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PubMed1988

New sensitivity test using flow cytometry.

Flow cytometry (FCM) has been used to evaluate not only the malignancy of tumor cells but also the effects of chemotherapy. Here a new application of FCM for selecting the best antineoplastic agent in the chemotherapy for brain tumors is reported. Through our preliminary study using established brain tumor cell lines, the system for this sensitivity test was developed. Antineoplastic agents were placed in contact with monolayer-cultured cells; then cell viability and changes in the DNA histogram were analyzed by FCM. Cell viabilities were measured with the fluorescein diacetate (FDA) staining method, and the DNA histogram was analyzed by the propidium iodide (PI) staining method. The best antineoplastic agent was determined based on changes in cell viability and cell cycle. In other words, when markedly decreased viability as compared with that of the control, is measured by FCM, then the agents can be considered to have had a cytocydal effect on the tumor cells, and thus the sensitivity of the agents is able to be evaluated. If the viability of the tumor cell is observed to be similar to that of the control, the cytostatic effects of the agents are able to be evaluated only if a marked change is observed in the DNA histogram. After the preliminary study, this system was applied clinically to malignant brain tumor cases, resulting in success in selecting the best antineoplastic agent for each individual case. Our sensitivity test using this FCM established in vitro system has much potential value for clinical use.

Brain Neoplasms↗
PubMed1992

[Studies on nuclear DNA content in testicular germ cell tumors using flow cytometry].

Flow cytometric DNA analysis was done in 76 patients of testicular germ cell tumors (GCTs) experienced in our clinic for evaluating the clinical relevance of DNA index (DI), clarifying those biological features and shedding some insights in the pathogenesis of testicular GCTs. Histological type and its incidences were seminomas in 35 adults, nonseminomatous germ cell tumors (NSGCTs) in 24 adults and prepubertal germ cell tumors (P-GCT) in 17 boys (9 in yolk sac tumors and 8 in teratomas). Totally 190 samples of paraffin embedded materials (with a mean of 2.5 samples per case) were histologically reconfirmed and employed the flow cytometry analysis with some modification of the Hedley's technique for analyzing the DNA ploidy and DIs. Coefficient of variation (CV) were acceptably ranged from 3 to 10%. In 58 evaluable cases with adult GCTs, 57 (98%) were revealed as DNA aneuploid, while DNA diploid tumor were observed in only one case with NSGCT. On the other hand, all specimens showed DNA euploid in P-GCT; DNA diploid in all teratomas and 6 yolk sac tumors, DNA tetraploid in other 3 yolk sac tumors. DNA aneuploid pattern was not apparently detected in children. Even in one adult case with pure yolk sac tumor and two adults with mature teratomas, all specimens were classified as DNA aneuploid. These results support the present hypothesis that the pathogenesis of P-GCTs is different from that of adult GCTs. DIs in adult NSGCTs (median DI = 1.56) are significantly lower than those in adult pure seminomas (median DI = 1.85) (p < 0.01). Although there was no significant correlation among the DIs in NSGCTs and the clinical staging of Japanese Urological Association, the distribution of DIs in NSGCT patients of the advanced extent were lower than that of the other extents of NSGCTs on the basis of Indiana University staging system (p < 0.05). In general, it has been postulated that the higher DI is paralleled to the more malignant nature of neoplasms. Nevertheless, this study conversely suggested that the lower DI in adult testicular GCTs is apt to related with high malignant potential determined by histological type and clinical stage. DNA heterogeneity was observed only in 4 of 23 cases with adult NSGCTs (17%), but 3 of these 4 cases (75%) with DNA heterogeneity were assigned as the advanced extent. These data suggest that the lower DI and the presence of DNA heterogeneity may have prognostic relevance for adult NSGCTs.

Adolescent↗
PubMed1979

DNA distribution in biopsy specimens from human cervical carcinoma investigated by flow cytometry.

Flow cytometry was used for the investigation of the DNA distribution in biopsy specimens from 51 patients with cervical carcinoma. Portio biopsy specimens from 9 pregnant women and from 10 patients with cancer of the breast served as controls. The results demonstrate that most specimens from patients suffering from cervical carcinoma contain considerable cell populations with increased DNA as compared with controls. The possible clinical significance of these findings is discussed.

