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Objective: To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma. Data Sources: Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks. Rationale: This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

Persistent cervical high-risk human papillomavirus (HR-HPV) infection results in high-grade cervical intraepithelial neoplasia (CIN2/3) and cervical carcinoma. The susceptibility of the cervix to HPV carcinogenesis and the importance of HPV18 in cervical carcinoma despite relative infrequency in CIN2/3 could be linked to HR-HPV infection of immature metaplasia (IM) at the squamocolumnar junction. Atypical IM (AIM) is an equivocal category used to describe changes in IM suggestive of high-grade neoplasia, which causes diagnostic and management problems. We used laser capture microscopy combined with polymerase chain reaction in 24 women with HPV18, HPV16, or other HPV infections on cytologic analysis and a cervical loop electrosurgical excision procedure to locate HR-HPV in cervical tissue. HPV18-positive AIM and CIN2/3 were present in 7/12 cases with HPV18 on cytologic analysis. In 2 cases with HPV18 and other HPV types, HPV18 was only present in AIM and not in CIN2/3. HPV16-positive AIM was present in 3/7 and HPV16-positive CIN2/3 in 5/7 cases with HPV16. No cases had HPV16 AIM without CIN2/3. Other HR-HPV-positive AIM and CIN2/3 cases were present, respectively, in 1/6 and 5/6 cases positive for HR-HPV types other than HPV16/18. In a subset, 94% HPV18 AIM regions showed CK17 and p16 positivity, and 41% were CK7 positive. CIN2/3 and AIM with other HR-HPVs showed similar patterns. AIM was a particular feature of HPV18 infection in women with CIN2/3. HR-HPV infection of CK7/17-positive AIM expressing p16 was particularly seen for HPV18 with and without classical CIN2/3 and should be regarded as a high-grade precancer.

Objective: Cytodiagnoses of specific malignancies are enabled through analyses of abnormal nuclear chromatin and cytoplasmic features in stained cells. Aim: The objective of this work was to explore the inception, development, and chemistry of the Pap stain method introduced in 1942 by Dr. G.N. Papanicolaou. Study Design: To achieve this, we carried out a review of the English literature. Results: Between 1914 and 1933, Papanicolaou first analyzed vaginal squamous cells in guinea pigs and later in human vaginal fluid samples using hematoxylin and eosin with limited color reactions, correlating the cell-type morphology with endocrinology and histology. The 5-dye Pap stain method evolved through 2 salient phases. The first, between 1933 and 1942, saw the introduction of alcohol-ether fixation and aqueous waterblue staining to enhance cellular transparency, aiding the distinction of cervical cancer cells from benign cells, with quantitative and qualitative assessment of squamous cell maturity. The second phase, between 1942 and 1960, saw the introduction and refinement of various alcoholic cytoplasmic counterstaining schemes with orange G and EA (light green, Bismarck brown, eosin) and phosphotungstic acid, allowing wider ranges of polychromasia and further enhancing cellular visualization, facilitating the distinction of cell types and improving diagnostic confidence. Conclusions: Development of the Pap stain method followed specific historical and scientific events. The staining method evolved following incremental improvements in cellular transparency achieved through tailored cellular fixation and cytoplasmic staining using variable dye and pH combinations.

Background. There are limited data on high-risk human papillomavirus (hr-HPV) genotypes among HIV-positive women in Africa, and little is known about their relationship with cervical cytology in these populations. Methods. We conducted a cross-sectional study among 194 HIV-positive women (143 from Tanzania, and 51 from South Africa) to evaluate HPV genotypes among HIV-positive women with normal and abnormal cytology. Cervical samples were genotyped for HPV types, and slides were evaluated for atypical squamous cell changes according to the Bethesda classification system. Results. Prevalence of high grade squamous intraepithelial dysplasia (HSIL) was 9%. Overall, more than half (56%) of women were infected with an hr-HPV type; 94% of women with HSIL (n=16), 90% of women with LSIL (n=35), and 42% of women within normal limits (WNL) (n=58) tested positive for hr-HPV. Overall, the most prevalent hr-HPV subtypes were HPV16 (26%) and HPV52 (30%). Regional differences in the prevalence of HPV18 and HPV35 were found. Conclusion. Regional differences in HPV genotypes among African women warrant the need to consider different monitoring programmes for cervical preneoplasia. HPV-based screening tests for cervical preneoplasia would be highly inefficient unless coupled with cytology screening of the HPV-positive sample, especially in HIV-positive women.

The Cellient™ Automated Cell Block System (Hologic) can be used to analyze cells of HPV-positive cervical scrapes staining positive with the biomarker p16. For this study fourteen cervical scrapes of Tanzanian women infected with HIV testing positive for HPV were selected. The paraffin Cellient sections were stained with the Papanicolaou method, with hematoxylin eosin (HE), and with the biomarker p16. This pilot study was limited to cases classified as atypical squamous lesion of unknown significance (ASCUS) and high-grade squamous lesion (HSIL) as diagnosed in the ThinPrep slide. The Cellient paraffin sections (cut from paraffin blocks prepared from the residual cervical sample) were classified into negative, atypical, CIN 1, CIN 2, and CIN 3. Multiple HPV genotypes were encountered in 79% of the scrapes. HPV16 was found in six scrapes and HPV52 in four. In the Papanicolaou sections, it was easy to detect dyskeratotic cells. Eleven of the 14 cases were p16 positive and five contained p16 positive dyskeratocytes. Of the 10 ASCUS scrapes, two contained p16 positive CIN 1 epithelial fragments. All four HSIL cases contained p16 positive CIN 3epithelial fragments. In HIV-positive HPV-positive women, the Cellient system resulted in high quality histology sections with perfect p16 images of dyskeratocytes.

