bluuebunny/crossref_metadata_2025 huggingface dataset:
Rapid fluorescence in situ hybridization with repetitive DNA probes: Quantification by digital image analysis
Authors: Celeda Dino , Aldinger Klaus , Haar Frank‐Martin , Hausmann Michael , Durm Markus , Ludwig Horst , Cremer Christoph
Published: June 2005

Fluorescence in situ hybridization (FISH) has become an important tool not only in cytogenetic research but also in routine clinical chromosome diagnostics. Here, results of a quantification of fluorescence signals after in situ hybridization with repetitive DNA probes are reported using a non‐enzymatic hybridization technique working with a buffer system not containing any formamide or equivalent chemical denaturing agents. Following simultaneous denaturation of both cells and DNA probes, the renaturation time was reduced to less than 30 min. For one of the DNA probes reasonable FISH‐signals were even achieved after about 30 s renaturation time. In addition, the number of washing steps was reduced drastically. As a model system, two repetitive DNA probes (pUC 1.77, D15Z1) were hybridized to human metaphase spreads and interphase nuclei obtained from peripheral blood lymphocytes. The probes were labelled with digoxigenin and detected by FITC‐antidigoxigenin. The hybridization time was reduced step by step and the resulting fluorescence signals were examined systematically. For comparison the pUC 1.77 probe was also hybridized according to a FISH protocol containing 50% formamide. By renaturation for 2 h and overnight two FISH signals per nucleus were obtained. Using shorter renaturation times, no detectable FISH signals were observed. Quantification of the FISH signals was performed using a fluorescence microscope equipped with a cooled colour charge coupled device (CCD) camera. Image analysis was made interactively using a commercially available software package running on a PC (80486). For the pUC 1.77 probe the major binding sites (presumptive chromosomes 1) were clearly distinguished from the minor binding sites by means of the integrated fluorescence intensity. For the two (pUC 1.77) or four (D15Z1) brightest spots on the metaphase spreads and in the interphase nuclei hybridized without formamide, integrated fluorescence intensity distributions were measured for different renaturation times (0.5, 15, 30 min). The intra‐nuclear variation in the intensity of the two brightest in situ hybridization spots appeared to be slightly higher (CV between 16 and 32%) than the corresponding variation on the metaphase spreads (CV between 10 and 19%). For the D15Z1 probe FISH signals were detected after hybridization without formamide and 15 min and 30 min renaturation. Always four bright spots were visible and tentatively assigned on the metaphase spreads (presumptive chromosome 15 and 9). The intensity variation of each pair of homologues in a metaphase spread showed a CV of 14 or 15%, respectively, for the presumptive chromosome 15, and 8 or 9%, respectively, for the presumptive chromosome 9. © 1994 Wiley‐Liss, Inc.
Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique
Authors: Deka Chiranjit , Sklar Larry A. , Steinkamp John A.
Published: June 2005

We have developed a method for fluorescence lifetime measurements in a flow cytometer based upon the amplitude demodulation of the fluorescence signals using digital data acquisition techniques. Amplitude demodulation is one of the two methods by which excited state life‐times may be investigated in the frequency domain. The other method involves the phase‐shift measurements. In frequency‐domain measurement techniques, the amplitude‐demodulation and phase‐shift data serve mutually complementary roles to enhance the analytical capabilities of the measurements. The purpose of having amplitude demodulation measurement capability is to obtain information that supplements, rather than replaces, that obtained by the phase‐shift method alone. Application of amplitude demodulation measurements has been widely explored in static, cuvette‐based, frequency domain systems. However, due to time dependence of the amplitude of the modulated fluorescence signal in a flow cytometer, the amplitude demodulation measurements in flow turns out to be more complicated than similar measurements in a static system. The goal of the present work is to explore the problems involved in amplitude demodulation measurements in flow (using digital method), through detailed theoretical modeling and use the model to develop a practical method that can be incorporated into a flow cytometer to measure amplitude modulation lifetimes. We experimentally verify the amplitude demodulation measurement capability of this method using fluorescent micro‐spheres. The experimental measurements show good agreement with static frequency‐domain measurements on microspheres in bulk suspensions. © 1994 Wiley‐Liss, Inc.
Improved sensitivity in flow cytometric intracellular ionized calcium measurement using fluo‐3/Fura Red fluorescence ratios
Authors: Novak Erik J. , Rabinovitch Peter S.
Published: June 2005

Measurment of changes in intracellular ionized calcium concentrations ([Ca2+]i) has proved to be of wide use in the study of cellular responses to activating stimuli. The fluorescent dye Indo‐1 has successfully been used in flow cytometry for this purpose, and when used as a ratiometric indicator it provides optimum sensitivity and accuracy. Unfortunately, this dye requires ultraviolet (UV) excitation which is often not available. We show here that similar results can be obtained using a ratio of green to red fluorescence from the simultaneous loading of the dyes Fura Red and fluo‐3. Both Fura Red and fluo‐3 are excited using the commonly available blue 488 nm laser line. With appropriate concentrations of the two dyes, the magnitude of response with the fluo‐3/Fura Red ratio is greater than that achieved with indo‐1, while the intercellular variation in measurement is similar to that seen with indo‐1. Analyses can be simultaneously combined with immunofluorescent detection of PE‐labeled antibodies to enable [Ca2+]i measurement within cell subsets. © 1994 Wiley‐Liss, Inc.
Evaluation of a method for quantitative immunohistochemical analysis of cisplatin‐DNA adducts in tissues from nude mice
Authors: Johnsson Anders , Olsson Christina , Anderson Harald , Cavallin‐Ståhl Eva
Published: June 2005

