Cytopathological characterization of ascites for the diagnosis of serous ovarian carcinoma / 中华肿瘤杂志

Objective: To investigate the cytomorphological and immunocytochemical features of tumor cells in the ascites of ovarian plasmacytoma (SOC). Methods: Specimens of serous cavity effusions were collected from 61 tumor patients admitted to the Affiliated Wuxi People's Hospital of Nanjing Medical University from January 2015 to July 2021, including ascites from 32 SOC, 10 gastrointestinal adenocarcinomas, 5 pancreatic ductal adenocarcinomas, 6 lung adenocarcinomas, 4 benign mesothelial hyperplasia and 1 malignant mesothelioma patients, pleural effusions from 2 malignant mesothelioma patients and pericardial effusion from 1 malignant mesothelioma. Serous cavity effusion samples of all patients were collected, conventional smears were made through centrifugation, and cell paraffin blocks were made through centrifugation of remaining effusion samples. Conventional HE staining and immunocytochemical staining were applied to observe and summarize cytomorphological characteristics and immunocytochemical characteristics. The levels of serum tumor markers carbohydrate antigen 125 (CA125), carcinoembryonic antigen (CEA) and carbohydrate antigen 19-9 (CA19-9) were detected. Results: Of the 32 SOC patients, 5 had low-grade serous ovarian carcinoma (LGSOC) and 27 had high-grade serous ovarian carcinoma (HGSOC). 29 (90.6%) SOC patients had elevated serum CA125, but the difference was not statistically significant between them and patients with non-ovarian primary lesions included in the study (P>0.05); The serum CEA was positive in 9 patients with gastrointestinal adenocarcinoma and 5 patients with lung adenocarcinoma, and the positive rate was higher than that in SOC patients (P<0.001); The serum CA19-9 was positive in 5 patients with gastrointestinal adenocarcinoma and 5 patients with pancreatic ductal adenocarcinoma, and the positive rate was higher than that in SOC patients (P<0.05). The serum CA125, CEA and CA19-9 were within the normal range in 4 patients with benign mesothelial hyperplasia. LGSOC tumor cells were less heterogeneous and aggregated into small clusters or papillary pattern, and psammoma bodies could be observed in some LGSOC cases. The background cells were fewer and lymphocytes were predominant; the papillary structure was more obvious after making cell wax blocks. HGSOC tumor cells were highly heterogeneous, with significantly enlarged nuclei and varying sizes, which could be more than 3-fold different, and nucleoli and nuclear schizophrenia could be observed in some cases; tumor cells were mostly clustered into nested clusters, papillae and prune shapes; there were more background cells, mainly histiocytes. Immunocytochemical staining showed that AE1/AE3, CK7, PAX-8, CA125, and WT1 were diffusely positively expressed in 32 SOC cases. P53 was focally positive in all 5 LGSOCs, diffusely positive in 23 HGSOCs, and negative in the other 4 HGSOCs. Most of adenocarcinomas of the gastrointestinal tract and lung had a history of surgery, and tumor cells of pancreatic ductal adenocarcinoma tend to form small cell nests. Immunocytochemistry can assist in the differential diagnosis of mesothelial-derived lesions with characteristic "open window" phenomenon. Conclusion: Combining the clinical manifestations of the patient, the morphological characteristics of the cells in the smear and cell block of the ascites can provide important clues for the diagnosis of SOC, and the immunocytochemical tests can further improve the accuracy of the diagnosis.

Evaluation of immunocytochemistry on destained giemsa stained smears as an alternative to conventional technique

Introduction: Protocol for immunocytochemical (ICC) staining in May-Grünwald Giemsa (MGG)–stained smears has been difficult to establish. It is the need of the hour to be able to use prestained slides for ICC in specific cases to deliver timely diagnoses and reduce inconvenience to patients. Aims and Objectives: To evaluate and compare the use of MGG-stained smears for the purpose of ICC, after de-staining and saline rehydration to that of routine standard ICC. Materials and Methods: A prospective study was conducted on 40 FNAC samples: 25 cases of breast disease and 15 cases of reactive lymphoid hyperplasia known to express pancytokeratin and leukocyte common antigen (LCA)/CD45, respectively. Air-dried smears of each case were stained by standard MGG stain and after the report was dispatched, one smear was selected and sent for ICC. The smears were analyzed to determine the overall result and grade each smear semi-quantitatively with respect to staining-intensity, stain-localization, staining-uniformity, counter-staining, and background-staining. Observations and Results: The proposed protocol was inferior to conventional ICC in all the parameters, more pronounced in pancytokeratin than LCA/CD45. Only 8% of air-dried smears stained for pancytokeratin showed optimal stain intensity (as opposed to 44% of wet-fixed smears), whereas only 14.3% of air-dried smears were optimally stained for LCA (as opposed to 85.7% of wet-fixed smears). Conclusion: The proposed protocol of de-stained Giemsa smears as an alternative to conventional technique for ICC was unsuccessful in giving satisfactory results.

