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.

THE SIGNIFICANCE OF EPIDERMAL GROWTH FACTOR RECEPTOR AND SURVIVIN EXPRESSION IN BLADDER CANCER TISSUE AND URINE CYTOLOGY OF PATIENTS WITH TRANSITIONAL CELL CARCINOMA OF THE URINARY BLADDER.

OBJECTIVE: To assess whether epidermal growth factor receptor (EGFR) and survivin immunostaining of tumour cells in urinary cytology and tissue of patients with bladder cancer has a prognostic significance. DESIGN: Prospective study SETTING: Department of Surgery (Division of Urology), Mubarak Al-Kabeer Teaching Hospital and Faculty of Medicine, Kuwait University, Kuwait SUBJECTS: Urine cytology smears obtainedpriorto cystoscopy in patients with transitional cell carcinoma (TCC) of the bladder were immunostained for EGFR and survivin. Bladder cancer tissue resected at surgery was also immunostained for EGFR and survivin expression. Tissue expression of EGFR and survivin in TCC of the bladder was compared to their expression in urine cytology and relationship to tumour grade and stage. RESULTS: 178 patients were studied (43 newly diagnosed bladder cancer, 58 with recurrent TCC and 77 in disease remission). Twenty five patients with normal urothelium served as controls. The mean sensitivity of urine cytology, tissue survivin immunohistochemistry (IHC) and tissue EGFR IHC was 30.5%, 62% and 59% respectively. The corresponding mean specificity was 95%, 79% and 38% respectively. For grades 1, 2 and 3 bladder tumors, tissue expression positivity for EGFR was 47.8%, 92.9%, 100% and for tissue survivin it was 27.8%, 18.2% and 33.3% respectively. For grades 1, 2 and 3 bladder tumors, urine expression positivity for EGFR was 35.7%, 40% and 67.7% and for urine survivin it was 8.3%, 42.9% and 33.3% respectively. CONCLUSION: Positive EGFR immunostaining of urine cytology specimen or tumour tissue increases with histological grade of TCC of the bladder. Survivin expression is less consistent in both urine cytology specimen and tissue samples. EGFR immunostaining may provide a useful tool in the grading of bladder TCC and aid in the selection of patients that may benefit from administration of EGFR inhibitors.

Survey of medical training in cytopathology carried out by the journal Cytopathology.

This report of the Editorial Advisory Board of Cytopathology gives the results of a survey of medical practitioners in cytopathology, which aimed to find out their views on the current situation in undergraduate and postgraduate training in their institutions and countries. The results show that training in cytopathology and histopathology are largely carried out at postgraduate level and tend to be organized nationally rather than locally. Histopathology was regarded as essential for training in cytopathology by 89.5% of respondents and was mandatory according to 83.1%. Mandatory cytopathology sections of histopathology were reported by 67.3% and specific examinations in cytopathology by 55.4%. The main deficiencies in training were due to its variability; there were insufficient numbers of pathologists interested in cytology and a consequent lack of training to a high level of competence. Pathologists without specific training in cytopathology signed out cytology reports according to 54.7% of responses, more often in centres where training was 3-6 months or less duration. Although 92.2% of respondents thought that specialist cytology should not be reported by pathologists without experience in general cytopathology, that practice was reported by 30.9%, more often in centres with small workloads. The survey report recommends that 6-12 months should be dedicated to cytopathology during histopathology training, with optional additional training for those wanting to carry out independent practice in cytopathology. Formal accreditation should be mandatory for independent practice in cytopathology. When necessary, temporary placements to centres of good practice should be available for trainees intending to practise independently in cytopathology. There should be adequate numbers of pathologists trained in cytopathology to a high level of competence; some of their time could be released by training cytotechnologists and trainee pathologists to prescreen cytology slides and assess adequacy of fine-needle aspiration samples when immediate diagnosis was not required. The survey demonstrated a clear need for European and international guidelines for training in cytopathology.

WHONET Software for the Surveillance of Antimicrobial Susceptibility.

