. The addition of ethanol to hypoxic stationary cultures dramatically increased the rate of cell death without further increasing the amount of peroxide produced, while glucose inhibited peroxide production and decreased the rate of cell death. Surprisingly, elevated peroxide levels of hypoxic/reaerated cultures were maintained upon addition of KH2PO4, although the cells remained viable for extended periods of time when compared to control and other test cultures. Similarities between our observations and those of other investigators using anoxic/reperfused organs suggest that hypoxic/reaerated yeast cultures may be a useful model system to study ischemia‐dependent tissue destruction of mammals. © 1993 Wiley‐Liss, Inc.
Evaluation of a dual‐color flow cytometry immunophenotyping panel in a multicenter quality assurance program
Authors: Schenker Eve Levine , Hultin Lance E. , Bauer Kenneth D. , Ferbas John , Margolick Joseph B. , Giorgi Janis V.
Published: August 2005

A basic immunophenotyping panel that employed dual‐color combinations of fluorescein isothiocyanate (FITC) and phycoerythrin (PE) conjugated monoclonal antibodies (mAb; FITC‐CD45/PE‐CD14, FITC‐IgG1/PE‐IgG2, FITC‐CD3/PE‐CD8, FITC‐CD3/PE‐CD4, FITC‐CD3/PE‐CD16+PE‐CD56, and PE‐CD19) was utilized in a quality assurance program to determine whether the 4 laboratories participating in a multicenter AIDS study obtained similar lymphocyte subset percentage values for T cells, B cells, NK cells, and CD4+ and CD8+ T cells. Over a 1½ year period, 78 shared peripheral blood specimens Were prepared and analyzed in each laboratory. The CD45brightCD14− percentage for each specimen was used to correct that individual's lymphocyte subset values. Interlaboratory coefficients of variation (CV) for the human immunodeficiency virus type I (HIV) seronegative (n = 38) and HIV‐seropositive (n = 40) specimens using this panel were <3% for total T cells; <5% for CD4+ T cells and CD8+ T cells; ≤17% for B and NK cells; and <8% for CD4T/CD8T ratios. The 6‐tube basic immunophenotyping panel has several notable features: (a) for clinical studies, it permits comprehensive evaluation of an individual's major lymphocyte subsets, i.e., T, B, NK, and CD4+ and CD8+ T cells; (b) for interlaboratory proficiency testing programs, it allows the detection of differences among laboratories in measurements of several functionally distinct cell populations; and (c) for within‐sample quality assurance, it provides several quality control checks, including the lym‐phosum, i.e., the sum of an individual's corrected T + B + NK values, a sum that was generally 100 ± 5% on the HIV‐seronegative specimens analyzed in this study. © 1993 Wiley‐Liss, INc.
Proposal for standardization of flow cytometric reticulocyte maturity index (RMI) measurements
Authors: Davis Bruce H. , Dicorato Michael , Bigelow Nancy C. , Langweiler Marc H.
Published: August 2005

Flow cytometric (FCM) reticulocyte analysis is more accurate, sensitive, and reproducible relative to previously employed manual microscopic methods in clinical laboratory hematology. FCM reticulocyte analysis using RNA binding fluorochromes additionally allows for the quantification of fluorescence intensity or population distribution of the reticulocyte RNA content. Viewed from the perspective of erythroid maturation, quantification of the fluorescence intensity distribution provides a reticulocyte maturation index (RMI). We performed a systematic study of 18 different methods to express thiazole orange stained reticulocyte fluorescence intensity, compared to standard mean fluorescence intensity quantification, using 185 anemic and non‐anemic human blood samples. The method best correlating with the mean fluorescence intensity RMI on 2 different FCM instruments (R2 = 0.93 and 0.86) was a ratio of the highly fluorescent reticulocytes, defined using a normal adult population, and the total number of reticulocytes (HFR%). In contrast to mean fluorescence intensity measurements, a HFR% RMI parameter can provide similar units of expression (0.01–1.00) with good correlation between different FCM instruments (R2 = 0.76). We conclude the HFR% method of RMI expression provides a superior means of interlaboratory standardization and clinical comprehension of this useful diagnostic parameter in clinical laboratory hematology. © 1993 Wiley‐Liss, Inc.
Detection of in situ hybridization to human chromosomes with the atomic force microscope
Authors: Putman C. A. J. , De Grooth B. G. , Wiegant J. , Raap A. K. , Van der Werf K. O. , Van Hulst N. F. , Greve J.
Published: August 2005

