This chapter is devoted to image cytometry instrumentation. In image cytometry a specimen or object of interest is placed on a glass slide or other rigid substrate and stained using fluorescenceor absorbance-based probes specific for the cellular substance or substances of interest. Analyses may be performed on tissue sections or individual cells. Image cytometry can be used to quantify probes (similar to flow cytometry) as well as to obtain morphometric and other information as permitted by its high optical resolution. Images may be acquired in two or three dimensions, permitting quantification and study of the distribution of substances throughout cells and tissues.

A cohort of 109 patients with primary transitional cell carcinomas, stages T2-T3, grade 2 or higher, was identified and further divided into two groups based on lymphatic metastasis at the time of cystectomy (n = 57 cases) or absence of detectable metastatic disease over a minimum of 5 years of follow-up after cystectomy (n = 52). Blocks corresponding to the primary tumor lesions were sectioned and distributed to different laboratories to be analyzed. Immunohistochemistry on deparaffinized tissue sections was conducted for evaluation of p53 nuclear overexpression (monoclonal antibody PAb1801), assessment of proliferative index (Ki-67 antigen-monoclonal antibody MIB1), and microvascular counts (factor VIII-related antigen). DNA content/ploidy studies were performed on material obtained from thick sections. A double-blinded strategy was used for the evaluation of laboratory data versus clinical parameters. The cutoff value for p53 nuclear overexpression was > or =20% of tumor cells displaying nuclear staining. The median values for MIB1 (> or =18% of tumor nuclear cell staining) and microvascular counts (> or =40 microvessels/area screened) were used as cutoff points for these two variables. The assessment of DNA content was conducted by classifying cases as diploid, tetraploid, or aneuploid. Statistical analyses were performed using the Fisher's Exact Test (2-tailed). Results revealed that none of the markers studied had a statistically significant correlation with the end point of the study, i.e., the presence of lymph node metastatic disease, in the cohort of patients studied, although an obvious trend for p53 was noted. It is concluded that alterations of p53, Ki-67 proliferative index, microvascular counts, and ploidy are not strongly associated with lymph node status in patients affected with high-stage, high-grade bladder cancer.

OBJECTIVES:To determine the sensitivity and specificity of combining fluorescence in situ hybridization (FISH) measurement of chromosome 9 and DNA cytometry of bladder irrigation specimens in the detection of bladder cancer.METHODS:Bladder irrigation specimens were obtained from 37 normal control patients and 317 bladder cancer patients during cystoscopic examinations. Bladder cancer patients were sampled in the absence of observable tumor (256 specimens) and concurrently with tumor (204 specimens). Chromosome 9 copy number was determined on a cellular basis by FISH, and cellular DNA content was determined by Feulgen DNA staining and image cytometry.RESULTS:Sensitivity of chromosome 9 FISH was 42% for all tumors and was not correlated to transitional cell carcinoma tumor grade, while the sensitivity of DNA cytometry was 55% and improved with increasing grade from 38% for grade 1 to 90% for grade 3 tumors. The results of FISH and DNA cytometry were combined, resulting in specificity of 92% and sensitivity of 69% for grade 1, 76% for grade 2, and 97% for grade 3 tumors.CONCLUSIONS:The lack of increase with grade in the percentage of positive specimens by FISH supports the hypothesis that chromosome 9 aberrations are critical events in bladder tumorigenesis for many patients. These data demonstrate the presence of cells in irrigation specimens with specific genomic lesions of chromosome 9 and DNA content. Combining FISH on chromosome 9 and DNA cytometry provides an increase in sensitivity to transitional cell carcinoma over either test alone.

Two laboratories equipped with CAS 200 (Becton Dickinson Image Cytometry Systems, San Jose, CA) instruments participated in this study of variability of DNA analysis of bladder tumor specimens. Formalin fixed paraffin embedded specimens were disaggregated and centrifuged onto microscope slides from ten bladder tumor specimens and two specimens of normal urothelium. Sources of variability considered were Specimen, Slide, Run, Laboratory, and Error. Slides were systematically scanned and 200 cells measured followed by the operator selecting 100 nuclei with abnormal morphology. DNA. index (DI) and hyperdiploid fraction (HDF) were calculated from the DNA frequency distributions. For systematic sampling, 92% of the variability was due to Specimen indicating that differences in HDF values between specimens reflect biological differences. With selective sampling, only 67% of the variability in HDF is due to Specimen differences. Other factors, Laboratory, Error, and Laboratory x Specimen interaction each accounted for approximately 10% of the variability. Similarly variability of DI with selective sampling was also higher, and less specimen dependent than systematic sampling. It is important that sampling schemes and selection criteria be carefully documented in order to control variability. Enriched Cor selective) sampling for abnormal cells has the potential to increase sensitivity but specimen classification based on these measurements must depend on determination of the frequency of such cells in the total population. (C) 1997 Wiley-Liss, Inc.