Biopsy↗
PubMed1999

Enumeration of marine viruses in culture and natural samples by flow cytometry

Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80 degreesC in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r = 0.937, n = 14, and r = 0.96, n = 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.

Journal Article↗
PubMed1978

High resolution dual laser flow cytometry.

Flow cytometers based on optical sensing utilize external light sources and fluorescent dyes to measure one or more specific components or properties of individual cells or subcellular particles in liquid suspension. To provide for independent excitation of two dyes used in double staining experiments we have constructed a high resolution flow cytometer that uses two laser beams to provide two wavelengths of excitation. These beams are separated spatially so that cells flow through them sequentially, with a time separation of about 20 musec. Since the dyes are excited sequentially their emission occurs at different times and their emission spectra may overlap without causing any difficulty in analysis. We have developed new light collection optics that permit up to four measurements to be made on each cell. This approach greatly increases the number of dye combinations that can be used in flow cytometry, thus removing a significant limitation of single illumination instruments.

Animals↗
PubMed1987

Analysis of heavy metal immunotoxicity by multiparameter flow cytometry: correlation of flow cytometry and immune function data in B6CF1 mice.

Bone marrow and spleen cells obtained from female B6C3F1 mice given a single i.p. exposure to cadmium acetate (0.9 mg/kg), lead acetate (12 mg/kg), or sodium acetate (12 mg/kg), were studied using flow cytometry, immunologic, and hematologic assays. Significant changes were detected in subpopulations of bone marrow cells using multiparameter flow cytometry within 1 day following treatment with cadmium or lead. Bone marrow cells obtained from B6C3F1 mice 5 days after treatment with cadmium or lead were found to have a decreased number of cells expressing Mac-1, 55-7.2, 14.8, and Lyt-1 antigens, suggesting a shift to immature cell types. An increase in the number of progenitor cells (CFU-C) obtained from the bone marrow of mice treated with heavy metals was also noted 5 days after exposure to cadmium or lead. A time-dependent suppression of the in vitro primary humoral immune response of spleen cells to SRBCs, TNP-Ficoll and TNP-LPS was produced by cadmium or lead treatment. Suppression of the mitogenic response of spleen cells to Con A, PHA, and LPS was also found to be time-dependent. Spleen cell surface marker expression (Mac-1, Lyt-1, Lyt-2 and 14.8) was altered in response to cadmium or lead treatments, but these changes did not appear to correlate with the humoral immunity or mitogen-induced proliferation data. These studies demonstrate that changes in cell surface markers on discrete subpopulations of lymphoid cells present in the spleens of heavy metal exposed mice may not correlate with alterations in the functional activity of these cells. However, changes in murine bone marrow surface markers in response to cadmium or lead treatment predicts a shift to immature cell types, which appeared to correlate with the increase in CFU-C activity.

Acetates↗
PubMed1997

Ki-67 labeling indices in non-small cell lung cancer: comparison between image cytometry and flow cytometry.

Expression of the proliferation-associated nuclear antigen Ki-67 was studied in 27 human non-small cell lung cancers (NSCLCs). We measured immunohistochemical Ki-67 labeling indices using image cytometry (ICM) and flow cytometry (FCM) and compared the indices determined with the two methods. Ki-67 labeling indices of tumor cells ranged from 2% to 59% with ICM, and from 3% to 56% with FCM, varying from case to case. There was a linear correlation of values between the two methods (r = 0.77, P < 0.05). To examine whether Ki-67 labeling indices represent tumor proliferative activity, we studied the relationship between the Ki-67 labeling index and the S-phase fraction determined by FCM. There was a positive correlation between the Ki-67 labeling index and the S-phase fraction (r = 0.91, P < 0.01). Comparison of Ki-67 labeling indices and clinicopathological parameters showed lower Ki-67 labeling indices in well-differentiated tumors than in moderate and poorly differentiated tumors (P < 0.05). No correlation was observed between Ki-67 labeling indices and p53 expression. It was concluded that the Ki-67 labeling index was in fact measured with both ICM and FCM, and that it represents tumor proliferative activity of NSCLCs.

