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L L Wheeless

Publications and source records attributed to L L Wheeless.

At least 19 recordsLinked to original sources
PubMed1991

DNA slit-scan flow cytometry of bladder irrigation specimens and the importance of recognizing urothelial cells.

DNA slit-scan flow cytometry was used to analyze 150 bladder irrigation specimens from 83 patients. Specimens were categorized into groups based on cystoscopy, histology, and cytopathology. Cells were stained for DNA with propidium iodide using a whole cell protocol. Non-specific fluorescence in the cytoplasm of some urothelial cells together with differential DNA staining of cell types in certain specimens was noted. DNA frequency distributions were analyzed using a semi-automated technique. Data were gated using slit-scan morphological features to remove cellular debris, multiple nuclei, and cells exhibiting nonspecific cytoplasmic fluorescence. Specimens were classified abnormal if they were aneuploid or had a hyperdiploid fraction (HDF) greater than 8%. The sensitivity to abnormality was 89% for grade 3 transitional cell carcinoma (TCC), 70% for grade 2 TCC, and 67% for grade 1 TCC. Specificity was 61%. Specimen data were then reprocessed using slit-scan morphological features to enrich for urothelial cells. The urothelial cells were identified by the ratio of nuclear diameter to cell diameter. This method was found to be in good agreement with immunofluorescent labeling of urothelial cells using the urothelium-selective T16 monoclonal antibody. The sensitivity to abnormality remained 89% for grade 3 TCC and 70% for grade 2 TCC, but fell to 52% for grade 1 TCC. Specificity for the urothelial cell enriched data increased to 77%. Reprocessing of data to enrich for urothelial elements resulted in 16 fewer specimens with an aneuploid DNA distribution and 2 fewer specimens with increased HDF.(ABSTRACT TRUNCATED AT 250 WORDS)

Aneuploidy↗
PubMed1991

Precision of DNA flow cytometry in inter-institutional analyses.

A Bladder Cancer Flow Cytometry Network study has been carried out to further identify and quantify sources of inter- and intra-laboratory variability. Replicate samples containing four mixtures of peripheral blood lymphocytes and aneuploid cell lines were distributed together with reference standards to six laboratories. The samples were stained for DNA using propidium iodide, with each laboratory using its own staining protocol. Two of each of the four sample types and a reference standard were analyzed by each laboratory on 3 separate days to obtain cellular DNA distributions. DNA index (DI) and hyperdiploid fraction (HDF) were calculated for each histogram using an automated technique. The results showed significant inter- and intra-laboratory differences. Results were evaluated by a two-way analysis of variance to estimate components of the overall variation attributable to individual sources. Error variation was found to be the major component of random variation. Specimen means were also compared for each laboratory. No significant differences were noted in mean DI for similar specimens; however, agreement in HDF between similar specimens was lacking in most laboratories. Prediction intervals were computed to estimate the range of values expected for a single specimen based on the analysis of the previous six. Prediction intervals for DI were quite good while those for HDF were troublesome due to wide variation. The results of these studies indicate that intra- and inter-laboratory variability are high enough that results for a single sample may not be sufficiently precise to allow comparison to results obtained in other laboratories.(ABSTRACT TRUNCATED AT 250 WORDS)

Analysis of Variance↗
PubMed1991

Bacillus Calmette-Guerin and interleukin-2 for treatment of superficial bladder cancer.

A total of 22 patients with bladder cancer received bacillus Calmette-Guerin (BCG) and interleukin-2. Significant bladder tumor remissions were noted in 15 of 17 patients (88%). Of 5 patients with carcinoma in situ 1 was noncompliant and he died of carcinoma in situ. The other 4 patients are in remission. BCG alone was instilled in 22 additional patients with superficial bladder cancer. The remission rates were encouraging. Of the 22 patients 13 (59%) had remission of the bladder tumor. A half dose of BCG (60 mg.) is adequate when given weekly for 6 weeks. Maintenance therapy is important as noted in both of our clinical arms. BCG and interleukin-2 therapy results in a higher remission rate.