Metodología para la evaluación automatizada de la expresión del receptor de vitamina D mediante técnica inmunocitoquímica / Methodology of automated Vitamin D receptor immunocytochemical expression análisis

Introducción: la citología permite examinar células de un tejido de manera mínimamente invasiva, sin embargo, la capacidad de realizar técnicas complementarias como la inmunocitoquímica (ICQ) no está exenta de dificultades. Es el objetivo de nuestro trabajo presentar una metodología que permita la utilización de ICQ automatizada asociada a un análisis automatizado mediante técnica de patología digital. Métodos: se incluyeron 5 sujetos sanos y se obtuvieron muestras de superficie ocular utilizando un citocepillo. La muestra fue procesada de manera automatizada mediante citología en fase líquida. Posteriormente se realizó ICQ automatizada para detectar la positividad nuclear del receptor de vitamina D. Para la evaluación, se utilizaron dos métodos: cuantificación directa bajo microscopio de luz y análisis automatizado usando analizador de imágenes en las diapositivas digitales obtenidas con un Scanner. El porcentaje de positividad encontrado con ambos métodos fueron comparados utilizando la prueba de Kappa. Resultados: todas las muestras presentaron una celularidad adecuada. En todos los casos fue posible realizar ICQ automatizada, más aún, todas las muestras presentaron una calidad óptima. Al comparar ambos métodos (manual versus automatizado) se observó un nivel de acuerdo sustancial (Kappa=0,69). Conclusiones: la metodología presentada en este manuscrito permite la evaluación automatizada de marcadores inmunohistoquímicos de la superficie ocular de manera mínimamente invasiva, siendo similar al conteo manual, pero más objetivo y reproducible. Esta técnica podría ser útil para el estudio proteómico en patologías como la enfermedad por ojo seco.

Introduction: Cytology tests use small amounts of tissue samples for diagnosis as a minimally invasive technique; however, the ability to perform complementary methods such as immunocytochemistry (ICC) is not without difficulties. The aim of our work is to present a method that allows the use of automated ICC associated with an automated image analysis using digital pathology. Methods: Five healthy subjects were included, and ocular surface samples were obtained using a cytobrush. The sample was processed as liquid-based cytology. Automated ICC was subsequently performed to detect vitamin D receptor nuclear positivity. Two methods were used for evaluation: manual counting under a light microscope and automated analysis using an image analyzer on digitized slides. The percentage of positivity found in both methods was compared using the Kappa test. Results: All samples presented adequate cellularity. In all cases, it was possible to perform automated ICC; moreover, all samples presented optimal quality. When comparing both methods (manual versus automated), a substantial level of agreement was seen (Kappa=0.69). Conclusions. The method presented in this manuscript allows the minimally invasive automated evaluation of ocular surface ICC markers, being like manual counting but more objective and reproducible. This technique could be useful for proteomic study in pathologies such as dry eye disease.

Immunocytochemical evidence of histamine 1 and histamine 2 receptors on mice sperm

Histamine is a biogenic amine which is synthesised by L-histidine decarboxylase enzyme (HDC). The histamine 1 and 2 antagonist administrations have been highly reported to cause detrimental effect on sperm parameters, which arisen the speculation of histamine 1 (H1R) and histamine 2 (H2R) receptors might be present in sperm. The present study was aimed to provide evidence on the localisation of H1R and H2R on mice sperm through immunocytochemistry. The sperm was harvested from cauda epididymis. After one hour of incubation, sperm suspension was smeared onto a poly-lysine-coated slide and allowed to dry before fixation and permeabilisation processes. The primary antibody encoded for receptors was exposed to the fluorescently tagged antibody; fluorescein isothiocyanate (FITC) conjugate followed by nuclear staining with 4?, 6-diamino-2-phenylindole dihydrochloride (DAPI). The testis, stomach, and skin were used as the positive controls. Our data showed that both receptors have been expressed on the midpiece and acrosome of mice. The present result was the first discovery of the presence and immunolocalisation of H1R and H2R on mice sperm. Therefore, present study proposes that these receptors could be involved in calcium regulatory mechanism and protein phosphorylation which are responsible for fertilisation-related processes.

