Comparison of cytology and histology results in English cervical screening laboratories before and after liquid‐based cytology conversion: do the data provide evidence for a single category of high‐grade dyskaryosis?

R. G. Blanks and R. S. Kelly
Comparison of cytology and histology results in English cervical screening laboratories before and after liquid‐based cytology conversion: do the data provide evidence for a single category of high‐grade dyskaryosis? Objective: To determine whether the difference between the positive predictive value (PPV) for cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+) of referral from moderate dyskaryosis and from severe dyskaryosis was reduced after laboratories converted from conventional to liquid‐based cytology (LBC). Furthermore, to explore the cytology/histology agreement after LBC conversion, and to determine post‐LBC whether there was increased support for the use of one single category of high‐grade dyskaryosis (equivalent to high‐grade squamous intraepithelial lesion). Methods: The association between cytology and histology has been examined using annual Korner return data (KC61 returns) collected by laboratories from the English National Health Service cervical screening programme. The study compares return data before and after LBC conversion. Results: The study examined data from 102 laboratories that converted from conventional cytology to LBC. Before conversion the PPV for CIN2+ of severe dyskaryosis was 88% and after increased to 90% (P = 0.003). For moderate dyskaryosis the PPV for CIN2+ increased from 70% to 72% (P = 0.06). The absolute difference of 18% between severe and moderate dyskaryosis was therefore the same pre‐ and post‐LBC conversion. The PPV of mild dyskaryosis for CIN2+ before and after conversion reduced from 23% to 19% (P < 0.001). The agreement between cytology and histology measured using a weighted Kappa statistic increased from 0.52 to 0.60 after conversion to LBC because of small increases in the proportions of severe dyskaryosis or worse with CIN3+ outcomes and mild dyskaryosis with CIN1 or less outcomes. Conclusions: Following LBC conversion there was evidence of a modest increase in the agreement between cytology and histology but no evidence of a change in the absolute difference in PPV for CIN2+ between moderate and severe dyskaryosis. The data support the conclusion that women referred with moderate dyskaryosis will on average have a lower risk of progression to invasive cancer than women referred with severe dyskaryosis. If the data were considered to support the categories of high‐grade dyskaryosis (moderate) and high‐grade dyskaryosis (severe) before LBC conversion then it can be strongly argued that they also support these categories after conversion.     

Vaginal vault cytology tests: analysis of a decade of data from a UK tertiary centre

H. Stokes‐Lampard, S. Wilson, C. Waddell and L. Bentley Vaginal vault cytology tests: analysis of a decade of data from a UK tertiary centre Objectives: To examine temporal trends in the use of vault cytology tests in primary and secondary care and the demographics of those women tested. Methods: Retrospective analysis of routinely collected data concerning women who had a vault cytology test processed during a 10‐year period (1 April 1995 to 31 March 2005) at Birmingham Women’s NHS Foundation Trust. Results: A total of 8457 vault cytology tests from 3164 women (range 1–17 tests, median = 2) were processed, representing approximately 2% of the cervical cytology workload of the Department of Cytopathology at Birmingham Women's Hospital. There was a significant reduction in annual numbers processed (Pearson correlation ?0.958, P < 0.001). Significant abnormalities (mild dyskaryosis or worse) were detected in 4.5%, with malignancy being detected in <0.1%. The unsatisfactory cytology test rate was 10.7% overall. There was a reduction in the numbers of vault cytology tests coming from the community, hospital outpatient clinics and operating theatres over time (χ² for linear trend = 139.53, 9 d.f., P < 0.0001). Tests originating from community settings had the lowest disease detection rates: no malignancies and only two severe abnormalities were detected from almost 4000 primary care samples; abnormal results represented 2.8% (n = 113), of which the majority (n = 73) were borderline results. All cancers (n = 8) were detected in samples taken in gynaecology and colposcopy clinics. Conclusions: Vault cytology test usage appears to be reducing, particularly from outpatient clinics and primary care. Community detection rates are very low. Further research is required to establish the true costs and benefits of vaginal vault cytology.     

