Papanicolaou-stained Slides Research Articles

Longitudinal clinical evaluation of a novel antibody cocktail for detection of high-grade squamous intraepithelial lesions on cervical cytology specimens.

Abstract Context.—Although cervical cancer screening by cytology is successful, the test continues to show relatively poor operating characteristics. Cell cycle markers may enhance detection of high-grade squamous intraepithelial lesions. Objective.—To determine the clinical usefulness of ProExC, an immunocytochemical assay for cell cycle components, performed on routine cervical cytology samples. Design.—Cervical cytology samples were collected using the SurePath method. Residual cells remaining after preparation of the Papanicolaou-stained slide were used to make a second slide for ProExC staining using an indirect polymer-based immunoperoxidase method. Only adequately cellular slides were evaluated for the presence of nuclear staining within cytologically abnormal epithelial cells. Results were correlated with clinical follow-up. Results.—Six hundred twenty-four samples were satisfactorily cellular and stained. Correlation with clinical follow-up for subsequent cervical intraepithelial neoplasia 2+ on ...

P63 differentiates subtypes of nonsmall cell carcinomas of lung in cytologic samples

Bevacizumab in combination with carboplatin and paclitaxel improves overall response and survival in patients with advanced or recurrent nonsmall cell lung carcinoma. However, this drug is not recommended in patients with squamous cell carcinoma or neoplasms with a dominant squamous component. Therefore, identification of squamous cell differentiation has therapeutic implications. In many instances, cytology is the diagnostic tool of choice; however, routine cytomorphology is limited in classification of nonsmall cell carcinomas into squamous and nonsquamous subtypes. The aim of this study was to identify the value of p63 immunocytochemical analysis in this distinction. Review of cytology records identified 51 consecutive pulmonary specimens (16 fine needle aspiration samples, 15 washes, 12 brushes, and 8 lavages) with the diagnosis of nonsmall cell lung carcinoma (9 carcinomas with squamous differentiation and 42 carcinomas without squamous differentiation). Histologically, they all proved to be nonsmall cell carcinomas, 26 with squamous differentiation and 25 without squamous differentiation. p63 immunocytochemical stain was performed on archival alcohol-fixed Papanicolaou-stained cytology slides using standard immunocytochemical methods. Twenty-three (88 %) of the 26 histologically proven squamous cell carcinomas were positive for p63 on cytologic smears. By using p63 immunocytochemistry, the authors detected 14 carcinomas with squamous differentiation not identified by cytomorphology. Smears from all histologically proven carcinomas with squamous differentiation were positive for p63. Sensitivity of cytology for the detection of nonsmall cell carcinoma of lung with squamous differentiation increased from 35% to 88% using p63 immunocytochemistry (P = .001; McNemar test). The squamous component in 4 carcinomas was detected only in cytologic and not in corresponding histologic samples when subsequent p63 immunostaining was performed. The authors concluded that p63 is a useful marker for the detection of nonsmall cell carcinomas of lung with squamous differentiation when used in cytologic pulmonary samples. p63 immunocytochemistry significantly increases the sensitivity for the identification of lung neoplasms with squamous differentiation from 35% to 88% (P = .001). Therefore, p63 immunocytochemistry may be used in pulmonary cytologic samples of nonsmall cell carcinomas to identify squamous differentiation and to improve therapeutic selection of patients with lung cancer.

Abstract B65: Atypia in ductal lavage: A reproducible marker of risk?

