bluuebunny/crossref_metadata_2025 huggingface dataset:
Increased accuracy and speed of absorption cytometric DNA measurements by automatic corrections for nuclear darkness
Authors: Allison David C. , Lawrence George N. , Ridolpho Paul F. , O'Grady Brian J. , Rasch Robert W. , Rasch Ellen M.
Published: August 2005

We have developed a method of calculating the average local absorbance (ALA) of a nucleus from the integrated nuclear absorbance and area. One can use the ALA, along with nuclear areas measured at different point absorbance thresholds, to determine whether a nucleus is stained too lightly or too darkly for accurate absorption measurements; this allows selection of an optimal light wavelength for the performance of these measurements. The ALA can also be used for automatic and instantaneous correction of integrated absorbance values from darkly stained cells. This allows the rapid measurement of the integrated absorbances of a large number of nuclei that are heterogeneous in stain intensity. Coefficients of variation of approximately 3% are obtained for the integrated absorbances of nuclei of nontransformed G0/G1 cells. This correction method can be applied with any image densitometer that generates both integrated absorbance and area values.
Estimation of sampling errors in a high‐resolution TV microscope image‐processing system
Authors: Harms H. , Aus H. M.
Published: August 2005

The basic postulate of this paper is that the commonly accepted sampling density of 2–4 pixels/μm in a high‐resolution TV microscope system is too low to digitize exactly and analyze the complex cellular detail found in stained cell images. Depending on the specific microscope system, the required sampling density is much higher, lying between 15 and 30 pixels/μm. This sampling density is derived from the aliasing error, the resolution loss, and computational limitations. The mathematical and optical methods and equipment used to obtain these results are described in detail.
Image processing for mitoses in sections of breast cancer: A feasibility study
Authors: Kaman E. J. , Smeulders A. W. M. , Verbeek P. W. , Young I. T. , Baak J. P. A.
Published: August 2005

This paper describes an image analysis technique for the counting of nuclei in mitosis in tissue sections. Five experienced pathologists scored mitoses in photographs of preselected areas of tissue sections of the breast. Objects consistently labelled as mitotic cells by all five pathologists were considered “mitoses” in the analysis. In total, there were 45 mitotic nuclei, 68 possible mitotic nuclei and 1,172 nonmitotic nuclei.

The image analysis procedure was designed to give priority to a low false negative rate, i.e., misclassification of mitoses. The procedure consists of three steps:

Segmentation of the image.

Reduction of the number of nonmitotic nuclei by using feature values based on the brightness histogram of the objects.

Fully automatic classification of the remaining objects using contour features.

The objects remaining after the first two steps were visualized in a composite display for interactive evaluation: 10% of the mitotic nuclei were missed, and 85% of the nonmitotic nuclei were eliminated. The result of the fully automatic procedure described in this paper is rather disappointing and gave a loss of 37% of the mitoses while 5% of the nonmitotic nuclei remained.
An automated image analysis system for the detection of rare autoradiographically labelled cells in the human lymphocyte HGPRT variant assay
Authors: Stark Margaret H. , Tucker J. H. , Thomson E. J. , Perry P. E.
Published: August 2005

This paper describes a fast and accurate method for the identification of autoradiographically labelled human hypoxanthine‐guanine phosphoribosyl transferase (HGPRT) variant lymphocytes on slide preparations using a high‐speed computer image analysis system–the Fast Interval Processor (FIP). The system has been developed primarily for the analysis of cultured human peripheral blood lymphocyte populations in which the frequency of labelled cells may vary from less than one to more than 2,000 in every 10,000 cells. Evaluation experiments have demonstrated the excellent performance of FIP in the counting of labelled and total cells over this frequency range.
Nuclear DNA cytophotometry in prostate carcinoma
Authors: Seppelt U. , Sprenger E.
Published: August 2005

Eighty patients with prostate carcinoma underwent fine‐needle aspiration biopsy for cytologic grading and DNA‐single‐cell fluorescence photometry before and at 6‐month intervals after endocrine treatment. The histograms of DNA values showed single peaks and bimodal and scattered distribution which correlated to the different tumor grades before therapy. The DNA values were significantly different from the controls with benign prostatic hypertrophy. After start of therapy, regressive changes of the DNA‐histograms were increases of diploid and hypodiploid DNA values and disappearance of secondary peaks. Progressive changes were increased scattering of DNA values and appearance of secondary peaks. Progressive changes in the histograms were closely related to clinical remission and stable disease, but related poorly to clinical progression. The survival correlated with the pretherapeutic DNA‐histograms and with the DNA‐median, third‐quartile, and maximum parameters.
Comparative flow DNA analysis of different cell suspensions in breast carcinoma
Authors: Chassevent A. , Daver A. , Bertrand G. , Coic H. , Geslin J. , Bidabe M.‐CL , George P. , Larra F.
Published: August 2005

