Formalin pre-fixation improves autopsy histology

Microscopic findings in key tissues are often critical to determine the cause of death in medical autopsies. The overall quality of histologic sections depends on numerous pre-analytic factors, among which are tissue section size and thickness. We designed a prospective quality improvement study to determine whether a simple intervention of formalin pre-fixation of myocardium, liver, and kidney tissues could improve the ease of cutting and quality of autopsy histologic sections as assessed by histotechnicians and pathologists. Of 46 autopsies included in the study, 21 were randomly assigned to formalin pre-fixation, and 25 underwent routine sectioning without formalin pre-fixation. A significant improvement in overall quality score by histotechnicians was detected in the sections from pre-fixed autopsy tissues compared to the control group (p=0.0327). There was no significant difference in quality score between the two groups as assessed by pathologists. Our autopsy quality improvement study demonstrates that a simple, low-cost intervention of formalin pre-fixation of fresh autopsy tissues for 90 minutes could significantly improve the overall quality of sections submitted for histologic processing.

Alcohol-based fixatives can better preserve tissue morphology than formalin / Fijadores a base de alcohol pueden preservar mejor la morfología tisular que la formalina

SUMMARY: Fixation is a crucial step in processing of tissue specimen for preservation of cellular architecture and composition of cells. Alcohol-based fixatives are considered some of the most promising alternatives to formalin. We evaluated the performance of alcohol-based fixatives (EthMeth and methacarn) and formalin as a comparator fixative in the research laboratory. Following 24 hours of fixation, tissue morphology and cellular details of the liver, spleen and brain (cerebral cortex) were evaluated. Morphological characteristics were evaluated by gross observations and analyzing cellular details, tissue architecture and overall staining characteristics (Hematoxylin and Eosin). EthMeth and methacarn fixation gave generally comparable and satisfactory results on the tissue morphology and subsequent identification of tissue characteristics. Particularly, tissues were well preserved and all nuclear as well as cytoplasmic details were clearly visible. However, formalin fixed tissues showed some peculiarity such as improper fixation, mild shrinkage, and alterations of tissue components. These results confirm that alcohol-based fixation is the superior alternative to formalin for preservation of tissue morphology. However, it is required to standardize the formalin-free methods and harmonize diagnosis in the laboratory worldwide.

RESUMEN: La fijación es un paso crucial en el procesamiento de muestras de tejido para preservar la arquitectura celular y la composición de las células. Los fijadores a base de alcohol se consideran algunas de las alternativas más prometedoras a la formalina. Evaluamos el rendimiento de los fijadores a base de alcohol (EthMeth y metacarn) y formalina como fijador comparativo en el laboratorio de investigación. Después de 24 horas de fijación, se observó la morfología del tejido y los detalles celulares del hígado, bazo y corteza cerebral. Se evaluaron las características morfológicas mediante observaciones generales y analizando detalles celulares, arquitectura de tejidos y características generales de tinción (hematoxilina y eosina). La fijación de EthMeth y metacarn dio resultados generalmente comparables y satisfactorios en la morfología del tejido y la posterior identificación de las características del mismo. Particularmente, los tejidos estaban bien conservados y todos los detalles nucleares y citoplasmáticos eran claramente visibles. Sin embargo, los tejidos fijados con formalina mostraron cierta peculiaridad, tal como una fijación inadecuada, la contracción leve y alteraciones de los componentes del tejido. Estos resultados confirman que la fijación a base de alcohol es la mejor alternativa a la formalina, para preservar la morfología del tejido. Sin embargo, es necesario estandarizar los métodos sin formalina y armonizar el diagnóstico en los laboratorios.

Brain structure morphology after being fixated with ethanol on electron microscope / Morfología de la estructura cerebral después de la fijación con etanol en microscopio electrónico

Fixation is one of the processes in preparing histology and pathology. The common material for fixation is buffered formalin including paraformaldehyde. However, the effect of the damaged cells, which is fixed for a long time, causes the research for other fixation materials to become necessary. In addition, paraformaldehyde is also harmful to human body and natural environment. Ethanol is one of the alternative fixation materials, which has been used for two hundred years. It has been used for many purposes, both in routine staining and immunohistochemistry. Nonetheless, no research confirms its effect on the electron microscope. The authors studied the effect of 50 % of ethanol on the cell membrane, organelles, and nucleus of Purkinje cells (Neuron purkinjense) observed on a light microscope and Transmitted Electron Microscope (TEM). Then it was compared to buffered formalin. In the light microscope, it shows that both of fixations have no different effects of the morphology of the cell membrane, cytoplasm, the nucleus of Purkinje cells and the neutrophils. We assume that our 50 % of ethanol concentration is almost the same as BF 10 % in the ability of hardening tissue and color absorption based on the previous study. In TEM, the structure of the cell membrane, organelles, and cytoplasm of Purkinje cell look broken in the cerebellum of 50 % of ethanol except for the nucleus. There was no significant difference diameter of the nucleus. It happened in general because of the shrinkage effect of ethanol. However, the authors recommend using 50 % of ethanol for routine staining.