Abstract B65 Background The majority of breast cancers originate in the ductal epithelium of the breast. Ductal lavage provides a minimally invasive tool for assessing epithelial cells and their microenvironment. Previous studies of pathological atypical hyperplasia on biopsy, and cytological aypia in nipple aspirate fluid (NAF) and random fine needle aspiration (FNA) have suggested that these cells are precancerous because of the subsequent increased risk of cancer. It has therefore been hypothesized that serial ductal lavage could be used to identify atypia and monitor the effects of chemoprevention over time. This report is part of a larger study examining the differences in hormone levels and cytology between ducts and their reproducibility. This report presents a cohort of women unselected for risk who voluntarily underwent ductal lavage of the same ducts on two occasions six months apart. Samples were analyzed for epithelial cells and macrophages. Methods A total of 107 women were recruited in this IRB-approved prospective study to undergo ductal lavage of three fluid or non-fluid-yielding ducts within a breast. Women were unselected for risk: the average Gail score was 1.67 and ten women had a history of contralateral breast cancer. The average age was 51 years. Fifty-four women were premenopausal, 4 were perimenopausal and 49 were postmenopausal. Samples were submitted in a blinded fashion to a central laboratory and were analyzed by duct for both macrophage and epithelial cell number on Papanicolaou-stained slides. If adequate epithelial cells were present (>10), a diagnosis of each duct (benign, mild atypia, or marked aytpia) was made by an experienced cytopathologist. Results Of the 107 women, 317 (99%) ducts were successfully cannulated without regard to nipple aspirate fluid (NAF) production. Thirty ducts in 23 women were diagnosed with mild or marked atypia on either of the two visits. Of these ducts, 16 were diagnosed at the initial visit and 13 at the second visit. Only one duct was diagnosed with mild atypia on both visits, in a woman with a Gail score of 1.4. Cellular atypia in ductal lavage was not reproducible at a six month interval (p=0.45). This absence of reproducibility was seen in women at all risk levels. Macrophage and epithelial cell numbers also showed a lack of reproducibility at the six month interval (p=0.31 and p=0.22, respectively). Conclusion Atypia on cytology in ductal lavage in women unselected for risk was not reproducible at a six month interval, suggesting that a single diagnosis of atypia on lavage should not be used as a marker for the identification of women at risk. Further studies are necessary to indicate whether persistent atypia over an extended interval could represent an elevated risk for breast cancer. In addition, ongoing studies are being performed to identify better cellular markers of premalignancy and subsequent risk. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B65.

KOC (K homology domain containing protein Overexpressed in Cancer) and S100A4‐protein immunoreactivity improves the diagnostic sensitivity of biliary brushing cytology for diagnosing pancreaticobiliary malignancies

Biliary tract brush cytology is one of the favored methods of evaluating lesions of the pancreatobiliary tract. However, although its specificity has been reported to be high (91-100%), the sensitivity is lower (30-88%). In this study we applied KOC and S100A4 protein immunocytochemistry to assess their potential use as adjunct markers in differentiating benign from malignant cells, and improve the diagnostic sensitivity of this method for pancreatobiliary malignancies. The authors examined KOC and S100A4 protein expression in 44 alcohol-fixed cytology specimens obtained by biliary brushings. Diagnoses included: (1) benign/atypical favor reactive (20 cases), (2) atypical/not diagnostic of malignancy (3 cases), and (3) suspicious for malignancy/malignant (21 cases). Alcohol-fixed Papanicolaou-stained slides (PAP) were stained with monoclonal antibody to KOC/L523S and polyclonal antibody to S100A4 protein. Results were recorded as negative or positive. Twenty-four cases were confirmed positive for adenocarcinoma and 20 cases were negative. The sensitivity and specificity of cytology was 83 and 95%, KOC showed a sensitivity of 92% and specificity of 95%. S100A4 protein showed a sensitivity of 79% and a specificity of 95%. The combined use of KOC and S100A4 protein showed a sensitivity of 100% and a specificity of 95%, respectively. The concurrent use of KOC and S100A4 protein improves the diagnostic sensitivity of biliary brushings cytology and demonstrates similar specificity as cytology alone in the diagnosis of pancreatobiliary malignancy.

