Validation of the use of Pap-stained vaginal smears for diagnosis of bacterial vaginosis

Papanicolaou-stained cervicovaginal smears (Pap smears) are used to screen for cervical cancer. Since there is a lack of consensus in published reports respecting the efficacy of Pap-stained smears in BV diagnostics, there is a need to validate their use for diagnosis of BV. Slides from the international BV00 workshop were Pap stained and independently analyzed by four investigators under a phase-contrast microscope. All workshop slides--whether Pap-stained, Gram-stained or rehydrated air-dried smears--were scored according to the same Nugent classification. The diagnostic accuracy of Pap smears for diagnosis of BV had a sensitivity of 0.85 and a specificity of 0.92, with a positive and negative predictive value of 0.84 and 0.93, respectively. The interobserver weighted kappa index was 0.86 for Pap-stained smears compared to 0.81 for Gram-stained smears, and 0.70 for rehydrated air-dried smears using the mean Nugent score as the criterion standard. Provided that the samples are taken from equivalent locations (the vaginal fornix) and analyzed according to the same scoring criteria, there is no discernable difference in the diagnostic accuracy of the three smear-staining methods. The Pap-stained vaginal smears can be used as a wholly adequate alternative to Gram-stained smears for BV diagnosis.     

Rehydration of air-dried smears with normal saline. Application in fine-needle aspiration cytologic examination

In fine-needle aspiration cytologic examination, nuclear features are often better assessed in hematoxylin and eosin (H and E) or Papanicolaou- (Pap) stained than Romanowsky-stained smears. However, both H and E and Pap stains require the use of immediately wet-fixed smears for cytomorphologic preservation. Some degree of air drying is usually inevitable. Placing air-dried smears in normal saline for 30 seconds before fixation in 95% alcohol is found to be a simple means of rehydrating the cells. The quality of the rehydrated smears is superior or identical to that of the immediately wet-fixed smears, provided that the period of drying does not exceed 30 minutes.     

Silver staining method for nucleolar organizer regions in cervical smears

The distribution of nucleolar organizer regions (NORs) was studied in Papanicolaou preparations of cervical smears in order to distinguish benign from preneoplastic lesions. Destained smears (six defined as normal, six as inflammatory with squamous metaplasia, six as CIN I, six as CIN II, and five as CIN III) were submitted to the Ag-NOR method after staining with Orange G and EA36. Ag-NOR count was performed in previously outlined fields on the smears. Statistically significant differences (P < .05) were found between the normal smears, inflammatory smears with squamous metaplasia, and each grade of CIN. We conclude that the Ag-NOR technique could be useful to evaluate cervical smears of doubtful interpretation, using previous demarcation of the abnormal fields/cells. Diagn. Cytopathol. 16:497–499, 1997. © 1997 Wiley-Liss, Inc.     

Immunostaining of estrogen receptor,progesterone receptor,MIB1 antigen,and c-erbB-2 oncoprotein in cytologic specimens: A simplified method with formalin fixation

An effective but simple fixation protocol for the immunocytochemical staining of cytologic smears for estrogen and progesterone receptors, the Ki-67 antigen (using MIB1 antibody), andc-erbB-2protein is described. One hundred twenty-seven smears from a variety of malignant and benign breast lesions showed good preservation of antigenicity when subjected to the following fixation protocol: Freshly made smears were air-dried for 20 min to 14 h at 22°C before immersing in 10% buffered formalin for 2–14 h. Immunostaining followed microwave-stimulated epitope retrieval. There was strong concordance of staining with corresponding tissue sections in 15 cases of malignant tumors (ER: r = 0.7381; PR: r = 0.6684; MIB1: r = 0.7234). Immunostaining staining, when delayed for 5–10 days in about half the smears, showed no noticeable difference in reactivity, attesting to effective storage of the formalin-fixed smears at room temperature. Diagn. Cytopathol. 17:127–133, 1997. © 1997 Wiley-Liss, Inc.     