La fijación es uno de los procesos en la preparación de muestras para histología y patología. El material más común para la fijación es la formalina tamponada. Sin embargo, el daño a las células que se mantienen en formalina durante mucho tiempo, hace necesario buscar otros materiales de fijación. Además, el paraformaldehido también es perjudicial para el cuerpo humano y el medio ambiente natural. El etanol es uno de los materiales de fijación alternativos que se ha utilizado durante muchos años, con diversos objetivos, tanto en la tinción de rutina como en la inmunohistoquímica. Sin embargo no se ha confirmdo su efecto con microscopio electrónico. Los autores estudiaron el efecto del 50 % de etanol sobre la membrana celular, los orgánulos y el núcleo de las células de Purkinje observados en un microscopio óptico y un microscopio de transmisión electrónico (TEM). Luego se comparó con la formalina tamponada. En el microscopio óptico se observó que ambas fijaciones no tienen efectos diferentes a la morfología de la membrana celular, el citoplasma, el núcleo de las células de Purkinje y los neutrófilos. Suponemos que nuestra concentración de 50 % de etanol es casi la misma que BF 10 % en la capacidad de endurecer el tejido y la absorción de color según el estudio anterior. En TEM, la estructura de la membrana celular, los orgánulos y el citoplasma de la célula de Purkinje presentaban daño en el cerebelo con un 50 % de etanol, a excepción del núcleo. No hubo diferencia significativa en el diámetro del núcleo. En general lo anterior se debió al efecto de contracción del etanol. En conclusión los autores recomiendan usar 50% de etanol para la tinción de rutina.

Tratamento das ptoses mamárias com implantes submusculares e pontos de fixação do tecido mamário ao muscular: aspectos técnicos e avaliação de resultado / Treatment of breast ptosis with submuscular implants and points of attachment of the breast to the pectoralis muscle: technical considerations and outcome assessment

Introdução: Mastopexia associada à inclusão de implante é uma situação desafiadora para o cirurgião plástico. O objetivo é descrever a colocação de implante submuscular com descolamento anatômico mais pexia firme do tecido glandular usando pontos de fixação do tecido mamário ao muscular e analisar os resultados estéticos das pacientes operadas. Método: Foram realizadas 23 mastopexias com implantes no período entre abril de 2015 e julho de 2017, pelo mesmo cirurgião, sendo as mamas das pacientes marcadas previamente, na posição sentada. Realizou-se incisão no sulco mamário e descolamento até o polo superior da mama no plano subfascial, fixação da glândula ao músculo peitoral maior com 9 a 12 pontos. A seguir, iniciou-se a dissecção do músculo peitoral maior através de sua origem costal e transição com os músculos reto abdominal e serrátil, liberando amplamente na porção inferior. Introduziu-se o implante e completou-se a mastopexia. Os tamanhos dos implantes variaram de 255ml a 355ml. Fotos das mamas de 12 pacientes foram avaliadas por dois cirurgiões plásticos e dois leigos, nos seguintes parâmetros: resultado estético, simetria das aréolas e grau de ptose mamária. As avaliações podiam ser Ruim, Razoável ou Bom. Resultados: A técnica cirúrgica mostrou-se reprodutível, apenas 1 caso de hematoma unilateral, nenhuma infecção, queixas de dor discretas. Apenas um caso foi considerado, por um único avaliador, como Razoável; as demais avaliações consideradas como Bom. Conclusão: O tratamento de ptoses mamárias com colocação de implante submuscular acrescido de pexia da glândula ao músculo peitoral é uma técnica reprodutível e com bons resultados estéticos.

Introduction: Mastopexy associated with implant placement is challenging for plastic surgeons. The objective is to describe the placement of a submuscular implant with anatomical detachment in combination with stable fixation of the breast tissue to the pectoralis muscle and analyze the aesthetic results. Method: Twenty-three mastopexy procedures with implants were performed from April 2015 to July 2017 by the same surgeon, and surgical markings were made in the breasts of the patients in a seated position. An incision was made in the inframammary fold, and the breast tissue was elevated to the upper pole in the subfascial plane and attached to the pectoralis major muscle using 9-12 stitches. Subsequently, the inferior margin of the pectoralis major muscle and the transition from the rectus abdominis muscle to the serratus muscle were dissected to expose the muscle. The implant was introduced and mastopexy was completed. Implant size ranged from 255 mL to 355 mL. Photographs of the breasts of 12 patients were evaluated by two plastic surgeons and two non-medical subjects, who considered the aesthetic results, symmetry of the nipple-areola complex, and degree of breast ptosis. The results were scored as unsatisfactory, satisfactory, or good. Results: The surgical technique was reproducible; there was only one case of unilateral hematoma, no implant infections, and only complaints of mild pain. Only one case was scored as satisfactory by one evaluator, whereas the results of the other cases were considered good. Conclusion: The treatment of breast ptosis with the placement of a submuscular implant in combination with fixation of the breast to the pectoralis major muscle is reproducible and yields good aesthetic results.

