Management of thyroid cytological material, preanalytical procedures and bio-banking.

Thyroid nodules are common and are increasingly detected due to recent advances in imaging techniques. However, clinically relevant thyroid cancer is rare and the mortality from aggressive thyroid cancer remains constant. Fine needle aspiration cytology (FNAC) is a standard method for diagnosing thyroid malignancy and the discrimination of malignant nodules from goitre. As the examined nodules on thyroid FNAC are often small incidental findings, it is important to maintain a low rate of undetermined diagnoses requiring further clinical work up or surgery. The most important factors determining the accuracy of the cytological diagnosis and suitability for biobanking of thyroid FNACs are the quality of the sample and availability of adequate tissue for auxiliary studies. This article discusses technical aspects (preanalytics) of performing thyroid FNAC, including image guidance and rapid on-site evaluation, sample collection methods (conventional slides, liquid-based methods, cell blocks) and storage (bio-banking). The spectrum of special studies (immunocytochemistry on direct slides or liquid-based cytology, immunohistochemistry on cell blocks and molecular methods) required for improving the precision of the cytological diagnosis of the thyroid nodules is also discussed.

FNA diagnosis of poorly differentiated thyroid carcinoma. A review of the recent literature.

Poorly differentiated thyroid carcinoma (PDTC) is a follicular cell-derived tumour that was recognised as a distinct entity by the World Health Organisation in 2004. The natural history and pathological features of PDTC are reported to be intermediate between those of well-differentiated and undifferentiated (anaplastic) thyroid carcinomas. Preoperative identification of PDTC could facilitate better initial patient management in many cases, namely more extensive surgery, without any delay. However, according to some experts, a diagnosis of PDTC can only be rendered on histologic specimens based on criteria recommended in the Turin proposal. Although high-grade features (namely necrosis and mitoses) can be recognised in FNA material, other cytomorphological features have limited value for the preoperative diagnosis of PDTC and specific features for a definitive diagnosis of PDTC have not yet been clearly defined. Here, we review the current status and future prospects for cytological recognition of PDTC; we emphasise the features that should raise suspicion of this rare condition in FNA cytology and provide an update on molecular features and management of PDTC. Despite proposed histological criteria for the diagnosis of PDTC, its recognition on routine thyroid cytology presents a notable challenge. Current and future advances in molecular testing could contribute to the cytological diagnosis of PDTC.

Molecular imaging with (99m)Tc-MIBI and molecular testing for mutations in differentiating benign from malignant follicular neoplasm: a prospective comparison.

PURPOSE: To compare mutation analysis of cytology specimens and (99m)Tc-MIBI thyroid scintigraphy for differentiating benign from malignant thyroid nodules in patients with a cytological reading of follicular neoplasm. METHODS: Patients ≥18 years of age with a solitary hypofunctioning thyroid nodule (≥10 mm), normal thyrotropin and calcitonin levels, and a cytological diagnosis of follicular neoplasm were prospectively enrolled. Mutation analysis and (99m)Tc-MIBI scintigraphy were performed and patients were subsequently operated on to confirm or exclude a malignant lesion. Mutations for KRAS, HRAS and NRAS and for BRAF and translocations of PAX8/PPARγ, RET/PTC1 and RET/PTC3 were investigated. Static thyroid scintigraphic images were acquired 10 and 60 min after intravenous injection of 200 MBq of (99m)Tc-MIBI and visually assessed. Additionally, the MIBI washout index was calculated using a semiquantitative method. RESULTS: In our series, 26 % of nodules with a follicular pattern on cytology were malignant with a prevalence of follicular carcinomas. (99m)Tc-MIBI scintigraphy was found to be significantly more accurate (positive likelihood ratio 4.56 for visual assessment and 12.35 for semiquantitative assessment) than mutation analysis (positive likelihood ratio 1.74). A negative (99m)Tc-MIBI scan reliably excluded malignancy. CONCLUSION: In patients with a thyroid nodule cytologically diagnosed as a follicular proliferation, semiquantitative analysis of (99m)Tc-MIBI scintigraphy should be the preferred method for differentiating benign from malignant nodules. It is superior to molecular testing for the presence of differentiated thyroid cancer-associated mutations in fine-needle aspiration cytology sample material.