Longitudinal Clinical Evaluation of a Novel Antibody Cocktail for Detection of High-Grade Squamous Intraepithelial Lesions on Cervical Cytology Specimens

Although cervical cancer screening by cytology is successful, the test continues to show relatively poor operating characteristics. Cell cycle markers may enhance detection of high-grade squamous intraepithelial lesions. To determine the clinical usefulness of ProExC, an immunocytochemical assay for cell cycle components, performed on routine cervical cytology samples. Cervical cytology samples were collected using the SurePath method. Residual cells remaining after preparation of the Papanicolaou-stained slide were used to make a second slide for ProExC staining using an indirect polymer-based immunoperoxidase method. Only adequately cellular slides were evaluated for the presence of nuclear staining within cytologically abnormal epithelial cells. Results were correlated with clinical follow-up. Six hundred twenty-four samples were satisfactorily cellular and stained. Correlation with clinical follow-up for subsequent cervical intraepithelial neoplasia 2+ on biopsy/high-grade squamous intraepithelial lesion on cytology (CIN 2+/HSIL) showed that 434 results were true negative, 78 true positive, 18 false-negative, and 94 false-positive, resulting in a sensitivity/specificity of 81%/82%. When ProExC results were combined with any level of cytologic atypia, sensitivity for CIN 2+/HSIL was 92% and specificity was 84%. ProExC shows promise as an aid in enhancing the sensitivity and specificity of cervical cytology for subsequent CIN 2+/HSIL and may be useful in identifying those cervical lesions most apt to progress.

Desmoplastic melanoma morphology on Thinprep: a report of two cases

BackgroundDesmoplastic melanoma is a variant of malignant melanoma that can range in appearance from sarcomatoid to scar-like. Cytomorphology of desmoplastic melanoma has been previously described on conventional smears; however, to our knowledge, detailed cytomorphology on ThinPrep has so far not been described. Herein, we describe the cytomorphology of two cases of desmoplastic melanoma on fine needle aspiration processed as ThinPrep slides and compare it to that seen on conventional smears. Pertinent immunocytochemical stains, performed on ThinPrep slides are also discussed.Case presentationThe first case is a woman with a history of desmoplastic melanoma of the scalp with previous local recurrences and lymph node metastasis with a new submandibular mass. The second case is a man with a previously resected desmoplastic melanoma with his first local recurrence. Conventional smears, including air-dried Diff-Quik-stained and alcohol-fixed Papanicolaou-stained smears, demonstrated aggregates of pleomorphic spindle cells admixed with fibrous stroma and single spindle cells. In both cases, nuclei were elongated and plump with irregular nuclear contours, deep grooves, and folds. Chromatin was dark and coarse with either inconspicuous or multiple prominent nucleoli. Cytoplasm was located at the nuclear poles and was fine, wispy, and delicate. The background was clean with no evidence of necrosis or melanin pigment. Papanicolaou-stained ThinPrep slides were prepared from needle rinses and demonstrated excellent correlation of nuclear and cytoplasmic detail of single spindle cells to that seen on conventional smears with the exception of only slight decrease in nuclear size; however, nuclear and cytoplasmic detail of spindle cells embedded in stroma was markedly attenuated. Confirmatory immunostain for S-100 protein in both cases was performed on ThinPrep slides demonstrating crisp cytoplasmic staining in the spindle cells.ConclusionThe cytomorphology of desmoplastic melanoma shows excellent correlation between cytomorphology of single spindle cells on conventional smears and on ThinPrep slides. The major difference noted on ThinPrep slides was attenuated nuclear and cytoplasmic detail of spindle cells embedded in fibrous stoma.

Vergleich der perioperativen Methylenblau-gefärbten Schnellzytologie mit der nach Papanicolaou gefärbten Permanentzytologie in der Detektion von Patienten mit High-grade-Urothelkarzinomen der Harnblase

The aim of the study was to investigate whether intraoperative methylene blue-stained and permanent Papanicolaou-stained urine cytologies show comparable accuracy in detection of high-grade urothelial carcinoma. The study included 130 patients; 50 patients were without malignancy (25 follow-up, 25 with hematuria). In 80 patients transurethral resection due to urothelial carcinoma was performed. Per patient two cytology specimens were prepared: one immediate methylene blue-stained specimen, which was evaluated by the surgeon, and one Papanicolaou-stained permanent cytology slide, which was blinded and evaluated by one urologist. Cytology results of all patients without malignancy were unsuspicious irrespective of the staining method. Of 80 patients with urothelial carcinoma, 50 showed a low-grade tumor. Sensitivity of tumor detection was 20 and 30% for methylene blue/Papanicolaou-stained slides, respectively. Among 30 patients with high-grade carcinoma, 10 were detected by methylene blue cytology and 30 by Papanicolaou-stained slides, corresponding to a sensitivity of 40 and 100%, respectively. The results of standard Papanicolaou-stained urine cytology in the detection of clinically relevant high-grade urothelial carcinoma are excellent. The quality of cytological tumor detection by methylene blue-stained cytology made by different evaluators is insufficient in our opinion.