Borderline nuclear change,high‐grade dyskaryosis not excluded: current concepts and impact on clinical practice

S. S. Hoo, A. Patel, H. Buist, K. Galaal, J. D. Hemming and R. Naik Borderline nuclear change, high‐grade dyskaryosis not excluded: current concepts and impact on clinical practice Objective: Borderline nuclear change, high‐grade dyskaryosis not excluded (B/HG) is a subcategory of the borderline category recommended by the British Society for Clinical Cytology as warranting direct referral to colposcopy. This subcategory is equivalent to the Bethesda category of atypical squamous cells, cannot exclude high‐grade squamous intraepithelial lesion (ASC‐H). The purpose of this study was to determine the validity and accuracy of using B/HG to identify potential cervical intraepithelial neoplasia (CIN) grade 2 or worse (CIN2+). Methods: Data were collected from the hospital pathology database for borderline, B/HG and high‐grade cytology (moderate dyskaryosis and above), and their respective histological and colposcopic outcomes. SPSS was used for data analysis. Results: Of the 84 799 total cytology samples screened between July 2006 and December 2009, 5225 (6.1%) were reported as borderline, 309 (0.4%) as B/HG and 1222 (1.4%) as high‐grade cytology. Thus, B/HG comprised 5.9% of the overall borderline category, in keeping with national guidelines (<10%). CIN2+ was confirmed in 86.6% of high‐grade, 40.8% of B/HG and 3.0% of borderline cytology. Of 309 women reported with B/HG cytology, 239 had colposcopy. Colposcopic appearances showed a positive predictive value (PPV) of 71.8% for detecting CIN2+ and a negative predictive value of 60.7%. Conclusions: The B/HG category was associated with a significantly higher incidence of CIN2+ compared with borderline cytology as a whole. This refining performance justifies its existence. Colposcopic appearances had a high PPV for detecting CIN2+. Therefore, colposcopy is recommended in patients with B/HG cytology and treatment should be offered if high‐grade colposcopic changes are seen.     

Use of the ThinPrep Imaging System for internal quality control of cervical cytology

T. Heard, A. Chandra, G. Culora, S.S. Gupta, A. Herbert and M. Morgan
Use of the ThinPrep Imaging System for internal quality control of cervical cytology Objective: To audit the use of the ThinPrep Imaging System (TIS) for internal quality control (IQC) in the place of rapid review (RR), and to compare its performance with routine primary screening. Method: During 9 months, 16 462 ThinPrep slides were processed by TIS. Slides were initially reviewed using the TIS review scope, as recommended by the manufacturer: 22 fields of view were observed and, if considered abnormal, a full microscopic review was conducted using the review scope. Different biomedical scientists (BMSs), working on each procedure in rotation, performed batches of TIS‐assisted quality control and routine primary screening independently on unmarked slides. Any slides with abnormalities detected by either method were referred to a consultant pathologist or advanced BMS practitioner for a final report. TIS results were compared with both previous records of RR and routine primary screening carried out on the same slides. We used the UK terminology in which ‘dyskaryosis’ is equivalent to squamous intraepithelial lesion (SIL) and borderline to atypical (including squamous and glandular cells). Results: TIS preview detected significantly more high‐grade dyskaryosis compared with RR during the previous 4 years: 2.0–4.2 compared with 0.1–1.8 detected per 1000 slides (P = 0.0001). TIS and routine screening were equivalent in sensitivity and specificity for the final cytology result, but BMSs were significantly more likely to classify slides as dyskaryotic rather than borderline when using TIS compared with routine screening. Referrals for potentially high‐grade abnormalities detected by TIS‐assisted IQC alone found 28 biopsies of at least cervical intraepithelial neoplasia grade 2 (CIN2+), whereas 15 CIN2+ biopsies were found on routine screening but missed using TIS. There was no significant change in the rates of inadequate tests, high‐ or low‐grade cytological abnormalities, or positive predictive value for CIN2+ when TIS was in use. Conclusions: Screening with TIS was more sensitive than RR for IQC, providing a rescreening method equivalent to routine primary screening in overall accuracy.     

