Fine-needle Aspiration Slides Research Articles

Thyroid nodules with DICER1 mutation or PTEN alteration: A comparative cytologic, clinical, and molecular study of 117 FNA cases.

DICER1 mutations and PTEN alterations are increasingly detected by thyroid fine-needle aspiration (FNA). Both are associated with nodular thyroid disease and cancer. The authors analyzed a large comparative thyroid FNA cohort with DICER1 mutation or PTEN alteration. A total of 117 thyroid FNAs with DICER1 or PTEN alterations were retrieved from the databases of two academic medical institutions. Demographic, clinical, and radiologic data were collected; FNA slides were analyzed for 29 cytomorphologic features. Of 117 thyroid FNAs, 36 (30.8%) had DICER1 mutation and 81 (69.2%) showed PTEN alteration. The DICER1 cohort had 33 (91.7%) females and three (8.3%) males (mean, 40.9 years); 61.8% had multinodular disease. FNAs were classified as atypia of undetermined significance (AUS), 23 (63.9%); follicular neoplasm (FN), 12 (33.3%); and malignant, 1 (2.8%). The PTEN subgroup had 66 (81.5%) females and 15 (18.5%) males (mean, 55.2 years) with increased multinodular disease (93.8%, p=.0016). PTEN FNAs had greater cytologic diversity: non-diagnostic, 2 (2.5%); benign, 5 (6.2%); AUS, 44 (54.3%); FN, 24 (29.6%); and malignant, 6 (7.4%). Both DICER1 and PTEN cases showed a range of resected tumor subtypes. The DICER1 cohort included thyroblastoma, and the PTEN group included anaplastic carcinoma. The cytomorphology of DICER1 and PTEN cases showed overlapping features, especially microfollicular patterns. Minor cytomorphologic differences included papillary patterns in DICER1 (p=.039), and oncocytic changes (p<.0001) in PTEN. DICER1 and PTEN FNAs reveal many cytologic similarities. DICER1 patients are younger, and PTEN patients had multinodular disease. Awareness of these genetic cohorts can identify patients at risk for thyroid cancer.

B-392 Standardization of Nucleic acid Extraction Method From Thyroid Fine Needle Aspiration (FNA) Cells on Stained and Fixed Slides for Next-Generation Sequencing Protocols

Abstract Background Thyroid Fine Needle Aspiration (FNA) cytopathology is the best method for histological classification of thyroid nodules. However, between 20%–30% of thyroid nodules show indeterminate classification and are often referred for surgery due to the malignancy risk. Post-surgical histological reports indicate that 84% of these cases are benign, highlighting a high rate of unnecessary surgeries. In this sense, molecular approaches using next-generation sequencing (NGS) have been useful to confirm benignity or malignancy. As important as the choice of a powerful methodology to evaluate different genes simultaneously such as NGS, is the standardization of nucleic acid extraction methods to allow sufficient quality recovery of these molecules. Pre-analytical steps involved in FNA slide preparation include fixation and stain methods that contribute to the low quality and poor recovery of nucleic acid, representing a great challenge to NGS analysis. Thus, this work aims to describe a standardization of nucleic acid extraction method from FNA slides for NGS protocols. Methodology Nucleic acid isolation from 30 FNA samples collected between 2016–2022 were performed by Trizol or RecoverAll kits, according to the manufacturer’s instructions (ThermoScientific). Initially, cells’ slides were counted in an optical microscope, considering cell clusters &amp;gt;10 (equivalent to 1 counter unit). Before extractions with Trizol, the slides were submerged in xylene for 15 min. For RecoverAll extractions, the varnished slides were previously exposed to dry ice for 5 min, while the slides containing coverslip were also exposed to dry ice and submerged in xylene for 15 min. In all cases, the slides were hydrated before scraping and then extracted. Subsequently, the extraction of whole slides and split slides was tested. To split slides, one-half of the slide was separated for RNA extraction, while the remaining half was for DNA extraction. Then, the DNA/RNA were quantified using the Qubit™ 4 Fluorometer equipment (ThermoScientific) and concentrated using Amicon® Ultra filters (Millipore). Results The DNA and RNA quantifications range from 0.0720–1.77 ng/µL and 0.508–7.43 ng/µL, respectively. Among samples, 12% (DNA) and 62.5% (RNA) presented concentration below the quantification limit (too low). Four samples extracted with Trizol did not obtain recovery of nucleic acid and this methodology was discontinued. The use of Amicon® filter improved all samples’ yield. The number of thyroid cell clusters was not proportional to the amount of DNA/RNA recovered. The split slides showed worst RNA/DNA recovery, being ideal to use at least two slides for each patient since co-extraction using the RecoverAll kit was not possible. Conclusion The isolations of nucleic acid from FNA slides are essential to performing subsequent molecular tests, such as NGS. However, specific protocols for nucleic acid extraction from FNA slides are hardly found in the literature. This work showed the combination of methodologies that allowed the scraping of material from FNA slides by exposure to dry ice in samples with varnish or coverslip. The nucleic acid recovery capacity described for this type of sample indicates a way of possibilities to improve the FNA extraction methodology.

