The role of liquid-based cytology in the investigation of thyroid lesions

Objective:  This study investigates the role of liquid-based cytology by ThinPrep^® technique in the detection of thyroid lesions.
Methods:  In all, 252 specimens from 157 patients for pre-operative evaluation of thyroid nodules, prepared by the ThinPrep^®, were examined. In all cases thyroidectomy followed the initial cytological evaluation. All cytological diagnoses were correlated to the histological ones.
Results:  According to our findings, a sensitivity of 87.80%, a specificity of 99.50%, a positive predictive value of 97.30%, a negative predictive value of 97.56% and an overall accuracy of 97.52% were observed in fine needle aspiration cytology in correlation to the histological diagnosis after thyroidectomy.
Conclusions:  ThinPrep^® technique is a valid method for the pre-operative cytological diagnosis of thyroid nodules, offering the possibility of ancillary techniques, such as immunocytochemical and molecular methods and can, therefore, be potentially complementary to histological evaluation for further investigation of follicular lesions.     

The effect of lubricant contamination on ThinPrep® (Cytyc) cervical cytology liquid-based preparations

Objectives:  To assess the extent of lubricant use by smear-takers and the effect of lubricant contamination of ThinPrep^® processed cervical cytology samples.
Methods:  All primary care smear-takers were sent a questionnaire on lubricant type and frequency of use. Fifty cervical cytology samples were then contaminated with incremental amounts of K-Y^® jelly, 50 samples contaminated with incremental amounts of Aquagel^® and ten non-contaminated vials were processed using the ThinPrep^® T2000 processor followed by Papanicolaou staining. The morphological appearances of lubricant contamination were described microscopically and formal cell counts performed on all slides.
Results:  Seventy of 94 (74.5%) primary care smear-takers indicated lubricant use of whom 9/70 (12.8%) used Aquagel^® and 61/70 (87.2%) used K-Y^® jelly. K-Y^® jelly appeared as mucoid blue deposits in the slide background whereas Aquagel^® appeared as pink stringy background material. Cell counting showed a significant difference between Aquagel^® and K-Y^® jelly contaminated slides compared to the original non-contaminated preparations for all fields and the average fields ( P  < 0.001) with a significantly higher count for the original non-contaminated slides than the lubricant contaminated groups.
Conclusion:  Lubricant contamination of ThinPrep^® cervical cytology samples may result in reduced cellularity of the subsequent slide. This study provides evidence-based data to support British Society for Clinical Cytology recommendations for no lubricant use when taking cervical samples.     

Collection fluid helps preservation in voided urine cytology

Objectives:  Degenerative change caused by delay in processing contributes to false-negative and false-positive diagnosis of urothelial carcinoma in cytology. The aim of the study was to see if the use of a collection fluid for urine samples made a significant difference to urine cytology diagnosis, and if one was better suited for routine use in the hospital laboratory. Three cell collection fluids were evaluated by analysing the preservation and degeneration of cells in urine samples, as was the routine preparation which did not use a collection fluid.
Methods:  In the design study 50 voided urine specimens were taken at random from the hospital haematuria clinic. Three commercially available collection fluids cytolyt^TM, cytospin^® and cytoRich^®Blue and the hospital's routine conventional preparation of urine were compared. The degree of degeneration, and so preservation, was assessed by a table of chosen criteria; then ranked and analysed by Friedman's nonparametric test, at P  = 0.05. A second table showing the cell content of each slide was also made.
Results:  These showed no significant diagnostic difference between the collection fluids, but there was a significant difference between the collection fluids and the routine preparation. Minor differences that do not affect diagnosis, such as crystals and ghost red blood cells, were noted in cytospin^® and cytoRich^®Blue.
Conclusion:  It is recommended that a collection fluid is used. This choice should be made after health and safety issues and cost are considered.     

