Peroxidase-antiperoxidase Technique Research Articles

Role of immunohistochemistry in clinicohistopathology-A rare case study

Disease affecting Lymph nodes form wide range of spectrum from simple to malignant pathology. Such cases are an important element to rule out malignancy in early stages. However Generalised Lymphedenopathy diagnosis become huge challenge. Hence, an insight into clinocohistopathological correlation with help of IHC markers becomes highly important to decide morphologic differential diagnosis.: To study lymph node lesions with clinicohistopathological correlation with the help of IHC study. This is a combined observational and prospective study held in District hospital Vijayapur during August 2022 to September 2023. Patient details were taken with the questionnaires. During his first visit clinical examinations and investigations, CBC, PS study, FNAC of left lower cervical region, Urine routine, chest X-ray & USG of abdomen performed and patient was analyzed & diagnosed as Chronic appendicitis and treated. During patient’s second visit, clinical examinations and investigations CBC, PS study, FNAC of left Axillary swelling, Urine routine, chest X-ray, USG of neck & abdomen, CT Neck & abdomen, USG guided FNAC of Liver, Bone marrow aspiration study, Biopsy of left lower Cervical lymph node for Histopathology study & IHC study performed. Case was studied using routine H&E, PAP stain slides of Biopsy material were evaluated by light microscopy. And for IHC, patient biopsy material referred to higher center, using specific monoclonal or polyclonal antibodies, paraffin sections were stained immunohistochemically (IHC) using a Peroxidase antiperoxidase (PAP) technique in referred higher center. Patient was diagnosed as B-cell Lymphoma as per WHO guidelines with the help of IHC markers.: From this study, concluded that IHC plays a significant role clinicopathology and helped in the definitive diagnosis and typing of tumours and appropriate treatment can be planned.

Infectivity assay for detection of SARS-CoV-2 in samples from patients with COVID-19.

Since the coronavirus disease 2019 (COVID‐19) outbreak, laboratory diagnosis has mainly been conducted using reverse‐transcription polymerase chain reaction (RT‐PCR). Detecting the presence of an infectious virus in the collected sample is essential to analyze if a person can transmit infectious severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). However, there have been no quantitative investigations conducted for infectious SARS‐CoV‐2 in clinical samples. Therefore, in the present study, a rapid and simple focus‐forming assay using the peroxidase‐antiperoxidase technique was developed to quantify infectious SARS‐CoV‐2 titers in 119 samples (n = 52, nasopharyngeal swabs [NPS]; n = 67, saliva) from patients with COVID‐19. Furthermore, the study findings were compared with the cycle threshold (Ct) values of real‐time RT‐PCR. The infectious virus titers in NPS samples and Ct values were inversely correlated, and no infectious virus could be detected when the Ct value exceeded 30. In contrast, a low correlation was observed between the infectious virus titers in saliva and Ct values (r = ‐0.261, p = 0.027). Furthermore, the infectious virus titers in the saliva were significantly lower than those in the NPS samples. Ten days after the onset of COVID‐19 symptoms, the infectious virus was undetectable, and Ct values were more than 30 in NSP and saliva samples. The results indicate that patients whose symptoms subsided 10 days after onset, with Ct values more than 30 in NSP and saliva samples, were less likely to infect others.