Objective: To report the prevalence of Gardnerella, Trichomonas and Candida in the cervical smears of 9 immigrant groups participating in the Dutch national cervical screening program. Study Design: Cervical smears were taken from 58,904 immigrant participants and 498,405 Dutch participants. As part of the routine screening process, all smears were screened for the overgrowth of Gardnerella (i.e. smears with an abundance of clue cells) and for the presence of Trichomonas and Candida. The smears were screened by 6 laboratories, all of which use the Dutch KOPAC coding system. The odds ratio and confidence interval were calculated for the 9 immigrant groups and compared to Dutch participants. Results: Immigrants from Suriname, Turkey and the Dutch Antilles have a 2–5 times higher prevalence of Gardnerella and Trichomonas when compared to native Dutch women. Interestingly, the prevalence of Trichomonas in cervical smears of Moroccan immigrants is twice as high, yet the prevalence of Gardnerella is 3 times lower than in native Dutch women. Conclusions: Immigrants with a high prevalence of Gardnerella also have a high prevalence of Trichomonas. In the context of the increased risk of squamous abnormalities in smears with Gardnerella, such slides should be screened with extra care.

Objective: The objective was to examine the use of a tailor-made DNA microarray containing probes representing the vaginal microbiota to examine bacterial vaginosis. Study Design: One hundred one women attending a health center for HIV testing in South Africa were enrolled. Stained, liquid-based cytology slides were scored for bacterial vaginosis. An inventory of organisms was obtained using microarray technology, probing genera associated with bacterial vaginosis in more detail, namely Gardnerella, Atopobium, Dialister, Leptotrichia, Megasphaera, Mobiluncus, Peptostreptococcus, Prevotella, and Sneathia. Results: Of 101 women, 34 were diagnosed positive for bacterial vaginosis. This condition was associated with an increased microbial diversity. It is no longer useful to base the diagnosis of bacterial vaginosis on Gardnerella alone. Rather, its presence with Leptotrichia and Prevotella species, and especially Atopobium was more indicative of an aberrant state of the vaginal flora. Conclusion: To understand the vaginal microbiota in more detail, microarray-based identification can be used after microscopic scoring. © 2011 Mosby, Inc. All rights reserved.

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.

Objective: The large set of ThinPrep slides prepared in the Leiden Cytology and Pathology Laboratory is exploited for calculating the impact of the transition from PAPNET neural network scanning to the Imager technology. Study Design: All cervical samples were suspended and fixed in the coagulant fixative BoonFix. We compared 57,541 ThinPrep slides which were scanned by PAPNET and 64,273 ThinPrep slides processed with the Imager: 99,157 cases originated from the Dutch population screening program of asymptomatic women (screenees) and the remaining 22,657 samples were of symptomatic women. In the PAPNET series, 23% were diagnosed by additional light microscopy; in the Imager method, all slides were studied light microscopically. The cytoscores (positive cytology per 1,000 samples) were calculated for normal, atypical squamous cells of undetermined significance (ASC-US), cervical intraepithelial neoplasia (CIN) grades I–II, and for CIN III+. The odds ratios (ORs) for the positive cytoscores were assessed for both the screenees and the symptomatic women. Results: The cytoscores, per 1,000 cases, for ASC-US varied from 17.77 to 40.59, for CIN I–II from 7.17 to 33.35, and for CIN III+ from 2.81 to 8.8. These 6 cytoscores were higher for symptomatic women than for screenees. We observe significantly elevated ORs for the Imager for ASC-US (1.26 and 1.23), CIN I–II (1.45) and for CIN III+ (1.58 and 1.45). These 3 ORs are higher for screenees than for symptomatic women. Conclusion: The Imager technology is more efficacious, particularly for handling screenee slides.

E. K. J. Risse, J. P. Holierhoek, E. M. Meijer-Marres, E. Ouwerkerk-Noordam and M. E. Boon 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.

Objective To analyze the impact of the 1995 revision of the Dutch cervical screening program guidelines (e.g., the introduction of more stringent criteria for cytologic diagnosis of atypical squamous cells of undetermined significance [ASCUS]) on the negative side effects of screening in Region West. Study design The data for this study were retrieved as two complementary datasets, both of which contained data on the invited screenees, including their cytology and histology follow-up. One dataset additionally included data on other smears. For invited screenees, we analyzed changes in cytoscores and histoscores between 1994, the last year before new screening guidelines were implemented, and 2003, the latest year for which follow-up to screening abnormalities was available in the retrieved data. Additionally, we analyzed changes in the total number of primary and repeat smears made. Results Between 1994 and 2003, the cytoscore for ASCUS decreased from 19.4% to 1.3% of screenee smears. The total number of smears taken decreased by 30%. The cervical intraepithelial neoplasia 3+ histoscore remained the same (OR = 0.97, p = 0.91). Conclusion The reduction of equivocal ASCUS diagnoses resulted in a decrease of costly repeat smears, without measurably decreasing the effectiveness of the screening program.