Comparison of SurePath® and ThinPrep® liquid‐based cervical cytology using positive predictive value,atypical predictive value and total predictive value as performance indicators

P. K. Wright, J. Marshall and M. Desai Comparison of SurePath ^® and ThinPrep ^® liquid‐based cervical cytology using positive predictive value, atypical predictive value and total predictive value as performance indicators Objective: Two liquid‐based cytology (LBC) systems are in widespread use in the UK: ThinPrep^® and SurePath^®. A number of studies have now compared LBC with conventional cytology in cervical screening. However, to date, we are aware of no studies that have compared ThinPrep^® with SurePath^® LBC. As the selection and use of specific diagnostic systems in a laboratory has significant clinical and economic implications, there is a clear need to compare directly existing LBC technology. The objective of this study was to compare ThinPrep^® with SurePath^® LBC in a single cytology laboratory using performance indicators. Methods: Data were collected for all cervical cytology samples processed at Manchester Cytology Centre over a 1‐year period. ThinPrep^® LBC was compared with SurePath^® LBC using positive predictive value (PPV), atypical predictive value (APV) and total predictive value (TPV), reflecting outcome of cervical intraepithelial neoplasia (CIN) grade 2 or worse for high‐grade dyskaryosis (PPV), low‐grade dyskaryosis or borderline (atypical) cytology (APV) and all (total) abnormal cytology (TPV). Results: 2287 (out of 56 467) (ThinPrep^®) and 586 (out of 22 824) (SurePath^®) samples showed borderline or worse cytology after exclusion criteria. PPV, APV and TPV were within acceptable ranges for both ThinPrep^® and SurePath^®. Conclusions: ThinPrep^® and SurePath^® were equivalent based on three performance indicators. We suggest that APV and TPV should be used as an adjunct to PPV and other methods of quality assurance for cervical screening.     

The number of dyskaryotic cells on an initial ThinPrep® cervical sample showing mild dyskaryosis has predictive value

OBJECTIVES: Recent National Health Service Cervical Screening Programme (NHSCSP) guidelines suggest referral for colposcopy following an initial result of mild dyskaryosis. The aim of this study was to investigate if the number of dyskaryotic cells counted on an initial ThinPrep cervical sample showing mild dyskaryosis has predictive value. METHODS: Cases of mild dyskaryosis on ThinPrep cervical samples from 2002 were retrieved from the cytology department records of St Luke's Hospital. A total of 123 sequential cases with a first-time result of mild dyskaryosis on ThinPrep slides with follow-up cytology available in the same institution were identified. While blinded to outcome, the number of dyskaryotic cells was counted in each case. Follow-up colposcopy/histology information was retrieved where indicated. The number of dyskaryotic cells counted on each slide was collated with outcome data. RESULTS: Of the 123 cases, six women were lost to follow-up. Seventy-three had a negative outcome, 27 had a low-grade outcome and 17 had a high-grade outcome. Only one of 17 high-grade outcome cases had < or = 15 dyskaryotic cells on the initial slide. The distribution of women with a negative/low-grade outcome and those with a high-grade outcome with >15 and < or = 15 dyskaryotic cells on the initial slide was tested using a chi-square test (P = 0.008). The negative predictive value for a high-grade outcome when < or = 15 dyskaryotic cells were present on the initial slide was 97.7%. CONCLUSION: The number of dyskaryotic cells on ThinPrep slides showing mild cervical dyskaryosis has predictive value. The number of dyskaryotic cells may be used to select women suitable for cytological rather than colposcopic follow-up.     

Liquid-based cytology and conventional smears compared over two 12-month periods.

BACKGROUND AND OBJECTIVE: Liquid based cytology (LBC) was introduced across the Scottish Cervical Screening Programme in 2003-2004. The objective of this study was to compare in a large cytopathology laboratory the results of cervical samples over two twelve-month periods, 2001-2002, when the great majority of smears were conventional, with 2003-2004, when all were LBC. METHODS: The results of smears in both periods were analysed to give overall reporting profiles, and correlated with results of cervical biopsies. The numbers of patients referred for colposcopy were compared. RESULTS: The percentage of unsatisfactory smears fell from 13.6% to 1.9%. Colposcopic referrals for repeated unsatisfactory smears fell from almost 25% to 0.5%. There was a decrease in overall smear numbers, but despite this there was an increase in the number of smears reported as showing dyskaryosis of any grade. There was an increase in positive predictive value for moderate dyskaryosis and above, from 79.5% to 86.1%. The outcome of biopsies from patients referred with mild dyskaryosis showed no decrease in accuracy of predicting a low grade histological lesion. Workload in the laboratory decreased, due to fewer smears received overall, more rapid primary screening times and fewer multi-slide cases. Primary screening backlogs all but disappeared, and reporting times greatly improved. CONCLUSIONS: Introduction of liquid based cytology led to improvements in unsatisfactory smear rates, with significant benefits to colposcopic referrals and laboratory turnaround times. Pick-up rates of dyskaryosis were maintained, and the positive predictive value of a dyskaryotic smear report was improved.     

