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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.

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.

A low resolution slit-scan flow system is described. Cells flow in solution through a thin "wall" of fluorescence excitation illumination where they are sequentially illuminated. Computer analysis of the resulting slit-scan contours provide a measure of cell diameter, nuclear diameter, nuclear to cell diameter ratio in percent, nuclear fluorescence, cytoplasmic fluorescence, and certain low resolution morphological information. The system, its potential, and remaining problems are discussed.

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.

A technique for Acridine Orange staining of cells for automated flow analysis involving post-staining fixation with Millonig's glutaraldehyde buffer is presented. Preliminary data indicate that with this protocol there is decreased background fluorescence and increased signal to noise ration in flow. In addition, stained cells may be stored for several months without alteration in cellular morphology or fluorescence characteristics. Finally, nuclear and cytoplasmic fluorescence do not appear to vary significantly with respect to cell concentration or distribution on a slide or filter when cells are stained according to the glutaraldehyde protocol.

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.

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.

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.

Squamous cells in fluid flow are photographed using a laser stroboscopic technique. The technique provides either dark field or phase contrast multiple images of a cell on a single frame of film at resolutions permitted by the thickness of the cell stream. Cell orientation, velocity and acceleration are obtained from these photographs for evaluation of flow system performance.

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.

A poststaining fixation technique was developed for acridine orange staining of cytologic specimens. A quantitative evaluation was performed to assess the suitability of this technique for routine use in analytic cytology and its potential for future use in automated screening equipment. Three major advantages of the poststaining fixation technique over conventional acridine orange staining methods were documented: (1) stained cells may be stored for several months without alteration of cellular morphology or fluorescence characteristics; (2) the protocol minimizes diffusion of stain out of cells; and (3) poststaining fixation reduces dependence of nuclear or total cell fluorescence on cell distribution or concentration on a slide or in flow.

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.

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.

A study was undertaken to assess the applicability of the slit-scan technique to automated prescreening of urinary tract cytology. Cells from voided and catheterized urines were stained with acridine orange and measured on a static cell slit-scan cytofluorometer. Analysis of data from the specimens indicates that nuclear fluorescence alone appears adequate for recognition of abnormal specimens. Remaining problems in the automation of urinary tract cytology prescreening are discussed.

Gynecologic cell orientation and dynamics in sheath flow for three flow nozzle geometries are studied and compared. Cell orientations are classified into four categories based upon visual inspection of dark field laser stroboscopic photographs of cells in flow. Results are discussed in the context of slit-scan cytofluorometry.

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.

Mechanical shear in the form of syringing was applied to human gynecologic cytology specimens in an effort to disperse cell clumps. The percentage of single squamous cells achieved with the optimum conditions is generally 70 to 100 per cent. Syringing with a constant pressure device produces a high yield of single squamous cells without the concurrent high cell loss as often observed using enzymatic or chemical procedures. Data presented demonstrate that the slide preparation and clump evaluation procedures used for this study yield reliable and reproducible data. Studies varying stroke number and plunger pressure were completed and conditions for maximum dispersal were established.

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.

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.

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.