Papel da arquitetura nuclear no manejo do carcinoma de tiroide / Role of nuclear architecture in the management of thyroid carcinoma

One of the biggest dilemmas facing a cytopathology slide is the differential diagnosis of follicular thyroid lesions, grouped as follicular pattern lesions, which include goiter, follicular adenoma and follicular carcinoma, follicular variant of papillary thyroid carcinoma and non-invasive follicular thyroid neoplasm with papillary like nuclear features. Such lesions share many characteristics, which makes the proper identification of malignant follicular lesions a challenge. The cytology obtained through fine needle aspiration puncture is the most effective standard method for diagnosis of thyroid nodules, but its diagnostic efficacy clearly decreases in lesions of thyroid follicular pattern. Thus, a series of auxiliary tools for diagnoses, such as morphometry and nuclear texture analysis, have been increasingly used in the pathologist's practice, as an objective and reproducible tool. These are techniques, which depend on the incorporation of software to digital image analysis and can add accuracy to classical morphological analysis and immunohistochemistry in the evaluation of follicular pattern lesions. In addition to immunocytochemistry and molecular techniques, morphometry allows the estimation of parameters identified in individual cells and represents a tool that, based on quantitative parameters, translates reliable parameters for objective classification of the malignancy. This study aims to review the nuclear characteristics and their role in the diagnosis of follicular thyroid lesions.

Um dos maiores dilemas diante de uma lâmina de citopatologia é o diagnóstico diferencial de lesões foliculares da tiroide agrupadas como lesões de padrão folicular e que incluem; bócio, adenoma e carcinoma foliculares, carcinoma papilífero variante folicular e a neoplasia folicular não invasiva com características nucleares papilares (Uno de los mayores dilemas que presenta una muestra de citopatología es el diagnóstico diferencial de las lesiones foliculares tiroideas reunidas como lesiones de patrón folicular, que incluyen: bocio, adenoma folicular, carcinoma folicular, variante folicular del carcinoma papilar y la neoplasia folicular no invasiva con características nucleares de tipo papilar). Tais lesões compartilham muitas características, o que faz com que a identificação adequada de lesões foliculares malignas represente um desafio. A citologia obtida através de punção aspirativa por agulha fina é o método padrão mais efetivo para diagnóstico em nódulos de tiroide, mas sua eficácia diagnóstica diminui nitidamente em lesões de padrão folicular da tiroide (La citología por punción y aspiración con aguja fina es el método estándar más eficaz para el diagnóstico de los nódulos tiroideos, pero su eficacia diagnóstica se ve notablemente reducida en las lesiones de patrón folicular de la tiroides). Assim, uma série de ferramentas auxiliares ao diagnóstico, como a morfometria e a análise de textura nuclear, têm sido utilizadas cada vez mais na prática do patologista, como ferramenta objetiva e reproduzível. São técnicas que dependem da incorporação de softwares para análise digital de imagens e podem agregar acurácia à análise morfológica clássica e à imunohistoquímica na avaliação de lesões de padrão folicular (para el análisis de imágenes digitales y puede agregar precisión al análisis morfológico clásico y la inmunohistoquímica en la evaluación de lesiones de patrón folicular). Somando-se à imunocitoquímica e às técnicas moleculares, a morfometria permite a estimativa de parâmetros identificados em células individuais e representam uma ferramenta que, a partir de parâmetros quantitativos, traduz parâmetros confiáveis para classificação objetiva de malignidade. O objetivo deste estudo é rever as características nucleares e seu papel no diagnóstico de lesões foliculares da tiroide (es revisar las características nucleares y su papel en el diagnóstico de las lesiones foliculares tiroideas).

Diagnostic utility of cytospin, cell block and immunocytochemistry in pleural effusion cytology

Background: With the introduction of cytospin, the sensitivity of diagnosing malignancies has increased mainly due to the increase in cellular yield. Cell block also gives the advantage of ancillary testing and allows for retrospective studies. Immunocytochemical markers are used to differentiate and subtype various malignancies in body effusions.Aim of the study was to compare the morphological features of both technique and to assess the diagnostic utility of cell block methods in the cytodiagnosis of pleural effusions.Methods: This was a Prospective observational comparative study of two cytopreparatory techniques. All samples were examined and processed by cytospin and cell block techniques. Continuous data were expressed as Mean±SD (standard deviation) while categorical data were expressed in number, percentage and compared by chi-square (χ2) test.Results: The final diagnosis of both cytospin (147 cases) and cell block (150 cases) techniques was divided into four broad categories: Inadequate, Benign, Suspicious and Malignant. The significant diagnostic cytospin (AUC=0.857, p<0.001) in discriminating positive and negative malignant cases with 75.00% sensitivity (95% CI=53.3-90.2) and 100.00% specificity (95% CI=86.7-100.0) and with 100.0% positive predictive value and 81.2% negative predictive value. In contrast, cell block also showed significant diagnostic but with higher accuracy (AUC=1.000, p<0.001) and sensitivity 100.00% (95% CI=86.7-100.0) and specificity 100.00% (95% CI=86.7-100.0) and 100.0% positive predictive value and 100.0% negative predictive value than cytospin technique.Conclusions: Cell block as a technique should be used in routine practice as it not only increases the diagnostic yield but ancillary test can also be done.

