Endometrial cells in liquid-based cervical cytology: a diagnostic pitfall solved by preparing cytohistology from the residual thin layer sample.

OBJECTIVE: It was our aim to assess the usefulness of cytohistology in cervical thin layer brush samples with problems in the differential diagnosis of endometrial cells. STUDY DESIGN: This study reveals the cytological, cytohistological and immunohistochemistry findings of 8 cases suspicious of adenocarcinoma in situ (AIS)/adenocarcinoma (AC) in cervical liquid-based cytology (LBC) preparations that turned out to be normal endometrial cells. RESULTS: All 8 cervical LBCs featured endometrial and atypical endocervical-like columnar cells with frequent ragged 'feathered' edge appearance and rosette formations. Overlapping atypical glandular cell groups were present on 2 ThinPrep slides as well. In cytohistology of 7 cases, the recognition of endometrial stroma with endometrial glands easily allowed the diagnosis of normal endometrium. In 1 case with very small loose tissue fragments without glands, the diagnosis could be established by positivity for CD10 marker (endometrial stroma) and without proliferative activity in the Ki-67 immunostaining. CONCLUSION: In cervical LBC preparations, nuclear hyperchromasia, pleomorphism and nucleoli in normal endometrial cells are more obvious than in conventional smears, and their arrangement is sometimes suggestive of AIS or AC. In the 8 cases presented, we could avoid a false-positive diagnosis of AIS or AC through cytohistology/immunohistochemistry, and in consequence, unnecessary colposcopical/histological examination.

Increased diagnostic accuracy of atypical glandular cells in cervical liquid-based cytology using cell blocks.

OBJECTIVE: The purpose of this study was to reduce the number of diagnoses of atypical glandular cells (AGC). Residual material from the cervical ThinPrep® samples (Hologic, Marlboruogh, MA, USA) was used for cell blocks (CB) and immunohistochemistry (IHC). METHODS: In 2007 there were 87 patients (0.12% of tests) with AGC on liquid-based cytology (LBC) in the Leiden Cytology and Pathology Laboratory (LCPL) using the Bethesda System 2001 (TBS). CB with IHC was used for 26 of these cases. The vials still containing the brush (Cervex-Brush(®) Combi) were placed in a shaker for 10 minutes to dislodge the material trapped between the bristles. The residual sampling fluid was used to prepare paraffin sections (Shandon Cytoblock(®)) stained with Papanicolaou and immunostaining. RESULTS: Four of five cases with AGC not otherwise specified (NOS) were diagnosed with CB/IHC as benign mimics (endometrium, tubal metaplasia, follicular cervicitis, microglandular hyperplasia) and one of four with AGC-favour neoplasia (FN) (endocervical polyp). In one of five cases with AGC-NOS and in two of seven with AGC-FN, CIN3 was found on subsequent histological biopsy. Of six cases diagnosed as adenocarcinoma in situ (AIS) on LBC with CB/IHC the diagnosis was confirmed in four; one was adenocarcinoma and one glandular atypia. Of eight cases diagnosed as adenocarcinoma on cytology and CB/IHC, the diagnosis was confirmed in three. The other five cases were found to be one each of AIS, squamous cell carcinoma, CIN3, CIN2 with glandular atypia, and cervical endometriosis. CONCLUSIONS: By reducing the number of benign mimics of AGC, we achieved a high proportion (16/26; 61.5%) of neoplastic or preneoplastic lesions (glandular or squamous) on histological outcome potentially avoiding colposcopy. Histological biopsy verification by the gynaecologist is needed for final diagnosis of AGC-FN, AIS and adenocarcinoma.

Preneoplastic and neoplastic cervical lesions as detected in Cytoblock sections: the importance of sampling women with bleeding symptoms.

PURPOSE OF INVESTIGATION: The purpose was to use cervical samples to prepare Cytoblocks and use the subsequent paraffin sections for additional immunostaining in our studies on angiogenesis. METHODS: Between January and April 2006, 261 women with bleeding complaints were selected of which 85 had gone to their general practitioner (GP) because of postcoital bleeding. The 261 cervical samples were processed by the Shandon Cytoblock Preparation System. On the subsequent prepared Papanicolaou-stained paraffin sections a histological diagnosis was rendered on the minibiopsies. RESULTS: In all (pre)invasive cases, the paraffin sections contained numerous cancerous minibiopsies. The (pre)invasive cases had many Ki-67 positive nuclei displaying an S-phase staining pattern. In the Ki-67 stained sections, the glandular architecture of the two AIS cases and the two adenocarcinoma cases was highlighted. CONCLUSION: Histologic paraffin sections provided enough minibiopsies to allow concise diagnosis including evaluation of proliferation. Signs of cervical angiogenesis, including postcoital bleeding, can be a strong argument to prepare cytoblocks from samples collected by sampling brushes.

How to improve cytologic screening for endocervical adenocarcinoma?

