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Results for “Flow Cytometry”

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PubMed1979

Flow cytometry: general principles and applications to selected studies in tumor biology.

The cell populations derived from normal tissues and solid tumors comprised many different cell types. Within each cell type there is a distribution of cells in different phases of the cell cycle and/or metabolic states (ie, differing rates of protein, RNA, and other macromolecular syntheses). Flow cytometry and companion instrumentation now promise to aid in rapid quantitative analyses of heterogeneous cell populations, thus finding broad applicability in many areas of cancer research and treatment. Since it is projected that this analytical technique will greatly expend our knowledge in tumor biology, it seems appropriate to review the basis principles of the methodology and to demonstrate recent applications in several areas of current research. After reviewing basis principles, a detailed description of one specific flow cytometer, the PHYWE-ICP-22, with its computer interface as developed in this laboratory is described. Subsequently, applications of this methodology to analyses of tumor cell kinetics, assays of blastogenesis, and studies of human colon cancer are presented as specific, current applications of flow cytometry. It is anticipated that this overview of flow cytometry along with some current applications will provide a background understanding for the inevitable rapid future developments in this area of research.

Animals↗

PubMed1997

Flow cytometry in squamous cell esophageal cancer and precancerous lesions.

Flow cytometry has also been used to study the nuclear DNA content (ploidy) and cell cycle kinetics of esophageal cancers. Studies of limited numbers of patients with Barrett's esophagus undergoing endoscopic surveillance suggested that aneuploidy may be a useful marker to identify subsets of patients at increased risk for malignancy. Few studies to date have evaluated premalignant tissues associated with the development of squamous-cell cancer of the esophagus. The present retrospective study comprises 80 surgical specimens of squamous-cell carcinoma of the esophagus from a high-incidence region of Thailand. All patients had surgery at the Department of Surgery, Prince of Songkla University, between March 1983 and December 1993. Sets of serial sections were cut every 0.5 cm starting from the proximal margin and down to the distal margin, and histopathology was confirmed to flow cytometric parameters (DNA content, S-phase fraction). Aneuploidy was found in 84% of squamous-cell carcinoma, 22.2% of carcinoma in situ, 28.6% of severe dysplasia, 11.0% of moderate dysplasia and 0% of mild dysplasia and normal esophageal mucosa specimens. The percentage was higher according to the level of severity or dysplasia. S-phase fraction was found to be 21.0 +/- 0.9% in squamous-cell carcinoma, 20.3 +/- 10.3% of carcinoma in situ, 20.9 +/- 5.3% of severe dysplasia, 12.9 +/- 9.7% of moderate dysplasia 7.6 +/- 0.8% of mild dysplasia and 8.9 +/- 3.2% of normal tissue. Similarly, the percentage of S-phase fraction tends to be higher according to the level of severity or dysplasia. These findings demonstrate that the aneuploidy and percentage of S-phase fraction tend to correlate with progression of esophageal premalignant tissues to invasive carcinoma. These measures may be clinically useful to identify patients at increased risk for esophageal malignancy.

Aged↗

PubMed1992

[Diagnostic value of measurement of RNA in platelets by fluorescence flow cytometry].

We analyzed the RNA in platelets by fluorescence flow cytometry after staining with thiazole orange(TO) in whole blood samples from hematologically normal subjects and patients with thrombocytopenia. The percentage of TO-positive platelets and their mean fluorescence channel number in 32 control subjects were 6.2 +/- 2.5% (mean +/- SD) and 6.9 +/- 0.7, respectively. In 11 patients with idiopathic thrombocytopenic purpura, the percentage of fluorescently labeled platelets was significantly elevated (p less than 0.05) to 21.5 +/- 14.3%. By contrast, the proportion of positively stained platelets in 14 patients with thrombocytopenia due to impaired platelet production did not significantly differ from that of the controls, whereas the absolute counts of TO-positive platelets were significantly lowered (p less than 0.05). In both patient groups, the mean fluorescence channel numbers of TO-positive platelets were significantly elevated to 16.1 +/- 16.8 and 6.9 +/- 0.7, respectively (p less than 0.05, 0.005). We conclude that flow cytometric analysis of platelets after staining with TO provides information on the thrombopoietic activity in thrombocytopenic disorders. The main advantages of this method for clinical use are its simplicity and the rapidity.

Blood Platelets↗

PubMed1996

Fluorescent probes and flow cytometry: new insights into environmental bacteriology.