Soft tissue sarcomas: Evaluation by immunohistochemistry

Soft tissue sarcomas represent a histologically heterogenous group of malignant tumors arising from the soft tissue compartments, i.e smooth muscles, skeletal muscles, adipose tissue, blood vessels, connective tissue mesenchyme, neural tissue and other tumors of uncertain histogenesis(1). Soft tissue sarcomas are relatively rare tumors, represent less than 1% of adult malignancies based on data report from American Cancer Society (2). The crude incidence rate in Iraq for soft tissue sarcomas was 219/ 100,000 population during 2014 with male: female ratio of 1.3:1, and highest peak age between 30-34 years (3). The histological subtype and microscopic criteria are two parameters that influence the tumor grade which is the best predictor for aggressiveness. ( 2)
 Objectives:
 A retrospective evaluation of soft tissue sarcomas, using immunohistochemical study of Myosin, Desmin, Smooth muscle Actin (SMA) and Vimentin, aiming to re-evaluate the primary diagnosis and whether IHC assessment in soft tissue sarcoma is mandatory to reach a final interpretation.
 Material and Patients: 
 A total of 50 cases soft tissue sarcomas were reviewed retrospectively from case archives of histopathology department/ Central Public Health Laboratory, for the period (2011-2018). The data for these cases were collected including the age, gender, site of the tumor and the preliminary biopsy report findings. Hematoxylin& Eosin (H&E) stain and IHC staining of unstained sections from formalin-fixed, paraffin-embedded tissue were performed using SMA, Vimentin, Desmin and Myosin.
 Methods: Age and gender distribution of the 50 cases was correlated. The IHC staining performed on 5 microns paraffin sections, applying peroxidase-Antiperoxidase technique and both positive and negative control slides were included for Smooth muscle actin (SMA), clone 1A4, Desmin (clone D33), Myosin (clone 2F12.A9) and Vimentin (Clone V9). The monoclonal antibodies used were from DAKO manufacturer as ready to use monoclonal antibodies while freeze-dried antibodies used for myosin with 1:20 dilution. Negative and positive control slides were used simultaneously. The staining intensity was assessed based on the percentage of positive cells expression of the four IHC monoclonal antibodies. Negative staining reported when no stain seen. Positive (+) means up to 30% of tumor cells with antigen expression. Positive (++) interpretation given when 31-60% of tumor cells show staining reaction, while Positive (+++) interpretation reported when over 60% of tumor cells showing staining reaction.
 Results:
 The staining interpretation of the four monoclonal antibodies were confirmatory for the original diagnosis in 40% of cases, while 60% of the final diagnoses were against the original interpretation. SMA was positive in 38/50 cases (76%). Desmin was positive in 8/50 cases (20%) Vimentin was positive in 42/50 of cases (84%), Myosin was positive in 30/50 of cases (71.4%).
 The current study focus on histological re-evaluation of different types of soft tissue sarcomas (including RMS-with its subtypes), MFH-storiform, pleomorphic and myxoid subtypes), fibrosarcoma, leiomyosarcoma, alveolar soft part sarcoma and synovial sarcoma depending on immunohistochemical study. The original diagnosis have been changed after the immunohistochemistry stain from fibrosarcoma to rhabdomyosarcoma and leiomyosarcoma , Malignant fibrous histiocytoma to rhabdomyosarcoma, alveolar rhabdomyosarcoma to Malignant fibrous histiocytoma, soft tissue sarcoma to rhabdomyosarcoma , pleomorphic MFH to pleomorphic rhabdomyosarcoma. Embryonal and alveolar rhabdomyosarcoma were confirmed by IHC as so. Leiomyosarcoma was confirmed as the same in one case. Embryonal Rhabdomyosarcoma changed to leiomyosarcoma by IHC positivity for desmin. Six cases of MFH were confirmed as malignant fibrous histiocytoma after IHC. Two cases of synovial sarcoma were re-evaluated as rhabdomyosarcoma. Vimentin revealed 100% positivity in 10 cases and regarded as a sensitive marker for mesenchymal tissue but it is not specific for certain types of sarcomas. The presence of desmin diffusely throughout a tumor is an indication of myoid differentiation and the presence of either SMA or desmin focally should not necessarily be equated with myoid lineage but rather with myofibroblastic lineage, which go in the same direction with Hirofumi N. et al. (1998).(4)
 Conclusions: The co-expression of SMA and Desmin helped in the diagnosis of leiomyosarcoma (LMS). Myosin expression was helpful in the diagnosis of rhabdomyosarcoma (RMS) and alveolar soft part sarcoma (ASPS). Smooth muscle actin (SMA) and vimentin were not specific for the diagnosis of malignant fibrous histiocytoma (MFH). High grade spindle cell sarcoma express SMA only without myosin or desmin can be categorized as pleomorphic myogenic sarcoma. By applying immunohistochemical markers, 60% of soft tissue sarcomas were different from the preliminary diagnosis while 40% of cases were supportive of the original H&E diagnosis.

Comparison of Immunocytochemistry and Immunohistochemistry on Breast Carcinoma: A Boon or a Bane?