High-risk HPV detection in specimens collected in SurePath preservative fluid: comparison of ambient and refrigerated storage

Objective:  With moves to introduce human papillomavirus (HPV) triage at sentinel sites in England, it is essential that optimal storage and transport conditions are determined for efficient HPV detection using residual liquid-based cytology specimens.
Methods:  Two cytology laboratories with comparable workloads sent residual cervical cytology specimens collected in BD Surepath™ Preservative Fluid to the Specialist Virology Centre for HPV testing. Storage and transport of specimens was at ambient (site A) or refrigerated (site R) temperatures. The effect of temperature on the ability to detect high-risk human papillomavirus (HR-HPV) using Digene Hybrid Capture^® 2 High-Risk HPV DNA Test (hc2) and Roche AMPLICOR^® HPV Test (AMPLICOR) was assessed. All specimens with discordant results were tested using Roche Linear Array HPV Genotyping test.
Results:  A total of 796 residual cytology specimens, with cytology ranging from normal to severe dyskaryosis, were provided (399 from site A and 397 from site R). Ambient storage and transit of cervical specimens in SurePath medium did not appear to affect significantly the suitability of the specimen for HPV testing, as measured by the concordance of the HR-HPV screening assays for ambient versus refrigerated specimens and by the proportion of specimens which tested invalid.
Conclusion:  Residual cytology specimens in SurePath medium, stored and transported at ambient temperature, appear suitable for HR-HPV detection by AMPLICOR beyond the manufacturer's recommended time and potentially up to four weeks.     

Using a quality control approach to define an 'adequately cellular' liquid-based cervical cytology specimen

Objective:  To define a minimum acceptable total squamous cellularity for (ThinPrep^®) liquid-based cervical cytology (LBC) specimens using quality control techniques.
Methods:  Two hundred LBC preparations were made containing varying numbers (<200) of severely dyskaryotic squamous cells and with varying total cellularities.
Results:  Ninety-eight per cent of the LBC preparations that were missed by one or more of three cytoscreeners had fewer than 16 abnormal objects (single dyskaryotic cells or clumps of cells) and 87 dyskaryotic cells. The minimum ratio of dyskaryotic to total squamous cells that, in a preparation of 5000 squamous cells has a probability of at least 0.98 that 87 or more dyskaryotic cells will be present is 1 : 47. Twenty-three preparations diagnosed as abnormal had ratios of dyskaryotic to total squamous cells of between 1 : 2.5 and 1 : 4596. There is thus no feasible minimum acceptable squamous cellularity that will give an acceptable probability of detection of all specimen vials containing abnormal cells in the observed proportions.
Conclusions:  It is suggested that the minimum acceptable cellularity for LBC specimens is set pragmatically by the screening programme to give a feasible percentage of repeat tests.     

Mg2+-free buffer elevates transformation efficiency of Vibrio parahaemolyticus by electroporation

Aims:  The ability to transform Vibrio spp. is limited by the extracellular nuclease that their cells secrete. The reported transformation efficiency of this organism is 10²–10⁵ transformants per microgram DNA. We tried different buffers and conditions, aiming to elevate its transformation efficiency.
Methods and Results:  MgCl₂ and sucrose are often included in the washing and/or electroporation buffers to stabilize the cell membrane. However, Mg²⁺ is required for production and activity of the extracellular nuclease. A simple electroporation buffer lacking Mg²⁺ was found to increase transformation efficiency dramatically, to levels 50-fold more than the buffers containing Mg²⁺. To maintain the stability of the cell membranes, Mg²⁺ was replaced with high concentrations of sucrose, from 272 to 408 mmol l⁻¹. With the new buffers, the transformation efficiency of Vibrio parahaemolyticus was increased to 2·2 × 10⁶ transformants per microgram DNA.
Conclusions:  Mg²⁺ in the buffer adversely affected transformation of V. parahaemolyticus by electroporation. The cell membranes of vibrio can be stabilized by high concentration of sucrose when Mg²⁺ is absent.
Significance and Impact of the Study:  A greater transformation efficiency can facilitate the genetic analysis of an organism and its pathogenicity. Buffers lacking Mg²⁺ can be used for other nuclease-producing organisms.     

