Intraoperative imprint cytology versus histological diagnosis for the detection of sentinel lymph nodes in breast cancer treated with neoadjuvant chemotherapy

OBJECTIVES: To compare imprint cytology and paraffin section histology for sentinel lymph node detection in women with breast cancer treated with neoadjuvant chemotherapy. METHOD: A cross-sectional study and report of the sentinel lymph node statuses of 64 patients with breast cancer who underwent intraoperative imprint cytology and neoadjuvant chemotherapy in a referral cancer institute in Rio de Janeiro, Brazil, between 2014 and 2016. RESULTS: The mean age was 51 years. The most common histological type was invasive ductal carcinoma (93.75%), and the most common differentiation grade was 2 (62.5%). Overall, 153 lymph nodes were identified, with a mean of 2.39/case. Thirty-four lymph nodes tested positive for malignancy by imprint cytology, and 55 tested positive by histology. Of the 55 positive lymph nodes, 41 (74.5%) involved macrometastases, and 14 (25.5%) involved micrometastases. There were 21 false negatives with imprint cytology, namely, 7 for macrometastases and 14 for micrometastases, resulting in a rate of 17.6%. The sensitivity of imprint cytology was 61.8%, with a specificity and positive predictive value of 100%, a negative predictive value of 82.4% and an accuracy of 86.3%. The method presented null sensitivity for the identification of micrometastases. CONCLUSIONS: The false-negative rate with imprint cytology was associated with the number of sentinel lymph nodes obtained. The rate found for complete response to neoadjuvant chemotherapy was comparable to the rates reported in the literature. The accuracy of imprint cytology was good, and its specificity was excellent for sentinel lymph node detection; however, the method was unable to detect lymph node micrometastases.

Increasing diagnostic effectiveness of thyroid nodule evaluation by implementation of cell block preparation in routine US-FNA analysis

ABSTRACT Objective Ultrasound-guided fine-needle aspiration (US-FNA) biopsy has proven to be an accurate and efficient tool in thyroid nodule evaluation. We evaluated whether cell block adds to the diagnostic accuracy of US-FNA. Subjects and methods Three hundred twenty-eight consecutive patients underwent US-FNA, cytology and cell block evaluation. Six slides were prepared for each patient and stained by Papanicolaou and Giemsa techniques. The residual hemorrhagic aspirate in the syringe and needle was fixed in 10% formalin and paraffin-embedded (cell block). The histological sections were examined as a complementary diagnostic tool to US-FNA. Results The study population comprised 89% females and the mean age was 57.4 ± 13.7 years. The mean nodule size was 2.3 ± 1.2 cm. US-FNA cytological results were as follows: Bethesda I, 17.1% (n = 56); Bethesda II, 61.6% (n = 202); Bethesda III, 9.5% (n = 31); Bethesda IV, 5.8% (n = 19); Bethesda V, 2.4% (n = 8), and Bethesda VI, 3.6% (n = 12). Cell blocks were obtained in 100% of cases and were considered diagnostic in 89.6%. Combined cytological and cell block (cyto-cell block) results were as follows: unsatisfactory, 4.3% (n = 14); benign, 72.6% (n = 238); indeterminate, 11.3% (n = 37); follicular lesion, 5.8% (n = 19); suspicious for malignancy, 2.4% (n = 8), and malignant, 3.6% (n = 12). The sensitivity and specificity for cyto-cell block was 100% and 90%, respectively, and the accuracy was 94%. Cyto-cell block analysis reduced the rate of unsatisfactory samples (p < 0.001). Conclusions The cyto-cell block interpretation improved the efficiency of US-FNA. This simple, fast and low-cost technique should be used as an adjunctive test in thyroid nodule evaluation. Arch Endocrinol Metab. 2016;60(4):367-73.

