Proffered papers14.00–15.00 Tuesday 16 September 2003

Background  ERCP-directed brush cytology is used to sample lesions of the pancreatic and biliary ducts and the ampulla of Vater. With conventional preparations, the sensitivity and specificity range from 44% to 63% and 80% to 98%, respectively, and increased N : C ratio, nuclear molding and loss of honeycombing are reliable features of malignancy. The performance and morphology of specimens prepared by ThinPrep, a liquid-based cytology technique is mostly unknown.
Methods  The laboratory information system was searched for all cases prepared by ThinPrep. Patient disease classification of benign or malignant was determined by linkage with the provincial cancer registry and was the gold standard against which sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. True positives and negatives were reviewed to identify predictive cytomorphologic features.
Results  Between 1996 and 2001, there were 149 ThinPrep specimens; 55 (37%) were reported as positive for malignancy and 94 (63%) as negative. Disease was classified as malignant in 86 (58%) patients and benign in 63 (42%). There were 42 false negative, 11 false positive, 52 true negative, and 44 true positive cytology results. Sensitivity was 51.2% (CI; 40.2 : 62.0), specificity 82.5% (CI; 70.5 : 90.6), and PPV and NPV 80.0% (CI; 66.6 : 89) and 55.3% (CI; 44.7 : 65.5), respectively. Cell groups with crowded, enlarged, irregular nuclei and nuclear features of vesicular chromatin and large, multiple irregular nucleoli correlated with malignant disease, while monolayered sheets of uniform columnar cells, regular nuclei and a finely granular chromatin correlated with benign disease.
Conclusions  The performance of ThinPrep brushings from this anatomic site equals conventional preparations. Cytomorphologic features of malignancy are more frequent and pronounced with ThinPrep.     

Proffered papers 14.00–15.00 Tuesday 16 September 2003 8 Ampullary,pancreatic and biliary duct exfoliative brush cytology prepared by the thinprep method: performance and morphology

Background ERCP‐directed brush cytology is used to sample lesions of the pancreatic and biliary ducts and the ampulla of Vater. With conventional preparations, the sensitivity and specificity range from 44% to 63% and 80% to 98%, respectively, and increased N : C ratio, nuclear molding and loss of honeycombing are reliable features of malignancy. The performance and morphology of specimens prepared by ThinPrep, a liquid‐based cytology technique is mostly unknown. Methods The laboratory information system was searched for all cases prepared by ThinPrep. Patient disease classification of benign or malignant was determined by linkage with the provincial cancer registry and was the gold standard against which sensitivity, specificity, positive (PPV) and negative predictive values (NPV) were calculated. True positives and negatives were reviewed to identify predictive cytomorphologic features. Results Between 1996 and 2001, there were 149 ThinPrep specimens; 55 (37%) were reported as positive for malignancy and 94 (63%) as negative. Disease was classified as malignant in 86 (58%) patients and benign in 63 (42%). There were 42 false negative, 11 false positive, 52 true negative, and 44 true positive cytology results. Sensitivity was 51.2% (CI; 40.2 : 62.0), specificity 82.5% (CI; 70.5 : 90.6), and PPV and NPV 80.0% (CI; 66.6 : 89) and 55.3% (CI; 44.7 : 65.5), respectively. Cell groups with crowded, enlarged, irregular nuclei and nuclear features of vesicular chromatin and large, multiple irregular nucleoli correlated with malignant disease, while monolayered sheets of uniform columnar cells, regular nuclei and a finely granular chromatin correlated with benign disease. Conclusions The performance of ThinPrep brushings from this anatomic site equals conventional preparations. Cytomorphologic features of malignancy are more frequent and pronounced with ThinPrep.     

