Salivary gland FNA cytology: role as a triage tool and an approach to pitfalls in cytomorphology.

OBJECTIVE: To highlight the importance of salivary gland fine needle aspiration (FNA) cytology as a triage tool for surgery and to determine its sensitivity and specificity. To discuss the diagnostic pitfalls and potential role of ancillary techniques in diagnosis and prognosis. METHODS: The study included a total of 920 cases of salivary gland FNAs received in the cytopathology department of University College London Hospital during December 2004 to December 2012. The cases with known histological outcomes were analysed to determine the sensitivity and specificity. RESULTS: Surgery was carried out on 180 (19.6%) of 920 patients. Excluding nine with inadequate/non-diagnostic cytology, the sensitivity of FNA cytology for a malignant outcome was 89% (33/37) and the specificity was 97% (130/134). Diagnostic pitfalls are discussed with respect to eight FNAs with discrepant histology. Histological outcome was not available for 740 cases (80.4%): excluding 88 non-diagnostic FNAs, 324 (49.7%) had non-neoplastic diagnoses (not indicating surgery) and 328 (50.3%) had neoplastic diagnoses, which included recurrences/metastases of known tumours. Patients with other neoplasms on FNA were lost to follow-up and may have had surgery elsewhere. Cases with clinical concerns were discussed at weekly multidisciplinary meetings. CONCLUSION: Salivary gland FNA is crucial in the distinction of non-neoplastic from neoplastic lesions, emphasizing the fact that FNA is an excellent triage tool for surgery. Salivary gland FNA has a high sensitivity and specificity. However, it is important to interpret the cytological diagnoses in the light of clinical findings and imaging. Diagnostic pitfalls are seen in a minority of cases and could potentially be overcome with the help of recently described diagnostic and prognostic markers.

Nasseem Husain: homage to a pioneer of cytology automation.

Dr Oliver Anthony Nasseem Husain, who died on 22 September 2014, aged 90 years, was one of the great names of European cytology, a pioneer of automated cervical screening and a founding member of both the British Society for Clinical Cytology (BSCC) and the European Federation of Cytology Societies (EFCS). The life of this one remarkable man involved much of the pioneering work, which is reviewed in this article, that has brought conventional cytology to the complex multimodal discipline it is today.

The role of the cytopathologist's interpretation in achieving diagnostic adequacy of head and neck fine needle aspirates.

OBJECTIVE: We aimed to assess the potential role of interpretation by cytopathologists on the level of diagnostic adequacy of head and neck fine needle aspirations (FNAs). METHODS: An audit ('first audit') was performed between 1 May 2007 and 30 April 2008 using data from three different hospitals (A, B and C). The specimens were interpreted by two cytopathologists with specific experience in head and neck pathology in hospitals A and B, and by any of the eight cytopathologists (only two of whom were experienced in head and neck cytopathology) in hospital C. Following the analysis of the initial findings, there was a change in practice in hospital C, after which specimens were also read only by two experienced cytopathologists. A new audit ('second audit') was then performed between 20 January 2011 and 20 December 2012 in the same three hospitals. RESULTS: During the first audit, the diagnostic adequacy of FNAs from hospital C was 84.2% compared with 96.6% in hospital A and 97.7% in hospital B (P = 0.000). No significant difference in the diagnostic adequacy rate of the FNAs performed in hospitals A and B was found when comparing the first and second audits. The FNA diagnostic adequacy for hospital C increased significantly in the second relative to the first audit (95.5% versus 84.2%, P = 0.000). CONCLUSIONS: Our study confirms that expert cytology interpretation is important in achieving optimal diagnostic adequacy of head and neck FNAs.

Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates.