Sentinel lymph node biopsy in rats. Comparison between paraffin and frozen section analysis

PURPOSE: To simulate a lymph node metastasis in an animal model using activated carbon, assess their identification in frozen section analysis and compare with histopathological examination in paraffin. METHODS: Thirty two adult female rats were used. They received the carbon injection on its hind legs. Half of the rats was sacrificed on day one, and the other half after 21 days. Thus, 64 lymph nodes were dissected and split longitudinally. One half of the lymph node was sent immediately to frozen section analysis. The other half was fixed in 10% formaldehyde to be cut in paraffin. Slides were divided into quadrants and classified by the presence of carbon in these four quadrants_ They were also classified by the carbon staining intensity. RESULTS: Comparing the slides obtained in the first day and 21 days, there was a tendency of carbon to spread over time, but without statistical significance. The intensity did not alter over time. CONCLUSION: There was no concordance between the two methods of pathological analysis, however the actived carbon was seen in all lymph nodes. .

Incidencia de la eritroleucoplasia en la cavidad bucal / Incidence of erythroleukoplakia in the oral cavity

Se efectuó un estudio descriptivo y transversal de 20 mujeres y hombres mayores de 19 años, adictos al tabaco, atendidos en la consulta estomatológica del Policlínico de Especialidades del Hospital Provincial Docente Saturnino Lora Torres de Santiago de Cuba, desde septiembre del 2007 hasta igual mes del 2010, con vistas a evaluar los resultados del diagnóstico clinicohistopatológico, obtenido del examen de la cavidad bucal y de la biopsia realizada a los pacientes con eritroleucoplasia. Las muestras fueron fijadas y procesadas por la técnica convencional de inclusión en parafina, con lo cual se confirmó la presencia de cambios tisulares. En la casuística prevalecieron el sexo masculino, la ausencia de manifestaciones clínicas y la mucosa de carrillo como el sitio de ubicación más susceptible. Asimismo, las alteraciones hísticas más comunes fueron la hiperqueratosis y el infiltrado inflamatorio subepitelial.

A descriptive and cross-sectional study of 20 women and men older than 19 years, who were heavy smokers, assisted in the Stomatology Department of the Specialties Polyclinic from Saturnino Lora Torres Provincial Teaching Hospital in Santiago de Cuba was carried out from September, 2007 to the same month of 2010, with the objective of evaluating the results of the clinico-histopathological diagnosis, obtained from the examination to the oral cavity and from the biopsy carried out to patients with erythroleukoplakia. The samples were fixed and processed through the conventional technique of inclusion in paraffin, which confirmed the presence of tissue changes. Male sex, the absence of clinical manifestations and the cheek mucosa as the most susceptible location prevailed in the case material. Also, the most common tissue alterations were the hyperkeratosis and the subepithelial inflammatory infiltrate.

Caracterización histopatológica de la leucoplasia bucal en La Habana durante 10 años / Histopathological characterization in the leukoplakia buccalis in Havana during 10 years

La displasia epitelial es considerada el más fuerte predictor histológico de la futura transformación maligna de la leucoplasia bucal. El objetivo del presente trabajo fue determinar la presencia y severidad de la displasia epitelial y su asociación con las alteraciones histológicas de los tejidos epitelial y conectivo de la mucosa en la leucoplasia bucal. Se efectuó una investigación descriptiva y transversal de 83 biopsias procesadas mediante la técnica de inclusión en parafina y coloreadas con hematoxilina y eosina, de pacientes con diagnóstico de leucoplasia bucal entre los años 1991 y 2001, procedentes de la Facultad de Estomatología de la Universidad de Ciencias Médicas de La Habana, Cuba. Se encontró que 92,8 por ciento de los pacientes con diagnóstico clínico de esta enfermedad tuvieron displasia epitelial, de estos y acorde a su grado de severidad, se encontró displasia epitelial ligera en 53,0 por ciento, displasia epitelial moderada en 36,4 por ciento y displasia epitelial severa en 10,4 por ciento. La paraqueratosis se manifestó como sigue: asociada a la displasia epitelial ligera en 43,9 por ciento de los pacientes, a la displasia epitelial moderada en 78,6 por ciento y a la displasia epitelial severa en 100,0 por ciento. La hiperplasia epitelial se presentó en 57,1 por ciento de los casos y el infiltrado inflamatorio en 79,2 por ciento de las muestras examinadas. Se concluyó que en la muestra de leucoplasia bucal estudiada existió un porcentaje elevado de displasia epitelial, con predominio de su grado ligero y una asociación entre el grado de severidad con los diversos patrones de queratinización y la intensidad del infiltrado inflamatorio, lo que se consideró importante al analizar su evolución ante un diagnóstico determinado y su pronóstico.