Mesothelioma Symposium 11.30–12.30 Tuesday 16 September 2003. Cytopathology of malignant mesothelioma

The diagnosis of malignant mesothelioma on the cytology of serous effusions is a two‐phase process. First is to determine that the effusion is malignant based on morphological features such as a highly cellular fluid with many large three dimensional cell aggregates, and/or the recognition of minor malignant criteria including prominent cell engulfment, uniformly present very prominent nucleoli, or the finding of very large (giant) cells. In cell block sections, strong positive staining with EMA often with cell membrane accentuation provides compelling support for a cytological diagnosis of malignancy. Second is to recognize that the malignant cells have a mesothelial phenotype and do not represent metastatic malignancy (usually adenocarcinoma). Criteria in support of mesothelioma include the lack of a ‘two cell’ population, that is one native (mesothelial) and one foreign (metastatic), cells with abundant dense staining cytoplasm, the presence of ‘windows’ where mesothelioma cells lie in close apposition and intracytoplasmic glycogen presenting either as small peripheral vacuoles on MGG stained smears or large yellow refractile crescents on Papanicolaou stained smears. In addition, mesothliomas often possess connective tissue stromal cores occurring as either well‐formed collagen within papillary aggregates or lying free as pink (MGG) or light green (Pap) amorphous material in the background of the smear or in loose association with mesothelioma cells. Finally small orange staining squamous‐like cells can occasionally be identified and sometimes this may be a very prominent finding and has resulted in the false impression of a squamous cell carcinoma. Almost certainly these cells represent apoptotic tumour cells. The connective tissue mucin hyaluronic acid may be found as a net‐like pattern in the smear background or as large hard‐edged magenta‐stained vacuoles on MGG‐stained smears. Cell block sections provide architectural information and it is usually possible to separate mesothelioma aggregates with their cuboidal cells, central nuclei and abundant dense cytoplasm arranged in solid, papillary or hollow clusters from those of adenocarcinoma with less dense, often foamy cytoplasm, often composed of columnar cells with elongated nuclei. Aggregate form in adenocarcinoma can be variable but true acini are a rare finding. These cell block sections provide an ideal medium for histochemistry (PAS with and without diastase digestion) and immunocytochemistry. By using a panel of antibodies (Calretinin and CK 5/6, BerEp4, CEA, B72.3) it is almost always possible to distinguish mesothelioma from metastatic adenocarcinoma. Calretinin and CK 5/6 positive staining and absent staining with BerEp4, CEA and B72.3 is considered diagnostic of mesothelioma.     

Proffered papers 9.30–10.00 Monday 15 September 2003 2 Clinicopathologic significance of the ‘borderline nuclear change – high grade dyskaryosis not excluded’ report

Both the original Bethesda system and the current UK classifications of cervical cytology have proved robust but each has a major weakness in the area of abnormalities of uncertain significance. Cytologists recognize that sometimes it is simply impossible to differentiate between reactive and dyskaryotic material. For this reason, the Australian version of the Bethesda system introduced a new category of ‘high grade inconclusive’ with a recommendation for referral to colposcopy. Approximately 60% of such cases are found to have high grade lesions at colposcopy (Schoolland M, Sterrett G, Knowles S et al.). The present UK system even with the proposed changes requires of the pathologist, a decision as to whether such cases are probably high grade (=a report of moderate dyskaryosis) or not (= a report of borderline). This continues to ignore the fact that sometimes you just cannot tell, even on review. We have taken a consecutive series of 50 referral smears, reported as moderate dyskaryosis, where the histological outcome (by loop cone) is known. These cases were rescreened and then reviewed blind by a pathologist with extensive experience of the Australian NH & MRC modified Bethesda system. On review, the material was reclassified along NH & MRC lines. The results were compared with the biopsy findings in order to determine whether the category of ‘inconclusive’ might be of value in the context of the NHSCSP.     

Proffered Papers 10.30–11.15 Monday 15 September 2003 4 An audit of the positive predictive value of high‐grade dyskaryosis for CIN 2 or worse on histology

Introduction Colposcopy Quality Standards state that more than 85% of excisional biopsies should show CIN 1 or worse. Data from the National KC65 reports show a large difference between units (50% to 100%). This study investigates the reasons for failure to meet the standard. Methods A review of 1158 consecutive new and 1043 follow‐up colposcopy examinations from a colposcopy database in East Somerset. Patients were treated at the time of initial examination if there was a clinically significant lesion, or at follow‐up depending on the results of investigations. Results Only 59% of excisional biopsies showed CIN 1 or worse. The possible reasons for failure to meet the standard were explored. Possible explanations explored are erroneous colposcopy, false negative histology, and false positive cytology. The cytology–histology correlation was compared between excisional and directed biopsies, and between the two local cytology screening departments. Conclusions The collection of meaningful national data has more to do with careful definition than clinical practice or data collection.     