Atomic force microscopy (AFM) permits one to generate a topographic representation of the sample under investigation with high spatial resolution. We assumed that cytochemical staining techniques, which yield reaction products which can be discriminated from the surrounding material on basis of their topographic properties, would be applicable in AFM. Here we show the validity of this assumption by employing an in situ hybridization technique in which the final label was the precipitated product of a peroxidase/diaminebenzidine reaction. After hybridization of the DNA probe pUC1.77 that recognizes the heterochromatic region of human chromosome 1 (1q12), the AFM clearly detects the sites of in situ hybridization. In situ hybridization with DNA probe p1‐79 results in clear marking of the telomere region 1p36. The diameter of the probe p1‐79 linked reaction product was 75–100 nm, indicating that resolution of 200 nm can readily be reached with this AFM approach of DNA mapping. This precision is directly linked with the amount of precipitated material. © 1993 Wiley‐Liss, Inc.
DNA content in eurasian sturgeon species determined by flow cytometry
Authors: Birstein Vadim J. , Poletaev Andrei I. , Goncharov Boris F.
Published: August 2005

The nuclear DNA content in 10 species of chondrostean fishes was measured by flow cytometry. The sterletAcipenser ruthenusblood cells were used as an internal standard. The sterlet DNA content was calculated on the basis of comparison with theXenopus laevisblood cells, 2C = 6.30 pg. In the tetraploidA. ruthenusandA. stellatusthe DNA content comprises 3.74 pg/nucleus and is practically invariant; inHuso dauricusit is almost the same, 3.74–3.81 pg; and inA. nudiventrisit is a little higher, 3.88–4.04 pg. In the oldest chondrostean,Pseudoscaphirhynchus kaufmanni, the nuclear DNA content is slightly lower, 2C = 3.46–3.48 pg, and in the American paddlefishPolyodon spathulait is lower still, 3.17 pg. In two octoploid sturgeons,A. baeriandA. gueldenstaedti, the DNA content is twice as high as that of the sterlet, 8.29–8.31 and 7.86–7.88 pg, respectively; a very similar amount, 8.24–8.42 pg, was determined in the hybridHuso huso×A. ruthenus. In the Sakhalin sturgeon,A. medirostris(=A. mikadoi), the DNA content is two times higher than in the octoploids, 13.93–14.73 pg; therefore its ploidy may be 16n and the number of chromosomes could be 500. © 1993 Wiley‐Liss, Inc.
Isolation and characterization of CD34+ hematopoietic stem cells from human peripheral blood by high‐gradient magnetic cell sorting
Authors: Kato Kimitaka , Radbruch Andreas
Published: August 2005

CD34+ cells, present at a frequency of 0.18 ± 0.052% among leukocytes from peripheral blood (PB), can be rapidly and efficiently (recovery of 39.0–74.0%) enriched to a frequency of 38.6–87.1% (54.4 ± 12.3%) by high‐gradient magnetic cell separation (MACS) for immunophenotyping, characterization in colony‐forming cell (CFU) assays, and further purification to homogeneity (>98%) by multiparameter fluorescence‐activated cell sorting (FACS). Enriching PB‐CD34+ cells for immunophenotyping allows the detection of small subpopulations, expressing the B‐cell antigens CD10, CD19, and CD20, the T‐cell antigens CD45RA and CD7, and a small subpopulation expressing high levels of CD34 (1.20 ± 0.12%), which mostly coexpress CD19 (91.9 ± 9.05%), CD20 (64.8 ± 14.4%), and CD38 (84.5 ± 10.3%). All PB‐CD34+ cells express elevated levels of CD71 (transferrin receptor), with a subpopulation of high expressing cells, and CD38. Some cells express CD33. MACS‐enriched PBCD34+ cells show “normal” hematopoietic colony formation in vitro. The ease and efficiency of purification of large numbers of CD34+ cells from PB by MACS is not only relevant for the characterization of migrating stem cells but also opens new possibilities for stem cell transplantation and genetic manipulation of the hematopoietic system. © 1993 Wiley‐Liss, Inc.
Forward scatter pulse width signals resolve multiple populations of endosomes
Authors: Hammond Timothy G. , Majewski Rebecca R. , Morré D. James , Schell Kathleen , Morrissey Lawrence W.
Published: August 2005

