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PubMed1987

On multiparameter data analysis in flow cytometry.

Increasing numbers of parameters that are accessible to simultaneous measurement in flow cytometric instruments, combined with the extremely large sample sizes common in flow cytometry, make it necessary to examine methods of multivariate statistics for their applicability to problems of visualization and quantitative analysis of flow cytometric data. This article describes some approaches to dimensionality reduction that appear well suited for data sets obtained by flow cytometry.

Flow Cytometry↗
PubMed1990

Flow cytometry.

Explore the source record for details and available documents.

Animals↗
PubMed1991

Flow cytometry of fine needle aspirations of the Sprague-Dawley rat testis: defining normal maturation and the effects of multiple biopsies.

Quantitative assessment of spermatogenesis by flow cytometry reproducibly correlates with histological examination. Flow cytometry of fine needle aspirates from the testis was used to analyze normal spermatogenesis of Sprague-Dawley rats. Aged matched male weanlings were divided into 3 groups: group 1-5 rats underwent bilateral percutaneous aspiration of the testes with a 22 gauge Chiba needle on postpartum days 19, 24, 30 and 40; group 2-5 rats aspirated on days 24, 30 and 40, and group 3-4 rats aspirated on days 30 and 40. This sequential approach allowed for evaluation of normal spermatogenesis and the effects of repeated fine needle aspiration on spermatogenesis. Deoxyribonucleic acid distribution analysis by flow cytometry correlates with haploid (1N), diploid (2N), tetraploid (4N) and S phase cell populations. These cell populations were evaluated and comparisons among each group were made at all biopsy times. No significant differences in mean 2N, 4N or S phase cell populations in single versus repeat biopsied testes were detected. However, there was a significant increase in the 1N population at 30 days post partum in the repeat biopsy group (p = 0.037), which normalized by day 40. This increase in 1N corresponds to the beginning of meiosis, which is maximal between postpartum days 30 and 40, and occurs earlier in the repeat biopsied testis. Repeat fine needle aspiration of the testis can be performed without significantly affecting spermatogenesis in the "weanling" rat. This provides a useful technique in the future investigations of the time related effects of varicocele, chemotherapy and toxicologic drugs on spermatogenesis.

Animals↗
PubMed2006

Detection of rabies virus antigen or antibody using flow cytometry.

BACKGROUND: Rabies is invariably a fatal encephalomyelitis that is considered to be a serious public health problem. Rabies diagnosis must be rapid and conclusive. Detection and quantification of antirabies antibodies is used for assessment of the effectiveness of rabies vaccines. Hence, computer-automated detection of fluorescence using flow cytometry was attempted to reduce the work time required to undertake the conventional rapid fluorescent focus inhibition test (RFFIT). METHODS: Pasteur virus (PV)-infected mouse neuroblastoma (MNA) cells were stained with rabies virus antinucleocapsid antibody, fluorescein isothiocyanate (FITC) conjugate, and the percentage of infected cells at 24, 48, and 72 h postinfection (PI) was determined using flow cytometry. Serum samples containing known antibody titres estimated by RFFIT in terms of IU/ml were used to neutralize 50 FFD50 dose of PV. The percentage of MNA cells infected by the un-neutralized virus was estimated by flow cytometry. Using the value of the percentage of cells infected in the presence of known negative serum as 100%, the infection inhibition caused by antibodies at each dilution of positive reference serum was calculated and a regression equation generated for the prediction of rabies virus antibody titres in test sera samples as equivalent units per ml (EU/ml). RESULTS: There was a significant increase in the percentage of infected cells between 24 and 48 h PI from 26.45 to 75.28%. The percentage of cells having high side scatter was also highest at 72 h PI (11.11%). Antibody titres predicted by flow cytometry and those estimated by RFFIT as IU/ml showed a correlation coefficient of 0.74. CONCLUSIONS: Thus, flow cytometry could be used to detect rabies virus antigen in infected cells and to predict serum antibody titres from a single dilution of serum tested with the potential advantages of automation, rapidity, and lack of subjectivity. It has the potential to replace the time-tested RFFIT in rabies serology in the years to come.

Animals↗
PubMed1999

Assessment of DNA flow cytometry as a surrogate end point biomarker in a bladder cancer chemoprevention trial.