Guest Lecture 8.45–9.30 Monday 15 September 2003 Endocervical Adenocarcinoma and its Precursors

The incidence of malignant and pre‐malignant endocervical glandular lesions is increasing. Part of this is an apparent increase due to a reduction in the number of invasive cervical squamous carcinomas but there is evidence that there is a real increase in malignant and pre‐malignant endocervical glandular lesions. Different terminologies are in use in the UK where the term cervical glandular intraepithelial neoplasia (CGIN) is commonly used and the rest of the world where pre‐malignant lesions are classified as glandular dysplasia and adenocarcinoma in situ (AIS) (WHO classification). It is well established that high‐grade CGIN (AIS in WHO terminology) is a precursor lesion of cervical adenocarcinoma but it is controversial whether a recognizable precursor to high grade CGIN (namely low‐grade CGIN) exists and criteria for diagnosing this are poorly established and poorly reproducible. Most cases of CGIN are of usual or endocervical type but other morphological subtypes described include endometrioid, intestinal, tubal and stratified mucinous intraepithelial lesion (SMILE). The presence of skip lesions and lesions high up the endocervical canal has been overemphasised in CGIN with most cases occurring close to the transformation zone. Treatment is on an individualized basis but local excision with negative margins and close cytological follow‐up may be employed. There is evidence in the literature that early invasive adenocarcinomas behave in a similar fashion to early invasive squamous carcinomas and that, on selected occasions, conservative therapy can be safely undertaken. However, further studies are needed to ascertain the behaviour and natural history of early invasive cervical adenocarcinoma. In 10%–15% of cases it may be impossible to ascertain whether a malignant endocervical glandular lesion is invasive or in situ. There are many benign mimics of CGIN and adenocarcinoma, including tuboendometrial metaplasia (TEM), endometriosis and microglandular hyperplasia (MGH). Although careful morphological examination usually allows confident distinction of these lesions, a panel of immunohistochemical stains including MIB1, bcl2 and p16 may assist.     

Guest Lecture 9.45–10.30 Wednesday 17 September 2003. Csp Colposcopy Guidelines

The success of the Cervical Screening Programme (CSP) is due in part to its management being underpinned by Quality Assurance. These measures ensure uniform standards across the country. Since 1992 Colposcopy Guidelines have been in place; these were updated in 1997 and have just been redefined. It is entirely consistent with the National CSP that colposcopy is governed by Guidelines. The aim of clinical practice guidelines is to raise the standard of care and improve outcomes. The objectives are, therefore:

• a) to develop evidence based guidelines;
• b) to ensure the guidelines are widely adopted.

The credibility of guidelines is crucial to their adoption and this depends far more on the demonstration of an evidence base than that the authors are ‘experts’. Development by a professional group or body who are seen as having a legitimate role is very important as is involvement of all ‘stakeholders’ in ensuring acceptability. In terms of their nature, guidelines should be valid i.e. they will achieve what they are intended to achieve, and they should be robust i.e. they will work when implemented by different individuals in different settings. Colposcopy lends itself well to guidelines because it is largely a routine practice, but substandard care can have serious consequences. In previous years there has been a set of Guidelines for Practice ^1, 1Duncan 1992 ² 2Duncan 1995 and a set of Quality Standards ³ 3Luesley 1996 . On this occasion these two components have been put together in a simple publication. It needs to be borne in mine that the new guidelines were being developed in the context of a number of potential changes which could interact with each other and impact on the Guidelines. These include: The process for the development of the Guidelines included an Editor, an editorial group, and a group of contributors to produce a draft set of evidence based guidelines across 18 areas. New areas covered included HIV +ve women, immuno suppressed women, and working practice. The draft has been available for comment for several months and amendments have been made. Clearly there are areas where evidence is lacking and where different views are expressed. The most contentious area not surprisingly is in the topic of managing mild dyskaryosis; controversy in this has persisted for many years. The quality standards are either attainable or within attainment and are a driver for rising standards. These programme practice guidelines and standards have earned UK colposcopy international respect. They provide a benchmark for QA assessment and will continue to require amendment as new developments come into being.     