Continuous surveillance of local antimicrobial susceptibility patterns is a must for combating emerging antimicrobial resistance. WHONET is an effective computerized microbiology laboratory data management and analysis program that can provide guidance for empiric therapy of infections, alert clinicians of trends of antimicrobial resistance, guide drug-policy decisions and preventive measures. The program facilitates sharing of data amongst different hospitals by putting each laboratory data into a common code and file format, which can be merged for national or global collaboration of antimicrobial resistance surveillance. The system can be implemented in hospital laboratories of Armed Forces at no additional cost. Cumulative analysis of surveillance data obtained from various hospitals of Armed Forces at higher centers may help in formulating health policies and control measures at various levels.

Could nuclear matrix protein 22 (NMP22) play a role with urine cytology in screening for bladder cancer?--experience at Kuwait University.

OBJECTIVES: This prospective study was undertaken to evaluate nuclear matrix protein (NMP22) compared to urine cytology in the detection of bladder cancer and also to determine whether indexing suspicious cytology to NMP22 could enhance the clinical utility of cytology. METHODS: Cytological findings of voided urine collected prior to a cystoscopic biopsy were correlated with urine NMP22 assay in 46 patients attending the urology clinic in Mubarak Al-Kabeer Hospital. The patients were clinically categorized into newly diagnosed cases of transitional cell carcinoma (TCC), recurrent TCC, TCC in remission and controls. RESULTS: Using histological diagnosis as the gold standard the sensitivity and specificity of NMP22 were 78% and 43% respectively and of cases with malignant urine cytology were 30% and 87% respectively. If suspicious and malignant cytology were combined as positive results the sensitivity increased significantly to 87% while the specificity decreased but not significantly to 74%. Suspicious or malignant cytology enhanced by positive NMP22 gave a sensitivity of 70% and specificity of 87% neither of which was significantly different from cytology alone. There were three false positive cases on cytology and 13 false positive cases on NMP22 assay. There were three false negative cytology and five false negative NMP22 cases but only one was false negative for both, resulting in a high sensitivity (96%) but low specificity (30%) if either positive NMP22 or malignant or suspicious cytology was taken as a positive result. CONCLUSION: Combining NMP22 with malignant or suspicious cytological result improved sensitivity for the detection of bladder cancer but with a major decrease in specificity, suggesting a potential role in screening rather than diagnosis.

The role of breast FNAC in diagnosis and clinical management: a survey of current practice.

Most participating countries have now adopted a triple assessment approach, i.e. clinical,imaging and pathology, to breast diagnosis, with FNAC as the first-line pathological investigation in both screening and symptomatic populations, with the exception of microcalcifications. Pathologists specialized in cytopathology are best qualified to collect and interpret FNAC samples, but this is not always possible or practical. Radiologists involved in breast imaging should ensure that they have the necessary skills to carry out FNAC under all forms of image guidance. Best results are achieved by a combination of both techniques, as shown in the image-guided FNAC in the presence of the cytopathologist. The majority of European countries use similar reporting systems for breast FNAC (C1-C5), in keeping with European Guidelines for Quality Assurance in Breast Cancer Screening and Diagnosis, although some still prefer descriptive reporting only. When triple assessment is concordant, final treatment may proceed on the basis of FNAC, without a tissue biopsy. ER and PR assessment can be done safely on FNAC material. However, not all institutions may have expertise in doing this. HER-2 protein expression on direct cytological preparations is insufficiently reliable for clinical use, although its use for FISH is possible, if expertise is available. The majority of participants practise a degree of one-stop diagnosis with a cytopathologist present in the out-patient clinic. Formal recognition of the importance of the time spent outside the laboratory, both for cytopathologist and cytotechnologist, is necessary in order to ensure appropriate resourcing. The use of core biopsy (CB) has increased, although not always for evidence-based reasons. CB and FNAC are not mutually exclusive. FNAC should be used in diagnosis of benign, symptomatic lesions and CB in microcalcifications, suspicious FNAC findings and malignancies where radiology cannot guarantee stromal invasion.

Divergent strains of human immunodeficiency virus type 1 circulating in India, subtyped by heteroduplex mobility assay.

Genomic variations in HIV-1 represent a major problem in understanding disease progression, studying drug resistance and developing effective vaccines. Heteroduplex Mobility Assay (HMA) was used for analyzing HIV-1 subtypes resulting from genetic similarity or divergence of C2 -V3 -V5 region of envelope gene between HIV-1 strains obtained from clinical samples in a tertiary care center at Pune. DNA from the PBMCs of infected individuals was amplified by nested PCR. Heteroduplexes were then formed by denaturing DNA from the unknowns with DNA from the reference strains. The results were analyzed by polyacrylamide gel electrophoresis. Out of 177 samples analyzed, 170 were of subtype C (96%). Four samples were found to be of subtype B (2.2%); in three samples, no definitive assignment of subtype was possible by HMA and these perhaps could be circulating recombinant forms (CRFs) of HIV-1. These findings may have significant implications toward development of a candidate vaccine for India.