Objective To describe recent trends in the prevalence of cytologic patterns of the vaginal flora (koilocytosis, Trichomonas, dys-bacteriosis, Candida, Gardnerella, Actinomyces, Chlamydia trachomatis) over the last decade. Study design From 1996 to 2005 > 500,000 cervical smears were screened in the context of the Dutch national screening program on a 5-year basis. Data from the first screening period were compared with those of the second screening period. Results Prevalences differed from 34.8 for dysbacteriosis to 0.2 for C trachomatis. Bacterial imbalance (dysbacteriosis, unequivocal Gardnerella and Trichomonas) showed a decline in all age groups. Cases of human papillomavirus (HPV)-related koilocytosis have dramatically increased among young women (30 and 35 years). Conclusion Bacterial imbalance of the vaginal flora has significantly decreased during the past decade in all age cohorts. Campaigns on consciousness of vaginal hygiene might have contributed to this amazing effect. We ought to be concerned about the increase in HPV-related koilocytosis.

Objective To exploit cervical thin layer brush samples through cytohistology in cases with invasive carcinoma with application of antibodies. Study design Fourteen cases from women with carcinoma diagnosed in 2006 were selected out of 29 invasive carcinomas. From these 14 cases liquid-based cervical cytology material was available to prepare cytohistology. Eight women had squamous cell carcinoma, 4 endocervical adenocarcinoma, 1 endometrial adenocarcinoma and 1 ovarian adenocarcinoma. The residual material from the thin layer sample, collected by brushes by general practitioners, was used to prepare paraffin sections. These were stained with the Papanicolaou method and for the biomarkers Ki-67 and p16 and, if desired, for differentiation markers, including carcinoembryonic antigen, vimentin, cytokeratin 7 and cytokeratin 20 to establish the immunoprofile of the carcinoma. Results The morphologic details in the cancer nuclei in the paraffin sections were excellent, while in all cases the thin layer cytology slide contained thick epithelial fragments with blurred nuclei. In 5 of the 6 adenocarcinomas, the glandular architecture diagnostic of adenocarcinoma was visible in the cytohistology, which was highlighted in the biomarker stainings, particularly so in the Ki-67 sections. With the exception of endometrial adenocarcinoma, all p16(INK4a) stainings were positive, as they were in the ovarian adenocarcinoma case. Conclusion Cytohistology is an adjunct to routine cervical cytologic examination of thin layer samples, allowing an unequivocal and refined diagnosis.

Objective To optimize the morphology of pl6-positive atypical and (pre)neoplastic cells in paraffin cytoblock sections with the aim to minimize equivocal diagnoses on cytology samples. Study design Patients with negative cytology results or results within normal limits (WNL) (n=38), atypical squamous cells of undetermined significance (ASCUS) (n=25) and high grade squamous intraepithelial lesion (HSIL) (n=16) were selected. The residual material of the cervical brush samples was processed to cytoblock paraffin sections and stained for pl1. The cytohistodiagnosis of the pl1-stained paraffin sections was based on the cytologic and histologic morphology. Results Of the 38 cytologically negative cases, only 4 contained afe w faintly positive pl61cells. Of the 25AS CUS cases, I 1as cytohistologically upgraded to low grade squamous intraepithelial lesion (LSIL). All 16 HSIL cases contained cells with outspoken diffuse positive immunostaining, highlighting chromatin clumping in the (pre)malignant cells. In the paraffin sections the tissue fragments showed architecture consistent with that of HSIL. The nuclear to cytoplasmic ratio in the HSIL was severely disturbed. Conclusion Cervical brush samples allow an unequivocal cytohistodiagnosis based on the (pre)malignant nuclear changes highlighted by the p16 staining of the paraffin sections.

Objective To evaluate cytologic diagnoses of dysbacteriosis and Gardnerella infection and to obtain insight into the diagnostic problems of Gardnerella. Study Design One hundred randomly selected samples of each of 3 diagnostic series were rescreened by 2 pathologists, resulting in 2 rescreening diagnoses and a consensus diagnosis. A smear was considered unequivocal when the original O code and the O code of the consensus diagnoses were equal and discordant when the flora diagnoses of the 2 pathologists differed. Results Discordance was highest in the dysbacteriotic series (20%) and lowest in the healthy group (4%). Unequivocal diagnoses were established in 65% of the dysbacteriotic smears, 80% of the Gardnerella smears and 93% of the healthy smears. Misclassification of Gardnerella occurred in the presence of clusters of bacteria mixed with spermatozoa. Conclusion Blue mountain cells in Gardnerella infection can be identified unequivocally in cervical smears. Because of the clinical importance of treating Gardnerella, such advantageous spin-offs of cervical screening should be exploited.

