Antonius G. J. M. Hanselaar

Ovarian stimulation and granulosa-cell tumour

Abstract Ovarian stimulation in the treatment of infertility is far from physiological because patients and their ovaries are exposed to high concentrations of gonadotropins. Many studies have focused on the two most common side-effects of ovarian stimulation—ie, hyperstimulation and multiple pregnancy. We describe 12 patients in whom granulosa-cell tumour was discovered after ovarian stimulation treatment with clomiphene citrate and/or gonadotropins. Although we cannot prove a causal link between the tumour and the medication, investigations in animals have shown a relation between gonadotropin exposition and the development of granulosa-cell tumours. The possible relation of ovarian stimulation and granulosa-cell tumours in human beings has not been published before. We postulate three explanations for this finding; first, the granulosa-cell tumour is present in the ovary, waiting for a hormonal trigger; second, increased follicle stimulating hormone concentrations are oncogenic to granulosa cell; and third, the onset of the granulosa-cell tumour during ovarian stimulation is coincidental. We recommend that ovarian stimulation is done only if there is a valid indication after proper assessment of the ovaries, and that women who have had ovarian stimulation are followed for longer than at present.

Short Fragment Polymerase Chain Reaction Reverse Hybridization Line Probe Assay to Detect and Genotype a Broad Spectrum of Human Papillomavirus Types : Clinical Evaluation and Follow-Up

The purpose of this study was to detect and genotype 16 different human papilloma virus (HPV) types simultaneously using a short fragment polymerase chain reaction (SPF) hybridization line probe assay (LiPA). 152 women who were referred to the gynecologist because of abnormal cervical smear underwent colposcopic examination and repeat cervical smear. In addition, the cervical scrapes were analyzed for the presence of HPV by a novel general HPV polymerase chain reaction assay followed by a single reaction genotyping assay allowing for a simultaneous detection and identification of 16 different HPV types. HPV DNA was detected in 38% of normal follow-up cervical scrapes, 51% of scrapes with atypical squamous cells of undetermined significance, 78% of scrapes with mild dysplasia (low grade squamous intraepithelial lesions), 86% of scrapes with moderate dysplasia (high grade squamous intraepithelial lesions), and in 88% of scrapes with severe dysplasia and carcinoma in situ. One case of invasive squamous cell carcinoma was positive for HPV 16. Overall, a single HPV type was detected in 56% of HPV positive scrapes, with HPV 16 being the most common and accounting for 45% of all single infections. Forty-four percent of the positive scrapes contained multiple HPV types, of which double infections prevailed. Follow-up results proved the reproducibility and reliability of SPF HPV LiPA. In conclusion, we have used and evaluated the SPF-HPV-LiPA system for the detection and genotyping of HPV infections. The combined detection-typing method proved to be sensitive, specific, simple, and fast, making mass screening of cervical scrapes accessible for routine practice and facilitating individual patient management.

Liquid-based cervical cytology†

The objective of the current study was to evaluate the applicability of liquid‐based cytology in the Netherlands population screening program for cervical cancer.

Clear cell adenocarcinoma of the vagina and cervix

The objective of this study was to update the registry of women in the Netherlands with clear cell adenocarcinoma (CCAC) of the cervix or vagina with or without intrauterine exposure to diethylstilbestrol (DES).

Criteria for organized cervical screening programs: Special emphasis on the Netherlands program

Based on the criteria of Wilson and Jungner and experiences in the population-based organized cervical screening program in the Netherlands, conditions for efficient and effective population screening for cervical cancer are described. The purpose of this paper is to determine if these criteria are met for cervical cancer screening and to give recommendations for improvement. Cervical cancer is still an important health problem; the present incidence reflects both background risk and screening activity during previous decades. A positive effect of screening is reached because of the long development time of the disease and the ability of the Pap smear test to detect precancer and early, symptomatic disease. Considerable reduction in the incidence and mortality of cervical cancers can be reached if all women attend and all detected lesions are adequately followed up. Common terminology and classification criteria for histology and cytology should be used. Whether newly developed techniques that may improve or replace cytology can be used in screening programs should be a multidisciplinary decision after clinical trials have given evidence-based information on the performance, cost-effectiveness and need of these techniques. When cervical cancer screening is undertaken, it should be offered in organized programs at the medical level closest to the patients, the general practitioner. High compliance is the most important factor in reducing cervical cancer incidence. Quality control and assurance must be performed at all levels. In the case of limited resources, the program should use a five-year interval and concentrate on the age range 25-60 years, with special attention to women who have never been screened or were screened > 10 years previously. Evaluation of medical and organizational aspects is mandatory. Cooperation between all involved parties is a prerequisite of creating a successful screening program.

Hue-Saturation-Density (HSD) Model for Stain Recognition in Digital Images From Transmitted Light Microscopy

BACKGROUNDnTransmitted light microscopy is used in pathology to examine stained tissues. Digital image analysis is gaining importance as a means to quantify alterations in tissues. A prerequisite for accurate and reproducible quantification is the possibility to recognise stains in a standardised manner, independently of variations in the staining density.nnnMETHODSnThe usefulness of three colour models was studied using data from computer simulations and experimental data from an immuno-doublestained tissue section. Direct use of the three intensities obtained by a colour camera results in the red-green-blue (RGB) model. By decoupling the intensity from the RGB data, the hue-saturation-intensity (HSI) model is obtained. However, the major part of the variation in perceived intensities in transmitted light microscopy is caused by variations in staining density. Therefore, the hue-saturation-density (HSD) transform was defined as the RGB to HSI transform, applied to optical density values rather than intensities for the individual RGB channels.nnnRESULTSnIn the RGB model, the mixture of chromatic and intensity information hampers standardisation of stain recognition. In the HSI model, mixtures of stains that could be distinguished from other stains in the RGB model could not be separated. The HSD model enabled all possible distinctions in a two-dimensional, standardised data space.nnnCONCLUSIONSnIn the RGB model, standardised recognition is only possible by using complex and time-consuming algorithms. The HSI model is not suitable for stain recognition in transmitted light microscopy. The newly derived HSD model was found superior to the existing models for this purpose.