Prevalence of extended-spectrum beta-lactamases among Escherichia coli and Klebsiella pneumoniae isolates in a tertiary care hospital.

Extended-spectrum beta-lactamases (ESBLs) continue to be a major problem in clinical setups the world over, conferring resistance to the expanded-spectrum cephalosporins. Knowledge about their prevalence is essential to guide towards appropriate antibiotic treatment. The aim of the present study is to determine the prevalence of ESBL producers among Escherichia coli and Klebsiella pneumoniae isolates at a tertiary care institution. A total of 357 clinical isolates comprising E. coli (n = 181) and K. pneumoniae (n = 176) were recovered from various clinical samples over a period of six months from April to September 2006. Antibiogram profile of these isolates was determined to commonly used antibiotics, along with screening for ESBL production by the screening test as recommended by the Clinical Laboratory Standards Institute (CLSI). Isolates which showed positive results with screening test were shortlisted for confirmatory tests of ESBL production. Two tests were performed: phenotypic confirmatory test with combination disk and the minimum inhibitory concentration (MIC) reduction test. Out of 357 isolates of E. coli and K. pneumoniae screened for ESBL production, 120 were found to be potential ESBL producers. Of these, 80 isolates were confirmed to be ESBL producers. Thus the prevalence of ESBL-producing isolates of E. coli and K. pneumoniae was found to be 22% (80 out of 357). This was significantly lower than the data available from other hospitals.

Morpho-Molecular Characterization of Two Ampelomyces spp. (Pleosporales) Strains Mycoparasites of Powdery Mildew of Hevea brasiliensis.

Powdery mildew disease of rubber affects immature green leaves, buds, inflorescences, and other immature tissues of rubber trees, resulting in up to 45% losses in rubber latex yield worldwide. The disease is often controlled by dusting the diseased plants with powdered sulfur, which can have long-term negative effects on the environment. Therefore, it is necessary to search for alternative and environmentally friendly control methods for this disease. This study aimed to identify mycoparasites associated with rubber powdery mildew species, and characterize them on the basis of morpho-molecular characteristics and phylogenetic analyses of ITS rDNA regions. We observed that the Ampelomyces fungus parasitizes rubber powdery mildew, and eventually destroys it. Furthermore, on the basis of phylogenetic analyses and morphological characteristics we confirmed that the Ampelomyces mycoparasite isolated from rubber powdery mildew is closely related to other mycohost taxa in the Erysiphe genus. A total of 73 (71 retrieved from GenBank and two obtained from fresh collections of rubber powdery mildew fungi) Ampelomyces spp. were analyzed using ITS rDNA sequences and 153 polymorphic sites were identified through haplotypic analyses. A total of 28 haplotypes (H1-H28) were identified to have a complex network of mutation events. The results from phylogenetic tree constructed on the basis of maximum likelihood analyses, and the haplotype network tree revealed similar relationships of clustering pattern. This work presents the first report on morpho-molecular characterization of Ampelomyces species that are mycoparasites of powdery mildew of Hevea brasiliensis.

Hepatitis B e Antigen Negative Chronic Hepatitis in Indian Patients : A Reality.

BACKGROUND: Hepatitis B e antigen negative chronic hepatitis (e(-) CHB) with detectable levels of hepatitis B virus DNA (HBV DNA) in serum has been reported in cases from Asia. This study was undertaken to find out prevalence e(-)CHB and to correlate its presence with the clinical status and severity of the illness in cases of chronic liver disease in India. METHODS: All patients of infective hepatitis, who were hepatitis B surface antigen (HBsAg) positive by enzyme-linked immunosorbent assay (ELISA), were evaluated with liver function tests and HBeAg and antiHBe antibody studies. Polymerase chain reaction (PCR) test was carried out to detect HBV DNA qualitatively. RESULT: Out of 2064 samples tested by ELISA, 429 (20.78 %) were HBsAg positive. HBV DNA (qualitative) was performed on all 429 patients and 74 (17.2%) were HBV DNA positive. Of these only 42 (56.75 %) tested positive for HBeAg. Overall, 8.3 % of HBeAg negative patients (32/384) were viraemic with evidence of chronic liver disease/clinical cirrhosis and alteration of transaminase levels, while three cases (0.84 %) HBeAg positive cases did not show presence of HBV DNA. CONCLUSION: This study shows e(-)CHB prevalence rate of 8.3% in patients with HBV infection in India. Since HBeAg negative patients had detectable levels of HBV DNA as seen in HBeAg positive patients, benefit of antiviral therapy should be given to them. Population studies on e(-) CHB cases are needed to determine its true prevalence, natural course and response to therapy.

