Koilocytosis and squamous (pre)neoplasia as detected in population-based cervical screening: practice and theory.

INTRODUCTION: Koilocytosis (cavitation of the cytoplasm due to active HPV infection) can be detected in the screening process for cervical carcinoma. OBJECTIVE: To report the practice of detection of koilocytosis and (pre)neoplasia in population screening and to exploit the collected data to propose an explanation for the relationship between HPV infection and nuclear precancerous changes. STUDY DESIGN: Centrally collected and stored (SBBW, Leiden, the Netherlands) data from all smears of six regional pathology laboratories (1995-2002), coded according to KOPAC (the national cervical smear coding system; S1: normal thru S9: invasive carcinoma) were accessed. Prevalences per 100,000 smears were calculated for koilocytosis and for squamous abnormalities after stratification for country of origin of screenees. The relative risk (RR) for the ethnic (age) groups was computed by dividing the prevalence of the relevant ethnic (age) group by the prevalence of all women. RESULTS: Surinamese women featured the highest prevalence of koilocytosis and of all squamous abnormalities. Moroccan women the lowest. The RR for koilocytosis was highest at 30 years (1.84) and lowest at 60 (0.26). RR dependence on age of S5-S9 lesions was similar. Compared to nonkoilocytotic smears, koilocytosis was 104 times more frequent in the 1,500 S4 smears, 36x more frequent in the 6,700 S2-S3 smears, and 24x more frequent in the 1,740 S5-S9 smears. In all three categories this difference is statistically significant. CONCLUSION: High prevalences for both koilocytosis and for preneoplasia were detected in Surinamese immigrants, however, it still does not exclude HPV infection as a confounder linked to sexual lifestyle. The presence of koilocytosis in cervical smears may serve to identify patients with an increased risk for cervical cancer and perhaps warrant more intensive surveillance than what is provided through five-yearly screening.

Legal issues in the treatment of a violent manic patient in a non-gazetted setting: a case report.

INTRODUCTION: Psychiatrists in non-gazetted treatment settings, like psychiatric wards in restructured general hospitals and private hospitals, face a major problem when psychiatric patients who require admission are either not competent or refuse to consent to admission and treatment, although they are clearly in need of such inpatient management. Admission to the state mental hospital is often refused by their relatives for a number of reasons, like the stigma attached to admission to such a hospital, and the fear that future employment prospects might be affected. CLINICAL PICTURE: Mr X, a manic, violent patient, had no insight into his disorder and refused admission and treatment for his manic episode. He was the head of a large corporation, and his relatives were apprehensive he would make decisions that could jeopardize the company. TREATMENT: He refused oral medication, could not tolerate parenteral haloperidol and had lithium nephrotoxicity. Inpatient electroconvulsive therapy had to be administered, after which he responded satisfactorily. CONCLUSION: The legal implications in this case, like consent for treatment and admission, and ethical issues, are discussed.

Microwave irradiation and cross-linking of collagen.

In a multifactorial experiment, dermal sheep collagen was treated in diluted glutaraldehyde solutions, 70% ethyl alcohol, Cialit 1:5000, and distilled water for 1, 3 and 5 min, respectively, in combination with microwave irradiation at different temperature settings. The shrinkage temperature indicating the degree of cross-linking achieved was then determined. Treatment in 0.65% glutaraldehyde with microwave irradiation setting for 60 degrees C resulted in the maximum shrinkage temperature within 1 min, whilst at the lower setting of 50 degrees C, the maximum shrinkage temperature for both glutaraldehyde solutions is only reached after 5 min. Neither microwave irradiation by itself, Cialit or ethyl alcohol induce cross-linking of collagen fibres. These findings are relevant for implant studies.

Microwave treatment of xenogeneic cartilage transplants.

Human rib cartilage was irradiated with microwaves according to six different methods and transplanted into rabbits. Untreated rib cartilage preserved in Cialit served as a control. After 12 and 40 wk of implantation, the microscopic appearance of these xenogeneic cartilage transplants was given a score in comparison with the transplants preserved in Cialit. The microwave treatment of the cartilage appeared to improve the results. The Cialit-preserved transplants showed progressive resorption by macrophages with central necrosis and fragmentation, which was not present in the microwave-treated grafts. Microwaves seem to stabilize the cartilage matrix and enhance the diffusion of fixatives. Irradiation in ethanol as an immersion fluid appeared to be the best method. The results of transplantation can benefit from the use of microwaves in the preservation of the cartilage. It is argued that, in addition, microwave irradiation might be used for inactivation of human immunodeficiency virus in human cartilage used for transplantation.

Major improvement in microscopic-image quality of cryostat sections. Combining freezing and microwave-stimulated fixation.

