IDENTIFICATION OF CANINE VISCERAL LEISHMANIASIS IN A PREVIOUSLY UNAFFECTED AREA BY CONVENTIONAL DIAGNOSTIC TECHNIQUES AND CELL-BLOCK FIXATION

After the report of a second case of canine visceral leishmaniasis (CVL) in São Bento da Lagoa, Itaipuaçu, in the municipality of Maricá, Rio de Janeiro State, an epidemiological survey was carried out, through active search, totaling 145 dogs. Indirect immunofluorescence assay (IFA), enzyme-linked immunosorbent assay (ELISA), and rapid chromatographic immunoassay based on dual-path platform (DPP(r)) were used to perform the serological examinations. The parasitological diagnosis of cutaneous fragments was performed by parasitological culture, histopathology, and immunohistochemistry. In the serological assessment, 21 dogs were seropositive by IFA, 17 by ELISA, and 11 by DPP(r), with sensitivity of 66.7%, 66.7% and 50%, and specificity of 87.2%, 90.2% and 94%, respectively for each technique. The immunohistochemistry of bone marrow using the cell-block technique presented the best results, with six positive dogs found, three of which tested negative by the other parasitological techniques. Leishmania sp. was isolated by parasitological culture in three dogs. The detection of autochthonous Leishmania infantum in Itaipuaçu, and the high prevalence of seropositive dogs confirm the circulation of this parasite in the study area and alert for the risk of expansion in the State of Rio de Janeiro.

Manipulation of a quasi-natural cell block for high-efficiency transplantation of adherent somatic cells

Recent advances have raised hope that transplantation of adherent somatic cells could provide dramatic new therapies for various diseases. However, current methods for transplanting adherent somatic cells are not efficient enough for therapeutic applications. Here, we report the development of a novel method to generate quasi-natural cell blocks for high-efficiency transplantation of adherent somatic cells. The blocks were created by providing a unique environment in which cultured cells generated their own extracellular matrix. Initially, stromal cells isolated from mice were expanded in vitro in liquid cell culture medium followed by transferring the cells into a hydrogel shell. After incubation for 1 day with mechanical agitation, the encapsulated cell mass was perforated with a thin needle and then incubated for an additional 6 days to form a quasi-natural cell block. Allograft transplantation of the cell block into C57BL/6 mice resulted in perfect adaptation of the allograft and complete integration into the tissue of the recipient. This method could be widely applied for repairing damaged cells or tissues, stem cell transplantation, ex vivo gene therapy, or plastic surgery.

Emprego do cell block de agarose como método complementar no diagnóstico citológico de tumores mamários caninos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparison of the efficacy of two commercially available media for culturing one-cell embryos in the in vitro fertilization mouse model

Objective: To examine the effects of two commercial media on the development of mouse ova fertilized in vitro to the blastocyst stage. Design: Animal model. Setting: Academic institution. Animal(s): Eight-week old, superovulated mice. Intervention(s): One-cell embryos cultured in vitro up to the blastocyst stage in potassium-enriched simplex optimized medium (KSOM) or G1/G2 medium. Main Outcome Measure(s): Blastocyst and hatching rates, total cell number count, and proportion of allocation of cells to the inner cell mass (ICM) and trophectoderm (TE). Result(s): The percentage of zygotes that developed to the blastocyst stage 96 and 120 hours after insemination was statistically significantly higher in the KSOM group. The percentage of blastocysts that partially or completely hatched by day 5 of culture was 84% and 71% for the KSOM and G1/G2 groups, respectively, showing a statistically significant difference between the groups. The mean number of ICM cells was 11.7 +/- 4.0 and 9.2 +/- 5.2 for the zygotes cultured in KSOM and G1/G2 media, respectively, revealing a statistically significantly higher cell number in the ICM of blastocysts derived from culture in KSOM medium. The ICM/TE ratio in the blastocysts cultured in KSOM or G1/G2 media was similar in both groups. Conclusion(s): Commercially available KSOM medium is superior to sequential G1/G2 media for culturing one-cell embryos up to the blastocyst stage in the mouse IVF model.