Adenocarcinoma↗
PubMed1993

Quality control study of the Italian group of cytometry on flow cytometry cellular DNA content measurements.

A quality control study on DNA flow cytometry, extended to 43 national laboratories, has been carried out by the Italian Group of Cytometry, using defined fixed suspensions of cultured human leukemia K562 cells and human blood lymphocytes. The participating laboratories were allowed to follow their own staining and measurement protocols. Aliquots of cellular suspension had to be measured three times on the same day and two other times on different days. A large heterogeneity of procedures emerged among participants. The average of mean DNA index laboratory values, from 36 laboratories who sent evaluable data, was 1.68, with a range from 1.49 to 1.97. The coefficients of variation ranged from 2.35 to 9.39% and from 2.79 to 8.5% for diploid and aneuploid peaks, respectively. Statistical analysis of the results showed quite good intralaboratory reproducibility, but statistically significant differences were observed among laboratories, for both DNA indices and coefficients of variation. These differences appear to be consistent. For standardization, it is essential that efforts should be made to identify the main sources of variation and to control them.

Analysis of Variance↗
PubMed1997

Quality control study of the Italian Group of Cytometry on flow cytometry DNA content measurements: II. Factors affecting inter- and intralaboratory variability.

A multicentric national quality control study has been organized under the auspices of the Italian Group of Cytometry to find a possible influence of some procedural steps in DNA flow cytometry measurements on DNA index (DI) values and to identify the main parameters affecting the interlaboratory variability. To 40 participating laboratories we provided suspensions containing unknown mixture of different cell types: an homogeneous thymocyte population used to check instrument linearity; one mixture composed of two cell types characterized by DI = 1.00 and 1.10; and another composed of three different cell types with relative DIs of 1.00, 1.26, and 1.62, respectively. Possible effects due to staining protocols were studied, allowing the participants to stain cellular DNA according to the procedure routinely adopted in each laboratory, in addition to a standardized procedure with a fixed PI solution. As far as the influence of instrument linearity on DI values is concerned, we did not find any correlation with the DI variability observed, even if the use of a standardized staining protocol could lead to a sensible gain in interlaboratory DI reproducibility. Twenty-five of 40 (65%) laboratories were able to discriminate the near-diploid subpopulation, and a coefficient of variation of less than 4% was the minimum condition necessary to recognize the DI = 1.1 population. In samples containing two aneuploid subpopulations, 25 of 35 (71.4%) laboratories showed a high reproducibility with the standard staining protocol and 22 of 38 (57.9%) with the free staining protocol. However, a sensible improvement in interlaboratory reproducibility emerged with respect to the previous trial.

Animals↗
PubMed2006

Identification and purification of classical Hodgkin cells from lymph nodes by flow cytometry and flow cytometric cell sorting.

We demonstrate the feasibility of using flow cytometry (FC) to identify the Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (CHL). Initial flow cytometric studies of the HRS cell line L1236 demonstrated potentially useful antigens for identifying HRS cells. L1236 cells spontaneously bound normal T cells, analogous to the T-cell rosetting of HRS cells seen in tissue sections of CHL, but these interactions could be blocked by using a cocktail of unlabeled antibodies to 4 adhesion molecules. Among 27 lymph nodes involved by CHL, FC enabled HRS cells to be identified in 89%, whereas none of 29 non-CHL neoplasms or 23 reactive lymph nodes demonstrated HRS populations. Of the CHL cases, 82% demonstrated interactions between HRS cells and T cells that could be disrupted with blocking antibodies. Flow cytometric cell sorting experiments demonstrated typical HRS cytomorphologic features among the purified cells. FC may offer an alternative to immunohistochemical analysis in confirming the diagnosis of CHL in certain cases, and, through cell sorting, it provides a means of rapidly isolating pure HRS cells.

Antibody Specificity↗
PubMed1994

Comparative DNA analysis by image cytometry and flow cytometry in non-small cell lung cancer.