The epithelial dysplasia is considered the stronger predictor of a future malignant transformation of the leukoplakia buccalis. The aim of present paper was to determine the presence and severity of this type of dysplasia and its association with the histological alterations of epithelial and connective tissues of mucosa in the leukoplakia buccalis. A cross-sectional and descriptive research was conducted in 83 biopsies processed by technique of paraffin inclusion and stained with Hematoxylin and Eosin of patients diagnosed with this entity between 1991-2001 from the Stomatology Faculty of the Medical Sciences University of La Habana. The 92.8 percent of patients with a clinical diagnosis of leukoplakia buccalis had slight epithelial dysplasia, from these and according to its grade of severity, there was found a slight epithelial dysplasia in the 53.0 percent, moderate epithelial dysplasia in the 36.4 percent and a severe epithelial dysplasia in 10.4 percent. The parakeratosis was manifested in the 43.9 percent associated with a slight epithelial dysplasia, 78.6 percent with a moderate dysplasia and in the 100 percent with the severe one. The epithelial dysplasia was present in the 57.1 percent of cases and the inflammatory infiltrate in the 79.2 percent of study samples. Authors conclude that in the study sample of leukoplakia buccalis there is a high percentage of epithelial dysplasia with predominance of its slight degree and a association between its severity degree with the different patterns of keratinization and the intensity of inflammatory infiltrate, something important in the analysis of its course in face a determined diagnosis y its prognosis.

Avaliação de combinações de técnicas alternativas de construção e montagem de blocos de tissue microarray / Evaluation of alternative technique combinations of building and preparation of tissue microarrays blocks

INTRODUÇÃO E OBJETIVOS: O objetivo deste estudo foi pesquisar diferentes métodos alternativos de tissue microarray (TMA) à técnica original e conduzir adaptações desses, combinando diferentes métodos de punção das amostras teciduais e de montagem dos blocos de TMA, de modo a introduzir no Laboratório de Patologia Bucal da Faculdade de Odontologia de Pernambuco da Universidade de Pernambuco (LPBFOP/UPE) técnicas de TMA facilmente operáveis, reproduzíveis e de baixo custo. RESULTADOS: Foram reproduzidas quatro técnicas de punção dos blocos doadores e duas de montagem dos blocos de TMA, resultando em oito combinações possíveis. Para cada combinação, foram confeccionados três blocos de TMA, contendo nove, 16 e 32 amostras, respectivamente, e avaliadas quanto a perda de amostras, custo, tempo de confecção e dificuldade. Para blocos com nove amostras, a combinação 2 mostrou-se a mais adequada; para blocos com 16, a combinação 6 foi constatada como a mais eficiente; e para blocos com 32, a combinação 1 apresentou o melhor custo-benefício. CONCLUSÃO: Foi concluído que a escolha da combinação a ser utilizada depende do número de amostras a serem colocadas nos blocos de TMA.

INTRODUCTION AND OBJECTIVES: The aim of this study was to investigate different alternative tissue microarray (TMA) techniques and to make adaptations, combining different tissue punch and TMA block construction techniques in order to introduce easily reproducible, operational and cost effective TMA techniques in the Oral Pathology Laboratory of Pernambuco College of Dentistry, State University of Pernambuco. METHODS: Four donor punch techniques and two TMA block construction techniques were performed, resulting in a total of eight possible combinations. For each combination three TMA blocks were made, containing 9, 16 and 32 samples, respectively. They were evaluated as to sample loss, cost effectiveness, construction time and difficulty. RESULTS: For blocks with 9 samples, combination 2 was the most appropriate; for blocks with 16, combination 6 was the most efficient; and for blocks with 32, combination 1 was the most cost effective. CONCLUSION: It was concluded that the combination choice depends on the number of samples to be put in TMA blocks.