The reproducibility of an immunohistochemical method for visualization and quantitation of the cytotoxic drug cisplatin (CDDP) in its active position, bound to nuclear DNA, was investigated in tissues from CDDP‐treated nude mice. Kidney, liver, and tumor sections were stained with a PAP technique using an antiserum elicited against CDDP‐DNA adducts. The resulting brown nuclear precipitate was quantitated using a CAS 200 image analyzer. The variance components of the errors in the staining procedure and in the image analysis were estimated. The method was found to be feasible for comparisons between slides of the same type of tissue, stained in the same batch, and measured by one observer on one occasion. The method should be a valuable tool for studies of CDDP pharmacodynamics, sensitivity prediction, and the effects of interactions between CDDP and other drugs and chemomodulators. © 1994 Wiley‐Liss, Inc.
Fully automatic chromosome segmentation
Authors: Ji Liang
Published: June 2005

This paper is concerned with automatic segmentation of high resolution digitized metaphases. This includes automatic detection and rejection of interphase nuclei, stain debris, and other “noise”; automatic detection and segmentation of touching and overlapping chromosome clusters; and automatic rejection of cells which are evaluated as being incomplete, or incorrectly segmented, or where the cell is otherwise unsuitable for further analysis.

In this paper, a rule‐based approach is described which treats the cell as a whole rather than as a series of individual chromosomes or clusters. The rules adapt classification and segmentation parameters for each cell. Initially, different sets of parameters are chosen according to the staining method of the cells, and the goal of the segmentation. A chromosome number predictor is used to guide the adaptation of the parameters and to estimate the performance. The adaptation is iterative, and the self‐adjustment will stop when either a satisfactory result is achieved or if the cell is rejected. The method was implemented on both a Sun workstation and a Cytoscan, a commercial machine for chromosome analysis. Seven hundred and thirteen cells from real data have been tested. A success rate of 90–95% has been achieved. The procedure has been implemented in an automatic aberration scoring system for routine use. © 1994 Wiley‐Liss, Inc.
Comparative flow cytometric analysis of Ki‐67 and proliferating cell nuclear antigen (PCNA) in solid neoplasms
Authors: Steck Kim , El‐Naggar Adel K.
Published: June 2005

The proliferation associated antigens, Ki‐67 and proliferating‐cell nuclear antigen (PCNA), have been widely used in studies assessing the growth fraction in human malignancies. It remains unclear, however, whether these markers yield similar assessments of proliferative activity in any given neoplasm. In this study, we compared Ki‐67 and PCNA expression in 93 malignant solid neoplasms using bivarite flow cytometric analysis of these antigens and DNA content. The growth fractions measured by Ki‐67 and PCNA were compared and correlated with acridine orange (AO) analysis and tumor grade. Our results indicate a significant difference between Ki‐67 and PCNA values in neoplasms of low and intermediate grade (P = 0.002); Ki‐67 values were significantly lower than those obtained by PCNA in this group. No statistical difference between Ki‐67 and PCNA values was found in high grade neoplasms (P = 0.38). Analysis of different cell cycle compartments indicates that the observed difference in the positivity of these markers was due to their differential expression in the G0/1 segment of the cell cycle. We conclude that Ki‐67 may better reflect the proliferative activity in solid neoplasms than does PCNA. © 1994 Wiley‐Liss, Inc.
Development of a method for measuring cell number: Application to CNS primary neuronal cultures
Authors: Volonté Cinzia , Ciotti Maria Teresa , Battistini Luca
Published: June 2005

In the present work we further develop a method for counting cell number that is totally independent from the permeability or uptake conditions of the cells, from the state of activation of intracellular enzymes, and from cellular metabolism. We provide a visual characterization of the method and show that it is highly suitable for cells not growing as monolayers as well as for cultures containing numberous aggregates. We also extend the applicability of this method to CNS primary neuronal cultures and show its direct comparison with alternative means for cellular quantification. The technique is fast, does not require tedious procedures or long washes, and offers advantages such as a high sensitivity and no background. © 1994 Wiley‐Liss, Inc.
Evaluation of a flow cytometric fluorescence quenching assay of phagocytosis of sensitized sheep erythrocytes by polymorphonuclear leukocytes
Authors: Van Amersfoort Edwin S. , Van Strijp Jos A. G.
Published: June 2005

A number of reports have been published describing phagocytosis assays for flow cytometric analysis. In some of these, the fluorescence quenching technique has been used to discriminate between adherent and ingested particles. In this report, we have evaluated the efficacy of a quantitative fluorescence quenching technique with crystal violet and trypan blue for application in a phagocytosis assay with polymorphonuclear leukocytes and sensitized sheep red blood cells. We set the requirements to a high quenching efficiency of the fluorescence of extracellularly bound particles and no intracellular quenching. The later was determined using polymorphonuclear leukocytes stained with the fluorescent nuclear dye hydroethidine. We observed that both trypan blue and crystal violet efficiently quench the fluorescence of PKH26 (a red fluorescent membrane‐associated dye) erythrocytes but that only crystal violet quenches intracellular fluorescence. In testing trypan blue and crystal violet from different manufacturers, there was no real difference between different brands of crystal violet, but only the trypan blue from Merck turned out to be an efficient quencher, whereas the other brands of trypan blue showed low quenching efficiency. Trypan blue at a concentration of 25–50 μg/ml proved to be a good quencher of the fluorescent erythrocytes and exerted minimal side effects: over 90% quenching of the erythrocytes, no intracellular quenching, moderate increase in autofluorescence of the polymorphonuclear leukocytes, and no cell loss. © 1994 Wiley‐Liss, Inc.
Detection of bromodeoxyuridine incorporation by alteration of the fluorescence emission from nucleic acid binding dyes using only an argon ion laser
Authors: Frey Tom
Published: June 2005