To determine whether image cytometry (ICM) is advantageous for clinical DNA analyses of tumor cells, nuclear DNA contents measured by ICM were compared with those by flow cytometry (FCM), using 46 samples of non-small cell lung cancers. ICM was performed on smear specimens of fresh materials (f-ICM) and cell suspensions obtained from paraffin-embedded tumors (p-ICM). The same cell suspensions were also analyzed by FCM (p-FCM). Aneuploid rates/coefficient of variation (CV) of f-ICM, p-ICM, and p-FCM were 76.1/4.90, 71.7/5.01 and 60.9/5.31%, respectively. There was a high correlation in the DNA indices between p-ICM and p-FCM (r = 0.80). In the comparative DNA analysis, there were seven discordant samples. Six of them were estimated as aneuploid by p-ICM, but they were miscounted as diploid or undefinable (impossible) by p-FCM. This was caused by measuring condensed nuclei or debris. All "impossible" samples in p-FCM were squamous cell carcinoma with necrosis. In cell cycle analysis, the S and S+G2/M phase fractions in diploid samples were higher in p-ICM than those in p-FCM (P < 0.005), because the G0/G1 phase (2N) fraction presented by FCM was composed of cancer and non-malignant cells in diploid cancers. In ICM, they can be separately measured by means of morphological selection. These findings indicated that ICM is superior to FCM, especially for the practical DNA measurement of a few cancer cells and in the evaluation of the proliferation rates.

Adult↗
PubMed1996

Toxic action of copper on the membrane system of a marine diatom measured by flow cytometry.

Flow cytometric measurements were used to investigate the toxic action of copper on some Phaeodactylum tricornutum membrane systems. Throughout the time of metal exposure, the percentage of viable cells decreased as copper concentration increased. The forward scatter signal increased as a result of copper exposure. After 72 h of metal exposure, cultures with 0.5 and 1 mg l-1 of copper showed an important increase in the peroxidase activity in comparison with control cells. Cells cultured with copper presented alterations in the membrane potential, increasing as copper concentration increased, after 96 h of metal exposure. Results obtained in this work showed that copper induced a degenerative process in P. tricornutum cells, closely related with alternations or disorders in membrane systems.

Cell Membrane↗
PubMed1983

Parametric analysis of histograms measured in flow cytometry.

Flow cytometric histograms frequently consist of several components that show various degrees of overlap. For many types of analysis it is of great importance to decompose the original histogram into its components. To that purpose, we investigated the maximum likelihood approach in detail. It is shown that the iterative method to solve the maximum likelihood equations is well behaved for a variety of initial values. Algorithms to obtain initial values are presented, and the performance of the method is tested when applied to the analysis of DNA measurements from heterogeneous cell populations that differ with respect to DNA content.

Animals↗
PubMed1987

Improved methodology for intracellular enzyme reaction and inhibition kinetics by flow cytometry.

Flow cytoenzymology is the determination of enzyme activities or concentrations in single intact cells. Using the flow cytometer built and designed in our laboratory and recent modifications to hardware and software, we have developed an improved dynamic flow cytoenzymological procedure for the assay of cellular enzyme kinetics. The reaction mixture is sampled continuously, and the computer clock incorporates time as a parameter for kinetic determinations. Conditions for cellular esterase analysis were optimized and the rates of hydrolysis of two fluorogenic substrates, fluorescein diacetate (FDA) and 4-methylumbelliferone acetate (MUA), by esterases in EMT6 mouse mammary tumor cells were studied. Reaction kinetics were characterized, and Km values of 19 and 72 microM were obtained for the hydrolysis of FDA and MUA respectively. The kinetics of the cellular efflux of fluorescein were investigated, and a half-life of 7.5 min obtained. Enzyme inhibition kinetics were investigated using the competitive substrates p-nitrophenyl acetate and phenyl acetate, and the carbamoylating agents physostigmine and n-butyl isocyanate. The latter was particularly potent with an I50 of 4.8 X 10(-5) M for FDA hydrolysis compared with 6.5 X 10(-3) M for physostigmine. The I50 of 8.8 X 10(-5) M for n-butyl isocyanate inhibition of MUA hydrolysis was similar to that obtained with FDA as substrate. By monitoring FDA and MUA reactions separately and simultaneously, we showed them to act as competitive substrates. A comparison of flow cytoenzymological and conventional spectrofluorimetric analysis was also made, and differences identified in some cases.