A comparative study of telomerase activity in sputum, bronchial washing and biopsy specimens of lung cancer.

The potential of telomerase, the ribonucleoprotein enzyme, as a non-invasive screening marker was studied in pre-bronchoscopy sputum (S), bronchial washings (W) and bronchoscopic biopsy (B) samples from individuals under evaluation for lung cancer. Out of the 52 cases studied, 42 were clinically suspected primary lung cancer patients and 10 had pulmonary disorders but had no clinical evidence of lung cancer. Fifteen (39.5%) S samples, 24 (63.1%) W samples and 32 (84.2%) B samples, which were cytologically/histopathologically positive were also positive for telomerase activity. Interestingly, 16 (42%) S samples, 20 (52.6%) W samples and 20 (52.6%) B samples, initially reported cytologically/histopathologically negative, showed detectable telomerase activity. Lung cancer was finally confirmed in these cases by repeat cytology/histopathology. However, telomerase activity was detected in 31 (81.6%) S, 26 (68.4%) W and 33 (86.8%) B samples of suspected lung cancer patients. Telomerase activity was negative in S, W, and B of four of the suspected cases, which ultimately turned out to be negative for lung cancer. Cytopathology/histopathology alone (including repeat attempts) identified 15 (39.5%) cases of sputum, 24 (63.1%) cases of bronchial washings and 32 (84.2%) bronchoscopic biopsy samples. Out of 10 controls, low telomerase activity was detected in only one (10%) of the bronchial washings, which later turned out to be due to large number of inflammatory cells. Telomerase activity assay of sputum carried sensitivity, specificity and diagnostic accuracy of 81.6, 100 and 86.5%, respectively, while that for bronchial washing was 68.4, 100 and 76.9%, respectively, and for bronchoscopic biopsy samples was 86.8, 100 and 88.1%, respectively. A positive correlation (P<0.01) was seen between age and telomerase activity in sputum, bronchial washing and biopsy samples but no significant correlation was seen between sex and telomerase activity or duration of smoking and telomerase activity. A significant positive correlation was observed between staging and telomerase activity in sputum (P<0.01), bronchial washing (P<0.01) and biopsy samples (P<0.01). Our findings indicate that telomerase is a specific marker for malignant lung disease and can complement cytology/histopathology in the diagnosis of lung cancer. Sputum telomerase assay holds the potential for early and non-invasive diagnosis of lung cancer.

Comparison of in house polymerase chain reaction with conventional techniques for the detection of Mycobacterium tuberculosis DNA in granulomatous lymphadenopathy.

AIMS: To evaluate the usefulness of the devR based polymerase chain reaction (PCR) in the detection of Mycobacterium tuberculosis in lymph node aspirates and tissues of lymphadenitis and to compare PCR with conventional diagnostic techniques. SUBJECTS AND METHODS: Coded specimens of fine needle aspirates and biopsies from 22 patients with tuberculous lymphadenitis, 14 patients with non-tubercular lymphadenitis, and nine patients with granulomatous lymphadenitis were processed and subjected to analysis by PCR, smear microscopy, M tuberculosis culture, histology, and cytology. RESULTS: Tuberculous lymphadenitis was correctly diagnosed by PCR in 18 patients, by culture in five patients, by histology in 13 patients, and by cytology in seven patients. PCR gave two false positive results in 14 patients with non-tubercular lymphadenitis. The sensitivity of the conventional techniques was significantly higher with biopsies (17 of 22 specimens; 77%) than with fine needle aspirates (nine of 22 specimens; 41%). However, the sensitivity of PCR was not significantly higher with biopsies (68%) in comparison with fine needle aspirates (55%). The sensitivity of either biopsy PCR or fine needle aspirate PCR was not significantly different from that of either histology combined with culture or cytology combined with culture. The overall combined specificity of PCR was 86%. Mycobacterium tuberculosis DNA was detected in six of nine patients with granulomatous lymphadenitis. CONCLUSION: PCR is the most sensitive single technique available to date for the demonstration of M tuberculosis in specimens derived from patients with a clinical suspicion of tuberculous lymphadenitis. The value of PCR lies in its use as an adjunct test in the diagnosis of tuberculous lymphadenitis, particularly in those patients where conventional methods fail. Because fine needle aspiration is not an invasive procedure, it is the procedure of choice, and PCR should be performed initially on these samples. Excisional biopsy histology and PCR should be recommended only for patients in whom fine needle aspirate PCR is negative or when there is discrepancy with the clinical impression.