A comparative study of fixatives for axolotl blastema tissue / Estudio comparativo de fijadores para el tejido blastema del axolotl

Regeneration is defined as tissue renewal and functional restoration process of the damaged parts of the body after an injury. Ambystoma mexicanum, commonly named the Axolotl, is one of the unique vertebrates, which has a remarkable ability to regenerate their extremities following the amputation. Although the process of regeneration includes several periods, it can be divided into two main phases; blastema formation and dedifferentiation. In the couple of hours following the amputation, wound closure occurs by migration of epithelial cells around the amputation site followed by macrophage infiltration and dedifferentiation of cells to turn into stem cells. Accumulated stem cells form a very authentic tissue type called blastema, which is crucial for successful regeneration. In order to evaluate this exceptional tissue and acquire high quality images, it is crucial to employ specific procedures to prepare the tissue for imaging. Here, in this study, we aimed to investigate success of various fixative solutions (Carnoy's, Bouin's, % 10 NBF, Clarke's, Alcoholic Formaline and AFA) to monitor the fixed blastema. Our data reveals that integrity of the blastema tissue differs among used fixatives and a significant difference is observed between the samples in terms of staining quality.

La regeneración se define como la renovación del tejido y el proceso de restauración funcional de las partes dañadas del cuerpo después de una lesión. Ambystoma mexicanum, comúnmente llamado Axolotl, es uno de los únicos vertebrados que tiene una notable capacidad para regenerar sus miembros después de una amputación. Aunque el proceso de regeneración incluye varios períodos, se puede dividir en dos fases principales: formación del blastema y desdiferenciación. En el par de horas después de la amputación, el cierre de la herida ocurre por la migración de células epiteliales alrededor del sitio de la amputación seguido por una infiltración de macrófagos y la desdiferenciación de las células para convertirse en células madre. Las células madre acumuladas forman un tipo de tejido muy diferenciado denominado blastema, que es crucial para una exitosa regeneración. Para evaluar este tejido y adquirir imágenes de alta calidad, es crucial emplear procedimientos específicos para la obtención de imágenes. En este estudio, se intentó investigar el éxito de varias soluciones fijadoras (Carnoy, Bouin, % 10 NBF, Clarke, Formalina Alcohólica y AFA) para monitorear la fijación del blastema. Nuestros datos revelan que la integridad del tejido del blastema difiere entre los fijadores utilizados y una diferencia significativa observada entre las muestras se da en términos de la calidad de tinción.

Fita microporosa para fixação de enxertos cutâneos / The use of a microporous tape for fixing skin grafts

Introdução: A fixação do enxerto cutâneo é essencial para sua integração no leito receptor. A literatura apresenta várias técnicas de fixação, porém, o uso da fita de microporosa é pouco relatado. O objetivo é demonstrar e divulgar o uso da fita microporosa na fixação do enxerto cutâneo Métodos: Estudo prospectivo, realizado de janeiro de 2014 a janeiro de 2016. Em 40 pacientes foi utilizada a fita microporosa esterilizada como método isolado para a fixação do enxerto Resultados: Enxertos cutâneos apresentaram resultado satisfatório sem mobilização e, consequentemente, boa integração. Conclusão: O uso da fita microporosa esterilizada é um excelente método para a fixação de enxertos cutâneos, por ser simples, rápido e seguro.

Introduction: Fixing a skin graft is essential to its integration in the recipient bed. The literature presents several fixation techniques. However, only few reports on the use of microporous tape are available. This study aims to demonstrate and promote the use of microporous tape in fixing skin grafts. Methods: A prospective study was performed from January 2014 to January 2016. In 40 patients, a sterilized microporous tape was used as an isolated method to fix skin grafts. Results: The use of skin graft immobilization showed satisfactory results and consequently good integration. Conclusion: The use of a sterilized microporous tape is an excellent method for fixing skin grafts because it is easy, fast, and safe to use.

Difference between actual vs. pathology prostate weight in TURP and radical robotic-assisted prostatectomy specimen

Introduction To investigate and highlight the effect of formaldehyde induced weight reduction in transurethral resection of prostate (TURP) and radical robotically-assisted prostatectomy (RALP) specimen as a result of standard chemical fixation. Materials and Methods 51 patients were recruited from January 2013 to June 2013 who either underwent a TURP (n=26) or RALP (n=25). Data was collected prospectively by the operating surgeon who measured the native, unfixed histology specimen directly after operation. The specimens were fixed in 10% Formaldehyde Solution BP and sent to the pathology laboratory where after sufficient fixation period was re-weighed. Results Overall mean age 64.78 years, TURP mean age 68.31 years RALP mean age 61.12years. We found that the overall prostatic specimen (n=51) weight loss after fixation was a mean of 11.20% (3.78 grams) (p≤0.0001). Subgroup analysis of the native TURP chips mean weight was 16.15 grams and formalin treated mean weight was 14.00 grams (p≤0.0001). Therefore, TURP chips had a mean of 13.32 % (2.15 grams) weight loss during chemical fixation. RALP subgroup unfixed specimen mean weight was 52.08 grams and formalin treated mean weight was 42.60 grams (p≤0.0001), a 19.32 % (9.48grams) mean weight reduction. Conclusion It has not been known that prostatic chips and whole human radical prostatectomy specimen undergo a significant weight reduction. The practical significance of the accurate prostate weight in patient management may be limited, however, it is agreed that this should be recorded correctly, as data is potential interest for research purposes and vital for precise documentation. .

