Training and practice of cytotechnologists: a discussion forum focused on Europe.

OBJECTIVES: To discuss the role and training of cytotechnologists (CTs) in Europe, to identify areas of good practice and to provide an informed opinion to those providing guidelines for training and practice in Europe. METHODS: All members of the Editorial Advisory Board of Cytopathology were invited to take part in a 'discussion forum' for which six topics were circulated in advance concerning the roles of CTs with regard to: (1) pre-screening slides; (2) 'signing out' reports; (3) carrying out ancillary techniques; (4) supervising laboratory staff; (5) taking part in rapid on-site evaluation (ROSE) of fine needle aspirates (FNAs); and (6) whether CTs were trained specifically in cytopathology or in general histopathology. Notes of the meeting were circulated by email and a final report was agreed by 22 participants from 17 predominantly European countries. RESULTS: Training for CTs throughout Europe was variable, especially for non-gynaecological cytology, which was inconsistent with the range of activities required. The participants recommended graduate entry, preliminary training in general laboratory technology, and subsequent training to take account of the probability and, in some centres, the reality of primary cervical cancer screening changing from cytology to human papillomavirus (HPV) testing. They further recommended that CTs should perform HPV tests and take part in ROSE for FNAs, and they supported the European Federation of Cytology Societies developing guidelines for training and practice. CONCLUSION: With CT training added to a university-based education in laboratory or biomedical science, a career in cytotechnology should be an attractive option involving a diverse range of laboratory and clinically based activities.

Effects of restraint stress on inoculated tumor growth and immune response in rats.

Rats were given injections s.c. of mammary adenocarcinoma cells which developed into undifferentiated carcinomas within a few days. The animals were either left alone or were stressed by restraint for 3 h a day for 11 days and then left for 12 days undisturbed to recover. During this schedule, some animals were sacrificed immediately after the 11-day stress period, whereas others were allowed the 12-day recovery period; unstressed animals were sacrificed as controls on these 2 days. Tumor burden was significantly increased during stress and markedly decreased after the recovery period as compared to unstressed rats. Higher mitotic activity was seen in the tumors of rats which recovered from stress. The immune system responded differently to stress in healthy and tumor-bearing animals. In the tumor-bearing animals, leukocytes were decreased by stress and increased after the recovery period. Lymphocytes were increased, and neutrophiles and large granular lymphocytes were decreased after the recovery period. Total T-cells and suppressor T-cells were decreased during stress and increased during recovery. The percentage of T-cell populations was unaffected by stress, but the percentage of suppressor T-cells increased during recovery. Natural killer cell activity was unaffected by stress but increased after the recovery period. These results indicate that (a) stress and recovery from stress differentially affect tumor development and growth, (b) stress and recovery from stress cause different effects on the immune system in healthy or tumor-bearing animals, (c) stress and recovery from stress stimulate or inhibit different parts of the immune system, and (d) a decreased lymphocyte count and total and suppressor T-cell numbers correlated best with enhanced tumor growth, whereas increased numbers of neutrophils, large granular lymphocytes, total and suppressor T-cells, natural killer cell activity, and a decreased percentage of T-suppressor cells correlated best with depressed tumor growth.

Prostate-specific antigen production in the female breast: association with progesterone.

Prostate-specific antigen (PSA) is produced by the female breast. Prior in vitro evidence suggests that PSA expression in breast epithelial cells is regulated by androgens and progestins but not estrogens. The purpose of this study was to determine whether (a) PSA expression in breast nipple aspirate fluid (NAF) and in serum is influenced by progesterone (PG); (b) the ability to obtain NAF decreases with repeated breast aspirations; and (c) PSA in NAF correlates with abnormal NAF cytology. Eight pre- and three postmenopausal women with no breast cancer risk factors were enrolled in a pilot study and had NAF and serum collected every 3-4 days for a month to evaluate the influence of serum PG, luteinizing hormone, estradiol, and follicle-stimulating hormone on PSA in serum and in NAF. NAF was obtained in 99% (112 of 113) of aspiration visits. Median, mean, and peak NAF but not serum PSA levels were higher in pre- than in postmenopausal subjects. NAF PSA levels were associated with the rise or peak in serum PG in seven of eight premenopausal women (seven of seven with a PG surge) and in zero of three postmenopausal women. Considering all 11 women, there was an association between NAF PSA and PG (P = 0.005) but not luteinizing hormone, estradiol, or follicle-stimulating hormone. NAF volume did not significantly change over time. Atypical hyperplasia (9%) and hyperplasia without atypia (36%) were identified in the NAF of a subset of the subjects. Median, mean, and peak levels of NAF PSA (P = 0.05, 0.05, and 0.10, respectively) were higher in subjects with normal versus hyperplastic cytology. PSA production in the breast increases in association with PG. With aspiration every 3-4 days, NAF volume does not significantly decrease over time. NAF cytology and PSA levels in NAF may help identify women at increased breast cancer risk. Changes in biomarkers of breast cancer risk in NAF (including PSA and cytology) may predate mammographic abnormalities. NAF may, therefore, be useful as a breast cancer screening tool for young women who are not recommended to undergo mammography and as an adjunct to screen women who have mammograms performed.