Recent trends in flow cytometry have established new techniques in bacteriology. Advances in fluorescent dye technology complement these improvements, offering probes for a variety of cellular functions. Bacterial ecology requires the application of new techniques to help answer questions unanswerable by traditional methods alone. Here we review some aspects of how coupling the two technologies has enabled researchers to directly study individual bacterial cells, and revealed the complexity and heterogeneity present in both laboratory cultures and in environmental samples. Results are discussed with respect to viability analysis, stress induced changes, specific cell detection and cell sorting.

Bacteriological Techniques↗

PubMed1992

Longer in vivo survival of CD59- and decay-accelerating factor-almost normal positive and partly positive erythrocytes in paroxysmal nocturnal hemoglobinuria as compared with negative erythrocytes: a demonstration by differential centrifugation and flow cytometry.

Three populations of erythrocytes have been shown by flow cytometric analysis on complement regulatory proteins: CD59 and decay-accelerating factor (DAF) on erythrocytes in paroxysmal nocturnal hemoglobinuria (PNH). CD59 and DAF in PNH may be completely deficient in CD59- and DAF-negative erythrocytes, they may be decreased varyingly in partly positive erythrocytes, and they may be approximately normal in almost normal positive erythrocytes. Control erythrocytes are always CD59- and DAF-normal positive. CD59- and DAF-negative erythrocytes have been shown to be most sensitive to complement lysis in vitro. However, it has not yet been elucidated whether CD59- and DAF-almost normal positive and partly positive erythrocytes in a patient have a longer in vivo survival than negative erythrocytes. Blood from controls and PNH patients was separated in five fractions by differential centrifugation. CD59 and DAF on the fractionated erythrocytes were determined by flow cytometry using specific antibodies. Ratios of CD59- and DAF-almost normal positive and partly positive cells to negative erythrocytes were increased progressively from the top fraction to the bottom. The erythrocytes in the top fraction are younger and reticulocyte-rich, while those in the bottom are older and reticulocyte-poor. Hence, the present results indicate that CD59- and DAF-partly positive erythrocytes as well as almost normal positive erythrocytes in patients may have a longer in vivo survival than negative erythrocytes.

Antigens, CD↗

PubMed1992

Flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin, its subunits, and fragments on human cancer cells.

A quantitative flow cytometry method for the analysis of membrane-associated human chorionic gonadotropin (hCG), its subunits, and fragments on human cancer cells was developed using a double-antibody reaction; a flow cytometry with a 2-W argon laser, standard settings, and filters for fluorescein isothiocyanate use; commercially available software; and the ectopic hCG producer CCL 2 HeLa cells from the American Type Culture Collection (ATCC) as a cell control to standardize the reagents and for overall quality control. Twenty-two monoclonal antibodies (MoAb) and immunoglobulin G fractions from three rabbit polyclonal antisera were tested for effects of antibody concentration (titration), reproducibility at different levels of epitope expression, and variability of epitope expression to select appropriate primary antibodies. Based on the results of the various tests, three polyclonal immunoglobulin G antibodies and a panel of nine MoAb directed to epitopes located in five different regions on the hCG molecule were selected as first antibodies. Their specificity was determined by using two unrelated MoAb of the same isotype at the same concentration to replace the primary MoAb and by a competition experiment. The unrelated MoAb also were used for the selection of the appropriate control fluorescence profile needed for the software. The unique characteristics of this method were: the use of living cells, standardized reagents, internal and external quality control, and the highest sensitivity, which could detect as few as 10(3) molecules of fluorochrome per cell. Serial analyses of the ATCC CCL 2 HeLa cells and two of its variants and of the eutopic hCG producer JEG-3 choriocarcinoma cells revealed the expression of membrane-associated epitopes of intact hCG, its subunits, and fragments by a high percentage of the cells, indicating that the expression of these sialoglycoproteins by these two different types of cancer cells is a common phenotypic characteristic.

Adenocarcinoma↗

PubMed2000

Quantitation of spermatogenesis by DNA flow cytometry from fine-needle aspiration cytology material.

DNA flow cytometry (FCM) was performed from fine-needle aspiration cytology (FNAC) of testis in 15 cases of male infertility to quantitate spermatogenesis. The results were correlated with FNAC findings. DNA FCM showed a ploidy relationship of haploid (1N) > diploid (2N) > tetraploid (4N) in cases of normal spermatogenesis. A ploidy relation of 2N > 1N > 4N was observed in cases of hypospermatogenesis or maturation arrest. In Sertoli cell-only cases, there were only 2N populations of cells. With the help of DNA FCM, a rapid and objective assessment of spermatogenesis is possible from FNAC of the testis.