AIM: A retrospective study was undertaken to investigate how to improve the diagnosis of endocervical adenocarcinoma in screening programs. MATERIAL AND METHODS: The study group consisted of 29 slides of women diagnosed with cancer but who had negative smears. The slides were subdivided in 12 smears taken less than one year before diagnosis by histology and 17 smears taken between one and 10 years prior to diagnosis. A hundred smears of healthy women were used for comparison. All smears were studied macroscopically after which both groups of smears were scanned by the Neural Network Scanner (NNS). Differences between groups were studied for statistical significance using Pearson's Chi-squared test. FINDINGS: The macroscopic parameter of these smears found to be present most frequently was a heavy admixture of blood. The presence of blood (lysed or not) in the smears was equally consistently highlighted by the NNS. Statistical significance of the association of this parameter, with the presence of cancer, was demonstrated. CONCLUSION: The awareness of blood as a background feature of adenocarcinoma of the cervix will help to select cases needing special attention. These difficult bloody smears, studied by light microscopy and by NNS images can also be selected for additional MiB-1 staining. With this approach, blood in smears, otherwise frequently leading to a compromise of classification, can become a blessing in disguise. The diagnosis of endocervical adenocarcinoma in screening smears will therefore be improved.

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.

PAPNET for analysis of proliferating (MIB-1 positive) cell populations in cervical smears.

In diagnostic tumor pathology, immunohistochemical detection of proliferating cell populations is increasingly used. With the event of microwave-antigen retrieval it has become possible to detect proliferating cells in cervical smears staining positive for MiB-1. We report that with the PAPNET system, using neural network computing, it is possible to collect from the smears the images with epithelial fragments containing positive nuclei. We used this system for quantification of staining results. Cases with carcinoma in situ contained many epithelial fragments having a large number of positive-staining nuclei and with labelling indices of 60 +/- 16. Dysplasias were often completely devoid of cells with positive nuclei. In addition, we could not detect differences between progressive and regressive dysplasias. Automatic prescreening of immunostained smears using PAPNET is useful when it is desired to quantify staining results.

Exploiting the "toothpick effect" of the Cytobrush by plastic embedding of cervical samples.

The introduction of the combined spatula-Cytobrush sampling method has increased the diagnostic accuracy of cervical cytology. However, the smears from a small number of cases contained epithelial fragments dislodged by the "toothpick effect" of the Cytobrush. Due to their thickness, these epithelial fragments in the smears are very difficult (if not impossible) to scrutinize and are thus undiagnosable. The presence of only such fragments in smears led to false-negative diagnoses in two cases of invasive carcinoma. To solve this problem, these epithelial fragments were embedded in plastic, with thin sections prepared from the blocks. This paper presents the morphologic features and diagnostic accuracy of 77 such problem cases (found among 50,000 cases with spatula-Cytobrush smears) to which this method was applied. In almost all cases, the diagnosis on the plastic-embedded sections matched the diagnosis on the colposcopically directed biopsy. Of these 77 patients, the biopsy diagnosis showed 5 severe dysplasias, 6 carcinomas in situ, 1 squamous-cell carcinoma, 2 adenocarcinomas in situ and 2 adenocarcinomas. In addition, data are presented concerning the nuclear-size and shape-factor differences in smears versus plastic-embedded sections, and the stereologic consequences of smearing and cutting these epithelial fragments are discussed. These plastic-embedded sections are well suited for use in quantitative microscopy, as well as for diagnostic purposes.

The application of a microwave technique for the preparation of cell blocks from sputum.

Microwaves stimulate diffusion in tissue and affect the proteins and membrane of the cell. These actions can be used to shorten the time required to fix and prepare cell blocks from sputum. The fixative containing ethyl alcohol and polyethylene glycol is particularly suitable for microwave enhanced fixation. Applying a commercially available domestic microwave oven, in which the temperature and power level can be set, cell blocks can be prepared from fresh sputum in 35 min. The microscopical results are of a superior quality because of the microwave-induced homogeneous distribution of the effects of the applied immersion fluids on the cells. The method is very successful for all specimens containing a large amount of mucous. Health hazards for laboratory personnel are nil in this cell blocking method; in contrast to when fresh sputum containing infectious agents is used for smear preparation.

Microwave-stimulated diffusion for fast processing of tissue: reduced dehydrating, clearing, and impregnating times.

In this paper we describe a method of preparing tissue blocks for paraffin sections within 30 min. The method is based on microwave-stimulated diffusion reducing the dehydrating, clearing, and impregnating times by a factor of 48. The developed technique was inspired by the experimentally observed sizable temperature-dependence of viscosity and other transport properties of liquids. It is clear that, by considering the theoretical aspects of diffusion and by analysis of the influence of the used chemicals in different tissue depths, histotechnical results can be optimized. The histotechnical microwave results are light-microscopically excellent and indistinguishable from those of the well-performed 'classical' method. The nuclear size of several cell types hardly differs in both methods. The new method is valuable in particular for individual cases in which a fast diagnosis is asked for, and in which a frozen-section diagnosis is thought to be too unreliable. In addition, this method can be used in small research laboratories processing small quantities of histological material. The only equipment needed to prepare tissue blocks of optimal quality is the microwave oven.