A method was developed that uses paired DNA dyes to detect the incorporation of bromodeoxyuridine (BrdUrd) into cellular DNA and requires only 488 nm excitation. The flurescence of thiazole blue, TO‐PRO‐3 and LDS‐751 was found to be enhanced by the presence of Brd Urd in DNA. Pairing LDS‐751, thiazole blue, or TO‐PRO‐3 with propidium iodide (PI) for flow cytometry allowed the differentiation of cells containing BrdUrd from BrdUrd unlabeled cells. LDS‐751 can be excited directly at 488 nm, and TO‐PRO‐3 or thiazole blue can be excited indirectly by resonance energy transfer from PI. The enhancement of fluorescence from these dyes is correlated with a decrease in PI fluorescence, suggesting an increased energy transfer from PI to the red emitting dye. Flurescence from other dyes, including thiazole orange, TO‐PRO‐1, rhodamine 800, oxazine 750, and 7‐aminoactinomycin D, was not altered by the presence of BrdUrd in DNA. Results also were obtained showing that BrdUrd detection using PI and TO‐PRO‐3 is compatible with immunofluorescence staining with FITC‐labeled antibodies. © 1994 Wiley‐Liss, Inc.
Carcinoma in situ of the testis detected by DNA flow cytometry of testicular fine‐needle aspirates
Authors: Hittmair Anton , Rogtsch Hermann , Feichtinger Hans , Hobisch Alfred , Mikuz Gregor
Published: June 2005

Testicular carcinoma in situ (CIS) is usually diagnosed histologically on surgical biopsies. The present study was performed to test an alternative approch—DNA flow cytometry (FCM) of testicular fine‐needle aspirates (FNA)—for the detection of this lesion. FNAs from 18 cases of testicular germ cell tumors (TGCT) and tumor adjacent parenchyma were analysed by DNA FCM. DNA histograms of all cell nuclei and histograms representing selectively the hyperdiploid compatment were analysed. The presence and extention of CIS were determined by histology and immunohistochemistry. In 16 to 18 cases, CIS was histologically present, whereas aneuploid peaks were detected in only 11 cases in DNA histograms of all cell nuclei. In the analysis of the histograms of the hyperdiploid region, 4 additional cases of CIS could be identified increasing the sensitivity to 15/16 cases (93.8%). In all but one case, the DNA indices of CIS and invasive tumors were identical. The study demonstrates that DNA FCM of testicular FNAs using the described data acquisition and analysis could also be applicable for detection of CIS in a clinical situation. © 1994 Wiley‐Liss, Inc.
An optimized method for routine HLA‐B27 screening using flow cytometry
Authors: Hulstaert F. , Albrecht J. , Hannet I. , Lancaster P. , Buchner L. , Kunz J. , Falkenrodt A. , Tongio M. , de Keyser F. , Veys E. M. , Noens L. , Mir N. , Costello C. , Becker R. , Strauss K.
Published: June 2005

Flow cytometry and monoclonal antibodies are promising tools for HLA‐antigen detection. Previous approaches have been hampered by the lack of a carefully standardized system for calibration and sample analysis. A new system for HLA‐B27 screening was developed using a FACScan flow cytometer, software for automated calibration and analysis, calibration beads, and the anti‐HLA‐B27‐FITC/anti‐Leu4‐PE (CD3) monoclonal antibodies. The median fluorescence channel result for the HLA‐B27‐FITC signal of CD3+ T lymphocytes is compared to a decision marker. Values lower than this threshold are read as HLA‐B27 negative and those above are recommended for retesting with the classic microcytotoxicity assay on the presumption of HLA‐B27 positivity. The anti‐HLA‐B27 antibody reacts with all six HLA‐B27 subtypes and shows a weaker binding to HLA‐B7. The screening test results were compared with those from the microcytotoxicity assay for HLA‐typing in studies involving several European centers. The observed sensitivity was 100% (95% Cl: 98.6–100) and the specificity was 97.4% (95% Cl: 96.4–98.3). Other performance studies verified the reproducibility and reliability of results obtained with the screening system. © 1994 Wiley‐Liss, Inc.
Two color flow cytometric analysis of concomitant acute myeloid leukemia and chronic lymphocytic leukemia
Authors: Tamul Karen R. , Meyers Dawn C. , Bentley Stuart A. , Folds James D.
Published: June 2005

During evaluation for macrocytic anemia and thrombocytopenia, a 74‐year‐old female was found to have a leukocytosis with two apparent populations of malignant cells identified in her peripheral blood smear and bone marrow aspirate. Morphologic characteristics of the two cell types were unusual, and cytochemical assays yielded ambiguous results. Two‐color flow cytometric analysis demonstrated two distinct cell populations with immunophenotyping patterns consistent with chronic lymphocytic leukemia (CD5 + /CD20 +) and acute myelocytic leukemia (CD33 + /CD34 +), detected concurrently. The concomitant appearance of CLL and AML has been reported only rarely. The use of two‐color flow cytometry to differentiate the populations demonstrates the utility of this technology in resolving unusual hematological malignancies. © 1994 Wiley‐Liss, Inc.
Phenotypic and functional abnormalities in the peripheral blood T‐cells of patients with primary Sjogren's syndrome
Authors: Aziz Karim Elias , McCluskey Peter J. , Wakefield Denis
Published: June 2005