Laser capture microdissection is a valuable tool in the preoperative molecular screening of follicular lesions of the thyroid: an institutional experience.

OBJECTIVES: The application of molecular tests to thyroid fine needle aspiration (FNA) has been shown to be a valuable tool to better refine the pre-operative malignant risk of patients with indeterminate cytology results. In this study, we investigated the feasibility of using the laser capture microdissection (LCM) technique to obtain DNA and RNA for molecular tests in routine thyroid FNA smears. METHODS: Nine coupled FNA and histological retrospective cases and 31 prospective FNA cases with a follicular neoplasm/suspicious for a follicular neoplasm (FN/SFN) diagnosis were included in this study. Both cytological and histological specimens were investigated by direct sequencing and reverse transcription-polymerase chain reaction (RT-PCR) for BRAF and RAS mutations and for PAX8/PPARG and RET/PTC rearrangements, respectively. RESULTS: LCM yielded good DNA and RNA quality in all cases (100%) in both series, irrespective of the staining used (Giemsa, Papanicolaou, immunostain for thyroglobulin) and the cytology technique (conventional or liquid-based preparations). Total mutations found in the FNA and in the corresponding histological specimen in both series were: one PAX8/PPARG rearrangement in a follicular carcinoma (FC), four NRAS mutations [in two FCs, one papillary carcinoma and one follicular adenoma (FA)] and one HRAS mutation in one FA. The sensitivity was 67% and the specificity was 91%. CONCLUSIONS: LCM is a valuable tool to obtain good quality DNA and RNA for molecular tests in cytological material from thyroid FNA, and can be a useful option in the management of patients with an FN/SFN FNA diagnosis.

Cytologic features of focal papillary thyroid carcinoma arising within follicular adenoma: a masked cytomorphologic analysis of 17 cases.

BACKGROUND/OBJECTIVE: Focal papillary thyroid carcinoma (PTC) arising within a follicular adenoma (PTCFA) represents a clinically significant, but rare, histopathologic subset of papillary carcinomas whose cytologic features have not been well described. This uncommon presentation of PTC may contribute to a subset of thyroid aspirates interpreted as 'atypia of undetermined significance/follicular lesion of undetermined significance' (AUS/FLUS). STUDY DESIGN: Seventeen fine-needle aspiration biopsy (FNAB) cases diagnosed as 'PTCFA' on corresponding surgical excision were identified from the archival records of 2 large academic medical centers. A control group of 40 FNAB comprised of 20 follicular adenomas (FA) and 20 PTC was identified (based on the corresponding surgical pathology diagnosis) for comparison. All 57 FNAB were reviewed in a masked fashion and scored for a series of 31 cytomorphologic features. The intraclass correlation between diagnostic categories and overall agreement between cytopathologists was statistically evaluated. RESULTS: Aspirates of PTCFA were originally diagnosed as 'negative' (n = 3), 'AUS/FLUS' (n = 7), 'suspicious for a follicular neoplasm' (n = 3), 'suspicious for malignancy' (n = 3), and 'malignant' (n = 1). On masked review, the most common cytomorphologic features of PTCFA were a nonmacrofollicular cytoarchitectural pattern (71%), medium-large cell size (74%), and micronucleoli (79%). Intranuclear pseudoinclusions and a papillary architecture were absent in 85 and 88% of the cases, respectively. Relative to the 2 control groups, the PTCFA cases demonstrated overlapping features between FA and PTC for the majority of the 31 examined cytomorphologic features. CONCLUSION: PTCFA represent a rare subset of PTC that is difficult to recognize as PTC by FNAB. Most cases exhibit overlapping features between a benign thyroid nodule and conventional PTC, and they are often interpreted as 'AUS/FLUS'.

[Fine needle aspiration of the thyroid: the value of on-site evaluation]. / Ponction à l'aiguille fine de la thyroïde: intérêts de l'évaluation cytologique immediate.

Thyroid nodules are very common in the general population and most of them are benign. Fine needle aspiration (FNA) of the thyroid is routinely used because it is a rapid, simple, accurate and cost-effective technique which allows the adequate selection of patients who should be managed surgically. However, the diagnostic yield of FNA is variable and depends of multiple factors including the one who performs FNA, the aspiration and cytological slide preparation techniques, and cytopathologist's experience. On-site evaluation of thyroid FNA material and close clinico-pathological collaboration offer many advantages and optimizes the yield of FNA.

