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.     

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.     

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.     

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.     

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)     

Use of cytomorphometry and cytology in the diagnosis of transitional-cell carcinoma of the upper urinary tract

Because of the rise in incidence of upper urinary tract tumors, there is a need for a simple and reliable method for diagnosing these tumors, especially in people in a "high-risk" group. This retrospective study showed the usefulness of cytology and cytomorphometry in making the diagnosis of transitional-cell carcinoma of the upper urinary tract. The study also emphasized that the methods of collection and processing are of the utmost importance: the cytologic evaluation of ureteral catheterized urine specimens gave 100% accuracy as compared with a 40% false-negative rate in the cytologic diagnosis of voided urine specimens. A higher accuracy of urinary cytology for the diagnosis of upper urinary tract lesions clearly requires selective catheterization of the ureter. Objective cytomorphologic grading of the urinary cytology specimens was shown to compare favorably with histologic grading. Cytomorphologic grading not only can offer important information in determining the prognosis and in planning treatment but can also assist in quality control of other diagnostic methods and can help to resolve apparent diagnostic discrepancies.     

Cytologic evaluation of urine after kidney transplantation

Over a ten-month period, 54 kidney transplant patients returning for routine clinic visits were prospectively evaluated utilizing urinary cytology; 101 urine specimens were examined. The incidence of undetected infection was low. Two patients were noted to have polyomavirus infections, and two had candiduria. No patient had urinary tract malignancy.     

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.     

The diagnostic accuracy of sputum and urine cytology

The diagnostic accuracies of sputum and urinary cytology were examined in series spanning more than 20 years. The sensitivity and respiratory tract cytology in 1,289 patients with subsequently proven lung cancer was 69% while that or urine cytology in 860 patients with urinary tract cancer was 77%. The specificities were 96% for lung cancer and 97% for urinary tract cancer. Neither procedure was widely used for routine screening, but diagnostic cytology played an important part in providing a definite morphologic diagnosis in many of these cases. Urinary cytology was also very effective in the follow-up of patients with treated bladder cancer because of its high sensitivity for detecting carcinoma in situ.     

Urothelial neoplasms of the upper urinary tract. A correlation between cytologic and histologic findings in 43 patients with urothelial neoplasms of the renal pelvis or ureter.

The present study elucidates the possibilities to diagnose and classify urothelial tumors of the upper urinary tract by means of exfoliative cytology on voided urine using a membrane filter method. In a series of 30 patients with renal pelvic tumors and 13 patients with ureteral tumors an overall agreement between cytology and histopathology was obtained in 25 cases (58%). None of the Grade 1 tumors or of the non-invasive Grade 2 tumors were regarded as positive by cytology whilst two out of five invasive Grade 2 tumors had positive cytologic reports. The series included 24 patients with poorly differentiated or anaplastic tumors, 17 of whom had positive cytology (71%). By excluding from the series 13 patients with obstructed urinary passages or radiologically non-functioning kidneys on the tumor side an agreement between cytology and pathology was reached in 83 per cent of the cases, regardless of tumor grade, and in 17 out of 18 patients with Grade 3-4 tumors (94%).     

Usefulness of bile cytology in the diagnostic management of patients with biliary tract obstruction

Eighteen patients with evidence of biliary tract obstruction had a total of 29 satisfactory bile samples submitted for diagnostic cytology during a two-year period. These 29 specimens were reviewed in order to determine if bile cytology is useful in the diagnostic management of patients with obstructive biliary tract disease. Twenty-one of the bile specimens were from patients with malignant biliary stricture, and eight were from patients with benign biliary obstruction. Bile cytology was positive for carcinoma in eight samples from patients with malignant stricture and was inconclusive for malignancy in two. There were no false positives. The diagnostic specificity of bile cytology was 100%, the diagnostic sensitivity was 48%, and the diagnostic accuracy was 62%. When carefully collected and promptly processed, bile proved an excellent specimen for cytologic evaluation and was a valuable adjunct to other diagnostic procedures for the detection of carcinoma causing biliary tract obstruction.     

Automation in cervical cytology: an overview.

Cancer of the uterine cervix is one of the more common female cancers and a major source of premature female mortality. UK deaths in 1987 exceeded 2000. To reduce these rates, national screening programmes have been introduced using the Papanicolaou method. In screening for cancer, the examination of specimens is an intensive and expensive task. Its high cost has led to a number of attempts to automate the process, either fully or partially. Over the last 30 years, various experimental prescreening systems have been developed for the diagnosis of cytological samples including the use of image processing techniques. This paper presents a historical overview of automation in cervical cytology and the status of current developments for automated cytological diagnosis.     

Slit-scan flow cytometry: separability properties of cell features

A model is presented to compare the separability of cell populations described by features measured in low resolution slit-scanning flow systems with their separability when the features are extracted from high resolution digitized cell images. The results show that although the accuracy of the feature measurements deteriorates for increasing slit width, this is not necessarily true for the discriminatory power of the features. Depending on their original position in the high resolution feature space, the cell populations may be located even farther apart in the space of low resolution slit-scan features for reasonably small widths of the slit. The results presented with high resolution images of cells from gynecological specimens and simulated slit-scan measurements can be explained by the model. For the features nuclear DNA content and diameter the abnormal populations are shifted closer to the normal populations in the slit-scan simulations as compared to the high resolution measurements. The cell classifier errors rates are unacceptably high.     

