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Biomedical subjects

L L Wheeless

Publications and source records attributed to L L Wheeless.

At least 37 records · Page 2Linked to original sources
PubMed1989

Technique for cellular fluorescence distribution analysis.

The usefulness of multidimensional slit-scan flow cytometry in whole cell measurements is dependent on extracting relevant features from the cellular fluorescence distributions (slit-scan contours). In addition, the extraction of these features must be rapid to allow for real-time data processing during acquisition. This paper describes two algorithms that have been used successfully to count the numbers of local maxima (peaks) and to find nuclear boundaries in a cellular fluorescence distribution. These routines are efficient, use only simple integer arithmetic, and have been implemented on several different microprocessors.

Algorithms↗
PubMed1989

Measurement variability in DNA flow cytometry of replicate samples.

A Bladder Cancer Flow Cytometry Network study has been carried out aimed at identification of the sources of inter- and intralaboratory variability. Replicate "cocktail" samples containing a mixture of peripheral blood lymphocytes and an aneuploid cell line and samples of peripheral blood lymphocytes serving as a DNA reference standard were distributed to five network laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Sets of these samples were analyzed by flow cytometry to obtain cellular DNA distributions. DNA index and hyperdiploid fraction were calculated for each histogram using an automated technique. Results were evaluated by analysis of variance to identify sources of variability. Three important sources of variation were found that affect flow cytometry in general and- the transportability of flow cytometry results to routine clinical use in particular. The significant variation among laboratories that is constant across time most probably represents stable differences in instrumentation, instrument set-up, and laboratory techniques. This variation can be compensated for, if it is known and stable, to develop transportable classification criteria. The second type of variation, termed the interaction component, represents differences among laboratories that are not constant across time. Sources of this variation include inconsistency in sample preparation, staining, and analysis. The elimination of this type of variation is required for meaningful comparison of data within and among laboratories and the creation of interlaboratory data-bases. The third type of variation represents pure measurement variability and affects the sensitivity of the technique.

Cell Line↗
PubMed1988

Interinstitutional variability in DNA flow cytometric analysis of tumors. The National Cancer Institute's Flow Cytometry Network Experience.

Flow cytometric DNA analysis of human urinary bladder specimens may be clinically useful for prognosis in transitional cell (urothelial) carcinoma and for detecting recurrence after treatment. However, many important methodological differences exist among institutions which have described this technique, and it has not previously been shown that data from different institutions are comparable. The National Cancer Institute has created a Flow Cytometry Network to address the need for technology assessment of flow cytometry. This report describes the independent flow cytometric analysis and interpretation of "unknown" paraffin-embedded bladder tumor specimens by the five Network institutions. Although important differences in method existed among the institutions, substantial agreement was achieved in actual data generated and their interpretation. This suggests that a consensus regarding acceptable laboratory performance of this technique could be reached, which should facilitate its more widespread clinical implementation.

Carcinoma, Transitional Cell↗
PubMed1988

Comparison of frequency distributions in flow cytometry.

A number of methods have previously been considered for the statistical comparison of flow cytometric frequency distributions. For two distributions, the foremost of these is the Kolmogorov-Smirnov (K-S) test, which has been criticized as "too sensitive." We discuss some alternative methods based on the Poisson distribution. The assumption of Poisson variation within channels allows the use of channel-by-channel confidence intervals and chi-square tests. These are simple and more appropriate for discrete data than the K-S test. Graphical displays of these and other techniques are presented. We also attempt to set the problem in an appropriate context. We argue that any statistical procedure must rest on a reasonable understanding of the nature of the variability in the system. This understanding takes the form of an appropriate probability model, which may be approximate but must provide a reasonably accurate description of the data. Incomplete understanding of the data can lead to inappropriate analysis. We discuss the assumptions that underlie our techniques and consider extensions to more complex situations.

Flow Cytometry↗
PubMed1988

An EPROM-based programmable contour generator for use in flow cytometry.

An erasable programmable read-only memory (EPROM) contour generator has been fabricated to produce contours for use in flow cytometry. Contours are analog waveforms representing the fluorescence or light-scatter intensity distribution along a cell or object. The generator has particular utility in the development and testing of slit-scan instrumentation and analysis algorithms. Contours are generated without the requirement of specimens or full operation of the flow instrumentation. The generator provides control of contour height, width, offset, and rate. The EPROM may be custom programmed to produce contours for specific test applications or for reproducing "real" contour events. The generator is useful in situations where constant repetitive contours of predetermined characteristics are required.

Electronic Data Processing↗
PubMed1988

Comparison of automated and manual techniques for analysis of DNA frequency distributions in bladder washings.