Ex Vivo Fluorescence Imaging of Normal and Malignant Urothelial Cells to Enhance Early Diagnosis

Urinary cytology is a noninvasive and unconstraining technique for urothelial cancer diagnosis but lacks sensitivity for detecting low-grade lesions. In this study, the fluorescence properties of classical Papanicolaou-stained urothelial cytological slides from patients or from cell lines were monitored to investigate metabolic changes in normal and tumoral cells. Time- and spectrally-resolved fluorescence imaging was performed at the single cell level to assess the spectral and temporal properties as well as the spatial distribution of the fluorescence emitted by urothelial cells. The results reveal quite different fluorescence distributions between tumoral urothelial cells, characterized by a perimembrane fluorescence localization, and the normal cells which exhibit an intracellular fluorescence. This is not caused by differences in the fluorescence emission of the endogenous fluorophores NAD(P)H, flavoproteins or porphyrins but by various localization of the EA 50 Papanicolaou stain as revealed by both the spectral and time-resolved parameters. The present results demonstrate that the use of single-cell endofluorescence emission of Papanicolaou-stained urothelial cytological slides can allow an early ex vivo diagnosis of low-grade bladder cancers.

Immunocytochemistry for hepatocyte nuclear factor‐1β (HNF‐1β): A marker for ovarian clear cell carcinoma

Recent microarray studies have shown that the expression of hepatocyte nuclear factor-1beta (HNF-1beta) was significantly up-regulated in clear cell carcinoma (CCC) of the ovary. HNF-1beta may be a useful marker for CCC in peritoneal fluid cytology. We designed an experimental study using three CCC cell lines to evaluate the influence of alcohol fixation or air drying on immunocytochemistry for HNF-1beta. Each cell line was cultured on chamber slides or transplanted into the abdominal cavity of nude mice, then the slides or ascites smears of nude mice were immunostained with or without microwave-mediated epitope retrieval. Immunoreactivity with HNF-1beta, which was either alcohol-fixed or air-dried, was noticeably improved after microwave heating. In contrast, two serous adenocarcinoma cell lines never showed immunoreactivity. Based on these results, 21 archival Papanicolaou-stained slides of peritoneal fluid (5 CCCs, 12 serous, 2 mucinous, and 2 endometrioid adenocarcinomas) were decolorized and immunostained under heating pretreatment. Five of 5 CCCs were distinctively positive for HNF-1beta, whereas all non-CCC ovarian cancers were negative for this protein. The present study clearly demonstrated that heating epitope retrieval improved the immunoreactivity for a nuclear protein in alcohol-fixed or air-dried cytology specimens. HNF-1beta is likely to be helpful for the diagnosis of CCC in the peritoneal fluid.

Evaluation of p16INK4a expression in ThinPrep cervical specimens with the CINtec p16INK4a assay