Two laboratories equipped with CAS 200 (Becton Dickinson Image Cytometry Systems, San Jose, CA) instruments participated in this study of variability of DNA analysis of bladder tumor specimens. Formalin fixed paraffin embedded specimens were disaggregated and centrifuged onto microscope slides from ten bladder tumor specimens and two specimens of normal urothelium. Sources of variability considered were Specimen, Slide, Run, Laboratory, and Error. Slides were systematically scanned and 200 cells measured followed by the operator selecting 100 nuclei with abnormal morphology. DNA index (DI) and hyperdiploid fraction (HDF) were calculated from the DNA frequency distributions. For systematic sampling, 92% of the variability was due to Specimen indicating that differences in HDF values between specimens reflect biological differences. With selective sampling, only 67% of the variability in HDF is due to Specimen differences. Other factors, Laboratory, Error, and Laboratory x Specimen interaction each accounted for approximately 10% of the variability. Similarly variability of DI with selective sampling was also higher, and less specimen dependent than systematic sampling. It is important that sampling schemes and selection criteria be carefully documented in order to control variability. Enriched (or selective) sampling for abnormal cells has the potential to increase sensitivity but specimen classification based on these measurements must depend on determination of the frequency of such cells in the total population.

Annals of the New York Academy of SciencesVolume 677, Issue 1 p. 82-85 The Clinical Utility of DNA Cytometry LEON L. WHEELESS, LEON L. WHEELESS Department of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York 14642Search for more papers by this author LEON L. WHEELESS, LEON L. WHEELESS Department of Pathology and Laboratory Medicine University of Rochester Medical Center Rochester, New York 14642Search for more papers by this author First published: March 1993 https://doi.org/10.1111/j.1749-6632.1993.tb38767.xCitations: 4AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat Citing Literature Volume677, Issue1Clinical Flow CytometryMarch 1993Pages 82-85 RelatedInformation

CytometryVolume 14, Issue 5 p. 478-481 Special ReportFree Access Consensus review of the clinical utility of dna cytometry in bladder cancer† Leon L. Wheeless, Corresponding Author Leon L. Wheeless Department of Pathology, University of Rochester Medical Center, Rochester, New York 14642Analytical Cytology Unit, Department of Pathology & Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Box 626, Rochester, NY 14642Search for more papers by this authorRobert A. Badalament, Robert A. Badalament Division of Urology, Ohio State University Medical Center, Columbus, OhioSearch for more papers by this authorRalph W. deVere White, Ralph W. deVere White Department of Urology, University of California, Davis, Sacramento, CaliforniaSearch for more papers by this authorYves Fradet, Yves Fradet L'Hotel-Dieu de Quebec, Laval University Cancer Research Center, Quebec, CanadaSearch for more papers by this authorBernhard Tribukait, Bernhard Tribukait Department of Medical Radiobiology, Karolinska Institute, Stockholm, SwedenSearch for more papers by this author Leon L. Wheeless, Corresponding Author Leon L. Wheeless Department of Pathology, University of Rochester Medical Center, Rochester, New York 14642Analytical Cytology Unit, Department of Pathology & Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Box 626, Rochester, NY 14642Search for more papers by this authorRobert A. Badalament, Robert A. Badalament Division of Urology, Ohio State University Medical Center, Columbus, OhioSearch for more papers by this authorRalph W. deVere White, Ralph W. deVere White Department of Urology, University of California, Davis, Sacramento, CaliforniaSearch for more papers by this authorYves Fradet, Yves Fradet L'Hotel-Dieu de Quebec, Laval University Cancer Research Center, Quebec, CanadaSearch for more papers by this authorBernhard Tribukait, Bernhard Tribukait Department of Medical Radiobiology, Karolinska Institute, Stockholm, SwedenSearch for more papers by this author First published: 1993 https://doi.org/10.1002/cyto.990140504Citations: 85 † These consensual guidelines and recommendations address the potential utility of DNA cytometry in characterizing human malignancies. They are provided to inform laboratory personnel, pathologists, and clinicians about DNA cytometry. For individual patients, use of DNA cytometry, selection of specific techniques, and interpretation and utilization of results remains the responsibility of the attending physicians. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume14, Issue51993Pages 478-481 ReferencesRelatedInformation

The National Cancer Institute-supported Flow Cytometry Network for Bladder Cancer concluded that when properly used, DNA flow cytometry of bladder irrigation specimens can be a clinically useful laboratory procedure to monitor patients with bladder cancer. It recommended the use of this technique in managing patients with low-stage disease, particularly flat carcinoma in situ. The method has limited value in managing patients with high-stage (i.e., muscle-invasive) carcinoma; it is not recommended for screening subjects in the absence of a clinical suspicion of or a history of bladder tumors. DNA histograms alone are not sufficient for diagnosis or clinical action but require correlation with other clinical information. Bladder irrigation specimens collected during cystoscopy or by vigorous barbotage via a number 18 Foley catheter may be processed for flow cytometric analyses. Criteria for sampling adequacy have been established and are presented in this report. Optimal results are obtained with fresh specimens that are stained and examined promptly after collection. Preservation procedures are described for cases in which fresh specimens cannot be evaluated. Used with appropriate quality-control measures, commercially available flow cytometers can provide clinically useful information. Staining protocols using propidium iodide are recommended by the Network. Staining protocols are described for isolated nuclei and whole cells (see Appendix). The presence of bladder cancer is signaled by the identification of cell populations with clearly aneuploid DNA content. Quality-control measures and issues of inter- and intralaboratory differences in histogram configuration and analysis must be considered in the interpretation of results. Although the Network participants recognize the potential value of additional markers, these were not evaluated.

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)

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)

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.

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.

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.

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.