Fine Needle Aspiration Cytology of Breast In Invasive Carcinoma of Tubular Type and In Radial Scar/Complex Sclerosing Lesions

Fine needle aspirates (FNA) from 31 invasive carcinomas of tubular type and 22 radial scar/complex sclerosing lesions (RS/CSL), diagnosed in Edinburgh between 1986 and 1991, were reviewed. the lesions in this study varied in size and palpability at presentation, but are of increasing interest in the differential diagnosis of non-palpable areas of increased mammographic density. In agreement with previously published information^1'5'8, it was found that the tubular cancers were usually selected for biopsy following aspiration, but less often definitively diagnosed as malignant. Nearly 50% of the RS/CSL group were correctly reported benign on cytology, but in 40%, biopsy was recommended to exclude malignancy. In addition, cytological features helpful in suggesting malignancy in aspirates from tubular cancers, also raised suspicion in aspirates from the RS/CSL group, with a risk of overdiagnosis of cancer.     

Diagnostic pitfalls in the evaluation of fine needle aspiration cytology of the thyroid: correlation with histopathology in 260 cases

Objectives:  Fine needle aspiration cytology (FNAC) of the thyroid is a non-invasive, cost-effective screening procedure that is valuable for distinguishing neoplastic lesions from non-neoplastic nodules. The aim of this study was to determine the diagnostic accuracy of FNACs performed at our institution by correlating FNAC results with histopathological diagnoses.
Methods:  Two hundred and seventy-one aspiration cytology specimens followed by thyroidectomy were included in the study, and the results of 260 adequate FNACs were compared with their histological diagnoses.
Results:  The sensitivity and specificity of thyroid FNAC for detecting neoplasia were 92.6% and 91.6%, respectively. There were 15 (5.7%) false positives and six (2.3%) false negatives.
Conclusions:  The results showed that follicular cells that exhibit some of the features of papillary carcinoma could be observed in a cytology slide of Hashimoto's thyroiditis, leading to a diagnostic pitfall. In addition, cellularity and overlapping cytological criteria in hyperplasia might lead to a false diagnosis.     

Deep-seated rectal/anal basaloid carcinoma: useful immunocytochemistry in rare squamous cell carcinoma variants

Objectives:  To report the cytological aspects of ano-rectal basaloid carcinoma (BC) variant in endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) conventional and liquid-based cytology (LBC), in a series of 10 cases of deep-seated squamous cell carcinomas (SCC), and to discuss the diagnostic difficulties in interpreting the morphology and immunocytochemical findings.
Methods:  Ten cases of EUS-FNA smears and LBC specimens of deep-seated pelvic masses were retrospectively collected from January 2001 to November 2006.
Results:  Ten EUS-FNA specimen cases were SCC, eight corresponding to usual SCC and two to BC-variant. Of these two cases, only one was correctly diagnosed by EUS-FNA specimen, whereas in the second case, the initial cytological diagnosis was poorly differentiated adenocarcinoma and the final diagnosis of basaloid carcinoma variant was established on surgical resection. Immunocytochemistry (ICC) using CK7, CK20 and CK34βe12 on FNA specimens confirmed the diagnosis retrospectively.
Conclusion:  The diagnosis of basaloid variant of SCC in a rectal location can be very difficult, both on account of the uncommon location and because of the low specificity of morphological aspects on EUS-FNA smears. The immunocytochemical technique, including a limited spectrum of keratins (CK7, CK20, CK34βe12, and p63) is necessary to avoid this diagnostic pitfall.     