This study compared three methods of dissociation of breast lesions for DNA flow cytometry. Eleven benign lesions and 66 cancers were dissociated using mechanical, Ficoll, or enzymatic methods. DNA flow analysis showed that the DNA index did not vary from one method of dissociation to another. All benign lesions were diploid and 67% of all cancers were aneuploid. Enzymatic dissociation gave a lower percentage of aneuploid cells with a diminution of the proportion of cells in the G2 + M phase (13.2% enzymatic against 17.6% Fi‐coll); on the other hand, it provided cell populations of greater viability than the other methods (32.6% enzymatic, 17.2% Ficoll; P < 0.01). The mechanical and Ficoll suspensions did not differ significantly when they were analyzed on the basis of their DNA content and their cellular viability. When compared with mechanical preparation, Ficoll suspension showed a lower recovery of tumor cells, but this inconvenience was compensated for by a more homogenous aspect where the contribution of aggregates and debris was clearly lessened. Therefore, this study led us to choose Ficoll suspension for subsequent flow analysis of breast tumors.
Estimation of the frequency of malformed sperm by slit scan flow cytometry
Authors: Halamka J. , Gray J. W. , Gledhill B. L. , Lake S. , Wyrobek A. J.
Published: August 2005

We have investigated the utility of Slit Scan Flow Cytometry (SSFCM) for measuring the frequencies of malformed sperm heads in control and mutagen treated B6C3F1/CRL mice. In SSFCM, fluorescence profiles of sperm heads stained with the DNA‐specific fluorescent dye acriflavine were recorded for sperm flowing lengthwise through a 2.5‐μ‐thick laser beam. Malformed sperm were detected as having fluorescence profiles that differed substantially from an average fluorescence profile for sperm from untreated mice. Specifically, a sum of squared difference (SSD) value was calculated for the fluorescence profile of each sperm according to the equation

where c(i) and t(i) are the ith values for the fluorescence profiles from control and test sperm, respectively. Profiles whose SSD exceeded a threshold value of 20 were considered to be from malformed sperm. We measured fluorescence profiles for 500 sperm per mouse from five control mice, five mice injected intraperitoneally daily for 5 days with a total of 375 mg/kg of body weight methyl methane sulfonate (MMS), and for 30 mice injected intraperitoneally daily for 5 days with total doses of procarbazine ranging from 125 mg/kg to 1,250 mg/kg.

Sperm were collected from the caudae epididymides 35 days after the last injection. Frequencies of malformed sperm in these samples were also estimated by visual analysis. All samples were analyzed in double blind fashion. The visual and SSFCM malformed sperm frequencies for the samples from control, MMS‐treated, and procarbazine‐treated mice were correlated (r = 0.83). A dose effect was seen with both the visual and SSFCM estimates for the sperm from the procarbazine‐treated mice.
Accessibility of DNA in situ to various fluorochromes: Relationship to chromatin changes during erythroid differentiation of friend leukemia cells
Authors: Darzynkiewicz Zbigniew , Traganos Frank , Kapuscinski Jan , Staiano‐Coico Lisa , Melamed Myron R.
Published: August 2005

Friend leukemia cells from exponentially growing or differentiated (DIMSO‐induced) cultures were permealized and their DNA was stained with 4′6‐diamidino‐2‐phenylinciole (DAPI), Hoechst 33342, acridine organge, ethidium bromide, propidium iodide, Quinacrine, 7‐amino‐actinomycin D, mithramycin, or chromomycin A3. Accessibility, of DNA to each of the above fluorochromes was compared in differentiated and nondifferentiated cells before and after nuclear proteins, mostly histones, were extracted with O. 1N HC1. A decrease in the accessibility of DNA to several dyes, especially pronounced in the case of some intercalators, was observed in differentiated cells. After extraction of nuclear proteins with HO there was an increase in DNA accessibility, of varying degree depending on the fluorochrome and the difference between differentiated and nondifferentiated cells was abolished for most of the intercalating dyes. The increase was the lowest for DAPI (45%), the highest for 7‐amino‐actinomycin D (13 fold), and in general was higher for the intercalating dyes that unwind DNA than for dyes binding externally to the double helix. The results are discussed in terms of the mode of interactions between DNA and the fluorochromes and factors associated with chromatin structure that may affect accessibility of DNA in situ in exponentially growing and differentiated cells.
Cytochemical analysis of the chromatin of liver cell nuclei and nuclei of oval cells by means of the feulgen hydrolysis curve in rats treated by thioacetcamide
Authors: Praet M. , Roels H.
Published: August 2005

The present study is an analysis of the Feulgen hydrolysis characteristics in nuclei of liver cells and oval cells in rats treated by thioacetamide (TAA) and of liver cells in control rats. The curves show a double‐peaked pattern. A slower hydrolysis is noted in the first peak after TAA treatment. This suggests an alteration of the acid‐labile part of the chromatin. The curve obtained in the oval cells is different from the one in the liver cells. The implications of these differences are discussed with respect to development of cholangiocarcinomas.
Carcinoembryonic antigen in cytological specimens of urothelial carcinoma
Authors: Holmberg Viveca , Wahren Britta , Esposti Pier‐Luigi
Published: August 2005