Changes in regulatory T‐cell subset (including the recently described CD4 helper inducers or suppressor inducers) balance in the peripheral blood may play a role in the pathogenesis of primary Sjogren's syndrome (SS). Direct immunofluorescence and flow cytometry were used to quantitate and analyse peripheral blood lymphocytes in 15 patients with primary SS and 15 control subjects. A reduction in the percentage of circulating CD4 lymphocytes was observed in patients with SS. There was no quantitative abnormality in the percentage of circulating CD4+ 2H4+ (suppressor inducer), CD4+ 4B4+ (helper inducer), CD2, CD3, CD8, CD8+ 2H4+, CD8+ 4B4+, CD25 (IL‐2R), CD19, CD16, CD57 lymphocytes in the patients. Circulating CD8 lymphocytes expressing the activation marker HLA‐DR were increased in the patients. The functional status of peripheral blood lymphocytes was assessed by PHA (phytohaemagglutinin) stimulation followed by monitoring their proliferative response by radiolabelled thymidine uptake and expression of CD25 (Inter‐leukin‐2 receptor). A reduction in the proliferative response of total, CD4‐depleted, and CD8‐depleted lymphocytes suspensions to PHA was demonstrated. The level of expression of CD25 (IL‐2 receptor) was similar in patients and controls before and after 24 h stimulation with PHA. We conclude that there is a disturbance in the functional properties of peripheral blood T cells that can contribute to the immunopathogenesis of SS. Meanwhile, the quantitative reduction of suppressor/inducer lymphocytes as defined by the CD4 2H4 phenotype can be precluded from a role in the development of such an autoimmune condition. © 1994 Wiley‐Liss, Inc.
Simultaneous expression of CD2 on a B‐cell, small lymphocytic neoplasm
Authors: Viciana Ana L. , Lancet Frederick C. , Chamizo Wilfredo , Voigt Walter , Ruiz Phillip
Published: June 2005

An unusual neoplasm, best classified as a B‐cell chronic lymphocytic leukemia on the basis of cytofluorographic, immunohistologic, and gene rearrangement analysis also co‐expressed the T‐cell associated marker CD2 (sheep erythrocyte receptor), but without other cell markers restricted to T cells. This case was associated with a more aggressive course than typically seen with B‐CLL or malignant lymphoma, small lymphocytic type, implying along with previous reports that CD2 expression may serve as a marker of small lymphocytic tumor cell behavior and the ultimate clinical course. Thus, surface phenotypic analysis of these particular hematopoietic neoplasms may serve not only for identification of the malignancy, but could also render prognostic information. © 1994 Wiley‐Liss, Inc.
Development and clinical evaluation of an amplified flow cytometric fluoroimmunoassay for Clostridium difficile toxin A
Authors: Renner E. D.
Published: June 2005

A rapid (2 h) amplified flow cytometric fluoroimmunoassay (AFCF) for Clostridium difficile toxin A was developed and compared with the cytotoxin assay (CTA) and culture of the organism from stool specimens from patients with suspected C. difficile‐associated gastrointestinal disease (CAD). For this assay polyclonal antitoxin A was attached to 10‐μm diameter and monoclonal antitoxin A was attached to fluorescent 0.1 μm‐diameter polystyrene microspheres. The microspheres and sample were reacted together as in a conventional double‐antibody sandwich assay. However, laser flow cytometric measurement allowed the omission of separation and washing steps by gating on light scattered by the larger microspheres and measuring only the fluorescence associated with these particles. The amount of fluorescence from the attached 0.1 μm microspheres was dependent on the concentration of toxin A in the sample. The AFCF detected purified toxin A at levels of 1 pg/ml and was linear from 1 to 40 pg/ml. The AFCF was compared with the CTA and culture of C. difficile for clinical use by comparing results from 198 stool specimens from patients with suspected CAD. The AFCF was 85.7% sensitive and 95.8% specific relative to the CTA, and 85.2% sensitive and 98.3% specific compared to the culture assay. If the isolation of toxigenic C. difficile or the patients clinical course was considered indicative of CAD, the sensitivities of the AFCF, CTA, and culture assay were 77.4%, 67.7% and 96.8%, respectively. The AFCF demonstrated a specificity of 98.8%, while both CTA and culture had a specificity of 100%. The AFCF for toxin A is a sensitive and rapid test, and in laboratories with a flow cytometer offers an alternative method for the laboratory diagnosis of CAD. © 1994 Wiley‐Liss, Inc.
Retrospective evaluation of flow cytometry as a platelet crossmatching procedure
Authors: Gates Karen , MacPherson Bruce R.
Published: June 2005

We performed a retrospective analysis of flow cytometry as a platelet crossmatching procedure. Sera from 17 alloimmunized refractory patients were tested against 32 donor platelets, which had been stored as platelet‐rich plasma for up to 36 months. Overall, 14/32 (44%) crossmatches were positive. The mean 1 h posttransfusion corrected count increments (CCIs) were 9,195 and 2,269 for a negative and a positive crossmatch, respectively. The predictive value of a positive crossmatch was 86%, whereas the predictive value of a negative crossmatch was 56%. When samples with low background fluorescence or with high panel‐reactive antibody (PRA) levels were evaluated separately, the accuracy of the crossmatch improved from 69% to 80%. When compared to the platelet adhesion immunofluorescence test (PAIFT) and the standard and antiglobulin‐enhanced lymphocytotoxicity tests for the detection of HLA antibodies, flow cytometry appeared to be more sensitive. We conclude that flow cytometry is a useful technique for platelet crossmatching, particularly for alloimmunized patients for whom HLA compatible platelets may not be readily available. © 1994 Wiley‐Liss, Inc.
Artifactual staining of monoclonal antibodies in two‐color combinations is due to an immunoglobulin in the serum and plasma
Authors: Nicholson Janet K. A. , Rao Patricia E. , Calvelli Theresa , Stetler‐Stevenson Maryalice , Browning Sandra W. , Yeung Lily , Marti Gerald E.
Published: June 2005

