bluuebunny/crossref_metadata_2025 huggingface dataset:
Preservation of B‐cell‐associated surface antigens by chemical fixation
Authors: Caldwell Charles W.
Published: June 2005

Usual methods of chemical fixation preclude examination of cells with most monoclonal antibodies due to alteration or destruction of the surface antigen itself. A method of chemical stabilization and preservation of human B‐cell‐associated surface antigens is described which facilitates retrospective flow cytometric analysis. This method involves pretreatment of the cells with protease enzyme inhibitors, followed by chemical crosslinking of surface proteins with 2% formalin, and finally blockade of non‐specific reactive groups with excess glycine. Once prepared, the expression of pertinent cellular antigens is stable on the cell surface for as long as 4 years. Such methodology could conceivably be used for preparation of cells for longitudinal quality control of monoclonal antibodies or archival storage of patient specimens for retrospective flow cytometric analysis. © 1994 Wiley‐Liss, Inc.
Detection of terminal transferase in acute myeloid leukemia by flow cytometry
Authors: Paietta Elisabeth , Meenan Bernadette , Heavey Claire , Thomas Dylan
Published: June 2005

The purpose of this study was twofold: (1) to develop an optimized, reliable method for the flow cytometric analysis of the intranuclear DNA polymerase, terminal deoxynucleotidyl transferase (TdT) in acute myeloid leukemia, and (2) to establish the usefulness of a novel, fluorescein‐isothiocyanate conjugated monoclonal anti‐TdT antibody (HT‐6) in double‐fluorescence staining for surface antigens in the characterization of leukemic cells. Inclusion of an aldehyde blocking buffer in the staining protocol reduced background fluorescence sufficiently to allow for the detection of the low‐level fluorescent TdT+ myeloblasts. When admixed to normal peripheral blood mononuclear cells, 0.4–0.5% of HLA‐DR+ or myeloid surface antigen+, TdT+ double‐stained myeloblasts could be reliably detected above background levels. Flow cytometric TdT measurements using the HT‐6 antibody in 55 patients with TdT+ acute lymphocytic or myelocytic leukemia or blast crisis of chronic myelogenous leukemia were equal or superior to the results obtained with a mixture of monoclonal anti‐TdT antibodies (anti‐HTDT‐Mix) and comparable to those obtained by the conventional slide method employing polyclonal rabbit anti‐human TdT antiserum. This flow cytometric TdT determination in combination with surface antigen staining using a novel anti‐TdT monoclonal antibody (HT‐6) allows for the recognition of minimal leukemic blast cells during clinical remission in acute myeloid leukemia. © 1994 Wiley‐Liss, Inc.
Counting, measuring, and mapping in fish‐labelled cells: Sample size considerations and implications for automation
Authors: Carothers Andrew D.
Published: June 2005

Statistical models are used to investigate the need for automation in several potential areas of application of FISH‐la‐belling techniques, including perinatal and tumour cytogenetics, genetic toxicology, and gene mapping. Predictions of the models, based on current estimates of likely error rates for spot‐counting and measuring, suggest that a fully automated system is a realistic prospect for detecting full or high‐level mosaic trisomies and that interactive systems have the potential to reduce substantially the human workload required to detect residual malignant disease or radiation‐induced chromosome aberrations. There appear to be no foreseeable limits to the requirements for speed and accuracy in such systems, since there is effectively no lower limit to the level of relevant biological detail that can be investigated with these techniques. © 1994 Wiley‐Liss, Inc.
Interactive computer‐assisted analysis of chromosome 1 colocalization with nucleoli
Authors: Léger Isabelle , Guillaud Martial , Krief Bruno , Brugal Gérard
Published: June 2005

The applications of DNA cloning and fluorescent in situ hybridization (FISH) techniques have strengthened the hypothesis of an ordered chromatin structure in interphase nuclei, strongly suspected to vary with functional state. The nonrandom distribution of the centromeres and their dynamic rearrangement during the cell cycle have been well documented. A close proximity of specific centromeres to nucleoli has also been reported, but the functional meaning of this association is still unknown. In order to investigate whether the chromosome 1 centromere region to nucleolus association depends on the cell cycle and chromosome status, we combined FISH of probes specific for the 1q12 region with Ki‐67 nucleolar antigen fluorescent immunocytochemical (FICC) detection on the MCF‐7 human breast cancer cell line and on the MRC‐5 normal fibroblastic cell line. Both FISH and FICC signals were interactively localized in a one‐step fluorescent microscopic observation and further analyzed using the Highly Optimized Microscope Environment (HOME) graphics microscope workstation, which provided computerized interactive marking of 1q12 to nucleolus associations (1q12‐nu) at the individual nucleus and nucleolus levels. This study confirms that centromeric regions, other than those adjacent to the major ribosomal cistrons, contribute to the perinucleolar chromatin and demonstrate that, during the cell cycle, the heterochromatic band 1q12 is dynamically rearranged with regard to both the nuclear volume and the nucleoli. A relationship between the association of the chromosome 1 pericentromeric region with nucleoli and the nucleolar transcriptional activity is also strongly suggested. © 1994 Wiley‐Liss, Inc.
Fast kinetic measurements and on‐line dilution by flow injection cytometry
Authors: Lindberg Walter , Scampavia Louis D. , Ruzicka Jaromir , Christian Gary D.
Published: June 2005