A peroxidase‐antiperoxidase (PAP) technique was developed for the detection of carcinoembryonic antigen (CEA) in urothelial transitional cells of 52 bladder cancer patients. The percentage of CEA‐containing malignant cells varied from 10% to 100%. As a mean, 65% of the malignant cells stained for CEA, while the corresponding figure for benign‐looking cells was 24%. The results were compared with cytological evaluations, flow cytophotometric results, and icnmunofluorescent (IF) staining for CEA. With increasing malignancy, more CEA was detected with the PAP technique, whereas the IF technique failed to show this trend. 18 of 20 malignant‐tumors had an aneuploid DNA pattern. The two diploid cases were moderately well differentiated. Samples from bladders with heavy inflammation should be avoided in the PAP technique, since the unspecific staining of granulocytes disturbed a correct evaluation of the transitional cells. The PAP technique used on cytological material is recommended for antigen determinations, since good morphology is obtained.
A count‐dependent filter for smoothing flow cytometric histograms
Authors: Schuette W. H. , Shackney S. E. , MacCollum M. A. , Smith C. A.
Published: August 2005

An adaptive count‐dependent algorithm for smoothing statistically limited histograms has been developed. It considers both the spatial frequency limitations of the measurement system (described by the measurement system point spread function) and the reliability of the measured data (indicated by the effective number of counts influencing each channel of the histogram).

Windows for smoothing flow cytometric histograms are derived from an assumed Gaussain‐shaped point spread function (PSF) with a constant coefficient of variation. The windows are developed by scaling the variances of the Gaussian functions inversely with the statistical reliability of the data contained in each channel of the measured histogram.

The reliability of this data is determined by taking the square root of the number of counts influencing the value tabulated for each channel.

Using the algorithm, a smoothed version of the measured histogram may be developed from a linear sum of the products of the individual scaled Gaussian functions and the original measured histogram.

Data are presented demonstrating the advantages of count‐dependent smoothing over non‐count‐dependent smoothing using synthesized DNA histograms as a function of sample size.
The application of perpendicular and forward light scatter to assess nuclear and cellular morphology
Authors: Benson Mitchell C. , McDougal Denise C. , Coffey Donald S.
Published: August 2005

Perpendicular and forward light scatter have been employed in a multiparametric approach to distinguish the various lines of the transplantable Dunning R3327 rat prostatic adenocarcinoma. Perpendicular light scatter has been shown to correlate with nuclear size and shape. The average intensity of forward light scatter and perpendicular light scatter signals was demonstrated to increase as cells became less well‐differentiated. The combination of perpendicular and forward light scatter allows for the discrimination of histologically indistinguishable tumors and may therefore be useful to establish a system of grading cancer.
Purification of beta cells from rat islets by monoclonal antibody‐fluorescence flow cytometry
Authors: Russell Thomas R. , Noel Jack , Files Nancye , Ingram Marylou , Rabinovitch Alexander
Published: August 2005

Fluoresceinated monoclonal antibody plus flow cytometry was used to purify beta cells from mixed pancreatic islet endocrine cell populations. A2B5, a monoclonal antibody to a glycolipid on the surface of cells of neuroen‐docrine origin, was incubated with single cells dissociated from rat pancreatic islets. Antibody‐bound cells were labeled with fluoresceinated goat F(ab')2 antimouse immuno‐globulin and highly fluorescent cells were separated from less fluorescent cells on a Coulter EPICS IV cell sorter. Sorted cell populations were analyzed by radioimmunoassay for insulin, glucagon, and somatostatin. The highly fluorescent cell population was enriched sixfold for insulin‐containing beta cells, indicating that islet beta cells are relatively enriched in A2B5 antigen and can be partially purified by this method.
Preparation of chromosome suspensions from cells of a solid experimental tumour for measurement by flow cytometry
Authors: Kooi M. W. , Aten J. A. , Stap J. , Kipp J. B. A. , Barendsen G. W.
Published: August 2005

A method is described by which metaphase chromosomes are isolated from cells of a transplantable rat sarcoma. The chromosomes are derived from cells in a suspension prepared by trypsinisation of tumours from rats that have been treated with vindesine 24 h before excision in order to accumulate cells in mitosis. Histograms obtained for the chromosomes of the solid tumour are compared with flow karyotypes of cells cultured for 20 h or for several generations in vitro.
A simple device to obtain high local concentrations of material sorted by flow cytometry for biochemical or morphological analysis
Authors: Métézeau Philippe , Bernheim Alain , Berger Roland , Goldberg Michel E.
Published: August 2005

A system is described which allows us to collect flow cytometry sorted objects onto a very small area, thus obtaining very concentrated particles. For that purpose, collection is made directly on a nitrocellulose filter while a vacuum is established below the filter. By comparison with collection onto a microscope slide, the local concentration is increased about 1,000‐fold. This efficient and simple system allows us to concentrate cell or cellular organelle populations for observation by microscopy of many objects in a single field, or to study the biological or biochemical properties of a small number of sorted particles.
Preparative dual‐beam sorting of the human Y chromosome and in situ hybridization of cloned DNA probes
Authors: Cremer C. , Rappold G. , Gray J. W. , Müller C. R. , Ropers H.‐H.
Published: August 2005

Bivariate Hoechst/chromomycin flow karyotypes for chromosomes from a Chinese hamster‐human hybrid cell line (CH‐Y‐VII) were established that have retained a human Y chromosome. These bivariate flow karyotypes showed the human Y chromo‐some to be completely separated from the peaks for the Chinese hamster chromosomes. In preparative dual‐beam sorting: experiments, 3 × 106 chromosomes were sorted from the Y peak into frozen petri dishes. An examination of Q‐banded samples of sorted chromosomes revealed that 82% ± 5% of them were human Y chromosomes.