Diagnostic Performance of Thyroid Core Needle Biopsy Using the Revised Reporting System: Comparison with Fine Needle Aspiration Cytology

BackgroundWe aim to validate the diagnostic performance of thyroid core needle biopsy (CNB) for diagnosing malignancy in clinical settings to align with the changes made in recently updated thyroid CNB guidelines.MethodsWe retrospectively analyzed 1,381 thyroid CNB and 2,223 fine needle aspiration (FNA) samples. The FNA and CNB slides were interpreted according to the Bethesda System for Reporting Thyroid Cytopathology and updated practice guidelines for thyroid CNB, respectively.ResultsCompared to FNA, CNB showed lower rates of inconclusive results: categories I (2.8% vs. 11.2%) and III (1.2% vs. 6.2%), and higher rates of categories II (60.9% vs. 50.4%) and IV (17.5% vs. 2.0%). The upper and lower bounds of the risk of malignancy (ROM) for category IV of CNB were 43.2% and 26.6%, respectively. The CNB subcategory IVb with nuclear atypia had a higher ROM than the subcategory without nuclear atypia (40%–62% vs. 23%–36%). In histologically confirmed cases, there was no significant difference in the diagnostic performance between CNB and FNA for malignancy. However, neoplastic diseases were more frequently detected by CNB than by FNA (88.8% vs. 77.6%, P=0.046). In category IV, there was no difference in unnecessary surgery rate between CNB and FNA (4.7% vs. 6.9%, P=0.6361).ConclusionThyroid CNB decreased the rate of inconclusive results and showed a higher category IV diagnostic rate than FNA. The revised guidelines for thyroid CNB proved to be an excellent reporting system for assessing thyroid nodules.

Interpretive Errors in Fine-Needle Aspiration of Thyroid: A 13-Year Institutional Experience in Lebanon

Introduction: Fine-needle aspiration (FNA) is a worldwide established diagnostic tool for the assessment of patients with thyroid nodules. All thyroid FNA interpretive errors (IEs) were reviewed at the American University of Beirut Medical Center over a 13-year period, in order to identify and analyze them. Materials and Methods: All FNAs and their corresponding pathology results are correlated yearly for quality assurance. Discrepant cases are segregated into sampling errors and IEs. All thyroid FNAs with IEs were collected from 2005 to 2017. FNA and pathology slides were reviewed by trained, board-certified cytopathologists, adhering to the latest Bethesda criteria. Reasons for erroneous diagnoses were studied. Results: There was a total of 11 IEs out of 340 thyroid FNAs followed by surgical resection. Five benign follicular nodules (BFNs) were misinterpreted as suspicious for carcinoma. Focal nuclear atypia in cyst-lining or follicular cells and a monotonous population of macrophages misinterpreted as Hurthle cells (HCs) were the causes of IEs in this category. Four Hashimoto’s thyroiditis (HT) cases were misinterpreted as suspicious for malignancy. Innate atypia of HCs and sampling misinterpretation were the causes of IEs in HT. One medullary and 1 follicular carcinoma were misinterpreted as suspicious for follicular neoplasm and BFN, respectively. Nine cases were better classified after review. Conclusion: Thyroid FNA IEs can be mitigated by meticulous screening and identification of all elements on FNA smears. Awareness of focal nuclear atypia in reactive cyst-lining and follicular cells in BFN, as well as HCs in HT, is highlighted. Adherence to The Bethesda System for Reporting Thyroid Cytopathology and consulting experienced cytopathologists significantly decrease IEs.