Two‐color whole blood lysis is the assay of choice for lymphocyte immunophenotyping because of the additional information it provides. Recently, artifactual double‐staining of some specimens has been observed with this assay. In these cases, the samples appear to be uncompensated for spectral overlap or to inappropriately coexpress two antigens simultaneously. This artifact can result in the apparent coexpression of CD4 and CD8 (observed in lymphoblastic processes) or of CD5 and CD20 (characteristic of chronic lymphocytic leukemia) in normal persons, leading to an erroneous diagnosis. Using plasma, serum, or immunoglobulin preparations from donors who exhibit this artifact we sought to determine 1) the source of the artifact and 2) ways to overcome it. This staining is apparently due to an immunoglobulin in the donors' serum and plasma which does not have specific reactivity with mouse immunoglobulin. Washing whole blood samples or blocking with mouse immunoglobulin is a convenient way of avoiding this artifact. © 1994 Wiley‐Liss, Inc.
Leukemia‐associated changes identified by quantitative flow cytometry: I. CD10 expression
Authors: Lavabre‐Bertrand Thierry , Janossy George , Ivory Kamal , Peters Rowajda , Secker‐Walker Lorna , Porwit‐MacDonald Anna
Published: June 2005

We have compared CD10 antigen expression in normal fetal bone marrow with that of B‐lineage acute lymphoblastic leukemia (ALL). Both quantitative indirect immunofluoresence (QIFI) and direct immunofluorescence (IF) tests with Quantum beads were used to convert median fluorescence intensity (MFI) values into numbers of antigen molecules expressed per cell (AgE). Lymphoid precursors in the fetal marrow and liver expressed 3–12.5 × 103 CD10 molecules/cell with an upper limit of 5 × 104/cell (MaxAgE). The median CD10 AgE in the different cases of acute B‐lineage ALL were variable and ranged from undetectable to very high values (> 1.8 × 105). In 24 of the 72 cases (33%) tested with QIFI the median CD10 AgE was above the highest values seen in normal samples (> 5 × 104/cell). An additional 23.6% of cases had higher median values than the normal median CD10 AgE. Next, CD10 antigen was quantitated in 78 cases during the routine multiparameter analysis of B‐lineage leukemia using CD10/class II/CD34 3‐color IF test or CD10/TdT 2‐color IF test. The aberrant overexpression was confirmed in 43.6% of ALL cases. The CD10bright display suggested ALL diagnosis even when few cells were available for study, e.g., in early relapse and in ALL masquerading as aplastic anemia. The levels of CD10 expression were maintained in relapse. In addition, different CD10 levels were associated with the various chromosomal alterations: high CD10 levels (> 3 × 104/cell) with hyperdiploidy, low CD10 levels (1.8–4 × 103/cell) with the t(1;19), and undetectable levels (< 1.2 × 103/cell) with the t(4;11) translocations. These findings show that while all these diseases are part of the precursor B‐cell spectrum, the CD10 changes are linked to disease‐associated alterations instead of truly reflecting the features of subtypes of normal B‐cell precursors. The quantitative CD10 assessment should therefore be part of the routine flow cytometric assessment in ALL, and further careful studies are warranted during the regeneration phase following chemotherapy. High CD10 expression alone during strong bone marrow regeneration may not be leukemia specific and both the multiparameter analysis described above and parallel studies for gene rearrangements will be necessary to establish the relative values of these assays in detecting minimal disease amidst regenerating marrow. © 1994 Wiley‐Liss, Inc.
Computerized image analysis and flow cytometric evaluation of ovarian bordeline tumors: A study of 24 cases
Authors: Esposito Michael J. , Fuchs Alexander
Published: June 2005

DNA content evaluation in the study of ovarian borderline tumors has been shown to be a useful adjunct to histopathologic diagnosis. This study compares flow cytometry and computerized image analysis (CIA) in evauating the DNA content of these lesions.

Twenty‐four cases of ovarian borderline tumors (15 serous, 9 mucinous) were studied utilizing formalinfixed paraffin‐embedded tissue. Flow cytometry of cell suspensions and CIA of cell suspensions and paraffin sections were compared in the evaluation of DNA content.

Twenty‐three tumors (96%) were diploid and only 1 (4%) was aneuploid. There was 100% correlation between flow cytometry and CIA of cell suspensions. Image analysis of Feulgen‐stained paraffin sections was found to be unreliable when compared with the use of cell suspensions in the evaluation of DNA content. Clinical follow‐up in 17 patients showed recurrent disease in two patients with diploid tumors and no evidence of disease in the patient with an aneuploid tumor.