Litigation cells: their incidence and classification in gynecologic smears

"Litigation cells" are defined as benign cells which may mimic dysplasia or cancer and might be used by plaintiffs' witnesses to imply that the cytotechnologist or pathologist "missed" cells of dysplasia or cancer. We reviewed 180 cervical smears from 166 patients who had hysterectomy for benign leiomyomas. All smears were performed within 12 months prior to hysterectomy. None of the uteri contained dysplasia or cancer on histologic examination. 90.6% of smears reviewed had at least one cell or cell group with atypia mimicking dysplasia or cancer. These "litigation cells" were classified as follows: parabasal cells, metaplastic squamous cells, degenerated endocervical cells, reactive endocervical cells, endometrial cells, neutrophils, histiocytes, and air-dried cells. Diseases mimicked by these cells included squamous cell carcinoma, high-grade squamous intraepithelial lesion, low-grade squamous intraepithelial lesion, adenocarcinoma, and glandular dysplasia. These "litigation cells" can be correctly classified by experienced cytotechnologists and cytopathologists and recognized as benign. We recommend that in all cases of alleged malpractice against cytotechnologists and/or pathologists the smears should be reviewed by a panel of individuals trained and experienced in cytopathology. The smears should be reviewed without knowledge of the clinical outcome and in an environment that simulates the normal screening practice.     

Clinical parameters associated with unsatisfactory specimens of conventional cervical smears

Although The Bethesda System 2001 attempted to standardize the criteria for specimen adequacy, much confusion still exists, which includes the significance of unsatisfactory smears, the causes and clinical conditions related to unsatisfactory smears, and the appropriate management of unsatisfactory smears. The aim of this study is to find out the clinical factors associated with unsatisfactory cervical smears. We reviewed the medical charts of patients who received conventional Pap smears between March 2006 and August 2006 in a tertiary care center. After excluding 378 cases with incomplete demographic data, the clinical data of 7,059 cases were processed for analysis. Clinical parameters retrieved included: history of pelvic malignancy, pelvic irradiation, conization, hysterectomy, pregnancy status, within 3‐months postpartum. Vaginal bleeding, abnormal vaginal discharge, intrauterine device, and cervical polyps found during pelvic examinations were also documented. The 1,397 cases with history of pelvic irradiation, pelvic malignancy, and hysterectomy were excluded. Finally, 5,662 cases were enrolled for data analysis. The relationship between clinical parameters and unsatisfactory smears were analyzed by Pearson's chi‐square test with Yates' continuity correction and multivariate binary logistic regression test. The incidence of unsatisfactory smears was 4.5% (252/5,662). Clinical parameters correlated with unsatisfactory smears were postpartum status (OR = 1.92, 95% CI = 1.23–3.01, P = 0.004), vaginal bleeding (OR = 2.02, 95% CI = 1.30–3.16, P = 0.002), and endocervical polyps (OR = 2.62, 95% CI = 1.39–4.947, P = 0.003). In conclusion, if any of these parameters are noted prior to obtaining a Pap smear, optimal collecting devices, better sampling techniques, and liquid‐based cytology should be considered to decrease the incidence of unsatisfactory smears. Diagn. Cytopathol. 2011;39:87–91. © 2010 Wiley‐Liss, Inc.     

Follow-up of Papanicolaou smears diagnosed as atypical squamous cells of undetermined significance

In the first year since the institution of the Bethesda system at UCDMC, 549/7,388 (7.43%) Papanicolaou (Pap) smears were diagnosed as having an epithelial abnormality. One hundred ninety-three of the 549 (35.1%) of the abnormal smears received an ASCUS diagnosis, representing 2.61% of the total volume. Follow-up was obtained on 124/193 (64.2%) and consisted of colposcopy with biopsy in 38.3%, one repeat Pap smear in 51.2%, and two or more repeat Pap smears in 10.5%. Follow-up revealed a squamous intraepithelial lesion (SIL) in 29.1%, ASCUS in 12.9%, and no evidence of an epithelial lesion in 58.0%. Review of the original ASCUS Pap smear from the group with no epithelial lesion on follow-up showed increased inflammation plus metaplasia and/or reactive changes in 69.5% and ASCUS in 19.5%. This study demonstrates that a significant percentage of ASCUS Pap smears represent SIL. However, many Pap smears with reactive, inflammatory changes are misclassified as ASCUS and would be best diagnosed under “Reactive/Reparative Changes” in the Bethesda system. Careful attention to criteria for ASCUS and inflammatory and reactive atypia is recommended to avoid misclassification and to make this category more meaningful to the clinician. Diagn Cytopathol 1996;14:20–24. © 1996 Wiley-Liss, Inc.     