Analysis of cervical liquid-based cytology results in Eskişehir, Turkey: correlation of cytology results with histology, immunocytocemical HPV Ab and HPV DNA results of 18404 women

Background: To analyze retrospectively the results of HPV DNA, immunocytochemical HPV antibody staining of gynecologic smear samples evaluated in a public hospital and to observe their compatibility with histologic diagnosis. At the same time, the contribution of ICC HPV Ab staining experience results to the morphological evaluation is discussed in this paper.Methods: In this study, liquid-based cytology test results of patients who applied to the gynecology between 2014 and 2017 were analyzed. The Ultravision Quanto Detection System was modified for immunocytochemical staining. HPV DNA tests were performed with the Qiagen Hybrid Capture test.Results: The 18404 test result was included in the research. The percentage of smear that epithelial cell atypia is seen was 3.4%, the rate of ASC/SIL was 1.89%. Compared to the first 3 years of the study, the increase in the rate of LSIL is seen with a partial decrease in ASCUS rate in year 2017 (p<0.05). The atypical positive test rate with histologic confirmation was 73.61%. Among 138 HPV Ab results, 58.7% of them were negative and 41.3% of them were positive. Sensitivity and specificity rates were determined 76.19% and 52.17% for SIL. Among 53 HPV DNA results (53% negative and 46.3% positive); sensitivity and specificity rates were determined 92.86% and 50% for SIL.Conclusions: İmmunocytochemical HPV Ab staining provided statistically significant contribution to LSIL (p<0.05). It is thought that it also will provide additional evidence for morphological findings while cytological evaluation and may help the clinician in managing the conditions for disease.

Evaluation of Effusion Fluid (Pleural and Peritoneal) with Special Reference To Cell Block Preparation and Immunohistochemistry for Differentiating Between Reactive Mesothelial and Malignant Effusion

Background: The serous cavities are lined by a single layer offlat mesothelial cells called the serosa. Normally these cavitiesare collapsed and contain only a small amount of fluid, enoughto lubricate the adjacent surfaces. Cytological examination ofserous fluid is of paramount importance. It reveals informationabout inflammatory conditions of serous membrane, infectionby bacteria, fungi, virus and presence of malignant cells.Differentiation of population of reactive mesothelial cells fromthose of malignant cells remains a diagnostic challenge inconventional cytological smear. To overcome this challenge,cell block technique along with immunocytochemistry gives abetter histoarchitectural pattern and support immensely forcategorising the effusion to be reactive or malignant.Aims and Objectives: To evaluate utility of cell blocktechnique in effusion fluid (pleural and peritoneal) using limitedimmunohistochemistry markers for differentiating betweenreactive mesothelial and malignant mesothelial cells.Materials and Methods: The present study was carried out inDepartment of Pathology at M.K.C.G MCH, Berhampur,Odisha over a time period from July 2016- July 2018. A total of90 cases were evaluated. The fluids were stained with routinecytological stains. Cases on evaluation of cytomorphology ifsuspicious for malignancy, cell block was prepared. Cell blockwere stained both for routine hematoxylin and eosin andimmunohistochemistry with EMA (Epithelial marker antigen) forepithelial cells and Calretinin for mesothelial cells.Results: A total of 90 cases were evaluated cytologically. 40cases showed benign features and 24 cases showedmalignant features on cytomorphology alone. 26 cases weresuspicious for malignancy which on cell block preparation andimmunocytochemistry were differentiated as benign (10 cases)or malignant (16 cases). EMA showed 97.5 % sensitivity and98% specificity. Calretinin showed 100 % sensitivity and 97.5%specificity.Conclusion: The use of cytopathology of pleural andperitoneal effusion is helpful for early diagnosis and treatment.The technique is cheap, easy to perform and produces speedydiagnosis. In the identification of malignant cells in effusion andits differentiation from cells showing reactive and degenerativechanges there were diagnostic difficulties in some of the cases.Immunocytochemistry is an important diagnostic tool ineffusion cytology.