Deep Learning Fast Screening Approach on Cytological Whole Slides for Thyroid Cancer Diagnosis.

Simple SummaryPapillary thyroid carcinoma is the most common type of thyroid cancer and could be cured if diagnosed and treated early. In clinical practice, the primary method for determining diagnosis of papillary thyroid carcinoma is manual visual inspection of cytopathology slides, which is difficult, time consuming and subjective with a high inter-observer variability and sometimes causes suboptimal patient management due to false-positive and false-negative results. This study presents a fast, fully automatic and efficient deep learning framework for fast screening of cytological slides for thyroid cancer diagnosis. We confirmed the robustness and effectiveness of the proposed method based on evaluation results from two different types of slides: thyroid fine needle aspiration smears and ThinPrep slides.Thyroid cancer is the most common cancer in the endocrine system, and papillary thyroid carcinoma (PTC) is the most prevalent type of thyroid cancer, accounting for 70 to 80% of all thyroid cancer cases. In clinical practice, visual inspection of cytopathological slides is an essential initial method used by the pathologist to diagnose PTC. Manual visual assessment of the whole slide images is difficult, time consuming, and subjective, with a high inter-observer variability, which can sometimes lead to suboptimal patient management due to false-positive and false-negative. In this study, we present a fully automatic, efficient, and fast deep learning framework for fast screening of papanicolaou-stained thyroid fine needle aspiration (FNA) and ThinPrep (TP) cytological slides. To the authors’ best of knowledge, this work is the first study to build an automated deep learning framework for identification of PTC from both FNA and TP slides. The proposed deep learning framework is evaluated on a dataset of 131 WSIs, and the results show that the proposed method achieves an accuracy of 99%, precision of 85%, recall of 94% and F1-score of 87% in segmentation of PTC in FNA slides and an accuracy of 99%, precision of 97%, recall of 98%, F1-score of 98%, and Jaccard-Index of 96% in TP slides. In addition, the proposed method significantly outperforms the two state-of-the-art deep learning methods, i.e., U-Net and SegNet, in terms of accuracy, recall, F1-score, and Jaccard-Index (). Furthermore, for run-time analysis, the proposed fast screening method takes 0.4 min to process a WSI and is 7.8 times faster than U-Net and 9.1 times faster than SegNet, respectively.

Constitutive Cytomorphologic Features of Medullary Thyroid Carcinoma Using Different Staining Methods.

(1) Background: Accurate preoperative identification of medullary thyroid carcinoma (MTC) is challenging due to a spectrum of cytomorphologic features. However, there is a scarcity of studies describing the cytomorphologic features as seen on fine-needle aspiration (FNA) smears prepared using different staining methods. (2) Methods: We performed a retrospective study on MTC cases with available FNA slides from 13 hospitals distributed across 8 Asia-Pacific countries. The differences in the constitutive cytomorphologic features of MTC with each cytopreparatory method were recorded. A comparative analysis of cytologic characteristics was carried out with appropriate statistical tests. (3) Results: Of a total of 167 MTC samples retrospectively recruited, 148 (88.6%) were interpreted as MTC/suspicious for MTC (S-MTC). The staining methods used were Papanicolaou, hematoxylin-eosin, and Romanowsky stains. Seven out of the eleven cytologic criteria can be readily recognized by all three cytopreparatory methods: high cellularity, cellular pleomorphism, plasmacytoid cells, round cells, dyshesive cells, salt-and-pepper chromatin, and binucleation or multinucleation. An accurate diagnosis was achieved in 125 (84.5%) of the 148 samples whose FNAs exhibited five or more atypical features. Conclusions: The present work is the first study on MTC to compare the morphological differences among the cytologic staining techniques. We investigated the constitutive features and the reliability of diagnostic parameters. A feasible scoring system based upon cytomorphologic data alone is proposed to achieve a high degree of diagnostic accuracy.