Evaluation of Feulgen reaction performed on unfixed smears.

Feulgen reaction--both the original one with Schiff reagent and the modified one with toluidine- or methylene blue, pH = 4.0--can be carried out on air-dried smears without fixing. Hydrolysis--5 n HCl at room temperature should be 15 to 20 min for the original and 5 to 10 min--for the modified reaction. Binding of the dyes is stoichiometric in both cases.     

Microglandular endocervical hyperplasia and tubal metaplasia: Pitfalls in the diagnosis of adenocarcinoma on cervical smears

The detection of atypical glandular cells of undetermined significance (AGUS) has risen recently due to the use of new endocervical canal sampling devices, in particular the cytobrush. From April 1993–June 1994, a diagnosis of AGUS ranging from adenocarcinoma in situ (AIS) to invasive adenocarcinoma was initially made on cervical smears from 6 women for whom histologic follow-up data were available. The purpose of this study was to determine if benign cervical glandular lesions can be reliably distinguished from adenocarcinoma on cytology. Review of the smears and histologic slides from 3 patients showed microglandular endocervical hyperplasia on cervical cone specimens. Cervical smears from 2 of these patients showed clusters of small-to-medium-sized cells with nuclei containing coarse, granular chromatin and prominent nucleoli. Cytoplasmic vacuoles and engulfment of neutrophils were present, findings suggestive of endometrial adenocarcinoma. The third patient's smear showed clusters of large cells with ample, vacuolated cytoplasm and vesicular nuclei containing prominent nucleoli, findings suggestive of endocervical adenocarcinoma, In 3 cases evaluated by cervical conization, histologic slides showed tubal metaplasia. The cervical smears showed clusters and sheets of cells with round-to-oval-shaped nuclei containing evenly distributed, finely granular chromatin and inconspicuous nucleoli. Pseudoglandular formation was present in 2 cases, a finding suggestive of AIS. Since the cytologic features of microglandular endocervical hyperplasia and tubal metaplasia overlap those of adenocarcinoma, a differential diagnosis is prudent on cytologic smears of AGUS. Diagn. Cytopathol. 16:168–173, 1997. © 1997 Wiley-Liss, Inc.     

Application of nucleolar organizer region staining technique to air-dried blood smears]

Silver staining of nucleolar organizer regions (NOR) was applied to air-dried peripheral and bone marrow smears of normal subjects and leukemic patients. Specimens were fixed in buffered acetone formalin. Even in smears kept for 2 years at room temperature, the stainability of Ag-NOR was well preserved. By dipping Giemsa-stained smears in 5% trichloracetic acid and then placing them in methanol for 5 minutes, the stain was leached out. After the dye had been removed, the smears were clearly stained by a Ag-NOR staining technique. The mean number of Ag-NOR per nucleus of mature granulocytes and mononuclear cells in normal peripheral bloods was 0.59 and 1.43 respectively. The mean number of Ag-NOR per nucleus of peripheral and bone marrow leukemic cells from patients with acute leukemia and chronic myelocytic leukemia in blastic crisis was 2.32 and 2.66 respectively. On the other hand, the mean number of Ag-NOR per nucleus of peripheral leukemic cells from patients with chronic lymphocytic leukemia was 1.48. These results suggest that acute leukemia cells possess a more active proliferating potential. The Ag-NOR staining technique is very simple and might be useful for investigation of hematologic cells.     