Introduction:Breast carcinoma is the most common cancer among women in the urban Indian population. Conventionally, immunohistochemistry (IHC) is done to determine the hormone receptor status of the tumor. Immunocytochemistry (ICC) on fine-needle aspiration cytology (FNAC) was carried out to determine the same hormone receptor status of the tumor.Objective:The study was undertaken to evaluate the diagnostic reliability of performing estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (Her2/neu) receptor status on FNAC by ICC and to compare the results with IHC.Materials and Methods:A 2 years 6 months prospective study conducted in the Department of Pathology, ESIC Medical College and PGIMSR and ESIC Model Hospital, Rajajinagar, Bengaluru, wherein 100 breast carcinoma patients' samples both cytology and histology were collected. IHC and ICC were done by peroxidase antiperoxidase technique. Validations of the receptor status were analyzed using sensitivity, specificity, positive and negative predictive values (PPV and NPV), and kappa statistics for agreements between ICC and IHC.Results:ICC was positive for ER, PR, and Her2/neu in 53, 50, and 22 cases, respectively. For ER, a cytohistologic correlation of 98%, with a sensitivity of 96.3%, specificity of 100%, and PPV and NPV being 100% and 95.7%. For PR, concordance of 97%, with a sensitivity of 94.3%, specificity of 100%, and PPV and NPV being 100% and 94%. Her2/neu had an agreement of 89%, with a sensitivity of 72%, specificity of 95.5%, and PPV and NPV being 85.7% and 90.1%.Conclusion:ICC has been a boon and can be a paramount diagnostic adjunct to the routine investigations.

Immuno Histochemical Study of Osteosarcoma Using Ki-67 as a Labeling Index in Naturally Fluoridated Community

Osteosarcoma is the most common primary malignant bone tumor which characterized microscopically by formation of osteoid or bone by the neoplastic cells (osteoblastic type) or production of fibrous tissue (fibroblastic type) and/or a cartilage (chondroblastic type). Ki-67 protein is present during all active phases of the cell cycle (G1, S, G2 and mitosis but is absent from resting cells (G0), makes it an excellent marker for determining the so called growth fraction of a given cell population. However, fluoride concentration in drinking water shows great variation between the costal-western region of Libya (mean fluoride concentration 1.38ppm) and Cairo-Egypt (0.4ppm). Therefore, the aim of this study is to evaluate and compare histopathologically the proliferative activity of Osteosarcomas in naturally fluoridated community (Libyan cases) and non-fluoridated community (Egyptian cases) as a retrospective case-control study using the labeling index Ki-67. 20 cases of Osteosarcoma from each country in a formalin-fixed embedded specimens and serial sections from the paraffin blocks for immuno histochemical examination were included in this study in which Peroxidase-antiperoxidase technique was performed. Assessment of Ki-67 proliferative activity was carried out using software image analysis and the area fraction was given by the percentage of immuno positive area to the total area of the microscope field. The result of this study showed that the mean value of area of fraction in Libyan cases was 5.33 and in Egyptian cases were 3.12. When comparing the area fraction of Ki67 immunopositivity of tumor cells in Lybian cases versus Egyptian cases, statistical analysis revealed a statistically significant difference (P value=0.023). It is concluded from this study that a high expression of Ki-67 of Osteosarcomas in fluoridated area compared to those of non-fluoridated area could explain the aggressive behavior of this tumor, in which fluoride ions may play a role as an underlying etiological factor.

Hormone receptor expression in breast carcinoma at our hospital: An experience

Background: Breast carcinoma is the most common cancer among women in the urban Indian population and second only to cervical cancer in the rural population based on cancer registry data. Prognosis and management of breast cancer are influenced by classic variables such as histologic type, grade, tumor size and lymph node status. More recently hormone receptor (HR) status of estrogen receptor (ER), progesterone receptor (PR), and HER-2/neu expression have opened a new gateway in the field of adjuvant hormonal and/or chemotherapeutic regimen. Objective: The objective of this study was to assess the ER, PR, and HER-2/neu reactivity pattern in breast carcinomas at our hospital. Materials and Methods : A study of 100 patient samples of breast carcinoma was carried out from June 2011 to June 2014 in the Department of Pathology, ESIC Medical College and PGIMSR, ESIC Model Hospital, Rajaji Nagar, Bengaluru. Brief demographic and clinical data were obtained. Immunohistochemistry (IHC) was done by peroxidase antiperoxidase technique for detection of ER, PR, and HER-2/neu receptor status. Details regarding histopathological diagnosis, pathological grading, staging, and HR status of breast carcinoma were collected. Obtained parameters were evaluated using descriptive statistical analysis and presented in terms of percentage. Results : The age of the patients ranged from 24 to 75 years. Majority of tumors were infiltrating ductal carcinomas-not otherwise specified and predominantly histological grade 2. By IHC 52% were ER+/PR+, 25% were HER-2/neu positive and 20% of triple negatives. Conclusion : Detection of hormone expression is of paramount importance since these are one among the classic variables needed for providing suitable adjuvant hormonal and/or chemotherapeutic options, targeted treatment, and predicting prognosis.