Quantitative methods for interpretation of flow cytometry DNA histograms are required for the widespread clinical use of this technology. The usefulness of a histogram analysis technique in this setting requires that it be operator independent, easy to implement in a clinical laboratory, and provide high sensitivity to the desired information. Additionally, the technique must be tolerant of the relatively low signal-to-noise ratios often found in DNA distributions obtained from clinical samples. Among the factors that have been used to assess the malignant potential of tumors are the presence of an aneuploid population, the proportion of hyperdiploid cells, the width of the G1 peak, the DNA index, and the fraction of cells in S. A computer-based method has been developed for extraction of the above-mentioned features from DNA histograms. The program detects peaks in the histogram and uses straight-line fits to the cumulative frequency distribution to define cell population bounds. A test set of 44 histograms compiled from bladder irrigation specimens obtained from patients with a present or past history of transitional cell carcinoma (TCC) was analyzed by five collaborating laboratories forming a Network sponsored by the National Cancer Institute (NCI). This test set was used to evaluate the performance of the computer-based method by comparing results with those of four expert observers. In this preliminary analysis, perfect agreement was found in the detection of aneuploid cell populations by all observers and the computer-based method. Correlation of percent hyperdiploid cell fraction was also excellent.(ABSTRACT TRUNCATED AT 250 WORDS)

Algorithms↗
PubMed1987

Improved model for specimen classification based on single-cell classifiers.

We consider probabilistic models for specimen classification procedures based on systems which classify individual cells as normal or abnormal. The models which we consider generalize those discussed previously by Castleman and White (Anal. Quant. Cytol. 2:117-122, 1980; Cytometry 2:155-158, 1981) and by Timmers and Gelsema (Cytometry 6:22-25, 1985). In particular, they include the biologically plausible possibility that the specimen contains cells which are intermediate between the extremes of normal and abnormal. We find that if these additional cells occur differentially in normal and abnormal specimens, then specimen classification can become substantially more efficient when the cell classifier has different error rates for these cells.

Cervix Uteri↗
PubMed1986

A protocol for Papanicolaou staining of cytologic specimens following flow analysis.

A protocol has been developed for restaining cytologic specimens that have been analyzed on a multidimensional slit-scan flow system. The technique involves Papanicolaou staining of cells on a membrane filter that has been previously stained with acridine orange and fixed with glutaraldehyde buffer. The specimen and staining solutions were sequentially added to a 5-micrometers pore size, 47-mm diameter Gelman "Metricel" filter while it remained in a glass filtration apparatus. The practice of retaining the filter in the filtration apparatus throughout the staining procedure minimizes cell loss and eliminates specimen cross contamination when compared with conventional filter dip staining. The availability of this postflow specimen Papanicolaou staining protocol permits accurate determination of the performance characteristics of a multidimensional slit-scan flow system and should be useful whenever staining of a limited number of cells with minimal cell loss is desired.

Acridine Orange↗
PubMed1986

A statistical analysis of prescreening alarms in a population of normal and abnormal gynecologic specimens.

A multidimensional slit-scan flow system has been developed to serve as an automated prescreening instrument for gynecological cytology. Specimens are classified abnormal based on the number of cells having elevated nuclear fluorescence (alarms). An alarm region in a bivariate histogram of nuclear fluorescence versus nuclear-to-cell-diameter ratio is defined. Alarm region probability arrays are calculated to estimate the probability that an alarm falling in a particular bin of the alarm region is either from a normal or an abnormal specimen. From these arrays, a weighted alarm index is generated. In addition, summary indices are derived that measure how the distribution of alarms in each specimen compares with the average distributions for the normal and abnormal specimen populations. These indices together with current features are evaluated with respect to their utility in specimen classification using a nonparametric classification technique known as recursive partitioning. Resulting classification trees are presented that suggest information in the distribution of alarms in the bivariate histogram. In addition, they validate the features and rules currently used for specimen classification. Recursive partitioning appears to be useful for multivariate classification and is seen as a promising technique for other applications.

Cytodiagnosis↗
PubMed1986

Multidimensional slit-scan detection of bladder cancer. Preliminary clinical results.

A multidimensional slit-scan flow system was developed for the automated recognition of abnormal cells derived from cancer of the uterine cervix and its precursors. It provides great sensitivity in both its ability to recognize cellular abnormality and to deal with the myriad potential causes of false alarms in an automated flow system. While its initial application was the automated recognition of the spectrum of neoplasia in gynecologic cytology samples, a preliminary study was carried out using specimens obtained from the urinary bladder. Cellular material was collected by bladder irrigation and stained with the fluorochrome acridine orange. One hundred fifty-three bladder irrigation specimens, including 115 abnormal specimens containing cells derived from neoplastic lesions of the bladder epithelium, were analyzed. For the purposes of this study, abnormal specimens from the urinary bladder included specimens containing cells derived from the following lesions of the urothelium: dysplasia (atypical hyperplasia), carcinoma-in-situ, and transitional cell carcinoma, grades 1-3. Approximately 50,000 cells were analyzed for most specimens. Of the 38 presumed normal specimens (specimens containing only normal urothelial components), four were instrument classified abnormal. For the 69 specimens containing cells derived from transitional cell carcinoma, grade 1, 1-2, 2, 66 were correctly classified as abnormal while three were classified as normal.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma in Situ↗
PubMed1984

Multidimensional slit-scan prescreening system: preliminary results of a single blind clinical study.