Evaluation of the CytoRich® technique for cervical smears

The aim of the study was to assess the ability of the CytoRich^® System to prepare optimal gynaecological smears for diagnosis. The diagnostic results obtained from evaluating 1,325 matched slide-pairs, prepared using conventional methods and thin-layer technology, were compared. Cytological material for study was obtained using the combined spatula–cytobrush sampling technique. An assessment of the pitfalls associated with the interpretation of these smears was also undertaken. Diagnostic agreement was achieved in 1,272 of the 1,325 matched slide-pairs (96.0%), and these included 1,172 negative, 50 atypical, 24 low-grade squamous intraepithelial lesion (LSIL), 24 high-grade SIL (HSIL), and two malignancies. A total of 1,309 cases showed the same diagnosis within one diagnostic grade for an agreement of 98.8%. Evaluation of the 53 discordant diagnoses revealed that the conventional smear identified a significantly greater number of abnormal smears than the CytoRich technique (P < .001). It is suspected that the use of the combined spatula–cytobrush sampling technique did not provide adequate residual specimen for CytoRich after conventional smear preparation. This limitation is evidenced by the fact that the CytoRich preparations showed a lower yield of endocervical cells (P < .001) and infectious organisms (P < .001) than was demonstrated on conventional smears. Despite a number of diagnostic pitfalls associated with the interpretation of thin-layer smears, these preparations were easier and faster to screen and showed well-preserved and evenly distributed cells. Thin-layer smears were also characterised by a marked reduction in thick cell groups, air drying artifact, and obscuring inflammation and blood. The results confirm the limitation of the combined spatula–cytobrush technique in these types of comparative studies. Diagn. Cytopathol. 1998;18:236–242. © 1998 Wiley-Liss, Inc.     

Demonstration ofChlamydia trachomatis in papanicolaou-stained gynecological smears

A fluorescent antibody staining method was developed to detectChlamydia trachomatis elementary bodies in Papanicolaou-stained cervical smears. Twenty-five known chlamydia-positive and 12 chlamydia-negative smears were correctly identified using this method. Smears from individuals with culture-positive chlamydia infections showed a high density of elementary bodies. Among 64 routine cervical smears, 27% (6/22) of smears that showed inflammatory changes were positive by fluorescent antibody staining for chlamydia, compared to 2.4% (1/42) of smears that were negative for inflammatory changes. This method should facilitate detection of chlamydia infections in populations undergoing cytological screening.     

Immunostaining of cell preparations: A comparative evaluation of common fixatives and protocols

Immunostaining of cytologic preparations has been beset by problems of inconsistency, high background staining, and the requirement of different fixatives for different antigens. This study sought to identify a universal fixative and a simple fixation protocol suitable for a wide range of tissue antigens commonly employed for cytologic diagnosis. In an analysis of 23 fixation protocols involving acetone, acetone/methanol, acetone/formalin, glutaraldehyde, ethanol, methanol, and formal saline, fixation in 0.1% formal saline overnight at 27°C followed by 10 min fixation in 100% ethanol produced the most consistent and optimal preservation of immunoreactivity which could be further enhanced by pre-treatment with microwaves for epitope retrieval. Blocking of endogenous peroxidase was not necessary with this fixation protocol. Provided the smears were well air-dried (for at least 14 hr) prior to immersion in formal saline, there was no need to employ adhesive-coated glass slides. The smears could be kept at 27°C (room temperature) for at least 7 days and at −70°C for 5 wk without loss of immunoreactivity as air-dried smears or after fixation in formal saline. One hundred percent acetone and 100% ethanol produced good morphology and immunoreactivity but a high level of background staining, whereas acetone-based mixtures resulted in inconsistent immunostaining. Diagn Cytopathol 1996;15:167–174. © 1996 Wiley-Liss, Inc.     