Identification of components of fibroadenoma in cytology preparations using texture analysis: a morphometric study

S. Singh and R. Gupta Identification of components of fibroadenoma in cytology preparations using texture analysis: a morphometric study Objectives: To evaluate the utility of image analysis using textural parameters obtained from a co‐occurrence matrix in differentiating the three components of fibroadenoma of the breast, in fine needle aspirate smears. Methods: Sixty cases of histologically proven fibroadenoma were included in this study. Of these, 40 cases were used as a training set and 20 cases were taken as a test set for the discriminant analysis. Digital images were acquired from cytological preparations of all the cases and three components of fibroadenoma (namely, monolayered cell clusters, stromal fragments and background with bare nuclei) were selected for image analysis. A co‐occurrence matrix was generated and a texture parameter vector (sum mean, energy, entropy, contrast, cluster tendency and homogeneity) was calculated for each pixel. The percentage of pixels correctly classified to a component of fibroadenoma on discriminant analysis was noted. Results: The textural parameters, when considered in isolation, showed considerable overlap in their values of the three cytological components of fibroadenoma. However, the stepwise discriminant analysis revealed that all six textural parameters contributed significantly to the discriminant functions. Discriminant analysis using all the six parameters showed that the numbers of pixels correctly classified in training and tests sets were 96.7% and 93.0%, respectively. Conclusion: Textural analysis using a co‐occurrence matrix appears to be useful in differentiating the three cytological components of fibroadenoma. These results could further be utilized in developing algorithms for image segmentation and automated diagnosis, but need to be confirmed in further studies.     

Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US,ASC‐H,LSIL and HSIL)

G. Bigras, J. Wilson, L. Russell, G. Johnson, D. Morel and M. Saddik
Interobserver concordance in the assessment of features used for the diagnosis of cervical atypical squamous cells and squamous intraepithelial lesions (ASC‐US, ASC‐H, LSIL and HSIL) Objectives: Given the well‐known poor reproducibility of cervical cytology diagnosis, especially for atypical squamous cells of undetermined significance (ASC‐US) and low‐grade squamous intraepithelial lesion (LSIL), this study surveyed reproducibility in the assessment of individual cytomorphological features. Methods: One hundred and fifty cells or groups of cells, with a variety of morphological appearances, including normal cells, high‐grade squamous intraepithelial lesion (HSIL), LSIL, ASC‐US and ASC cannot exclude HSIL (ASC‐H), were precisely marked on 150 different liquid‐based cytological preparations. They were analysed by 17 observers who assessed 17 cytological features including nuclear features (chromatin texture, nuclear outline, nuclear shape, etc.), cytoplasmic features (cell shape, cytoplasmic staining, cytoplasmic clearing, etc.) and group characteristics (nuclear polarity, cellular density, etc.). A total of 43 350 data scores were collected in a database using a web‐based survey. Kendall’s W and relative entropy indexes were utilized to compute concordance indexes of respectively ordinal and nominal variables. Results: Nuclear features have significantly lower reproducibility (0.46) compared with other cytological features (0.59). The feature with least agreement is assessment of chromatin texture. A small but significant difference in concordance was found between two subsets of observers with different levels of experience. Conclusion: Most previous studies assessing reproducibility of cytological diagnoses show, at best, moderate reproducibility among observers. This study focused on agreement regarding the presence of constituent morphological features used to recognize dyskaryosis and various grades of squamous intraepithelial lesions. A map of reproducibility indexes is presented that highlights, for daily practice or teaching, the robustness of features used for cytological assessment, recognizing that diagnosis is always based on a combination of features.     