Proffered Papers 16.00–16.30 Monday 15 September 2003 6 Development of a multiplex PCR protocol for rapid screening of ThinPrep® cervical samples for HPV and Chlamydia trachomatis

Human papillomaviruses and Chlamydia trachomatis are two of the most commonly found sexually transmitted infections in cervical Pap smears. They are often asymptomatic and if left untreated can progress to cause serious complications such as pre‐cancerous lesions and tubal factor infertility, respectively. The aim of this study was to develop a rapid multiplex PCR for the simultaneous detection of HPV and C. trachomatis in ThinPrep^® liquid cytology samples. Two multiplexes were optimized. (A) For the detection of C. trachomatis using primers for the cryptic plasmid and for a chromosomal gene (Hsp60); (B) for the simultaneous detection of HPV and C. trachomatis using consensus primers for HPV and plasmid primers for C. trachomatis. Both multiplexes included a set of primers for a human housekeeping gene‐β‐globin. DNA from 34 ThinPrep^® cervical samples was extracted using the QiAmp DNA Mini Kit (Qiagen Ltd, UK). All 34 samples were previously confirmed positive for C. trachomatis using another nucleic‐acid based test. Using multiplex A.for the detection of C. trachomatis, 33 of 34 samples were positive for C. trachomatis by either the plasmid or chromosomal gene primer set. All samples were positive for β‐globin. Ten of the 34 C. trachomatis positive samples were known positives for HPV. Using the combined HPV and C. trachomatis multiplex 10 of 10 were positive for both HPV and C. trachomatis. These simple multiplexes are cost‐effective, rapid and have potential for rapid screening of cervical ThinPrep samples simultaneously for both HPV and C. trachomatis.     

Proffered Papers 10.30–11.15 Monday 15 September 2003 3 5 year outcome of women with borderline and mild dyskaryosis smears 3 5 year outcome of women with borderline and mild dyskaryosis smears

Aims

• 1 To identify the outcome status of women with borderline and mild dyskaryosis smears.
• 2 To determine whether the presence or absence of koilocytosis influences the outcome status.
• 3 To identify the proportion of women with borderline smears showing koilocytosis.

Materials and methods Borderline and mild dyskaryosis cervical smears diagnosed during January to March 1997 were identified from the laboratory database. Each slide was reviewed by two researchers independently, who then agreed a final consensus diagnosis. All slides were classified according to the presence or absence of koilocytosis. Slides were excluded from the study if the review diagnosis was negative, inadequate or high‐grade dyskaryosis. The outcome status was classified according to the worst lesion identified histologically and/or cytologically during the 5‐year follow‐up period. Results 1974 women were identified with borderline or mild dyskaryosis cervical smears of which 1597 were included in the study. Table 1 shows the outcome status of these women.

Table 1. . The outcome status of these women

Posters. P7 Development of a web‐based resource pack for cytology training officers

The NHSCSP Cervical Cytopathology Training Log and accompanying Workbook are central to the education and training of new recruits in cytology laboratories in the UK. These are currently available only in paper format and do not fully address the needs of training officers and trainees in Wales. A web‐based resource pack, which complements the NHSCSP training documents, has been developed by the Welsh Cytology Training School to address these problems. The pack consists of the following sections and has recently been posted on the Welsh intranet:

• ? guide for training officers;
• ? teaching aids, including handouts and ‘powerpoint’ tutorials;
• ? image library;
• ? worksheets; and
• ? competency sheets.

The electronic format allows for instant updating and simultaneous availability to all training officers in Wales. It has been devised in an effort to standardize the training of new cytology recruits in laboratories throughout Wales, thereby ensuring that all new trainees receive the same high‐quality education and training wherever they are employed. CPD modules in gynaecological and non‐gynaecological cytology are being developed. Samples from the resource pack will be presented.     

