Comprehensive assessment of 12 commercial DNA-binding dyes as alternatives to ethidium bromide for agarose gel electrophoresis.

Staining and visualization of the nucleic acid bands on agarose gels using ethidium bromide (EB) has been a widely used technique in molecular biology. Although it is an efficient dye for this purpose, EB is known to be mutagenic and genotoxic in humans. This led to the emergence of various alternative dyes, which were claimed to be safer and more efficient than EB. However, these dyes portray varied sensitivity and interference with the electrophoretic mobility of nucleic acids. This work aimed at assessing ten nucleic acid-binding dyes and two prestained dyes for these properties by three staining techniques, such as precasting, preloading, and poststaining. Of these, preloading was not suitable for any of the dye while poststaining worked optimal for most of them. Precasting was suitable for only four dyes viz. DNA Stain G, SYBR™ safe, EZ-Vision® in-gel, and LabSafe™. Poststaining was, in general, a costlier method than precasting. The work gives a comprehensive understanding of the performance of nucleic acid-binding dyes for routine molecular biology experiments.

Application of multiple laboratory tests for Mycobacterium avium ssp. paratuberculosis detection in Crohn's disease patient specimens.

The difficulties involved in detecting and enumerating Mycobacterium avium subsp. paratuberculosis (MAP) as a pathogen potentially involved in Crohn's disease (CD) are well known. This study aimed to improve this situation through the application of multiple laboratory diagnostic tests to detect and isolate this bacterium from different specimens collected from CD-patients and non-CD subjects as controls. A total of 120 samples (terminal ileum and colon biopsies, blood and stool) were obtained from 19 CD-patients and from 11 individuals who did not have a clinicopathological diagnosis of CD (non-CD controls) attending for ileocolonoscopy. All samples were processed by staining techniques, culture on both solid and liquid media, and Insertion Sequence 900/F57 real-time PCR. The MAP frequency in CD-patients was found in a significantly greater proportion than in non-CD subjects; the most positive samples were biopsies from CD-patients tested by real-time PCR. MAP detection in biopsies, and in the other samples, by applying multiple and validated laboratory diagnostic tests, could be a marker of active infection, supporting MAP involvement in CD.

Cell-death assessment by fluorescent and nonfluorescent cytosolic and nuclear staining techniques.

Apoptosis, a genetically programmed cellular event leads to biochemical and morphological changes in cells. Alterations in DNA caused by several factors affect nucleus and ultimately the entire cell leading to compromised function of the organ and organism. DNA, a master regulator of the cellular events, is an important biomolecule with regards to cell growth, cell death, cell migration and cell differentiation. It is therefore imperative to develop the staining techniques that may lead to visualize the changes in nucleus where DNA is housed, to comprehend the cellular pathophysiology. Over the years a number of nuclear staining techniques such as propidium iodide, Hoechst-33342, 4', 6-diamidino-2-phenylindole (DAPI), Acridine orange-Ethidium bromide staining, among others have been developed to assess the changes in DNA. Some nonnuclear staining techniques such as Annexin-V staining, which although does not stain DNA, but helps to identify the events that result from DNA alteration and leads to initiation of apoptotic cell death. In this review, we have briefly discussed some of the most commonly used fluorescent and nonfluorescent staining techniques that identify apoptotic changes in cell, DNA and the nucleus. These techniques help in differentiating several cellular and nuclear phenotypes that result from DNA damage and have been identified as specific to necrosis or early and late apoptosis as well as scores of other nuclear deformities occurring inside the cells.

Detection, Isolation and Quantification of Myocardial Infarct with Four Different Histological Staining Techniques.