In this article the authors describe a procedure to obtain cryostat sections of superior quality using microwave stimulation of a fixation reagent with suitable microwavability properties. In fact, the light microscopic images are of a quality that is comparable to that of the best conventional paraffin technique. The new procedure requires less time than the existing cryostat technique. The only additional piece of equipment needed in this procedure is a relatively inexpensive commercially available microwave oven.

Accelerated immunogold silver and immunoperoxidase staining of paraffin sections with the use of microwave irradiation. Factors influencing results.

With the use of microwave irradiation, incubation times for the immunotechniques used in immunomicroscopy can be shortened significantly. The authors found that factors influencing results are irradiation time, power output, and presence of gold label. Precise temperature control is essential, and techniques to achieve this are discussed. For beta-human chorionic gonadotropin (beta-HCG), the results with the immunoperoxidase technique were inferior to those of the conventional technique, and with the immunogold silver technique an enhancement of labeling results was achieved. Immunogold silver staining for beta-HCG on paraffin sections can be performed with the use of microwave irradiation within one hour.

Microwave irradiation of human brain tissue: production of microscopic slides within one day.

A three step method using microwave irradiation enabled microscopic slides of human brain tissue to be obtained within one working day: steps 1 and 2 hardened and solidified brain tissue; step 3 completed formalin fixation. The efficacy and precision of the method was compared with slides of conventionally processed brain tissue that had been fixed in formalin for six weeks. The microscopic quality of the sections was excellent with good presentation of brain tissue and equalled that of conventionally processed slides.

Programming the microwave oven.

Microwaves can be used to stimulate chemical bonding, diffusion of reagents into and out of the specimen, and coagulation processes in preparatory techniques. Temperature plays an important role in these processes. There are several ways of controlling the temperature of microwave-exposed tissue, fluids and solids that are used in these preparatory techniques. In this paper we describe our accumulated experience with monitoring and controlling microwave-oven parameters with the help of a personal computer (PC). A specially designed microwave oven was used with an advanced temperature probe. The oven was connected to an IBM-compatible PC. This enabled us to analyze the quality of temperature control, and the relationship between displayed and actual temperature. We show that for microwave-transparent materials the addition of a microwave-absorbent plate to the material results in better temperature control.

Immunocytochemistry on free-floating sections of rat brain using microwave irradiation during the incubation in the primary antiserum: light and electron microscopy.

We studied the effects of microwave irradiation during the incubation of free-floating brain sections with primary antibodies against gamma-aminobutyric acid (GABA), enkephalin and vasopressin. Vibratome sections of perfusion-fixed rat brain were incubated: (a) overnight at room temperature (20-22 degrees C), (b) during various periods of time under microwave irradiation, such that the induced temperatures did not exceed 10 degrees C, (c) same as (b) but with induced temperatures not exceeding 40 degrees C, (d) without microwave irradiation, at 4-10 degrees C (temperature control for (b)), (e) same as (d) but at 40 degrees C (temperature control for (c)). During the incubation-irradiation we continuously monitored the temperature and controlled it by cooling and by manipulating the energy output of the magnetron. The peroxidase immunocytochemical procedure was completed using for all sections the same incubation parameters. Selected GABA-immunoreacted sections were examined in the electron microscope. Incubation at 10 degrees C in the primary antiserum as short as 30 min, with or without microwave irradiation, already results in (weak) binding of the antibodies to immunoreactive structures. One or 2 h of incubation in the primary antiserum in the microwave oven at 40 degrees C or at the same temperature outside the microwave oven results in excellent staining of GABA-immunoreactive structures and of good staining of enkephalin- or vasopressin-immunoreactive structures. The ultrastructural details were much better preserved in incubated-irradiated sections than in sections incubated overnight and only slightly less preserved than in the other control sections. There is no improved penetration of the antibodies into the sections. We conclude that by using microwave technology or by raising the temperature of the incubation medium, the time of incubation, at least in these antisera, can be shortened drastically, whereas the ultrastructural details remain well preserved.

Microwave-cryostat technique for neuroanatomical studies.

A novel method to process nervous tissue for neuroanatomical studies is presented. In this cryostat method, the brain tissue is first treated with microwave irradiation after perfusion with physiological saline. Cutting quality of the microwave-treated brain tissue was excellent. The sections were stained with the Nissl staining, the Klüver-Barrera method, and the Bodian method, respectively. In addition, immunocytochemistry using the RAM/PO method was performed. For the latter a monoclonal antibody against neurofilament (NF90), recognizing the 200, 150, and 70 kD units was used. Excellent results were obtained. The advantage of the microwave-cryostat technique for processing of nervous tissue is not merely a substantial reduction of time to obtain microscopical slides of good quality. This method allows in addition the estimation of the effect of fixation by formaldehyde in immunocytochemistry.

