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Biomedical subjects

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

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↗
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

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↗
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↗
PubMed1979

False alarms in a slit-scan flow system: causes and occurrence rates. Implications and potential solutions.

A slit-scan technique was developed as a basis for an automated prescreening system for gynecologic cytology. A flow system based on this technique was fabricated and tested and results indicated that false alarms (misclassification of objects or events from normal specimens as abnormal) are the greatest remaining obstacle to development of an automated prescreening instrument. A dual view correlation system was fabricated to provide exact image-contour correlation in flow and permit precise determination of causes and occurrence rates of false alarms. This paper presents data from correlation analyses of 23 normal cytologic specimens. Major causes of false alarms and their implications to automated prescreening are discussed. A technique that would eliminate the majority of false alarms in flow is presented.

Cervix Uteri↗
PubMed1979

Cytofluorometric and cytochemical comparisons of normal and abnormal human cells from the female genital tract.

Acridine orange staining of exfoliated cells from epithelial tissues facilitates discrimination between normal and abnormal cells: abnormal cells develop highly elevated nuclear fluorescence. Comparisons of acridine orange (AO) staining with propidium iodide (PI) or Feulgen staining have shown that: (a) PI staining also provides highly elevated nuclear fluorescence from abnormal cells; (b) the distributions of nuclear fluorescence following AO or PI staining were usually not significantly different as judged by the Kolmogorov-Smirnov test; (c) fluorescence emission spectra from AO and PI stained cells are consistent with the hypothesis that both fluorochromes bind to DNA within cell nuclei; (d) DNAse treatment of AO stained normal cells eliminates the nuclear fluorescence peak from slit-scan contours; RNAse treatment has no effect on nuclear fluorescence; (e) the distribution of abnormal cell nuclear fluorescence after AO staining is usually, but not always, significantly different from the distribution of abnormal cell nuclear absorbance after Feulgen staining, with relative nuclear fluorescence being greater than relative nuclear absorbance. The hypothesis currently most consistent with these results is that elevated Feulgen DNA content can account for only part of the discrimination provided by AO staining, and that the chromatin within abnormal cells is altered so as to increase accessibility of DNA to intercalating dyes.

Cell Nucleus↗
PubMed1978

A comparison of acridine orange and Feulgen cytochemistry of human tumor cell nuclei.

Specimens of cells derived from tumors of the human female genital tract plus normal cells as standards have been divided into aliquots and stained according to acridine orange or pararosanilin:Feulgen procedures. Acridine orange-stained cells were slit-scanned for 535 nm nuclear fluorescence; Feulgen-stained cells were comb-scanned for 580 nm nuclear absorbance. For each specimen examined, the tumor cell:normal cell ratio of mean nuclear fluorescence following acridine orange staining was greater than the tumor cell:normal cell ratio of mean nuclear absorbance following Feulgen staining. The tumor cell:normal cell ratio of mean nuclear fluorescence ranged from 2.3 for a nonkeratinizing squamous cell carcinoma to 3.9 for a keratinizing squamous cell carcinoma. The tumor cell:normal cell ratio of mean nuclear absorbance ranged from 1.4 for a mixed mesodermal sarcoma to 2.3 for a small cell squamous cell carcinoma. These results indicate that the elevated nuclear fluorescence intensity from acridine orange-stained tumor cells cannot be explained solely on the basis of elevated Feulgen:DNA content. An alternative hypothesis, consistent with these results, is that DNA is the principal binding substrate for intranuclear acridine orange and that the DNA of certain tumor cells is more accessible to acridine orange than is the DNA of normal cells.

Acridines↗
PubMed1977

Application of Fraunhofer diffraction theory to feature-specific detector design.

Light scatter from epithelial cells in a slit-scan flow system is modeled using the Fraunhofer condition of scalar diffraction theory. Power spectra are calculated for successive positions of model cells in the line focus of a laser beam with a Fourier transform computer program. Using the calculated power spectra, detector configurations are designed to detect specific cell structures of interest. Detector configurations are tested in a static slit-scan scatter apparatus. Data indicating the ability to detect boundaries and cell orientation are discussed.

Cell Nucleus↗
PubMed1977

Imaging systems for correlation of false alarms in flow.

False alarms, arising from a variety of sources, are the greatest remaining obstacle to development of an automated prescreening system for gynecologic cytology. This paper describes two correlation systems under development at the University of Rochester and discusses their utilization in the study of false alarms in slit-scan cytofluorometry. Both systems permit imaging of objects in flow and correlation between images and corresponding slit-scan contours. Correlation systems will permit a detailed study of false alarm causes and aid in the search for new features to assist in their recognition.

Autoanalysis↗