Flow cytometric determination of estrogen receptors in intact cells.

Flow cytometry has been used to detect estrogen receptor (ER) in intact cells, using 1-(N)- fluoresceinyl estrone thiosemicarbazone ( 17FE ), a ligand shown to have sufficient affinity and specificity to identify high-affinity receptors. For each concentration of 17FE , two fluorescent distributions were obtained: (a) "total" fluorescence due to 17FE alone; and (b) diethylstilbestrol-inhibited fluorescence ("nonspecific"). The mean fluorescence intensity for both the "total" (MT) and "nonspecific" (MN) distributions was calculated at each ligand concentration by obtaining the total brightness of each sample, normalized to the number of cells analyzed. A specific binding curve was constructed over a broad range of 17FE concentrations (1.5 X 10(-9) to7 .5 X 10(-7) M) by plotting the mean specific fluorescence intensity (MS = MT-MN) at each 17FE concentration. Saturable binding was demonstrated for a known ER-positive cell line (MCF-7) at low ligand concentrations (5 to 10 X 10(-8) M), while no specific binding was obtained for a known ER-negative cell line (MDA-231). The Kd was estimated from the specific binding curve of MCF-7 cells as the concentration of 17FE at half-maximal binding. When corrected for the binding activity relative to estradiol, a Kd of 18 X 10(-10) M was obtained. The flow cytometer-generated distributions give a qualitative estimate of the proportions of ER-rich and ER-poor cells. Quantitation of ER heterogeneity will require a mathematical algorithm expressing the difference between the "total" and "nonspecific" distributions. These studies demonstrate that flow cytometry and an appropriate ligand can detect and partially describe ER heterogeneity in intact cells.

Cell sorter analysis of carcinogen metabolites in human tissues.

A fluorescence -activated cell sorter (FACS-II) was used to examine biochemical parameters in a heterogeneous population of cultured human lymphocytes. Incubation of cells in the presence of benz(a)anthracene (BA) during culture was employed to induce the enzyme system which metabolizes carcinogens. Carcinogen metabolism was assayed directly by measuring the phenolic metabolites of cells exposed to benzo(a)pyrene (BP). Metabolism of benzo(a)pyrene was measured in single cells and was determined to be greater in the larger cells than in the smaller cells of the cultures. For a given size of cells, the enzyme activity was greater in those exposed to benz(a)anthracene during culture. In some studies, viable cells were first sorted by size and subpopulations assayed for the o-deethylation of the compound, ethoxyresorufin, which measures more specifically the activity of cytochrome P-448. Larger cells had higher levels of enzyme activity than smaller cells in agreement with the direct determinations above. It is possible to measure carcinogen metabolism in other tissues by using the techniques described here.

Automatic cell identification and enrichment in lung cancer. I. Light scatter and fluorescence parameters.

Two physical parameters were investigated to automatically recognize cells in sputum from human squamous cell carcinoma of the lung and to separate them for preparation by the Papanicolaou methods, for human interactive identification and for automated high resolution image analysis. The two parameters, 0.5-15.0 degrees forward argon-ion laser light scatter to estimate total cell size and 546 nm Acridine orange fluorescence to approximate total cell DNA content, were measured in a flow-through fluorescence activated cell sorting system. Enrichment for neoplastic cells in three cases of squamous cell carcinoma of the lung averaged 7.8-fold over the original sputum when only green fluorescence was used and 10.5-fold using green fluorescence and forward light scatter. The average enrichment for neoplastic cells was 65.6-fold relative to polymorphonuclear deenrichment.

Automatic cell identification and enrichment in lung cancer. III. Light scatter and two fluorescence parameters.

Two fluorescence parameters and size are used in a flow through system to enrich sputum specimens for cancer cells. Human cells in sputum which are stained with acridine orange show a characteristic distribution of red and green fluorescence from which cancer cells can be localized. The peak enrichment is obtained by selectively sorting cells with the largest values of red and green fluorescence. Cancer cells located in other distribution regions having smaller fluorescence intensities show progressively diminished nuclear and cytoplasmic tinctorial features by Papanicolaou stain, consistent with the decreased intensity of red and green fluorescence.