The distribution of serotonin-immunoreactive cells in larynx, trachea and bronchi of goat (Capra aegagrus) and bovine (Bos taurus)

Summary The respiratory tract contains numerous peptides secreted from special pulmonary epithelial cells which are called endocrine cells. The function of neuroendocrine cells is modulated by amines. Serotonin has strong vasomotor and bronchomotor effects in the airway mucosa. The objective of this study was to determine presence and distribution of serotonin-positive endocrine cells in respiratory tract of goat and bovine via PAP (peroxidase anti-peroxidase) technique. As a result, while serotonin-immunoreactive cells were found in glands of larynx and bronchial epithelium in bovine, no positive cells were detected in goat. The present study suggests that the distribution of serotonin-immunoreactive cells may show differences between both respiratory tract regions and mammalian species.

Albumin Localization in the Testis of Adult Golden Hamsters by Use of Immunohistochemistry: Die Lokalisation von Albumin im Hoden adulter Goldhamster mit Hilfe der Immunhistochemie

There is an increasing interest in different biochemical roles of albumin (Alb) in respect to testicular function. For this reason, Alb was localized by the peroxidase-antiperoxidase technique in the testis of adult golden hamsters. A strong Alb immunoreactivity occurred in the interstitial space and on the surface of Leydig cells. A few Sertoli cells showed a strong Alb response under the appearance of so-called leakages traversing Sertoli-Sertoli cell junctions. Spermatogonia reacted strongly, while spermatids and sperms showed rather a mild, or moderate response. Spermatocytes remained unstained. When similar maturation stages of seminiferous tubules were compared, the number of reacting cells varied strikingly. Our results support the assumption that Alb or a protein with an Alb-like immunoreactivity exerts diverse functions of the testis.

Commentary on in-transit lymph node metastases in breast cancer: a possible source of local recurrence after Sentinel Node procedure

A comparative study of beta enolase and myoglobin as markers of muscle differentiation in rhabdomyosarcoma was carried out, using an immunoperoxidase peroxidase antiperoxidase technique. Material from 26 cases of childhood...

Estudio histopatológico e inmunohistoquímico sobre neosporosis en fetos bovinos procedentes de matadero

Objetivo. Determinar la presencia de lesiones, formas evolutivas y antígenos compatibles con Neospora caninum, a través de estudios histopatológicos e inmunihistoquímicos en el sistema nervioso central de fetos bovinos procedentes de matadero. Materiales y métodos. Se estudiaron muestras de cerebro, cerebelo y médula de 108 fetos mayores de cinco meses, provenientes de un frigorifico del municipio de Montería – Córdoba. Los estudios histopatológicos se realizaron mediante coloración con hematoxilina - eosina y los estudios inmunohistoquímicos a través de la técnica de peroxidasa anti-peroxidasa. Resultados. El estudio estableció que de los 108 fetos examinados por histopatología, el 27,8% presentaron lesiones compatibles con el protozoario. Estas lesiones, fueron observadas principalmente en cerebro (24.1%) y en menor número a nivel medular (3.7%). Las lesiones compatibles observadas consistieron en gliosis, meningitis, congestión y hemorragia focal severa. Los estudios inmunihistoquímicos, utilizados para detectar antígenos tisulares del agente etiológico, resultaron negativos. Conclusiones. Con base en los resultados, se puede afirmar que existen alteraciones histopatológicas en el sistema nervioso central de fetos bovinos procedentes de matadero, compatibles con infecciones por el protozoario N. caninum. La coloración inmunohistoquímica para detección del parásito fue negativa, no obstante, no se descarta la presencia del agente por lo que se sugiere la realización de técnicas más específicas como PCR.