Natural history of SARS-CoV-2 infection in healthcare workers in Northern Italy.

At present, the time-frame used for the quarantine of individuals with coronavirus disease 2019 (COVID-19) is the entire duration of symptoms plus 14 days after symptom recovery; however, no data have been reported specifically for healthcare workers (HCWs). In the study population of 142 HCWs with COVID-19, the mean time for viral clearance was 31.8 days. Asymptomatic subjects cleared the virus more quickly than symptomatic subjects (22 vs 34.2 days; P<0.0001). The presence of fever at the time of diagnosis was associated with a longer time to viral clearance (relative risk 11.45, 95% confidence interval 8.66-14.25; P<0.0001). These findings may have a significant impact on healthcare strategies for the future management of the COVID-19 pandemic.

Diverse glandular pathologies coexist with high-grade squamous intraepithelial lesion in cyto-histological review of atypical glandular cells on ThinPrep specimens.

OBJECTIVE: To identify in cytology, high-grade squamous intraepithelial lesions with endocervical glandular extension in cases previously diagnosed as atypical glandular cells (AGC), analyse possible reasons for the diagnostic pitfall and document the frequency of glandular pathology coexisting with high-grade cervical intraepithelial lesion in histology. METHODS: Thirty-nine ThinPrep cervical smear (Pap) tests reported as AGC of undetermined significance and showing high-grade lesions on histology [cervical intraepithelial neoplasia (CIN) 2 or 3, endometrial or extrauterine adenocarcinoma] were reviewed retrospectively to identify the cases of high-grade squamous intraepithelial lesion with endocervical glandular extension, using the Bethesda 2001 system. Cyto-histological correlation was performed. RESULTS: A high frequency of diverse glandular pathologies coexisted with high-grade cervical intraepithelial lesions on histology. This included endocervical glandular extension in 63%, benign glandular pathology in 33% and pre-neoplastic or malignant glandular pathology (endocervical glandular dysplasia, adenocarcinoma in situ and metastatic breast carcinoma) in 17% cases. On cytology, the sensitivity was 40%, specificity was 80% and positive predictive value was 86% for endocervical gland extension in high-grade squamous intraepithelial lesions. CONCLUSIONS: Special efforts to recognize endocervical glandular extension in high-grade squamous intraepithelial lesions and glandular neoplasia coexisting with squamous intraepithelial lesions from the heterogeneous category of AGC can contribute to increasing the diagnostic accuracy. The identification of endocervical glandular extension on cervical cytology would alert the gynaecologist to perform a thorough assessment of the endocervix during colposcopy. This could also help to decide on the need to perform deeper conization rather than loop electrosurgical excision procedure to ensure negative margins when colposcopic biopsy shows CIN 2 or 3.

[Clinical isolates of Pseudomonas aeruginosa producers of extended spectrum beta-lactamases at a private institution in Cordoba]. / Aislados clínicos de Pseudomonas aeruginosa productores de beta-lactamasa de espectro extendido en un centro privado de Córdoba.

Extended spectrum beta-lactamases (ESBL) are capable of inhibiting the action of extended spectrum cephalosporins and monobactams. The objective of this work is to describe six isolates of ESBL producing Pseudomonas aeruginosa, retrieved from intensive care patients. The susceptibility test was performed by diffusion. For the phenotypic detection of ESBL, the following was assessed: the difference between ceftazidime ceftazidime/clavulanic acid (CAZ-CAC) and the synergy between imipenem-ceftazidime (IMI-CAZ) and cefepime-ceftazidime/clavulanic acid -ceftazidime (FEP-CAC-CAZ). The presence of metallo-beta-lactamases (MBL) was discarded through the double disc imipenem-EDTA/mercaptoacetic-meropenem (IMI-EDTA/SMA-MER) method. Molecular characterization of ESBL was performed by polymerase chain reaction (PCR) with blaGES primers. Synergy IMI-CAZ was observed in the studied strains; ESBL type GES was confirmed in five of them. The strategic location of the discs and the evaluation of alert signals for the detection of ESBL is essential, thus contributing to the correct recommendation of treatment in the clinical report.