The technique of pulse width analysis, developed to optimize cell size resolution in cell cycle kinetics, has not previously been applied to small particles such as endosomes. Offset is used to subtract a portion of the beam diameter from forward scatter pulse width signals to optimize visualization and discrimination of small particles. We identify multiple endosomal populations by offset pulse width of light scatter parameters. Specifically, linear forward scatter pulse width measurements reveal at least two populations of endosomes in the rat renal cortex, the rat renal papilla, and the luminal endothelium of the toad urinary bladder. Logarithmically amplified forward scatter pulse width measurements display the full dynamic range of these signals, resolving additional populations not manifest with linear amplification.

To confirm that the endosomes observed were resolved from optical and electronic noise, we examined physiological function. The endosomes acidified after supplying ATP to the intrinsic membrane H+‐ATPase present. Further, electron microscopy of sorted endosomal populations from the toad urinary bladder confirmed identity and homogeneity of the fraction.

Flow cytometric analysis of endosomal populations by multiparametric techniques including pulse width analysis of structural parameters and pulse height analysis of fluorescence from entrapped fluorophores allows identification, isolation, and quantification of multiple endosomal populations. © 1993 Wiley‐Liss, Inc.
Signal processing in slit‐scan flow cytometry of cell conjugates
Authors: Bakker Schut Tom C. , Florians Alex , Radošević Katarina , de Grooth Bart G. , Greve Jan
Published: August 2005

The design and implementation of a real‐time signal processing system for slit‐scan flow cytometry is described. The system is used to measure the separate scatter and fluorescence peak heights of 2 adherent cells. Preliminary measurements of changes in the membrane potential induced by interactions between natural killer (NK) cells and their target cells are presented. © 1993 Wiley‐Liss, Inc.
Flow cytometric method for simultaneous detection of lymphocyte‐K562 conjugates and immunophenotyping of the conjugate forming cells
Authors: Radošević Katarina , de Grooth Bart G. , Greve Jan
Published: August 2005

A flow cytometric method for the simultaneous quantification and immunophenotyping of conjugates formed by human peripheral blood lymphocytes (PBL) and K562 cells has been developed. The method uses three fluorescent probes. One of the fluorescent probes (F‐18) is used for labeling of PBL prior to incubation with K562 cells. After incubation the cells are treated with monoclonal antibodies labeled with phycoerythrin and Red613, respectively. The combination of F‐18 fluorescence and light scattering signals enables identification and quantification of the conjugates while the fluorescence of the monoclonal antibodies provides information about the phenotype of the conjugate forming cells. Results obtained using different monoclonal antibodies are presented. The highest conjugate forming capacity has been found in the CD56+CD8+ population while the CD4+CD8− population has shown the lowest capacity to form conjugates. The influence of a washing step on the conjugate formation is discussed. The possibility to use the method in combination with a cytotoxicity assay is indicated. © 1993 Wiley‐Liss, Inc.
Dna ploidy status and DNA content instability within single tumors in renal cell carcinoma
Authors: Bringuier P. P. , Bouvier R. , Berger N. , Piaton E. , Revillard J. P. , Perrin P. , Devonec M.
Published: August 2005

In order to investigate the relationship between genetic instability and DNA aneuploidy of malignant cells in human solid tumors, we studied the variations of DNA index within a single tumor. Multiple sampling (mean of 6.4 samples per tumor) was performed in 24 renal cell carcinomas (RCC). Based on the variations in DNA indices within a single tumor, RCC were divided into three groups: (1) tumors with stable DNA indices (variation within the range of the measurement error), including all DNA diploid tumors (n = 8), all hyperdiploid tumors (n = 3), a hypodiploid tumor (n = 1), and only 2 of 11 tetraploid and hypotetraploid tumors; (2) tumors with moderate variations in DNA indices, which were all close to tetraploidy (n = 5); and (3) tumors with large variations of DNA indices. In this last group, subclones present within a tumor varied widely in their DNA indices from the tetraploid to the triploid region, reflecting the DNA content instability within the tumor cell population. These results suggest that DNA aneuploidy can arise by two different mechanisms: (1) loss or gain of chromosomes leading to hypodiploid or hyperdiploid tumors with no apparent increase in DNA content instability, and (2) doubling of the chromosome set followed by random loss of chromosomes as suggested by the DNA indices ranging from tetraploid to triploid region found in three tumors. Differences in DNA indices within one tumor characterize subclones which may arise by chromosome loss or gain. © 1993 Wiley‐Liss, Inc.
Quality control study of the italian group of cytometry on flow cytometry cellular DNA content measurements
Authors: Danesi D. Tirindelli , Spanòg M. , Altavista P.
Published: August 2005