Clinical study of brush liquid-based cytology combined with automatic immunohistochemistry in the classification and diagnosis of lung cancer / 中华肿瘤杂志

Objective@#To explore the feasibility of bronchoscopic brushing liquid-based slide cytology combined with automatic immunocytochemistry (ICC) for pathological typing of lung cancer.@*Methods@#A liquid-based thin-prep was prepared from 171 bronchoscopic brushing specimens of patients with pulmonary lesions. ICC was detected by automatic immunohistochemistry instrument while cytomorphological diagnosis was made. The results were compared with those of histopathological diagnosis.@*Results@#Among 171 patients, 130 (76.0%) could be classified by cell morphology alone, including 31 squamous cell carcinomas, 44 adenocarcinomas and 55 small cell carcinomas; 162 (94.7%) could be classified by cell morphology combined with ICC, including 38 squamous cell carcinomas, 61 adenocarcinomas and 63 small cell carcinomas (P<0.001). According to the gold standard of histopathological diagnosis, the coincidence rate of cytomorphology combined with ICC was higher than that of cell morphology alone. The coincidence rate of squamous cell carcinoma was increased from 85.2% to 97.1% (P=0.093), adenocarcinoma from 92.5% to 98.0% (P<0.001), and small cell carcinoma from 96.1% to 98.3% (P=0.465).@*Conclusion@#The combination of liquid-based thin-prep cytology and automatic immunohistochemistry can effectively improve the accuracy of pathological typing of brushing specimens under fiberoptic bronchoscopy, and provide more objective diagnostic results for clinical treatment.

Lucy-tag promotes expression of sea lamprey (Petromyzon marinus) olfactory receptor on cell membrane / 中国药理学通报

Aim To elucidate the effect of Lucy-tag polypeptide label on the expression of olfactory receptor membrane in sea lamprey ( Petromyzon marinus). Methods Immunocytochemis-try, double luciferase system and calcium flow detection were used to verify the effect of polypeptide label Lucy-tag on olfactory receptors of sea lamprey. Results Polypeptide label Lucy-tag promoted the membrane expression of 13 olfactory receptors out of 20 olfactory receptors, and Lucy-tag did not affect the intrinsic activity of olfactory receptors, nor did it affect the IP3 signaling pathway of G protein coupled receptor (GPCR). Conclusions It provides a favorable technique for finding the ligand of olfactory receptor and lays a good theoretical foundation for the further study of the function of the GPCR and ligand.

Immunocytochemistry Study on Pulmonary Cytology Samples in Lung Cancer

There is an increasing need to subtype non-small cell lung carcinoma (NSCLC) into squamous cell carcinoma and adenocarcinoma of lung, and this subtyping guides the treatment with the advent of targeted therapies that are available for cases of adenocarcinoma of lung, by which prognosis can be improved. In cases of lung cancer, many a times, cytology may be the only material available to render a diagnosis; cases that are poorly differentiated on morphology are challenging to subtype and the immunocytochemical markers can be applied on these cytosmears for further subtyping of NSCLC. Aims and Objectives This study was carried out to check the expression of p63 and TTF-1 in diagnostically difficult cases, such as NSCLC, poorly differentiated lung carcinomas on pulmonary cytology samples, and to further subtype them into squamous cell and adenocarcinoma. Materials and Methods The study period was from 1 September 2013 to 31 August 2015; during this span, the total number of malignancies that were diagnosed on cytology was 585 cases. Prestained slides (haematoxylin and eosin or H&E, Papanicolaou or Pap) were collected over a span of these two years and these were destained using 1% acid alcohol with intermittent washing followed by running of p63 and TTF-1 as the immunocytochemical markers. Observations and Results The smears that are positive for p63 are labelled as squamous cell carcinoma and those that are positive for TTF-1 are labelled as adenocarcinoma, as these markers are highly sensitive and specific for the diagnosis of squamous cell and adenocarcinoma, respectively; neuroendocrine markers were used in the cases that were positive for TTF-1 to rule out small cell carcinoma. Conclusion: By the positive expression of p63 and TTF-1, the cases were labelled as squamous cell carcinoma and primary adenocarcinoma of lung, with due considerations given to the clinical and radiological parameters.