Clinicopathological study of Papanicolaou (Pap) smears for diagnosing of cervical infections

Cervical infections are not uncommon in our population especially in young and sexually active women. One thousand samples of married women, aged between 20 and 70 years, were studied by conventional Papanicolaou smears. These samples were examined in the Department of Pathology, King Edward Medical University, Lahore from January 2007 to June 2009. Only cases without (pre)neoplastic cytology were included. Six types of infections were diagnosed cytologically. The overall frequency of normal, inadequate, neoplastic, and infective smears was 50%, 1.8%, 10.2%, and 38.3%, respectively. Most of the patients (67%) were in the reproductive age group with mean age 34.7 ± 2.6 years. The commonest clinical sign seen in 354/383 (92%) cases and symptom in (349/383; 91%) cases were vaginal discharge and pruritis vulvae. Among the infective smears, 290 cases (75.7%), the cytologic diagnosis was nonspecific inflammation. Most of these 290 smears contained clue cells (indicating Gardnerella infection) and a lack of lactobacilli. Such smears are predominant in patients suffering from bacterial vaginosis (BV). Twenty-eight smears (7.3%) were positive for Trichomonas vaginalis, 27 cases (7%) were smears with koilocytic change pathognomonic of human papilloma virus infection. Twenty-five smears (6.5%) were positive for fungal infection. Seven cases (1.8%) were diagnosed as herpes simplex virus infection. Finally, there were six cases (1.5%) with atrophic vaginitis. We conclude that the cervical smear is well suited for diagnosing cervical infections. It is clear that Gardnerella, known to be associated with bacterial vaginosis, is a major problem in our Pakistani population.     

Should we report cervical smears lacking endocervical component as unsatisfactory?

The purpose of the study is to correlate the accuracy of diagnosis of cervical smears with the presence/absence of an endocervical component. The referral smears and the smears obtained during colposcopic examination of 84 patients with biopsy-proven squamous lesions were evaluated for the presence of an endocervical component, and the cytologic interpretations were compared with the histologic findings. Of the 136 smears containing an endocervical component, 111 (81.6 percent) had good correlation with histology and 25 (18.4%) showed a discrepancy. Of the 30 smears lacking an endocervical component, 24 (80%) had good histologic correlation and 6 (20%) were discrepant. Our data showed no significant difference in the detection of squamous lesions in those smears with an endocervical component from those without. We conclude that, although the presence or absence of an endocervical component should be documented in the cytology report, its absence should not be an indication to report the cervical smears as unsatisfactory.     

Automated screening for quality control using PAPNET: A study of 638 negative Pap smears

To determine if the PAPNET screening system can be used for quality control to lower false-negative rates for Pap smears 638 manually screened. “negative” Pap smears were subjected retrospectively to the PAPNET screening system. Twenty-nine of the smears came from 18 patients who subsequently had biopsy-proven high-grade squamous intraepithelial lesions (SIL). The remaining 609 negative smears were arbitrarily selected as controls. One hundred twenty-eight (128) of the retrospectively reviewed smears (20%) were selected by PAPNET for microscopic referral because of potential abnormalities. Abnormalities were confirmed on 14 of these smears upon microscopic evaluation. Five of these 14 smears were from smears obtained from four of the 18 women with high-grade SIL. The incidence of manually screened false-negatives detected by PAPNET rescreening was 14/638 or 2.2% for the entire patient population in this study and 5/29 or 17.24% for the targeted patients known to have subsequently developed high-grade lesions. The 2.2% decrease in the false-negative rate in this experiment may be partly artificial as this study group was seeded with false-negative cases at high risk for containing missed abnormalities. The implementation of the PAPNET system for quality control may lower false-negative rates for Pap smears. Diagn Cytopathol 1996;14:316–320. © 1996 Wiley-Liss, Inc.     

Cervical endometriosis: A potential pitfall in the evaluation of glandular cells in cervical smears

The use of the cytobrush and other endocervical sampling instruments has resulted in an increasing rate of detection and attention to glandular abnormalities of the cervix. Lesions that have been identified as look-alikes to endocervical gland dysplasia or adenocarcinoma in situ include squamous carcinoma in situ, atypical squamous metaplasia involving glands, and tubal metaplasia. In this report, we describe our recent experience with another condition that can mimic glandular abnormalities—cervical endometriosis. An in-depth review of the features seen in cervical smears from patients with cervical endometriosis is presented. Diagn. Cytopathol. 16:274–280, 1997. © 1997 Wiley-Liss, Inc.     