Decrease in numbers of glandular cell groups in post-LLETZ liquid-based cytology preparations

Objective:  Large loop excision of the transformation zone (LLETZ) has become standard of care in the management of cervical squamous neoplasia and with cone biopsy glandular intraepithelial neoplasia. Controversy remains about the long-term effects of this traumatic procedure. The aim of this study was to count and compare the number of endocervical glandular cell groups in pre- and post-LLETZ cervical preparations using liquid-based cytology to establish a cyto-morphological correlate of destruction of the transformation zone.
Methods:  The cytology/histology correlation audit records of the Cytopathology Department of St Luke's Hospital in 2003 and early 2004 were used to select patients with a cytological diagnosis of high grade dyskaryosis followed by LLETZ. Only those cases with post-LLETZ cytological follow-up were selected. Cases using conventional smears were excluded. One hundred and twenty slides (60 pairs of slides) in total were retrieved. The cases underwent review and all groups of >3 glandular cells in each slide were counted by AM while blinded as to whether smears were pre- or post-LLETZ. Medians were compared using a Mann–Whitney U -test.
Results:  The median number of groups of endocervical glandular cells of the pre-treatment group was 5.5 and of the post-treatment group was 2.0. There were significantly fewer endocervical glandular cell groups in the post-LLETZ population ( P  = 0.03).
Conclusions:  The number of endocervical glandular groups in cervical cytological preparations decreases significantly following LLETZ procedure. This suggests that cytological follow-up may not be as useful in glandular neoplasia cases. Few or absent glandular cell groups in post-LLETZ preparations may have implications for adequacy assessment.     

Higher diagnostic accuracy with the ThinPrep method in a simulated intraoperative environment

Objective:  To compare the accuracy of intraoperative fine needle aspiration cytology samples prepared by the ThinPrep method to conventional cytological methods. Specimen adequacy and turn around time (TAT) were also assessed.
Methods:  Fifty consecutive fresh tumours submitted for histological analysis were aspirated and each prepared as follows: (i) direct smear with H&E stain, (ii) direct smear with Pap stain, (iii) ThinPrep slide with H&E stain, and (iv) ThinPrep slide with Pap stain. The slides were randomly distributed to three cytopathologists for interpretation. The quality of the preparation, the diagnosis and the time needed for interpretation were recorded.
Results:  Accuracy was measured as the percentage of absolute agreement between the cytological and the histopathological diagnoses of the lesions. Histologically, there were 43 malignant and six benign lesions and one atypical lipoma. The TAT began when the slides/cytolyte specimens arrived at the lab and ended with the pathologist's diagnosis.
Conclusions:  In terms of accuracy and specimen adequacy, ThinPrep slides with Pap stain is the best procedure. This advantage however is offset by the longer testing time.     

Long-term effects of successive Ca(OH)2 and CaCO3 treatments on the water quality of two eutrophic hardwater lakes

1. Whole-lake experiments were conducted in two hardwater lakes (Halfmoon and Figure Eight) in Alberta, Canada, to investigate the effectiveness of repeated lime (slaked lime: Ca(OH)₂ and/or calcite: CaCO₃) treatments (5–78 mg L^–1) for up to 7 years.
2. Randomized intervention analysis of intersystem differences between the experimental and three reference lakes demonstrated a decline in euphotic total phosphorus and chlorophyll a concentrations in the experimental lakes after repeated lime treatments.
3. After the second lime application to Halfmoon Lake, mean winter total phosphorus release rates (TPRR) decreased to < 1 mg m^–2 day^–1 compared with 3.6 mg m^–2 day^–1 during the winter after initial treatment. In the final year of lime application, mean summer TPRR decreased to 4.5 mg m^–2 day^–1 compared with 7.6 mg m^–2 day^–1 in the pre-treatment year.
4. Mean macrophyte biomass declined and species composition was altered at 1 and 2 m depths in Figure Eight Lake during lime application. Over the first 6 years of treatment, macrophyte biomass at 2 m declined by 95% compared with concentrations recorded during the initial treatment year. In the last year of the study, macrophyte biomass at 2 m reached initial treatment concentrations, which coincided with the greatest water transparency. Over the treatment period, macrophyte species shifted from floating to rooted plants.
5. Multiple lime applications can improve water quality in eutrophic hardwater lakes for periods of up to 7 years.     