Prostate-specific antigen levels in nipple aspirate fluid correlate with breast cancer risk.

Despite the fact that breast cancer is the most common non-cutaneous cancer and a leading cause of cancer deaths in women, accepted markers of breast cancer risk miss up to 40% of these tumors. Moreover, screening methods involving the analysis of tissue or cells are limited by the need for a surgical biopsy. Nipple aspiration is a quick, efficient, noninvasive method to obtain breast epithelial cells, the cells at risk for transformation to carcinoma. Prostate-specific antigen (PSA), a protein thought to be specific to the prostate but recently found in a subset of breast tumors, has been correlated with improved survival. The purpose of this study was to measure PSA in a group of women with increasing breast cancer risk (no risk or family history of breast cancer, precancerous mastopathy, and invasive cancer) and determine if PSA correlates with risk. Nipple aspirate fluid was obtained from the intact breast and from surgical specimens using a modified breast pump. PSA was then measured in the fluid using a highly sensitive and specific immunofluorometric procedure. PSA was found at levels ranging from 0-13,423 ng/g of total protein, and there was a significant relationship between PSA level and breast cancer risk (P = 0.001). That is, all women with no risk factors and 90% of those with a family history had high PSA levels, whereas 68% of subjects with precancerous mastopathy or invasive cancer had low PSA levels. PSA was higher in premenopausal subjects (P = 0.002). After adjusting for the effect of menopausal status, there remained a significant association between PSA and breast cancer risk. These findings suggest that PSA in nipple aspirate fluid may be a useful marker of breast cancer risk.

Choroidal metastasis as the initial manifestation of a pigmented neuroendocrine tumor.

We report the case of a 77-year-old woman in whom choroidal metastasis was the initial manifestation of a primary neoplasm presumed to be a pigmented pulmonary carcinoid tumor. The tumor initially was misdiagnosed cytologically and pathologically as a choroidal melanoma because it contained intrinsic melanin pigment. Positive immunoreactivity for cytokeratin, synaptophysin, chromogranin, and calcitonin and the presence of dense-core neurosecretory vesicles disclosed by electron microscopy established that the metastasis was a neuroendocrine tumor. Findings from systemic evaluation suggested that the primary tumor was located in the lung. The patient subsequently developed an intradural paraspinal metastasis, which also contained melanin pigment. The latter observation confirmed that the melanin in the uveal metastasis was intrinsic and did not represent secondary phagocytosis by tumor cells. Metastases from pigmented tumors of nonmelanocytic derivation are exceedingly rare but present a major diagnostic challenge to ocular pathologists and cytopathologists if the diagnosis is not suspected. Confirmatory immunohistochemical analysis should be obtained when a pigmented choroidal tumor thought to be a melanoma has atypical features. Arch Ophthalmol. 2000;118:841-845

Multiple myeloma involving the iris.

Intraocular invasion by neoplastic plasma cells is a rare ophthalmic manifestation of plasma cell dyscrasias. A 67-year-old man who had documented multiple myeloma presented with an infiltration of the iris that simulated a nongranulomatous uveitis. Cytologic examination of the aspirate obtained by anterior chamber paracentesis demonstrated that the infiltrate was composed of neoplastic plasma cells.

Cytology of granulosa cell tumor of the ovary.

Granulosa cell tumor of the ovary may create a diagnostic challenge in cytologic preparations. Our experience with needle aspiration material, ascitic fluid, and peritoneal washings indicates that in fluids, despite some similarity between the tumor cells and reactive mesothelial cells, a correct diagnosis can be achieved by careful examination. The paucity of the cytoplasm and an intense indentation of nuclear membrane are the most helpful features in distinguishing the granulosa cells from mesothelial cells. The literature is reviewed, and the controversies regarding needle aspiration of ovarian neoplasms are discussed.

Aspiration cytology of papillary cystic neoplasm of the pancreas.