E2/E6 ratio and L1 immunoreactivity as biomarkers to determine HPV16-positive high-grade squamous intraepithelial lesions (CIN2 and 3) and cervical squamous cell carcinoma / 부인종양

OBJECTIVE: Human papillomavirus (HPV) 16 is the most carcinogenic HPV genotype. We investigated if HPV16 L1 capsid protein and E2/E6 ratio, evaluated by cervical cytology, may be used as biomarkers of ≥cervical intraepithelial neoplasia (CIN) 2 lesions. METHODS: Cervical specimens were obtained from 226 patients with HPV16 single infection. Using cytology specimen, L1 capsid protein and E2/E6 ratio were detected and the results were compared with those of the conventional histologic analysis of cervical tissues (CIN1–3 and squamous cell carcinoma [SCC]) to evaluate the association. RESULTS: The L1 positivity of CIN2/3 was significantly lower than that of normal cervical tissue (p < 0.001) and SCC demonstrated significantly lower L1 positivity than CIN1 (p < 0.001). The mean E2/E6 ratios of specimens graded as SCC (0.356) and CIN2/3 (0.483) were significantly lower than those of specimens graded as CIN1 (0.786) and normal (0.793) (p < 0.05). We observed that area under the receiver operating characteristic curve (AUC) for E2/E6 ratio (0.844; 95% confidence interval [CI]=0.793–0.895) was higher than that for L1 immunochemistry (0.636; 95% CI=0.562–0.711). A combination of E2/E6 ratio and L1 immunocytochemistry analyses showed the highest AUC (0.871; 95% CI=0.826–0.917) for the prediction of ≥CIN2 lesions. CONCLUSION: To our knowledge, this is the first study to validate HPV L1 capsid protein expression and decreased HPV E2/E6 ratio as valuable predictive markers of ≥CIN2 cervical lesions. Cervical cytology may be analyzed longitudinally on an outpatient basis with noninvasive procedures as against invasive conventional histologic analysis.

Clinical significance of liquid-based cytology and cell paraffin blocks combined with immunocytochemistry in cytopathologic diagnosis of hydrothorax and ascites / 重庆医学

Objective To investigate the clinical value of liquid-based cytology and cell paraffin blocks combined with immu-nocytochemical examination in cytopathologic diagnosis of hydrothorax and ascites.Methods One hundred and fourteen cases of hydrothorax and ascites sample were collected.The detection positive rates were compared between the liquid-based cytology and cell paraffin blocks combined with immunocyochemical examination.The suspected positive and positive cases were performed the immunocytochemical examination for further judging benign and malignancy and histological source.Results 114 cases of hydro-thorax and ascites samples,36 cases(31.58%)of positive were diagnosed by liquid-based cytologyamong and 55 cases(48.24%)of positive were diagnosed by cell paraffin blocks combined with immunocytochemical examination,the difference was statistically sig-nificant(P<0.01).There were 55 cases of malignant hydrothorax and ascites,45 cases were hydrothorax and 10 cases were ascites, in hydrothorax,there were 32 cases of lung adenocarcinoma,3 cases of small cell lung cancer,4 cases of breast cancer,1 case of en-dometrial carcinoma,1 case of lymphatic hematopoietic system tumor and 4 cases of unknown origin;in ascites,there were 2 cases of ovarian cancer,1 case of digestive system tumor,1 case of endometrial cancer,1 case of lymphatic hematopoietic system tumor and 5 cases of unknown origin.Conclusion The cell paraffin blocks combined with immunocytochemistry can significantly improve the positive detection rate of hydrothorax and ascites,and helps to judge the source of tissue.

Patologia morfológica e molecular aplicadas à inovação diagnóstica e vigilância da leishmaniose visceral / Morphological and molecular pathology applied to diagnostic innovation and surveillance of visceral leishmaniasis

O conceito de Saúde Única surgiu para ressaltar a união indissociável entre a saúde animal, humana e ambiental. Nesse contexto, a leishmaniose visceral americana (LVA) é considerada uma importante doença de saúde pública no Brasil, devido a sua crescente expansão geográfica e aumento na incidência de casos humanos. A LVA é uma doença parasitária, zoonótica, causada pela Leishmania (Leishmania) infantum (syn. chagasi) e transmitida por flebotomíneos do gênero Lutzomyia. Os cães são considerados os principais reservatórios do parasito nas áreas urbanas. O diagnóstico da LVA é baseado em aspectos epidemiológicos, clínicos e laboratoriais. A demonstração da presença do parasito através de exames diretos em tecidos biológicos do hospedeiro é o diagnóstico de escolha, principalmente, em municípios em que a transmissão de LVA ainda não tenha sido confirmada. Diversas metodologias podem ser aplicadas com essa finalidade. O objetivo desse trabalho é apresentar as técnicas citológicas, anatomo-patológicas e moleculares em amostras fixadas em formalina e incluídas em parafina para o diagnóstico da leishmaniose visceral em humanos e cães. Esses dados são complementares à apresentação realizada no I Simpósio Internacional de Leishmaniose Visceral, realizado nos dia 23 e 24 de Abril de 2018, e organizado pelo Instituto Adolfo Lutz em São Paulo-SP, Brasil.