Precise quantification of myocardial infarction is crucial for evaluating the therapeutic strategies. We developed a robust, color-based semi-automatic algorithm capable of infarct region detection, isolation and quantification with four different histological staining techniques, and the isolation and quantification of diffuse fibrosis in the heart. Our method is developed based on the color difference in the infarct and non-infarct regions after histological staining. Mouse cardiac tissues stained with Masson's trichrome (MTS), hematoxylin and eosin (H&E), 2,3,5-Triphenyltetrazolium chloride and picrosirius red were included to demonstrate the performance of our method. We demonstrate that our algorithm can effectively identify and produce a clear visualization of infarct tissue for the four staining techniques. Notably, the infarct region on a H&E-stained tissue section can be clearly visualized after processing. The MATLAB-based program we developed holds promise in the infarct quantification. Additionally, our program can isolate and quantify the diffuse fibrotic elements from an MTS-stained cardiac section, which suggested the algorithm's potential for evaluating pathological cardiac fibrosis in diseased cardiac tissues. In conclusion, we demonstrate that this color-based algorithm is capable of accurately identifying, isolating and quantifying cardiac infarct regions with different staining techniques, as well as the diffuse and patchy fibrosis in MTS-stained cardiac tissues.

Papillary adenoma of the lung: A case report and literature review.

Papillary adenoma of the lung, a rare and benign tumor, is easily confused with other primary benign or malignant lung tumors and especially with lung adenocarcinoma that has a papillary growth pattern. Enhanced understanding and an accurate diagnosis of papillary adenomas of the lung are crucial for clinical treatment and prognostic assessment. A 61-year-old man who presented with an opportunistic finding in relation to a left lower lobe lung nodule during an examination was admitted to The First Hospital of China Medical University (Shenyang, China) for further treatment. Computed tomography (CT) revealed a well-circumscribed left lower lobe nodule (diameter, ~1 cm), comprising branched papillae with a fibrovascular core and no other structural components. The tumor cells appeared relatively uniform in shape and well arranged with round or oval nuclei. No nucleoli or mitotic figures were observed. Immunohistochemically, the papillary structures of the tumor cells were strongly and diffusely positive for cytokeratin (CK), CK7, Napsin-A and thyroid transcription factor 1. The Ki-67 index was ~1%. A pathological diagnosis of primary papillary adenoma of the lung was made based on these findings. A left lower-lobe wedge resection was performed and the patient's postoperative course was uneventful. Surgical resection is the preferred treatment. Papillary adenoma of the lung is very rare, and its clinical manifestations and CT images are non-specific. It is important to avoid misdiagnosing of papillary adenoma of the lung as another type of lung tumor, especially adenocarcinoma. A clear understanding of the morphological and immunohistochemical features of papillary adenomas is important for the diagnosis of this rare lung tumor.

Ciliated Peritrichous Protozoa in a Tezontle-Packed Sequencing Batch Reactor as Potential Indicators of Water Quality.

The presence of colonial and solitary ciliated peritrichous protozoa was determined in a Sequencing Batch Reactor system filled with tezontle, a volcanic rock, economic, and abundant material that can be found in some parts of the world, like Mexico. The presence of these protozoa was related to the removal efficiencies of organic matter. Also, two novel staining techniques are proposed for staining both colonial and solitary peritrichous protozoa. The results show that tezontle promotes the growth of solitary and colonial ciliated peritrichous protozoa, which, once identified, could be used as indicators of the efficiency of the wastewater treatment process. Additionally, the staining techniques established in the current study allowed the precise observation of protozoan nuclei. They can represent a useful complementary methodology for identifying protozoan species present in water treatment processes, along with the already existing identification techniques. The number and variety of protozoa found in the system may be considered potential bioindicators of water quality during biological treatments.

First report and molecular characterization of Cryptosporidium spp. in humans and animals in Khartoum state, Sudan.

BACKGROUND AND AIM: Cryptosporidium is recognized to infect several mammalian species as well as humans, causing substantial economic losses and serious public health concern. Infected animals can be a source of environmental contamination and human infections. In general, the occurrence of Cryptosporidium species in animals and human in Sudan and zoonotic importance is not well documented. This study aimed to identify Cryptosporidium spp. infecting different animal species and humans and to compare between different isolates obtained. MATERIALS AND METHODS: To provide molecular information about Cryptosporidium in animals and humans, both modified Ziehl-Neelsen (MZN) specific stain and molecular assay were used. Concentration techniques followed by three protocols of DNA extraction were carried out. After microscopic screening of 263 fecal samples (goats [n=197], cattle [n=12], sheep [n=12], and human [n=42]), 61 positive and 30 negative, randomly selected samples were used in nested polymerase chain reaction (PCR) targeting part of the 18S RNA. RESULTS: Nested PCR amplification confirmed 91.8% (56/61) of microscopic-positive samples. 8.2% (5/61) of negative samples by PCR (positive by microscopy) were considered false negatives. Sequencing followed by alignment of the 14 isolates indicated that all samples were identical (100%) and belonged to Cryptosporidium parvum. CONCLUSION: MZN staining procedure is reliable for the routine diagnosis of Cryptosporidium; cetyltrimethylammonium bromide extraction buffer and nested PCR targeting 18S rRNA gene are reliable and useful in epidemiological studies of this parasite.