Automatic cell identification and enrichment in lung cancer. II. Acridine orange for cell sorting of sputum.

Fluorescence spectra were obtained from cells from sputum and pleural effusions stained with different fluorescent dyes and fixed by alternate methods. The spectra were referenced to a standard allowing for fluorescence comparisons of unstained and stained cells under various conditions. The metachromasia of acridine orange-stained cells offers nuclear/cytoplasmic differentiation in a single stain; mithramycin and propidium iodide do not. Unstained cells have an appreciable amount of green (546 nm) fluorescence, as does Carbowax in Saccomanno's preservative. Cytoplasm stained with acidine orange also has appreciable green fluorescence. Consequently, cells with much cytoplasm have high total fluorescence. Cytoplasmic fluorescence is negligible with mithramycin or propidium iodide. The metachromasia of acridine orange-stained cells is altered by alcohol and Carbowax levels in fixatives, keeping other factors constant.

Technology for automated analysis of maize pollen used as a marker for mutation: 1. Flow-through systems.

Maize pollen is used as a monitor for environmental pollutants. Mutant pollen grains (induced by environmental pollutants) are detectable above a background frequency of 5 or less in 10(5). To enumerate a satisfactory number of mutant grains, it is necessary to count 10(6) grains in a sample, a laborious, time-consuming process which should be amenable to automated analysis techniques. High resolution image analysis technology has been used in the morphologic assessment of rare cells in a sample, provided a suitable training set could be devised to instruct the computer on the characteristics of the rare cells. On the other hand, flow cytometry uses primarily cytochemical means for detection and has been shown to detect rare events. Hence, the two technologies, which may be viewed as complementary, are suitable for the task. Alternatively, a hybrid technology employing both cell sorter and image analysis techniques may be extremely desirable for this problem. The potential for archival storage of analyzed samples is very attractive when considering the possibility of an adversary relationship between a putative regulator and polluter.

Specifications for a satellite based wide area network.

To provide rural America with modern telemedicine requires very high bandwidth resources that may take decades to appear. Satellites provide the natural choice for communication between the rural primary care centers and the tertiary care hospital. However there are problems if TCP, as required for Internet access, is to be the protocol of choice.

Accuracy of electronic deposition of cells onto microscope slides using a cytometric positioning system.

The Cell Deposition System (CDS) analyzes and deposits biological cells to specific locations on microscope slides. This research will determine the accuracy of the CDS by comparing the stored computer data with the cells placement on the microscope slide. Two independent maps (Control 1 and Control 2) of cell placement for each slide were made, each map to provide a control for the other. The fluorescence data (cell sequence data) was compared to each of the control maps. For the slide, CT4, the error between maps was 13.6% mismatches, between Control 2 and the fluorescence data 32.1% mismatches. Comparing the maps 3/16 rows were perfectly matched; Whereas, comparing fluorescence data to maps 1/16 express perfect matches. Depositing cells in this manner is remarkable accurate. Nevertheless work remains to prevent the "disappearance" of cells, their adjacent positional invasion, and their clustering. This technique provides a superior preparative method for sample archival storage and for further automated multiparametric microscope assessment.

Use of computer algorithms to reduce viral quasispecies sequence space.

A virus may express multiple simple mutations producing a set of viral subspecies called quasispecies: the quasispecies are considered the same species as the original virus. We are interested in reducing the point mutation space to enumerate that sequence space. We form a point mutation by applying a single bit mutation to a strand of viral DNA. How many differing viruses are possible if we allow any of the base pairs to change along the strand? For a strand of arbitrary length n we see that there is a possible sequence space of 4n-1.4 = 4n combinations. We can further remove identical sequences due to redundant amino acid codon encoding. This requires the use of a computer, but this time the complexity is a product: the number of possible amino acids times the number of codons. This substantial reduction from an exponential complexity O(4n) to a product O(n.amino-acid-number) gives us the complete list of mutant viral entities which are one mutation away from the original. Further reduction is possible, but requires biological insight regarding the viability of the mutation. By recognizing the possible sequence space, prediction can be made toward identifying future viral strains of HIV and influenza (to name two important viral particles), and perhaps develop a predictive intervention.

Quantitation of neutrophil phagocytosis, using fluorescent latex beads. Correlation of microscopy and flow cytometry.