Experimental model for the study of soft tissue fixation methods on skin-pericranium flaps in rats

PURPOSE: To develop an experimental model to study and radiologically monitor displacement of skin flaps in the pericranium of rats subjected to traction and surgical fixation using suture anchored in a skull bone tunnel or with N-butyl-2-cyanoacrylate (HistoacrylTM) surgical adhesive. METHODS: Radiological markers were placed in the subcutis of Wistar rats undergoing subperiosteal detachment of the pericranium with pulling and fixation of the flap. We performed radiography on postoperative days 3, 7, 14, 21, and 45. A p-value of <0.05 was considered significant. RESULTS: Qualitative analysis of the data indicated that the flaps in the surgical adhesive group remained in place with no change from the immediate postoperative position. However, the flaps in the suture anchored in the skull bone tunnel group and in the control group showed similar healing, with a loss of attachment of 9.7% and 22%, respectively, compared with the immediate postoperative position. There was no quantitative difference between the groups. CONCLUSIONS: This experimental model created acceptable experimental conditions for testing different soft tissue fixation methods. The use of tissue fixatives contributed to better placement of pericranium-cutaneous flaps, and surgical adhesive was superior to suture anchor in the skull bone tunnel for fixation of pericranium-cutaneous flaps.

Effect of fixatives' temperatures on subsequent histochemical staining

Fixation is complex series of chemical events which differs for the different group of chemical substances found in tissues. Some chemical reactions, including those involved in fixation occur more rabidly at higher temperature. To assess the effect of varying fixatives' temperature on the quality of subsequent histochemical staining. Rabbit samples were collected including tongue tissue to demonstrate collagen fibers using Van Geison's stain, and liver tissue to demonstrate cell morphology using Erlich's haematoxylin. Specimens were divided into pieces; each sample was fixed in the following fixatives: formal saline, neutral buffer formalin [NBF], Carnoy's and Bouin's fixative in different temperatures as follow 4°C, 25°C, 37°C and 60°C. There after, tissues were embedded in paraffin and cut sections into 5 micron and stained with Ehrlich's hematoxylin and Van Gieson histochemical stains. For Erlich's heamtoxylin, formal saline gave the best result for tissues fixed at 60°C; NBF gave the best results at 37°C and 60°C. For Van Geison stain, formal saline and NBF the best results obtained at 37°C. The study concluded that using 10% NBF, 10% Formal saline, Carnoy's and Bouin's fixatives applying different temperatures include 4°C, 25°C, 37°C and 60°C affect the subsequent histochemical staining of Ehrlich's hematoxylin, and Van Gieson

Efeito da descelularização com SDS na prevenção da calcificação em pericárdio bovino fixado em glutaraldeído: estudo em ratos / Effect of SDS-based decelullarization in the prevention of calcification in glutaraldehyde-preserved bovine pericardium: study in rats

OBJETIVO: Avaliar a descelularização com SDS como tratamento anticalcificante em pericárdio bovino fixado em glutaraldeído. MÉTODOS: Peças de 0,5 cm² foram implantadas em modelo subcutâneo de 18 ratos por até 90 dias. Foram formados quatro grupos: grupo GDA: pericárdio fixado em glutaraldeído 0,5% (GDA), grupo GDA-GL: pericárdio fixado em GDA + ácido glutâmico (GL) 0,2%, grupo D-GDA: pericárdio descelularizado (D) com SDS 0,1% e fixado em GDA e grupo D-GDA-GL: pericárdio descelularizado + GDA + ácido glutâmico 0,2%. Cada animal recebeu enxertos dos quatro grupos. Os explantes foram realizados com 45 e 90 dias. As avaliações foram: análise histológica com as colorações hematoxilina-eosina e alizarina-red, análise morfométrica e quantificação de cálcio por espectrometria de absorção atômica. RESULTADOS: O padrão de infiltrado inflamatório foi o mesmo nos quatro grupos, sendo mais intenso nos grupos GDA e GDA-GL aos 45 dias, ficando mais evidente aos 90 dias. O conteúdo de cálcio aos 45 dias foi de 32,52 ± 3,19 µg/ mg no grupo GDA; 22,12 ± 3,87 µg/mg no grupo GDA-GL; 1,06 ± 0,38 µg/mg no grupo D-GDA e 3,99 ± 5,78 µg/mg no grupo D-GDA-GL (P< 0,001). Aos 90 dias, foi de 65,91 ± 24,67 µg/mg no grupo GDA; 38,37 ± 13,79 µg/mg no grupo GDA-GL; 1,24 ± 0,99 µg/mg no grupo D-GDA e 30,54 ± 8,21 µg/mg no grupo D-GDA-GL (P< 0,001). O grupo D-GDA foi o único que não apresentou progressão da calcificação de 45 para 90 dias (P=0,314). CONCLUSÃO: A descelularização com SDS reduziu o processo inflamatório e inibiu a calcificação em pericárdio bovino implantado em modelo subcutâneo de ratos até 90 dias.