Ultrasound-guided fine needle aspiration cytology and ultrasound examination of thyroid nodules in the UAE: A comparison

Thyroid nodules are a common clinical finding and most are benign, however, 5-15% can be malignant. There is limited regional data describing the accuracy of ultrasound-guided fine needle aspiration (FNA) cytological examination compared to ultrasound examination of thyroid in patients who have undergone thyroid surgery. A retrospective analysis of ultrasonographic (US) reports, FNA cytology reports and histopathology reports of 161 thyroid nodules presented at the endocrine center at Tawam hospital in Al Ain city, the United Arab Emirates during the period 2011-2019 was performed. US reports and images with FNA cytopathology reports and slides were reviewed by an independent radiologist and pathologist. In total, 40 nodules were reported as benign by US examination, while very low suspicious, low suspicious, intermediate suspicious and highly suspicious categories were reported in 21, 41, 14 and 45 nodules respectively. In addition, 68 nodules were reported as benign (Bethesda category II), while atypical follicular cells of unknown significance (Bethesda category III), follicular neoplasm (Bethesda category IV), suspicious for malignancy (Bethesda category V), and malignant (Bethesda category VI) categories were reported in 33, 9, 24 and 27 nodules respectively. The risk of malignancy for US benign nodules was 5%, while the risks of malignancy in very low suspicious, low suspicious, intermediate suspicious and highly suspicious nodules were 52%, 36%, 100% and 87%, respectively. The risk of malignancy for Bethesda category II was 3%, while the risks of malignancy in category III, IV, V and VI were 58%, 67%, 96% and 100%, respectively. Thyroid FNA cytological examination and ultrasonography are key tools in predicting malignancy in thyroid nodules. Thyroid nodules with the diagnosis of Bethesda category III & IV run a high risk of malignancy thus more vigilance is required.

Cytologic diagnosis of medullary thyroid carcinoma in the Asia-Pacific region.

The accurate preoperative identification of medullary thyroid carcinoma (MTC) is challenging due to the rarity of tumor and variable cytologic appearance. The Asian experience with diagnosing MTC by fine-needle aspiration (FNA) was scarcely reported. Cases of MTC with available FNA slides were enrolled from 13 hospitals representing 8 Asia-Pacific countries. Clinicopathological information, including sample preparation technique, staining method, original cytologic diagnosis and review diagnosis were collected. Of a total of 145 MTC cases retrospectively recruited, 99 (68.3%) were initially interpreted as MTC/suspicious for MTC (S-MTC). The distribution of original FNA diagnostic categories was not associated with the staining method or sample preparation technique. The staining methods used were Papanicolaou, hematoxylin-eosin and Romanowsky stains. Liquid-based cytology (LBC) was used only in three countries. After reviewing all cases, the diagnostic rate of MTC/S-MTC increased to 91.7% (133/145). Cases with initially unrecognized MTC had either marked pleomorphism or cytology mimicking papillary carcinoma or follicular neoplasm. Although LBC provided certain benefits, there was no significant difference in diagnostic accuracy between conventional smear and LBC. Immunocytochemistry was available in 38 cases (26.2%), all of which were correctly recognized as MTC. Our report summarizes how MTC is handled in contemporary Asian thyroid FNA practice. Although the detection rate of MTC by cytology alone is less satisfactory, integration with ancillary tests could achieve an excellent performance. The recognition of constitutive cytomorphologic features is needed for each cytopreparatory method, which may result in a lower threshold to initiate further workup for MTC.

Noninvasive follicular thyroid neoplasm with papillary-like nuclear features: An interobserver study of key cytomorphological features from a large academic medical centre.