MIB1, A PROMISING MARKER FOR THE CLASSIFICATION OF CERVICAL INTRAEPITHELIAL NEOPLASIA

Formalin‐fixed and paraffin‐embedded tissue specimens of normal and dysplastic cervical epithelia (five CIN1, seven CIN2, five CIN3, and five normal) were assessed by an immunoperoxidase technique, using the monoclonal antibody MIB1, regonizing a formalin‐fixation‐resistant epitope on the cell proliferation‐associated Ki‐67 antigen. An image analysis system was used to measure four parameters associated with proliferative activity: the Ki‐67 labelling index (LI), the number of Ki‐67‐positive nuclei per unit length of basement membrane, and the maximum value and 90th percentile of the relative distances of Ki‐67‐positive nuclei from the basement membrane. All these four proliferation‐related parameters were highly correlated with the grade of dysplastic change in the epithelium (0·90

AgarCyto: A Novel Cell-processing Method for Multiple Molecular Diagnostic Analyses of the Uterine Cervix

SUMMARY In diagnostic cytology, it has been advocated that molecular techniques will improve cytopathological diagnosis and may predict clinical course. Ancillary molecular techniques, however, can be applied only if a sufficient number of preparations are made from a single cell sample. We have developed the AgarCyto cell block procedure for multiple molecular diagnostic analyses on a single scraping from the uterine cervix. The optimized protocol includes primary fixation and transport in ethanol/carbowax, secondary fixation in Unifix, and embedding in 2% agarose and then in paraffin according to a standard protocol for biopsies. More than 20 microscopic specimens were produced from a single AgarCyto cell block, and standard laboratory protocols have been successfully applied for H&E staining, immunohistochemistry for Ki-67 and p53, and in situ hybridization for the centromere of human chromosome 1 and human papilloma virus Type 16. In addition, single AgarCyto sections yielded sufficient input DNA for specific HPV detection and typing by LiPA-PCR, and the protocol includes an option for DNA image cytometry. The AgarCyto cell block protocol is an excellent tool for inventory studies of diagnostic and potentially prognostic molecular markers of cervical cancer.

Development and validation of a computerized cytomorphometric method to assess the maturation of vaginal epithelial cells.

BACKGROUNDnAfter menopause, declining levels of estrogens may cause vaginal discomfort, or so-called vaginal atrophy. Evaluation of therapies for vaginal atrophy may be performed using the so-called maturation index. The maturation index is expressed as the percentage of (para)-basal, intermediate, and superficial epithelial cells in a vaginal smear. Manual assessment of the maturation index is subject to inter- and intraobserver variations. In this study, assessment of the maturation of cells in vaginal smears using automated image analysis was investigated.nnnMATERIALS AND METHODSnAutomated assessment, using a commercially available image analysis system, was performed on hematoxylin-eosin-stained cytospin specimens. A training set was constructed by an experienced cytotechnologist, based upon visual classification of stored grey value images. From this, two discriminant functions (DFs) were calculated capable of classifying cells in one of the three types. These cell classifiers were capable of classifying 97% of the cells correctly. Data from automated assessment were compared with those of classical manual counting. Specimens of 13 mature and 6 atrophic vaginal specimens were assessed in duplicate, both manually and by image analysis, using the DFs.nnnRESULTSnNo significant interobserver effect was found for image analysis, whereas a significant effect was found for manual counting. Both methods were able to distinguish between matured and atrophic specimens.nnnCONCLUSIONSnIt was concluded that for assessment of vaginal maturation, the use of automated image analysis systems is recommended. Besides increased reproducibility, image analysis systems yield additional data describing the size and shape of the cytoplasm and nucleus of cells, which might increase discriminating power.

Spectral imaging of multi-color chromogenic dyes in pathological specimens

We have investigated the use of spectral imaging for multi‐color analysis of permanent cytochemical dyes and enzyme precipitates on cytopathological specimens. Spectral imaging is based on Fourier‐transform spectroscopy and digital imaging. A pixel‐by‐pixel spectrum‐based color classification is presented of single‐, double‐, and triple‐color in situ hybridization for centromeric probes in T24 bladder cancer cells, and immunocytochemical staining of nuclear antigens Ki‐67 and TP53 in paraffin‐embedded cervical brush material (AgarCyto). The results demonstrate that spectral imaging unambiguously identifies three chromogenic dyes in a single bright‐field microscopic specimen. Serial microscopic fields from the same specimen can be analyzed using a spectral reference library. We conclude that spectral imaging of multi‐color chromogenic dyes is a reliable and robust method for pixel color recognition and classification. Our data further indicate that the use of spectral imaging (a) may increase the number of parameters studied simultaneously in pathological diagnosis, (b) may provide quantitative data (such as positive labeling indices) more accurately, and (c) may solve segmentation problems currently faced in automated screening of cell‐ and tissue specimens. Figures on http://www.esacp.org/acp/2001/22‐3/macville.htm.