Nabothian cyst content: A potential pitfall for the diagnosis of invasive cancer on Pap test cytology.

Nabothian cysts are common, benign findings of the squamocolumnar junction of the adult cervix. These cysts are filled with mucus and can also contain proteinaceous material, neutrophils, or neutrophil debris. Nabothian cysts can be broken by the spatula during smear taking, may stick to the brush and be smeared onto slides in conventional cytology or dissolved in the preserving solution for liquid-based cytology (LBC) preparations. The granular content of Nabothian cysts may be mistaken for the tumor diathesis (TD) pattern associated with invasive carcinoma. In the case described, the patient presented a high-grade squamous intraepithelial lesion associated with granular material (Nabothian cyst content) that we considered erroneously on LBC to be TD-like material, thus, raising the suspicion of invasive carcinoma. To the best of our knowledge, this is the first report showing that Nabothian cyst content may present a potential pitfall in the diagnosis of invasive carcinoma on LBC.

Predictive factors of vascular intima media thickness in HIV-positive subjects.

BACKGROUND: The predictive factors of intima media thickness (IMT) in the HIV-infected population are still poorly understood. PATIENTS AND METHODS: We studied three groups of subjects, aged 30-50 years, to find potential predictive factors of carotid and/or femoral thickening (IMT > 1 mm in at least one area): healthy controls (G1, n = 54), HIV-infected naive (G2, n = 53) and highly active antiretroviral treatment (HAART)-treated subjects (G3, n = 133). All the subjects underwent ultrasonography of the carotid and femoral vessels to evaluate IMT. RESULTS: Demographic characteristics of the three groups were comparable, except for gender (G1 had a higher percentage of females) and lipid levels (higher in G3). A total of 115 subjects (47.9%) had carotid and/or femoral IMT: 26 in G1 (48.1%), 21 in G2 (39.6%) and 68 in G3 (51.1%). Independent predictive factors of carotid and/or femoral IMT were older age (OR: 2.81, 95% CI: 1.95-4.04, P < 0.01, for each additional 5 years), triglycerides >or=150 mg/dL (OR: 2.66, 95% CI: 1.27-5.57, P < 0.001), serum glucose >or=110 mg/dL (OR: 5.24, 95% CI: 1.02-27.05, P = 0.04), high homocysteinaemia (OR: 2.75, 95% CI: 1.17-6.46, P = 0.02) and high body mass index (OR: 1.10, 95% CI: 1-1.22, P = 0.05 for each additional unit); females had a lower risk (OR: 0.38, 95% CI: 0.18-0.79, P < 0.01 versus males). HAART use was not associated with IMT (OR: 0.64, 95% CI: 0.27-1.53, P = 0.32 and OR: 0.80, 95% CI: 0.30-2.13, P = 0.20 for G3 and G2 versus G1, respectively). CONCLUSIONS: This study demonstrates that traditional risk factors for cardiovascular diseases overshadow the role of HAART in determining premature vascular lesions.

Cytology of benign multicystic peritoneal mesothelioma in peritoneal washings.

OBJECTIVE: To describe the cytological aspect of peritoneal washings in benign multicystic peritoneal mesothelioma (BMPM). METHODS: Three peritoneal washing specimens stained by standard cytological and histological procedures and analysed by light microscopy. RESULTS: The specimens showed an abundance of monomorphous mesothelial cells devoid of atypia or mitoses. The mesothelial cells were calretinin positive. They also showed numerous squamous metaplastic cells arranged in flat sheets or isolated cells. The background contained some inflammatory cells. CONCLUSION: The combination of cytology of the peritoneal washing, histology (cell block and surgical specimen) and clinical history allow differentiation of BMPM from other cystic lesions (cystic lymphangioma and malignant mesothelioma).

Detecting N-RAS Q61R Mutated Thyroid Neoplasias by Immunohistochemistry.

