Comparative evaluation of the quality of Papanicolaou staining at different intervals of fixation times using 96% ethyl alcohol / Avaliação comparativa da qualidade da coloração de Papanicolaou em diferentes tempos de fixação em álcool etílico a 96%

ABSTRACT Introduction: Fixation of cytological smears consists of immediate immersion in appropriate fixative, in order to preserve cellular morphological characteristics, it is essential for the microscopic examination and diagnostic interpretation. Objective: To evaluate the influence of fixation times on the morphological and staining characteristics of samples fixed in ethanol and stained by the Papanicolaou method. Method: Experimental, quantitative and qualitative research was carried out on 99 samples of the jugal mucosa scrapings from 33 participants, fixed in 96% ethyl alcohol in three different times. Group A: 15 minutes; group B: 30 minutes; group C: seven days. The quality of staining was categorized in Optimal, Good, Regular and Poor, with subsequent recategorization at optimal and non-optimal. To verify the association among the groups and the categories, Fisher's exact test was performed, with significance level of 0.05. Results: From the 99 stained slides, 19 were discarded due to acellularity, remaining 80 slides. From these, 28 in group A, 26 in group B and 26 in group C were evaluated. In Group A, optimal quality was found in 60.7% (n = 17), good in 28.6% (n = 8), regular in 10.7% (n = 3) and poor in 0% (n = 0). In group B optimal was found in 61.5% (n = 16), good in 30.8% (n = 8), regular in 7.7% (n = 2) and poor in 0% (n = 0). In group C, optimal was found in 92.3% (n = 24), good in 7.7% (n = 2), regular in 0% (n = 0) and poor in 0% (n = 0). In the three groups, there was no representation of the Poor category. Conclusion: The results suggest that there is a significant difference in the staining quality (p-value = 0.01) according to the fixation time.

RESUMEN Introducción: La fijación de extensiones citológicas consiste en la inmersión inmediata en fijador adecuado para preservar la morfología celular, siendo esencial para el análisis microscópico y la interpretación diagnóstica. Objetivo: Evaluar la influencia de los tiempos de fijación en las características morfológicasy de tinción de muestras fijadas con metanoly tenidas con el método de Papanicolaou. Método: Se realizó una investigación experimental, cuantitativa y cualitativa de 99 muestras de raspado de la mucosa yugal de 33 participantes, fijadas con etanol al 96% en tres tiempos distintos. Grupo A: 15 minutos; grupo B: 30 minutos; grupo C: 7 días. La calidad de la tinción fue categorizada en óptima, buena, regular y mala, con posterior reclasificación en óptima y no óptima. Para determinar la asociación entre los grupos y las categorias, se realizó la prueba exacta de Fisher, con un nivel de significación del 0,05. Resultado: De las 99 muestras tenidas, 19 fueron desechadas por acelularidad, quedando 80 para ser analizadas. De estas muestras, 28 fueron evaluadas en el grupo A, 26 en el grupo B y 26 en el grupo C. En el grupo A, hemos encontrado calidad óptima - 60,7% (n =17); buena - 28,6% (n = 8); regular -10,7% (n = 3) y mala - 0% (n = 0). En el grupo B, óptima - 61,5% (n = 16); buena - 30,8% (n = 8); regular - 7,7% (n = 2); y mala - 0% (n = 0). En el grupo C, óptima - 92,3% (n = 24); buena - 7,7% (n = 2); regular - 0% (n = 0) y mala - 0% (n = 0). En los tres grupos no hubo representación en la categoria mala. Conclusión: Los resultados sugieren que hay diferencia significativa en la calidad de la tinción (p = 0,01) de acuerdo con el tiempo de fijación.

RESUMO Introdução: A fixação dos esfregaços citológicos consiste na imersão imediata em fixador adequado para preservar as características morfológicas celulares, sendo essencial para a análise microscópica e a interpretação diagnóstica. Objetivo: Avaliar a influência dos tempos de fixação nas características morfológicas e tintoriais de amostras fixadas em álcool etílico e coradas pelo método de Papanicolaou. Método: Realizou-se pesquisa experimental, quantitativa e qualitativa de 99 amostras de raspado da mucosa jugal de 33participantes, fixadas em álcool etílico 96% em três tempos diferentes. Grupo A: 15 minutos; grupo B: 30 minutos; grupo C: sete dias. A qualidade da coloração foi categorizada em ótima, boa, regular e ruim, com posterior recategorização em ótimo e não ótimo. Para verificar a associação entre os grupos e as categorias, realizou-se teste exato de Fisher, com nível de significância de 0,05. Resultado: Das 99 lâminas coradas, 19 foram desprezadas por acelularidade, restando 80 lâminas para serem analisadas. Destas, foram avaliadas 28 no grupo A, 26 no grupo B e 26 no grupo C. No grupo A, foi encontrada qualidade ótima - 60,7% (n = 17); boa - 28,6% (n = 8); regular - 10,7% (n = 3) e ruim - 0% (n = 0). No grupo B, ótima - 61,5% (n = 16); boa - 30,8% (n = 8); regular - 7,7% (n = 2); e ruim - 0% (n = 0). E no Grupo C, ótima - 92,3% (n = 24); boa - 7,7% (n = 2); regular - 0% (n = 0); e ruim - 0% (n = 0). Nos três grupos não houve representação na categoria ruim. Conclusão: Os resultados sugerem que há diferença significativa na qualidade da coloração (p = 0,01) de acordo com o tempo de fixação.