Although conventional cytology represents the most widely performed cytometric analysis of bladder cancer cells, DNA flow cytometry has, over the past decade, been increasingly used to evaluate cell proliferation and DNA ploidy in cells from bladder washings. We have investigated whether DNA flow cytometry and conventional cytology of epithelial cells obtained from bladder washings provide reliable surrogate endpoint biomarkers in clinical chemoprevention trials. We used cytometric and clinical data from a chemoprevention trial of the synthetic retinoid Fenretinide on 99 patients with superficial bladder cancer. A total of 642 bladder washing specimens obtained from the patients at 4 month intervals was analyzed. Intra-individual agreement and correlation of flow cytometric DNA ploidy (diploid vs. aneuploid), DNA Index, Hyper-Diploid-Fraction (proportion of cells with DNA content higher than 2C), and conventional cytologic examination, as assessed by kappa statistics and Spearman's correlation test, were poor from baseline through 24 months. Moreover, no correlation was found between DNA ploidy and cytology at each time point. The same results were obtained when the analyses were stratified by treatment group. In addition, the association between the results of bladder washing (by either DNA flow cytometry or cytology) and concomitant tumor recurrence was significant only for abnormal cytology, while neither biomarker was predictive of tumor recurrence at the subsequent visit. During the time of this study only four patients progressed to muscle-invasive bladder cancer, indicating the "low-risk" features of the patient population. We conclude that DNA flow cytometry and conventional cytology on epithelial cells obtained from bladder washings do not appear to provide suitable surrogate endpoint biomarkers during the early stages of bladder carcinogenesis.

Antineoplastic Agents↗
PubMed2000

[Differential malignant lymphoproliferative syndrome (LPS) diagnosis with flow cytometry: a study of 100 patients].

The clinical utility of flow cytometry in diagnosis of chronic lymphoproliferative disorders (LPD) is well established. Accurate diagnosis of related but still distinct entities is relevant to therapeutic decisions. We report on the immunophenotypic findings of 100 patients with a new diagnosis of LPD established by two-color flow cytometry. A panel of > 15 monoclonal antibodies was regularly applied. The characteristic immunophenotype of B-cell chronic lymphocytic leukemia (CD5+, CD23+, FMC7-) was found in 74 patients including one with Richter's transformation. Hairy cell leukemia (CD5-, CD11c+, CD103+) was diagnosed in 6, and B-cell Non-Hodgkin lymphoma (B-NHL) in 13 patients, respectively. 6 of the B-NHLs belonged to the entity of splenic lymphoma with villous lymphocytes (CD5-, CD23-, FMC7+) and 6 were identified as mantle cell lymphomas (CD5+, CD23-, FMC7+). One B-NHL was typed as follicular center cell lymphoma (CD5-, CD10+, FMC7+). Three B-cell LPDs without a characteristic marker profile were histologically further classifiable. With a total of 4 patients T-cell LPDs were much less frequent. Sézary syndrome (CD4+, CD8-, CD56-) and T gamma lymphoproliferation (CD4-, CD8+, CD16-, CD56-, CD57+) were diagnosed twice. In 17 patients with a characteristic marker profile (1 Richter's transformation, 5 hairy cell leukemias, 3 splenic lymphomas with villous lymphocytes, 4 mantle cell lymphomas, 4 T-cell proliferations) a further histological or molecular investigation confirmed the immunophenotypic diagnosis in all cases. Clinical presentation with lymphadenopathies and B-symptoms was mainly associated with the diagnosis of mantle cell lymphoma, whereas splenomegaly and infection were suggestive of hairy cell leukemia. 94% of the B-CLL patients were diagnosed at an early clinical stage with still conserved hematopoiesis, 32% of the LPDs were diagnosed following a routine hematogramm.

Antigens, Differentiation, B-Lymphocyte↗
PubMed2006

Comparison of disruption procedures for enumeration of activated sludge floc bacteria by flow cytometry.