OBJECTIVE: The aim of study was to investigate the SDS-based decellularization process as an anticalcification method in glutaraldehyde-preserved bovine pericardium in subcutaneous rat model. METHODS: Pericardium samples with 0.5 cm² area were divide in four groups: group GDA: 0.5% glutaraldehydepreserved pericardium (GDA); group GDA-GL: GDA + 0.2% glutamic acid (GL); group D-GDA: decellularized (D) pericardium with 0.1% SDS + GDA and group D-GDA-GL: decellularized pericardium + GDA + 0.2% glutamic acid. After this samples were implanted in 18 rats in subcutaneous position till 90 days. Each animal received samples of the four groups. The explants were performed at 45 and 90 days. The explants were subjected to histology in glass slides stained with hematoxilin-eosin and alizarin red, morphometry evaluation and the calcium content was measured by flame atomic absorption spectrometry. RESULTS: The inflammatory infiltrate was the same in all groups, however more intense in GDA and GDA-GL groups in 45 days, increasing at 90 days. The calcium contents for 45 days were: 32.52 ± 3.19 µg/mg in GDA group; 22.12 ± 3.87 µg/ mg in GDA-GL group; 1.06 ± 0.38 µg/mg in D-GDA group and 3.99 ± 5.78 µg/mg in D-GDA-GL (P< 0.001). For 90 days were 65.91 ± 24.67 µg/mg in GDA group; 38.37 ± 13.79 µg/mg in GDA-GL group; 1.24 ± 0.99 µg/mg in D-GDA group and 30.54 ± 8.21 µg/mg in D-GDA-GL (P< 0.001). Only D-GDA did not show increase rates of calcium at 45 to 90 days (P=0.314). CONCLUSION: SDS-based decellularization process reduced the inflammatory intensity and calcification in bovine pericardium in subcutaneous rat model for 90 days.

Minimally invasive lift of the middle third of the face using musculoaponeurotic suspension with periosteal fixation technique: a review of 50 cases / Suspensão musculoaponeurótica com fixação periostal minimamente invasiva do terço médio da face: revisão de 50 casos

BACKGROUND: At present, demanding workplaces in our society cause patients to search for less invasive procedures with diminished morbidity and more rapid healing to meet their cosmetic requirements. A combination of several new noninvasive procedures allows significant facial changes, achieving a youthful and healthy appearance without traditional surgical procedures. OBJECTIVE: The purpose of this study is to describe the minimally invasive lift of the middle third of the face using a musculoaponeurotic suspension with periosteal fixation technique. METHODS: Fifty patients (age, 39 to 68 years; all female) who underwent an operation from December 2008 to June 2010 were enrolled in this study. The patients underwent a minimally invasive facelift technique for the middle third of the face, based on a thread lift of the temporal region and musculoaponeurotic suspension with periosteal fixation, inside the hairline. RESULTS: During the follow-up period of up to 18 months after the procedure, satisfactory results were observed. The patient satisfaction degree, especially in the first 6 months after the procedure, was extremely high (88 percent). CONCLUSIONS: The procedure offers good and immediate results, without incisions or a recovery period. The association of this procedure with other procedures is a good option for patients who cannot undergo or do not want to undergo traditional surgical procedures. The procedure is very different from current techniques that use threads because the suspension is musculoaponeurotic and does not invade the face. Therefore, morbidity and recovery time are decreased.

INTRODUÇÃO: Na sociedade atual, em decorrência das demandas profissionais, os pacientes cada vez mais procuram por procedimentos menos invasivos, com baixa morbidade, rápida recuperação, e que atendam a suas preocupações estéticas. Uma combinação de vários novos procedimentos não-invasivos permite mudanças faciais significativas e aparência jovem e saudável, sem a utilização de procedimentos cirúrgicos tradicionais. OBJETIVO: O objetivo deste estudo é a descrição de técnica de suspensão musculoaponeurótica com fixação periostal minimamente invasiva do terço médio da face. MÉTODO: Foram incluídos nesse estudo 50 pacientes, com idades entre 39 anos e 68 anos, todos do sexo feminino, operados no period de dezembro de 2008 a junho de 2010. As pacientes foram submetidas à realização de facelift minimamente invasivo do terço médio da face, com suspensão musculoaponeurótica com fixação periostal, baseado na tração com fio passado na região temporal, dentro da área do cabelo. RESULTADOS: No acompanhamento das pacientes, até 18 meses após a realização do procedimento, verificaram-se resultados satisfatórios. O grau de satisfação das pacientes com os resultados obtidos, principalmente nos primeiros seis meses após a realização do procedimento, foi extremamente alto (88 por cento). CONCLUSÕES: O procedimento ofereceu bons e imediatos resultados, sem incisões ou período de recuperação. Associado a outros procedimentos de rejuvenescimento facial, trata-se de boa opção a pacientes que não podem ou não querem se submeter a procedimentos cirúrgicos tradicionais. O procedimento difere significativamente das técnicas atuais que usam fios, pois a suspensão é musculoaponeurótica e não invade a área da face, o que diminui a morbidade e o período de recuperação.

Preservation of Haemal Nodes Fixed by Means of Two Different Aldehyde Fixatives: a Scanning Electron Microscopic Study / Preservación de Nodos Linfáticos Hemales Fijados por Dos Fijadores de Aldehído Diferentes: un Estudio de Microscopía Electrónica de Barrido

Glutaraldehyde is the fixative most commonly used in electron microscope studies of biological tissues, however it is often necessary to use samples which were not fixed in this fixative, even with the usual uncertainty of the results that may be obtained. The fixation is the more delicate step of the sample processing. Therefore in this work, the quality of preservation of haemal nodes fixed with two classic aldehyde fixatives: formaldehyde and glutaraldehyde we have compared under the scanning electron microscope. Our results showed that both fixatives were successful in preserving the morphology of haemal nodes components; however glutaraldehyde conferred satisfactory results mainly in the preservation of parenchymal cells, whereas formaldehyde was better for preservation of stromal fibres.