4D confocal microscopy for visualisation of bone remodelling.

Until recently it was very time consuming and difficult to make three-dimensional (3D) images of newly formed bone. With the advent of confocal technologies and increased computer power, 3D imaging is greatly facilitated. In this paper we demonstrate that enhanced confocal visualisation of newly formed bone is possible when bone is labelled in vivo sequentially with two osteotropic markers (xylenol orange and tetracycline). Computer-assisted reconstruction of the confocal optical sections was achieved through the use of the CONVEX Application Visualisation System (AVS). The computerised image data provides the researcher with ample flexibility in displaying the results. It was found that CSLM combined with AVS is excellent for visualising the remodelling process in three and four dimensions, in which the fourth dimension is time. With this approach visualised bone remodelling has become possible in a manner not easily achieved by other techniques.

Microwaves for immunohistochemistry.

Microwaves are now widely used in immunohistochemistry for fixing and stabilizing tissue prior to embedding and cutting, for antigen retrieval and for immunoincubations. These techniques can be used for frozen sections and for material embedded in paraffin and plastic. Material prepared in this way shows high contrast in light microscopy. In principle, these microwave methods can also be used for electron microscopy. To be successful in the application of these techniques, insight into the physics of exposure to microwaves and the effects of microwaves on the material is a must. Microwave immunohistochemistry depends on optimal temperature control. To guarantee this, special measures should be taken and dedicated laboratory ovens should be used. The recently developed Coverplate units facilitate immunoincubations in the microwave oven. We show that the total microwave approach, combining microwave fixation, embedding and immunoincubations, is very useful for confocal microscopy.

Boon's disease: hemorrhagic cystitis in conjunction with massive exfoliation of degenerated urothelial cells (apoptosis?) during intercontinental flights in an otherwise healthy person.

Hemorrhagic cystitis is a well-defined clinical emergency, usually occurring in the course of treatment with toxic agents such as cyclophosphamide. We present a case of hemorrhagic cystitis in an otherwise completely healthy female. The three documented attacks were severe and started during intercontinental flights. This type of hemorrhagic cystitis as a disease proved to be a boon, treatable by drinking large amounts of water, and was diagnosed by and in Dr. Boon; thus was the appellation Boon's disease coined. Cellular changes in the urine specimen taken after onset of the disease indicated massive exfoliation of degenerated urothelial cells with morphological features suggestive of apoptosis. It seems likely that this process can be initiated by any event which is associated with compromise of vitality of the urinary bladder lining, such as may occur in hypovolemia. This type of hemorrhagic cystitis is most probably not uncommon in susceptible individuals during intercontinental flights.

Neural network processing can provide means to catch errors that slip through human screening of pap smears.

The problem of the false-negative smear deserves the attention of the cytologic community. We found that by using the PAPNET system, equipped with neural network programming, cancer cells can be detected in repeatedly misdiagnosed Pap smears. The correct diagnosis of these false-negative smears depended on the skills of the diagnostician to recognize the cells on one or more of the 128 tiles of the video display as abnormal, and to make the proper decision when to turn to light microscopy. In 8 of the 10 tested false-negative smears, the cancer cells were found exclusively in epithelial fragments; in two cases there were no more than five abnormal cells in the smear (which were detected by PAPNET). This study gave us insight into the nature of the false-negative problem and showed us that the application of neural network processing can provide means to catch errors that slip through human screening of Pap smears.

Semiautomatic PAPNET analysis of proliferating (MiB-1-positive) cells in cervical cytology and histology.

With the event of microwave-antigen retrieval it has become possible to detect proliferating cells (staining positive for MiB-1) in cervical smears containing epithelial fragments and in paraffin sections containing cancerous cervical epithelium. The PAPNET system, using neural network computing, is able to collect from the slides epithelial fragments with positive-staining nuclei. The nuclei in epithelial fragments are semiautomatically quantitated using the PAPNET-digitized images. The parameter PPN (proportion-positive nuclei), in which the nuclear area of the positive nuclei is taken into account, prove to be superior to the proliferation index for distinguishing moderate dysplasia from carcinoma in situ. In repair cells, all four quantitative parameters are close to those of moderate dysplasias, indicating that this method is unfit for distinguishing these two entities. However, we show that MiB-1 staining is valuable for the analysis of "false-negative" and "false-positive" smears, and for quantifying proliferation in sections of carcinoma in situ.

Analysis of three-dimensional architecture in Feulgen-stained fine-needle liver aspirates, using confocal scanning laser microscopy.