Because of the indolent nature of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) and potential requisite for a more conservative treatment, it is crucial to identify features of this entity preoperatively. Our group recently published findings that there are several cytomorphological features that may be used as clues to distinguish NIFTP, papillary thyroid carcinoma (PTC) and follicular adenoma (FA) on fine needle aspiration. Therefore, we aimed to determine the interobserver reproducibility of these findings. Presurgical fine-needle aspiration slides from NIFTP (n=30), classic PTC (n=30) and FA (n=30) collected from 1/2013-8/2016 were reviewed by seven cytopathologists blindly. Presence of selected cytomorphological features was recorded and compared to determine percent agreement and inter-rater reliability among study cytopathologists using Gwet's AC1 statistics. For all the cytomorphological features, the overall percent agreement amongst the pathologists ranged between 65.1% and 86.8% (Gwet's AC1 0.30-0.80). There was substantial or almost perfect agreement (Gwet's AC1>0.60) in seven cytomorphological features in the classic PTC group, in six features in the NIFTP group and in five features in the FA group. There were no features with poor agreement (Gwet's AC1<0.0). The current study supports the reproducibility of our previous findings. The high level of agreement amongst pathologists for these groups, and particularly the NIFTP group, supports the notion that when viewed in combination as a cytological profile, these cytomorphological features may assist the cytopathologist in raising the possibility of NIFTP preoperatively. This can potentially aid clinicians in deciding whether more conservative treatment may be appropriate.

Recurrent Primary Angiosarcoma of Breast–A Rare Entity

Angiosarcoma of the breast is a rare tumour with a reported incidence of only 0.04% of all breast malignancies. A 34-year-old lady presented with a lump in her right breast. In view of haemorrhage and few stromal fragments on Fine Needle Aspiration Cytology (FNAC), a diagnosis of phylloides was given. Simple mastectomy was done which showed a tumour with areas of haemorrhage. On microscopy anastomosing irregular vascular channels filled with RBC’s were noted. Sheets of tumour cells with high mitotic activity was observed at places. There were areas of haemorrhage and ischaemic necrosis. A diagnosis of angiosarcoma was made which was confirmed by immunohistochemistry (CD 34 and factor VIII). Eight months later she presented with a recurrent lump in her right chest wall following which lumpectomy was done. Microscopy showed similar features as in the earlier mastectomy specimen. On reviewing the fine needle aspiration slides we realised that the endothelial fragments were mistaken for stromal fragments.The importance of this case report lies in the fact that primary breast angiosarcoma is a rare tumour which can develop without radiation exposure/history of previous breast surgery. The presence of spindle cells on fine needle aspiration slides necessitates careful examination and evaluation to rule out malignancy. It is important that the cytopathologist is aware of this possibility, to enable an early diagnosis

Analytical validity of a microRNA-based assay for diagnosing indeterminate thyroid FNA smears from routinely prepared cytology slides.

BACKGROUNDThe majority of thyroid nodules are diagnosed using fine‐needle aspiration (FNA) biopsies. The authors recently described the clinical validation of a molecular microRNA‐based assay, RosettaGX Reveal, which can diagnose thyroid nodules as benign or suspicious using a single stained FNA smear. This paper describes the analytical validation of the assay.METHODSMore than 800 FNA slides were tested, including slides stained with Romanowsky‐type and Papanicolaou stains. The assay was examined for the following features: intranodule concordance, effect of stain type, minimal acceptable RNA amounts, performance on low numbers of thyroid cells, effect of time since sampling, and analytical sensitivity, specificity, and reproducibility.RESULTSThe assay can be run on FNA slides for which as little as 1% of the cells are thyroid epithelial cells or from which only 5 ng of RNA have been extracted. Samples composed entirely of blood failed quality control and were not classified. Stain type did not affect performance. All slides were stored at room temperature. However, the length of time between FNA sampling and processing did not affect assay performance. There was a high level of concordance between laboratories (96%), and the concordance for slides created from the same FNA pass was 93%.CONCLUSIONSThe microRNA‐based assay was robust to various physical processing conditions and to differing sample characteristics. Given the assay's performance, robustness, and use of routinely prepared FNA slides, it has the potential to provide valuable aid for physicians in the diagnosis of thyroid nodules. Cancer Cytopathol 2016;124:711–21. © 2016 Rosetta Genomics. Cancer Cytopathology published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

Metastatic HPV-related head and neck squamous cell carcinoma to the lung and mediastinal lymph nodes in aspirated cytology material: A diagnostic pitfall.