Animals↗
PubMed1989

Discrimination of eukaryotic phytoplankton cell types from light scatter and autofluorescence properties measured by flow cytometry.

Flow cytometric methods for recognizing several groups of eukaryotic marine phytoplankton were tested using 26 laboratory cultures. Each culture was divided into three aliquots, and these samples were analyzed for 1) Coulter volume; 2) light scatter (magnitude and polarization properties of forward scattered light and magnitude of right-angle scattered light) and autofluorescence emission (phycoerythrin and chlorophyll); and 3) autofluorescence excitation (by 488 nm and 515 nm light). Three kinds of cells could be easily distinguished from others in the culture collection: 1) The two cryptophytes and the rhodophyte had high phycoerythrin/chlorophyll ratios; 2) the two coccolithophores depolarized forward scattered light; and 3) the two pennate diatoms scattered only a relatively small amount of light in the forward direction compared with that at right angles. Mean chlorophyll fluorescence excited by blue light relative to that excited by green light was highest in the four chlorophytes, but there was overlap between some of these and some other kinds of cells. Unresolved cell types included centric diatoms, dinoflagellates, and naked coccolithophores. Forward light scatter and Coulter volume were closely related (except for the pennate diatoms) over a range of about 0.01 to 30 pL (equivalent spherical diameter about 3 to 40 microns), according to a logarithmic function.

Cells↗
PubMed1991

Pertussis toxin effects on chemoattractant-induced response heterogeneity in human PMNs utilizing Fluo-3 and flow cytometry.

Flow cytometric methods were utilized to determine N-formylpeptide-induced cytosolic calcium levels in human polymorphonuclear leukocytes (PMNs) detected with the calcium indicator Fluo-3. Fluo-3 was readily loaded into PMNs as the acetoxymethyl ester. At room temperature Fluo-3 extrusion was minimal (less than 10%) over a 2 h time period. Flow cytometric histograms yielded symmetric distributions indicating homogeneous labelling of the cells. Stimulation of the cells with N-formyl-met-leu-phe (FMLP) caused homogeneous activation of all cells as indicated by a shift of the fluorescence distribution to higher fluorescence levels while still maintaining a symmetrical distribution. Resting values or FMLP-induced cytosolic calcium levels were similar in cells loaded over a 20-fold range of Fluo-3-acetoxymethyl ester. The effect of graded pertussis toxin (PT) treatment on the calcium response was determined by incubating cells with different concentrations of pertussis toxin for a time period that yielded a range of ADP ribosolation levels inside the cells. When these cells were activated with FMLP, the fluorescence histograms showed that pertussis toxin treatment resulted in a conversion of cells from responders to nonresponders. The responding cells responded with maximum calcium elevations similar to controls. This behavior may reflect heterogeneous insertion of the A-protomer of PT or a very sharp threshold of coupled G-proteins required to transduce the responses.

Aniline Compounds↗
PubMed1991

Measuring potential doubling times of murine tumors using flow cytometry.

Flow cytometric analysis of bromodeoxyuridine (BrdUrd) labeled cells has led to the description of various methods for determining population potential doubling times (Tpot) from samples obtained at a single time point. There have been several reports of results from human tumor samples using these various methods of analysis, but little documentation of the validity of these approaches in experimental tumor models. Recently, we reported results using in vitro cell cultures and determined the most reliable method for these. In this paper we report the results of this methodology when applied to two in vivo murine tumor models (MCaK and FSA). The analytic approach tested is summarized as follows: The relative movement (RMlu(t], a measure of the mean DNA fluorescence of BrdUrd-labeled cells that have not undergone division at the time of sampling, and a quantity v, a function of the fractions of BrdUrd-labeled divided and undivided cells, were measured for a series of time points following labeling. From each value of RMlu(t) an estimate of the length of S-phase (TS) was computed and from each v and TS a value for Tpot was found. These results were compared to the values of TS and Tpot obtained by fitting all the values simultaneously. Tumor MCaK values of TS and Tpot of 9.6 and 28.0 h were obtained from fitting all of the data. Tumor FSA gave values of 16.8 and 42.3 h for TS and Tpot, respectively. The results of this analysis show that single time point measurements can give reliable estimates of TS and Tpot.

Animals↗