The One Health concept emerged to highlight the inseparable link between animal, human and environmental health. In this context, American Visceral Leishmaniasis (AVL) is considered an important public health disease in Brazil, due to its increasing geographic expansion and in the incidence of human cases. AVL is a parasitic and zoonotic disease caused by Leishmania (Leishmania) infantum (syn. chagasi) and transmitted by sandflies of the genus Lutzomyia. Dogs are considered the main reservoirs of the parasite in urban areas. The diagnosis of AVL is based on epidemiological, clinical and laboratory aspects. The demonstration of the presence of the parasite through direct examinations in biological tissues of the host is the diagnosis of choice, mainly in municipalities where the transmission of AVL has not yet been confirmed. Several methodologies can be applied for this purpose. The objective of this work is to present the cytological, anatomopathological and molecular techniques in formalin fixed and paraffin embedded samples for the diagnosis of visceral leishmaniasis in humans and dogs. These data are complementary to the present study at the First International Symposium on Visceral Leishmaniasis, held on April 23 and 24, 2018, and organized by Adolfo Lutz Institute in São Paulo, Brazil.

Study on immunocytochemistry p16/Ki-67 double staining combined with DNA ploidy analysis for analyzing and predicting cervical high grade squamous intraepithelial lesion / 重庆医学

Objective To investigate the diagnostic value of DNA ploidy analysis combined with immunocytochemistry p16/ki-67 double staining in cervical high grade squamous intraepithelial neoplasia(HSIL) and cervical squamous cell carcinoma(SCC).Methods A total of 73 cases of cytological tests were randomly collected.Among them,53 cases were small DNA ploidy abnormal cells and 20 cases were DNA ploidy negative.The p16/Ki-67 results were detected by immunocytochemistry double staining.With the pathological results as the golden standard,the diagnostic values of DNA ploidy analysis and DNA ploidy analysis combined with p16/Ki-67 double staining in HSIL + was contrastively analyzed by pathologic results.Results Among 20 samples of DNA ploidy negative,the p16/Ki-67 double staining results all were negative.The positive predictive value of DNA ploidy analysis for HSIL + was 34.62％.The sensitivity of DNA ploidy analysis combined with p16/Ki-67 double staining for HSIL + was 84.62％,and its specificity was 92.31％,the positive predictive value was 78.57％ and the negative predictive value was 94.74％,which were significantly higher than those of DNA ploidy analysis(P＜0.05).Conclusion p16/Ki-67 double staining can significantly im prove the prediction value of HSIL.The DNA ploidy analysis combined with p16/Ki-67 double staining is an effective method for predicting HSIL +,which is suitable for the implementation in the areas with lack of medical resources.

Study of predictive value for detection of high-grade cervical intraepithelial neoplasia by p16/Ki-67 dual-stained liquid-based cytology / 临床与实验病理学杂志

Purpose To study of predictive value for detection of high-grade cervical intraepithelial neoplasia (CIN2 +) by p16/Ki-67 dual-stained liquid-based cytology.Methods Random collection of 123 women including 103 samples of atypical squamous cell of undetermined significance (ASC-US) and above with results of high-risk human papillomavirus (HR-HPV)testing and cervical biopsy,20 samples of negative for intraepithelial lesion or malignancy (NILM) by using immunocytochemical p16/Ki-67 dual-stained and the morphology assessment.Results In normal control group,the expression of p16/Ki-67 dual-stained in squamous epithelial cells were negative.Sensitivity of p16/Ki-67 dual-staind cytology for biopsy-confirmed CIN2 + was 66.67％ (ASC-US),91.67％ (LSIL) and 92.86％ (HSIL),specificity rates were 95.92％ (ASC-US),95.00％ (LSIL) and 0 (HSIL),positive predictive value were 50.00％ (ASC-US),91.67％ (LSIL) and 92.86％ (HSIL),negative predictive value were 97.92％ (ASC-US),95.00％(LSIL) and 0 (HSIL),respectively.Condusion p16/Ki-67 dual-stained cytology are improved obviously the predictive value for detection of CIN2 +,p16/Ki-67 dual-stained cytology may efficiently complement HPV-based screening programs to prevent cervical cancer.

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer / 中国病理生理杂志

AIM:To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b + myeloid cells in hepatic metastases from colorectal cancer.METHODS:Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion,and then the CD11 b + myeloid cells were isolated by flow cytometry.The sorted cells were identified by immunocytochemistry.The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining.The cell purity was evaluated by flow cytometry.RESULTS:Sufficient numbers of CD11b+cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion.The purity of the cells was confirmed by statistical analysis (P ＜ 0.05).The positive rates of the cells before and after sorting were significantly different (P ＜0.01).The positive cells were verified by immunocytochemical method.Meanwhile,the sorted cells had complete morphology and good activity.CONCLUSION:The CD11b + myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity,good activity and complete morphology.