Chromogenic and fluorescent ‘turn‐on’ chemodosimeter for fluoride based on a F–‐triggered cascade reaction

We developed a new chromogenic and fluorescent ‘turn‐on’ chemodosimeter 1 based on a F^–‐triggered cascade reaction. This system displayed significant changes in UV/vis absorption and fluorescence emission intensities selectively for F^– over other anions in a mixture of CH₃CN/H₂O (95 : 5, v/v) and in acetonitrile. Copyright © 2014 John Wiley & Sons, Ltd.     

Structural and conformational modifications of α-MSH/ACTH4–10 provide melanotropin analogues with highly potent behavioral activities

Previous studies have identified the (4–10) heptapeptide sequence as the central core of α-MSH/ACTH peptides required for mediation of important biological activities. In the present study, the structure-activity relationships of Nle⁴-substituted and -bridged cyclic α-MSH analogues, which were previously shown to exhibit a wide range of melanotropic potencies from weak agonism to super potency, were examined for grooming behavioral activity in the rat following intracerebroventricular injections. The results showed that stepwise C-terminal elongation of the linear Nle⁴-substituted Ac-α-MSH_4–10-NH₂ increased grooming potencies of the peptides in a manner similar to their actions on melanocytes. The most interesting finding was the observation that cyclization of the inactive linear “central (4–10) core” of α-MSH (Ac-α-MSH_4–10) to form Ac-[ ]-α-MSH_4–10-NH₂ resulted in a super potent agonist in the grooming assay. However, while cyclization of the (4–10) heptapeptide produced potent agonists on grooming behavior, the structure-activity relationships were different than the frog skin bioassay. These findings support the hypothesis that appropriate structural and confirmational modifications of α-MSH-related peptides can produce profound effects on the bioactivities of the peptides, and suggest that different structural-conformational requirements exist for α-MSH interactions with its various receptors.     

Hydrothermal synthesis for high‐quality glutathione‐capped CdxZn1 – xSe and CdxZn1 – xSe/ZnS alloyed quantum dots and its application in Hg(II) sensing

High‐quality Cd_xZn_{1 – x}Se and Cd_xZn_{1 – x}Se/ZnS core/shell quantum dots (QDs) emitting in the violet–green spectral range have been successfully prepared using hydrothermal methods. The obtained aqueous Cd_xZn_{1 – x}Se and Cd_xZn_{1 – x}Se/ZnS QDs exhibit a tunable photoluminescence (PL) emission (from 433.5 nm to 501.2 nm) and a favorable narrow photoluminescence bandwidth [full width at half maximum (FWHM): 30–42 nm]. After coating with a ZnS shell, the quantum yield increases from 40.2% to 48.1%. These Cd_xZn_{1 – x}Se and Cd_xZn_{1 – x}Se/ZnS QDs were characterized by transmission electron microscopy, X‐ray diffraction, X‐ray photoelectron spectroscopy and Fourier transform infrared (FTIR) spectroscopy. To further understand the alloying mechanism, the growth kinetics of Cd_xZn_{1 – x}Se were investigated through measuring the fluorescence spectra and X‐ray diffraction spectra at different growth intervals. The results demonstrate that the inverted ZnSe/CdSe core/shell structure is formed initially after the injection of Cd²⁺. With further heating, the core/shell structured ZnSe/CdSe is transformed into alloyed Cd_xZn_{1 – x}Se QDs with the diffusion of Cd²⁺ into ZnSe matrices. With increasing the reaction temperature from 100 °C to 180 °C, the duration time of the alloying process decreases from 210 min to 20 min. In addition, the cytotoxicity of Cd_xZn_{1 – x}Se and Cd_xZn_{1 – x}Se/ZnS QDs were investigated. The results indicate that the as‐prepared Cd_xZn_{1 – x}Se/ZnS QDs have low cytotoxicity, which makes them a promising probe for cell imaging. Finally, the as‐prepared Cd_xZn_{1 – x}Se/ZnS QDs were utilized to ultrasensitively and selectively detect Hg²⁺ ions with a low detection limit (1.8 nM).