Non-small cell lung cancer: the prevalence of oncogenic driver mutations in Switzerland

Background. Predictive molecular marker analyses are standard of care in order to select non-small cell lung cancer (NSCLC) patients for targeted therapies. The aim of this study was to determine the prevalence of targetable oncogenic driver mutations including EGFR, KRAS, BRAF, HER2, ALK and ROS1 in Switzerland. Methods. Eight Swiss pathology institutions provided retrospective and anonymized data on their predictive molecular marker results performed on NSCLC from January 2012 to December 2014. Clinico-pathological data were recorded including age, gender, histological NSCLC-subtype and specimen type (biopsy, conventional cytology and cell block, respectively) used for molecular analyses. The prevalence of oncogenic mutations were calculated and compared between the centres. Results. A total of 4187 NSCLC were included into the study. The median age was 67 years and 55% were male patients. The tumor specimens for molecular analysis were mostly derived from biopsies (69%), 26% were from conventional cytology specimens and only in 5% from cell blocks. The most prevalent gene mutation was KRAS with 30.6% (range: 27.3-33.9%), followed by EGFR, BRAF and HER2 mutations in 12.2% (range: 10.2-13.1%), 3.9% (range: 2.5-5.6%) and 1.1% (range: 0.9-4.0%), respectively, without significant differences between the eight centers. Concomitant EGFR and KRAS mutations were detected in only 3/2027 NSCLC. In contrast the prevalence of ALK (mean 6.5%, range: 2.8-11.7%) and ROS1 (mean 2.4%, range: 1.5-6.2%) rearrangements varied significantly between centers. Conclusions. The Prevalence of EGFR, KRAS, BRAF and HER2 mutations are well in line with data from other West European populations. Concomitant EGFR, KRAS, BRAF or HER2 mutations are exceptional. ALK FISH results vary significantly between the eight centres. Concomitant ALK FISH positive results in NSCLC harbouring other oncogenic driver mutation have only been observed in two smaller centres, highlighting the difficulty in ALK-FISH interpretation.

Fine needle aspirate cell blocks are reliable for detection of hormone receptors and HER-2 by immunohistochemistry in breast carcinoma

Aims: To evaluate the reliability of fine needle aspirate cell blocks in the assessment of oestrogen receptor (ER), progesterone receptor (PR) and HER-2/neu proteins by immunohistochemistry in comparison with surgical specimens. Materials and methods: This is a retrospective study of 62 cases of breast carcinoma diagnosed by fine needle aspiration cytology (FNAC) and confirmed using the surgical specimen. Immunohistochemical tests were performed to assess the presence of oestrogen receptor (ER), progesterone receptor (PR) and HER-2/neu proteins in cell blocks and the corresponding surgical specimens. The cell block method used alcohol prior to formalin fixation. Cases with 10% or more stained cells were considered positive for ER and PR. Positivity for HER-2/neu was assessed on a scale of 0-3+. The criterion for positivity was a score of 3+. Results: Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and accuracy of the cell blocks in the investigation of ER, PR and HER-2/neu protein (3+) were (%): ER, 92.7, 85.7, 92.7, 85.7 and 90.3; PR, 92.7, 94.7, 97.4, 87.0 and 93.5; HER-2/neu, 70.0, 100.0, 100.0, 94.5 and 95.2. Discrepancies were seen in cell blocks in the 1+ and 2+ HER-2/neu staining scores: two of 12 cases scoring 2+ and one case of 26 scoring 1+ on cell blocks scored 3+ on surgical specimens. The correlation index between cell block and corresponding surgical specimen varied from 90% to 94%. Conclusion: Cell blocks provide a useful method of assessing ER, PR and HER-2/neu, mainly for inoperable and recurrent cases, but consideration should be given to carrying out FISH analysis on 1+ as well as 2+ HER-2/neu results. © 2012 Blackwell Publishing Ltd.

Anaplastic Thyroid Carcinoma Morphologic Findings and PAX-8 Expression in Cytology Specimens

Objective To evaluate the morphologic findings most encountered in anaplastic thyroid carcinomas (ATCs) and evaluate for the expression of PAX-8. Study Design The cytology specimens from 21 cases of ATC were evaluated for the following several cytologic criteria, cell morphology, pleomorphism, presence or absence of multinucleated cells, neutrophilic infiltrate and well-differentiated component. Immunohistochemical studies for PAX-8 were performed on cell blocks in selected cases. Results The most common morphology present was epithelioid, followed by spindle, multinucleated giant cell and rhabdoid, with 75% demonstrating more than 1 cell morphology. Marked pleomorphism (81%), neutrophilic infiltrate (90%) and necrosis (63%) were frequent. No nuclear grooves, colloid or well-differentiated component was idenrifled in any case. Immunocytochemical stains for PAX-8 were negative in 5 cases in which a cell block was available. Conclusion ATC is a tumor with diverse morphologic characteristics, and more than 1 cell morphology is usually present A neutrophilic infiltrate is a common finding and represents a clue to the correct diagnosis. (Acta Cytol 2010;54:668-672)