Although HPV-related head and neck squamous cell carcinoma (HNSCC) has good prognosis, a small proportion of patients develop distant metastases and have worse outcomes. Such metastases can be particularly difficult to diagnose in the lung and mediastinum, where they show extensive morphologic overlap with primary pulmonary neoplasms. This case series discusses metastatic HPV-related HNSCC in lung and mediastinal fine needle aspiration (FNA) cytology. The cytopathology archives were searched for lung and mediastinal FNA specimens of patients with HNSCC. Specimens were included if either the index FNA or the patient's original HNSCC was positive for HPV by DNA in-situ hybridization (ISH). Ten such cases were identified. Patient demographics and primary tumor details were tabulated from the electronic medical record. All FNA slides and stains were reviewed. The ten patients (mean age of 58.2 years) included 4 smokers. Smears from all cases were hypercellular and hyperchromatic, with focal keratinization in 6/10 (60%). Core biopsies and cell blocks showed basaloid morphology with variable amounts of necrosis. All ten FNAs were diffusely positive for p16 (100%) and 7/9 cases stained (77.8%) were positive for HPV DNA ISH. Metastatic HPV-related HNSCC to the lung and mediastinal lymph nodes share the characteristic basaloid, minimally keratinizing morphology seen in Primary HNSCC cytology. Poorly differentiated pulmonary squamous cell carcinoma and neuroendocrine carcinomas are the primary differentials. Although primary lung neoplasms are not HPV-related, p16 positivity can be seen in both squamous cell and small cell lung carcinomas. HPV ISH allows definitive diagnosis of metastatic HPV-related HNSCC.

Fine Needle Aspiration Cytology of Breast Pure Squamous Cell Carcinoma: Cytologic Differential Diagnosis from Mammary Lesions Associated with Aberrant Squamous Cells

Fine needle aspiration (FNA) cytology of two cases of breast pure squamous cell carcinoma (SCC) and one invasive carcinoma mixed with SCC is reported. Cytopathologic findings of breast cancer vary greatly because mammary carcinomas are composed of many subtypes. Squamous cells, both malignant and benign, are occasionally seen in the breast FNA smears. Many differential diagnoses including various neoplastic and non-neoplastic diseases should be raised when squamous cells are observed in the mammary FNA smears. Squamous cells endowed with severe grade atypia obviously indicate malignancy. However, apparently benignlooking squamous cells are seen in the FNA specimens of both malignant and benign breast disorders. Accurate interpretation of cytologic findings is essential for discriminating malignant diseases from benign conditions. As prognosis and therapeutic options depend on tumour subtypes, deducing tumour histologic subtypes from cytologic specimens is useful for the appropriate treatment planning. FNA samples of pure SCC and invasive carcinoma mixed with SCC were examined in detail in an attempt to clearly define the cytopathologic characteristics of pure breast SCC. Careful review of the FNA slides of these cases helped us to recognize cytomorphologic findings useful in the differential diagnoses and predicting of histologic pictures of mammary disorders containing squamous cells.

Utility of BRAF mutation detection in fine-needle aspiration biopsy samples read as "suspicious for papillary thyroid carcinoma".

The purpose of this study was to evaluate the diagnostic utility of BRAF mutation testing on thyroid nodules "suspicious for papillary thyroid carcinoma" (PTC) cytology. A chart review of patients with fine-needle aspiration (FNA) results "suspicious for PTC" with subsequent thyroidectomy was performed. Corresponding archived FNA slides underwent BRAF mutation testing. Sixty-six patients with FNA "suspicious for PTC" underwent thyroidectomy. Forty-two (63.6%) had PTC diagnosed on final histopathology, whereas 21 (31.8%) had benign findings. Thirty-five patients (83%) with histologically proven PTC underwent total thyroidectomy, whereas 7 (17%) underwent hemithyroidectomy. BRAF mutation was detected in 17 of 49 samples (34.6%) available for testing and had 45.5% sensitivity, 87.5% specificity, 88.2% positive predictive value (PPV), and 43.8% negative predictive value (NPV) for diagnosing PTC. Two of 4 patients (50%) who underwent hemithyroidectomy with subsequent completion thyroidectomy had mutated BRAF detected. BRAF testing is a useful adjunct to improve PPV for patients with "suspicious for PTC" cytology.

Fine-needle aspiration in a low-resource setting.

Fine-needle aspiration in a low-resource setting.