Comparative validation of c-kit exon 11 mutation analysis on cytology samples and corresponding surgical resections of gastrointestinal stromal tumours

Objective:  Studies have shown that c-kit mutation analysis of gastrointestinal stromal tumours (GISTs) obtained by endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) can be routinely performed. We validated c-kit exon 11 mutational analysis on cell block material obtained from fine needle aspiration cytology (FNAC) for diagnostic purposes and compared it with the same analysis in formalin-fixed paraffin-embedded full sections of the corresponding resection specimens.
Methods:  c-kit mutation analysis was done on cell block material obtained from ten cases encountered in our department from 1999 to 2008 on which FNAC was attempted pre-operatively. The findings were compared with analysis on full paraffin section of the corresponding resected tumours in seven cases where patients opted for resection. c-kit exon 11 was examined via bidirectional nucleic acid sequencing.
Results:  Our results showed 100% concordance for the presence and type of exon 11 mutation in the resected and aspirated tumours in all seven cases. These mutations had diagnostic value when compared with other neoplasms that are part of the cytomorphological differential diagnosis, such as leiomyosarcoma or gastric adenocarcinomas.
Conclusion:  Molecular cytopathology is a powerful tool that can complement morphology and immunohistochemical assessment of cytological material in routine practice for the diagnosis and prognostication of GISTs. We briefly discuss the advantages and limitations of the fine needle method of obtaining tissue for the diagnosis and prognostication of GISTs, and its current therapeutic strategies.     

Response of plankton communities to whole-lake Ca(OH)2 and CaCO3 additions in eutrophic hardwater lakes

1. The impact of whole-lake lime (slaked lime, Ca(OH)₂, and/or calcite, CaCO₃) addition on plankton communities was evaluated in eutrophic hardwater lakes on the North American Boreal Plain.
2. Two lakes received a single treatment of lime (Ca(OH)₂ at 74 or 107 mg L^–1), two lakes received multiple treatments with Ca(OH)₂ and/or CaCO₃ (5–78 mg L^–1), and four lakes were untreated and served as reference systems.
3. Over the long-term (> 1 year), phytoplankton biomass was reduced in multiple-dose lakes, but not in single-dose lakes. Cyanobacteria typically dominated the algal community in the years before, during and after lime treatment in both single- and multiple-dose lakes.
4. In the single-dose lakes, randomized intervention analysis showed no significant change in the biomass of zooplankton after lime addition.     

Growth and volatile compound production by Brettanomyces/Dekkera bruxellensis in red wine

Aims:  Brettanomyces / Dekkera bruxellensis is a particularly troublesome wine spoilage yeast. This work was aimed at characterizing its behaviour in terms of growth and volatile compound production in red wine.
Methods and Results:  Sterile red wines were inoculated with 5 × 10³ viable cells ml⁻¹ of three B. bruxellensis strains and growth and volatile phenol production were followed for 1 month by means of plate counts and gas chromatography-mass spectrometry (GC-MS) respectively. Maximum population levels generally attained 10⁶–10⁷ colony forming units (CFU) ml⁻¹ and volatile phenol concentrations ranged from 500 to 4000 μg l⁻¹. Brettanomyces bruxellensis multiplication was also accompanied by the production of organic acids (from C₂ to C₁₀), short chain acid ethyl-esters and the 'mousy off-flavour' component 2-acetyl-tetrahydropyridine.
Conclusions:  Different kinds of 'Brett character' characterized by distinct metabolic and sensory profiles can arise in wine depending on the contaminating strain, wine pH and sugar content and the winemaking stage at which contamination occurs.
Significance and Impact of the Study:  We identified new chemical markers that indicate wine defects caused by B. bruxellensis. Further insight was provided into the role of some environmental conditions in promoting wine spoilage.     