BACKGROUND. The objective of this study was to investigate whether the presence of vaginal Candida or dysbacteriosis predisposes women to an increased susceptibility for (pre)neoplasia over time. METHODS. A retrospective, longitudinal, cohort study was performed and was conducted in a population of 100,605 women, each of whom had 2 smears taken over a period of 12 years as part of the Dutch Cervical Screening Program. From these women, a cohort of 1439 women with Candida and a cohort of 5302 women with dysbacteriosis were selected as 2 separate study groups. The control cohort consisted of women who had completely normal cervical smears (n = 87,903 women). These groups were followed retrospectively over time. The odds ratios (OR) for squamous abnormalities in the follow-up smear for the women in these 3 cohorts were established. RESULTS. The dysbacteriotic cohort was significantly more likely to have low-grade squamous intraepithelial lesions (LSIL) and high-grade squamous intraepithelial lesions (HSIL+) in their follow-up smear (OR, 1.85; 95% confidence interval [95% CI], 1.28–2.67 and OR, 2.00; 95% CI, 1.31–3.05, respectively) compared with women in the control group. In contrast, the Candida cohort had no significantly increased or decreased risk of developing SIL. The equivocal diagnosis ‘atypical squamous cells of undetermined significance’ was rendered significantly more often in the follow-up smear of both study cohorts (Candida cohort: OR, 1.42; 95% CI, 1.03–1.95; dysbacteriotic cohort: OR, 1.44; 95% CI, 1.22–1.71). CONCLUSIONS. The results from this study indicated that the presence of Candida vaginalis was not associated with an increased risk for SIL over time. In contrast, women with dysbacteriosis had a significantly increased risk of developing (pre)neoplastic changes. These findings should be taken into account in further research concerning predisposing factors for cervical carcinogenesis. Cancer (Cancer Cytopathol) 2007. © 2007 American Cancer Society.

In a prospective cohort study, 10 symptomatic women with recurrent vulvovaginal candidiasis were taught how to prepare vaginal smears of their own vaginal fluids on days 7, 14, 21, and 28. The 40 smears were stained with the PAS-method and examined by three different cytopathologists for presence of Candida. Thereafter, the smears were restained with Giemsa-stain to determine presence of lactobacilli, Gardnerella vaginalis (“clue cells”) and neutrophils. All three cytopathologists unequivocally established Candida blastospores and (pseudo)hyphae in 27 out of the 40 PAS-stained vaginal smears, whereas in the remaining 13 smears Candida was not found. All 10 patients had Candida in their smears during the second half of their menstrual cycle. Self sampled smears prove to be reliable for establishing the presence of Candida in symptomatic patients with candidiasis. Candida is associated with a lactobacillus-predominated vaginal flora, but with the absence of Gardnerella vaginalis. Further studies may be directed towards the interaction between the various members of the vaginal flora. This study should open molecular methodology for determining the possible interactions of lactobacilli and Candida. Diagn. Cytopathol. 2007;35:635–639. © 2007 Wiley-Liss, Inc.

OBJECTIVE To determine the accuracy of the microscopic diagnosis of vulvovaginal candidiasis (presence of [pseudo] hyphae and blastospores) in stained vaginal smears in clinical practice. STUDY DESIGN General practitioners trained in diagnosing vulvovaginal candidiasis performed microscopy of 324 stained vaginal smears. These smears were sent to the pathologist for confirmation of the microscopic diagnosis of the clinician; cytologic diagnosis by the pathologist was considered the gold standard. RESULTS In 104 of the 342 cases Candida was established by the pathologist. The clinicians made 24 false positive and 50 false negative diagnoses of Candida. Sensitivity and specificity of the microscopic diagnoses of the clinicians were 52% and 89%, respectively. The most frequent reason for a false positive diagnosis was presence of hairs, whereas the most frequent reason for a false negative diagnosis was understaining of the smear. CONCLUSION This study shows that even in stained smears it is difficult for clinicians to recognize blastospores and (pseudo)hyphae. Efforts are clearly needed to improve the quality of the clinical diagnosis of vulvovaginal candidiasis.

The objective of this study was to establish the relationship between Candida vaginalis and (pre)neoplasia and the prevalence of Candida and (pre)neoplasia related to age and ethnicity. Data were collected from 445,671 asymptomatic women invited for mass screening between 1995 and 2002 and coded according to the Dutch cervical smear coding system (KOPAC) with six grades for (pre)neoplastic changes. Prevalence and relative risks (RRs) were established for Candida and squamous abnormalities in Dutch women and four groups of immigrants. The prevalence of Candida is significantly higher in the cohort of 30-year-old women and lower in the cohorts of 45-, 50-, 55-, and 60-year-old women. The RR of having Candida was higher for Surinamese women (1.24; CI 1.08-1.42). Furthermore, the RR of having mild dysplasia was higher for Surinamese women (1.47; CI 1.14-1.89) and for women born in other countries than in The Netherlands, Turkey, and Morocco (1.36; CI 1.13-1.62). No statistically significant relationship between (pre)neoplasia and Candida was observed. C. vaginalis is more frequent among Surinamese women. Presence of Candida is not associated with an increased risk for squamous abnormalities; therefore, women carrying Candida are not at an increased risk of developing cervical cancer.

The vaginal/cervical smears of a group of Moroccan immigrants were used to compare vaginal dysbacteriosis (i.e., a bacterial population change with a decrease in lactobacilli and an increase of coccoid bacteria in vaginal/cervical smears) with Dutch women.From our archives, 779 smears from Moroccan immigrants were compared with 1,060 smears of age-matched Dutch women. For bacterial flora, Jones-Marres silver stains here used to define four groups. Koilocytosis and cervical intraepithelial neoplasia (CIN) were also recorded.The bacterial vaginal flora of Moroccan immigrants and Dutch women was different. The Moroccan women had a lower dysbacteriosis than Dutch women (3% vs. 24%). Koilocytosis and CIN were less frequent in the immigrant population.The possible synergy of a disturbed vaginal flora with human papillomavirus (HPV), HIV, or cervical preneoplasia indicates that vaginal hygiene and a normal flora may have positive effects on the uterine cervix.