An algorithm for microscopic specimen delineation and focus candidate selection

In this paper, we compare four field-of-view (FOV) metrics that, when applied to a low-resolution image of a microscope slide, are capable of both accurately delineating the specimen and selecting a subset of focus candidate FOVs required for construction of high-resolution focus map. The metrics evaluated are: threshold index (TI) that measures image intensity; normalised auto-correlation index (NACI) that measures spatial image similarity; auto-phase correlation index (APCI) that measures image phase diversity; and entropy index (EI) that measures the predictability of image intensities. Experiments are undertaken on a data set of forty slides including PAP stained Thin-prep cervical cytology and breast fine-needle aspiration slides and haematoxylin and eosin (HE) stained histology slides. These slides were scanned on an automated bright-field microscope and chosen to be indicative of a variety pathology specimens, containing artefacts such as excess coverslip glue and ink markers. Results are presented on the performance of each metric for correct ranking/segmentation of foreground (specimen) from background, and subsequently selecting focus candidate FOVs characteristic of the specimen's focal plane(s). The experimental results demonstrate that while NACI, APCI and EI are all effective at specimen delineation, only APCI is capable of effectively selecting superior focus candidates and ignoring artefacts.

Left parotid mass in a 71-year-old woman

CASE PRESENTATION In October 2012, a 70-year-old black woman presented to her ophthalmologist, who noted that she had a left parotid mass. She was subsequently referred to a hospital to have the mass evaluated. It was unclear how long the mass was present, but there were no symptoms. The patient reported no family history of malignancy, and her health was significant for type 2 diabetes mellitus, non-insulin-dependent. The patient had smoked 2 packs a day for 22 years, but she stated that she had quit 40 years ago. She stated she drank 1 to 2 alcoholic beverages per month. At an outside hospital, ultrasonography found a heterogeneously hypoechoic mass with irregular margins containing focal necrosis with possible deep extracapsular extension measuring 1.96 2.49 cm. No increased flow signal was seen on Doppler mode. A fine-needle aspiration (FNA) of the parotid mass was suspicious for acinic cell carcinoma. Within the same month, the patient was sent to Memorial Sloan Kettering Cancer Center. A computed tomography (CT) scan of the neck found an ill-defined, enhancing soft tissue mass in the left parotid tail with no deep parotid involvement, measuring 2.3 1.9 2.0 cm (Figure 1). No abnormal lymphadenopathy was noted in the neck. A second FNA taken of the left parotid found a salivary gland neoplasm with a cylindromatous pattern, which favored pleomorphic adenoma (Figure 2). The FNA slides from the previous hospital were also reviewed, and ultimately the diagnoses encompassed a differential diagnosis that included both acinic cell carcinoma and pleomorphic adenoma. A positron emission tomographyecomputed tomography scan found a 1.3 1.5-cm left parotid nodule with a maximum standardized uptake value of 2.7 (Figure 3). No hypermetabolic lymphadenopathy was noted in the neck.

Targeted next‐generation sequencing using fine‐needle aspirates from adenocarcinomas of the lung

Molecular testing of cancer is increasingly critical to medicine. Next-generation sequencing (NGS) provides comprehensive, unbiased, and inexpensive mutation analysis of multiple genes with a single test. However, to the authors' knowledge, the usefulness of NGS in fine-needle aspiration (FNA) specimens, which may be the only specimens available, is unknown. Non-small cell lung cancer (NSCLC) is an ideal model in which to evaluate cytopathologic applications of NGS because FNA is used for diagnosis and staging and specific molecular therapeutic targets in NSCLC are known. Herein, the performance and quality of targeted NGS in FNA specimens from a small series of lung adenocarcinomas is evaluated. Sequence data were generated from FNA specimens and paired formalin-fixed paraffin-embedded (FFPE) tissues from 5 patients with lung adenocarcinoma. DNA was isolated from FNA aspirate smears and cores of FFPE tissue. Multiplex sequencing of 27 cancer-related genes was performed after hybrid capture enrichment. Read-quality metrics and single-nucleotide variant calls were compared. The overall concordance of total reads across specimens was > 99% and the average concordance of single-nucleotide variants was 99.5%. The total reads generated, as well as the percentages of mapped, on-target, and unique reads were statistically indistinguishable (P > 0.05) between FFPE and FNA preparations. There also was no difference in the depth of sequencing coverage, including exon-level coverage in known lung cancer mutation hotspots. DNA isolated from FNA slides yields comprehensive, accurate, and statistically indistinguishable sequence information compared with that obtained from FFPE tissue. These results support the integration of NGS technologies into the standard cytopathology workflow. Cancer (Cancer Cytopathol) 2014;122:104-13. © 2013 American Cancer Society.