An improved flow injection cytometry (FIC) system suitable for fast cellular kinetic measurements and on‐line dilution is described. The instrument allows for measurements from 1.2 s (± 0.05 s) after the initiation of mixing, up to any time thereafter. Crucial factors in determining fast kinetic measurements, such as the displacement volume for sample introduction, rate of transport to cytometric interrogation point, and the mixing speed are evaluated and discussed. By varying the volume aspirated, this instrument can facilitate the dilution of cells and/or reagent over a range of one order of magnitude. The fast kinetic and on‐line dilution capabilities were demonstrated by on‐line staining of DNA in trout erythrocytes with 4′, 6‐diamidino‐2‐phenylindole (DAPI). The utility of the instrument for measurement of enzyme kinetics was illustrated using human lymphocytes, measuring the glutathione S‐transferase (GST) catalyzed reaction between monochlorobimane (MCB) and glutathione (GSH). © 1994 Wiley‐Liss, Inc.
Reversing the effect of formalin on the binding of propidium iodide to DNA
Authors: Overton W. Roy , McCoy J. Philip
Published: June 2005

Formalin, an excellent preservative of cellular morphology, is a commonly used fixative for tissue specimens in hospital pathology laboratories. This preserved material is a potential source of tissue for diagnostic and retrospective research studies on DNA using flow cytometry. Unfortunately, formalin interferes with the binding of propidium iodide (PI) and other fluorescent dyes to DNA, thus altering the measurement of DNA content by flow cytometry or image analysis. This interference has been attributed to the cross‐linking of histones by formalin. Since formalin alters the measurement of DNA content in formalin‐fixed and formalin‐fixed, paraffin‐embedded tissues, this study was designed to explore the use of various physicochemical methods to reverse the effect of the formalin on the binding of PI to DNA. This study demonstrates that resuspending formalin‐fixed cells in PBS and heating them at 75°C for at least 1 h prior to staining with PI restores the staining of the DNA to approximately the same fluorescence intensity as that of fresh tissue. © 1994 Wiley‐Liss, Inc.
Pattern sorting: A computer‐controlled multidimensional sorting method using k‐d trees
Authors: Bigos M. , Parks D. R. , Moore W. A. , Herzenberg L. A. , Herzenberg L. A.
Published: June 2005

Multidimensional binary trees provide a memory efficient and general method for computing sorting decisions in real time for a flow cytometer. Their fundamental advantage over conventional lookup table sorting techniques is that sort criteria in the full N‐dimensional data space which cannot be described by projections onto two‐dimensional parameter planes can be effectively implemented. This becomes particularly relevant when multidimensional analysis methods such as principal components or clustering are employed. We describe a prototype implementation of this method and point out other possible implementations. © 1994 Wiley‐Liss, Inc.
A flow cytometric method using Hoechst 33342 and propidium iodide for simultaneous cell cycle analysis and apoptosis determination in unfixed cells
Authors: Belloc Francis , Dumain Patrice , Boisseau Michel R. , Jalloustre Claudine , Reiffers Josy , Bernard Philippe , Lacombe Francis
Published: June 2005

A flow cytometric method to detect apoptotic cells is described. This method is based on the detection of differences in chromatin condensation with Hoechst 33342 as a probe and the detection of dead cells with propidium iodide as a probe for membrane damage. By this method it was possible to detect, in the same sample and at the same time, intact cells, cells undergoing apoptosis, and dead cells resulting from apoptotic and/or necrotic processes. The method was successfully applied to the detection of apoptotic cells in two human cell models: cultured polymorphonuclear cells and the U937 cell line treated with antitumoral drugs. Staining specificity for apoptotic cells was controlled by cell sorting of the presumed apoptotic population, followed by morphologic examination or DNA analysis of the sorted populations. The usefulness of such a method is discussed in terms of applications in the analysis of heterogeneous clinical samples, populations with low DNA degradation during apoptosis, and cell cycle position of the apoptotic cells. © 1994 Wiley‐Liss, Inc.
The macro flow planktometer: A new device for volume and fluorescence analysis of macro plankton including triggered video imaging in flow
Authors: Hüller Reinhold , Glossner Ewald , Schaub Stefan , Weingärtner Johann , Kachel Volker
Published: June 2005

A newly developed macro flow planktometer is described for measuring plankton organisms with a size of 100–2,000 μm by the flow principle in time‐saving and noncontact fashion. Based on the method of changes in electric resistance (Coulter principle), the device allows determination of the body volume of living organisms in their natural medium (fresh or seawater) at a measuring rate of up to 50 organisms per second. Simultaneous, laser‐excited fluorescence measurement permits quantitative detection of fluorescent substances within the organisms. Controlled by the multiparametric measurement, video images of organisms of particular interest can be taken in the flow chamber using a stroboscopic imaging system. Valuable morphological information on the material under test is thus available in addition to the analytical measurement. Data acquisition and imaging, the total system control, and the fast data evaluation are performed with an IBM‐AT compatible computer using an extensive software package. The easy‐to‐handle desktop unit can be used both in land laboratories and on ships. The modular structure of the system permits any desired combination of individual components to adapt it to various requirements. The operability of this new measuring system is demonstrated by several applications. © 1994 Wiley‐Liss, Inc.
A disposable‐chamber temperature‐regulation system for the study of intracellular calcium levels in single live T cells using fluorescence digital‐imaging microscopy
Authors: Boltz R. C. “Dutch” , Sirotina Anna , Blake Tom , Kath Gary , Uhrig Brian , McKeel John , Quinn Colleen
Published: June 2005