Dysbacteriosis is a microscopical diagnosis. In women with dysbacteriosis, an overgrowth of coccoid bacteria and almost a complete absence of lactobacilli are observed in the (stained) vaginal smear. The aim of this study was to determine the accuracy of this microscopic diagnosis in clinical practice. The analysis concerned 342 consecutive cases in which the microscopy of the stained smears was performed by general practitioners trained in diagnosing dysbacteriosis. These smears were sent to the pathologist for confirmation of the microscopical diagnosis of the clinician. The cytological diagnoses of the pathologist, sometimes performed on restained slides when the quality of the staining was substandard, were considered as the “gold standard.” In 92 of the 342 cases, dysbacteriosis was unequivocally established by the pathologist. Sensitivity and specificity of the microscopical diagnoses of the clinicians were 40% and 85%, respectively. There were 37 false-positive and 54 false-negative diagnoses of dysbacteriosis rendered by the clinicians. The most frequent reason for a false-negative diagnosis was an excess of lactobacilli in the smear. This study shows that even in stained smears it is difficult for clinicians to render a correct evaluation of the status of the vaginal flora. Diagn. Cytopathol. 2006;34:686–691. © 2006 Wiley-Liss, Inc.

Objective To assess, in a longitudinal study in women diagnosed with high grade squamous epithelial lesion (HSIL), the progression over time of proliferative activity in reserve cells using population screening cervical cytology specimens. Study Design Twenty consecutive, unselected patients with HSIL lesions were part of the national cervical screening program. From the archives, for each patient, the last prior normal population screening smear was included in the study. Concurrent sets of cervical smears from 80 age-matched women without pathology formed the controls. The original slides were stained using MIB-1 monoclonal antibody. The fraction of MIB-1-positive reserve cells was assessed using systematic random sampling and running progressive means assessment to ensure a sufficient sample size. Results The proliferation fraction in reserve cells of HSIL patients was significantly raised (mean, 65.0%; range, 53.5-94.1 %; p < 0.01) as compared with that in concurrent controls (mean, 12.8%; range, 1.9-45.4%). Prior smears from HSIL patients, although without morphologic abnormalities, had abnormally high proliferation fractions (mean, 59.1%; range, 1.0-94.7%), significantly raised over those from concurrent controls (mean, 9.4%; range 1.6-41.%). Conclusion In population-based cervical smear screening, HSIL patients already have abnormally raised proliferation fractions of reserve cells, even without morphologic changes in squamous cells, 1-5 (mean, 3.6) years prior to diagnosis.

Introduction: Sunlight's UV B component, a known cellular immunosupressant, carcinogen, and activator of viral infections, is generally seasonally available. Venereal human papillomavirus (HPV) transmission, at least in part, causes cervical cancer. We have previously inspected the monthly rates of venereal HPV infection and sunlight fluency in Southern Holland over 16 consecutive years. Both peak in August with at least 2-fold seasonality. The amount of available sunlight and the rate of Papanicolaou (Pap) smear screen–detected HPV are positively correlated. We now investigate whether premalignant and malignant cervical epithelial changes are also seasonal and related to seasonal sunlight fluency. Methods: We have studied >900,000 consecutive, serially independent, interpretable screening Pap smears obtained by a single cervical cancer screening laboratory in Leiden, Holland, during a continuous 16-year span from 1983 through 1998. The average monthly rates of premalignant and malignant epithelial change were inspected and the annual patterns contrasted to the annual pattern of sunlight fluency at this global location and to monthly average HPV infection rate. Because HPV is venereally transmitted, Dutch seasonal sexual behavior was evaluated by assessment of the annual pattern of Dutch conception frequency as a competing cause for cervical cancer seasonality. Results: (a) Twice as many premalignant and malignant epithelial changes were found among Pap smears obtained in the summer months, with an August peak concurrent with histopathologic evidence of HPV infection and sunlight fluency in Southern Holland. (b) Monthly sunlight fluency is correlated positively with both the monthly rates of Pap smear–detected cervical epithelial dysplasia and carcinomatous histopathology, as well as HPV. (c) Conception frequency, in this location, peaks in Spring not summer, and has a 4.8% annual amplitude. Conclusions: (a) Cervical epithelial HPV infection and HPV-induced cervical epithelial dysplasia and carcinomatous change may each be novel sun exposure risks and thereby behaviorably avoidable. (b) Because screening Pap smears uncover many abnormalities that resolve spontaneously (false positives), these data may argue for screening and follow-up Pap smear examinations in seasons other than summer in the Northern Hemisphere, to diminish the false-positive smear rate. Global data are available to confirm and further test each of these conclusions.

Polymerase chain reaction to detect high- risk human papillomavirus has been suggested as a gold standard for cytology. The Netherlands and Surinam were prospectively compared in regard to the proportions of Negative, Atypical Squamous Cells of Undetermined Significance, and Squamous Intraepithelial Lesion smears that had detectable high- risk human papillomavirus. For the Netherlands, 14 600 negative, 270 Atypical Squamous Cells of Undetermined Significance and 120 Squamous Intraepithelial Lesion smears were evaluated by polymerase chain reaction. For Surinam, 150 negative, 50 Atypical Squamous Cells of Undetermined Significance, and 150 Squamous Intraepithelial Lesion smears were evaluated by polymerase chain reaction. In all, 4% of Dutch and 80% of Surinamese negative smears had detectable high- risk human papillomavirus ( chi(2) = 1313, P 0.75). Human papillomavirus DNA testing may not be a suitable gold standard in general because its use would make specificity and sensitivity prevalence- dependent. A new statistic, the percent of Negative pap smears with detectable high- risk human papillomavirus, is posited, which may be important if human papillomavirus DNA testing is used clinically.

