A Dutch view on American cytology in the headlines.
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Cytodiagnosis↗
Biomedical subjects
M E Boon
Publications and source records attributed to M E Boon.
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.
Antigens, Nuclear↗
Collagen implants remain supple not allowing fibroblast ingrowth.
Dense, reconstituted collagen membranes, using an optimized cross-linking protocol, were implanted subcutaneously into rats. After implantation, only a mild inflammatory response in the surrounding tissues was observed. The morphology of the fibroblasts around the membranes was seen to go through characteristic stages of proliferation and synthesis. In the 7 week follow-up it was found that these membranes did not allow any fibroblast ingrowth into their substance and remained completely intact and supple without signs of calcification, whereas the non-cross-linked controls proved to be completely degraded. The cross-linked reconstituted collagen may thus be considered for use in situations where neither incorporation nor dissolution of the biomaterial is desired.
Adipose Tissue↗
Transience of cervical HPV infection in sexually active, young women with normal cervicovaginal cytology.
Human papillomavirus DNA was detected in cervical specimens from 366 sexually active young women with cytomorphologically normal cervices using the polymerase chain reaction. In 93% (25/27) of initially infected women, the same viral type was not detected upon re-examination four menstrual cycles later. These results suggest that the majority of HPV infections in young women are transient.
Adolescent↗
Handling of large brain specimens: the use of microwaves. A review.
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Brain↗
Combining microwave stabilization and microwave-stimulated fixation of brain tissue with microwave-stimulated staining.
Microwave stabilization of rat brain tissue using saline was compared with microwave-stimulated fixation using either formaldehyde or Kryofix. Sections were stained according to the microwave-adapted Nissl, Klüver-Barrera, and Bodian methods. Depending on the preparative choice the microwave staining method had to be adjusted. Optimal combinations of preparative techniques and staining methods are feasible. In most cases the microwave saline-stabilized and microwave Kryofix-treated sections were better suited for application in neurohistology than the microwave formaldehyde-treated sections for light microscopy.
Animals↗
Ultrarapid vacuum-microwave histoprocessing.
A novel histoprocessing method for paraffin sections is presented in which the combination of vacuum and microwave exposure is the key element. By exploiting the decrease in boiling temperature under vacuum, the liquid molecules in the tissues have been successfully extracted and exchanged at relatively low temperatures during each of the steps from dehydration, clearing, and impregnation. In this vacuum-microwave method, an extremely short time suffices for the preparation of optimal-quality paraffin blocks. No xylene (but isopropanol instead) was used as the intermediate solvent. Thirty biopsies (thickness 2-4 mm) can be processed in 40 min. In addition, this approach can be used to produce large sections of giant blocks (4 x 6 x 1 cm3) which can be easily cut on a routine microtome due to the optimal paraffin impregnation. These giant blocks do not shrink during this vacuum-microwave histoprocessing.
Biopsy↗
Recognition of atypical reserve cell hyperplasia in cervical smears and its diagnostic significance.
In this study, the histological, cytological, and electron microscopical features of cervical atypical reserve cell hyperplasia are presented. The most important feature of atypical reserve cells in smears is the absence of cytoplasm. Thus, they must be recognized on the absence and not on the presence of a feature, which makes identifying these cells a controversial issue. These stripped nuclei are erroneously believed to be degenerated cylindrical cells, and accordingly are ignored. The atypical reserve cell nuclei are easily damaged in the smear process; however, the MIB-1 staining shows that these disrupted nuclei are derived from proliferating cells. In a follow-up histological study of cases diagnosed as mild dysplasia in a smear, it was found that the presence of MIB-1 positive staining atypical reserve cells was closely related to the development of carcinoma in situ. Recognizing the atypical reserve cells and observing their proliferating activity in a smear might prove to be more important than focusing on the better-known dysplastic cells.
Antibodies, Monoclonal↗
The two-step vacuum-microwave method for histoprocessing.
When microwaving and vacuum is combined, decrease of boiling temperature can be exploited in the histoprocessing procedures allowing a completely novel approach for impregnating tissue with paraffin. We found that, if the pressure is sufficiently low in the paraffin step, no ethyl-alcohol step is necessary for the dehydration. In that case, the fixed tissue blocks can be directly placed into the isopropanol and, after that, in the hot paraffin, in which the final dehydration of the tissue takes place. The full histoprocessing procedure can thus be shortened into two steps only, with a processing time (for 72 biopsies) of a mere 40 min. Working completely without ethyl alcohol might be of interest for countries in which ethyl alcohol is difficult or expensive to obtain. We conclude that vacuum-microwave histoprocessing allows us to omit ethyl-alcohol dehydration and to replace xylene, resulting in a two-step 'green' (ecological) method.
Histological Techniques↗
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.
Adenocarcinoma↗
Proliferating cell nuclear antigen (PCNA) in herpes virus infected cells.
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Autoantigens↗
Chemical and physical properties of collagen implants influence their fate in vivo as evaluated by light and confocal microscopy.