Death by electrocution: Histological technique for copper detection on the electric mark.

The current observation of deaths by electrocution, both for domestic and work-related accidents as well as those in other contexts, has deepened the scope of investigation into electric marks, especially from the histological point of view. This is one of the few investigation tools that may lead to the diagnosis of death by electrocution in this distinct area, bearing in mind the diagnostic difficulties that this type of fatality presents. Our attention has been placed on the phenomenon of metallization. In particular, we focused on using the Timm's method [1] to locate the copper deposits. The phenomenon of metallization, usually could be caused by the copper deposit, this happens due to the copper debris released onto the skin by the live conductor. To date, this technique has only been used in the pathological field. Nevertheless, we tried to assess its application in seven selected cases, after partially modifying the technique, comparing it with the most common staining detection techniques and analysing the specificity, sensitivity as well as the potential for its application in the routine.

Using cytology to increase small animal practice revenue.

Diagnostic cytology is a useful, noninvasive test with practical foundations in high-quality medicine and applications to practice building. Cytology will generate practice revenue whether assessed in-house or sent to a clinical pathologist. Thorough in-house evaluation is adequate in some cases, but expert opinion is important in many cases. Specimen slides should at least be reviewed in-house for assessment of cellularity and potential artifacts before submission to a reference laboratory. Reference laboratories also provide special stains and advanced molecular diagnostics to help further characterize many neoplastic processes, search for organisms, identify pigments, and address other important aspects of the lesion.

COMPARISON OF PERMANENT STAINING METHODS FOR THE LABORATORY DIAGNOSIS OF TRICHOMONIASIS

Trichomonas vaginalis is the etiologic agent of trichomoniasis, the most common non-viral sexually transmitted disease (STD) in the world. The diagnosis is based on wet mount preparation and direct microscopy on fixed and stained clinical specimens. The aim of this study was to compare the performance of different fixing and staining techniques used in the detection of T. vaginalis in urine. The smears were fixed and submitted to different methods of permanent staining and then, the morphological aspects of the parasites were analyzed and compared. The Papanicolaou staining with ethanol as the fixative solution showed to be the best method of permanent staining. Our data suggest that staining techniques in association with wet mount examination of fresh specimens contribute to increase the sensitivity in the diagnosis of trichomoniasis.

Padronização do diagnóstico de Blastocystis hominis por diferentes técnicas de coloração / Standardization of Blastocystis hominis diagnosis using different staining techniques

INTRODUÇÃO: O presente trabalho foi realizado no período de março a maio de 2008, com o objetivo de avaliar a eficácia de diferentes técnicas para diagnóstico de Blastocystis hominis em uma amostra da população atendida pelo Laboratório de Biomedicina da Universidade Feevale, Novo Hamburgo/RS. MÉTODOS: Foram estudadas 100 amostras de fezes de crianças e adultos, que foram coletadas e submetidas às técnicas de sedimentação espontânea (HPJ), sedimentação em formol-éter (Ritchie) e de coloração por Gram e May-Grünwald-Giemsa (MGG). RESULTADOS: A presença de Blastocystis hominis foi observada em 40 amostras, quando utilizadas técnicas de coloração (MGG e Gram), enquanto que as técnicas de sedimentação se mostraram menos eficientes (32 amostras positivas para a técnica de Ritchie e 20 amostras positivas para a técnica de HPJ). CONCLUSÕES: Nossos resultados demonstram que o HPJ foi menos eficiente que outros métodos, indicando a necessidade de se incluir na rotina do laboratório técnicas que permitam a identificação deste parasita.

