BioPEPR: a system for the automatic prescreening of cervical smears.

A feasibility study has indicated that a Prescion Encoding and Pattern Recognition (PEPR) cathode ray tube prescreening system for cervical smears can be both accurate and fast. Smears are prepared using a syringing technique and are stained with a Feulgen-type nuclear stain and a protein counter-stain. The use of film as an intermediate step between the cells and Bio PEPR allows the scanning of fields as large as 8 x 8 mm. The morphological features of the cells are measured as directed by a hierarchical decision strategy. Additional programs detect artifacts, overlaps, and leukocytes. For clean samples, false positive and false negative rates on the cell level have been obtained that will allow acceptable smear level rates (10% false positive, 1% false negative). These rates have been reached without compromising the required speed goals of 120 to 180 smears per hr. The efficiency of the system is dependent on the quality of the smears. Measurements on a set of 192 routinely prepared smears indicate acceptable false negative rates and a false positive rate of about 18%. A reduction of this rate is expected with small improvements in cell preparation and measuring software, leading to the overall system efficiency required for commercial feasibility.     

Rapid (partial) prescreening of cervical smears: the quality control method of choice?

Rapid rescreening of all negative and inadequate smears is the quality control method of choice in the UK. The sensitivity of primary screening of laboratory and individual screeners are major indicators of screening quality and are dependent on the number of false negative smears found by rapid screening for their calculation. High sensitivity may indicate good quality primary screening or poor quality rapid review. Quantifiably high quality rapid rescreening is essential if these sensitivity figures are to be meaningful. A 12-month study was undertaken in routine practice using the prescreening mode to ascertain the sensitivity of rapid (partial) screening in our department. The final results of smears were compared with those of rapid prescreening. The calculated sensitivity ranged from 92-54% for high-grade abnormalities and 75-33% for all grades, revealing a wide range of performance between individual prescreeners. Rapid prescreening can identify individuals best suited to rapid screening in routine practice. By using these prescreeners only, the sensitivity of cervical screening could be raised. Rapid (partial) prescreening should be considered as the quality control method of choice.     

Flow cytometric classification of biopsy specimens from cervical intraepithelial neoplasia

The distribution of single-cell DNA content was investigated in biopsy specimens from the human cervix of 121 women suspected of having intraepithelial neoplasia. Comparison of the results of the histopathological examination with the ploidy level showed that all normal specimens were diploid. Thus, no false-positive results occurred. Most of the specimens classified as mild and moderate dysplasia were diploid as well. Aneuploid cell populations occurred in 78% of the lesions classified as severe dysplasia and carcinoma in situ. The ploidy level distribution permitted a natural division of the aneuploid cell populations into two groups with DNA indices either above or below 1.5. The importance of the aneuploidy in carcinogenesis is discussed.     

Telomerase activity in cervical smears.

It is well known that almost all carcinoma cells including those of the uterine cervix have re-established their telomerase activity. However, until now there is no conclusive picture on the telomerase activity in cervical dysplasias and about their relationship to HPV infection. To investigate this question, material from 34 patients (15 with normal epithelium, 11 with LGSIL, 8 with HGSIL) obtained by conventional cervical brushing was used and subjected to non-radioactive TRAP-ELISA (Boehringer Mannheim). The HPV analysis was performed by PCR on formalin-fixed, paraffin-embedded biopsy material obtained after cytological investigation. We could show that telomerase activity is detectable in normal cervical epithelium, and that an gradual increase exists for both telomerase activity and HPV positivity from normal epithelium to HGSIL. However, HPV infection and telomerase activity appear to be independent of each other. The high frequency of telomerase positivity in patients with normal cervical epithelium indicates that telomerase activity is not a useful differential diagnostic aid. Whether patients with telomerase-positive dysplasias have a higher probability to progress into an invasive carcinoma remains to be clarified by follow-up studies.     

Cytophotometric studies in suspicious cervical smears.