Factors contributing to false‐negative and potential false‐negative cytology reports in SurePath™ liquid‐based cervical cytology

N. Gupta, D. John, N. Dudding, J. Crossley and J. H. F. Smith
Factors contributing to false‐negative and potential false‐negative cytology reports in SurePath ? liquid‐based cervical cytology Objectives: The characteristics of false‐negative conventional cervical cytology smears have been well documented, but there is limited literature available for liquid‐based cytology (LBC), especially SurePath? samples. We aimed to assess the characteristics of false‐negative SurePath LBC samples. Methods: Over a period of 5 years, an audit of false‐negative reports in SurePath cervical cytology was undertaken. In a workload of 183, 112 samples, 481 (0.3%) false negatives were identified using two routes: those detected by routine laboratory internal quality control (rapid pre‐screening) (n = 463) and those reported as normal (true false negatives) with concurrent high‐grade cervical histology (n = 18). Ninety‐five false‐negative cases with a subsequent biopsy reported as at least cervical intraepithelial neoplasia grade 2 (CIN2+) were reviewed for a number of different cytomorphological features. Results: Of 95 samples with subsequent CIN2+, 30.5% predominately contained microbiopsies/hyperchromatic crowded cell groups (HCGs), 27.3% sparse dyskarytotic cells, 4.2% pale cell dyskaryosis, 6.3% small dyskaryotic cells; 3.2% were misinterpreted cells, 8.4% contained other distracting cells, 7.4% were low contrast, 5.3% were unexplained and 7.4% were true negatives. The mean number of microbiopsies/HCGs in that category was 4.6. The mean number of abnormal cells in the sparse dyskaryotic cell category was 13.8. Conclusions: Microbiopsies/HCGs were the commonest reason for false negatives. They were usually present in sufficient numbers to be detected but interpretation could be problematic. Dispersed single abnormal cells were usually not identified because of their scarcity or the presence of distracters.     

Analysis of intraspecific seed diversity in Astragalus aquilanus (Fabaceae), an endemic species of Central Apennine

• This work aims to study seeds of the endemic species Astragalus aquilanus from four different populations of central Italy. We investigated seed morpho‐colorimetric features (shape and size) and chemical differences (through infrared spectroscopy) among populations and between dark and light seeds.
• Seed morpho‐colorimetric quantitative variables, describing shape, size and colour traits, were measured using image analysis techniques. Fourier transform infrared (FT‐IR) spectroscopy was used to attempt seed chemical characterisation. The measured data were analysed by step‐wise linear discriminant analysis (LDA). Moreover, we analysed the correlation between the four most important traits and six climatic variables extracted from WorldClim 2.0.
• The LDA on seeds traits shows clear differentiation of the four populations, which can be attributed to different chemical composition, as confirmed by Wilk's lambda test (P < 0.001). A strong correlation between morphometric traits and temperature (annual mean temperature, mean temperature of the warmest and coolest quarter), colorimetric traits and precipitation (annual precipitation, precipitation of wettest and driest quarter) was observed.
• The characterisation of A. aquilanus seeds shows large intraspecific plasticity both in morpho‐colorimetric and chemical composition. These results confirm the strong relationship between the type of seed produced and the climatic variables.

     

Posters. P11 Positive predictive values for high and low‐grade cytological abnormalities as surrogates for specificity and sensitivity

Introduction Positive predictive value (PPV), measuring the percentage of moderate dyskaryosis or worse confirmed as CIN2 or worse, is used as a measure of accuracy in cervical screening. However, it relates more to specificity than sensitivity because the denominator includes false positives rather than false negatives. Low values reflect over‐reporting of high‐grade dyskaryosis but high values may reflect under‐reporting. Sensitivity is impossible to measure from correlation of cytology with outcome because women with negative cytology are rarely referred for colposcopy. Rates of CIN3 resulting from referrals for low‐grade cytology may be used as a surrogate for sensitivity, as high values may reflect under‐reporting (ref). Study design Outcome of colposcopy referrals was monitored during a period of 4 years, using a fail‐safe database. Results PPV at Guy's & St Thomas rose from 54% in 1998/1999 to 69% in 2001/2002. The former was below the NHSCSP recommended range. During the same period of time CIN1 rates for moderate dyskaryosis fell from 37% to 24%, reflecting the main source of discrepancy. While specificity increased (as reflected by increasing PPV) sensitivity remained constant in that CIN3 rates for mild dyskaryosis and borderline remained below 6%: average rates in England have fallen over the last 3 years and were 7.4% in 2000/2001 (ref). CIN2 rates for mild dyskaryosis also remained constant at 11% to 12%. Conclusion Correlation of biopsy results with high‐ and low‐grade cytological abnormalities is a useful method of monitoring accuracy of cytology reporting, and can be used to measure over‐ and under‐reporting as surrogates for specificity and sensitivity.     