The reported rate of aneuploidy in borderline tumors varies depending on the diagnostic method employed in evaluating DNA content. This study shows good correlation between flow cytometry and image analysis of cell suspensions and supports the low rate of aneuploidy found in these lesions by other authors utilizing these method. The majority of borderline ovarian tumors have been shown to have a diploid DNA content; however, the finding of aneuploidy in histologically classified borderline tumors warrants close clinical follow‐up since aneuploidy in these tumors has been associated with an adverse prognosis. © 1994 Wiley‐Liss, Inc.
DNA image cytometric analysis of paraffin‐embedded sections of small renal cortical neoplasms
Authors: Farnsworth William V. , Derose Patricia B. , Cohen Cynthia
Published: June 2005

DNA ploidy analysis of neoplasms is most frequently performed by flow cytometry of single nuclear suspensions or image analysis of Feulgen‐stained cytologic specimens. To assess the prognostic significance of ploidy in renal adenomas (less than 3 cm in diameter), nuclear DNA content of 21 renal cortical neoplasms was analyzed by image cytometry (CAS 200, Cell Analysis Systems, Inc., Elmhurst, IL) on Feulgen‐stained 5‐μM fixed embedded tissue sections. Five (36%) of 14 small neoplasms (so‐called renal adenomas; (mean diameter, 7.2 mm; range, 2–24 mm) were in the aneuploid range. Four (57%) of seven carcinomas (mean diameter, 6.3 cm; range, 3.5–10 cm) were aneuploid, a frequency not significantly diifferent from that in the adenomas (P = 0.4). Mean DNA index (1.38) for aneuploid adenomas and carcinomas was identical. Diploid renal adenomas (mean diameter, 5.9 mm) were smaller, although not significantly so (P > 0.05), than the aneuploid ones (mean, 9.7 mm). Size of diploid (mean, 6.7 cm) and aneeuploid (mean, 6.1 cm) renal carcinomas was similar. Clinical follow‐up studies to evaluate utility of DNA ploidy and proliferative activity in prognosis and management of small renal cortical neoplasms are indicated. © 1994 Wiley‐Liss, Inc.
Detection of terminal deoxynucleotidyl transferase by flow cytometry: A three color method
Authors: Horvatinovich Joseph M. , Sparks Sara D. , Borowitz Michael J.
Published: June 2005

This short communication describes a three‐color flow cytometric analysis for terminal deoxynucleotidyl transferase (TdT). The method combines CD45‐gating principles and a new permeabilization method based on a commercially prepared lysing reagent. While traditional permeabilizing fixatives are often cumbersome to use, distort cell light scatter, and fail to retain the fluorochrome peridin chlorophyll alpha protein (perCP), this simple method retains light scatter and does not affect perCP, making it ideal for three‐color analysis. The procedure simply involves staining whole blood or bone marrow with CD45 perCP and an appropriate phycoerythrin (PE) conjugated monoclonal antibody (MoAb) and lysing with FACS lysing solution (Becton Dickinson, San Jose, CA). After washing the cells, fluorescein isothiocyanate (FITC) conjugated anti‐TdT is added. The cells are washed, fixed with formaldehyde, and acquired on a flow cytometer compensated for three‐color analysis. Display of CD45 perCP vs. right angle light scatter (RALS) allows identification of blasts. Dual color expressionof anti‐TdT FITC and a PE conjugated MoAb identifies TdT useful for TdT analysis but may also prove to be a valuable tool for looking at expression of other cytoplasmic antigens in combination with surface antigens on CD45‐defined blast populations. © 1994 Wiley‐Liss, Inc.
CD3+, CD56+ non‐MHC restricted cytotoxic T lymphocytes in two fraternal aids patients: A case report
Authors: Warzynski Michael J. , Barbour Stephen D. , Winkler‐Pickett Robin T. , Ortaldo John R.
Published: June 2005

We describe two brothers with hemophilia and AIDS, with an unusually large percentage of CD3+, CD56+ lymphocytes. They experienced no major complications with opportunisitic infections (OI) and infrequent secondary infections, even though they had nearly 0% CD4 lymphocytes for 3 years. Both patients died in 1991 of progressive cardiomyopathy. The patients' lymphocytes were immunophenotyped by flow cytometry and analyzed for functional cytolytic activity against K562 and HIV infected HUT 78 cell lines. Single color CD4 counts were 2–9% for 4 years. Dual color CD4 counts at our laboratory demonstrated 0–1% CD4 for the last 6 months. When CD3+ lymphocytes were examined, both patient 1 and patient 2 demonstrated a significantly higher proportion and absolute number of CD3“bright”+, CD56+ double‐positive cells, 47% and 22%, respectively, compared to other HIV‐positive children with hemophilia (≤2%). Functional studies with could not be augmented by in vitro addition of IL‐2, whereas patient 2 had no NK activity unless IL‐2 was added. Functional studies with HIV‐infected HUT‐78 cells demonstrated patient 2 had cytolic activity against HIV‐infected cells and patient 1 had high nonspecific cytolytic activity even against uninefected HUT‐78 cells, whereas controls had minimal activity to HUT‐78 cells or HIV‐infected HUT‐78 cells. The case report raises a speculative question requiring a larger database, whether the anti‐HIV activity and/or unuasal clinical course without typical 0.1. of some AIDS patients may be related to the presence of CD3“bright” +, CD56+ lymphocytes of their immune system. © 1994 Wiley‐Liss, Inc.
Computer image analysis of comparative genomic hybridization
Authors: Piper Jim , Rutovitz Denis , Sudar Damir , Kallioniemi Anne , Kallioniemi Olli‐P. , Waldman Frederic M. , Gray Joe W. , Pinkel Dan
Published: June 2005