BACKGROUND: In a wastewater treatment plant, the degradation process is performed by a variable and mixed community of microorganisms in an aerobic aquatic environment. The activated-sludge process is based on the formation of strong microbial flocs where many bacteria are attached to sludge flocs. METHODS: Cytometric analysis requires an homogeneous cell suspension and so detachment of bacteria from flocs is required. In this study, sonication and homogenization were compared to find the most adequate pretreatment method for bacterial cytometric analysis in activated sludge samples. Bacterial viability was tested with a nucleic acid double-staining (NADS) protocol (Barbesti et al., Cytometry 2000;40:214-218) and on flow cytometry. RESULTS: Each method showed a good efficiency in terms of bacterial detachment; thus finally, the choice of which could be the best treatment method was based on both viability results and analysis rapidity. On the basis of the degree of cell detachment and viability, the maximum value was obtained by sonication (2 x 45''). CONCLUSIONS: The use of flow cytometry in conjunction with fluorescent dyes and an adequate pretreatment represents a useful method to rapidly detect and enumerate bacteria in activated sludge samples.

Bacteria↗
PubMed1996

Comparison of absolute CD4+ lymphocyte counts determined by enzyme immunoassay (TRAx CD4 test kit) and flow cytometry.

Currently, CD4+ lymphocyte counts are one of the most widely used surrogate markers for monitoring disease progression in and initiating therapy for human immunodeficiency virus-infected individuals. However, the process of obtaining lymphocyte subset counts can be complex and expensive, often rendering the test inaccessible to many patients. In contrast to standard laser-based flow cytometry, the TRAx CD4 Test Kit utilizes an enzyme-linked immunoassay format to provide CD4+ lymphocyte counts by a simple and more cost-effective means. In order to evaluate the utility of the TRAx CD4+ assay in comparison with flow cytometry, heparinized blood samples were drawn from 188 infected and uninfected adult patients and 24 infected pediatric patients and evaluated by both assays. The correlation coefficient for all adult individuals tested was 0.94, and the mean absolute counts (in cells per milliliter, +/- standard deviation) were 510 +/- 358 for TRAx and 480 +/- 361 for flow cytometry. The correlation for the pediatric group was 0.93, with mean absolute counts of 956 +/- 767 for TRAx and 1,521 +/-a 1,438 for flow cytometry. Overall the TRAx CD4 Test Kit performed well in comparison to flow cytometry, and its lower cost and ease of use make it an encouraging alternative for the routine determination of CD4+ lymphocyte counts.

Adult↗
PubMed1993

Flow cytometry: a useful technique in the study of multidrug resistance.

Flow cytometry has recently become a very important tool in the study of the mechanisms of multidrug resistance (MDR). This technique allows rapid access to information regarding two characteristics related to the MDR phenotype: i) the level of overexpression of the P-glycoprotein; and ii) its functional aspect in expulsion of the cytotoxic agents to which the cell is exposed. In pharmacology, flow cytometry also allows evaluation of the capacity of modulating agents to reverse this resistance, and contributes a deeper insight into the mechanism of action of new molecules.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1998

Analysis of anti-IgE and allergen induced human basophil activation by flow cytometry. Comparison with histamine release.

OBJECTIVE AND DESIGN: On the basis of flow cytometric methods previously described for the analysis of human basophil activation, we present here a bi-color anti-IgE FITC, anti CD63 PE method and the correlation with histamine release. MATERIALS AND SUBJECTS: Subjects allergic to grass pollen were selected by their clinical history, skin tests and specific IgE. METHODS: Basophils gated in the lymphocyte region of the side scatter (SSC)/forward scatter (FSC) pattern were selected by their high IgE epitope density. Percentage of cells expressing CD63 marker, upregulated on activated basophil membrane, was calculated by the cytometer. Histamine released into the supernatants was measured by RIA. RESULTS: In these conditions, flow cytometric analysis of blood leukocytes showed that the selected cells had the phenotype CD14-, CD19-, CD45+, IgE++ and CD63- or + which is related to human basophil phenotype, the isotype controls being negative. The use of an anti-CD41 FITC antibody also showed the presence of aggregated platelets on the basophil membrane, CD63 antigen being, however, expressed by basophils themselves and not by platelets. Moreover, no statistical difference was observed between histamine release and flow cytometry after passive sensitization of blood donor leukocytes. CONCLUSION: Flow cytometry, as a popular method often used in the immunology and haematology departments of clinical laboratories may represent a new alternative for allergy diagnosis and basophil pharmacology.