The DNA from the sorted chromosomes (approximately 250 ng) was isolated and used to establish a genomic library (vector λ gt WES.λB). Three clones (YACG 45, 52, 54) of this library containing inserts of repetitive human DNA were used for chromosomal localization by means of in situ hybridization to metaphase spreads of male human lymphocytes and of CH‐Y‐VII cells. In all three cases, a significant binding to the human Y chromosome was observed.

A more detailed study of the chromosomal distribution of sequences homologous to the insert of YACG 45 suggested the existence of minor binding sites on several human autosomes. Southern blot analysis revealed the existence of other human specific sequences without Y chromosome specificity.
A kinetic study of membrane immunoglobulin capping by flow cytometry
Authors: Cuchens Marvin A. , Buttke Thomas M.
Published: August 2005

A flow cytometric procedure has been developed for performing kinetic studies on the capping of membrane immunoglobulin (mIg) on B lymphocytes. Mouse B cells were stained with fluorescein‐conjugated antimouse‐Ig antisera and subjected to pulse‐shape (width, peak, and area) analyses prior to, during, and after ligand‐induced redistribution of mIg. It was found that ring‐stained, patched, and capped cells could be discriminated based on the width of the electronic signal curve generated as the cells passed through the laser beam. Additionally, endocytosis and or shedding of the cap could be correlated with a change in the area under the curve. Using these two parameters (width and area), the effects of temperature, cross‐linking, and several pharmacological agents on the capping process were examined. Through the use of flow cytometry, the inhibitory effects of various perturbants could be localized to discrete stages of the capping process.
Recognition of central nervous system leukemia by flow cytometry
Authors: Redner Arlene , Andreeff Michael , Miller Denis R. , Steinherz Peter , Melamed Myron
Published: August 2005

Cerebrospinal fluid of 24 patients with acute leukemia was studied by DNA/RNA flow cytometry. In six of 15 patients with central nervous system (CNS) relapse, the spinal fluid cells had abnormal DNA stemlines, ranging from near haploid to hyperdiploid. In two additional cases, leukemic cells were identified by a abnormally high RNA content only. One patient had two different aneuploid cell populations in spinal fluid not distinguished by cytologic morphology. Another patient with initial diploid leukemia had CNS relapse characterized by the same DNA stemline long after a triploid DNA stemline emerged in the marrow. DNA/RNA flow cytometry detected leukemic cells that were not identified (“uniform” or “reactive”) by cytological criteria in 5/6 patients studied and in addition differentiated lymphoblastic from nonlymphoblastic cell types according to low and high RNA content.
Analysis of DNA synthesis rate of cultured cells from flow cytometric data
Authors: Bertuzzi A. , Gandolfi A. , Germani A. , Spanò M. , Starace G. , Vitelli R.
Published: August 2005

The rate of DNA synthesis along S phase is estimated from flow cytometric histograms on the basis of a mathematical model of a cell population. In the absence of loss, the model expresses the population kinetics in terms of DNA synthesis rate, S‐phase influx, and population size. A single histogram is sufficient to determine the DNA synthesis rate when the population is in balanced exponential growth. Two suitably chosen histograms are necessary if the S‐phase influx is exponential in a time interval longer than the S‐phase duration. The analysis procedure was tested on published autoradiographic data and applied to three cultured cell lines (CM‐S, 3LL, and M14 cells) that show various patterns of DNA distribution. In each case the cell‐cycle fractions, the DNA synthesis rate, and the S‐phase duration were obtained.
Flow cytometry of postmortem human testicular tissue in cases of atherosclerosis
Authors: Klein Richard , Pfitzer Peter
Published: August 2005

From forty‐seven autopsy cares of atherosclerosis flow cytometry (FCM) of DNA and histology of both testes are compared with the histological sections of their supplying vessels arteriae testiculares and arteriae ductus deferents at different levels. By this method, changes of spermatogenesis are judged separtely for each side and the results can be related to the local conditions of blood supply. Four young men, dead after traffic accidents, served as control. In the majority of cases, the computer‐assisted evaluations of the meiotic DNA histograms show no differences between the right and left testis, even which differences of the arterial diameters are found by histology. On the other hand, cases with distinct differences in the histograms can show insignificant pathological alterations of the vessels. Though most excessive forms of macroscopic and microscopic atherosclerosis do not necessarily leas to a significant reduction in spermatogenesis, some cases with moderate forms show a strong reduction or even a total loss. This discrepancy can best be explained by superposition of other diseases.
Design of flow chamber with electronic cell volume capability and light detection optics for multilaser flow cytometry
Authors: Schuette W. H. , Shackney S. E. , Plowman F. A. , Tipton H. W. , Smith C. A. , MacCollum M. A.
Published: August 2005

A multibeam optical detection system has been developed with a high optical efficiency, achieved through a reduction in the number of optical interfaces employed in the system. This reduction is made possible by a combination of employing simple lenses, gluing the objective lens directly upon the face of the flow cuvette and the extraction of only one fluorescence signal from each laser beam. A modified flow chamber is also described that includes fluidic resistance elements for the elimination of most of the electric shielding normally associated with electronic cell volume measurements.
Identification of inverted duplicated #15 chromosomes using bivariate flow cytometric analysis
Authors: Lalande Marc , Schreck Rhona R. , Hoffman Robert , Latt Samuel A.
Published: August 2005

A dual laser FACS IV cell sorter has been used to obtain bivariate flow histograms of human metaphase chromosomes stained with the DNA‐specific dyes, 33258 Hoechst and chromomycin A3. Approximately twenty distinct chromosomal fluorescence populations can be resolved using this double staining technique and the flow cytometer which has been modified only by the substitution of a specially designed air‐spaced achromat for the standard focusing lens.