Morphological findings in lungs of the horses with chronic obstructive pulmonary disease (COPD)

The frequency and characteristics of chronic obstructive pulmonary disease (COPD) based on morphological and cytological changes in equine lungs were studied in this paper. Lungs obtained from 51 horses of different age and sex were examined grossly and tissue samples were collected for pathohistological examination. Cytological examination was done on impression smears from the tracheal bifurcation. Pathohistological preparations were stained with hematoxylin eosin (HE), toluidine blue (TB), Giemsa staining, PAS staining and Pearls reaction for iron detection. The peroxidase anti-peroxidase technique was used to demonstrate CD3+ (T lymphocytes) and CD79+ (B lymphocytes). Tracheal bifurcation cytology impression smears were stained with hematoxylin eosin (HE), Giemsa staining and PAS staining. Pulmonary emphysema and lack of pulmonary collapse were the most common gross lesions whereas alveolar emphysema was described in 70.59% of all examined horses, more frequently in the distensive form (54.90%) and less frequently in the destructive form (15.69%). Pathohistological chronic bronchitis/bronchiolitis, with characteristic changes in the lumen, mucosa, submucosa and smooth muscle layer was described in all examined horses. Increased immunoreactivity was described in the lungs. The most common lesions seen on cytology impression smears from the tracheal bifurcation were thick, viscous, PAS positive mucus which forms Curschmann's spirals. The dominant cell population consisted of desquamated airway epithelial cells, as well as eosinophils, neutrophils, mast cells, erythrocytes and alveolar macrophages. Primary pulmonary pathogens as well as potential contaminants and secundary infection agens were isolated bacteriologically from lung samples. All the above-mentioned findings correlate pointing to the fact that chronic bronchitis/bronchiolitis represents the basic substrate of chronic obstructive pulmonary disease (COPD) which has a combined inflammatory and immunological etiology. Emphysema being secondary to airway obstruction.

Chronic exposure to estrogen and tamoxifen regulates synaptophysin and phosphorylated cAMP response element-binding (CREB) protein expression in CA1 of ovariectomized rat hippocampus

We report here the in vivo effects of estrogen (E2) on modulation of synaptic plasticity and the agonistic (estrogen-like) role of selective estrogen receptor modulator (SERM), tamoxifen (TAM) in the CA1 of the rat hippocampus. Effects on synaptophysin (SYP), a presynaptic vesicular protein, and phosphorylated cyclic AMP responsive element- binding (p-CREB) protein, a signal transduction pathway molecule, were studied using the ovariectomized (OVX) experimental rat model. Bilateral ovariectomy was performed on 40 rats and these were divided into 4 groups based on the treatment they received (at 2 weeks post-ovariectomy, a subcutaneous injection daily for 4 weeks) viz., OVX + E2 (0.1 mg/kg body weight), OVX + TAM (0.05 mg/kg body weight), OVX + vehicle and one group served as OVX control. An additional 10 animals served as the ovary intact control group. At the end of the treatment schedule, five animals/group were used for immunohistochemical staining of SYP and p-CREB using specific antibodies with peroxidase anti-peroxidase technique on paraformaldehyde-fixed cryostat sections. Protein estimation and Western blot analysis coupled with densitometric analysis (using gel-documentation system and image analysis software) were performed on unfixed hippocampus collected from rest of the five animals/group. Serum estradiol levels were estimated with radioimmunoassay prior to sacrifice. The results revealed that ovariectomy reduced SYP and p-CREB expression whereas E2 or TAM administration resulted in their upregulation. Serum estradiol levels of E2 administered animals were comparable with the ovary intact group whereas those of TAM administered group persisted in the range of OVX controls. To conclude, long-term estrogen therapy modulates the synaptic plasticity of hippocampal neurons and presumably, the agonist biocharacter of TAM as observed in the present investigations, may in the long run have a potential in the treatment and prevention of various estrogen-related disorders.