Allergens↗
PubMed2005

[Use of flow cytometry in the diagnosis of gestational trophoblastic disease].

INTRODUCTION: Histopathologic analysis of aborted tissue in molar pregnancy is frequently complicated by scarcity of tissue as well as by the fact that ultrasound and biochemistry permit the clinical diagnosis of gestational trophoblastic disease at the very beginning of pregnancy, when all classical morphologic features have not yet developed. Flow cytometry is today a widely used method that can help the pathologist reach a correct diagnosis, having implications for the patient. AIM: To show the role of flow cytometry in addition to classical histopathologic methods in the early diagnosis of gestational trophoblastic disease, i. e. molar pregnancy. PATIENTS AND METHODS: A total of 103 consecutively received placental tissue samples from spontaneous/medically induced abortions were histopathologically examined and submitted to flow cytometry analysis. RESULTS: The patient mean age was 31 (range 19-50) years, and mean gestational age was 10 (range 5-19) weeks. Residual placental tissue with or without degenerative changes was found in 51.5% of the samples; in 8.7% embryonal/fetal tissues were identified as well. The histopathologic diagnosis of partial hydatiform mole was made in 34% and of complete hydatiform mole in 5.8% of cases. The difference in mean age of women according to histopathologic findings or placental ploidy was not significant. Flow cytometry revealed 42.7% of diploid samples, 58.3% of aneuploid samples and 1% of tetraploid samples. Of 53 samples showing normal morphology or degenerative hydropic changes, 60.4% were diploid and 39.6% aneuploid; of 9 samples containing fetal tissues 44.4% were diploid and 55.5% aneuploid (NS). A significant difference (p < 0.01) was found between the placental tissue ploidy with histopathologic diagnosis of residual placental tissue, and with histopathologic diagnosis of partial hydatiform mole (p < 0.001); when fetal tissues were present the difference was not significant CONCLUSION: This study confirmed the partial hydatiform mole to be a common but clinically underdiagnosed condition. Except for the knowledge whether the abortion was caused by cytogenetic factors, flow cytometry helps the pathologist reach an accurate diagnosis and has a place in daily practice.

Adult↗
PubMed1992

The current status of flow cytometry in gynecologic oncology.

In recent years, the use of flow cytometry has expanded from its initial research role to include the detection of clinically relevant abnormalities in the nuclear DNA content of tumor cells and variations in cell-growth kinetics. Retrospective studies have shown that DNA ploidy, and possibly the proliferative or S-phase fraction, is a valuable prognostic indicator in patients with gynecologic malignancies, including gestational trophoblastic disease, early- and late-stage ovarian cancer, squamous-cell cervical cancer and its precursor lesions, and endometrial adenocarcinoma. Further prospective studies are needed to establish the exact value of flow cytometry for gynecologic and other tumors. However, the prognostic value of this information certainly will influence the clinical management of patients with these malignancies in the near future.

Aneuploidy↗
PubMed1991

[Analysis of tumor DNA by flow cytometry on prostate cyto-aspiration product. Value of routine cytologic evaluation].

In this prospective study, flow cytometry DNA profile of 169 stage D2 prostatic carcinomas were compared with conventional cytologic data. Two transrectal fine-needle aspiration biopsies were performed in each patient. Aspirates were immediately fixed in 2% polyethylene glycol 1500* alcoholic solution (Merck). Suspensions were mixed and studied by a conventional cytologic technique (Papanicolaou) and by flow cytometry (propidium iodide stain on isolated nuclei). A highly significant correlation was found between the DNA profile and the cytologic grade (p less than 0.001). The DNA profile was bimodal in 14% (3/21) of aspirates containing benign or atypical cells, 18% (3/17) of grade I aspirates, 30% (13/43) of grade II aspirates and 71% (24/34) of grade III aspirates. Routine cytologic evaluation of cell suspensions evaluated by flow cytometry is important in clinical practice since both falsely unimodal and falsely bimodal profiles occur. Leukocytes or benign epithelial cells can interfere with the tumor cell population. In fine-needle aspirates, the broad range of non-malignant contaminating cells limits the value of immunocytochemistry and emphasizes the usefulness of routine conventional cytologic evaluation.

Adenocarcinoma↗
PubMed1989

DNA flow cytometry in solid tumors: practical aspects and clinical applications.