Morphological diagnosis of parvovirus B19 infection. A cytopathic effect easily recognized in air-dried, formalin-fixed bone marrow smears stained with hematoxylin-eosin or Wright-Giemsa.

Readily identifiable intranuclear inclusions characterize parvovirus B19 cytopathic effect in formalin-fixed, paraffin-embedded material. Such inclusions are not apparent in air-dried smears of bone marrow aspirates. Brief formalin fixation of bone marrow smears, followed by either hematoxylin-eosin or Wright-Giemsa staining, permitted easy detection of parvovirus B19 inclusions in material from a renal transplant recipient with parvovirus B19 infection documented serologically and by electron microscopy. Formalin fixation of bone marrow and peripheral blood smears before hematoxylin-eosin or Wright-Giemsa staining may simplify the morphological diagnosis of parvovirus B19 infection.     

Cytologic correlates of benign versus dysplastic abnormal keratinization

The purpose of this study was to determine the cytologic and histologic features that differentiate benign from squamous intraepithelial lesion (SIL)-associated cervical abnormal keratinization, defined as hyperkeratosis, parakeratosis, or individual cell dyskeratosis. Fifty-four cervical Papanicoloau (Pap) smears that contained abnormally keratinized cells were reviewed without knowledge of the concurrent biopsy. Twenty-three Pap smears were diagnosed as SIL and the corresponding biopsy showed SIL in 21 (91%) of these cases. Of the 23 Pap smears diagnosed as negative for SIL, the corresponding biopsy in 20 cases (87%) showed benign (SIL negative) abnormal keratinization. Eight Pap smears showed squamous atypia, of these 5 showed SIL on biopsy, and the other 3 revealed benign keratinization. The Pap smear correlates of the 25 biopsies that were negative for SIL included marked hyperkeratosis (18/25–72 vs. 5/29–17% for biopsies with SIL) and regular nuclear membranes (16/18–89% cases with nucleated dyskeratotic cells vs. 5/29–17% for biopsies with SIL). The cytologic correlates of the 29 biopsies that showed SIL included irregular chromatin clumping (27/29–93% vs. 3/18–17% for biopsies without SIL), and a disorganized growth pattern (24/29–83 vs. 5/25–20% for biopsies without SIL). It is concluded that the cytologic distinction between benign and SIL-related Pap smears with abnormal keratinization can be reliably made by the degree of hyperkeratosis, nuclear chromaticity pattern and contour, and the growth pattern of the dyskeratotic cells. Diagn. Cytopathol. 1997;17:447–451. © 1997 Wiley-Liss, Inc.     

DNA cytometry in postirradiation cervical-vaginal smears.

Benign radiation change (BRC) in cervical-vaginal smears may be difficult to distinguish from postirradiation dysplasia (PRD) or recurrent cervical carcinoma. The utility of DNA analysis in postirradiation smears was evaluated retrospectively in 71 patients. Representative Papanicolaou smears were restained with a Feulgen method and 100 to 250 cells were analyzed for DNA content using the CAS 200 image analysis system. Thirty-three control irradiated patients had negative smears with a minimum 3-year follow-up. Thirty controls (91%) had diploid histograms with a mean coefficient of variation of 8.2% and an average of 6.8% of cells in S and G2/M phase. Three control patients had atypical nondiagnostic histograms. Twenty-three patients had abnormal smears and subsequent local recurrence; 21 (91%) had abnormal histograms, with seven showing polyploidy and 14 showing aneuploidy. The remaining 15 patients had abnormal smears diagnosed as PRD but no evidence of recurrent carcinoma. Five were polyploid, six were aneuploid, one was diploid, and three were atypical but nondiagnostic. Interactive DNA cytometry is useful in differentiating BRC from PRD and recurrent cancer. Aneuploidy is rarely, if ever, seen in negative smears with BRC. However, BRC may be associated with broad diploid peaks and increased proliferating cells. An abnormal histogram can be seen with PRD and does not always correlate with recurrent disease.