Phagocytic particle uptake was calculated from data obtained by microscopy and flow cytometry utilizing commercially available fluorescent beads of uniform (2 micrometer diameter) size. PMNs collected from a Ficoll-Hypaque density gradient were incubated with the fluorescent beads in human plasma and gently washed in buffer to remove most of the extracellular beads. The cells were observed and analyzed by fluorescence microscopy (sample size = 300 cells) and flow cytometry (sample size = 50,000 cells) to determine the percent phagocytic cells, number of beads per phagocytic cell, and beads per 100 PMNs. Correlation of these indices for microscopic and machine counts was in high agreement, with r values greater than 0.80. Since flow cytometry increases the sample size 500-fold above that attained by manual microscopic counts, the coefficient of variation is proportionately reduced. Unlike other automated techniques for phagocytosis such as the use of radiolabeled particles, flow cytometry yields information on the cell population such as percent of phagocytic cells and the distribution of intracellular fluorescent beads per cell as well as particle uptake. Thus the advantages of speed, greater statistical accuracy, and the ability to accumulate information on the cell population indicate that flow cytofluorometry has unique applications for the study of phagocytosis. Application of the technique to in vitro stimulation of blood neutrophil phagocytic uptake is included.

Strategies for automated placement of cells for microscopy.

Computer-software-controlled placement of cells onto microscope slides is advantageous for quantitative optical microscopy. Cell detection, indentification, and placement have been implemented on a software-controlled flow sorter. Computer hardware timing latencies that comprise the dead times of the system are sufficiently short (10 micros) to allow adequate dealy of cells for sorting. Operating system timing latencies (150 micros) are also short enough for low-rate sorting. As a result of the low cost of computers and because formats for cell placement are still controversial, software control is desirable somce it provides the flexibility to implement multiple variations for cell detection, identification, amd sorting.

Rural telemedicine: satellites and fiber optics.

Rural America Telemedicine requires very high bandwidth to provide timely transmission of large data sets. These resources may take decades to appear because of the economics of low population densities and costly installation, and the historically low rate of bandwidth improvement available from the common communication providers. Satellites provide the natural choice for communication between the rural primary care centers and the tertiary care hospital. Furthermore recent improvements in technologies have substantially reduced the costs of ground stations. A network of satellite ground stations with symmetric bandwidth connected by satellite is the architecture of choice. Analysis of multi-station satellite access clearly argues for distributed non-random methods and hence for appropriate handling of TCP data streams. However the overhead in delay of Satellite based TCP, as required for Internet access, substantially increases the transmission time and hence cost. Simulations of TCP/IP data over satellite links show a substantial reduction in transmission times. Initial business models show that the transmission cost per second is 60 times that of telephone lines while the increase in speed is nearly 3000 fold, effecting a 50 fold cost savings. But over decades, the infrastructure can be expected to improve. In particular speculative fiber optic installations in power lines and along major highways are betting on future traffic. These so-called dark fibers take advantage of synergistic installations. Their small size, ease of manipulation and gigantic bandwidths (in terabytes) allows for economic installation in anticipation of future use. Thus for rural America a strategy can evolve in which satellites provide an intermediate solution to high speed data communication while the terrestrial fiber-optic infrastructure catches up.

Automatic cell identification and enrichment in lung cancer: V. Adenocarcinoma and large cell undifferentiated carcinoma.