OBJECTIVE: To evaluate the outcome of population based cervical screening at 5-year intervals. STUDY DESIGN: Results from the west region of the Netherlands (population 2 million) were used. The 1995-2000 round was compared with the first 2 years of the second (2001-2002). All results were prospectively collected in a central database. Positive cytologies and histoscores per 1,000 screened for preinvasive squamous cervical intraepithelial neoplasia (CIN) lesions and invasive squamous cell carcinoma were calculated. RESULTS: In the first round, 378,081 women were screened; in the second round, 100,561 women were screened. In both rounds the youngest screenees had the highest cytoscores. Cytoscores in the first round did not differ significatly from those in the second. The histoscore for CIN 1 and 2 was 1.42 per 1,000 in the first round and 1.18 per 1,000 (NS, P CONCLUSION: Population-based screening at 5-year intervals in the Netherlands may result in stabilization of positive cytology and of the incidence of CIN and (histologic) invasive squamous cell carcinoma. The program seems more cost effective than that of 2 decades ago, with a screening interval of 3 years.

The large Dutch data sets acquired as a result of population-based cervical smear screening programs can be further exploited to obtain an urbanization-weighted score to gain insight into the quality of the performance of the individual cytology laboratories. Based on the first four digits of the postal code of the screenees, the data are stratified according to urbanization. Urb 1 corresponds to (semi)rural, which includes villages and small townships with less than 20,000 inhabitants; Urb 2, to towns with between 20,000 and 250,000 inhabitants; and Urb 3, to big cities, in this case, The Hague. From the postal code data of the screenees, the urbanization profiles of the laboratories can be calculated. The urbanization degree proved to have a substantial effect on the cytologic scores in the four laboratories. The number of expected, urbanization-weighted patient cases is calculated. Accordingly, the laboratories could be compared with respect to performance. We conclude that laboratories in our screening program were quite similar in performance for the cytologic diagnosis leading to referral to the hospital, with little difference between the actual and the expected, urbanization-weighted number of cases. It is evident that the equation for calculating the expected scores for S5-S9 is relevant for control of quality of care provided by laboratories and regions, but also for the quality of these assessments. Diagn. (C) 2004 Wiley-Liss, Inc.

MiB-1 immunostaining may facilitate recognition of developing adenocarcinoma in cervical smear screening. In a retrospective analysis of prospectively collected data of 170 patients with atypical endocervical glandular cells and with repeat smears, archival Papanicolaou-stained smears were restained for MiB-1 and classified for the presence of preneoplastic changes of MiB-1 positive epithelial fragments. The results of classification based on MiB-1 positive epithelial fragments corresponded "roughly" with cytomorphological diagnoses. Of the 38 patients in which the primary smear was found to be MiB-1-positive, 12 of these persisted in the repeat smear. Thirty-eight of the 132 repeat smears of patients originally negative for MiB-1 pathology were positive. Of the total 50 MiB-1 positive repeat smears, four showed adenocarcinoma in situ on cytomorphological grounds. MiB-1 staining enhances detection of (pre)neoplastic changes. This approach does not destroy the morphology of the original smear and can be applied to routine material.

Cytological cervical screening is rather inefficient because of relatively high proportions of false negative and false positive smears. To evaluate the efficiency of high-risk human papillomavirus (hrHPV) testing, by GP5+/6+ PCR-enzyme immunoassay (EIA), in conjunction with cytology (Intervention Group) to that of the classical cytology (Control Group), we initiated the Population Based Screening Study Amsterdam (POBASCAM). POBASCAM is a population-based randomized controlled trial for implementation of hrHPV testing in cervical screening. The outcome measure is the proportion of histologically confirmed ≥CIN3 lesions in each study arm up to and including the next screening round after 5 years. We present the design, methods and baseline data of POBASCAM. When, in the next 5 years, the follow-up will be completed, the data obtained will be used in model studies, including a cost-effectiveness study, to advise the Dutch Ministry of Public Health in deciding whether cervical screening should be based on combined hrHPV and cytology testing instead of cytology alone. Between January 1999 and September 2002, 44,102 women (mean age = 42.8 years; range = 29-61) that participated in the regular Dutch screening program were included in our study. In the Intervention Group the distribution of cytology and hrHPV by cytology class was as follows: normal cytology 96.6% (3. 6% hrHPV positive); borderline and mild dyskaryosis (BMD) 2.5% (34.6% hrHPV positive); and moderate dyskaryosis or worse (>BMD) 0.8% (88.3% hrHPV positive), i.e., 0.4% moderate dyskaryosis (82.9% hrHPV positive), 0.3% severe dyskaryosis (92.5% hrHPV positive), 0.1% carcinoma in situ (95.2% hrHPV positive), BMD, i.e., 0.4% moderate dyskaryosis, 0.3% severe dyskaryosis, 0.1% carcinoma in situ, BMD of 13.7% among women with a positive hrHPV test was not age-dependent. Our study indicates that large-scale hrHPV testing by GP5+/6+ PCR-EIA in the setting of population-based cervical screening is practically feasible, is accepted by both participating women and general practitioners and yields highly reproducible results.

To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma.Cytopathologists with an interest in the field involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC) contributed to this update. Reference material includes peer-reviewed publications and textbooks.This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in Cape Town.