Utilizing flow cytometry, we previously demonstrated that the potassium channel blocker margatoxin (MgTX) inhibits the[Ca2+]i transient involved in T‐cell activation. We wished to extend these studies to single‐cell transients using florescence digital‐imaging microscopy (DIM). However, the most currently available temperature‐regulation chambers reuse part or all of the apparatus and introduce compounds via perfusion. Thus, these apparatuses are not suitable for studies involving compounds that are particularly sticky. We have designed a dual‐temperature regulation system that will maintain Nunc, eight‐well, coverglass‐bottom, disposable chambers, and three disposable addition pipets at 37°C for physiological studies on an inverted digital‐imaging microscope. We have demonstrated that calcium transients of human T lymphocytes can be initiated and monitored reproducibly during the addition of three distinct chemical species. The DIM results correlate with flow cytometry measurements in the number of responding cells and the heterogeneity of the response in both control and MgTX‐inhibited cultures. Additionally, DIM revealed that the [Ca2+]i transient is more rapid than the flow‐cytometric measurement indicated. The correlation between flow cytometry and DIM permits the interpretation of studies on the regulation of T‐cell activation. © 1994 Wiley‐Liss, Inc.
Textural differences between AA and SS blood specimens as detected by image analysis
Authors: Robinson Roy D. , Benjamin Lennette J. , Cosgriff Janice M. , Cox Christopher , Lapets Oleg P. , Rowley Peter T. , Yatco Edward , Wheeless Leon L.
Published: June 2005

High‐resolution image analysis was employed in the analysis of round (discoid) erythrocytes from hematologically normal (AA) individuals, AA individuals with nonspecific anemia, individuals with sickle cell trait (AS), individuals with SC disease (SC), and individuals with sickle cell anemia (SS). The shape feature Form Factor (4πArea/Perimeter2) was used to select round cells and to exclude sickle and other abnormal cells. Textural features extracted from round cells of SS and SC patients were found to differ from those derived from cells of normal andanemic AA individuals. Two textural features, Standard Deviation of Run Length Matrix Counts and Rotation Moment of the Cooccurrence Matrix, discriminated between patient mean values from AA samples and those from SS samples. The ability of textural features to separate round cells into classes based on genotype suggests that high resolution image analysis may be an effective tool in the study and monitoring of sickle cell disease. © 1994 Wiley‐Liss, Inc.
TO‐PRO‐3 iodide: A novel HeNe laser‐excitable DNA stain as an alternative for propidium iodide in multiparameter flow cytometry
Authors: Van Hooijdonk Candida A. E. M. , Glade Conrad P. , Van Erp Piet E. J.
Published: June 2005

A new red emitting fluorophore, TO‐PRO‐3 iodide (TP3), which is best excited by an HeNe laser (633 nm), has been compared with propidium iodide (PI) for measuring relative DNA content. TP3, which has a peak absorbance at 642 nm and emission at 661 nm, has been tested on peripheral blood lymphocytes (PBL) and keratinocytes in a two‐laser system. As an example, we present a three‐color flow cytometric application utilizing TP3 in combination with fluorescein‐isothiocyanate (FITC) and phycoerythrin (PE) conjugated to monoclonal antibodies in this paper.

A subequilibrium concentration of 1 μM TP3, most preferably used in combination with RNase treatment, proved to be a powerful alternative for DNA amount determination. In human‐ and mouse‐Balb/MK‐keratinocyte populations with different S‐phase fractions, PI and TP3 showed a good correlation. Finally, in the triple labelling experiment we clearly demonstrated that TP3 is readily applied to the analysis of binding of two antibodies and relative DNA content simultaneously. © 1994 Wiley‐Liss, Inc.
Highly sensitive, specific, and stable new fluorescent DNA stains for confocal laser microscopy and image processing of normal paraffin sections
Authors: Tekola Paulos , Baak Jan P. A. , Beliën Jeroen A. M. , Brugghe Jane
Published: June 2005

The area, volume, shape, DNA content, and chromatin pattern of nuclei can be important for the diagnosis and prognosis of cancers. Confocal laser scan microscopy can be useful to obtain such information by optical slicing and three‐dimensional (3‐D) reconstruction of nuclei in thick paraffin sections. To retrieve individual quantitative features from the section, highly sensitive, specific, and stable fluorescent stains are required. Two new nuclei acids stains (TOTO‐1 iodide and YOYO‐1 iodide) can detect picogram amounts of nucleic acids in gels. Despite their high sensitivity to detect DNA, they have not been used to stain nuclei in paraffin embedded tissue sections (as routinely applied in surgical cancer pathology). We have developed a technique to stain nuclei in 4% buffered formaldehyde fixed paraffin tissue sections using TOTO‐1 iodide and YOYO‐1 iodide. The technique developed gives bright specific staining of the nuclei (with nearly zero background intensity, much less than with acriflavine). Moreover, TOTO‐1 iodide and YOYO‐1 iodide stains both give fluorescent signals only when they interact with DNA. Thus washing off the excess stain left on the stained specimen is not necessary (washing of excessive stain is necessary with acriflavine). Care has to be taken that the deparaffinizing liquids (xylol etc.) are not polluted with eosin (a frequently used counterstain in surgical pathology specimens), as this gives undesired fluorescence of cytoplasm and connective tissue at the same wavelength as TOTO‐1 iodide and YOYO‐1 iodide. Contrary to acriflavine, bleaching of TOTO‐1 iodide and YOYO‐1 iodide fluorescence is minimal, even after 30 min continuous laser light excitation. Morevover, integrated TOTO‐1 iodide and YOYO‐1 iodide fluorescence shows good linearity with the DNA content of diploid, tetraploid, and octaploid liver nuclei. The sections stained with TOTO‐1 iodide and YOYO‐1 iodide are particularly suitable for two‐dimensional morphometric and cytometric assessments of nuclei in sections, and also for the 3‐D reconstruction and visualization of nuclei by confocal microscopy. The high intensity and stability of the fluorescent image of the nuclei and the low uniform intensity of the background will decrease the work needed in image segmentation of nuclei and help to increase the accuracy of cytometric assessments. © 1994 Wiley‐Liss, Inc.
Simultaeous detection of β‐galactosidase activity and surface antigen expression in viable haematopoietic cells
Authors: Berger Christoph N. , Tan Seong‐Seng , Sturm Karin S.
Published: June 2005