El glutaraldehido es el fijador que se utiliza con más frecuencia en estudios en los tejidos biológicos a través microscopía electrónica. Sin embargo, a menudo es necesario utilizar muestras que no han sido fijadas con este fijador, aún con la incertidumbre de los resultados que se puedan obtener. La fijación es el paso más importante en el procesamiento de los tejidos. Por lo anterior, hemos efectuado este estudio comparando la calidad de conservación de nodos linfáticos hemales fijados con formaldehido y glutaraldehido. Los resultados muestran que ambos fijadores conservaron adecuadamente la morfología de los componentes de los nodos linfáticos hemales, sin embargo, el glutaraldehido conservó en mejores condiciones, principalmente, las células del parénquima, pero el formaldehido conservó mejor las fibras del estroma en nodos linfáticos.

SNP typing of formalin-fixed tissue in individual identification / 法医学杂志

OBJECTIVE@#To explore the method for DNA typing in formalin-fixed tissue by detecting SNP markers on X chromosome.@*METHODS@#Genomic DNA was prepared from formalin-fixed tissue. In the event that typing using Sinofiler and MiniFiler kits had failed, 51 SNPs were amplified with multiplex PCR and typed with matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). RESULTS Full profiles of X-SNP loci were obtained from the formalin-fixed tissue, while the STR and miniSTR genotyping failed.@*CONCLUSION@#SNP genotyping technique can be used to obtain more information for formalin fixed tissues.

Influence of amplicon size on the polymerase chain reaction of Parvovirus B19 genome in formalin-fixed specimens / Influência do tamanho do amplicon na reação em cadeia da polimerase na detecção do genoma do PB19 em amostras fixadas em formalina

The polymerase chain reaction (PCR) has provided diagnosis of archival material, but some fixation methods such as formalin damage DNA and, subsequently, affect PCR analysis, particularly paraffin-embedded tissues. PCR is known due to its high specificity and sensitivity, although some difficulties arise when formalinfixed and paraffin-embedded tissue is used. Not only does this occur due to protein cross-linking, which increases with longer fixation time, but it also happens due to the direct damage that formalin causes in the DNA itself. PCR was used to analyze placenta and fetal organs from 34 samples with suspected Parvovirus B19 infection. It was not possible to amplify Parvovirus B19 DNA using nested-PCR, probably due to the size of the amplicon generated with the first set of primers. We approached this problem by using only the second set of primers. Two out of 34 tissue samples (5,9 percent) were positive by PCR. However, PCR performed on corresponding fetal organs was negative in one of the two. We also observed a negative relation between the thickness of the tissue fragment and the positivity of the samples. In conclusion, although PCR is highly specific and sensitive in fresh or ideally fixed material, a careful standardization of PCR assays is necessary when using formalin fixed paraffin-embedded tissues by applying primers that require smaller DNA fragments for amplification.

A reação em cadeia da polimerase (PCR) tem fornecido diagnóstico de material de arquivo, mas alguns métodos de fixação, tais como formalina, provocam danos ao DNA e subsequentemente afetam sua análise, particularmente tecidos embebidos em parafina. A PCR é conhecida pela sua alta especificidade e sensibilidade, embora algumas dificuldades ocorram quando o material utilizado foi fixado em formalina e embebido em parafina. Isso não se deve somente pela formação de cross-linkings com proteínas, a qual aumenta com o maior tempo de fixação, mas também pelo dano direto que a formalina causa no DNA. PCR foi usada para analisar placenta e órgão fetais de 34 amostras com suspeita de infecção pelo Parvovírus B19 (PB19). Não foi possível amplificar o DNA do PB19 usando nested-PCR, provavelmente devido ao tamanho do amplicon gerado com o primeiro passo dos primers. Adequamos o problema utilizando somente o segundo par de primers e pudemos observar que das 34 amostras, duas eram positivas para PCR (5,9 por cento). Entretanto, a PCR dos órgãos fetais foi negativa em um de dois casos. Observamos também uma relação negativa entre a espessura do corte dos materiais com a positividade das amostras. Em conclusão, embora a PCR seja altamente específica e sensível em amostras a fresco ou idealmente fixadas, uma padronização cuidadosa para análise com a PCR é necessária quando se utiliza tecidos fixados em formalina e embebidos em parafina utilizando primers que requerem menor fragmento de DNA para a amplificação.

Improved nitric acid digestion method for detecting diatom in autopsy tissue / 法医学杂志

OBJECTIVE@#To investigate the advantages of nitric acid digestion method and its differences with the traditional method.@*METHODS@#Ethanol was used to fully fix the testing sample. About 80-100 g of the testing samples were cut into pieces and digested with nitric acid. It was then centrifuged and washed to remove organic components. Smears were prepared and examined under the light microscope.@*RESULTS@#The diatom had been identified with clear striations, counted conveniently and classified easily.@*CONCLUSION@#The improved nitric acid digestion method is not only simple with a higher successful rate of detection, but also can prevent interference from contamination. It can improve the stability of the experimental results, avoid harm to human and environment, and provide higher safety in the course of experiment.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples / Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10 percent non-buffered or 10 percent buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10 percent non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10 por cento ou formalina tamponada a 10 por cento e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e, separadamente, para amplificação da sequência de inserção IS6110, específica do complexo M. tuberculosis. O resultado da amplificação do gene da beta-actina foi positivo em todas as amostras. Não foram gerados amplicons na PCR-IS6110 em amostras de tecido pulmonar fixadas usando formalina não tamponada a 10 por cento e incluídas em parafina com cera de abelha. Em conclusão, fatores inibitórios combinados interferiram na detecção de M. tuberculosis em material de arquivo. É importante controlar estes fatores inibitórios para poder implementar o diagnóstico molecular em laboratórios de patologia.