Investigators are currently using techniques of DNA flow cytometry to measure ploidy status (DNA content) and proliferative potential (S phase fraction) in a wide variety of solid tumors. These measurements have shown relevance for diagnosis, prognosis, and treatment for patients with cancer. National cooperative group studies are beginning to evaluate these measurements in controlled clinical trials to further define their clinical utility as well as limitations. The purpose of this report is to discuss both practical aspects of DNA flow cytometry and clinical applications of these measurements in a variety of solid tumors. We describe in detail our methods for sample handling, processing, and interpretation of DNA histograms. Although there are multiple ways of processing samples and interpreting histograms, we present methodology and interpretations that have proven efficient and reproducible. The review of clinical applications of flow cytometry to solid tumors focuses on breast cancer, but includes a discussion of other solid tumors.

Breast Neoplasms↗
PubMed1999

Analysis of subclonal expansion of colorectal carcinomas by flow cytometry.

DNA heterogeneity of colorectal carcinomas has been investigated by flow cytometry, most studies have focused on the clinical usefulness of DNA ploidy analysis. Since cancers consist of predominant subclones with proliferative advantage due to clonal expansion, we attempted to analyse the clonal expansion of colorectal carcinomas within a tumour by measuring DNA ploidy. The DNA ploidy and heterogeneity of multiple fresh samples obtained from 164 colorectal adenocarcinomas were analysed by flow cytometry. Each tumour was divided into an average of six specimens, which were analysed separately. For 146 of the tumours (89%) at least one DNA aneuploid population was found within the cancer tissue examined. DNA multiploidy was detected in 26 cases (17.8%) among the cancers with aneuploidy. Based on the DNA index (DI), hypertriploid aneuploidy (1.7<DI<1.8) was found most frequently in the aneuploid colorectal cancers examined. DNA ploidy heterogeneity was seen in 75 (51.4%) of the DNA aneuploid tumours. There were only 3 cases with more than three subclones including a diploid line. The present results indicate that colorectal carcinomas consist of a few dominant subclones and have a DNA content (hypertriploid aneuploid) that confers a proliferative advantage.

Adenocarcinoma↗
PubMed2003

Importance of CD117 in the evaluation of acute leukemias by flow cytometry.

BACKGROUND: The issue of which specific antibodies need to be used when evaluating acute leukemias by flow cytometry is controversial. METHODS: Recent studies have suggested that antibodies against CD117 or c-kit are not essential for the assignment of blast lineage by flow cytometry, even though CD117 appears to be a very specific marker for myeloid lineage acute leukemias. We report a case of acute myeloid leukemia M2 subtype with an 8:21 translocation, where the leukemic blasts expressed CD117, CD19, and CD15 but did not show definitive expression of the myeloid markers CD13 or CD33. RESULTS AND CONCLUSIONS: This study highlights the importance of CD117 when evaluating acute leukemias by flow cytometry, which was necessary in this case to suggest that the blasts were phenotypically abnormal myeloblasts. In addition, this case presented an unusual acute myeloid leukemia phenotype that will likely be encountered by others and could be difficult to interpret.

Acute Disease↗
PubMed1985

A new standard fluorescence microsphere for quantitative flow cytometry.

A standard fluorescence microsphere has been prepared for quantitative flow cytometry. A uniformly sized (6.53 +/- 0.34 micron in diameter) and perfectly spherical plastic microsphere, Micropearl-FT, was covalently conjugated with fluorescein isothiocyanate (FITC). The characteristic of this microsphere to lyse completely in strong alkaline solutions made it possible to determine the amount of bound FITC easily and reproducibly. The amount of bound FITC depended on the concentration of FITC and the incubation time at pH 9.5 and 37 degrees C. In flow cytometry, the fluorescence intensity of the FITC-conjugated microsphere (FCMS) showed linear dependence on the amount of bound FITC. The fluorescence intensity of FCMS was stable for at least 3 months even in aqueous solutions. Using FCMS, an Epics V was calibrated and the minimum measurable fluorescence intensity was examined. From analysis using this system, the number of FITC-conjugated OKT3 molecules bound to human T lymphocytes was shown to average 4.4 X 10(4) per cell at saturation.

Adult↗