The quantitation of intracellular β‐galactosidase activity has been described for viable cells. By using the fluorogenic substrate fluorescein‐di‐β‐D‐galactopyranoside (FDG)in conjunction with flow cytometry, the proportion of positive cells as well as the level of expression can be determined. In this paper we describe β‐galactosidase expression in lymphoid and myeloid cells from transgenic mice that widely express β‐galactosidase from an inserted lacZ transgene. Both foetal and adult haematopoietic tissues are able to express β‐galactosidase. The intracellular fluorescene reflecting β‐galactosidase activity can be readily combined with fluorescently labelled antibodies against cell surface antigens. Thus, β‐galactosidase can be used as a marker in transplantation experiments to study the development of lymphoid and myeloid precursor cells. © 1994 Wiley‐Liss, Inc.
Reliable method for the simultaneous detection of cytoplasmic and surface CD3ε expression by murine lymphoid cells
Authors: Franek Karl J. , Wolcott R. Michael , Chervenak Robert
Published: June 2005

During their development, T‐cell precursors (pre‐T cells) undergo a variety of changes with respect to their expression of specific surface proteins. Among the most critical of the surface markers acquired by developing T cells is the T‐cell receptor (TCR)/CD3 complex. Prior to the assembly and transport of complete TCR/CD3 multimeric complexes to the plasma membrane, the individual constituent subunits are expressed in the cytoplasm (ER‐Golgi). In order to study the expression of the T‐cell receptor TCR/CD3 complex during pre‐thymic T‐cell differentiation, we have developed a flow cytometric technique for the simultaneous detection of surface (sCD3ϵ) and cytoplasmic CD3ϵ (cCD3ϵ). This technique, which employs fixation in 1% paraformaldehyde and permeabilization with 1% saponin and 0.025% digitonin, features reliable internalization and low nonspecific binding of anti‐CD3ϵ in murine lymphoid cells, as well as tissue culture cell lines. The combination of saponin and digitonin treatment was also compatible with the staining of sCD3 and other lymphocyte surface antigens such as Thy1, CD4, CD8, B220, and IgM. In contrast, permeabilization of cells with the detergents Tween 20 and Triton X‐100 was shown to remove surface‐bound anti‐CD3ϵ. The present technique permitted the detection of discernible sCD3ϵ and cCD3ϵ double and single positive lymphocytes and may prove useful in defining bone marrow‐resident pre‐T cells. © 1994 Wiley‐Liss, Inc.
Flow cytometric quantitation of C‐myc and P53 proteins in bovine papillomavirus type 1‐transformed primary mouse fibroblasts
Authors: Agrawal Reitu S. , Agrawal Yash P. , Mäntyjärvi Rauno A.
Published: June 2005

Bovine papillomavirus type 1 (BPV‐1)‐transformed mouse fibroblast cell lines were analyzed via flow cytometry (FCM) for expression of p53 and c‐myc proteins along with their DNA content. In comparison to the nontransformed control cell line, significantly elevated levels of both the p53 and the c‐myc protein were present in some but not all of the transformed cell lines. Quantitation of p53 and c‐myc proteins in cell lines containing BPV‐1 DNA revealed that the tumorigenic cell lines expressed higher levels of both the p53 (P = 0.0034; Mann‐Whitney U test) and the c‐myc protein (P = 0.0039; Mann‐Whitney U test) as compared to the nontumorigenic cell lines. On average, at least 9,000–10,000 p53 or c‐myc protein molecules per cell were detected in the transformed tumorigenic cell lines. These results show that quantitative FCM can be reliably used to detect very low levels (3,000 molecules per cell) of specific protein, and FCM is a useful tool to study the virus‐induced changes in the levels of nuclear proteins within a cell population and in tumorigenesis. © 1994 Wiley‐Liss, Inc.
A knowlewldge‐based system for the interpretation of flow cytometry data in leukemias and lymphomas
Authors: Diamond Lawrence W. , Nguyen Doyen T. , Andreeff Michael , Maiese Russell L. , Braylan Raul C.
Published: June 2005