The authors present a case of papillary and cystic neoplasm of the pancreas (PCN) in which fine-needle aspiration was performed intraoperatively. Only a few reports of fine-needle aspiration of this rare tumor have been published. The features most helpful in reaching a diagnosis of PCN were a monotonous population of tumor cells, round to oval bland-appearing nuclei, scanty to moderate ill-defined cytoplasm, and the presence of numerous capillaries. Despite the lack of branching papillary clusters described in previous reports, it was possible to suggest the correct diagnosis by identifying these other characteristic features. In addition, estrogen and progesterone receptor levels were measured and found to be negligible. A review of the literature with emphasis on cytologic features also is presented.

The value of transformed lymphocyte count in subclassification of non-Hodgkin's lymphoma by fine-needle aspiration.

No established criteria exist for predicting lymphoma grade or transformation in cytologic material. We counted transformed lymphocytes in fine-needle aspiration (FNA) biopsy specimens to determine whether the percentage of these cells in the smear could predict the histologic grade, the biologic behavior, or both. The percentage of transformed lymphocytes out of total lymphoid cells was determined on Papanicolaou-stained smears. Afterward, a cytodiagnosis was based on clinical information available at the time of the FNA, cytomorphologic data, and flow cytometry data. Results were correlated with results of examination of the surgical biopsy specimen, clinical behavior of the lymphoma, or both. The percentage of transformed lymphocytes was 10% or less in all low-grade or indolent lymphomas. Aspirates with transformed lymphocyte counts of 20% or greater were aggressive lymphomas. We also report our experience in the diagnosis of non-Hodgkin's lymphoma by FNA using cytomorphologic examination and immunophenotyping by flow cytometry at a cancer referral hospital. This is a preliminary study, and larger series may help establish the ranges of transformed lymphocyte counts that correlate with the lymphoma subtype.

Ureteroscopic biopsy in the upper urinary tract.

OBJECTIVES: To determine the optimal instruments and techniques for biopsy of upper urinary tract lesions through the small working channel in flexible and small semirigid ureteroscopes. METHODS: Urinary tract filling defects and other lesions were accessed with rigid or flexible ureteroscopy and biopsies were done using one or more devices: a 3 F cup biopsy forceps, 2.5 F and 3 F baskets, 2.5 F and 3 F graspers, 3 F snare, brush, and aspiration catheters. All samples, including the cup forceps samples, were sent for cytopathologic study, with those containing grossly visible tissue particles processed as a cell block. Only those specimens reported definitely positive or negative were considered diagnostic, whereas the others were grouped as nondefinitive. Not all sampling techniques could be used in every patient because of the size of the lesion and the technical limitations, including bleeding and instrument position during biopsy. RESULTS: There were 55 procedures in 43 patients. The indications included hematuria, filling defect, abnormal cytology, and periodic surveillance. A basket was used in 22 procedures and gave unequivocal results in 15. The other samples were equivocal, nondiagnostic, or unsuitable. The biopsy forceps provided a definitive result in 16 of 21 samples and the grasper was definitive in 5 of 6 samples. We could not obtain a suitable specimen using a snare in 2 cases and the brush gave a definitive result in only 5 of 11 cases. Samples of aspirate and washings were definitive in less than 50% of instances but detected some tumors for which other techniques were equivocal. CONCLUSIONS: For the best results, the largest biopsy specimen possible should be obtained. Aspiration or wash alone is often not diagnostic but can give a diagnosis in some patients. Tissue sampling devices, such as the forceps and basket, have an advantage in obtaining a larger sample. Cytopathologic techniques are particularly useful for handling and interpreting the small specimens obtained with ureteroscopic biopsy techniques.

Choroidal melanoma in an African-American albino.

PURPOSE: To report the rare occurrence of choroidal melanoma in an African-American albino. METHODS: A 68-year-old African-American man with oculocutaneous albinism developed an amelanotic choroidal mass in his left eye. A transvitreal fine-needle aspiration biopsy was performed to confirm the diagnosis, and the patient was treated with iodine 125 plaque radiotherapy. RESULTS: The cytology of the needle biopsy showed spindle cells with nuclear atypia and prominent nucleoli. The cells showed positive immunoreactivity for HMB-45, supporting the diagnosis of choroidal melanoma. CONCLUSION: To our knowledge, this is the first reported case of a choroidal melanoma occurring in an African-American albino.

Extramedullary plasmacytoma confined to the choroid.