Papanicolaou stained smears of various cervical lesions diagnosed as "suspicious" by routine cytology were reviewed with regard to different cell types leading to this diagnosis. The smears were then submitted to Feulgen hydrolysis and redyed by Acriflavin-SO2 for fluorescence-cytophotometry. In nine of 14 cases measured, the DNA content of all types of "suspicious" cells was increased with DNA modes at euploid levels of 2 n, 4 n and 8 n indicating that the "suspicious" cells in those cases are polyploid. However, in five cases aneuploid DNA-distribution patterns were found similar to those observed in carcinoma in situ or severe dysplasia. Since polyploidization may be considered as a cellular response to higher functional requirement (i.e. inflammation or regeneration) a "suspicious" cervical smear with a polyploid DNA-distribution pattern may reverse to normal cervical epithelium after normal conditions are restored. However, a "suspicious" smear with an aneuploid DNA-distribution pattern should be considered more seriously as being related to a precancerous lesion requiring immediate surgical treatment.     

Flow cytometric analysis of porcine preadipocytes.

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal-vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta (TGF-beta). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD-1 cells surface antigen was greater in cultures supplemented with 2% pig serum and 10 nM dexamethasone than in other media. In cultures, GH, TNF-alpha and TGF-beta each inhibited lipid deposition, whereas TNF-alpha and TGF-beta, but not GH, inhibited AD-1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF-alpha and TGF-beta was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.     

Flow cytometric study of cultured mammalian cells.

Flow cytometry provides a rapid, sensitive and accurate analytical means to monitor hybridoma cell cultures. The use of flow cytometry has enabled us to study the changes in DNA, RNA, protein, IgG, mitochondrial activity and cell size that take place during the growth cycle of batch culture. The temporal changes in the levels of these analytes and their heterogeneity have been related to the growth/death kinetics. The maximum proportion of S-cells was reached early in the growth phase while a population of low fluorescence cells with lower polidy than G1, dead cells and fragmented nuclei emerged during the death phase. Supplementation with amino acids during the exponential phase prolonged the growth cycle by enhancing cell proliferation. The fraction of S/G2 cells was much reduced by a reduction in serum concentration but was maintained during the prolonged non-proliferating "stationary" phase. The magnitude of Rhodamine 123 staining showed a consistent and general decrease during late exponential and decline phases. This trend was accompanied by an increase in the fraction of the Propidium Iodide-stained population which reflected the deteriorating metabolic and membrane integrity. Decrease in mean fluorescence intensity for DNA, RNA, protein and intracellular IgG was noted at the decline phase. Intracellular immunofluorescence was a more reliable indicator of antibody productivity than surface immunofluorescence.     

Quality grading for cervical smears

Three thousand five hundred and eighty cervical smears were taken in 1990–1992 at a Genitourinary Medicine Clinic with various spatula or spatula brush combinations. the unsatisfactory rate and the detection of cellular abnormalities showed some relation to spatula type. However, the satisfactory smears screened in the laboratory are routinely assigned a quality grade-good, fair or poor. Analysis shows higher rate of detection of cellular abnormalities in good quality smears, the detection of dyskaryosis being twice as high, in contrast to the fair or poor quality smears. It is suggested that quality grade is a better way of classifying smear quality in the cervical screening programmes rather than the presence or absence of endocervical and/or metaplastic cells.     