Estudo comparativo de três diferentes procedimentos para extração de RNA a partir de amostras fixadas em formalina e embebidas em parafina / Comparative study of three different procedures for RNA extraction from formalin-fixed paraffin-embedded samples

INTRODUÇÃO: O desenvolvimento de métodos de extração de RNA a partir de amostras fixadas em formalina e embebidas em parafina (FFEP) possibilitou estudos retrospectivos de biologia molecular. Objetivos: Comparar a quantidade e a qualidade do RNA extraído de amostras FFEP a partir de três kits disponíveis comercialmente. MATERIAL E MÉTODO: Utilizando-se três diferentes procedimentos, o RNA total foi extraído de 14 blocos de parafina contendo fragmentos de carcinomas mamários, todos arquivados há 10 anos. A quantidade do RNA foi expressa em pg/µl; e a qualidade, pelo número de integridade do RNA (NIR), utilizando-se o Bioanalyzer da Agilent com o Pico LabChip. O RNA de maior NIR extraído de cada uma das 14 amostras foi amplificado por reação em cadeia da polimerase com transcrição reversa (RT-PCR) utilizando-se o gene G6PD, com primers designados para gerar fragmentos de 67, 151 e 242 pares de bases (pb). RESULTADOS: A média e a mediana da quantidade do RNA extraído para os três protocolos foram, respectivamente, 42,91 e 31,31 pg/µl. A média e a mediana do NIR foram, respectivamente, 1,8 e 2. Em todas as amostras, o gene G6PD foi amplificado para fragmentos de RNA de 67 e 151 pb. DISCUSSÃO: Como houve grande variação individual na quantidade e na qualidade do RNA extraído para cada amostra, os dados do presente estudo indicam que, se não for possível extrair RNA de uma determinada amostra na primeira tentativa, uma segunda extração deve ser realizada antes de se descartar essa amostra para testes de biologia molecular. CONCLUSÕES: Nos três procedimentos utilizados foi possível extrair RNA de qualidade aceitável para amplificação por RT-PCR (com NIR > 1,4).

BACKGROUND: The development of methods for RNA extraction from formalin-fixed paraffin-embedded (FFPE) samples has allowed retrospective studies of molecular biology. OBJECTIVES: To compare the quantity and quality of RNA extracted from FFPE samples using three commercially available kits. Material and methods: Using three different procedures, the total RNA was extracted from 14 paraffin blocks containing fragments of mammary carcinomas, which had been archived for 10 years. The quantity of RNA was expressed in pg/µl; and the quality in RNA integrity number (RIN), by using the Agilent Bioanalyser with Pico LabChip. The RNA with higher RIN extracted from each of the 14 samples was amplified by reverse transcriptase-polymerase chain reaction (RT-PCR) using the G6PD gene with primers designed to create fragments with 67, 151 and 242 base pairs (bp). RESULTS: The mean and median of RNA quantity extracted for the three procedures were respectively 42.91 and 31.31 pg/µl. The mean and median of RIN were respectively 1.8 and 2. In all the samples, the G6PD gene was amplified for RNA fragments with 67 and 151 bp. DISCUSSION: Due to the significant individual variation in quantity and quality of the extracted RNA from each sample, the data from the present study show that, if it is not possible to extract RNA from a given sample in the first attempt, a second extraction should be performed before excluding this sample. CONCLUSION: It was possible to extract RNA with acceptable quality for amplification by RT-PCR (RIN > 1.4) in the three procedures used.

Comparison of multiplex PCR and acid fast and auramine -rhodamine staining for detection of Mycobacterium tuberculosis and non tuberculosis mycobacteria in paraffin- embedded pleural and bronchial tissues with granulomatous inflammation and caseous necrosis