Isolated lymphadenitis due to Histoplasma capsulatum diagnosed by fine-needle aspiration biopsy and immunohistochemistry

In the disseminated form of histoplasmosis, isolation and further identification of Histoplasma capsulatum can be performed by several methods, namely, bone marrow aspiration, blood culture, and liver biopsy. Lymph node disease usually is diagnosed by excisional biopsy. Although fungal stains can identify this fungus, detection of specific antigens by immunohistochemistry shows a higher specificity and sensitivity. This approach can use the cell block method when the material is not sent to fungal cultures or fresh staining.

Comparison study of procedures for aspiration of biopsy samples

Introduction: The samples obtained from fine needle aspiration in liquid base cytology (FNAC) are often limited by scarce cellularity compared to the amount of colloid and presence of blood. Accordingly, it was important to test alternative technical procedures so as to maximize the cellularity of each sample. Objective: To compare the morphological features and cellularity of the three procedures in the FNAC cytodiagnosis of the thyroid. Methods: A total of 31 cases were each subjected to a cell block and ThinPrep preparation as well as a routine smear. The observation and analysis was performed using an optical microscope. Cytological diagnosis of each cell block case was objectively analysed for cellularity, presence of background and cellular preservation. Each smear and ThinPrep case was analysed for the presence or absence of cells. The data was analysed with Microsoft Excel (Office 2010) and SPSS (Statistical Package of Social Science) version 15.0 for Windows. Results: Of 31 cases, only 20 had thyroid cells in the cell block and ThinPrep preparations, however, all smear cases contained thyroid cells. Some background was found in 30 Cell block cases with only 5 of these containing well preserved cells for cytodiagnosis. Conclusions: As indicated by the results, smear is the most appropriate procedure for FNAC of the thyroid.

Accuracy of routine diagnostic tests used in paracoccidioidomycosis patients at a university hospital

The identification of appropriate laboratory measures to confirm clinical hypotheses is important in routine paracoccidioidomycosis medical care. The clinical records and laboratory reports of 401 paracoccidioidomycosis patients attended at the Tropical Diseases Area, Faculdade de Medicina de Botucatu, from 1974 to 2008 were reviewed. Direct mycological (DM), cell block (CB), histopathological (HP), and double immunodiffusion (DID) tests were evaluated before treatment. Typical Paracoccidioides brasiliensis yeast forms were observed in clinical specimens of 86% of the patients, but 14% were detected only by serological test. DM of 51 different tissue specimens produced 74.5% sensitivity, and 62.5% sensitivity was observed in 112 sputum samples. CB in 483 sputum samples generated 55.3% sensitivity. HP performed in 239 samples from different tissues revealed 96.7% sensitivity. Serology carried out in 351 patients and 200 healthy controls provided 90.0% sensitivity, 100.0% specificity, 100.0% positive predictive value, 85.1% negative predictive value and 93.6% accuracy. Comparisons of laboratory measurements performed in the same patient showed that sensitivity decreases from HP to DID to CB and DM, with the last two assays providing similar sensitivities. This study demonstrated that P. brasiliensis identification by HP, CB, and/or DM associated with DID is sufficient to establish the laboratorial diagnosis of paracoccidioidomycosis in practically all cases. (C) 2011 Royal Society of Tropical Medicine and Hygiene. Published by Elsevier Ltd. All rights reserved.

Sputum cytology in the diagnosis of pulmonary paracoccidioidomycosis

The presence of Paracoccidioides brasiliensis was determined in sputum samples from 50 patients with paracoccidioidomycosis using four different techniques: (a) cell-block preparations stained with silver methenamine, (b) direct microbiologie examination, (c) smears stained with Shorr, and (d) smears stained with silver methenamine. Overall, cell-block preparations and smears stained with silver methenamine proved to be the most sensitive techniques, followed by smears stained with Shorr and direct microbiologic examination in decreasing order of sensitivity. Sputum cytology tended to be less positive in patients with interstitial pulmonary lesions as determined by chest X-ray than in patients with alveolar lesions. In addition to its high sensitivity, cell-block preparation technique allows storage of blocks and slides for further studies. © 1991 Kluwer Academic Publishers.

Diagnóstico da paracoccidioidomicose em pacientes atendidos em serviços de rotina de hospital universitário

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo comparativo morfológico e imunoistoquímico entre citoinclusão e espécime cirúrgico de carcinoma primário da mama

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)