The aim of this study was to examine p16(INK4a) protein expression in ThinPrep (Cytyc Corporation, Marlborough, Mass) cervical specimens by using the CINtec p16(INK4a) Cytology Kit (Dako, Glostrup, Denmark). The ability of this assay to accurately identify underlying high-grade lesions was assessed by using follow-up biopsies and comparing these results with Hybrid Capture 2 (Digene, Gaithersburg, Md) high-risk HPV (hc(2)) results. Three hundred ninety-eight residual ThinPrep samples were collected, and histological follow-up data were retrieved for abnormal cytology specimens. After preparation of a Papanicolaou-stained slide, a second slide was processed in preparation for p16(INK4a) immunostaining. High-risk human papillomavirus testing (hc(2)) was also performed. Of the 163 cytologically abnormal samples, 6-month biopsy follow-up data were available for 45% of the specimens. At initial blinded evaluation, 21 of the 26 cases with cervical intraepithelial neoplasia (CIN) II/III follow-up were positive for p16(INK4a), yielding an overall diagnostic sensitivity of 81%; 29 of the 47 cases diagnosed as CIN I or less were p16(INK4a) negative, yielding a diagnostic specificity of 62%. In comparison, the hc(2) test results indicated a diagnostic sensitivity of 100% with a diagnostic specificity of 15%. After review of selected cases with CIN II/III follow-up, 25 of 26 slides were deemed to be positive for p16(INK4a), increasing the diagnostic sensitivity to 96%. The CINtec p16(INK4a) Cytology Kit, in combination with ThinPrep cervical samples, allowed clear evaluation of p16(INK4a) protein overexpression. Diagnostic specificity of the CINtec p16(INK4a) assay was significantly improved relative to hc(2). To increase p16(INK4a) immunostaining in abnormal cells, a modified kit version with improved staining performance has been developed and is currently being evaluated.

Grading follicular lymphomas in fine-needle aspiration biopsies

The World Health Organization (WHO) recommends a 3-tier grading system (Grades 1-3) for follicular lymphomas (FLs) based on the absolute number of centroblasts per high-power microscopic field (HPF) in 10 neoplastic follicles. Grades 1 and 2 FL are still managed as indolent FLs, whereas Grade 3 FL is thought to behave more aggressively. In this study, the feasibility of grading FL using ThinPrep (TP) slides and flow cytometry (FC) was evaluated. Fifty-three cases of lymph node fine-needle aspiration (FNA) from patients with histologically confirmed FL (20 Grade 1, 17 Grade 2, and 16 Grade 3) were included. The number of centroblasts present in 300 lymphoid cells and in 10 HPF in TP Papanicolaou-stained slides was evaluated. The percentage of CD10-positive small cells was calculated with FC results. Statistical analysis was performed with the Jonckheer-Terpstra nonparametric trend test and the Wilcoxon rank sum test. The statistical analysis demonstrated a significant upward trend in the number of centroblasts as the grades increased. Also, all 3 methods had statistically significant results to distinguish different grades of FL, except when FC was used to distinguish Grade 2 from Grade 3 FL. Counting centroblasts, either in 300 lymphoid cells or per 10 HPF in TP slides, represented a statistically significant method to separate different grades of FL in FNA samples. Analysis of cell size by FC was not as reliable to distinguish different grades of FL, especially Grade 2 from 3.

FISH detection of t(14;18) in follicular lymphoma on Papanicolaou-stained archival cytology slides

The t(14;18)(q32;q21) translocation is present in about 85% of follicular lymphomas (FL) and can be identified using fluorescence in situ hybridization (FISH). In the diagnostic laboratory setting, the cytologic archival material consists of stained slides, and only rarely is material saved for molecular testing. The authors proposed FISH for FL using Papanicolaou-stained archival cytology material as a practical ancillary technique for diagnosing FL. Cases included 35 FL, 6 small lymphocytic lymphomas/chronic lymphocytic leukemias (SLL/CLL), 4 mantle cell lymphomas (MCL), 4 marginal zone lymphomas (MZL), 1 lymphoplasmacytic lymphoma (LPL), and 10 reactive lymphoid tissues (RLT). FISH was performed on Papanicolaou-stained archival cytology slides using probes for immunoglobulin heavy chain (IGH) on chromosome 14 and BCL2 on chromosome 18. In all, 25 of 32 (81%) FL cases exhibited the t(14;18) translocation, whereas 7 of 32 (19%) lacked the translocation. No cases of non-FL were positive for t(14;18). This series shows a sensitivity of 81% and specificity of 100% for detecting the t(14;18) translocation as a diagnostic tool in FL. When performed on Papanicolaou-stained cytology slides, FISH for t(14;18) is relatively sensitive and quite specific for FL. These findings are similar to those reported on other specimens, such as paraffin-embedded tissue and unstained cytology slides. The authors proposed that their technique would allow the pathologist and clinician the flexibility to utilize previously stained fine-needle aspiration slides for FISH evaluation.