Interpreting microbiopsies in cervical smears. A cytohistologic approach

OBJECTIVE: To assess interobserver variation in the diagnosis of thick tissue specimens (microbiopsies) in cytology smears and histologic sections taken from them, to evaluate the applicability of MIB-1 in histologic sections from microbiopsies and to evaluate whether processing microbiopsies in inconclusive smears has additional diagnostic value. STUDY DESIGN: Cytologic smears were selected in which there were diagnostic disagreements between pathologists and cytologists and microbiopsies were present. Interobserver variation among three pathologists and three cytologists in the diagnosis of these microbiopsies was investigated. The smears were processed for histologic sections, and interobserver variation between pathologist diagnoses were analyzed. An additional histologic slide stained for MIB-1 was used for consensus diagnosis. The consensus diagnosis was compared with available follow-up and its sensitivity and specificity determined. The value of applying the microbiopsy technique in slides diagnosed as inadequate or atypical squamous cells of undetermined significance (ASCUS) was analysed. RESULTS: From a series of 62,334 cervical smears, 49 with microbiopsies were selected. It was possible to derive histologic slides from 38 cases. Interobserver variability in the diagnosis of microbiopsies and histologic sections from them was moderate--kappa = .44 (SE = .06) and kappa = .44 (SE = .09), respectively. In the consensus meeting for all cases, a conclusive diagnosis was reached. The Pearson correlation coefficient between the consensus diagnosis and MIB-1 staining was r = .62. The sensitivity of the consensus diagnosis for the follow-up diagnosis was 71% and the specificity 60%. Diagnosis on approximately 50% of slides diagnosed as inadequate or ASCUS could be made. CONCLUSION: The histotechnical workup of microbiopsies is not difficult; however, their diagnosis can be a problem. Adequate diagnostic criteria are not available. Aided by MIB-1 staining, histologic sections from microbiopsies can be diagnosed, and the diagnoses correlated with follow-up in most cases. Processing of microbiopsies in smears with an inconclusive cytologic diagnosis or a diagnosis of ASCUS allowed correct diagnosis in 50% of cases in this study.     

Tumor marker studies of cervical smears. Potential for automation

The distribution of three tumor markers, epithelial membrane antigen (EMA), carcinoembryonic antigen (CEA) and prekeratin (PK) was studied in exfoliated epithelial cells in cervical smears using an immunoalkaline phosphatase staining technique to demonstrate the antigens. EMA was expressed by abnormal cells in a consistent and reproducible fashion whereas the other two markers were variably expressed by both normal and abnormal cells. Our results suggest that immunocytochemical staining for EMA could be of value not only for the diagnosis of cervical intraepithelial neoplasia but also for the automated screening of cervical smears.     

Flow cytometric analysis of microorganisms

The application of flow cytometry to microorganisms is as old as the technique itself, but it has historically been underexploited for microbial applications. This is now being reversed and microbiologists are ideally placed to benefit from recent technological advances. While earlier papers demonstrated the use of flow cytometry for studies of viability and taxonomy, recent developments in bioinformatics and reporter gene technologies are leading to novel applications in microbiology. Variants of green fluorescent protein have been used for the study of conditional microbial gene regulation in medically important host-pathogen interactions and fluorescence-activated cell sorting is being applied to the isolation of novel mutants in directed evolution studies. This paper reviews the reasons for the delay in the application of flow cytometry to microbial problems, the range of applications, and their limitations and considers the progress made in developing new strategies for use in microbiological investigations.     

Flow cytometric analysis of adriamycin-perturbed mouse mammary tumors.

The effects of a single intraperitoneal injection of adriamycin (10 mg/kg) on a fast-growing C3H mouse mammary tumor (S102F) have been analyzed volumetrically, biochemically, autoradiographically and flow cytometrically. Mathematical simulation of the data was also used to aid in the interpretation of the recovery kinetics. This dose of adriamycin did not induce regression in tumor volume but did inhibit the growth rate for 4-5 days. 3H-TdR incorporation was gradually inhibited to reach a low of 20% of control at 24 and 36 hr and then recovered back to control by 96 hr after adriamycin treatment. The flow cytometric analysis also showed a marked reduction in the relative fraction of cells in the S-phase with a minimum of 23% of control at 72 hr; however, in contrast to the 3H-TdR incorporation data, the fraction of cells in the S-phase was only at 39% of control at 96 hr after the adriamycin injection. Since the 3H-TdR incorporation data disagreed with the flow cytometry data, autoradiographic analysis was also done at selected times after the adriamycin injections, and qualitatively, this analysis confirms the flow cytometry data in that the labeling index was 29% of control at 96 hr after adriamycin. The mitotic index also dropped from 8 to 1%, respectively, for controls and at 96 hr posttreatment. The degenerate index was about 1% in control tumors and no increase was observed in treated tumors. Adriamycin-induced cell-cycle delay occurs predominately in G1 and G2 but there is also an apparent minor delay in the transit across the S-phase and some apparent cytotoxicity in G2 and/or M. The long delay in volumetric growth appears to be due to the extended cell-cycle delay rather than extensive cell killing.