Morphometric and immunohistochemical patterns of duodenal CCK-cells in rats with portacaval shunts

End-to-side portacaval shunt (PCS) performed in rats is an appropriate experimental model of chronic liver insufficiency. Functional hepatic injury followed by numerous metabolic and endocrine disorders develops, as well. Due to impaired functional hepatic activity in PCS and to the well known role of the liver in CCK metabolism and elimination, delayed CCK bloodstream elimination may be responsible for increased CCK-A receptor activity. Therefore, we investigated immunohistochemical and morphometric features of duodenal CCK-cells in rats with PCS. Male Wistar rats (180 - 250g body weight) were used in this experiment. The animals were divided into two groups: PCS-animals (experimental group (n=27) which underwent an operation) and C - unoperated animals [control group (n=11)]. End-to-side portacaval anastomosis was created using the technique of Lee and Fisher, modified by Bismuth et al. Eight weeks, after the operation, animals were sacrificed. Pancreatic and hepatic weights were measured and samples taken from appropriate segments of the small intestine were used for morphometric and immunohistochemical CCK-cells determination. IMMUNOHISTOCHEMICAL TECHNIQUE: Peroxidase-antiperoxidase (PAP) technique described by Stenberger was applied for detection of intestinal CCK-immunoreactive cells. Our results indicate that eight weeks after operation liver weight was reduced in animals with PCS compared to control animals (p<0.01). To the contrary, in comparasion with control animals, the pancreatic wight was increased (p<0.01). In the exocrine pancreas of rats with PCS there were lipid droplets and hypertrophy of the acinus cells. The morphometric analysis of CCK-immunoreactive cells in different small intestine segments indicates that in the duodenum of animals with PCS there was a significant cell increase compared to control, unoperated animals. In liver cirrhosis, plasma CCK-8 concentration increases. In PCS, performed in rats, as an experimental model of chronic liver insufficiency, proliferation of CCK-cells in the small intestine leads to pancreatic hypertrophy and hyperplasia.

Characterization of monoclonal antibody to L-homocysteic acid and its immunohistochemistry in rat hippocampus.

L-homocysteic acid (HCA) and other amino acids were conjugated to rat brain material (extracted rat brain protein) with glutaraldehyde to form HCA- and amino acids-brain material conjugates. The specificity of monoclonal antibody (McAb) was tested on serial dilution test and absorption test on enzyme-linked immunosorbent assay (ELISA) using these conjugates as antigens instead of amino acids-BSA (bovine serum albumin) conjugates used previously. The characterized McAb was applied for immunohistochemical staining using PAP (peroxidase antiperoxidase) technique in combination with silver enhancement of diamino-benzene (DAB) products. The results indicated that McAb to L-HCA reacted with L-HCA-brain material conjugates, but not with other amino acids-brain material conjugates so far tested. McAb absorbed with L-HCA-brain material abolished or decreased immunoreactivity of L-HCA-brain material with McAb. The antibody selectively stained subpopulation of cells and processes in the hippocampus fixed with glutaradehyde. Absorption of McAb with L-HCA-brain material abolished immunohistochemical staining. These results suggested that McAb was specific for L-HCA-brain materials and could be used for imuno-histocytochemistry. This would provide a new tool for immunohistochemical visualization and localization of L-HCA in the nervous system.

Diagnóstico da pneumonia enzoótica suína pela técnica da imunoperoxidase

Avaliou-se a técnica da imunoperoxidase como método auxiliar para a detecção de Mycoplasma hyopneumoniae em suínos naturalmente infectados. Foram colhidos 80 fragmentos de pulmões de 40 animais provenientes de granjas consideradas negativas e 40 de granjas com diagnóstico positivo de pneumonia enzoótica. Com a utilização de soro policlonal específico (IgG de coelho anti- M. hyopneumoniae) observou-se correlação positiva de 77% entre os diagnósticos microscópicos e imunoistoquímicos, enquanto que a correlação entre os diagnósticos macroscópico e imunoistoquímico foi de 49%. Nas granjas consideradas negativas observou-se presença de discreta imunorreação em 22,5% dos casos, o que poderia indicar a existência de reação cruzada com outros microrganismos. Nas granjas com diagnóstico positivo para pneumonia enzoótica a técnica da peroxidase-anti-peroxidase (PAP) revelou diferentes graus de intensidade, variando de fraca imunomarcação até espesso depósito amarronzado no epitélio ou na luz das vias aéreas, ou ainda no interior de macrófagos, com relação direta entre a intensidade das lesões e da imunorreação. A técnica imunoistoquímica possui sensibilidade de 95% e especificidade de 77,5%, podendo ser recomendada como ferramenta auxiliar, rápida e de baixo custo para o diagnóstico de pneumonia enzoótica suína em laboratórios de rotina em histopatologia.