To provide practical guidelines for the cytopathologic diagnosis of malignant mesothelioma (MM). Cytopathologists involved in the International Mesothelioma Interest Group (IMIG) and the International Academy of Cytology (IAC), who have an interest in the field contributed to this update. Reference material includes peer-reviewed publications and textbooks. This article is the result of discussions during and after the IMIG 2012 conference in Boston, followed by thorough discussions during the 2013 IAC meeting in Paris. Additional contributions have been obtained from cytopathologists and scientists, who could not attend these meetings, with final discussions and input during the IMIG 2014 conference in cape town. During the previous IMIG biennial meetings, thorough discussions have resulted in published guidelines for the pathologic diagnosis of MM. However, previous recommendations have stated that the diagnosis of MM should be based on histological material only.[12] Accumulating evidence now indicates that the cytological diagnosis of MM supported by ancillary techniques is as reliable as that based on histopathology, although the sensitivity with cytology may be somewhat lower.[345] Recognizing that noninvasive diagnostic modalities benefit both the patient and the health system, future recommendations should include cytology as an accepted method for the diagnosis of this malignancy.[67] The article describes the consensus of opinions of the authors on how cytology together with ancillary testing can be used to establish a reliable diagnosis of MM.

Objective: An overgrowth of coccoid bacilli in the absence of lactobacilli (bacterial vaginosis) is considered a sign of a “disturbed” vaginal ecologic system. The aim of this study was to establish the baseline prevalence of genital infections and of a disturbed vaginal ecologic system and their relationship to urbanization. Study Design: More than 190,000 cervical smears were screened by 1 cytology laboratory in the context of the Dutch national screening programs. The women who were symptom free and had been registered in the statutory files of municipal units were screened by invitation. Urbanization (place of residence) was derived from the postal code. To investigate a trend in prevalence in relation to urbanization, Schaafsma's method was used. Results: Bacterial vaginosis had the highest score per 1000 screened women (36.1), followed by Candida (12.6). Higher prevalence of human papillomavirus, bacterial vaginosis, and Trichomonas was related to urbanization as was, whereas Candida was not. Conclusion: Human papillomavirus and Trichomonas are more frequently found in women who live in the large cities, which in The Netherlands also harbor the high-risk population for premalignant cytologic evidence. Our findings concerning the baseline prevalence pattern of bacterial vaginosis support the hypothesis of the importance of a disturbed bacterial ecologic system in cervical carcinogenesis. (Am J Obstet Gynecol 2002;187:365-9.)

BACKGROUND. Neural network technology has been used for the daily screening of cervical smears in The Netherlands since 1992. The authors believe this method might have the potential to demarcate diagnoses of Grade 1-2 cervical intraepithelial neoplasia (CIN 1-2). METHODS. Of 133,196 women who were screened between 1992-1995, there were 2236 CIN 1-2 smears; 1128 of which were detected by means of neural network screening (NNS) (n = 83,404 women) and 1108 of which were diagnosed by conventional screening (n = 49,792 women). Cytologic and clinical outcomes (first cytologic or histologic follow-up diagnosis) were retrieved for all the women in the study population (n = 1920). Stratification based on clinical outcome resulted in the cases being grouped as overdiagnosed, concordant, or underdiagnosed. The smears were performed by general practitioners, whereas the biopsies were obtained by gynecologists. RESULTS. The prevalence rate for CIN 1-2 was 1.15% (95% confidence interval [95% CI], 1.08-1.23%) for NNS and 1.92% (95% CI, 1.80-2.04%) for conventional diagnosis (P

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. Diagn. Cytopathol. 24:373–377, 2001. © 2001 Wiley-Liss, Inc.

Neural network-based screening (NNS) of cervical smears can be performed as a so-called "hybrid screening method," in which parts of the cases are additionally studied by light microscope, and it can also be used as "pure" NNS, in which the cytological diagnosis is based only on the digital images, generated by the NNS system. A random enriched sample of 985 cases, in a previous study diagnosed by hybrid NNS, was drawn to be screened by pure NNS. This study population comprised 192 women with (pre)neoplasia of the cervix, and 793 negative cases. With pure NNS, more cases were recognized as severely abnormal; with hybrid NNS, more cases were cytologically diagnosed as low-grade. For a threshold value > or = HSIL (high-grade squamous intraepithelial lesions), the areas under the receiver operating characteristic (ROC) curves (AUC) were 81% (95% CI, 75-88%) for pure NNS vs. 78% (95% CI, 75-81%) for hybrid NNS. For low-grade squamous intraepithelial lesions (LSIL), the AUC was significantly higher for hybrid NNS (81%; 95% CI, 77-85%) than for pure NNS (75%; 95% CI, 70-80%). Pure NNS provides optimized prediction of HSIL cases or negative outcome. For the detection of LSIL, light microscopy has additional value.