OBJECTIVES: To collect data on the variability of immunocytochemical (ICC) procedures used to detect oestrogen/progesterone receptors (ER/PR) on cytological material; to test the reproducibility of results; and to identify the crucial points in the ICC procedures that affect the result. METHODS: Ten laboratories from eight countries participated in a two-part study. In the first part, one of the participants (the coordinator) prepared and distributed cytospins from a fine needle aspirate of a primary breast carcinoma. Laboratories performed ICC staining for ER/PR according to their own methods on the test slides and in-house positive controls. Slides were returned to the coordinator together with information on the preparation of positive control slides and the ICC methodology used. In the second part, obligatory methods of fixation and antigen retrieval were specified. Evaluation of results included grading the number of positive cells, staining intensity, background staining, cytoplasmic staining, sample condition and cellularity. Participants evaluated their own results, which were subsequently evaluated by the coordinator. RESULTS: There was great variability in the preparation of slides for in-house controls and ICC methodology. The outcome of ICC staining of in-house control slides was excellent in two laboratories, adequate in three, sub-optimal in four and inadequate in one. Only six obtained a positive reaction on the test slides and not all were of a high quality. Results of the second run were greatly improved in terms of cellularity of in-house positive control slides, and scores for the percentage of stained cells and staining intensity of control and test slides. Cytospins and monolayer (ThinPrep(®)) preparations were superior to direct smears; methods of fixation and antigen retrieval were the key points in the staining process. CONCLUSIONS: Our experience points to the need for guidelines for hormonal receptor determination and external quality control on cytological material, in order for cytological methods to be used in routine clinical practice with a suitable degree of confidence.

Serous fluid cytology of multicentric Castleman's disease and other lymphoproliferative disorders associated with Kaposi sarcoma-associated herpes virus: a review with case reports.

OBJECTIVE: The aim of this study is to describe and review the cytological features of Kaposi sarcoma-associated herpes virus (KSHV) related entities, such as multicentric Castleman's disease (MCD), plasmablastic-lymphoma (PBL) and primary effusion lymphoma (PEL), which all may present as body cavity effusions. Serous fluid cytology of MCD and PBL has not, to our knowledge, thus far been described. Although different in nature, MCD, PBL and PEL are characterized by similar morphological features. MATERIALS AND METHODS: Body cavity effusions from four different patients with previously known or unknown KSHV-related lymphoproliferations have been examined by routine cytology, immunocytochemistry (IC) and polymerase chain reaction (PCR). RESULTS: MCD, PBL and PEL are all characterized by increased cellularity, comprising mainly lymphoid and plasmacytoid cells with variable proportions of immunoblasts. Immunocytochemistry and PCR results show the MCD to be CD138 and KSHV positive, CD30 negative, IgM, IgH and lambda restricted but IgH polyclonal. PBL was CD138 positive, kappa restricted, weakly positive with VS38 and over 80% positive with MIB 1. PEL was CD45, EMA, CD138, KSHV, p53 and CD3 positive, CD20, EBV, CD30, CD2, CD4, ALK1, epithelial and mesothelial markers negative, and PCR monoclonal B-cell expanded (Ig-kappa bands). CONCLUSION: Cytological examination of effusions in KSHV-related lymphoproliferative disorders may show similar morphological features but clonality studies and immunocytochemistry are very helpful in distinguishing between these rare benign and malignant lymphoproliferative diseases.

Diagnostic terminology for reporting thyroid fine needle aspiration cytology: European Federation of Cytology Societies thyroid working party symposium, Lisbon 2009.

A European Federation of Cytology Societies (EFCS) working party of 28 members from 14 European countries met at the European Congress of Cytology in Lisbon in September 2009, with two observers from the USA, to discuss the need for standardising thyroid FNA nomenclature in the light of the National Institute of Cancer (NCI) recommendations resulting from the State of the Science conference in Bethesda in 2007. The data were obtained through two questionnaires sent by email and a transcript of the live discussion at the congress, which is presented in full. The surveys and discussion showed that there were currently no national terminologies for reporting thyroid FNA in the different European countries except in Italy and the UK. Personal, 'local', surgical pathology and descriptive terminologies were in use. All but one of the working party members agreed that thyroid FNA reporting should be standardised. Whilst almost a third would adopt the NCI Bethesda terminology, which offers the advantages of a 'risk of cancer' correlation and is linked to clinical recommendations, more than half favoured a translation of local terminology as the first step towards a unified nomenclature, as has been done recently in the UK. There was some disagreement about the use of: a) the six-tiered as opposed to four or five-tiered systems, b) the use of an indeterminate category and c) the 'follicular neoplasm' category, which was felt by some participants not to be different from the 'suspicious of malignancy' category. The conclusions will be passed to the different national societies of cytology for discussion, who will be asked to map their local terminologies to the Bethesda classification, observe its acceptance by clinicians and audit its correlation with outcome.

Head and neck cancer in the UK: what is expected of cytopathology?