Immunocytochemistry of collagenase expression in transitional cell carcinoma of the bladder

OBJECTIVE: To evaluate the usefulness of collagenase immunocytochemistry as well as its immunohistochemistry in assessing the correlation with prognostic factors in transitional cell carcinoma (TCC) of the urinary bladder. STUDY DESIGN: We investigated the expression of collagenase in catheterized urine and histologic specimens from 38 patients with TCC and 20 cases with benign lesions of the urinary tract. RESULTS: Thirteen (34.2%) and 17 (44.7%) patients with TCC showed positive expression of collagenase on cytologic and histologic specimens, respectively, whereas in no cases with benign lesions was such expression found (P < .01). Invasive and nonpapillary TCC had higher positive rates than noninvasive and papillary TCC. Grade 3 TCC was positive at a higher rate than was grade 2, whereas there were no positive cases with grade 1. Collagenase expression did not correlate significantly with stage. CONCLUSION: Collagenase expression in urinary TCC correlated well with tumor growth pattern, pathologic grade and invasiveness of the carcinoma; all are known to be prognostic factors. The application of collagenase immunostaining to urinary cytology is very useful for assessing prognosis in TCC.     

Differential immunocytochemical staining patterns of uroplakin observed on neoplastic and nonneoplastic tissue fragments obtained from upper urinary tract brush specimens

OBJECTIVE: To discern any differences in the distribution of uroplakin expression on neoplastic and nonneoplastic upper urinary tract lesions. STUDY DESIGN: Thirty-seven representative 95% ethanol-fixed direct smears of brush specimens, which were subsequently diagnosed histologically as 10 reactive and 27 transitional cell carcinomas (TCCs), were stained with polyclonal uroplakin antibodies utilizing the avidin-biotin-peroxidase method. In order to ascertain any differences in diagnostic accuracy between conventional cytomorphology and uroplakin immunocytochemical staining, the results were compared to the original final cytologic diagnoses for all 37 cases. RESULTS: The linear staining pattern on the luminal surface of umbrella cells was the dominant pattern expressed on tissue fragments from all 10 reactive lesions. Tissue fragments from low grade TCC demonstrated a weaker and less continuous superficial membrane staining pattern along with a variably intense, diffuse, membranous staining pattern throughout the tumor cell groups. This staining pattern was seen in all 17 (sensitivity = 100%) histologically confirmed low grade TCCs, of which only 13 of the 17 (sensitivity = 76.5%) were diagnosed as TCC on the original final cytology report. Tissue fragments from 10 high grade TCCs lacked the superficial linear staining pattern seen in reactive cell groups. Instead, all 10 high grade TCCs displayed a strong diffuse membrane staining pattern in all the cells in the fragment and also demonstrated microluminal structures within the tumor cell groups. CONCLUSION: The distinctive patterns of uroplakin antigen expression observed in nonneoplastic and neoplastic upper urinary tract lesions in the present study can greatly enhance the accuracy of diagnostic interpretation of upper urinary tract lesions in conventional cytologic specimens.     

Intermediate cell markers for malignancy. Consistency of expression

Studies of samples from a larger patient population confirmed the consistency of expression of the previously reported markers for malignancy in normal-appearing intermediate cervical cells in samples from patients with abnormal cytology (moderate dysplasia and severe dysplasia/carcinoma in situ). Based on samples of only 30 cells per case, a false-negative rate of 10% to 30% was estimated. The expression of the marker features thus provides a clear indication of uterine abnormal cytology; the lack of expression, however, does not entirely rule out the possibility of uterine abnormalities. The use of larger sample sizes and better staining protocols could further enhance the usefulness of marker feature studies in the prescreening for cancer.     

Tissue and serum TPA in subjects at risk for bladder cancer

We tested the presence of tumour polypeptide antigen (TPA) in lower urinary tract cells from 59 workers exposed to known bladder carcinogens and from 30 control subjects. We then correlated immunocytological expression and serum TPA levels. Lower urinary tract cells from 31 subjects gave either moderately or strongly positive immunocytological stains. Five also had high serum TPA. The detection of TPA by cytology and in serum differed significantly in workers exposed to cancer agents and the control group.     

Nephrogenic adenoma of the urinary bladder. Histologic and cytologic observations in a case

The histologic and cytologic features of multiple tissue and voided urine specimens from a man with nephrogenic adenoma of the urinary bladder are described. The urinary cytology showed papillary fragments, with cells showing palisade formation. The cells were round to oval, with a centrally placed nucleus with a fine chromatin pattern. These cells were identical to those seen in a low-grade papillary transitional-cell carcinoma. Tissue biopsy is needed to separate these two very different pathologic conditions.     

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.     

Multiprobe fluorescence in situ hybridization (UroVysion) for the detection of urothelial carcinoma - FISHing for the right catch

Urinary cytology has a well-established role in the detection and monitoring of urothelial carcinoma. The main strength of cytology is the high specificity for high-grade urothelial carcinoma and carcinoma in situ, but it has a low sensitivity for low-grade, non-invasive tumors. There are several other limitations of cytology. Cytology of the upper urinary tract and after intravesical therapy with bacillus Calmette-Guerin is notoriously difficult to interpret. In addition, there is a poorly defined but commonly used category of atypical cytology of uncertain significance. The UroVysion multiprobe fluorescence in situ hybridization has emerged as a helpful tool to address these limitations. It consists of fluorescently labeled DNA probes to detect increased copy numbers (polysomy) of the chromosomes 3, 7 and 17 and deletion of 9p21, the site of the P16 tumor suppressor gene. Multiple studies have shown that fluorescence in situ hybridization in voided urine and washing specimens can help in patient management due to its superior sensitivity over cytology in different situations. It can be particularly useful to clarify equivocal cytological findings. However, some aspects remain to be further addressed including cost efficiency, optimal cut-off values and the true performance under real-life conditions.