Flow cytometry immunophenotyping and analysis of DNA ploidy and proliferative activity have become extremely helpful techniques for diagnosing and subclassifying hematopoietic cell populations in a modern, full‐service hematopathology laboratory. The number of physicians with special training in the interpretation of these studies is limited. A knowledge‐based computer system has been designed to aid in the interpretation of immunophenotyping and DNA flow cytometry results in hematopoietic disease. The system, known as “Professor Fidelio,” is a heuristic classification system that reasons on the basis of defined diagnostic patterns. In this study, Fidelio was tested as a stand‐alone system on 366 specimens from two large tertiary medical centers. Fidelio's interpretation was considered to be appropriate in all cases. In 300 of 366 (82%) specimens, the system's interpretation agreed with the diagnosis of record. Many of the disagreements could be traced to errors in the recording of the original diagnosis and minor differences in diagnostic criteria between Fidelio's knowledge base and the criteria in use at the medical centers. When used in a stand‐alone mode, Fidelio's interpretation was less specific than the diagnosis of record in certain lymphoproliferative disorders that require morphologic information for subclassification. Professor Fidelio is one module in a workstation for the diagnostic hematology laboratory. This workstation is designed for interpretive reporting, education, and database functions for clinical research. Clinical and morphologic information are shared between Fidelio and the other modules for peripheral blood analysis, bone marrow morphology, and lymph node interpretation by means of a relational database. The system will be useful in hospitals that lack individuals specially trained in flow cytometry. The consistency of interpretation from case to case will also be valuable for education and research. © 1994 Wiley‐Liss, Inc.
Optical and mathematical corrections of micropipette measurements of red blood cell geometry during anisotonic perifusion
Authors: Engström K. Gunnar , Meiselman Herbert J.
Published: June 2005

Accurate knowledge of red blood cell (RBC) membrane surface area and cellular volume is critical to understanding the geometric factors that affect RBC deformability. A useful method to determine RBC geometry is via micropipette aspiration in which the cell partially enters the tip of a glass pipette with a diameter of about 2 μm. Measurement of the cell while in the pipette allows calculation of cell geometry, but such measurements are subject to several artifacts. Herein, we analyze the effects of three artifacts on calculated RBC geometry during experimental osmotic manipulation. (1) The pipette internal diameter, as measured optically, requires correction for refractive index and focusing errors; (2) the pipette barrel is tapered rather than cylindrical; (3) the observed RBC outline is affected by a diffraction phenomenon. These optical and mathematical errors were all found to influence the calculated RBC geometry; both absolute values and relative changes during osmotic manipulation were affected. For RBC in isotonic medium, use of the above corrections significantly (p < 0.001) reduced the calculated area by 3.7%, the volume by 7.0%, and the minimum cylindrical diameter by 3.9%. The calculated membrane area dilation for cells exposed to hypotonic media was also significantly reduced; at 200 mOsm, the dilation was 2.8% ± 0.2% without correction vs. 1.5% ± 0.2% when all corrections were employed (p < 0.01). We therefore suggest that micropipette aspiration of RBC requires use of these correction techniques to obtain accurate values for RBC geometry and that such correction methods are of particular importance when RBC of different sizes are compared. © 1994 Wiley‐Liss, Inc.
Display and correction of flow cytometry time‐dependent fluorescence changes
Authors: Kusuda Leo , Melamed M. R.
Published: June 2005

The resolution of measurements of cellular attributes by flow cytometry, based on differences in labeling by fluorescent markers, may be affected by variations in staining and sample stream flow that can occur during the course of data acquisition. We describe a method of display and correction of time‐dependent changes by looking at the list mode data in chronological order. © 1994 Wiley‐Liss, Inc.
Utilization of commercial antisera and flow cytometry in HLA‐B27 typing
Authors: Orr Ken , Thomson Glen T. D. , Alfa Michelle
Published: June 2005

The study of large groups of individuals (from reactive arthritis epidemics, for example) for epidemiologic and genetic data has been hampered by the techniques by which HLA typing is performed. Microlympho‐cytotoxicity is costly not only in materials but also in the time required to perform a given assay and thus does not lend itself to large scale testing. To study such epidemics, in the best way, one must be able to respond as soon as possible after the epidemic is recognized and on a large scale.

We thus examined HLA‐B27 typing by flow cytometry and commercially available monoclonal antibodies. Two commercially available monoclonal HLA‐B27 antibodies were compared. We conclude that the utilization of monospecific HLA‐B27 antibodies and flow cytometry allows for the determination of HLA‐B27 and HLA‐B7 phenotype of a large number of individuals over a short period of time, thereby making the study of a large group both economical and feasible. © 1994 Wiley‐Liss, Inc.
Effects of whole blood lysis and fixation on the infectivity of human T‐lymphotropic virus type 1 (HTLV‐I)
Authors: Ericson Julie G. , Trevino Alex V. , Toedter Gary P. , Mathes Lawrence E. , Newbound Garret C. , Lairmore Michael D.
Published: June 2005

Whole blood lysis and fixation methods for flow cytometric (FCM) analysis were tested for their ability to reduce the infectivity of human T‐lymphotropic virus type 1 (HTLV‐I). Our goals were to: (1) determine the effects of 1.0 and 2.0% paraformaldehyde (PF) fixation on HTLV‐I infected cell lines lines and (2) assess the infectivity of blood samples containing HTLV‐I‐infected cells following processing with 5 commercially available products (Immuno‐lyse, ImmunoPrep/Q‐Prep, FACS lysis solution, GenTrak lyse and fix reagent, and Ortho‐mune lysing reagent) compared to ammonium chloride lysis with either 0.1 or 1.0% PF fixation. Infectivity was determined by monitoring HTLV‐I p24 antigen production in cocultures of treated leukocytes with uninfected peripheral blood mononuclear cells (PBMC). Each method effectively reduced the viability of treated leukocytes. Commercial lysis/fixation methods significantly reduced HTLV‐I infectivity compared to prepared ammonium chloride/PF‐based methods. For all preparations, increasing the time of fixation (e.g., 60 min) effectively reduced viral infectivity. Taken together, these data suggest that commercially available fixatives greatly reduce, but do not eliminate the risk of HTLV‐I infection during processing of viral‐infected cells for FCM analysis. © 1994 Wiley‐Liss, Inc.
Computing the central location of immunofluorescence distributions: Logarithmic data transformations are not always appropriate
Authors: Coder David M. , Redelman Doug , Vogt Robert F.
Published: June 2005