Objectives. Karyometric analysis (Quanticyt) has proved of value as a cytologic marker for bladder cancer. This study was conducted to identify diagnostic and prognostic factors in a high-risk Quanticyt population to predict the prognosis of transitional cell carcinoma (TCC) of the bladder. Methods. Quanticyt is a karyometric system for quantitative bladder wash cytologic findings based on two nuclear features: the 2c-deviation index (2cDI) and the mean of nuclear shape. Samples are scored as low, intermediate, or high risk. Before 1995, 109 patients with high-risk quantitative bladder wash cytologic findings were identified at our clinic. Four patients with previous invasive tumors were excluded. Results. Histologically proven malignancy was found in 54 of 105 patients at first high-risk quantitative bladder wash cytologic findings. Invasive TCC was found in 16 patients, and another 10 patients had progression during a median follow-up of 3.7 years. In univariate analysis, the presence of carcinoma in situ (CIS), highest tumor grade, 2cDI, and highest tumor stage were significant predictors of progression. The presence of CIS proved to be the only predictor of progression in the multivariate analysis. A 2cDI of 2.00 c2 or higher was a significant predictor of CIS, invasive TCC, and progression. At follow-up analysis after negative cystoscopy, 2cDI showed a tendency toward predicting progression. Conclusions. These data confirm earlier findings that CIS is an important marker of progression. 2cDI as assessed by quantitative cytology is a practical tool to predict CIS, invasive TCC, and subsequent progression. A 2cDI of 2.00 c2 can be used to further stratify high-risk quantitative bladder wash cytologic findings.

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.

For this study, bladder washings of 923 patients with a follow-up for superficial bladder cancer were used. For the prognostication of each case (follow-up period up to 20 mo), two parameters were used: tumor recurrence and the event of (progressing) invasive growth. Each sample was classified as negative, low grade or high grade, based on confocal-aided architectural cytology. The feature architecture was based on the images of the epithelial fragments as seen in the Feulgen stain, which also could be visualized in confocal microscopy. The same three classes were also used for grading the samples according to the QUANTICYT system, in which DNA and morphometry of nuclei were automatically, assessed. The 532 cases graded as QUANTICYT high had a worse prognosis than the 391 cases with QUANTICYT low. In addition, architectural cytology contributed significantly for the prognostication of cases classified as QUANTICYT high, the 228 nonconcording cases faring better than the 304 concording ones. For QUANTICYT low, the 156 nonconcording cases had also a lower recurrence rate than the 235 concording ones, but this difference was only significant in patients followed for up to 8 mo. These findings indicate that QUANTICYT grading supplemented by architectural cytology supplies the clinician with valuable prognostic information which can be used to limit cystoscopies.

OBJECTIVE: To study the rescreen and PAPNET effects on enriched material derived from smears screened routinely using PAPNET and conventional microscopy. STUDY DESIGN: A series of 432 smears (containing 122 atypical squamous cells of undetermined significance [ASCUS] plus 44 at least dysplastic squamous intraepithelial lesion-positive [SIL+] ones), screened routinely with the conventional method, were rescreened using the PAPNET system. Another series of 461 smears (containing 140 ASCUS + 52 SIL + ones) screened routinely with PAPNET were rescreened conventionally. The rescreen effect, defined as the effect of differences between the rescreen and routine screening situation, was investigated by comparing the rescreen results in both series of smears with the routine results in both series. The effect of using either method of screening was studied by comparing the PAPNET results in both series with the results of conventional screening in both series. RESULTS: The rescreen effect was statistically significant both for a higher number of smears classified as negative (less than ASCUS) and a higher number of smears classified as high grade SIL or more. PAPNET-assisted screening resulted in a significantly higher number of smears classified as high grade SIL+, although for this latter finding there is an unexplained significant difference between conventional and PAPNET-screened cases in the changes made by the cytopathologist in the cytotechnologists' diagnoses. CONCLUSION: The rescreen effect should not be ignored when enriched material is used.

When an unsatisfactory smear (thick inflammatory infiltrate or blood influencing the staining characteristics of the epithelial cells) is restained for MiB-1, the diagnostic proliferating cells are visualized, and the MiB-1-positive smears can be thus upgraded as borderline, grade I, II, and III, corresponding with the cytologic diagnoses of, respectively, ASCUS, CIN I, II, and > or = III. In a period of 18 months, 2,068 unsatisfactory smears out of a material of 84,817 smears were restained for MiB-1. In the unsatisfactory group, significantly more abnormal smears were detected than in the satisfactory group. Seventy-five of the unsatisfactory group were biopsied because of the MiB-1 findings: Three women proved to have severe dysplasia, four had carcinoma in situ, and three had invasive carcinoma. The per mileage for invasive cervical carcinoma was ten times larger in the unsatisfactory group than in the satisfactory group; thus the MiB-1 method has further enhanced our diagnostic acumen in this difficult type of smears.

Objectives Quantitative cytology by image-analysis techniques enables objective interpretation of nuclear features in light microscopic images. QUANTICYT, a quantitative karyometric cytology system, was used in the follow-up of patients with superficial bladder cancer. Methods From 1992 to 1995, 41 37 samples from 1412 patients were obtained. At 1-year follow-up after the initial bladder washing, a tumor recurrence rate of 21% was found. In this period, tumor progression to invasive disease occurred in 1.6% of patients. Scoring of tumor by the QUANTICYT system was based on two nuclear features: the 2c deviation index and the mean of a nuclear shape feature: MPASS. Results The method was found to be reproducible and superior to visual cytologic interpretation. QUAN-TICYT analysis of the bladder washings resulted in a score of low, intermediate, and high risk. In a multivariate analysis, highest grade of earlier tumor and QUANTICYT risk score were the best predictors of tumor recurrence and progression. For the easy application of QUANTICYT analysis in daily routine, a report form that included patient history and DNA histogram was developed. Conclusions QUANTICYT karyometric analysis of bladder-wash material proved a useful, clinically applicable grading tool in the follow-up of patients with superficial bladder cancer, with sufficient power to be used in decision-making in the individual patient.