The aims of this study were to develop a protocol for the identification and enrichment of cancer cells from sputum obtained from patients with adenocarcinoma of the lung (n = 6) and large-cell undifferentiated carcinoma of the lung (n = 2), and to compare these findings with the results from our previous studies on other cell types from lung cancer. The hypotheses tested were: Cancer cells in sputum can be preserved following flow sorting. Enrichment for cancer cells from acridine orange (AO)-stained specimens can be achieved. Discrimination of cancer cells from noncancer cells is by AO green fluorescence and discrimination of lymphocytes from other cell types is by AO red fluorescence. Cancer cells are consistently enriched in the AO high green and red fluorescence region, although, for a given cell type, maximal enrichment is patient-dependent. Finally, cancer cell enrichment and lymphocyte exclusion can be done simultaneously. Cells from sputum were initially fixed, stained with AO, sorted on a dual parameter flow sorter, and classified into six groups corresponding to two ranges of green and three ranges of red fluorescence intensities. Cells of each region were stained by the method of Papanicolaou and differential counts were performed to determine the relative frequencies (i.e., purities) of leukocytes, macrophages, squamous cells, and cancer cells, in sorted and unsorted (i.e., control) samples. The average purity of leukocytes (81%), macrophages (6%), squamous cells (11%), and cancer cells (2%) varied markedly from sample to sample. However, the largest enrichment values (i.e., ratio of purity of a cell type in a sorted sample to its purity in the unsorted control sample) achieved for cancer cells consistently occurred for each patient sample in the region corresponding to high green and high red fluorescence intensities. Experimentally, a cancer cell average enrichment of sixteen-fold was obtained by this method. Additionally, fluorescence intensity ranges which increased the enrichment for macrophages by cell sorting typically excluded leukocytes and squamous cells, and vice versa. Finally, red fluorescence intensity was the primary discriminatory parameter for all cell types studied, although the additional use of green fluorescence intensity significantly increased cancer cell enrichment rates.(ABSTRACT TRUNCATED AT 400 WORDS)

Immune depression in trypanosome-infected mice. II. Characterization of the spleen cell types involved.

Spleen cells from Trypanosoma congolense-infected mice showed a drastic depression in their capacity to respond to B and T lymphocyte mitogens and to allogeneic spleen cells in mixed lymphocyte cultures. Spleen cells from infected mice were also poor stimulators in mixed lymphocyte cultures. The poor responsiveness or stimulation capacity was not due simply to dilution of relevant B or T lymphocytes by the large number of null cells found in the spleens of infected animals. These null cells expressed approximately eight times more H-2 antigen than spleen cells from normal (uninfected) mice and were devoid of Ia antigens.

Microfluorometric quantitation of size, deoxyribonucleic acid and viral antigenic determinants in cells productively infected with HSV-2.

The interaction of herpes simplex virus type 2 (HSV-2) with the eukaryotic human cell line (HEp-2) was investigated by flow microfluorometric analysis. The three parameters that were quantitated include cell size, deoxyribonucleic acid content and the expression of virus-specific antigens. Productively infected cells are always smaller than uninfected ones, and they resolve into two populations with respect to viral antigenic content. Consistent with viral replication, one of these two populations, displaying a higher viral antigenic content, shows morphologic features characteristic of cell degeneration. On the other hand, the second population, with a relatively lower content of viral antigens, displays morphologic features consistent with increased cell growth. Indeed, microfluorometric measurements of the HSV-2-infected cells with respect to DNA content and expression of viral antigenic determinants resolves four cell populations. Of them, one displays the lowest level of viral antigens with the highest (equivalent to 8N) DNA content, features consistent with transformation. The results demonstrate the great potential of this technique for the detection, separation and quantitation of statistically significant cell populations not otherwise identified and for the analysis of virus-host cell interactions resulting in different pathologic outcomes.

Fluorescence-activated separation of cervical abnormal cells using herpesvirus antigenic markers.

Antisera to total HSV-2 (G) viral antigens (Ra-2) and to increasingly purified viral antigenic fractions ("crude" and "pure" AG-e) specifically stain HSV-2-(G)- or HSV-1-(F)-infected HEp-2 cells in indirect immunofluorescence. Anti-"crude" AG-e sera induced by HSV-1 (F) or HSV-2 (G) produce a single precipitin band of identity when reacted in gel immunodiffusion against soluble antigenic mixtures from HSV-2-infected cells (HSV-2 (G) SAM). These reactivities are lost following adsorption of the sera with HSV-1 or HSV-2-infected cells or with pelleted or dextran gradient purified virions. Ra-2 and anti-"crude" or "pure" AG-e sera stain exfoliated cervical cells from patients with herpetic cervicitis as well as atypical cells from patients with atypia, carcinoma in situ or invasive cancer. Normal squamous cells do not stain. HSV-2 viral antigens recognized by the Ra-2 and anti-AG-e sera appear to constitute specific and sensitive markers for the separation of atypical cells in a fluorescence-activated cell sorter.