The idea of the “average” intensity of immunofluorescence data is often poorly defined, with such terms as average, mean, and peak used interchangeably. In addition, the common use of logarthmic amplifiers with immunofluorescence data further complicates the problem. Log amplifiers permit the display of a wider range of fluorescence intensities. At the same time, they effect a log transformation of the data. This transformation decreases the variance resulting in narrower fluorescence distributions, which are assumed to approximate normal distributions. When the log transform is used, the distribution mean is the geometric mean of the untransformed data, which is computed simply as the mean of the channel values. This mean value serves as a simple indicator of the population center. Despite the prevalence of log transformations in flow cytometry, this transformation may not yield normally distributed immunofluorescence data, whereas the square root or other fractional power transformations can yield normal distributions. © 1994 Wiley‐Liss, Inc.
Low level CD20 expression on T cell malignancies
Authors: Warzynski Michael J. , Graham David M. , Axtell Richard A. , Zakem Michael H. , Rotman Richard K.
Published: June 2005

Although initially thought to be a B lineage restricted antigen, low “density” or antibody binding capacity (ABC) CD20 has recently been detected on subset(s) of normal T lymphocytes (Hultin et al.: Cytometry 14:196–204, 1993). We report low ABC CD20 expression in three (two children, one adult) cases of T‐acute lymphoblastic leukemia (T‐ALL). CD20 and other pertinent antigens were detected using a direct dual color method with a Becton Dickinson FACScan® flow cytometer and Simulset® software. Only one cell population based on light scatter was noted in each case that immunophenotypically represented almost a pure population of malignant cells expressing T lymphocyte antigens (for example, CD7 98%, 92%, and 100%, respectively). A total of 95%, 87%, and 79% of the cells from the three cases expressed CD20 with an unusual low ABC compared to the customary “bright” CD20 expression on normal B lymphocytes. Other B lymphocyte associated antigens, such as CD19, CD22, Dr, and immunoglobulin light chains, were negative. Eleven other T lymphocytic malignancies from 1991 to 1993 were CD20 negative, including three other case of T‐ALL (one adult and two children). One unusual case of intestinal small lymphocytic non‐Hodgkin's lymphoma with a natural killer/T lymphocytic immunophenotype not described in this report appeared to be CD20“dim”+. Low ABC CD20 expression by T lymphocytic malignancies may provide a more unique immunophenotypic “fingerprint” to help support the diagnosis of T cell neoplasia vs. normal/reactive T cells (for example, low ABC CD20 cells represent only 2.4 ± 1.5% of normal peripheral blood lymphocytes). This characteristic might also facilitate monitoring patients for residual or recurrent disease. The prognostic significance of CD20 co‐expression is uncertain and must await further studies. © 1994 Wiley‐Liss, Inc.
Automated HLA‐B27 testing using the FACSPrep/FACScan system
Authors: Reynolds Wendy M. , Evans Philip R. , Lane Andrew C. , Howell W. Martin , Wilson Peter J. , Wong Raymond , Smith John L.
Published: June 2005

Tissue typing can help in the diagnosis of the seronegative arthropathy ankylosing spondylitis. Using an automatic sample preparation system and flow cytometry (FACSPrep/FACScan) we have developed a test for HLA‐B27 screening using whole blood which is both rapid, reproducible, and permits simultaneous screening of large numbers of samples. We have used both indirect (248 cases) and direct (126 cases) monoclonal antibody staining techniques. Results were assessed using median channel shift (CS) from the negative control and relative fluorescence intensity. There is known cross‐reactivity between HLA‐B27 and HLA‐B7 with the monoclonal antibody (MoAb) HLA‐ABC‐m3. Using this antibody and indirect staining, HLA‐B7 samples had a significantly lower CS value than HLA‐B27 samples. In 60% of these cases there was a clear distinction between HLA‐B27 and HLA‐B7. All HLA‐B27 and HLA‐B7 negative samples had a CS of 0 (P < 0.001). Using direct dual staining with CD3 and HLA‐B27, all HLA‐B7 negative samples had a low CS value (15 maximum), HLA‐B7 samples had an intermediate CS value (20–85), and HLA‐B27 samples had the highest CS values (70 upward). This system permits large scale rapid negative screening of samples with the elimination of over 60% as negatives (CS < 15). Limitations as a definitive test for HLA‐B27 are due to MoAb cross‐reactivity with HLA‐B7 which necessitates other confirmatory techniques. The substitution of the HLA‐ABC‐m3 with the recently available HLA‐B27 specific MoAb FD705 would substantially increase the value of this technique for routine HLA‐B27 typing. © 1994 Wiley‐Liss, Inc.
Quality control in flow cytometry for diagnostic pathology: II. A conspectus of reference ranges for lymphocyte immunophenotyping
Authors: McCoy J. Philip , Overton W. Roy
Published: June 2005

Immunophenotyping, as many other clinical assays, is interpreted only in the context of reference values obtained from healthy control individuals. While the use of these reference values, or ranges, has been commonplace in the clinical flow cytometry laboratory for well over a decade, there has been little consensus in standardizing how these values should be obtained, analyzed, or expressed. This report reviews the variables to be considered in establishing reference ranges and statistical methods which can be used. Additionally, examples are given of previously published reference ranges for a variety of specimens often submitted for immunophenotyping. © 1994 Wiley‐Liss, Inc.
Semiautomated DNA probe mapping using digital imaging microscopy: I. System development
Authors: Mascio Laura N. , Verbeek Piet W. , Sudar Damir , Kuo Wen‐Lin , Gray Joe W.
Published: June 2005