Metaphase chromosomes from two different cell lines bearing inverted duplicated #15 autosomes have been subjected to bivariate chromosome analysis. In both cases, the inverted duplicated #15 chromosomes have been identified in the bivariate flow histogram. This identification was supported by experiments in which doubly stained chromosomes were counterstained with either netropsin or distamycin A, resulting in a relative increase in the 33258 Hoechst fluorescence intensity of the structurally abnormal #15 chromosomes, compared with the other chromosomes, as predicted by cytological studies.

The possibility of identifying and separating small abnormal autosomes using commercially available instrumentation should facilitate the use of recombinant DNA techniques for the construction of libraries which are highly enriched for DNA sequences from limited autosomal subregions important in the study of chromosomal abnormalities such as deletions, translocations and inversion duplications.
Light‐scattering changes during chemotactic stimulation of human neutrophils: Kinetics followed by flow cytometry
Authors: McNeil Paul L. , Kennedy Amy L. , Waggoner Alan S. , Lansing Taylor D. , Murphy Robert F.
Published: August 2005

The light‐scattering properties of human neutrophils were compared on a cell‐by‐cell basis before and after stimulation with chemotactic peptide using flow cytometry. Between 20 and 180 sec after peptide addition, side (90°) scatter declined by up to 4% and forward scatter increased up to 6%. Between 3 and 15 min, side scatter increased up to 15% and forward scatter decreased up to 5%. Association of a fluorescence chemoattractant with neutrophils was most rapid during the initial phase of increasing forward and decreasing side scatter, and association saturated before the maximum increase in side scatter. Evidence is presented that the observed changes in scatter were not a consequence of chemoattractant‐induced cell‐cell adhesion or neutrophil degranulation. Rather, the early phases of light‐scattering changes are interpreted to represent membrane ruffling by the stimulated neutrophil; the later phases polarization of the neutrophil morphology.
Two‐dimensional impedance studies of BSA buoyant density separated human erythrocytes
Authors: Leif R. C. , Schwartz S. , Rodriguez C. M. , Pell‐Fernandez L. , Groves M. , Leif S. B. , Cayer M. , Crews H.
Published: August 2005

Combined DC (Coulter Volume) and radio frequency impedance studies were performed on human erythrocytes which had been separated by buoyant density in linear, neutral, isotonic bovine serum albumin gradients. The individual buoyant density fractions showed no reproducible shift in volume with buoyant density but did show a shift with opacity, radio frequency impedance divided by dc impedance. This new electronic parameter of opacity can be related to cell age, since both it and cell age are directly related to buoyant density. This increase in opacity with buoyant density is correlated with a change in shape.
A model for the classification of specimens containing random proportions of abnormal cells
Authors: Timmers T. , Gelsema E. S.
Published: August 2005

The effect of abnormal cell proportion on the performance of an automated cervical prescreening system is discussed in K.R. Castleman and B.S. White, Cytometry 2:155–18. The model employed assumes fixed proportions of abnormal cells, both in the design stage and in the test stage. In the present paper, an extended model is developed that allows for random variability of this proportion. It is shown, that there is a fundamental, non‐zero lower limit to the false‐negative specimen error rate, which depends only on the coefficient of variation. This limit may be reached even for moderate values of the coefficient of variation, which implies that a satisfactory prescreening system may not be feasible.
Flow cytometric determination of DNA ploidy level in nuclei isolated from paraffin‐embedded tissue
Authors: Schutte B. , Reynders M. M. J. , Bosman F. T. , Blijham G. H.
Published: August 2005

Nuclei, isolated from paraffin‐embedded tissue, were stained with propidium iodide (PI) and found suitable for DNA analysis by flow cytometry (FCM). DNA‐derived fluorescence intensity, however, was always decreased and had a much higher intersample variability as compared to results obtained with fresh material. Using chicken red blood cells (CRBC) as a model system, we found the lower fluorescence intensity to be due to the formalin fixation step in tissue processing. The intersample variability was found to be at least partly caused by variations in the duration of fixation. Overnight trypsinization improved the fluorescence intensity but did not reduce the intersample variability. Under all conditions tested PI binding to CRBC appeared to be saturable. Since fresh diploid or red blood cells could not be used to standardize DNA histograms, an alternative approach was developed in which nuclei from paraffinembedded normal and tumor tissue of the same specimen were mixed. With this method DNA indices (DI) of 24 colorectal cancers were found to be closely correlated (r = 0.9877, P < 0.001) with DI obtained with fresh tumor tissue from the same patients. The correlation of the percentages of S‐phase nuclei between paraffin‐extracted and fresh samples (r = 0.5875, P < 0.05) was as high as could be expected, taking sampling differences into account. This method is an important tool for the retrospective analysis of FCM‐derived DNA parameters in relation to diagnosis and prognosis of neoplasms.
Validation of a mathematical procedure for computer analysis of flow cytometric DNA data in human tumors
Authors: Del Bino G. , Bruni C. , Koch G. , Mazzini G. , Costa A. , Silvestrini R.
Published: August 2005