Improved immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) by flow cytometry

Background: The immnunophenotyping of lymphocytes in bronchoalveolar lavage fluid (BALF) is of particular importance in the differential diagnosis of interstitial lung disorders. The standard method of lymphocyte phenotyping is peroxidase–anti-peroxidase technique (PAP). However, it was time-consuming and experience-dependent. Flow cytometric (FCM) analysis of BALF lymphocytes was introduced to overcome these disadvantages. Unfortunately, when the number of cells counted was small, FCM could not distinguish lymphocytes from other cells and particles in BALF by light scattering. Methods: We established a tri-color flow cytometric approach to phenotyping of lymphocytes in BALF. FITC-CD45/PE-CD14 antibodies were used to gate lymphocytes and exclude other contamination. Propidium iodide (PI) was introduced to distinguish lymphocytes from debris. Forty-three BALF species were tested by flow cytometer as well as peripheral blood as a control group by conventional PAP method. Results: The variation of FCM (CV<1.0%) was much lower that that of PAP method (CV>9.8%). Meanwhile, we found that BALF had more clinical significance than peripheral blood in T subset analysis ( p<0.01). There were characteristic changes in some lung diseases. Both CD3 and CD4 were significantly increased with decreasing CD8 in sarcoidosis ( n=14). Idiopathic pulmonary fibrosis ( n=16) demonstrated the reverse tendency: CD8 rose but both CD3 and CD4 dropped. As for lung cancer ( n=7), CD3 was normal but the CD4/CD8 ratio declined. Tuberculosis of the lungs ( n=6) showed a normal CD3, CD4 and CD8. Conclusions: The high precision and reliability of tri-color flow cytometric approach to phenotyping of lymphocytes in BALF suggested that it should be used as a routine test, especially of BALF, which was often contaminated by inorganic particles.

Parvalbumin and calbindin D-28K immunoreactive neurons in area MT of rhesus monkey.

The chemical characteristics of the neurons of the motion sensitive visual area, area MT, remain to be established. We studied the distribution pattern of two calcium binding proteins, parvalbumin (PV) and calbindin D28K (CB) in this area, using specific monoclonal antibodies and the peroxidase-antiperoxidase (PAP) immunohistochemical technique. Aldehyde fixed 30-micron-thick cryostat sections from area MT of five animals were processed free floating for immunohistochemical staining. Besides studying the morphological characteristics of PV and CB positive neurons, quantitative analysis was carried out to determine their (1) perikaryal area (Pa) and diameter, (2) numerical densities (NV)/mm3 cortical tissue, (3) absolute number (NC) in a column of cortex under 1 mm2 cortical surface along with (4) layerwise absolute number (NL) under 1 mm2 cortical surface and (5) laminar percentage distribution of immunoreactive (IR) neurons. Quantitative analysis was carried out using a Leica QMC 500 image analysis system connected to a DMRE microscope. The results showed that both types of IR neurons were localized to all cortical layers except layer I. The PV +ve neurons were equidistributed between the supra- and infragranular layers, with the highest percentage being present in layer III (45%) followed by layer V (21%). The CB +ve neurons, on the other hand, were predominantly localized in supragranular layers, with the highest percentage being in layer III (54%) and the next highest percentage in layer II (18%). The average Pa and diameter of PV +ve neurons were found to be 96.90 +/- 28.43 micron 2 and 11.01 +/- 1.61 microns respectively. The CB +ve neurons were significantly smaller in size than the PV +ve neurons, with average Pa and diameter of the former being 92.23 +/- 26.18 micron 2 and 10.39 +/- 1.23 microns respectively. The NV for PV and CB +ve neurons showed ranges of 3157-3894 and 2303-2585, with means of 3347 +/- 285 (+/- SD) and 3436 +/- 100 respectively. The values for NC showed ranges of 5230-5444 and 4020-4268 with means of 5378 +/- 85 and 4167 +/- 95 for PV and CB neurons respectively. Variations in size together with the differential distribution of these neurons in the cortical layers may indicate their involvement in different functional circuitaries.