Algorithms have been developed to help automate the mapping of DNA sequences along metaphase chromosomes using fluorescence in situ hybridization (FISH). Custom algorithm computationally define chromosome boundaries and compute chromosomal medial axes. A dynamic regional thresholding (DRT) algorithm is described that allows reliable detection of hybridization domains, even when they differ substantially in size and intensity. Chromosomal locations are calculated by determining the fractional location of each hybridization probe along the medial axis of a metaphase chromosome relative to the short arm (FLpter). These algorithms were tested on simulated data and by analysis of the location of probes that had been previously mapped by other techniques. These algorithms allow probes to be mapped rapidly along human chromosomes with a precision of 2–3 Mb. © 1995 Wiley‐Liss, Inc.

This article is a US Government work and, as such, is in the public domain in the United States of America.
Prognostic significance of DNA aneuploidy in diffuse malignant mesothelioma
Authors: Isobe Hiroshi , Sridhar Kasi S. , Doria Raul , Cohen Felicia , Raub William A. , Saldana Mario , Krishan Awtar
Published: June 2005

DNA ploidy of pepsin digested preparations of 48 paraffin‐embedded specimens from 19 patients with histologically confirmed malignant mesothelioma was determined by laser flow cytometry. Eight of the 19 tumors (42%) were diploid and 11 (58%) were aneuploid. Of the aneuploid tumors, only one showed multiploidy. The median survival time of the patients with diploid tumors was 19, 16, and 14 months from the onset of symptoms, diagnosis, and treatment, respectively. The median survival in patients with aneuploid tumors was 8, 7, and 7 months from the onset of first symptoms, diagnosis, and treatment. Thus, patients with diploid tumors lived longer than patients with aneuploid tumors. These results suggest that DNA ploidy analysis may be of prognostic value in malignant mesothelioma. © 1995 Wiley‐Liss, Inc.
Is reduced accumulation of Hoechst 33342 in multidrug resistant cells related to P‐glycoprotein activity?
Authors: Lahmy Sylvie , Viallet Pierre , Salmon Jean Marie
Published: June 2005

Although bisbenzimidazole‐DNA interactions have been studied in solution, little information has been available in living cells. The reduced accumulation of the nuclear dye Hoechst 33342 (H342) in cells with multidrug resistant (MDR) phenotype suggested its possible use in a functional test for detection of these cells. We performed experiments to elucidate the mechanisms involved in the H342‐exclusion from resistant cells. As contradictory results have been reported in literature, we compared the entire fluorescence spectra of H342 in solution and in intact living cells under different experimental conditions. The study was performed by fluorescence image cytometry. This technique allow accurate quantification of the amount of H342 bound to DNA in living cells. The dye uptake was followed in sensitive and resistant cells, a lymphoblastoid cell line, CCRF‐CEM, and its resistant variant selected with vinblastine CEM/VLB100 under conditions that could modulate H342‐cell binding. Competition experiments with sodium azide, verapamil, and vinblastine indicated that resistant cells did not differ in the number of possible binding sites for H342. The obtained results ruled out the possibility of discriminating cells on the basis of a spectral shift. Two modes of binding, differing in their affinity for the dye, seem to co‐exist in intact cells. Although it clearly appeared that the P‐glycoprotein expressed in MDR cells was mainly responsible for the H342‐exclusion, other mechanisms might also be involved. © 1995 Wiley‐Liss, Inc.
Time‐related effects of enzymatic disaggregation on model human lung carcinomas
Authors: Howard Randy B. , Shroyer Kenneth R. , Marcell Tere , Swanson Lucille E. , Pagura Marco E. , Mulvin David W. , Cowen Michael E. , Johnston Michael R.
Published: June 2005

Enzyme cocktails used to prepare tumor cell suspensions may influence yield, viability, and cytology, thus time‐related cocktail effects on model human lung carcinomas were examined. A549, NCI‐H125, and NCI‐H460 carcinomas were completely disaggregated at 25°C over 2 h with either (mg/ml) collagenase/DNAse (C/D, 1/0.1), collagenase/hyaluronidase/DNAse (C/H/D, 1/0.1/0.1), or polymyxa protease/DNAse (PP/D, 3/0.1). Trypan blue viabilities, total yields, viable yields, and flow cytometric percent tumor cells (TC) were measured every 20–30 min (n = 4–7 per tumor type). The final percentages of TC, mononuclear cells (MN), polymorphonuclear cells (PMN), lymphocytes, and necrotic cells were determined by cytology (n = 4–5 per tumor type). The time‐dependent measurements showed that (1) disaggregation was progressive and complete with all cocktails; (2) viability was stable or increasing with all cocktails; (3) percent TC was stable for all cocktails, but lower for PP/D than C/D in final suspensions; and (4) PP/D gave lower final total yields, higher final viabilities, but the same final viable yields as the C cocktails, suggesting selective elimination of dead cells by PP/D. Final cytology measurements showed that PP/D gave a lower percent MN and a higher percent PMN than C cocktails. Cocktail effects may importantly influence cell suspension properties. © 1995 Wiley‐Liss, Inc.
Luminescence quenching by long range electron transfer: A probe of protein clustering and conformation at the cell surface
Authors: Matko Janos , Jenei Attila , Wei Taiyin , Edidin Michael
Published: June 2005