The aim of this study was to compare the sensitivity and specificity of Acid fast and Auramine-Rhodamine staining and Multiplex PCR for the detection of Mycobacterium tuberculosis complex and non tuberculosis Mycobacteria on formalin fixed paraffin embedded tissues [FFPE]. Forty cases of FFPE pleural and bronchial tissue with chronic granulomatous inflammation and caseous necrosis and 10 cases with bronchogenic carcinoma as controls were investigated. We designed a Multiplex PCR DNA amplification method with two targets: 123bp DNA fragment from IS6110, which is present only in mycobacterium tuberculosis complex and 162bp DNA encoding Ag 85complex which is present in all of mycobacteria. The FFPE also stained by Acid fast and Rhodamine-Auramine staining method. In 26 samples [65%] 123 bp and 162 bp DNA fragments were detected together [12 in bronchial samples and 14 in pleural samples].The 162 bp fragment wasn't detected alone. The sensitivity of PCR was 65% and the specificity was 100%. Eleven cases were positive for Acid fast staining. There was 27.5% sensitivity and 100% specificity. Thirteen cases were positive for Auramine-Rhodamine staining [A-R-S]; there was 32.5% sensitivity and 100% specificity. All of the 10 controls were negative for 123 bp, 162 bp DNA fragments, for Acid fast and Auramine-Rhodamine staining. Multiplex PCR is a sensitive, specific and rapid method for detection of M tuberculosis in FFPE tissues

Avaliação do protocolo PCR4 de Marchetti em tecidos parafinizados para o diagnóstico da tuberculose cutânea e ganglionar / Evaluation of Marchetti PCR4 amplification assay to the diagnosis of cutaneous and lymph node tuberculosis from formalin-fixed paraffin-embedded tissue

INTRODUÇÃO: A tuberculose cutaneoganglionar (TbCG) corresponde a 25,4 por cento dos casos de tuberculose (Tb) extrapulmonar no estado do Amazonas. Os métodos tradicionais, bacteriológicos e histopatológicos envolvem algumas dificuldades diagnósticas, e a reação em cadeia da polimerase (PCR) surge como método alternativo, podendo propiciar resultados específicos e em menor tempo. Nesse sentido, verificou-se a acurácia do protocolo PCR4 de Marchetti et al. no diagnóstico da TbCG comparativamente aos métodos bacteriológicos e histopatológicos. MATERIAIS E MÉTODOS: Realizou-se o nested-PCR com oligonucleotídeos para a IS6110 do complexo do M. tuberculosis em 83 amostras parafinizadas, sendo 52 cutâneas e 31 ganglionares, de pacientes clinicamente suspeitos de TbCG. Todos os casos foram avaliados pelos métodos bacteriológicos e histopatológicos. Foi realizada análise da acurácia entre os resultados obtidos na PCR em relação ao cultivo e à histopatologia. RESULTADOS E DISCUSSÃO: A positividade da PCR em todos os casos estudados foi de 50,6 por cento (42/83), sendo de 59,6 por cento (31/52) em amostras cutâneas e de 35,5 por cento (11/31) nas ganglionares. Em ambos os grupos foram observados resultados falso-positivos e falso-negativos. Algumas hipóteses que podem justificar estes resultados estão relacionadas à presença da IS6110 em micobactérias ambientais da região amazônica e à não-padronização da amostra de DNA amplificado. CONCLUSÃO: O protocolo em avaliação apresentou positividade em percentual semelhante a diferentes protocolos existentes na literatura. Sugere-se o uso da PCR em tecidos parafinizados associada com o cultivo ou com a histopatologia para o diagnóstico definitivo de Tb ganglionar. Para as lesões cutâneas continua sendo necessária a busca de protocolo que amplie a acurácia do método.

BACKGROUND: Cutaneous lymph node tuberculosis (CLTb) represents 25.4 percent of all cases of extra-pulmonary Tb in the state of Amazonas. The current methods of diagnose including bacteriological and histopathological assays involve some technical difficulties, and the polymerase chain reaction - PCR arise as an alternative method allowing specific results faster than the others. In this context the accuracy of PCR4 Marchetti et al. protocol was compared with traditional methods. MATERIAL AND METHOD: Nested-PCR for IS6110 (123 pb) were applied on 83 CLTb suspicious formalin fixed and paraffin embedded samples of tissues (52 cutaneous and 31 lymph node), obtained from 1997 to 2002. All cases were evaluated by bacteriological and histopathological methods. Accuracy analyses were carried out between the PCR amplification results and those related on bacteriological and histopathological methods. RESULTS AND DISCUSSIONS: Positive results of PCR4 were about 50.6 percent (59.6 percent in cutaneous samples and of 35.5 percent in lymph nodes samples). In both groups were observed false-negative and false-positive results. Some hypotheses that explain those results are related to the presence of IS6110 in environmental mycobacterias in the Amazon region and the absence of standardized DNA concentration to amplification assays. CONCLUSIONS: The proposed protocol was as positive as others ones available in the literature. Definitive Tb diagnostic can be obtained on lymph node paraffin embedded PCR in association with bacteriological or histopathological method. A better accuracy of an amplification assay applied to cutaneous Tb suspicious lesions has to be still under research.