Perda de amostras em tissue microarray: comparação entre técnicas com uso de fita adesiva comercial, lâminas silanizadas pelo método tradicional ou por método modificado / Loss of tissue samples in the tissue microarray: comparison between techniques using commercial adhesive tape, silane-coated microslides by the conventional method and by a modified technique

Introdução/objetivo: O microarranjo tecidual, ou tissue microarray (TMA), permite avaliar múltiplas amostrasde tecido em um único bloco. Um dos problemas do TMA é o descolamento dos cortes teciduais, por isso, para reduzir essa perda, tem-se utilizado fita adesiva especial comercial. Não há relatos comparando o uso dessas fitas adesivas com a técnica de silanização modificada. O objetivo desse estudo foi comparar as perdas de cortes entre lâminas usando fitas adesivas comerciais, lâminas silanizadas por técnica convencional elâminas silanizadas por técnica modificada, com menor consumo de acetona. Material e método: O TMA foiconstruído com blocos de tecido hepático, em dispositivo de base fixa, colocando-se 32 cilindros de 2 mmde diâmetro em duplicata e espaçamento de 2,2 mm. Quinze secções de 4 μm foram colocadas em lâminas silanizadas a 4% por técnica convencional (grupo 1), 15 em lâminas silanizadas com técnica modificada (6%de silano e com uso mínimo de acetona) (grupo 2) e 15 em lâminas com fita adesiva comercial de acordo comas recomendações do fabricante (grupo 3). Todas as lâminas foram processadas por imuno-histoquímica para citoqueratina 18, com recuperação antigênica em tampão citrato pH 6, em microondas. As perdas de amostrasforam quantificadas e expressas como: perda total (≥ 80%), quase total (75% a 79%) ou parcial (50% a 74%).Resultados: A perda de tecidos foi semelhante nos três grupos: com silanização tradicional, modificada oufita adesiva comercial (4,9 vs. 3,1 vs. 8,1, respectivamente) (análise de variância [ANOVA], p = 0,3654). Umadas lâminas com a fita adesiva apresentou descolamento artefatual de todos os tecidos e outra de 20 tecidosem um dos lados. Nenhuma das lâminas silanizadas apresentou tal artefato. Conclusão: Lâminas silanizadas têm resultados satisfatórios, requerem menos treinamento técnico e reduzem os custos da utilização do TMA justificando seu uso em pesquisa...

Introduction/objective: The tissue microarray (TMA) technique allows the evaluation of multiple tissue samplesin a single block. One of the problems of TMA is the ungluing of tissue sections, thus commercial adhesive tape has been used to reduce this loss. There are no reports comparing the use of the commercial adhesive tape with the use of the modified silane-coated technique. The objective of this study was to compare section loss in slides using commercial adhesive tape, silane-coated microslides with the conventional technique or with the modified technique. Material and method: The TMA was constructed with hepatic tissue blocks embedded in paraffin, using a fixed base device, placing 32 cylinders of 2 mm in diameter in duplicate and 2.2 mm apart from each other. Fifteen 4-μm sections were placed on conventional silane-coated microslides at 4% (Group 1), 15 on silane-coated microslides with a modified technique (6% of silane and minimum use of acetone) (Group 2), and 15 on slides using commercial adhesive tape, according to the manufacturer's recommendations (Group 3). All microslides were processed by immunohistochemistry for cytokeratin 18, with antigen retrieval accomplished by incubation with citrate buffer pH 6.0 with microwave enhancement. Samples loss was quantified and expressed as: total (≥ 80%), almost complete (75% to 79%) or partial (50% to 74%). Results: The loss of sections was similar in all three groups (4.9 vs. 3.1 vs. 8.1, respectively) (analysis of variance [ANOVA], p = 0.3654). One slide usingcommercial adhesive tape showed artifactual ungluing of all sections and another one showed loss of 20 sampleson one side of the slide. None of the silane-coated microslides showed such artifact. Conclusions: Silane-coated microslides show adequate results, require less technical training and reduce the cost of TMA procedure, whatjustifies their use in research...

Anatomía microscópica de las glándulas salivales por medio de una técnica histológica convencional y no convencional / Microscopio anatomy of the salivary glands for mean of conventional and not-conventional histologic technique

Las técnicas de fijación y conservación permiten detener los procesos de desorganización de los tejidos y son necesarios para analizar la anatomía microscópica de ellos. El propósito de este estudio fue analizar las características histológicas de las glándulas parótida y submandibular obtenidas a partir de tres cadáveres humanos fijados y conservados mediante: a) solución conservadora en base a formaldehido (muestra I) y b) cámara de frío por 12 horas (muestra II), ambas muestras procesados para hematoxilina-eosina (H-E); c) plastinación con resina epóxica (muestra III) y procesado para H-E y con azul de metileno- eosina sin inclusión previa. Se analizaron las características de los adenómeros y sistema de conductos glandulares. Las mejores características se encontraron en la muestra II, con un buen nivel de detalle en el parénquima glandular, una mayor basofilia se presentó en la muestra I. La muestra III presentó un bajo nivel de detalle a la observación microscópica, los mejores resultados se obtuvieron utilizando azul de metileno. Las mayores dificultades en el procesamiento histológico de las piezas plastinadas se encontraron en el corte y en el tiempo necesario para la tinción. Los resultados sugieren que es posible obtener preparaciones histológicas a partir de necropsias en cadáveres fijados y conservados para la docencia e investigación anatómica.