We describe and evaluate the image‐processing and analysis techniques we have developed for the quantitative analysis of comparative genomic hybridization (CGH; Science 258:818, 1992). In a typical CGH application, two genomic DNA samples are simultaneously hybridized to metaphase chromosomes and detected with different fluorochromes. The primary data in CGH are contained in the intensity ratios of the fluorochromes as a function of position on the chromosomes, which reflect variation in DNA copy number ratio between the two DNA samples. Analysis involves chromosome segmentation, intensity normalization, background corrections, and calculation of the fluorescence intensity profiles and the ratio profile along the chromosome's length. Profiles from several copies of the same chromosome in different metaphases are averaged to reduce the noise. Confidence intervals are calculated and displayed for the mean profiles. The techniques were evaluated by examining the variability found in comparisons of two normal genomic DNAs, where the ratio was expected to be constant, and by measuring the ratios obtained for cell lines with cytogenetically documented copy number changes involving several chromosomal segments. The limits of sensitivity of CGH analysis were investigated by simulation. Guidelines for the interpretation of CGH data and indications of areas for future development of the analytical techniques are also presented. © 1995 Wiley‐Liss, Inc.
Quantitative analysis of comparative genomic hybridization
Authors: du Manoir Stanislas , Schröck Evelin , Bentz Martin , Speicher Michael R. , Joos Stefan , Ried Thomas , Lichter Peter , Cremer Thomas
Published: June 2005

Comparative genomic hybridization (CGH) is a new molecular cytogenetic method for the detection of chromosomal imbalances. Following cohybridization of DNA prepared from a sample to be studied and control DNA to normal metaphase spreads, probes are detected via different fluorochromes. The ratio of the test and control fluorescence intensities along a chromosome reflects the relative copy number of segments of a chromosome in the test genome. Quantitative evaluation of CGH experiments is required for the determination of low copy changes, e.g., monosomy or trisomy, and for the definition of the breakpoints involved in unbalanced rearrangements. In this study, a program for quantitation of CGH preparations is presented. This program is based on the extraction of the fluorescence ratio profile along each chromosome, followed by averaging of individual profiles from several meta phase spreads. Objective parameters critical for quantitative evaluations were tested, and the criteria for selection of suitable CGH preparations are described. The granularity of the chromosome painting and the regional inhomogeneity of fluorescence intensities in metaphase spreads proved to be crucial parameters. The coefficient of variation of the ratio value for chromosomes in balanced state (CVBS) provides a general quality criterion for CGH experiments. Different cutoff levels (thresholds) of average fluorescence ratio values were compared for their specificity and sensitivity with regard to the detection of chromosomal imbalances. © 1995 Wiley‐Liss, Inc.
Optical method for measurement of number and shape of attached cells in real time
Authors: Ramsden J. J. , Li S.‐Y. , Heinzle E. , Prenosil J. E.
Published: June 2005

A novel, on‐line method of accurately parametrizing the shape of spreading cells is described. The substrate on which the cells are deposited serves as an optical waveguide. The method is based on the interaction between the evanescent part of the guided waves and the cells. No special labelling of the cells is required. A mathematical framework for interpreting the results is derived, and some illustrative results are presented. © 1995 Wiley‐Liss, Inc.
Study of surface carbohydrates on isolated Golgi subfractions by fluorescent‐lectin binding and flow cytometry
Authors: Guasch Rosa M. , Guerri Consuelo , O'Connor José‐Enrique
Published: June 2005

The Golgi complex is a functionally heterogeneous subcellular structure that plays a key role in the synthesis, maturation, and sorting of newly synthesized glycoproteins. Fluorescent lectins have been used extensively to analyze surface glycoproteins by flow cytometry in whole cells and more recently in isolated subcellular organelles, such as mitochondria and chloroplasts. We report here the use of several fluorescein‐isothiocyanate‐conjugated lectins to detect and quantify specific surface sugars by flow cytometry on isolated elements from purified cis and trans‐Golgi fractions from rat liver. Our results show that this approach may be useful to study Golgi composition and function, since it may reveal the intensity of specific binding of different lectins to each Golgi fraction and the percentage of elements binding the lectins specifically. Thus we show here that Golgi elements appear homogeneous in mannose and fucose, whereas galactose and N‐acetyl‐glucosamine residues are more abundant in the trans‐Golgi elements. © 1995 Wiley‐Liss, Inc.
DNA staining changes associated with apoptosis and necrosis in blood lymphocytes of individuals with HIV infection
Authors: Echaniz Pilar , de Juan Maria Dolores , Cuadrado Emilio
Published: June 2005

We used flow cytometry to quantitate cells that die by apoptosis or necrosis. The method uses low concentrations of two DNA binding dyes that allow one to establish selective regions for live, apoptotic, and necrotic cells in a rat thymocytes model. Quantitative analysis of blood lymphocyte death in individuals with HIV infection by this technique shows the presence of nonviable cells that exhibit a spectrum of changes in staining by DNA binding dyes. These changes range from typical features of cells undergoing programmed cell death or apoptosis to changes observed in cells that die by accidental death or necrosis. The proportion of cells exhibiting these lethal changes increases significantly in patients who progress to AIDS, but, although cells with staining features associated with apoptosis and necrosis were both found to be increased in in vitro‐activated cells from AIDS patients, spontaneous in vivo activation preferentially leads to apoptotic changes without a significant increase of cells exhibiting the staining changes associated with necrosis. © 1995 Wiley‐Liss, Inc.
Combined light and fluorescent microscopical imaging of nucleolar organizer regions and cellular rRNA as detected by fluorescent in situ hybridization
Authors: Pajor László , Honeyman Thomas W.
Published: June 2005