OBJECTIVE: This review highlights the role of cytopathology in cancer management within UK Head and Neck Cancer Networks and informs on the issues raised by recent UK Department of Health documents and other UK professional guidance. UK guidance requires the formal involvement of cytopathologists within multidisciplinary cancer teams, with medical and non-medical cytopathology staff setting up and running rapid access lump clinics, and support for image-guided fine needle aspiration cytology (FNAC) services. UK guidance also makes recommendations for training, resources and quality control. This review also highlights the resource gap between best practice evidence-based guidance for head and neck (HN) cancer services and existing UK provision for cytopathology, as evidenced by lack of availability of experienced staff and adequacy of training and quality control (QC). Finally, it stresses the importance in the UK of the Royal College of Pathologists' guidance, which defines the need for training, the experience needed for new consultants, the requirements for audit and QC. The implications for the additional resources required for HN cancer cytopathology services are discussed. Recent professional guidance specifying the provision of HN cancer services in the UK includes a cytopathology service for cancer networks, such as rapid access FNAC clinics. Although these clinics already operate in some institutions, there are many institutions where they do not and where the provision of cytopathology services would have to be restructured. This would need the support of local cancer networks and their acceptance of the detailed requirements for cytopathology, including resources, training and QC. The standards are not defined locally, as Strategic Health Authorities and Primary Care Trusts have been instructed by the Department of Health to support, invest and implement them.

BSCC Code of Practice--fine needle aspiration cytology.

The British Society for Clinical Cytology Code of Practice on fine needle aspiration cytology complements that on exfoliative cytopathology, which was published in the last issue (Cytopathology 2009;20:211-23). Both have been prepared with wide consultation within and outside the BSCC and have been endorsed by the Royal College of Pathologists. A separate code of practice for gynaecological cytopathology is in preparation. Fine needle aspiration (FNA) cytology is an accepted first line investigation for mass lesions, which may be targeted by palpation or a variety of imaging methods. Although FNA cytology has been shown to be a cost-effective, reliable technique its accurate interpretation depends on obtaining adequately cellular samples prepared to a high standard. Its accuracy and cost-effectiveness can be seriously compromised by inadequate samples. Although cytopathologists, radiologists, nurses or clinicians may take FNAs, they must be adequately trained, experienced and subject to regular audit. The best results are obtained when a pathologist or an experienced and trained biomedical scientist (cytotechnologist) provides immediate on-site assessment of sample adequacy whether or not the FNA requires image-guidance. This COP provides evidence-based recommendations for setting up FNA services, managing the patients, taking the samples, preparing the slides, collecting material for ancillary tests, providing rapid on-site assessment, classifying the diagnosis and providing a final report. Costs, cost-effectiveness and rare complications are taken into account as well as the time and resources required for quality control, audit and correlation of cytology with histology and outcome. Laboratories are expected to have an effective quality management system conforming to the requirements of a recognised accreditation scheme such as Clinical Pathology Accreditation (UK) Ltd.

Development and evaluation of a real-time PCR assay for detection of Pneumocystis jirovecii DNA in bronchoalveolar lavage fluid of HIV-infected patients.

BACKGROUND: Pneumocystis pneumonia (PCP) is conventionally diagnosed by identifying Pneumocystis jirovecii in lower respiratory tract samples using cytochemical stains. Molecular diagnosis of PCP is potentially more sensitive. METHODS: A study was undertaken to use an extensively optimised real-time polymerase chain reaction (PCR) using primers designed to hybridise with the P. jirovecii heat shock protein 70 (HSP70) gene to quantify P. jirovecii DNA in bronchoalveolar lavage (BAL) fluid from HIV-infected patients with and without PCP, and to compare this assay with conventional PCR targeting the P. jirovecii mitochondrial large subunit rRNA gene sequence (mt LSU rRNA). RESULTS: Sixty-one patients had 62 episodes of PCP (defined by detection of P. jirovecii in BAL fluid by cytochemical stains and typical clinical presentation). Quantifiable HSP70 DNA was detected in 61/62 (range approximately 13-18,608 copies/reaction; median approximately 332) and was detectable but below the limit of quantification (approximately 5 copies/reaction) in 1/62. Seventy-one other patients had 74 episodes with alternative diagnoses. Quantifiable HSP70 DNA was detectable in 6/74 (8%) episodes (range approximately 6-590 copies/reaction; median approximately 14) and detectable but below the limit of quantification in 34/74 (46%). Receiver-operator curve analysis (cut-off >10 copies/reaction) showed a clinical sensitivity of 98% (95% 91% to 100%) and specificity of 96% (95% CI 87% to 99%) for diagnosis of PCP. By contrast, clinical sensitivity of mt LSU rRNA PCR was 97% (95% CI 89% to 99%) and specificity was 68% (95% CI 56% to 78%). CONCLUSION: The HSP70 real-time PCR assay detects P. jirovecii DNA in BAL fluid and may have a diagnostic application. Quantification of P. jirovecii DNA by real-time PCR may also discriminate between colonisation with P. jirovecii and infection.