Asymptomatic visceral leishmaniasis in a child with acute lymphoblastic leukaemia.

Visceral leishmaniasis was detected incidentally in a patient with acute lymphoblastic leukaemia in remission, during maintenance therapy. Absence of fever, a normal haemogram, normal serum globulins, a negative serology and testicular involvement were the hallmarks of the case. Treatment with sodium stibogluconate (20 mg/kg for 55 days) failed. Subsequent therapy with pentamidine resulted in complete parasite clearance. Prolonged therapy with pentavalent antimony compounds or a higher dose of second line drugs such as pentamidine are recommended for complete clearance.

The role of fine needle aspiration cytology of breast lumps in the management of patients.

The accuracy of diagnosis with fine needle aspiration cytology (FNAC) was assessed in 3230 patients with breast lumps between 1978 and 1986. The results for sensitivity, specificity, positive predictive value and negative predictive value were 97.6, 99.4, 99.5 and 97.2 per cent respectively; the overall accuracy was 98.4 per cent. Results reveal that a positive cytologic report by an experienced cytopathologist need not wait for histologic confirmation and the patient can be given definitive treatment based on the cytology report.

Diagnostic accuracy of immediate interpretation of fine needle aspirates.

A total of 197 fine needle aspirates (FNA) from various body sites was subjected to immediate interpretation to determine its diagnostic accuracy. For the entire series, the immediate interpretation revealed a sensitivity of 97 per cent, specificity of 96 per cent, a positive predictive value (PV) of 92 per cent, a negative predictive value of 98 per cent and an efficiency of the test of 96 per cent. The false positive rate was 1.5 per cent and false negative rate 0.5 per cent. The diagnostic accuracy of immediate interpretation was comparable for aspirates from different sites. Our results indicate that immediate interpretation of FNA is ideal for determining the adequacy of material and providing a rapid basic diagnosis. This technique could offer useful substitute to frozen sections particularly when facilities for the latter are not available.

'Forgotten' intrauterine device leading to actinomycotic pyometra--13 years after menopause.

A case of a 'forgotten' intrauterine device (IUD) in a postmenopausal woman leading to Actinomyces israelii infection and pyometra is described. The patient was asymptomatic for 22 years after IUD insertion and developed the complications 13 years after menopause. To prevent such serious complications, it is important to ensure that intrauterine devices, once they have served the purpose and even if they are asymptomatic, are removed.

Aspiration cytology diagnosis of echinococcosis.

We report five cases of echinococcosis diagnosed on material obtained by fine-needle aspiration. The sites were the liver in three cases, the thigh in one case, and the retroperitoneum in one case. No complications were observed.

Medullary carcinoma of the thyroid: a cytological, immunocytochemical, and ultrastructural study.

The cytomorphological features of 38 cases of medullary carcinoma of the thyroid on FNAC were studied. These cases were retrieved from the files of the cytopathology laboratory. In addition, staining for calcitonin (19 cases), thyroglobulin (5 cases), neurone specific enolase (7 cases), and carcinoembryonic antigen (8 cases) was done. Electron microscopy was done in two cases. On morphology, the presence of amyloid together with cytoplasmic granules was found to be diagnostic. The plasmacytoid cell type and the mixed cell populations were most commonly observed. Pseudononuclear inclusions were seen in 58% of cases. Calcitonin was present in 74% of cases. NSE was noncontributory, whereas CEA was positive in one of the calcitonin negative cases.