Biopsy, Needle↗

PubMed1994

DNA index and S-phase in primary brain tumors. A comparison between fresh and deparaffinized specimens studied by flow cytometry.

Forty-nine primary intracranial tumors (33 astrocytomas, eight acoustic schwannomas, five oligodendrogliomas, and three miscellaneous tumors) were studied by flow cytometry. Each tumor specimen was divided into two portions: one was studied as an unfixed suspension stained with propidium iodide and the other after formalin fixation and paraffin embedding. The 50-microns sections from the paraffin blocks were deparaffinized, rehydrated, and enzymatically disintegrated, and the cells in suspension were stained with propidium iodide. Flow cytometry of the two portions showed a significant correlation between fresh and fixed specimens regarding the S-phase (r = 0.87) and a very close correlation fo the deoxyribonucleic acid (DNA) index (r = 0.95). When the 33 astrocytomas were analyzed separately, similar results were obtained (r = 0.86 for S-phase and r = 0.93 for DNA index, respectively). This study demonstrated a high correlation between fresh and fixed specimens for DNA ploidy and S-phase both in primary intracranial tumors in general and also in the selected subgroup of astrocytomas.

Adolescent↗

PubMed1993

Flow cytometry and RNA studies.

RNA was less studied by flow cytometry (FCM) in comparison to other cellular components. Moreover, most of these studies were applied to the measurement of total RNA. Nevertheless, such a quantification, essentially by the use of acridine orange, but also other dyes or dye combinations, permitted the acquisition of important information about the general metabolism, and the characterization of different tumoral cell types. More recently, FCM has been used to quantify specific mRNA, in two different approaches: the first one employs the sorting followed by RNA extraction and/or detection by specific radiolabeled probes; in the second approach, the use of fluorescence in situ hybridization (FISH) on cells in suspension allowed the direct quantification of specific mRNA by FCM.

Acridine Orange↗

PubMed2004

A simple assay for frequency of chromosome breaks and loss (micronuclei) by flow cytometry of human reticulocytes.

Exposure to environmental stress, such as radiation, poor nutrition, or smoking, can cause hazardous lesions in DNA, including double-strand breaks. In red blood cells, a DNA fragment or lagging chromosome forms a micronucleus when left behind after the main nucleus is extruded to form the mature reticulocyte during erythropoiesis. Reticulocytes with micronuclei in human peripheral blood are not generally available for analysis because the spleen removes aberrant cells. We have developed a simple and rapid method to isolate and analyze immature reticulocytes in the peripheral blood for the presence of micronuclei before these cells are removed by the spleen. This method applies single-laser flow-cytometry to measure micronuclei in an enriched transferrin-positive reticulocyte population. Abramsson-Zetterberg et al. (Abramsson-Zetterberg, L., Zetterberg, G., Bergqvist, M., and Grawe, J. Environ. Mol. Mutagen. 36, 22-31, 2000) have described a method to measure micronuclei in an enriched reticulocyte population using a dual-laser flow cytometry. Unlike the beads used in their magnetic-immunoseparation procedures, the beads used in this study do not require a prelabeling step and are compatible with the flow cell, sparing the need to release the cells from the beads and avoiding the potentially confounding DNase-treatment step. Dertinger et al. (Dertinger, S. D., Torous, D. K., Hall, N. E., Murante, F. G., Gleason, S. E., Miller, R. K., and Tometsko, C. R. Mutat. Res. 515, 3-14, 2002; Dertinger, S. D., Chen, Y., Miller, R. K., Brewer, K. J., Smudzin, T., Torous, D. K., Hall, N. E., Olvany, K. A., Murante, F. G., and Tometsko, C. R. (2003) Mutat. Res. 542, 77-87, 2003) further improved the scoring of micronuclei to enable the use of bench-top instruments in analyzing samples of unenriched reticulocyte-populations. The present method is distinct from flow cytometric assays, such as reported by Dertinger et al., which enable scoring of limited numbers of reticulocytes per sample and require lengthy data acquisition times. We assessed DNA damage in smokers using this novel flow-cytometry based micronuclei-assay. The results show that this assay can effectively detect micronuclei in human blood samples. This method, unlike available micronuclei assays, allows rapid evaluation of a large number of cells and therefore should prove to be useful in monitoring of human populations for genetic damage.