BCG Vaccine↗
PubMed1990

System for acquisition and real-time processing of multidimensional slit-scan flow cytometric data.

The high-speed sampling requirements of multidimensional slit-scan signals (cell contours) have typically required custom hardware. This specialized hardware has often lacked the flexibility to adapt to varying instrument setups and experimental requirements. A hardware and software system capable of sampling multiple slit-scan cell contours at rates of up to 40 MHz with 10-bit resolution is described. It utilizes commercially available CAMAC transient recorders, a Digital Equipment Corp. PDP-11/83 computer, and custom hardware for signal conditioning and trigger generation. The modular design of the software system allows various hardware options with minimal additional coding. Real-time digital processing checks each cell contour for multiple peaks; extracts morphological features such as width, height, and area; accumulates gated histograms of these data; and optionally saves the derived data, selected contours, or both into list mode files on disk.

Cervix Uteri↗
PubMed1989

Ethanol fixation of bladder irrigation specimens for flow cytometric analysis. A multiinstitutional study from the bladder cancer flow cytometry network.

Ethanol preservation of voided and catheterized urine has long been the standard for urinary cytologic study. In this report ethanol preservation of bladder irrigation specimens was evaluated for flow cytometric analysis in a multiinstitutional study requiring specimen transport. Specimens from ten patients obtained at one center were preserved for varying periods of time in 50% ethanol, then distributed by express mail to four other participating centers, up to 3000 miles distant. On receipt, from 1 to 3 weeks after collection, the samples were processed and examined by flow cytometric study using propidium iodide as the fluorochrome. Forty-six of the 50 (92%) analyzed specimens gave satisfactory histograms. In 41 of the 46 adequate samples (89%), an aneuploid peak in the alcohol preserved (propidium iodide stained) specimen correlated well with the fresh (acridine orange stained) specimen. However, there was variable loss of DNA stainability with a broadening of the coefficient of variation in some alcohol-preserved specimens and an increase in cellular debris so that measurements of DNA index and percent hyperdiploid cells were considered unreliable. The authors conclude that ethanol preservation of bladder irrigation specimens for short periods of time may be a feasible alternative when flow cytometric analysis cannot be carried out on fresh specimens, but that this is not optimal fixation for specimens that must be transported and further studies of other fixatives are recommended.

Aneuploidy↗
PubMed1989

Check samples for laboratory self-assessment in DNA flow cytometry. The National Cancer Institute's Flow Cytometry Network experience.

The National Cancer Institute's Flow Cytometry Network (NCI-FCN) is attempting to facilitate the transfer of flow cytometry (FCM) of exfoliated bladder cells from the research laboratory to the clinical laboratory. Demonstrating interinstitutional consistency in FCM analysis of replicate specimens simulating clinical barbotage specimens, fixed to allow easy transportation and storage at room temperature was one specific objective. Simulated barbotage specimens were prepared by mixing cultured aneuploid bladder carcinoma cells with normal or mitogen-stimulated peripheral blood mononuclear cells in different ratios. The samples were fixed in 10% formalin for 30 minutes, stored in buffer, and enucleated with pepsin, pH 1.5, before staining with propidium iodide for FCM DNA analysis. Preservation in ethanol or other common DNA cytochemical reagents was found to be unsatisfactory. In contrast, the formalin-fixed samples showed excellent preservation of quantitative DNA fluorescence and coefficient of variation of histogram peaks for over 2 weeks. Exchange of eight fixed specimens among five network laboratories that analyzed them as "unknowns" showed good overall agreement on histogram data and interpretation, although some noteworthy interlaboratory differences were found. This technique could be used for self-assessment surveys of clinical laboratory performance in DNA FCM of bladder barbotage specimens.

Cell Count↗
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↗