Diagnostic Utility of Galectin-3 in Aspirates of Thyroid Follicular Lesions

To investigate the expression of galectin-3 in various thyroid follicular lesions, including diffuse hyperplasia, nodular hyperplasia, and benign and malignant follicular neoplasms, to clarify the diagnostic utility of galectin-3 in aspirates of follicular lesions.A total of 146 follicular lesions diagnosed cytologically, obtained from patients who had undergone thyroidectomy for either benign or malignant nodules, were evaluated using Papanicolaou-stained slides and cell blocks with galectin-3 immunostaining. We primarily categorized the aspirated specimens as benign, indeterminate or suspicious for a follicular neoplasm based on cellularity, architectural arrangement of the follicular cells and presence or absence of colloid. Galectin-3 immunostaining was evaluated according to the proportion and intensity of positively stained cells. Cytologic diagnoses were correlated with the results of galectin-3 and categorized into 3 groups (benign, indeterminate for malignancy, suspicious for malignancy) and compared with the corresponding histologic diagnoses.When the histologic diagnoses were compared with the cytologic diagnoses, the accuracy in the distinction between benign and malignant cases was 79.5% except for 8 cytologically and 3 histologically indeterminate cases. Except for 11 indeterminate cases, there were 18 (12.3%) discrepant cases. False positive cases included 8 of 62 (12.9%) nodular hyperplasias and 1 of 42 (2.4%) follicular adenomas. Of 9 false negative cases, 4 minimally invasive carcinomas and 2 widely invasive carcinomas were included. Three follicular tumors of uncertain malignant potential were cytologically categorized as malignant, and all cases showed positivity for galectin-3.Galectin-3 could be used as a useful supplementary marker for cytologic diagnosis, although it was not an absolute marker in determining whether a lesion was benign or malignant.

Author reply

We thank Drs. Elsheikh and Corbin for their interest in our article1 and for sharing their additional experience with this technique. They performed immunohistochemistry on cell-transferred touch imprints that had been stained using Diff-Quik (American Scientific Products, McGraw Park, IL) or Papanicolaou staining using various antibodies, with and without destaining and with and without antigen retrieval. They found a 100% concordance in staining results between Papanicolaou-stained cell-transferred pieces and formalin-fixed tissue (the gold standard) using antigen retrieval. When no antigen retrieval was used, false-negative results were encountered in approximately 15% of cell-transferred pieces. Similar results were noted in our previous study with estrogen receptor staining.2 However, we did not perform antigen retrieval for markers other than estrogen receptor because of the fact that putative utilization of antigen retrieval in nonformalin-fixed cytologic samples remains controversial and to the best of our knowledge, well controlled validation data for other markers are still lacking. It is likely that omission of the antigen retrieval step might have contributed to the negative staining results observed in some cell-transferred pieces in our study. Elsheikh and Corbin also reported that approximately 5–10% of cell-transferred pieces were lost or damaged during the destaining process. We routinely apply destaining on Papanicolaou-stained slides before immunocytochemical staining with all markers except estrogen receptor. This practice was based on the results of our study demonstrating that destaining may give rise to false-negative staining with estrogen receptor.2 It appears to us that omitting destaining, coupled with applying antigen retrieval, might yield better staining sensitivity. However, a well controlled study is needed to validate this observation. The reliability of air-dried, Diff-Quik-stained tissue for immunostaining is limited. The high rate of false negativity and high background staining reported by Elsheikh and Corbin attest to this finding.