A method for combined light and fluorescent microscopic imaging of nucleolar organizer regions and cellular rRNA is described. Nucleolar organizer regions were detected by silver staining (Ag‐NOR), and rRNA was detected by fluorescent in situ hybridization (FISH). MG‐63 human fibrosarcoma cells were silver stained prior to in situ hybridization. To quantitate Ag‐NOR within individual cells, brightfield images were digitized, and the total Ag‐NOR area/nucleus was determined. Fluorescent images were digitized, and the total cellular fluorescence was calculated after correction for nonuniformity of illumination. By using this method, it was shown that the Ag‐NOR procedure did not significantly affect the fluorescence intensity related to FISH. Furthermore, the hybridization procedure did not interfere with quantitation of Ag‐NOR. With this method, both Ag‐NOR and rRNA product can be quantitated within the same cell. Because the relationship of rRNA content to cell proliferation is well established, correlation to quantitative Ag‐NOR parameters within individual cells will contribute to the better definition of the relationship of quantitative Ag‐NOR indices with cellular proliferation. © 1995 Wiley‐Liss, Inc.
Cell membrane‐dependent chromatin condensation
Authors: Vinogradov Alexander E.
Published: June 2005

It is shown by means of flow cytometry that during several seconds after cell membrane damage by a non‐ionic detergent in physiologically relevant buffer solution, the chromatin of mouse thymocyte nuclei undergoes a drastic decondensation, which is revealed by a sharp increase of binding of DNA‐specific fluorochromes (olivomycin or propidium iodide) and of DNA accessibility to DNAse I digestion. A similar change is observed in dead cells. Roughly half of this decondensation can be prevented by lowering the pH of the outside medium to the level known to be inside the cells; the other half remains thus far unexplained (divalent cations and the difference between small anion species seem not to be involved). The approach is based on a novel observation that fixation by formaldehyde conserves chromatin structure before the action of detergent. Flow cytometric assay is proposed for monitoring these condensation/decondensation events in media of different composition. In addition, a new approach to viable/dead cell determination, which has the advantage of immediately fixing the cell state and preserving it for a reasonably long time, is proposed. © 1995 Wiley‐Liss, Inc.
Flow cytometric detection of micronuclei by combined staining of DNA and membranes
Authors: Wessels Jurina M. , Nüsse Michael
Published: June 2005

A new staining method is presented for flow cytometric measurement of micronuclei (MN) in cell cultures and human lymphocytes using membrane‐specific fluorescent dyes in addition to DNA staining. Several combinations of fluorescent membrane and DNA dyes were studied for a better discrimination of MN from debris in a suspension of nuclei and micronuclei. For staining of membranes, the lipophilic dyes 2‐hydroxyethyl‐7,12,17‐tris(methoxyethyl)porphycene (HEPn) and 1,6‐diphenyl1,3,5‐hexatriene (DPH) were used in combination with ethidium bromide (EB), proflavine (PF), and Hoechst 33258 (HO). Due to their spectral properties, HO or EB combined with HEPn were not as suitable for the discrimination of MN from debris as was HEPn in combination with PF. With HEPn in combination with PF, however, additional noise was found at low fluorescence intensities, probably due to free fluorescent dye molecules in the solution. The optimal simultaneous staining of membranes and DNA was obtained using a combination of DPH and EB. The induction of MN in Chinese hamster and mouse NIH‐3T3 cells by UV‐B illumination was studied with this new staining technique. UV‐B illumination (280–360 nm) induced MN in both cell lines. Chinese hamster cells were found to be more sensitive to these wavelengths. Illumination with wavelengths above 360 nm did not induce MN in either cell line. The results obtained from human lymphocytes using the combination of EB and DPH were comparable to the results obtained with the combination of EB and HO. © 1995 Wiley‐Liss, Inc.
High‐speed photodamage cell selection using a frequency‐doubled argon ion laser
Authors: Keij Jan F. , Groenewegen Ad C. , Dubelaar George B. J. , Visser Jan W. M.
Published: June 2005

A flow cytometer was developed for the high‐speed “sorting” of desired cells by selectively irradiating (zapping) the undesired cells from a population. After previous efforts to photoinactivate cells with photosensitizers had failed, it was decided to exploit the photosensitivity of the cell's DNA at 257 nm. It was shown that a 257 nm laser output power of 20–100 mW was sufficient to induce a 4.5 log cell kill after the cells were processed through a focused 257 nm laser beam. Experiments proved that the photodamage flow cytometer (ZAPPER) could selectively photoinactivate cells at rates over 22,000 events/s, and selection purities ranged from 81% to 100%.

The yields of the desired cells depended on the selection mode. In the Enrichment mode, the zap laser was not aimed at the jet, and only undesired cells were exposed to a brief ultraviolet (UV) pulse after modulation of the UV laser beam. The yields of desired cells ranged from 95% to 105%. In the Purge mode, the zap laser beam was aimed onto the jet, and only desired cells were allowed to pass after deflection of the UV laser beam; the yields of desired cells ranged from 12% to 52%. The cause of the reduced yields in the PURGE mode was traced to the fact that the Electro‐Optic Modulator was used to modulate the zap laser proved too slow for the intended application.

The lifetime of the frequency‐doubling crystal used for the generation of the 257 nm beam was found to be limited to several days. These technical limitations could theoretically be overcome by the application of large argon ion lasers capable of emissions at 257–275 nm and an Acousto‐Optic Modulator to deflect the zap laser. © 1995 Wiley‐Liss, Inc.