Diagnostic yield of fine-needle aspiration cytology in HIV-infected patients with lymphadenopathy in the era of highly active antiretroviral therapy.

Fine-needle aspiration (FNA) cytology has an established role in the investigation of lymphadenopathy in HIV-infected patients. However, changes in the spectrum of disease have been observed since the introduction of highly active antiretroviral therapy (HAART). The aim of the study was to establish whether FNA cytology remains a useful investigative tool in the post-HAART era and to determine whether the cytology results reflect the changing patterns of disease. Retrospective search of the cytopathology database at University College London Hospitals identified 73 FNA cytology procedures performed in 62 patients between January 1998 and December 2006. FNA cytology showed significant disease in 90% of adequate samples. The most common diagnoses were persistent generalized lymphadenopathy (PGL, 50%), infection (22%) and malignancy (18%). Diagnoses could not be made in 31% of patients because of inadequate sampling. An open lymph node biopsy was subsequently performed in 27% of patients. FNA cytology remains an important initial investigation in the post-HAART era, particularly in the diagnosis of PGL, infection and malignancy. Difficulties in diagnosis of Castleman disease and Hodgkin's lymphoma by FNA cytology are recognized.

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.

EUS-guided tissue sampling: comparison of "dual sampling" (Trucut biopsy plus FNA) with "sequential sampling" (Trucut biopsy and then FNA as required).

BACKGROUND AND STUDY AIMS: Both endoscopic ultrasound- (EUS-) guided tissue sampling techniques, fine-needle aspiration (FNA) and Trucut biopsy, have advantages and limitations. The aim of this study was to develop a strategy of combining these two EUS-guided sampling techniques in order to maximize the diagnostic accuracy and minimize duplication. PATIENTS AND METHODS: In this multicenter study we performed "dual sampling" (i. e. with both FNA and Trucut biopsy) in 95 patients during phase 1 of the study and "sequential sampling" (i. e. performing FNA only when Trucut biopsy tissue cores were macroscopically inadequate) in 72 patients during phase 2. RESULTS: During the study period, 167/401 patients referred for EUS-guided sampling were eligible for the study; only solid lesions were included. In 143/167 patients (86 %), sampling was performed via the transesophageal or transgastric routes. When the dual sampling strategy was used, an accurate diagnosis was achieved in 78/95 patients by FNA, compared with 85/95 by Trucut biopsy ( P = 0.21). The combined accuracy of the dual sampling strategy was higher than FNA alone (88/95 vs. 78/95, P = 0.048), but was not significantly higher than Trucut biopsy alone (88/95 vs. 85/95, P = 0.61). Using the sequential sampling strategy, an accurate diagnosis was achieved in 66/72 patients (92 %) compared with 88/95 (93 %) for dual sampling ( P = 1.0), and 8/72 patients (11 %) had to undergo FNA after Trucut biopsy failed to obtain an adequate sample. One patient with mediastinal tuberculosis developed a cold abscess following Trucut biopsy. CONCLUSION: A sequential sampling strategy, in which EUS-guided Trucut biopsy is attempted first, and FNA performed only when Trucut biopsy fails to obtain a macroscopically adequate sample, achieves a diagnostic accuracy of 92 %, with 11 % of patients requiring both sampling procedures.

Best Practice No 185. Cytological and molecular diagnosis of lymphoma.