Morphometric sex determination of young Ospreys Pandion haliaetus using discriminant analysis

Capsule Discriminant functions based on morphometric variables provide a reliable method for sex identification of free‐living and hacked young Ospreys.

Aims To describe an easy, accurate and low‐cost method for sex determination of fully grown nestling and fledgling Ospreys Pandion haliaetus based on morphometric measurements.

Methods Four different measurements were taken in 114 birds (40–73 days old) and a DNA analysis, using PCR amplification, was carried out for sex identification. A forward stepwise discriminant analysis was performed to build the best explanatory discriminant models, which were subsequently validated using statistics and external samples.

Results Our best discriminant function retained forearm and tarsus as the best predictor variables and classified 95.1% of the sample correctly, supported also by external cross‐validations with both hacked and free‐living birds. Moreover, a discriminant function with only forearm as predictor showed a similar high correct classification power (93.4%).

Conclusions These discriminant functions can be used as a reliable and immediate method for sex determination of young Ospreys since they showed high discriminant accuracy, close to that of molecular procedures, and were supported by external cross‐validations, both for free‐living and hacked birds. Thus, these morphometric measurements should be considered as standard tools for future scientific studies and management of Osprey populations     

ATR microspectroscopy with multivariate analysis segregates grades of exfoliative cervical cytology

Although cervical cancer screening in the UK has led to reductions in the incidence of invasive disease, this programme remains flawed. We set out to examine the potential of infrared (IR) microspectroscopy to allow the profiling of cellular biochemical constituents associated with disease progression. Attenuated total reflection-Fourier Transform IR (ATR) microspectroscopy was employed to interrogate spectral differences between samples of exfoliative cervical cytology collected into liquid based cytology (LBC). These were histologically characterised as normal (n = 5), low-grade (n = 5), high-grade (n = 5) or severe dyskaryosis (? carcinoma) (n = 5). Examination of resultant spectra was coupled with principal component analysis (PCA) and subsequent linear discriminant analysis (LDA). The interrogation of LBC samples using ATR microspectroscopy with PCA-LDA facilitated the discrimination of different categories of exfoliative cytology and allowed the identification of potential biomarkers of abnormality; these occurred prominently in the IR spectral region 1200 cm(-1) - 950 cm(-1) consisting of carbohydrates, phosphate, and glycogen. Shifts in the centroids of amide I (approximately 1650 cm(-1)) and II (approximately 1530 cm(-1)) absorbance bands, indicating conformational changes to the secondary structure of intracellular proteins and associated with increasing disease progression, were also noted. This work demonstrates the potential of ATR microspectroscopy coupled with multivariate analysis to be an objective alternative to routine cytology.     

Transformation zone sampling rate is a useful performance indicator for practitioners collecting cervical samples using SurePathTM liquid-based cytology system

An analysis of the reports of 53 982 liquid-based cytology (LBC) samples processed at the Royal Victoria Infirmary, Newcastle, reveals a significant relationship between the transformation zone (TZ) sampling rate observed in specimens submitted from different practices and their overall detection rate for dyskaryosis. The observed correlation (R = 0.184; P = 0.033, CI = 0.017 to 0.397) is very similar to that described previously using conventional cervical smears and confirms the potential relevance of TZ sampling rates as an indicator of consistently effective specimen collection. The correlation between unsatisfactory sample rates and detection rates for high-grade dyskaryosis (R = -0.188, P = 0.030, CI = -0.865 to -0.045) also shows a significant relationship for LBC samples which was not previously seen in conventional smears. The essential role of smear taker feedback, the use of transformation zone sampling and unsatisfactory smear rates as key indicators and the practical issues relating to routine reporting of transformation zone components are discussed.     

Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology

A. Evered and N. Dudding Accuracy and perceptions of virtual microscopy compared with glass slide microscopy in cervical cytology Objective: To evaluate virtual microscopy in terms of diagnostic performance and acceptability among practising cytologists. Methods: Twenty‐four experienced cytologists were recruited to examine 20 SurePath® cervical cytology slides by virtual microscopy. Diagnostic accuracy was compared with glass slide microscopy using an unbiased crossover experimental design. Responses were allocated a score of one for a correct identification of normal or abnormal (borderline/atypical changes in squamous or glandular cells or worse) and a score of zero for an incorrect response (a normal slide reported as abnormal or vice versa). Perceptions of virtual microscopy were assessed by questionnaire analysis. Results: Participants yielded a total of 285 responses for the virtual slide set and 300 for the glass slide set. The approximate time to screen a virtual slide was 18 minutes, compared with 8 minutes or less for a glass slide. Overall there was no significant difference between virtual microscopy and glass slide microscopy in terms of diagnostic accuracy (P = 0.22). Virtual microscopy under‐performed when images were captured over a narrow focal range (P = 0.01). Diagnostic accuracy of virtual microscopy equalled that of glass slide microscopy when participants were able to focus through the full thickness of the slide images (P = 0.07). The most common difficulties experienced by participants with virtual microscopy were freezing of the computer screen during image download, slow response of the computer during slide movement and, in some instances, ‘fuzzy’ images. Cytologists have a strong preference for glass slides over virtual microscopy despite the overall equal diagnostic performance of the two viewing modalities. Conclusions: Diagnostic accuracy of virtual microscopy can equal that of glass slide microscopy. However, without good computer network connections, wide focal range and software that permits effortless navigation across virtual slides, cytologists are unlikely to be convinced of the utility of this technology for cytology screening and diagnosis.     

Morphological variation in Bosmina longirostris (O.F. Müller, 1785) (Crustacea: Cladocera): consequence of cyclomorphosis or indication of cryptic species?

Samples of the polymorphic cladoceran Bosmina longirostris were collected in several lakes and ponds in Western Germany between 1993 and 1999. The shape of the first antennule was used as a qualitative criterion to classify 1241 individuals to three morphs (`longirostris', `cornuta', `pellucida') according to Lieder (Crustacea: Cladocera: Bosminidae. Stuttgart, Germany: Gustav. Fischer Verlag, 1996). Eight morphometric variables were subsequently measured in each individual to quantify size‐ and shape‐related features. Principal component and discriminant analyses were used to describe the morphological variation within and among the three morphs. Seasonal environmental changes modified several traits and thus, cyclomorphosis accounted for a high proportion of within‐morph variation. However, there was no gradual change from one morph to another. As well among‐morph variation was considerably greater than within‐morph variation. Consequently, the disagreement between the qualitative pre‐classification and the assignment basing on morphometric discriminant functions was low (4.6–12.0%). Considering that each morph is morphologically well‐defined, and that the different morphs coexisted over several generations in the same lake we conclude that they represent different species rather than variants of one polymorphic species. Therefore, we recognize beside the nominal taxon Bosmina longirostris ( 26 ) two more species: Bosmina cornuta ( 10 ) and Bosmina pellucida 30 .     

Contribution of cervical cytology in the diagnostic work‐up of patients with endometrial cancer

Introduction

Abnormal cervical cytology in patients with endometrial cancer (EC) has been associated with poor outcome. The aim of this study was to evaluate whether cervical cytology could contribute to an improved preoperative identification of high‐grade EC (serous, clear cell, carcinosarcoma, high‐grade endometrioid EC) in final histology.

Methods

A retrospective cohort study was performed in five hospitals in the Netherlands. A total of 554 patients with EC that underwent primary surgical treatment between 2002 and 2010 were included. Primary outcome was defined as the contribution of abnormal cervical cytology in the preoperative identification of high‐grade EC. As secondary outcome, recurrence‐free survival (RFS) and disease‐specific survival were determined based on preoperative cervical cytology, and compared to the currently established risk factors: myometrial invasion, high‐grade and lymph vascular space invasion.