Recently, the immunohistochemistry (IHC) for N-RAS Q61R has been developed and commercialized for clinical practice. Here, we investigated the reliability of IHC to identify N-RAS Q61R mutated thyroid neoplasia. A series of 24 consecutive thyroid lesions undergone surgery following indeterminate cytology were enrolled. Paraffin sections were stained for IHC using the rabbit monoclonal anti-human N-RAS Q61R, clone SP174. N-RAS mutations in codon 61 were also investigated by automated sequencing. At histology, 12 cases of follicular carcinoma, cytologically defined as follicular lesions, 1 papillary cancer, 7 follicular adenomas, and 4 hyperplastic nodules were found. Of these, 4 showed a positive IHC for anti N-RAS antibody where N-RAS expression was detected mainly at cytoplasmic level with similar intensity of reaction. The remaining cases had negative IHC. A 100% concordance between IHC and molecular analysis for N-RAS Q61R was observed. In conclusion, this study shows high reliability of IHC to identify N-RAS Q61R mutated thyroid lesions with high cost-effectiveness. These data indicate the reliability of IHC to identify N-RAS Q61R mutated thyroid neoplasia and suggest to adopt this approach for a more accurate management of patients, when indicated.

DIAGNOSIS OF ENDOCRINE DISEASE: High-yield thyroid fine-needle aspiration cytology: an update focused on ancillary techniques improving its accuracy.

Thyroid fine-needle aspiration (FNA) cytology is a fast growing field. One of the most developing areas is represented by molecular tests applied to cytological material. Patients that could benefit the most from these tests are those that have been diagnosed as 'indeterminate' on FNA. They could be better stratified in terms of malignancy risk and thus oriented with more confidence to the appropriate management. Taking in to consideration the need to improve and keep high the yield of thyroid FNA, professionals from various fields (i.e. molecular biologists, endocrinologists, nuclear medicine physicians and radiologists) are refining and fine-tuning their diagnostic instruments. In particular, all these developments aim at increasing the negative predictive value of FNA to improve the selection of patients for diagnostic surgery. These advances involve terminology, the application of next-generation sequencing to thyroid FNA, the use of immunocyto- and histo-chemistry, the development of new sampling techniques and the increasing use of nuclear medicine as well as molecular imaging in the management of patients with a thyroid nodule. Herein, we review the recent advances in thyroid FNA cytology that could be of interest to the 'thyroid-care' community, with particular focus on the indeterminate diagnostic category.

Prefrontal D1 and ventral hippocampal N-methyl-D-aspartate regulation of startle gating in rats.

BACKGROUND: Sensorimotor gating, as measured by prepulse inhibition of the startle reflex, is deficient in schizophrenia patients, and in rats after specific manipulations of limbic cortico-striato-pallido-thalamic circuitry. For example, prepulse inhibition in rats is disrupted after D1 blockade in the medial prefrontal cortex, and after N-methyl-D-aspartate infusion into the ventral hippocampus. In the present study, we examined whether these two substrates form part of an integrated circuit regulating sensorimotor gating, which might contribute to the loss of prepulse inhibition in patient populations. METHODS: Prepulse inhibition was assessed in male Sprague-Dawley rats after systemic or intra-medial prefrontal cortex administration of the D1 antagonist, SCH 23390. Separate rats received intra-medial prefrontal cortex infusion of the retrograde transported label Fluoro-Gold. In rats with sham or electrolytic lesions of the medial prefrontal cortex, prepulse inhibition was tested after infusion of N-methyl-D-aspartate or vehicle into ventral hippocampus regions that were determined to send projections to the medial prefrontal cortex. RESULTS: Prepulse inhibition was disrupted after systemic SCH 23390 treatment and after infusion of SCH 23390 into medial prefrontal cortex sites within the prelimbic and cingulate cortices. Fluoro-Gold infusion into these medial prefrontal cortex sites labeled cells in the ventral hippocampus complex, including regions CA1 and entorhinal cortex. N-methyl-D-aspartate infusions into these ventral hippocampus regions disrupted prepulse inhibition in rats after sham but not electrolytic lesions of the medial prefrontal cortex. CONCLUSIONS: Prepulse inhibition appears to be regulated by interacting substrates within the ventral hippocampus and MPFC. Specifically, NMDA activation of the ventral hippocampus appears to disrupt prepulse inhibition in a manner that is dependent on the integrity of infralimbic or cingulate cortical regions that also support a D1-mediated regulation of prepulse inhibition. Conceivably, dysfunction within these hippocampal-frontal circuits may contribute to sensorimotor gating deficits in schizophrenia.

Cloning and characterization of two satellite DNAs in the low-C-value genome of the nematode Meloidogyne spp.