PURPOSE: To report a case of an extramedullary plasmacytoma confined to the choroid. METHODS: Interventional case report with cytopathologic correlation. RESULTS: A 76-year-old asymptomatic woman presented with 2 globular orange and amelanotic choroidal lesions in the right eye. Fine needle aspiration biopsy of one of the choroidal lesions followed by immunocytochemical studies revealed monoclonal plasma cells, diagnostic of plasmacytoma. Systemic evaluation was negative, except for increased serum gamma globulin with immunoglobulin kappa spike on electrophoresis. The patient was managed by ocular external beam radiotherapy. At 9 years follow-up, she was free of local disease and never developed multiple myeloma. CONCLUSION: Extramedullary plasmacytoma confined to the choroid is rare. It may present simulating other common amelanotic choroidal lesions. Fine needle aspiration biopsy and proper systemic evaluation are critical in establishing the diagnosis.

Effusion cytology.

Cytologic evaluation is the best way to detect the presence of malignancy in body cavity fluids. Although a positive diagnosis is highly reliable, a negative result does not rule out a malignant cause. Adenocarcinomas, well-differentiated squamous carcinomas, small-cell carcinomas, malignant melanomas, large-cell lymphomas, and acute leukemias are accurately classified when present in effusions. The definitive diagnosis of malignant mesothelioma, small-cell lymphomas, and chronic leukemias, and subclassification of sarcomas and poorly differentiated neoplasms are difficult and may require additional diagnostic techniques. With a few exceptions, the exact causes of benign effusions cannot be determined by cytologic methods.

Detection of Chlamydia trachomatis in cervical smears from pregnant population.

Specimens were obtained for chlamydial cultures and for routine cytologic studies from 281 consecutive pregnant women who presented for their first prenatal visit. Forty patients (14%) had positive cultures for Chlamydia trachomatis. The cervical smears from all patients with positive cultures and from an equal number of age-matched patients with negative cultures were screened for cytologic features reported to be associated with Chlamydia infection. The following features were noted to be more common in the patients with positive cultures: squamous metaplasia, cytoplasmic vacuolation, nuclear abnormalities, human papillomavirus-associated changes, and a greater degree of inflammation. Cytoplasmic inclusions were present in only a single cell in each of 4 (10%) of the patients with positive cultures. None of the cytologic findings was considered to be specific enough to allow a diagnosis of Chlamydia infection. In our experience, the evaluation of cervical smears is of limited value in the detection of Chlamydia infection in pregnant women.

Differential distribution of the neuron-associated class III beta-tubulin in neuroendocrine lung tumors.

OBJECTIVE: To study the immunoreactivity profile of the neuron-associated class III beta-tubulin isotype (beta III) in epithelial lung tumors. DESIGN: One hundred four formalin-fixed, paraffin-embedded primary and metastatic lung cancer specimens were immunostained with an anti-beta III mouse monoclonal antibody (TuJ1) and an anti-beta III affinity-purified rabbit antiserum. Paraffin sections from fetal, infantile, and adult nonneoplastic lung tissues were also examined. RESULTS: In the fetal airway epithelium, beta III staining is detected transiently in rare Kulchitsky-like cells from lung tissues corresponding to the pseudoglandular and canalicular but not the saccular or alveolar stages of development. beta III is absent in healthy, hyperplastic, metaplastic, and dysplastic airway epithelium of the adult lung. In contrast, beta III is highly expressed in small cell lung cancer, large cell neuroendocrine carcinoma, and in some non-small cell lung cancers, particularly adenocarcinomas. There is no correlation between expression of beta III and generic neuroendocrine markers, such as chromogranin A and/or synaptophysin, in pulmonary adenocarcinomas. Also, focal beta III staining is present in primary and metastatic adenocarcinomas (to the lung) originating in the colon, prostate, and ovary. beta III is expressed to a much lesser extent in atypical carcinoids and is rarely detectable in typical carcinoids and squamous cell carcinomas of the lung. The distribution of beta III in small cell lung cancer and adenocarcinoma metastases to regional lymph nodes and brain approaches 100% of tumor cells, which is substantially greater than in the primary tumors. CONCLUSIONS: In the context of neuroendocrine lung tumors, beta III immunoreactivity is a molecular signature of high-grade malignant neoplasms (small cell lung cancer and large cell neuroendocrine carcinoma) although its importance in atypical carcinoids must be evaluated further. In addition, beta III may be a useful diagnostic marker in distinguishing between small cell lung cancers and certain non-small cell lung cancers (poorly differentiated squamous cell carcinomas), especially in small biopsy specimens. To our knowledge, beta III is the only tumor biomarker that exhibits a substantially more widespread distribution in poorly differentiated than in better differentiated pulmonary neuroendocrine tumors. However, the significance of beta III phenotypes in non-small cell lung cancer, particularly adenocarcinoma, with respect to neuroendocrine differentiation and prognostic value, requires further evaluation.