The determination of tritiated thymidine labeling index and the percentage of cells with S phase DNA content was performed on cell suspensions obtained from 69 patients with non‐Hodgkin's lymphoma. The distributions of cells in the cell cycle by computer analysis of flow cytometric data were obtained by two mathematical procedures: the widely adopted Fried model and a new one proposed by Bruni et al. A significant agreement was observed by checking the Spearman index (rs) between the percentages of cells in the different cell cycle phases (G0/1, rs = 0.76; S, rs = 0.60; and G2+M, rs = 0.43; p < 0.001) determined by the two procedures. Similarly, a good correlation was observed between the labeling index (LI) and the S phase values obtained by the Fried (rs = 0.45, p < 0.001) and Bruni (rs = 0.69, p < 0.001) models, but with a higher agreement for the latter one. The S phase by the Bruni model was also superior in predicting LI: in fact, by employing the S cutoff value of 12%, a better agreement between low LI and low S phase or high LI and high S phase was observed with the Bruni procedure (90%) than with the Fried model (72%). Finally, the analysis of the prognostic significance of the different kinetic variables confirmed the prognostic relevance of LI at any time; the S phase percentage as determined by Bruni et al. was discriminant of survival only at shorter times, and no prognostic significance could be ascribed to S phase according to the Fried procedure.
Helpful hints in flow cytometry and sorting
Authors: Dean Phillip N.
Published: August 2005

Most users of flow cytometers and sorters become proficient in their use through direct operation of the instruments. There are no text books or operation manuals that describe all of the problems that will be encountered and that may not be apparent. We have discovered many of these problems through finding an explanation for unexpected data and in resolving difficulties in achieving and maintaining high resolution and stable operation of the instruments. This paper presents a summary of problems encountered and solutions found at this laboratory and others.
Linearity and noise sources in flow cytometry
Authors: Ubezio Paolo , Andreoni Alessandra
Published: August 2005

A model of pulse formation in flow cytometers is presented that demonstrates the proportionality between the area (or the peak height) of the fluorescence signal produced by the photomultiplier and the number of fluorochrome molecules present in the cell that cause the signal. The model clarifies the possible instrumental origins of inaccuracy in this linearity that results in a broadening of the histograms obtained. A comprehensive formula for the coefficient of variation of the unimodular histograms of an homogeneous population is presented that clearly discriminates among the different contributions of staining, possible inhomogeneity of the examined population, photon statistics, and instrumental instabilities.

Finally, some experimental data are presented that show the agreement with the proposed formula.
Inexpensive upgrading of a FACS I and isolation of rare somatic variants by double‐fluorescence sorting
Authors: Weichel Walter , Liesegang Bernhard , Gehrke Klaus , Göttlinger Christoph , Holtkamp Bodo , Radbruch Andreas , Stackhouse Thomas K. , Rajewsky Klaus
Published: August 2005

We have modified a FACS I by addition of a tunable dye laser and an optical system for fluorescence detection that allows physically independent measurement of green and red immunofluorescence. These modifications are inexpensive and should be applicable to most single‐laser system. By using the modified machine and double‐fluorescence activated selection with H‐2Kk‐specific monoclonal antibodies labelled with FITC or Texas Red, we have isolated from a murine T‐cell lymphoma line variant subclones expressing structurally altered histocompatibility class I (H‐2Kk) molecules.
Dual‐parameter scatter‐flow immunofluorescence analysis of bacillus spores
Authors: Phillips A. P. , Martin K. L.
Published: August 2005

Using a commercial flow cytometer (Cytofluorograf), narrow‐forward‐angle (NFA) light‐scatter signals were detected for spore preparations of Bacillus anthracis Vollum, B. anthracis Sterne, B. cereus NCTC 8035, and B. subtilis var niger. In the flow immunofluorescence (FIF) analysis of spores stained with fluorescein‐conjugated hyperimmune antibody to B. anthracis Vollum spores, fluorescence histograms could be acquired by selecting on NFA scatter. Fluorescence data selected on ninety degree scatter were rather noisier. Fluorescence analysis by dual parameter NFA scatter‐FIF techniques was shown to have several advantages over the subtraction FIF method reported earlier. The implication from FIF analysis of spore suspensions and corresponding cell‐free supernatants that the peak in the fluorescence histogram was caused by signals from fluorescing spores, was confirmed by use of the cell sorter and subsequent microscopy of the sorted samples. Although a proportion of spore aggregates was present in samples sorted from the right‐hand tail of the fluorescence histogram, it was demonstrated that the majority of the observed distribution of fluorescence was not due to the formation of aggregates but was rather an expression of variation in the degree of staining of individual spores.
Evaluation of colony‐forming ability experiments using normal and DNA repair‐deficient human fibroblast strains and an automatic colony counter
Authors: Thielmann Heinz Walter , Hagedorn Roland , Freber Werner
Published: August 2005