Quenching of luminescence from fluorescent and phosphorescent probes by nitroxide spin labels with a long range electron transfer (LRET) mechanism (44,45) has been tested as a tool to monitor association/clustering and conformational changes of cell surface proteins. The membrane proteins were labeled with monoclonal antibodies or Fab fragments conjugated with luminescent probes or water‐soluble nitroxide spin labels. The method was tested as a probe of 3 different aspects of protein‐protein association involving class I MHC molecules: (1) interaction between the heavy and light chains of the MHC molecules, (2) clustering, self‐association of MHC molecules, (3) proximity of MHC molecules to transferrin receptors of fibroblasts or surface immunoglobulin molecules of B lymphoblasts. The extent of quenching upon increasing the fractional density of the quencher was sensitive for protein association in accordance with earlier immunoprecipitation and flow cytometric Förster‐type energy transfer (FCET) data obtained on the same cells. These data suggest that the LRET quenching can be used as intra‐ or intermolecular ruler in a 0.5–2.5 nm distance range. This approach is simpler (measurements only on donor side) and faster than many other experimental techniques in screening physical association or conformational changes of membrane proteins by means of spectrofluorimetry, flow cytometry, or microscope based imaging. © 1995 Wiley‐Liss, Inc.
Kinetic analysis of glutathione in anchored cells with monochlorobimane
Authors: Barhoumi Rola , Bailey R. Hartford , Burghardt Robert C.
Published: June 2005

A method for the measurement of intracellular glutathione content and glutathione S‐transferase activity with monochlorobimane in adherent cells is described. The method involves the kinetic analysis of monochlorobimane conjugation to glutathione over a relatively short period of time. This permits extrapolation over time for determination of equilibrium fluorescence intensity (relative glutathione level) from scan intensity data that follows first‐order kinetics, minimizing problems commonly associated with the use of monochlorobimane. By using measured fluorescence intensity values from glutathione standards, a suspension calibration curve was generated and, subsequently, was used to determine the photomultiplier tube saturation rate. A theoretical intracellular calibration curve was then generated to quantify glutathione content in cells. This method was also applied to study the changes in glutathione in a variety of rodent and human cell lines and in selected cocultures of cells exhibiting similar or different glutathione levels. Comparison of the glutathione levels obtained with monochlorobimane and a standard colorimetric method (GSH‐400) indicated good correlation between the two methods. These studies support the use of laser cytometry for measuring intracellular glutathione with monochlorobimane as well as changes in glutathione occurring in cells that establish physical contacts with other cells. Laser cytometric analysis of glutathione in anchored cells also provides opportunities to monitor individual cellular responses to a variety of experimental manipulations, such as responses to various toxic insults or the protective effects of gap junction‐mediated intercellular communication. © 1995 Wiley‐Liss, Inc.
DNA and RNA content analysis by flow cytometry in the pathobiologic assessment of bone tumors
Authors: El‐Naggar Adel K. , Hurr Kenneth , Tu Z. Nora , Teague Kim , Raymond Kevin A. , Ayala Alberto G. , Murray John
Published: June 2005

Studies of simultaneous DNA and RNA contents by flow cytometry in hematologic and some solid neoplasms have been shown to provide information that may be useful in the pathobiological evaluation of these neoplasms. We contend that similar analysis may be equally valuable in assessing bone tumors. Our data revealed significant statistical differences in DNA ploidy and proliferative fraction between benign and malignant bone neoplasms. Benign tumors manifested predominantly DNA diploidy and low proliferative activity, whereas the majority of malignant tumors were DNA aneuploid and showed high proliferation rate. No significant difference in the RNA content between different histopathologic categories was found. We observed, however, a distinct and consistently high RNA content pattern in giant cell tumors, aneurysmal bone cysts, and chondroblastomas that may be useful in their differential diagnosis. Analysis of different prognostic factors in malignant tumors indicated that histologic grade and DNA content are a significant prognostic factors. Further analysis of malignant tumors showed that a correlation between the proliferative activity and the clinical outcome in the low grade category and between RNA content and patients' survival in osteosarcomas. Our study also showed that preoperative treatment significantly impacted on the extent of the proliferative fraction in malignant tumors. We conclude that DNA/RNA analysis of bone tumor may assist in: (1) the differential diagnosis of certain bone tumors, (2) evaluation of treatment response, and (3) the biological assessment of osteosarcomas. © 1995 Wiley‐Liss, Inc.
Flow cytometric measurement of NK cell immunoconjugates by pulse width processing
Authors: Manogaran P. S. , Kausalya S. , Pande Gopal
Published: June 2005

Pulse width analysis, in flow cytometry, has been widely used for optimal cell size resolution in cell kinetics analysis. Pulses, generated by scattered light or fluorescence of cells, are electronically analyzed for their height and width. The information generated from these two properties of the pulses is utilized to distinguish signals from single cells vs. signals from cell clumps or aggregates. Pulse width, unlike pulse height, is more sensitive to differences in cell diameter, and therefore can discriminate very small differences in it, which pulse height cannot. We have exploited this property of pulse widths to measure immunoconjugates between NK cells and their targets. Discrimination of the free target cells from the conjugated ones is possible by the pulse widths of only light scatter signals, both forward and/or orthogonal. This resolution was not obtained if pulse height of the same signals was visualized. Using this resolution it was possible to distinguish single cells from the aggregates between target and effector cells. We propose that this is a better method for distinguishing conjugates than the method in which prior vital staining of cells is used. © 1995 Wiley‐Liss, Inc.