Sensitivity of HPV Tests on Stained vs. Unstained Cervical Smears

To examine the effect of Pspanicolaou staining of cervical smears on the sensitivity of molecular biologic HPV tests.Two sensitive HPV tests were used, HPV DNA sequence analysis after polymerase chain reaction (PCR) amplification and the Hybrid Capture II method (HC II) (Digene Diagnostics Inc., Silver Spring, Maryland, USA). Papanicolaou-stained and unstained smears taken simultaneously were examined from 265 women readmitted for examination due to an atypical squamous cells of undetermined significance diagnosis.After an HPV test with the PCR method on unstained slides, 66% of the women were HPV positive, whereas the same women were HPVpositive in 54% when Papanicolaou-stained slides were analyzed. However, this difference was not statistically significant (p > 0.1). With the HC II method, 55% of unstained smears were HPV positive whereas 29% were HPV positive, when Papanicolaou-stained slides were examined. This difference was significant (p < 0.001). The same strong differences in sensitivity were observed when both the PCR and HC II methods were studied on the same Papanicolaou stained glass slides, whereas on unstained slides no significant difference was found.The results demonstrate that Papanicolaou staining of a cervical smear significantly decreases the sensitivity of an HPV test performed with the HC II method, whereas the PCR method is less affected. With the Papanicolaou method, the hematoxylin bath is followed by HCl treatment, and strong acid treatment destroys DNA.

Endocrine Biomarkers in Ductal Lavage Samples from Women at High Risk for Breast Cancer

Ductal lavage is a method of minimal epithelial sampling of the breast, with potential utility for repeat sampling and biomarker analysis in chemoprevention studies. We report here the baseline findings from a study designed to assess the utility of ductal lavage in this setting. Tamoxifen-eligible, high-risk women underwent ductal lavage; epithelial cell number (ECN) and morphology were assessed on Papanicolaou-stained slides. Additional slides were immunostained for estrogen receptor (ER) alpha, Ki-67, and cyclooxygenase-2, and the labeling index (LI) was established by counting negative and positive cells. The ductal lavage supernatant (DLS) was assayed for estradiol, several of its precursors, progesterone, cathepsin D, interleukin-6, and epidermal growth factor (EGF). One hundred sixty-eight women have entered the study (mean age, 51 years; mean 5-year Gail score, 2.8). Ductal lavage was accomplished in 145 (86.3%) women. Data were analyzed by duct and by woman (averaging data across all ducts). Mild atypia was seen in 43 of 145 (29.6%), whereas severe atypia was seen in 2 (1.4%) of women. We observed significant positive correlations between ECN and cytologic atypia, ER LI, cyclooxygenase-2 LI, and Ki-67 LI. EGF levels in supernatant were significantly associated with estrogenic precursors, ER LI and ECN. A factor representing the DLS hormone and protein variables explained 36% of the variance; total ECN was highest when factor score and ER LI were high and was lowest when both were low (P for interaction = 0.001). Biomarker analyses in epithelial cells and DLS are feasible. The significant associations of EGF with other markers suggest a possible role in increasing epithelial cell mass.

The establishment of sperm morphology satellite training laboratories in Africa

Summary Discrepancies in the results of semen analysis can be caused by a variety of factors, for example, a microscope for semen analysis can be situated in the practice of an urologist, cytologist or microbiologist, or even in a health care office in the countryside in a developing country. The study aimed to evaluate the (i) sperm morphology assessment skills of the participants who attended WHO seminology workshops and (ii) to appraise the possibility of using trained technologists to set up satellite training facilities in their own area. The Centre for Research in Reproductive Health, Ogun State University Teaching Hospital (Nigeria) initiated training sessions in their area. Subsequently, 16 technologists were trained in three sessions. Papanicolaou-stained slides, supplied by Tygerberg were used as test material. Pre- and post-training results were used to calculate the mean percentage of normal cells as well as the percentage deviation from the reference laboratory. The mean percentage normal spermatozoa recorded by the Nigerian trainees during pre-training evaluations differed by 38% from the reference value, compared with a post-training difference of 16% (P < 0.001). Tertiary academic hospitals can play an important educational role in the region. The present results underline the power of long distance educational programmes. Similar laboratories are currently being set up in Kenya and Zambia.