An automated procedure for evaluation of colony‐forming ability experiments has been developed using normal and xeroderma pigmentosum fibroblast strains. Technically this consists of an image analyzer equipped with a contrast‐intensifying video camera, monitor, and a desk‐top computer. Complex structures composed of overlapping colonies could be reduced to the consitituent single colonies by special correction programs. Eighteen experiments were evaluated, and the D0 values for colony‐forming ability were determined. A systematic but correctable deviation between visual and automated counting was found. The source of this deviation is described and a solution for correction is provided. The automatic procedure reduced the evaluating time by a factor of five to ten.
Quantitation of anthracyclines in human hematopoietic cell subpopulations by flow cytometry correlated with high pressure liquid chromatography
Authors: Speth Paul A. J. , Linssen Peter C. M. , Boezeman Jan B. M. , Wessels Hans M. C. , Haanen Clemens
Published: August 2005

The major white cell subpopulations present in bone marrow and peripheral blood can be discriminated by forward and perpendicular light scatter two‐parameter flow cytometry (FCM). Fluorescent properties of anthracycline antibiotics allow measurement of the concentration of these cytotoxic drugs in hematopoietic cells by FCM as a third parameter.

Analysis of scatter‐gated fluorescence histograms provides quantitative information about the cellular concentration of at least four cell categories in human blood and bone marrow cells. A good correlation was found between the mean cellular fluorescence measured by FCM and the overall cellular concentration of adriamycin, daunomycin, and their main metabolites determined with high‐pressure liquid chromatography (HPLC).

In incubation experiments with human hematopoietic tissues, the final concentration of various anthracyclines in subpopulations of white cells appeared to be dependent on cell density, incubation time, temperature, and type of compound and its concentration.

FCM analysis is a rapid, sensitive, and quantitative method for measurement of cellular anthracycline concentrations in subpopulations and therefore provides an useful new tool in monitoring chemotherapy.
Single‐cell analysis of the relationship among transferrin receptors, proliferation, and cell cycle phase in K562 cells
Authors: Rudolph Natalie S. , Ohlsson‐Wilhelm Betsy M. , Leary James F. , Rowley Peter T.
Published: August 2005

Multiparameter single‐cell analysis by flow cytometry was used to distinguish between size‐related changes in K562 cell transferrin receptor (TfR) expression and changes in membrane receptor density throughout the cell cycle and over time in culture. Light‐scatter pulse‐width time‐of‐flight, a direct and readily calibrated measure of cell diameter, was used to calculate receptor density as the average number of receptors per unit cell surface area. Cell surface TfRs were unimodally distributed over the cell population and were present throughout the cell cycle. The number of receptors increased as cells progressed through the cell cycle, but cell cycle phase was also correlated with cell volume. However, when size heterogeneity was factored out by reanalysis of listmode data, there was a clear cell‐cycle effect: among cells of the same size, both the number of receptors per cell and the receptor density increased from G1 to S to G2/M. TfR expression was also followed over time in culture after dilution into fresh medium. A decrease in growth rate after four days was preceded by one to two days by a decrease in both number of TfRs per cell and mean receptor density, indicating that decreased TfR expression represented true “down‐regulation” and not just decreased cell size or an increase in the proportion of smaller G1 cells. This type of analysis is generally applicable for resolving the effects of cell size heterogeneity and cell cycle on membrane protein distribution and for other studies of ligand‐receptor interaction.
Denaturation and condensation of DNA in situ induced by acridine orange in relation to chromatin changes during growth and differentiation of friend erythroleukemia cells
Authors: Darzynkiewicz Zbigniew , Traganos Frank , Kapuscinski Jan , Melamed Myron R.
Published: August 2005

DNA in situ is progressively denatured when the cells or nuclei are treated with increasing concentration of acridine orange (AO). This transition can be monitored by flow cytometry as a decrease in green fluorescence. The complexes of denatured DNA and AO undergo immediate condensation and aggregation; this step is manifested by appearance of red luminescence and formation of precipitates that can be detected by electron microscopy. The precipitates form preferentially in heterochromatin as well as in ribosomes and polysomes. Their formation and further aggregation affects cellular light scatter properties in both the forward and right‐angle direction.

The AO‐induced DNA denaturation and condensation was studied in nuclei of Friend erythroleukemia cells from exponentially growing, differentiated or quiescent cells. The DNA in nuclei of quiescent cells, from plateau‐phase cultures, was the most sensitive to denaturation; it denatured (measured by changes in luminescence) at an AO concentration between 50 and 80 μM with the midpoint of the transition (Cd) at 70 μM. DNA in nuclei of differentiated cells (dimethyl‐sulfoxide‐induced erythroid differentiation) was more resistant (Cd = 77–83 μM), whereas DNA in exponentially growing cells was the most resistant (Cd = 86 μM). Extraction of proteins with 0.1 M HCl at 0°CC abolished the differences between the cells and shifted the transition to a lower AO concentration (Cd = 46 μM). For comparison, the midpoint transitions representing condensation of free, nucleic acids measured as light scatter changes occurred at 13, 22, 31 and 53 μM of AO, for rRNA, tRNA, and denatured and native‐calf thymus DNA, respectively.