Nucleic acid recovery from thyroid fine-needle cytology slides

M uch interest has been focused in nucleic acid isolation from tissue samples stored for long periods and their utilization on molecular biomarkers research. However, sample fixation induces chemical modifications in macromolecules (1) that result in a laborious and difficult protocol to extract nucleic acid for molecular analysis. In this context, the article recently published by Kizys and cols. (2) contributed to show efficient methods of nucleic acid recovery from archived formalin-fixed/ paraffin-embedded (FFPE) and fine-needle aspiration (FNA) samples of thyroid tissue. Despite the efforts, the issue that remains a challenge in the most effective protocols is to obtain better yield and quality of DNA/RNA due to the damaging effect of the fixation process, particularly for RNA analysis. In contrast with the difficulty in obtaining conserved messenger RNA from archived samples, the fraction of microRNA (miRNA) is less affected by fixation and storage time (3,4). Mature miRNA is a single-stranded noncoding small RNA of ~19-22 nucleotide length that regulates gene expression at the post-transcriptional level (5). These molecules pair to 3’UTRs of target mRNA and thereby cause their silencing or degradation. The miRNA might be a promising prognostic and diagnostic biomarker for thyroid cancer (6). In this context, we have performed, in archived FNA-stained slides, the analysis of BRAFT1799A mutation (7) along with miRNAs expression, miR-146b (8) and let7-f (9), which were modulated in papillary thyroid carcinoma (PTC) (10,11). The acid guanidinium thiocyanate-phenol-chloroform method (12) using TRIzolTM enabled the RNA/DNA co-isolation from the same archived FNA, despite the reduced number of cells in the cytological slides. From the extracted RNA, we performed real-time PCR to amplify miRNA, and observed high expression of miR-146b in PTC compared with goiter (normalized expression miR-146b/RNU6B: PTC 21.55 ± 5.83* vs. goiter 0.18 ± 0.06, * P < 0.05), and no change in let7-f expression (normalized expression let7-f /RNU6B: PTC 0.30 ± 0.13 vs. goiter 0.07 ± 0.02, ns). In addition, DNA obtained was suitable for BRAF mutation analysis by RFLP-PCR (Restriction Fragment Length Polymorphism -PCR) in all FNA slides tested. Using this approach, we observed that it is possible to access both nucleic acids, RNA and DNA, from the same archived thyroid FNA-stained slide. Furthermore, DNA was adequate to perform mutation analysis, and the quality of total RNA and its amount was satisfactory to investigate several miRNAs from the same sample. In conclusion, this procedure could be useful in detecting molecular biomarkers and drawing correlations with cytological analysis, which might improve the accuracy of FNA as a diagnostic tool of thyroid nodule. 1 Department of Cell and Developmental Biology, Institute of Biomedical Sciences, Universidade de Sao Paulo (USP), Sao Paulo, SP, Brazil

A canine hemangiopericytoma case: Fine needle aspiration (FNA) cytology and histopathological findings

Veterinary cytology is gaining importance in diagnostics of skin and subcutaneous masses of dogs. A case of canine-hemangiopericytoma was described by cytopathological and histopathological findings on Fine needle aspiration (FNA) and excisional biopsy. A 13-year-old female terrier dog had a subcutaneous lesion on right leg. FNA was performed by the cytopathologist using 27-gauge needle and 10 ml. syringe attached to a syringe-holder. FNA and imprint slides from FNA aspirates and stained with May-Grunwald Giemsa,Papanicalau, Hematoxylin Eosin were prepared. Cyto-diagnosis was encountered with spindle cells. FNA can often provide information for a good diagnostic and management plan, especially for discrete, non-ulcerated lesions, allowing veterinarians to make more informed therapeutic choices