Real-time quantification of Pseudomonas fluorescens cell removal from glass surfaces due to bacteriophage varphiS1 application

Aims:  To study the efficacy of the lytic phage φS1 in eliminating Pseudomonas fluorescens in the early stage of biofilm formation, using an in situ and real time methodology for cell quantification.
Methods and Results:  Cell adhesion and phage infection studies were carried out in a parallel plate flow chamber under laminar conditions. Cells were allowed to adhere until reaching 1·7–1·8 × 10⁶ cells cm⁻² and phage infection was performed with two different phage concentrations (2 × 10⁹ PFU ml⁻¹ and 1 × 10¹⁰ PFU ml⁻¹). Phage concentration clearly affects the speed of infection. The less concentrated phage solution promoted a three times slower rate of cell removal but did not affect the overall percentage of cell removal. In fact, after a longer infection period the less concentrated phage solution reached the same 93% cell removal value.
Conclusions:  Phages are efficient in the eradication of bacterial cells at the early stage of biofilm formation and their presence at the surface did not allow bacterial recolonization of the surface.
Significance and Impact of the Study:  To date, no published studies have been made concerning in situ and real time quantification of cell removal from surfaces due to phage action.     

Histological examination of clots from cytological specimens: is this a worthwhile procedure?

A comparison of conventional cytology and clot histology was made on 174 serous fluids, fine needle aspirates (FNA) and other non‐gynaecological specimens. In eight cases (4.5%) this clot material contained malignant cells or cells suspicious of malignancy despite the absence of suspicious or malignant cells in conventionally prepared smears from the same specimen. In 11 cases (6.3%) the clot was negative, although conventional smears contained malignant or suspicious cells. A χ² test showed that there was no statistically significant difference between the number of positive diagnostic scores in each group with χ² of 0.223 (degrees of freedom=1), P=0.637. Examination of clot material from serous fluids and FNA aspirates is as effective as examination of conventional cytological preparations. Processing of clots from cytological aspirates for histological examination should be more widely adopted, and is applicable in all cytopathology laboratories.     

Multidrug resistance gene deficient (mdr1a–/–) mice have an altered caecal microbiota that precedes the onset of intestinal inflammation

Aim:  To compare caecal microbiota from mdr1a ^–/– and wild type (FVB) mice to identify differences in the bacterial community that could influence the intestinal inflammation.
Methods and Results:  Caecal microbiota of mdr1a ^–/– and FVB mice were evaluated at 12 and 25 weeks of age using denaturing gradient gel electrophoresis (DGGE) and quantitative real-time PCR. DGGE fingerprints of FVB and mdr1a ^–/– mice (with no intestinal inflammation) at 12 weeks revealed differences in the presence of DNA fragments identified as Bacteroides fragilis , B. thetaiotaomicron , B. vulgatus and an uncultured alphaproteobacterium. Escherichia coli and Acinetobacter sp. were only identified in DGGE profiles of mdr1a ^–/– mice at 25 weeks (with severe intestinal inflammation), which also had a lower number of total bacteria in the caecum compared with FVB mice at same age.
Conclusions:  Differences found in the caecal microbiota of FVB and mdr1a ^–/– mice (12 weeks) suggest that the lack of Abcb1 transporters in intestinal cells due to the disruption of the mdr1a gene might lead to changes in the caecal microbiota. The altered microbiota along with the genetic defect could contribute to the development of intestinal inflammation in mdr1a ^–/– mice.
Significance and Impact of the Study:  Differences in caecal microbiota of mdr1a ^–/– and FVB mice (12 weeks) suggest genotype specific colonization. The results provide evidence that Abcb1 transporters may regulate host interactions with commensal bacteria. Future work is needed to identify the mechanisms involved in this possible cross-talk between the host intestinal cells and microbiota.