Denaturation and condensation of DNA, which can be induced by AO either in isolated nuclei or viable permealized or fixed cells provides a new approach to discriminate cell sub‐populations with different chromatin structure by flow cytometry. The molecular mechanisms of this phenomenon are discussed.
Flow cytometric detection of a two‐step cell death induced by hyperthermia
Authors: Böhmer Ralph‐M.
Published: August 2005

A human leukaemic cell line (REH) was subjected to various temperatures °C42 °C for various time intervals; the cells were stained with a mixture of ethidium bromide and acridine orange, and red and green fluorescence were analysed by flow cytometry. Nontreated cells appeared as one cluster (V) in the biparameter histograms, but with time of heat treatment, two further discrete clusters (D1,D2) of cells appeared successively. They were distinguished by both the degree of red and green fluorescence. The kinetics of transit from one cluster to the other was dependent on temperature, the time lag between both steps becoming shorter with higher temperatures.

It was shown previously that the same effect occurred during incubation with various cytostatic agents, and that only the D2 stage correlated with the stage of cell death monitored by the usual trypan blue exclusion test. Therefore the ethidium bromide technique seems to monitor an earlier stage of cell death.

The decrease in the number of dye‐excluding (V) cells during heat exposure occurred in two phases. After an initial decrease a plateau of number of dye‐excluding cells was reached; the duration and level of this plateau depended on the temperature. The plateau was followed by a second phase where the remaining cells ceased to exclude the dye.
The cytodisk: A cytometer based upon a new principle of cell alignment
Authors: le Grooth B. G. , Geerken T. H. , Greve J.
Published: August 2005

A new method is described for one‐dimensional alignment of small particles such as biological cells. A drop of the particle suspension is spread out on a flat disk or plate equipped with V‐shaped grooves such as are present on a gramophone disk. After drying, the particles are located on the bottom of the grooves and are thus aligned in a one‐dimensional array. The new alignment procedure is demonstrated with a suspension of fluorescent polystyrene micropheres (diameter 3.8 μm) and a suspension of the unicellular algae chlorella vulgaris (diameter about 3 μm). It appears that the alignment of cells and spheres is very good.

When using microspheres, more than 95% of the particles in the grooves are located within ±2 μm of the centre line of the groove. Based upon this cell‐alignment principle, a new cytometer, named the cytodisk, is proposed. The proposed system has a number of advantages over the flow cytometer, among which is the unique ability of relocating a previously measured cell for further measurement or visual examination.

A prototype of a cytodisk, developed for initial test measurements, was built in our laboratory. The apparatus, constructed from a record player and ordinary long‐playing records, uses a simple mechanical tracking system and a single optical fiber for fluorescence excitation and detection. With this apparatus it is demonstrated that a cytodisk can indeed perform quite well: A histogram of fluorescing microspheres could be measured with a coefficient of variation of 4.1%. The performance of this prototype is limited by the quality of the mechanical tracking system and the optical system used. It is expected that considerable improvements may be obtained by using a more sophisticated optical detection system such as the tracking system in use in optical disk players.
Preparation of monolayer smears from paraffin‐embedded tissue for image cytometry
Authors: van Driel‐Kulker A. M. J. , Mesker W. E. , van Velzen I. , Tanke H. J. , Feichtinger J. , Ploem J. S.
Published: August 2005

A method is described for the preparation of monolayer smears from paraffin‐embedded tissue suitable for automated image analysis and DNA measurements. The proposed technique uses enzyme treatment and syringing for cell dispersal. Slide preparation is performed by centrifugal cytology. After Feulgen staining the quality of the monolayer smears is sufficiently high to enable visual morphologic evaluation. Automated DNA measurements using the Leyden television analysis system (LEYTAS) show coefficients of variation (CV) of 4.5% for the diploid cell population of the suspended tissue. This is approximately the same as the CV in fresh material from the same tumor. Formalin fixed trout red blood cells are used as reference cells. By applying image cytometry to paraffin‐embedded tissue this method allows retrospective studies of, for instance, the significan of DNA content with regard to the behavior of a tumor.
Differential of light‐scattering detection in an arc‐lamp‐based epi‐illumination flow cytometer
Authors: Steen Harald B. , Lindmo Tore
Published: August 2005

Light‐scattering histograms of blood cell suspensions were recorded for various ranges of scattering angles by means of an arc‐lamp‐based flow cytometer (AL‐FCM). The results were compared with those obtained with a conventional, laser‐based flow cytometer (L‐FCM) with forward scattering (2–20°) and scattering at right angles. Measuring with the AL‐FCM in the angle range upward from 13°, the relative light‐scattering intensities of lymphocytes, monocytes, and granulocytes were essentially independent of scattering angle and closely similar to the values measured as right‐angle scattering in the L‐FCM. With a range of scattering angles upward from 2° the AL‐FCM yielded histograms similar although not identical to that of the forward‐scattering detector in the L‐FCM. Differentiation between live and dead cells in this mode of operation was similar in the two instruments.
Automated identification of subpopulations in flow cytometric list mode data using cluster analysis
Authors: Murphy Robert F.
Published: August 2005

The application of K‐means (ISODATA) cluster analysis to flow cytometric data is described. The results of analyses of flow cytometric data for mixtures of fluorescent microspheres and samples of peripheral blood mononuclear cells are presented. A method for simultaneously displaying list mode data for any number of parameters, which had previously been applied to a continuous set of parameters such as multi‐angle light scattering data, is used to present the results of cluster analysis on physically unrelated parameters; this method allows rapid evaluation of the success of subpopulation identification. The factors that influence automated identification of subpopulations are examined, and methods for determining optimal values for these factors are described.