Results

Abnormal cervical cytology was present in 45.1%. For patients with preoperative inconclusive and high‐grade histology, the presence of abnormal cervical cytology contributed to an improved identification of high‐grade EC in final histology (odds ratio [OR] 6.40 [95% confidence interval {CI}: 1.92‐21.26]; OR 2.86 [95% CI: 1.14‐7.14]), respectively. Patients with abnormal cervical cytology had a significant worse 5‐year median RFS. Abnormal cervical cytology was independently related to RFS (hazard ratio 1.67 [95% CI: 1.04‐2.68]) and disease‐specific survival (hazard ratio 3.15 [95% CI: 1.74‐5.71]).

Conclusions

Abnormal cytology contributes to the preoperative identification of patients with high‐grade EC, and is associated with compromised outcome. Future studies are warranted to determine whether cervical cytology could be incorporated into preoperative prediction models for lymph node metastasis.     

Proposed Sheffield quantitative criteria in cervical cytology to assist the grading of squamous cell dyskaryosis, as the British Society for Clinical Cytology definitions require amendment

OBJECTIVE: In 1986, the British Society for Clinical Cytology (BSCC) published quantitative criteria to assist diagnosis in a three-tier grading system of squamous cell dyskaryosis. In dyskaryotic cells, area nuclear to cytoplasmic (NC) ratios below 50%, between 50% and 66% and over 66% were defined as equating with mild, moderate and severe grades respectively. Following the Terminology Conference in 2002, however, the BSCC recommended on their website that the three-tier model should be replaced by a new two-tier system of low- and high-grade squamous abnormalities. The latter broadly equate with the two-grade Bethesda System (TBS) for reporting squamous intraepithelial lesions. The purpose of this study was to assess the accuracy and reproducibility of the BSCC three-tier quantitative definitions, to investigate if they were applicable to liquid-based cytology (LBC) and to see how they related to the proposed new two-tier BSCC system. METHODS: Quantitative image analysis was undertaken on illustrations from the 1986 BSCC terminology publication and on microscope slides from external quality assessment and Cytology Training Centre teaching sets. RESULTS: Analysis of mean NC ratios showed that mild, moderate and severe dyskaryosis exist as statistically different populations. Overlap of NC ratio ranges, however, limits their practical application in the three-tier model, although interestingly no overlap was noted between mild and severe dyskaryosis. No grade of dyskaryosis had a mean area NC ratio over 50%, indicating that the BSCC quantitative definitions are incorrect. The mean diameter NC ratios for mild, moderate and severe dyskaryosis were found to be 40%, 49% and 66% respectively. Accordingly it is possible that those reporting cervical cytology could be interpreting the BSCC NC ratios as meaning diameter rather than area. Amalgamation of the three-tier results into the proposed two-tier model shows that the resulting mean NC area and diameter ratios identify statistically different low- and high-grade populations. The reduced degree of overlap, however, of NC ratio ranges in the two-tier model implies that NC ratios could have a useful practical role in the separation of the low- and high-grade categories. The two categories were reasonably well separated by mean area and diameter NC ratios of 25% and 50% respectively. A two-tier model combining mild with moderate rather than severe dyskaryosis was found to be a statistically valid alternative but gave rise to NC ratios that would be difficult to use in practice. Except for moderate dyskaryosis, no significant differences were identified between the mean NC ratios of either conventional and LBC preparations or LBC preparations using two different commercial methodologies (SurePath and ThinPrep). Differences, however, were noted in area measurements between SurePath and ThinPrep and this has potential implications for classifications (such as TBS) using area comparisons as their basis. In addition, it was found that the increased NC ratio, associated with higher grades of dyskaryosis is more a consequence of progressive cytoplasmic area reduction rather than nuclear area increase. The similar NC ratios of borderline nuclear changes associated with human papilloma virus and mild dyskaryosis support the BSCC proposal that these can be combined to constitute a low-grade category. This study shows that the BSCC area NC ratio criteria of grading squamous cell dyskaryosis require amendment. In addition, this study supports the new BSCC recommendation of low- and high-grade squamous cell categories. CONCLUSIONS: The study proposes Sheffield quantitative criteria to assist the grading of squamous cell abnormalities. Quantitative diameter NC ratio measurements, however, must always be accompanied by detailed assessment of qualitative morphological features and in particular those relating to nuclear chromatin. This is equally relevant to both two- and three-tier models.