Adult↗

PubMed1989

"Benign" metastasizing giant cell tumor: evaluation of nuclear DNA patterns by flow cytometry.

Nuclear deoxyribonucleic acid (DNA) ploidy was determined by flow cytometry for nine histologically benign giant cell tumors that developed systemic metastases and for eight tumors that did not metastasize. Specimens from the primary tumor, local recurrences, and pulmonary metastases were evaluated. No feature of the DNA ploidy pattern was identified to distinguish giant cell tumors that metastasized from those that did not. The mean percentage of diploid (G0/G1 peak, 2C) cells was 81% in the metastasizing group and 80% in the nonmetastasizing group. The DNA ploidy pattern of the primary tumors was not different from that of their metastases. No DNA aneuploid patterns were observed among the benign tumors.

Cell Nucleus↗

PubMed1994

Ploidy of 36 stromal tumors of the gastrointestinal tract. A comparative study with flow cytometry and image analysis.

DNA ploidy was investigated by flow cytometry (FC) and image analysis (IA) in paraffin-embedded tissue sections from 36 stromal tumors of the gastrointestinal tract. The results of both techniques were correlated with pathologic features of the tumors and survival. Ten (27.8%) tumors were aneuploid by FC and IA. Most of the diploid tumors were identified by both techniques, but FC appeared to be superior to tissue section IA for identification of aneuploid tumors (25% vs. 13.8%). Aneuploidy by FC correlated with pathologic grade and mitotic index (P < .05), and a trend to short survival was also detected (P < .1). No similar correlation was found by IA. Enlargement and variation of nuclei may explain the discrepancy between FC and IA.

Flow Cytometry↗

PubMed1991

DNA flow cytometry in the prognosis of primary malignant melanoma.

DNA flow cytometry was carried out on 804 primary melanomas. The data were analyzed with a follow-up of 24-96 months. 57% of the cases were diploid, 32% had one abnormal cell population, and 11% were multiclonal. In 8% of the aneuploid tumors there were cell lines in the hypertetraploid range. A reliable S phase determination was possible in 524 cases. Among these 11% had an S phase exceeding 15%. Using an increased tumor thickness, relapse rate and mortality as criteria of tumor progression, aneuploidy and multiclonality, the occurrence of hypertetraploid cell lines and a high S phase (greater than 15%) proved to be correlated with a poor prognosis.

Adolescent↗

PubMed2005

Flow cytometry of fine-needle-aspiration biopsies: a new method to monitor the intrahepatic immunological environment in chronic viral hepatitis.

SUMMARY: Information about the character and grade of the intrahepatic immune response in viral hepatitis is important for the evaluation of disease stage and effect of therapy. Complications like haemorrhage limit the frequent performance of tissue-needle biopsies (TB), and the cells of peripheral blood have to be used as surrogate markers instead. Fine-needle-aspiration biopsy (FNAB) of the liver represents a safe and atraumatic method that allows frequent cytological sampling. Our aim was to investigate whether flow cytometry of FNAB specimens allows co-analysis of phenotype, function and specificity of key populations of liver-infiltrating lymphocytes (LIL). In 20 consecutive patients with chronic viral hepatitis [10 hepatitis B virus (HBV), 10 hepatitis C virus (HCV)], flow cytometry was performed on FNAB cytology, and simultaneously on lymphocytes isolated from a TB and peripheral blood mononuclear cells (PBMC). The ratio of CD8+/CD4+ lymphocytes in FNAB correlated well with LIL from TB (r =0.78, P < 0.05) but differed from PBMC (mean ratio: 2.6, 2.1 and 0.7, respectively). Similarly, a correlation was observed for percentage CD56+ natural killer (NK) cells (mean %: 29.9, 32.3 and 14.5, respectively; r = 0.69, P < 0.05). The percentage of interferon (IFN)-gamma-producing CD3+ lymphocytes in both FNAB and TB was higher than in PBMC (mean %: 41, 44 and 22, respectively; P < 0.05). Furthermore, tetrameric complexes allowed analysis of HBV-specific T cells in FNAB specimens. In conclusion, flow cytometry of FNAB allows easy, atraumatic and reliable analysis of lymphocytes obtained from the intrahepatic compartment. Therefore, the FNAB is a valuable tool in the study of the immunopathology of viral hepatitis, and it may contribute to the improved clinical evaluation of chronic viral liver disease.