DNA extraction from formalin fixed and paraffin embedded tissues / 法医学杂志

Owing to the DNA degradation induced by formalin and the obstruction of paraffin to DNA extraction, it is difficult to recover high-quality DNA from Formalin fixed and paraffin embedded tissue (FFPET). In recent years, a lot of researches indicate that the DNA extraction from FFPET can be developed by improving the pretreatment, optimizing the digestion condition of proteinase, simplizing the procedures of the DNA extraction, purifying the extracted DNA and so on, which may pave a way for popularizing FFPET in DNA analysis.

Receptores estrogénicos: estandarización de un método para su demostración en muestras incluídas en parafina / Estrogen receptors: standardization of a method for it demonstration in paraffin included samples

Detecting estrogenic receptors on histological samples of breast tumors has a strong significance on the prognosis and treatment of this disease. The clasical detection method involves the incubation of the tissue with a monoclonal antibody from mouse directed against the estrogenic receptor (primary antibody) and a second incubation with another antibody that is directed against with an anti mouse I g G antibody. Afterwards this reaction is identified by an enzyme (generally horseradish peroxidase) that acquires a characteristic color that allows the identification of the cells that have the receptor. We demonstrate here that is possible to simplify the method modifyng qualitatively the treatment of the tissue samples before its exposure to the primary antibody and also shortening the incubation times with this antibody

A utilidade da inclusäo em parafina como técnica complementar no diagnóstico dos aspirados de tiróide / The usefulness of the inclusion in paraffin as complementary technique in the diagnosis of the thyroid aspirates

OBJETIVO. Estabelecer, em portadores de doenças tiroidianas, a importância da inclusäo em parafina, no diagnóstico de punçäo biópsia aspirativa com agulha fina (PBAAF), comparando seus resultados com aqueles obtidos por meio do esfregaço citológico convencional. METODOS. Foram analisados 100 aspirados de tiroidopatias e comparados os resultados fornecidos por esta técnica com aqueles obtidos por meio de esfregaços citológicos convencionais. Em nove casos, foi ainda possível cotejar estes resultados com os dados anatomopatológicos de peça cirúrgica. RESULTADOS. Em 93 dos 100 aspirados, pôde-se estabelecer, por meio da PBAAF com pelo menos uma das técnicas empregadas, a natureza da doença. Em quatro dos casos, a tiroidopatia foi diagnosticada somente pela análise do esfregaço, em 17 exclusivamente no material incluído e em 72 pelas duas técnicas (inclusäo e esfregaço, associadamente. Em 63 destes últimos 72 casos, o diagnóstico foi o mesmo, tanto no esfregaço como na inclusäo; nos restantes nove casos, os resultados no esfregaço divergiram dos obtidos na inclusäo. Em 70 das 100 PBAAF, o diagnóstico foi idêntico, independentemente de ser conclusivo ou inconclusivo. A natureza da doença teria sido estabelecida em 95,7 por cento dos casos, se se tivesse empregado como meio diagnóstico unicamente a técnica da inclusäo. Por outro lado, exclusivamente com esfregaços citológicos, ter-se ia uma percentagem de diagnóstico de 81,7 por cento. O estudo estatístico indica que a técnica de inclusäo em parafina levou a uma proporçäo de resultados conclusivos maior que a do esfregaço. CONCLUSäO. Os achados mostram que para diagnóstico das tiroidopatias por meio da PBAAF, sempre que possível, deve-se empregar, associadamente, as técnicas da inclusäo do aspirado e do esfregaço citológico convencional