Identification and establishment of sorting method for isolating CD11b+ myeloid cells in human hepatic metastases from colorectal cancer / 中国病理生理杂志

AIM:To establish a method for obtaining specific cells in solid tumor tissue by sorting of CD11b + myeloid cells in hepatic metastases from colorectal cancer.METHODS:Tumor tissues were prepared into single cell suspension by mechanical method combined with enzyme digestion,and then the CD11 b + myeloid cells were isolated by flow cytometry.The sorted cells were identified by immunocytochemistry.The viability and morphologiy of the sorted cells were evaluated by Giemsa and Typan blue staining.The cell purity was evaluated by flow cytometry.RESULTS:Sufficient numbers of CD11b+cells with high purity were isolated by sorting with flow cytometry from the single cell suspension prepared by mechanical and enzyme digestion.The purity of the cells was confirmed by statistical analysis (P ＜ 0.05).The positive rates of the cells before and after sorting were significantly different (P ＜0.01).The positive cells were verified by immunocytochemical method.Meanwhile,the sorted cells had complete morphology and good activity.CONCLUSION:The CD11b + myeloid cells in solid tumor tissue can be isolated by flow cytometry from the machine-enzyme digestion suspension with high purity,good activity and complete morphology.

Preparation of monoclonal antibody against human KIAA0100 protein and Northern blot analysis of human KIAA0100 gene / 药物分析学报

Monoclonal antibodies (MAbs) are important tools for the study of proteins′ function and structure. But there has been no report on the preparation of MAbs against human KIAA0100 protein up to date. Here, first, we generated the mouse MAb against human KIAA0100 protein using purified recombinant 6×Histidinc (6×His)- tagged human KIAA0100 protein segment (1557–2234) as an antigen; then, the mRNA expression of human KIAA0100 gene was detected in U937 cells using Northern blot analysis. The results showed that the mouse MAb against human KIAA0100 protein could sensitively recognize the human KIAA0100 protein using Western blot analysis and immunocytochemistry analysis. Besides, Western blot analysis revealed that human KIAA0100 gene possibly encoded two different protein products (254 kDa and < 250 kDa) in U937 cells. Moreover, Northern blot analysis confirmed that human KIAA0100 gene might produced two different mRNA products (6000–10000 bp and 5000–6000 bp) in U937 cells. The results provide a basis for large-scale production of the MAb against human KIAA0100 protein, which will be useful for the study of human KIAA0100 protein′s function/structure and MAb-targeted drugs in the future.

Comparison of the sensitivity and specificity of p16/Ki-67 dual staining and HPV DNA testing of abnormal cervical cytology in the detection of histology proven cervical intraepithelial neoplasia grade 2 and above (CIN 2+)

Introduction: Human papillomavirus (HPV) testing is used as a means of triaging cervico-vaginalsmears with low grade squamous abnormalities or as part of co-testing with cytology. While HPVtesting has a high sensitivity, it has a low specificity in detecting cervical intraepithelial neoplasiagrade 2 and above (CIN 2+) leading to unnecessary colposcopy referrals. We investigate the accuracyof the p16/Ki-67 dual immunocytochemical stain in determining the presence of CIN 2+ lesions onhistology and its potential as a superior biomarker for triage. Methods: Liquid based cervico-vaginalcytology specimens with squamous abnormalities and corresponding histology from 97 women withsubsequent colposcopy and biopsy were included. The specimens were then subjected to the dual stainand Roche Cobas 4800 multiplex real time PCR HPV DNA testing. The sensitivity and specificity ofthe dual stain and HPV testing were calculated using CIN 2+ on histology as a reference standard.Results: The sensitivity and specificity of the dual stain in detecting histology proven CIN 2+ was93.7% and 76.5% while HPV testing was 85.7% and 14.7% respectively. Of the 44 women withASCUS or LSIL on cytology, the dual stain also reduced the number of unnecessary colposcopyreferrals from 27 to 7 when used as a triage marker compared to HPV testing. Conclusion: p16/Ki-67dual stain was more sensitive and specific than HPV testing in determining the presence of CIN 2+on histology. It could triage low grade cervico-vaginal specimens more effectively and potentiallyhelp women avoid unnecessary colposcopies. Future studies are needed to further evaluate its rolein cervical cancer screening programmes.