A multidimensional slit-scan flow system was developed to serve as an automated prescreening instrument for gynecological cytology. A 2-year single blind clinical study was carried out to evaluate system performance. Cellular material was collected by scraping the uterine cervix and stained in suspension with acridine orange. Seven hundred and forty specimens (701 patients) including 156 abnormal specimens representing a broad spectrum of abnormality were analyzed. Approximately 50,000 cells were analyzed for each specimen. The system false-positive rate was 17.6% while the false-negative rate was 2.8%. All misclassified abnormals were specimens with cellular changes consistent with a slight dysplasia of nonkeratinizing type. The instrument in its present configuration appeared sensitive to the entire spectrum of abnormality existing in the female genital tract and it classified as abnormal any specimen containing on the order of 0.1% (or greater) abnormal cells.

Diagnostic Errors↗
PubMed1982

EMOSS: an epiillumination microscope objective slit-scan flow system.

An epiillumination microscope objective slit-scan flow system has been fabricated utilizing two dimensional slit scanning with hydrodynamic sample stream focussing. Low resolution (4 micron) analysis of cellular fluorescence is facilitated by the definition of a stabilized flow plane through hydrodynamic focussing. Coincidence of the region of stabilized flow with the focal plane of the microscope objective will allow for the collection and subsequent imaging of fluorescence from cells oriented along this plane. Two orthogonal slit-scan contours are generated as a cell traverses the excitation region. It is hoped that the need for a three dimensional system will be precluded by preferential orientation of the cells in the region of stabilized flow. Cellular fluorescence is collected by a high numerical aperture epiillumination optical system and imaged onto two orthogonal slits. Two photomultiplier tubes are used to detect fluorescence. It is anticipated that the epiillumination microscope objective slit-scan flow system will be used with a variety of fluorescent stains and markers, as well as extended to the research of light scattered by cells. (Steen, H.B., Cytometry 1:26-31, 1980.

Cell Nucleus↗
PubMed1981

Syringing as a method of cell dispersal. II. Effect on abnormal cells.

The generation of turbulent shear force using a constant pressure syringing device has been demonstrated to be a simple, effective method for the dispersal of intermediate and superficial squamous cells. This paper reports results of an evaluation of the effects of syringing on the dispersal of abnormal cells. Gynecologic cell samples obtained from preinvasive and invasive lesions were syringed. Overall cell loss, as well as degree of dispersion, was evaluated. Doublet, triplet and larger abnormal cell groupings remained in most syringed aliquots regardless of pressure. Although some cell loss was observed in the majority of cases, the percentage of single abnormal cells was increased in 96% of the syringed aliquots.

Adenocarcinoma↗
PubMed1979

A multidimensional slit-scan flow system.

A new slit-scan type flow system is described which provides three (X, Y, and Z) orthogonal one-dimensional projections of cell fluorescence. A photomultiplier tube and two semiconductor array detectors are used to obtain the three slit-scan contours from cells traversing a single fluorescence excitation beam. A high speed, dedicated preprocessor analyzes the three contours in parallel, extracting certain features useful for rejecting cells from which an accurate measurement of nuclear fluorescence cannot be obtain. Contour data is buffered and transferred to a PDP-11/40 computer where nuclear fluorescence is measured and cells are classified. It is anticipated that this new instrument will provide a significant reduction in false alarm rate when applied to prescreening of gynecologic cytology specimens.

Computers↗
PubMed1979

Predicted performance of single- versus multiple-slit flow systems.

Flow systems utilizing multiple orthogonal excitation slits have been proposed as a means of reducing some types of false alarms in prescreening systems for gynecologic cytology. Such false alarms include those caused by orientation-dependent events, such as passage of binucleate or overlapping cells through the measurement region with both nuclei entering the excitation slit simultaneously. This paper presents distributions of optimal projection angles for randomly oriented nuclei passing through one, two, and three slit excitation regions. The results are used to compute observed nuclear spacing of binucleate cells and to compare performance of one, two, and three slit systems in recognition of binucleate and overlapping cells.

Cell Nucleus↗
PubMed1979

Imaging in flow.

Imaging in flow has been valuable in investigating discrepancies in flow cell measurements due to cell orientation and flow dynamics. This paper discusses optical consideration in flow imaging, slit and full field imaging systems and various cell motion arresting techniques from the standpoint of image plane exposure and suitable detector choices. It concludes with an explanation of the slit-imaging techniques employed in a multidimensional slit-scan flow system and slit-scan correlation system.

Cell Nucleus↗