A quality control study on DNA flow cytometry, extended to 43 national laboratories, has been carried out by the Italian Group of Cytometry, using defined fixed suspensions of cultured human leukemia K562 cells and human blood lymphocytes. The participating laboratories were allowed to follow their own staining and measurement protocols. Aliquots of cellular suspension had to be measured three times on the same day and two other times on different days. A large heterogeneity of procedures emerged among participants. The average of mean DNA index laboratory values, from 36 laboratories who sent evaluable data, was 1.68, with a range from 1.49 to 1.97. The coefficients of variation ranged from 2.35 to 9.39% and from 2.79 to 8.5% for diploid and aneuploid peaks, respectively. Statistical analysis of the results showed quite good intralaboratory reproducibility, but statistically significant differences were observed among laboratories, for both DNA indices and coefficients of variation. These differences appear to be consistent. For standardization, it is essential that efforts should be made to identify the main sources of variation and to control them. © 1993 Wiley‐Liss, Inc.
Increased membrane permeability of apoptotic thymocytes: A flow cytometric study
Authors: Ormerod Michael G. , Sun Xiao‐Ming , Snowden Roger T. , Davies Reginald , Fearnhead Howard , Cohen Gerald M.
Published: August 2005

We have recently developed a method for the separation and quantification of viable apoptotic cells without the need for permeabilisation or fixation of the cells. The method is based on the observation that apoptotic rat thymocytes fluoresce more brightly than normal cells after a brief incubation with the DNA binding dye, Hoechst 33342. In order to understand these differences, we have investigated the uptake of Hoechst 33342 by normal and apoptotic thymocytes. By staining with fluorescein diacetate, we have shown that the efflux of fluorescein from apoptotic cells is more rapid than that from normal thymocytes. This result demonstrated an increase in the permeability of the plasma membrane of the apoptotic thymocytes and it is this change which probably results in the more rapid uptake of Hoechst 33342. The data also revealed two populations of apoptotic thymocytes. © 1993 Wiley‐Liss, Inc.
The use of flow cytometry for the investigation of cell death
Authors: Afanasyev Vladimir N. , Korol Boris A. , Matylevich Natalya P. , Pechatnikov Vladimir A. , Umansky Samuil R.
Published: August 2005

Flow cytometry is more and more widely used for investigations of cell death, predominantly in the study of DNA degradation in cells dying by apoptosis. There are different interpretations of changes observed in DNA histograms of these cells. We describe an approach based on extraction of chromatin degradation products from fixed cells and subsequent staining with DNA specific dyes. Apoptotic cells containing fragmented DNA are observed in < 2C DNA region of DNA histograms. DNA histograms of irradiated thymocytes dying in vitro and stained without extraction of fragmented DNA do not differ from control. Under the same staining conditions DNA histograms of lymphocytes dying in thymus of irradiated animals reveal fluorescent material in < 2C DNA region, most likely due to formation of apoptotic bodies (cell fragments, some of them contain fragments of nuclei). Similar changes are observed in thymocytes dying upon glucocorticoid treatment. Our present results and other data indicate that reduced amount of DNA in dying cells is the main reason for changes of DNA histograms. Examples of application of the method described for the investigations of cell death modifiers are presented. © 1993 Wiley‐Liss, Inc.
Application of artificial neural networks to chromosome classification
Authors: Errington Phil A. , Graham Jim
Published: August 2005