Distribution of delta sleep-inducing peptide in the newborn and infant human hypothalamus: an immunohistochemical study.

The distribution of delta sleep-inducing peptide immunoreactive cell bodies, fibers, and terminal-like structures was investigated in the normal human hypothalamus during the first postnatal year, using immunohistofluorescence and peroxidase anti-peroxidase techniques. Immunolabeled perikarya were relatively few and were mostly scattered through the anterior (preoptic) and mediobasal regions (infundibular nucleus) of the hypothalamus. DSIP-immunoreactive fibers and terminal-like fibers were observed throughout the entire rostrocaudal extent of the hypothalamus. They exhibit high densities in the preoptic region, the organum vasculosum of lamina terminalis, infundibular nucleus and median eminence. Moderate to low densities of DSIP-immunoreactive fibers were observed in the other hypothalamic structures, located in the anterior and mediobasal regions of hypothalamus, such as periventricular, paraventricular, suprachiasmatic, ventromedial, dorsomedial and parafornical nuclei. In the present study, the analysis of the immunohistochemical pattern of DSIP-immunoreactive neuronal elements in the human infant hypothalamus during the first postnatal year provided evidence of the presence of several differences. We have found qualitative age-related changes in the density of DSIP immunoreactivity in several hypothalamic structures such as the anterior region and the median eminence.

Cutaneous papillomas of green turtles: a morphological, ultra-structural and immunohistochemical study in Brazilian specimens

Eleven juvenile green turtles (Chelonia mydas) from Atlantic Ocean, Brazil, with multiple cutaneous papillomatosis were examined. Histologically, the papillomas exhibit stromal hyperplasia proliferation and epithelial proliferation. The epithelial cells had nuclear changes suggestive of viral infection and severe nuclear pleomorphism. A large nuclear halo was present in the cases of epithelial proliferation; in these cells, nuclear features were frequently dyscariotic, without inclusion. All fibropapillomas examined were negative for papillomavirus group-specific antigens (BPV) and herpesvirus group-specific antigens (HSV1 / HSV2) by the peroxidase-antiperoxidase technique. Electronic microscopy investigation was negative for papillomaviruses and herpes-viruses particles.

Cholesterol emboli to the kidney: an immunoperoxidase study

Cholesterol emboli (CE) are an increasingly recognised cause of renal impairment in the elderly population, especially following diagnostic vascular procedures and aortic surgery. They can present as part of a multisystem disease which can mimic many conditions, depending on the site of the emboli. As a pathological entity, it was described by Florey in 1945. Relatively few cases have been reported in the literature. At this stage there is no accepted treatment protocol for CE induced renal failure. Little is known about the precise nature of the cells involved in the proliferating tissue surrounding CE in the kidney. To date, all studies on CE have involved routine Haematoxylin and Eosin (H&E) stains. By studying the cellular interactions, hopefully this will contribute further to our understanding of CE in the kidney. Nine (n = 9) out of 1150 consecutive renal biopsies over a six year period were analysed. Standard three- and four-layered peroxidase-antiperoxidase techniques were employed. A panel of antibodies to specific cells were used. The particular cells analysed were the myofibroblast, smooth muscle, endothelium, macrophage, neutrophil, T cell and B cell. All vessels in the biopsy specimen containing CE were analysed. T cell, B cell, macrophage and neutrophil infiltrates were counted and expressed as cells/mm2 using a 0.022 mm2 graticule under 400 (10x40) magnification. The vessel and perivascular space were analysed. The myofibroblast, smooth muscle and endothelial cell proliferation were graded semiquantitatively. Vessels without evidence of CE were used as controls. The data were subjected to statistical analysis using the unpaired non-parametric Mann-Whitney Two Sample test. P values <0.05 were accepted as significant. Histological sections demonstrated the host response in the vessel to CE involve a significant response including the myofibroblast, endothelium, T cell and macrophage. The B cell response was absent and the smooth muscle cell response was not significantly different. The perivascular responses were not different for the cells studied. This study has characterised the host response to CE in the human kidney by demonstrating the presence of the myofibroblast, macrophage T cell and endothelial cell response. The myofibroblast is a cell which is increasingly being recognised in the host response of both granulation tissue and pathological tissue. The population at risk for CE is growing and the disease is increasingly iatrogenic in origin. Currently our only treatment is prevention.