Porcine collagen membranes having a rough and a smooth side were used for subcutaneous implantation studies in rats. Two tanning protocols were used for the membranes, a new one involving microwaving and glutaraldehyde treatment (NEWGA), and the other, a conventional method using glutaraldehyde treatment at room temperature (OLDGA). Untreated membranes (NONGA) were also implanted. Sections of the implants were examined by light microscopy and with the confocal laser scanning microscope focusing on neovascularization and incorporation of the implant. At 64 days, the smooth sides of NEWGA and OLDGA implants were not well incorporated, with scarring subjacent to the surface and dystrophic calcification of that side of the membrane. At the same time, the rough sides of the NEWGA and OLDGA were not calcified with a giant cell reaction around the porcine collagen. The best incorporation was found in the NONGA membranes with no dystrophic calcification, excellent neovascularization of all layers, and complete remodeling at day 64. After 5 months, the completely remodeled NONGA membrane still could be identified, and the NEWGA and OLDGA membranes were calcified with a giant cell reaction having a dense fibrous capsule. It is concluded that if cross-linking is deemed necessary, the microwave cross-linking method is advisable because in the early stages there is less reactive inflammation around it, and the implant surfaces should be rough with an open structure, making calcification of cross-linked collagen unlikely.
Cell Differentiation↗
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.
Ethanol↗
Glutaraldehyde crosslinking of collagen: effects of time, temperature, concentration and presoaking as measured by shrinkage temperature.
Experiments were carried out to study the effect on the degree of crosslinking of: (a) short term (1 or 5 min) high (50 degrees C) temperature glutaraldehyde (GA) fixation of native collagen membrane, (b) a combination of GA presoaking at low temperature [0 degree C or room temperature (rt)] followed by short time (< 3 min) heating of synthetic collagen fleece in a multilayer diffusion model. As a measure for the degree of crosslinking the shrinkage temperature (Ts) was determined. Short time (1 or 5 min) high temperature (50 degrees C) fixation using 0.1% GA solution caused the shrinkage temperature to increase to 80% and 93% respectively, of the maximum attainable Ts employing GA crosslinking (ca 91 degrees C). Fixation with 0.01% GA for 5 min at 50 degrees C appeared equally as effective as 1 min with 0.1% GA. Although an elevated fixation temperature (from rt to 45 degrees C) was found to produce a substantial increase in Ts of the collagen sheets, a homogeneous distribution of cross links was not obtained by this method. Presoaking the samples at rt (1 h) or at 0 degree C (3 h) with subsequent short time heating to 45 degrees C caused an almost equal rise in shrinkage temperature in Ts throughout the collagen samples.
Animals↗
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.
Antigens↗
Proliferation profile of benign and premalignant cervical epithelium as established by PCNA staining pattern.
Since immunohistochemical detection of proliferating cell nuclear antigen (PCNA) is closely related to the cell cycle, this method can be used to visualize proliferative activity in paraffin sections of benign and premalignant cervical lesions. This was done in two types of benign lesions, immature and mature metaplasia, and the three types of CIN (I, II, and III). PCNA staining was assessed as heavy, faint, or negative nuclei in the deep, middle, and superficial layers of the epithelium. In the analysis of variance, significant differences between the three layers and between the five groups were found. The PCNA values can be helpful to distinguish immature metaplasia from CIN I. The values for PCNA-positive (heavy+faint) for an individual lesion at the three layers constitute its proliferation profile. The PCNA profiles of the CIN lesions differ fundamentally from the metaplasia profiles. It is conceivable that for an individual CIN lesion, PCNA staining might be prognostically more valuable than its CIN grade.
Cell Division↗
Profile of the malignant melanoma excised in general practice.
An analysis of 27 malignant melanomas diagnosed in a Dutch laboratory specializing in the provision of a cytology and pathology service to general practitioners is reported. The median age of the patients was 39 years, and was lower in women (30 years) than in men (54 years). In the national data the mean age was 52 years for both sexes. There were twice as many women as men. There were no tumours from the head and neck region. The ratio of tumours on the limbs to those on the trunk was 3:2. The melanomas were very small (74% with a diameter of < or = 6.0 mm), and they were also very thin (74% with a Breslow thickness < or = 1.0 mm), compared with the national data in which 62% were < or = 1.0 mm. Not surprisingly, the estimated mean 5-year survival was favourable (95% for women and 76% for men). After a period of follow-up ranging from 2 to 32 months, none of the patients had any evidence of residual disease. This study demonstrates that in skin biopsies performed by general practitioners melanomas are mainly detected by chance, and have a very good prognosis.
Adult↗
Computer-assisted primary screening of cervical smears using the PAPNET method: comparison with conventional screening and evaluation of the role of the cytologist.
The PAPNET method is an interactive computer-assisted screening procedure. The diagnostician selects the abnormal video tiles out of the 128 and decides which smears need additional light microscopy. The original diagnoses of 1494 archival smears were compared with the PAPNET analysis of the same smears. The general trend observed was that the PAPNET-assisted diagnoses were of a higher grade than those assigned by the primary screener, thus less cases were signed out as negative. In addition, the PAPNET method was used for primary screening of 2971 randomly selected smears, whilst in the same period 5797 smears were conventionally screened. Using the PAPNET method, significantly fewer smears were signed out as negative. Seventy-three percent of the cases were diagnosed on the basis of the information provided by the 128 video tiles, 11% had to be screened completely by the light microscope, and the remaining cases needed additional light microscopy of a part of the smear. As a result, PAPNET-assisted screening was approximately two times faster. The great advantage of the method is that it is much less tiring for the eyes than conventional screening, making fatigue-related errors less likely, and if a smear contains only a few abnormal cells, these are easier to find.
Female↗