Early and accurate diagnosis of hepatocellular carcinoma in liver nodules using fine-needle aspiration biopsy can still pose difficulties, particularly in cirrhotic nodules containing dysplastic hepatocytes. Loss of architectural clues, which may aid diagnosis, can be overcome by using confocal scanning laser microscopic examination of tissue fragments in smears without the need for further processing of slides. The basic fuchsin of the Feulgen method resulted in excellent confocal images without the need for further processing of the smears. Clear morphological differences in three-dimensional reconstructions of optically sectioned tissue fragments were demonstrated in normal, hyperplastic, and malignant smears as an aid to diagnosis.

Confocal optical sectioning and three-dimensional reconstruction of carcinoma fragments in Pap smears using sophisticated image data processing.

Carcinoma fragments found in Pap smears contain important diagnostic information not available to the light microscopist because of their thickness and consequent blurring. Optical sectioning by the confocal microscope allows us to reclaim the mitotic figures, glandular architecture, and abnormal chromatin patterns in the restained original smears. The high spatial resolution of the confocal microscope can be further exploited by processing the digital images with the sophisticated Application Visualization System (AVS) on a CONVEX computer. Serial sections in which the fluorescent signals are color coded by this software package and three-dimensional reconstructions of the nuclei and mitotic figures expand our knowledge of these malignant epithelial fragments.

A model for quantitative follow-up studies of cervical lesions.

A model for follow-up studies of cervical lesions is described in which the specimen were taken in a noninvasive way using Cytobrushes and in which automatic measurement of the abnormal nuclei in the sampled epithelial fragments was possible because 2-microns thin Feulgen-stained plastic sections were prepared. The planimetric parameters AREA, PERIMETER, FORM PE, and FORM ELL, and the densitometric parameters optical density (OD) and integrated optical density (IOD) were assessed. The mean total volume and the mean total DNA were calculated using stereological methods. The moderate dysplasias differed from the carcinoma in situs for all the densitometric parameters except for OD, and from the invasive carcinomas for IOD and 5cER. In the moderate-dysplasia group, there were three types of DNA histograms: a highly abnormal type resembling the histograms of the carcinoma group, a normal type, and an intermediate type. Changes in DNA histograms can be established during follow-up studies of dysplasias without having disturbed the lesion due to the efficient and elegant noninvasive sampling method that was not used in earlier quantitative studies.

Presence of proliferating (MiB-1-positive) cells in cervical smears of women infected with HIV is associated with clinical outcome: a study of Brazilian women.

The hypothesis tested was that there is an association between the presence of proliferating (MiB-1-positive) cervical cells and clinical outcome of women infected with human immunodeficiency virus (HIV). Female partners (attending the Gynecology Outpatients Clinic of the University Hospital of Rio Grande, Brazil) of known HIV-positive (HIV+) men were used for this pilot study. Among these women, 25 were also HIV+. Papanicolaou smears of these 25 HIV+ women and of 44 HIV- women were graded as negative, CIN I, CIN II, or CIN III, using neural network screening. MiB-1 grading and HPV identification were also performed. The immune status of patients was determined using the current Centers for Disease Control classification. In agreement with the scientific literature, in these Brazilian women both CIN and HPV were associated with HIV. In the HIV+ women, the immune status tends to correlate with MiB-1 grading. Also, in the one case in whom progression from CIN I to invasive cervical carcinoma was observed, the smear contained many MiB-1-positive cells. Staining cervical smears of HIV+ women is a simple procedure to get an indication of clinical outcome of the patient.

Neural network-based digitized cell image diagnosis of bladder wash cytology.

In this pilot study, we tested whether it is possible to apply neural network-based diagnostics on bladder washings to detect urothelial cell carcinoma of the bladder. Eighty-five bladder wash (BW) samples were chosen at random from our own database. Cystoscopy, histology, and follow-up data concerning tumor recurrence were available. Each slide was scanned by the neural network-based digitized cell image system. The neural network-based diagnosis (NNBD) was based on 128 digitized cell images provided by the system. The light microscopic diagnosis (LMD) was rendered by an experienced cytopathologist using the same terminology, i.e., negative, low-grade tumor, and high-grade tumor. Finally, an automatic QUANTICYT analysis was performed on the same material, with as classification low, intermediate, and high risk. The sensitivity for diagnosing a histologically confirmed tumor was for NNBD 92%, for LMD 50%, and for QUANTICYT 69%. For the three methods, receiver operating characteristic (ROC) curves were made for the thresholds low grade/intermediate risk and high grade/high risk. For the prediction of a positive cystoscopy, the highest area under the curve (AUC) was found for NNBD, being 0.71. The AUC for LMD was 0.58. QUANTICYT analysis had the highest AUC value (0.62) for predicting tumor recurrence after a negative cystoscopy, with a lower value for NNBD (0.50). These findings indicate that neural network-based diagnosis of bladder washing samples is highly promising.