The techniques of fixation and conservation allow to stop the processes of tissues disorganization and they are necessary to analyze the microscopic anatomy of them. The purpose of this study was to analyze the histologic characteristic of the parotid and submandibular glands obtained from three human cadavers fixed and conserved by means of: a) conservative solution based on formaldehyde (Sample I) and b) camera of cold for 12 hours (Sample II), both samples processed for hematoxilin-eosin (H-E); c) plastination with epoxic resin (Sample III) and processed for H-E and with methylene blue - eosin without previous inclusion. The characteristics of the adenomer and glandular ducts system were analyzed. The best characteristics were in the sample II, with a good detail level in the glandular parenchyma, a greater basophilia was presented in the sample I. The sample III it presented a low detail level to the microscopic observation, the best results were obtained using methylene blue. The biggest difficulties in the histologic process of the plastinated specimens were in the cut and time for tintion. The results suggest that it is possible to obtain histologic preparations from autopsies in fixed cadavers and conserved for teaching and anatomical investigation.

Procesamientos histológicos convencional y en microondas: comparación histológica, histoquímica e inmunohistoquímica / Conventional and microwave histological processing: a histological, histochemical and inmunohistochemical comparison

El horno microondas (HM) fue introducido en Anatomía Patológica en los años 80, siendo utilizado en varios procesos dentro del laboratorio. Se realizó la comparación de algunos parámetros histológicos, histoquímicos e inmunohistoquímicos en muestras de tejido normal, procesados paralelamente de manera convencional y en HM. Se seleccionaron muestras en duplicado de diversos tejidos; se procesaron convencionalmente (PC) en un procesador automático y en HM utilizando un protocolo estandarizado previamente. Se realizaron tinciones histológicas e histoquímicas (H-E, PAS y Azul Alcián) e inmunohistoquímicas (CD45, CD3, CD20 Citoqueratinas AE1/AE3, 5/18, 20, S-100, Receptores de Estrógeno y Progesterona). Las técnicas se escogieron según tipo de tejido. Se evaluó la calidad de los preparados (tinción nuclear, tinción citoplasmática y calidad general), la intensidad de las tinciones histológicas, histoquímicas e inmunohistoquímica de marcadores escogidos. El HM posibilitó la entrega de la lámina histológica en un tiempo mucho menor que el PC, disminuyendo el tiempo de procesamiento de 12 a 1 hora. Se observó una contracción mayor en las muestras procesadas en HM que en sus pares procesadas convencionalmente: tonsila palatina (54,8 por ciento v/s 46,6 por ciento), intestino grueso (33,3 por ciento v/s 22,2 por ciento), piel (24,1 por ciento v/s 13,7 por ciento) y mama (15,1 por ciento v/s 26,3 por ciento). La calidad del corte histológico fue mayor en las muestras PC, observando en los tejidos HM una tinción menos homogénea, con agrietamientos y desprendimientos más notorios, siendo igualmente apropiadas para el diagnóstico histológico. En cuanto a las tinciones histoquímicas, no se observaron cambios significativos. Se observó una mayor intensidad de tinción inmunohistoquímica de proteína S-100 y citoqueratina AE1/AE3 en piel. Se concluyó que el HM convencional puede ser utilizado en el procesamiento de biopsias sin inconvenientes para el diagnóstico...

The microwave oven (MO) was introduced in Pathology practice in the eighties and has been used in several processes within the laboratory. It was achieved a comparison between conventional and microwave processing. Histological, histochemical and inmunohistochemical parameters were observed. Samples of four kinds of tissues were taken in duplicate, and were processed on an automatic processor and into a microwave oven using a previous used protocol. H-E, PAS and Alcian Blue stains ware used in addition to inmunohistochemicals stains (CD45, CD3, CD20, Keratins AE1/AE3, 5/18 and 20, S-100 protein and estrogens and progesterone receptors). Techniques were chosen according to the tissue. The slides were evaluated for two blind professionals, whom considered: nuclear and cytoplasm stain, specificity and intensity of histochemical and inmunohistochemical stains. MO made possible a quick delivery of the slides, shorting the time from 12 to 1 hour. Shrinking was observed to be higher in MO process than conventional process: tonsils (54.8 percent v/s 46.6 percent), intestine (33.3 percent v/s 22.2 percent), skin (24.1 percent v/s 13.7 percent) y breast (15.1 percent v/s 26.3 percent). Qualification was found lightly higher on conventional processing. No changes were observed on histochemicals stains. Higher intensity of inmunostains was observed just in AE1/AE3 and S100 protein. It was concluded that MO can be used without inconveniences in the Pathology Laboratory, allowing a rapid diagnose of biopsy samples.