The cytologic diagnosis of mesothelioma.

The cytologic features of malignant mesothelioma cells in serous effusions are presented. Carcinomatous mesotheliomas are characterized by abundant neoplastic cells occurring singly and in clusters. The optically dense cytoplasm with lacy peripheral vacuoles, scalloped borders of cell clusters, intercellular spaces, "cell-in-cell" arrangement, and frequent multinucleation of cells are features of malignant mesothelioma, but none is pathognomonic of this tumor. A positive cytoplasmic staining of tumor cells with periodic acid-Schiff (PAS) after diastase digestion, and with mucicarmine stain after hyaluronidase treatment are against the diagnosis of mesothelioma, while positive staining with alcian blue, which becomes negative after the treatment with hyaluronidase is strongly suggestive of mesothelioma. The tumor cells react with antibodies to cytokeratin and vimentin, and do not react with carcinoembryonic antigen. Ultrastructurally, mesothelioma cells are characterized by long slender branching microvilli and numerous pinocytotic vesicles. They lack mucin vacuoles and intracellular lumens. An accurate diagnosis of mesothelioma depends on a full knowledge of the clinical history and radiologic findings, and proper application of histochemical, immunodiagnostic, and electron microscopic techniques.

Intrapericardial instillation of sodium hydroxide: failure to produce pericardial symphysis.

Previously reported clinical studies have suggested sodium hydroxide as an effective agent in causing pleural symphysis. To explore the sclerosing potential of this agent, we instilled 0.5% sodium hydroxide into the pericardium of four dogs. Normal saline was instilled into the pericardium of one control dog. Echocardiograms, 24 hour EKG monitoring and intracardiac pressures were recorded. One dog was sacrificed at the end of 24 hours, another at the end of seven days and three other dogs were sacrificed at the end of 28 days. ST elevations and prolonged episodes of supra-ventricular and ventricular tachycardia were noted during and within 24 hours after the intrapericardial instillation of sodium hydroxide in all the dogs. No arrhythmias were noted in the control dog. None of the dogs showed pericardial symphysis. These results suggest that sodium hydroxide causes serious cardiac arrhythmias when instilled intrapericardially and is of limited value in causing pericardial symphysis.

Utility of polyclonal and monoclonal antibodies against carcinoembryonic antigen in hepatic fine-needle aspirates.

To assess the usefulness of polyclonal and monoclonal antibodies against carcinoembryonic antigen (CEA) in the differential diagnosis of hepatocellular carcinoma (HCC) vs. metastatic adenocarcinoma (MA), we studied 25 cases of fine-needle aspirates (FNA) of hepatic lesions. The material consisted of 9 primary HCCs, 8 MAs, and 8 benign hepatic aspirates. For immunostaining, the avidin-biotin complex technique was performed on paraffin sections of cell blocks, using a standardized automatic immunostainer. Specific bile canalicular immunostaining with polyclonal CEA (pCEA) antibody was present in five of eight (5/8) benign hepatic aspirates and eight of nine (8/9) HCCs. Diffuse cytoplasmic immunostaining with pCEA antibody was present in four of eight (4/8) MAs. None of the aspirates showed any positive immunostaining with monoclonal CEA (mCEA) antibody. We conclude that: (1) pCEA antibody is useful in the evaluation of hepatic FNAs. Diffuse cytoplasmic staining is seen in MAs, whereas canalicular immunostaining pattern is an indication of benign or malignant hepatocytes. (2) Paraffin-embedded cell blocks made from hepatic aspirate material are suitable for immunostaining with polyclonal CEA antibody. (3) mCEA antibody has no value in the diagnosis of HCC.

Liesegang rings in renal cyst fluid.

Peculiar ring-like structures identified as Liesegang rings (LRs) were found in renal cyst fluid from three patients with benign renal cysts. They ranged in size from 5 to 820 mu. Most had a double-layer outer wall with equally spaced radial cross-striations and an amorphous central nidus. Special stains were performed in one case, and the results are discussed. Reports of LRs in cystic or inflamed tissues have recently appeared in the literature. Some LRs have been mistaken for eggs or mature components of the giant kidney worm, Dioctophyma renale. We propose that cytologic assessment of renal cyst fluid in conjunction with histologic examination decreases the likelihood of misdiagnosis of LRs.