Quantitation of Ki-67 Expression in the Differential Diagnosis of Reserve Cell Hyperplasia vs. Small Cell Lung Carcinoma

To investigate whether the detection of proliferation-associated Ki-67 antigen may be of value in differentiating between reserve cell hyperplasia (RCH) and small cell lung cancer (SCLC). Retrospectively, 20 Papanicolaou-stained bronchial brushes or washings from 20 patients were selected. Ten were diagnosed as RCH (and had no SCLC in follow-up) and the other 10 as SCLC (histologically confirmed). All 20 Papanicolaou-stained slides were restained with the monoclonal antibody MIB1, directed against Ki-67 antigen; that simple and reliable procedure was described recently. In each specimen 5 coherent cell groups were identified, corresponding to RCH or SCLC, respectively; photographed; and studied for Ki-67 antigen expression after MIB1 staining of the slides. At least 3 cell groups remained in each specimen. The Ki-67 labeling index (LI) of the specimens was determined as the number of MIB1-positive cells divided by the total number of cells in the remaining cell groups. All cases of SCLC showed a mean Ki-67 LI of at least .415 (mean .684, SD .151), whereas in the cases with RCH the mean Ki-67 LI never was more than .158 (mean .048, SD .049). The difference was highly significant (P<.001, Student's t test). Linear discriminant analysis resulted in a classifier with which we were able to discriminate correctly between SCLC and RCH in 100% of the 20 bronchial brushings and washings. The results clearly demonstrate that measuring proliferative activity in Papanicolaou-stained bronchial brushings and washings by MIB1 restaining of the slides may be of great practical value in accurately discriminating RCH from SCLC. The method is simple and can be performed in any laboratory that is able to carry out immunocytochemical staining. However, an additional (prospective) study with a series of difficult cases is necessary to confirm these findings.

Monitoring technologist reading skills in a sperm morphology quality control program

Objective The value of sperm morphology as a predictor of a man’s fertilizing potential has often been challenged because of different classification systems used to distinguish between normal and abnormal spermatozoa. The study aimed to monitor the reading skills of 53 laboratory technologists who are enrolled in a continuous quality control program for sperm morphology assessment. Design Prospective analytical study. Setting Academic hospital and academic institution setting. Patient(s) Sperm samples from the sperm donor program and andrology technicians from sub-Saharan Africa. Intervention(s) Papanicolaou-stained sperm slides were prepared and shipped on a quarterly basis to participating laboratories. Papanicolaou-prestained sperm morphology slides were used as test material for 21 months. Main outcome measure(s) Percentage normal spermatozoa. Result(s) A new statistical model was developed to record reading skills of the participating technicians. Reading skills were classified as marginal (5.7% of cases), good (11.3% of cases), and excellent (83% of cases). Participants maintained their morphological reading skills and agreed with the reference laboratory by not exceeding a SD limit of 0.2 to set stringent standards for the program. Conclusion(s) Technician proficiency can be monitored using the results of a quality control program. A continuous quality control program can be initiated only after intensive training, because baseline values at the onset of the quality control program serves as an internal reference value.

Correlation between morphology and telomerase activity in cells from exfoliative lung cytologic specimens.

Telomerase is a ribonucleoprotein that compensates for the erosion of telomeres (chromosomal termini). Telomerase activity is detected in more than 85% of cancerous lesions and is therefore considered a novel marker of cancer. The authors compared cytologic morphology and telomerase activity at the cellular level to obtain further insight into their association. The authors used bronchial washing and brushing materials obtained from 18 patients with lung carcinomas (6 squamous cell, 8 adenocarcinoma, 2 large cell, 1 small cell, and 1 metastasis from colon carcinoma) and 20 patients with nonmalignant disease. An in situ telomeric repeat amplification protocol (TRAP) assay was performed, and routine Papanicolaou-stained slides using the same sample were assessed. Nuclear fluorescent signals at the nuclear area, corresponding to telomerase activity, shown by the in situ TRAP assay were only detected in samples containing morphologically malignant cells. No nuclear fluorescence was seen in the keratinizing component of well-differentiated squamous cell carcinoma. Nuclear staining was not seen in metaplastic or basal hyperplastic cells. Cytoplasmic fluorescence was only found in macrophages and polymorphonuclear leukocytes. Nuclear fluorescence corresponding to telomerase activity was not demonstrated in metaplastic or basal hyperplastic cells, thus indicating that detection of telomerase activity is closely associated with the presence of malignant cells, but not premalignant lesions, in lung carcinoma patients. Moreover, in some samples with cancer, cells failed to show telomerase activity, suggesting the limitation of this method for the detection of malignant cells in certain lung carcinoma patients.