Two highly reiterated StyI satellite DNAs have been cloned from two nematode species: one from Meloidogyne hapla and another from M. incognita. The monomeric units of these two satellites have a repeat length of 169 and 295 bp, respectively. These StyI repeated element families constitute 5% of the M. hapla and 2.5% of the M. incognita haploid genomes. The A + T content is elevated in both families (i.e., 68% and 77%, respectively). Nucleotide methylation and transcriptional activity are negative. No similarity was found between the two satellites, nor to other known highly repetitive elements. These StyI satellite DNAs are quite homogenous in sequence, showing on average 3% and 3.5% divergence from their respective calculated consensus sequence. An internal subrepeating unit of about 11 bp is observed in the StyI satellite monomer sequences of M. hapla, suggesting that it could have evolved from a shorter sequence. Because of the small size of the Meloidogyne genome (51 Mb) and the abundance of repeated sequences, this genus approaches a limit in terms of coding fraction.

Evaluation of erythrocyte washing using the Haemonetics Model 102 Cell Washer.

The Haemonetics Model 102 Cell Washer was evaluated for efficiency of leukocyte removal and erythrocyte recovery. Groups of 25 units of blood were washed using five different protocols, including the methods recommended by the manufacturer. Mean erythrocyte recovery ranged from 83% to 91% and median residual leukocytes ranged from 1% to 4% for the five protocols. The mean erythrocyte recovery for units washed with 30 sec of increased pump speed was 95%, while it was 84% for units washed with 90 sec of increased pump speed (P less than 0.001). The median residual leukocytes was 1% for units washed after buffy coat removal and 4% for all other units (P less than 0.001). There was no other significant difference among the different protocols. These findings could not be explained on the basis of age of units, prewash erythrocyte mass, or prewash total leukocytes.

Assessment of necrosis and hypoxia in ductal carcinoma in situ of the breast: basis for a new classification.

Modern classifications of ductal in situ carcinoma (DCIS) of the breast suffer from unsatisfactory reproducibility in inter-observer circulation analyses. Ducts in DCIS are markedly enlarged in the range of 360 microm in diameter. Since the diffusion of oxygen from peri-ductal vessels is limited to 100 microm, cells in the center of DCIS are poorly oxygenated and become either necrotic or remain hypoxic but viable. There is evidence that such alternative fate is dictated by the biological characteristics of the neoplastic cells. Therefore, determination of presence or absence of necrosis in ducts up to 360 microm in diameter might represent a simple, reproducible, and biologically sound criterion to classify DCIS. In the present work, following this criterion, we classified 32 cases of intra-ductal lesions as either "necrotic" or "hypoxic" and tested the reproducibility of such classification using K statistics. These cases had already been circulated among a group of European pathologists, who classified the lesions using five different classifications. The K statistics value obtained with the presently proposed system was extremely high (0.91). It remains to be established whether the classification "necrotic/hypoxic" withstands large inter-observer circulation analyses, whether it is predictive of the clinical evolution of DCIS, and whether it might constitute a reproducible basis for selecting appropriate treatments.

Solitary fibrous tumour of the adrenal gland associated with pregnancy.

Solitary fibrous tumour (SFT), first described as a pleural lesion, has been reported in several extrathoracic sites over the past 10 years. We describe a SFT of the left adrenal gland incidentally discovered in a 23-year-old, 22-week pregnant woman and characterised by a rapid growth during the third trimester of pregnancy. Elevated serum and urinary levels of cortisol and elevated blood levels of delta 4 androstendione and 17-OH progesterone were observed. After spontaneous delivery, the patient underwent laparoscopic resectioning of the mass and of the left adrenal gland from which the tumour was apparently originating. The kidney was not involved, and no other abdominal tumours were found. Histological and immunohistochemical features were typical of SFT of pleura and other locations. Only one case of adrenal SFT is on record, and the adrenal gland is to be added to the long list of extrathoracic locations of SFT. The association with pregnancy was a previously unrecognised event in SFT. The focal expression of progesterone receptors in the tumour cells may be related to pregnancy. This observation prompted an analysis of steroid hormone receptors in SFT of classical sites (pleura). Two of five cases had focal progesterone receptors too, a finding which deserves further investigations in a much larger series of SFTs.