Biopsy, Fine-Needle↗

PubMed1987

Solid tumor preparation for flow cytometry using a standard murine model.

The application of flow cytometry (FCM) to solid human tumors has been hindered by the difficulty in producing high yield, viable, unaltered single cell suspensions. Carcinomas containing a high desmosomal content, such as well-differentiated squamous cell (SCC) cancers of the head and neck (H&N) region, are particularly difficult to prepare. The desire to employ FCM to study cellular DNA parameters of these tumors led to the use of a 3-methylcholanthrene induced murine SCC for the comparative testing of preparative techniques. Dissociation techniques, including mechanical, enucleation, chemical, single and combination enzymes methods, were comparatively tested. Of these, the combination enzyme treatment employing trypsin and collagenase produced the highest cell yields in the shortest time with the highest dye exclusion viability and the least expense. Several fixation systems including glutaraldehyde, paraformaldehyde, acetic acid, and ethanol were comparatively tested using percent of cell loss and quality of the DNA histograms produced as end points. Ethanol-water systems with added fetal calf serum provided minimal cell loss and high quality histograms which were stable for extended periods of time. A murine tumor, closely mimicking the histology of the human tumor of interest, may be used as a model for the determination of optimum techniques of solid tumor preparation for flow cytometric analysis.

Animals↗

PubMed2005

Rapid identification of Candida species and other clinically important yeast species by flow cytometry.

Two rapid diagnostic assays, utilizing two different Luminex flow cytometry methods, were developed for identification of clinically important ascomycetous yeast species. Direct hybridization and allele-specific primer extension methods were both successful in establishing a DNA-based assay that can rapidly and accurately identify Candida albicans, Candida krusei, Candida parapsilosis, Candida glabrata, and Candida tropicalis as well as other clinical species. The direct hybridization assay was designed to identify a total of 19 ascomycetous yeast species, and the allele-specific primer extension assay was designed to identify a total of 34 species. Probes were validated against 438 strains representing 303 species. From culture to identification, the allele-specific primer extension method takes 8 h and the direct hybridization method takes less than 5 h to complete. These assays represent comprehensive, rapid tests that are well suited for the clinical laboratory.

Candida↗

PubMed2002

Cytohistologic discrepancies: a means to improve pathology practice and patient outcomes.

The use of cytohistologic discrepancies to investigate and reduce error seldom is studied. All gynecologic discrepancies (n = 283; 0.87% and 7.37% of all cytologic and histologic cases, respectively) and nongynecologic discrepancies (n = 146; 2.26% and 0.44% of all cytologic and histologic cases, respectively) for 26 months were classified as sampling or interpretive. Specimen type and pathologist discrepancy percentages, effect of discrepancies on patient outcome, and interobserver agreement of discrepancies were evaluated. Discrepancies were interpretive in 67% and 34% of gynecologic and nongynecologic cases, respectively. Statistically significant associations were seen between individual pathologist and discrepancy percentages. Breast (1.2%) and bronchial (0.8%) cytologic diagnoses had the highest discrepancy percentages. The kappa scores ranged from 0.02 to 0.45 for pairwise agreement of discrepant cases. Of nongynecologic interpretive discrepancies available for review, 63% (27/43) and 14% (6/43) were of no or minor clinical significance, respectively. Cytohistologic correlation is a useful tool to monitor performance and to identify specimen types prone to error.

Biopsy↗

PubMed1988

Urine: a suitable sample for deoxyribonucleic acid flow cytometry studies in patients with bladder cancer.

Deoxyribonucleic acid flow cytometry of bladder washings has proved to be a valuable procedure for the diagnosis and followup of patients with transitional cell carcinoma. However, for this procedure to gain maximal acceptance, it should be possible to use voided urine specimens instead of bladder washings. To evaluate this possibility we compared histogram findings for 114 bladder washings and 122 concomitantly obtained urine samples (voided and catheterized) from 89 consecutive patients who had active or a history of transitional cell carcinoma. Unsatisfactory histograms were obtained in 4 per cent of the urine samples and in 2.6 per cent of the bladder washing samples. The satisfactory rate for voided or catheterized urine samples was the same. We conclude that in this patient population satisfactory deoxyribonucleic acid histograms can be obtained from samples of voided urine.

Carcinoma, Transitional Cell↗