INTRODUCTION: The present study was carried out from March to May 2008, with the aim of evaluating the effectiveness of different techniques for diagnosing Blastocystis hominis in a sample of the population attended at the Biomedicine Laboratory of Feevale University, Novo Hamburgo, Rio Grande do Sul. METHODS: On hundred feces samples from children and adults were evaluated. After collection, the samples were subjected to the techniques of spontaneous sedimentation (HPJ), sedimentation in formalin-ether (Ritchie) and staining by means of Gram and May-Grünwald-Giemsa (MGG). RESULTS: The presence of Blastocystis hominis was observed in 40 samples, when staining techniques were used (MGG and Gram), while sedimentation techniques were less efficient (32 positive samples using the Ritchie technique and 20 positive samples using the HPJ technique). CONCLUSIONS: Our results demonstrate that HPJ was less efficient than the other methods, thus indicating the need to include laboratory techniques that enable parasite identification on a routine basis.

Avaliação da punção aspirativa por agulha fina (PAAF) em cavidade oral e região de cabeça e pescoço em diferentes técnicas de coloração / Evaluation of fine needle aspiration biopsy (FNAB) in oral cavity and head and neck region with different techniques stains

O presente estudo teve como objetivo avaliar a Punção Aspirativa por Agulha Fina (PAAF) em diferentes técnicas de coloração, em lesões nodulares de cavidade oral e região de cabeça e pescoço, quanto a sua sensibilidade, especificidade e acurácia, nas colorações de Panótico, Papanicolau e Hematoxilina-Eosina . Foram selecionados 46 pacientes consecutivamente que procuraram a Clínica da Disciplina de Estomatologia Clínica da FOUSP, portadores de lesões nodulares em cavidade oral e região de cabeça e pescoço. Como critérios de inclusão foram selecionados pacientes de ambos os sexos, todas as etnias, acima dos 5 anos de idade, sem restrição de comorbidades e que foram realizadas PAAF com confirmação diagnóstica pela biópsia. Como critérios de exclusão da pesquisa estão os pacientes abaixo dos 5 anos de idade e pacientes que foram somente submetidos a PAAF sem confirmação diagnóstica pela biópsia. O material obtido pela PAAF foi enviado em 6 lâminas diferentes, corados pelo método de Panótico, Papanicolau e Hematoxilina-Eosina a um mesmo patologista apenas com o diagnóstico clínico. Após a emissão do laudo da PAAF, o laudo do anátomo patológico era emitido, servindo como padrão ouro. Após os cálculos, o resultado da sensibilidade, especificidade e acurácia para o método de coloração com o Panótico foram de 28,6%, 76%, 15,4, respectivamente, para o método de coloração com o Papanicolau foram de 71,4%, 76,7%, 23,3%, respectivamente e para o método de coloração com a Hematoxilina-Eosina foram de 82,1%, 23,3%, 28,6%, respectivamente...

The present study aimed to evaluate the Fine Needle Aspiration Biopsy in different staining techniques in nodular lesions of the oral cavity and head and neck region, as their sensitivity, specificity and accuracy, staining with Panoptic, Papanicolaou and Hematoxylin-Eosin. 46 patients who sought the Clinic of the Discipline of Clinical Stomatology at FOUSP were selected consecutively, with nodular lesions in the oral cavity and head and neck region. Inclusion criteria were patients of both sexes, all ethnicities, above 5 years-old, with no restriction of comorbidities and FNAB performed with confirmation by biopsy. Exclusion criteria were patients under 5 years-old and patients who only underwent FNAB without confirmation by biopsy. The material obtained by FNAB was sent on 6 different slides, stained by the method of Panoptic, Papanicolaou and Hematoxylin-Eosin, to the same pathologist only with the clinical diagnosis. After the final report of FNAB, the biopsy report was issued, serving as gold standard. After the calculations, the results of sensitivity, specificity and accuracy for Panoptic staining were 28.6%, 76% and 15.4%, respectively...