With the advances in molecular pathology, the cell as a morphological and functional unit has become essential in the diagnosis of lymphoma. Conventional staining, preparation, and interpretation of cells, as seen in fine needle aspiration cytology (FNAC), often used as a first line investigation of lymphadenopathy, is being supplemented with an array of immunocytochemical and molecular analyses, aimed not only at a more precise disease definition, but also at recognising factors that can predict prognosis and response to treatment. Accepting the pitfalls of conventional cytomorphology, this review looks at molecular changes characteristic to particular lymphomas and explores the currently available technology for their detection, with particular reference to cytological material. Future protocols for the diagnosis and management of patients with lymphadenopathy should include FNAC as an initial investigation, followed by immunocytochemistry and molecular investigations. Tissue biopsy, the conventional method of diagnosis, may be avoided in selected cases.

Percutaneous fine needle aspiration cytology of the pancreas: advantages and pitfalls.

Fine needle aspiration of the pancreas was performed in 62 patients with radiological suspicion of malignancy. All fine needle aspirates were taken under computed tomography or ultrasound guidance. Fine needle aspirates were positive in 31 of 41 patients with histologically or clinically confirmed pancreatic carcinoma. There were no false positive results. The sensitivity of this method for detecting malignant disease was 86%. Cytology was not able to provide conclusive results of benign conditions. Difficulties were encountered in diagnosing well differentiated carcinoma and neuroendocrine tumours and distinguishing them from reactive epithelium and islet cell hyperplasia, respectively. This resulted in a 12.1% false negative rate. There were no complications in our series. Percutaneous fine needle aspiration proved a reliable method of diagnosing pancreatic carcinoma.

Contribution of fine needle aspiration cytology to diagnosis and management of thyroid disease.

AIMS: To determine the role of fine needle aspiration cytology (FNAC) in the diagnosis and management of thyroid disease. METHODS: Clinical histories of 144 patients who had undergone FNAC of the thyroid were analysed. Clinical presentation, non-invasive investigations including hormone assays, ultrasound, and isotope scan procedures were compared with FNAC diagnoses in all cases and with histological diagnosis in the 28 cases (19%) that had undergone surgery. Clinical management was decided upon combining all of the above investigations. The relative contribution of the FNAC was divided into: essential, additional and non-contributory, misleading. RESULTS: FNAC diagnoses included: 29 (16%) benign colloid goitre, 56 (39%) benign cystic goitre, 24 (17%) thyroiditis, and 22 (15%) neoplasms. Nineteen (13%) of the specimens were unsatisfactory. When compared with clinical diagnoses based on non-invasive diagnostic investigations FNAC represented no improvement on the diagnosis of benign colloid/cystic goitre (55% v 54% respectively). It represented an improvement on the diagnosis of thyroiditis (9% v 17% respectively). FNAC decreased clinically suspicious lesions in which 22 neoplasms were diagnosed from 37% to 15%. Eleven patients with neoplasms underwent surgery and neoplasms were confirmed histologically. Others including lymphoma, metastatic carcinoma, and analplastic carcinoma were managed conservatively. There were four false negative FNAC diagnoses (3%) in clinically suspicious lesions, found on histology to be benign follicular adenomas. CONCLUSIONS: FNAC had an essential role in the diagnosis and management of 23% of our patients, a confirmatory role in 61% of patients, a non-contributory role in 13% when specimens were inadequate, and was misleading in 3% where results were false negative. The positive identification of thyroiditis and neoplasia stands on its own as a justification for FNAC.

AUA1: new immunocytochemical marker for detecting epithelial cells in body cavity fluids.

Further to detailed genetic and biochemical characterisation of AUA1 as a surface glycoprotein present on epithelial cells, the antibody against AUA1 was used as an immunocytochemical marker of epithelial cells in body cavity fluids in an attempt to improve the diagnosis made on routine staining. AUA1 was initially tested in 144 morphologically clear cut effusions. It was positive in 46 of 52 (88%) carcinomas and negative in 82 of 84 (98%) benign effusions, including technically inadequate or poorly cellular preparations. There were no false positive results. AUA1 was subsequently used more selectively--that is, in 42 of 175 (24%) of morphologically difficult fluids. AUA1 provided essential diagnostic information in 15 of 42 (36%) and confirmed diagnosis in 17 of 42 (40%), thus enabling accurate diagnosis in a further 32 of 42 (76%) of the difficult cases. The total diagnostic accuracy was therefore 94.3%. AUA1 is a reliable immunocytochemical marker for detecting epithelial cells in body fluids. Its use improves diagnostic accuracy of morphological assessment in difficult cases.