This work presents an approach to the automatic classification of metaphase chromosomes using a multilayer perceptron neural network. Representation of the banding patterns by intuitively defined features is avoided. The inputs to the network are the chromosome size and centromeric index and a coarsely quantized representation of the chromosome banding profile. We demonstrate that following a fairly mechanical training procedure, the classification performance of the network compares favourably with a well‐developed parametric classifier. The sensitivity of the network performance to variation in network parameters is investigated, and we show that a gain in efficiency is obtainable by an appropriate decomposition of the network. We discuss the flexibility of the classifier developed, its potential for enhancement, and how it may be adapted to suit the needs of current trends in karyotyping. © 1993 Wiley‐Liss, Inc.
Pure populations of Dictyostelium discoideum prespore and prestalk cells obtained by flow cytometry have different redevelopment characteristics at their cell surfaces
Authors: Browne Lois H. , Williams Keith L.
Published: August 2005

The multicellular slug stage of Dictyostelium discoideum consists of two major differentiated cell types: prespore and prestalk cells, which become, respectively, the spores and the stalk of the fruiting body. It is known that these cells, although expressing cell‐type‐specific proteins, remain totipotent, and experimental disruption of slugs results in redifferentiation taking place. We looked at what happens to cell‐type‐specific surface molecules when a cell changes from one type to another. Using monoclonal antibodies and flow cytometry we were able for the first time to obtain pure populations of single cells of each cell type. These were analysed during redevelopment. The initial hypothesis was that a proportion of each cell type would redifferentiate to reestablish the original proportions. However, it was found that the two cell types responded quite differently. Whereas almost all prestalk cells retained their prestalk surface antigen, in contrast, all prespore cells redifferentiated. During this process redifferentiating prespore cells simultaneously expressed surface determinants of both cell types, an event not seen in normal development. © 1993 Wiley‐Liss, Inc.
Double‐staining artefact observed in certain individuals during dual‐colour immunophenotyping of lymphocytes by flow cytometry
Authors: Ekong Theresa , Gompels Mark , Clark Colin , Parkin Jackie , Pinching Anthony
Published: August 2005

An unusual double‐staining artefact has been repeatedly observed in certain individuals (both HIV‐positive and healthy laboratory controls) during 2‐colour immunophenotyping of lymphocytes by flow cytometry after red cell lysis. This artefact can falsely suggest co‐expression of CD4 and CD8, as well as some of the other monoclonal antibody pairs commonly used in the typing of lymphocytes. It is caused by a serum factor associated with the ammonium‐sulphate‐precipitated and gamma‐globulin fractions. It appears to be fairly common in the population (being found in 6 out of 105 of our healthy laboratory controls and in 42 out of 247 of our HIV‐1 seropositive subjects); it may be genetically determined, but may be acquired in some. It is a potential source of error in lymphocyte immunophenotyping, which can be avoided by simple adjustments to the recommended techniques. © 1993 Wiley‐Liss, Inc.
Evaluation of flow cytometry for multidrug resistance detection in low resistance K562 cells using daunorubicin and monoclonal antibodies
Authors: Van Acker Koen L. , Van Hove Luc M. , Boogaerts Marc A.
Published: August 2005

We compared the effectiveness of two flow cytometric methods for the detection of the multidrug resistance (MDR) phenotype. The sensitivity of both methods depended on the ability to discriminate low resistance cells from sensitive ones. Therefore, K562 cells with decreasing vinblastine (VLB) resistance levels were examined, the lowest resistance level being nonmeasurable with a colorimetric MTT assay. The fluorescent drug daunorubicin (DNR) was measured in combination with two modulators of MDR, cyclosporin‐A (CsA) and verapamil (Vp) in a functional flow cytometric assay. When compared to sensitive cells, DNR uptake levels at steady state were reduced in all resistant cell lines, except for the lowest resistant cell line. The effect of modulator, CsA, on DNR uptake was seen in all resistant sublines, compared to sensitive cells, except for the lowest resistant cells. In another assay, the P‐glycoprotein (Pgp) expression was analysed with monoclonal antibodies, MRK16 and C219. MRK16 was found to be the most sensitive antibody to screen for MDR + cells, since we could show Pgp hyperexpression in all resistant cells. C219 reactivity became evident in cells possessing resistance factors higher than 5. These results indicate that both the functional assay and the Pgp assay are sensitive to be used for screening of MDR + cells. © 1993 Wiley‐Liss, Inc.