Histological Study- The Effects Of Various Fixatives On Kidney

Fixation is the critical step in processing tissues. Following tissue removal from the body, autolysis begins and proceeds more or less quickly depending on many factors, including the level of enzymes or microorganism present in the tissue. We studied effect of five different types of fixatives. An essential part of all histological and cytological techniques is preservation of cells and tissues as they naturally occur. The aim of the current study is to see the effect of following fixatives namely 10% formalin, Buffered 10% formalin, Bouin’s fluid, Zenker’s fluid, Carnoy’s fluid on liver tissues and to observe the optimum result in a particular fixative in H&E sections. There is no single fixative which can be considered as best fixative for all purposes. Best fixatives for architectural preservation are Carnoy’s fluid and Zenker’s fluid. Best fixative for study of nuclear details is Bouin’s fluid.

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.

Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues

Abstract Most Departments of Pathology around the world have a considerable archive of formalin-fixed paraffin-embedded (FFPE) tissue suitable for molecular assessment. This article points out the potential DNA damage that may occur if basic steps are not followed during processing and storage of these samples. Furthermore, it hopes to establish parameters to optimize quality and quantity of DNA extracted from FFPE tissues.

Resumo A maioria dos Departamentos de Patologia em todo o mundo têm um considerável acervo de tecidos embebidos em parafina e fixados em formalina, que são passíveis para análises moleculares. Este artigo apresenta os danos ao DNA que podem ocorrer se passos básicos não forem seguidos durante o processamento e armazenamento destas amostras. Além disso, procura estabelecer parâmetros para otimizar a qualidade e quantidade do DNA extraído de tecidos FFPE.

Incapability of Formalin Based Fixative to Kill Maggot in Mutilated Human Carrion.

Background: Embalming is a process used to temporarily preserve a human cadaver to forestall decomposition and make it suitable for display at funerals; thus, are agents that prevent autolysis and putrefaction. The outbreak of maggots from a heap of inadequately embalmed bodies due to deep cuts and bodies involved in inferno, necessitated the need to re-investigate the efficacy of formalin based embalming fluid and its inability to kill maggots. Methodology: Various strength of Formaldehyde, Xylene, Kerosene, and, Lime fluid, Isopropanol, Gamalin 20, Potassium ferrocyanide, and Physiological saline as control were used in the investigation. In the present investigation, Two maggots under the same atmospheric condition were put in each of the ten selected chemical reagents/solutions, including Lime, Kerosene, and the Gamalin 20 that are naturally available were initially dispensed into ten glass universal containers. Maggot movements in each reagent solution were critically observed. Result: Maggots death occurred within the first ten minutes in test number three groups III that contains Concentrated Formalin and Xylene andMaggots died after fifteenminute of the experiment, butmaggots did not died until about eight hours after the test in two of the experiment. Discussion: Results of this investigation showed clearly thatMaggots were not killed as soon as expected by the embalmerwhen ordinary ten percent alcoholic formalin embalming fluid is used. Equal volume of concentrated formalin plus Xylene was found out to be effective at killing maggot instantly. Conclusion: It is therefore advisable to use Xylene plus Conc. Formalin when preserving cadaver infested with Maggot and this could at the same time prevent the occurrence ofMaggots Infestation and better preservation of mass of burnt mutilated corpses in our Mortuary.

Lesión severa de mano por proyectil de arma de fuego de alta energía con artrodesis del carpo e injerto sintético de hueso más fijadores externos: Reporte de un caso / Severe hand injury due to a high-energy gunshot projectile treated with arthrodesis of the carpus, synthetic bone graft and external fixators.: Case report

Antecedentes: La incidencia de heridas por proyectil de arma de fuego en mano de pacientes civiles se ha incrementado en los últimos años, lo cual es causa de incapacidades prolongadas o definitivas con un impacto personal, social y económico. Las lesiones severas de mano incluyen: afección del arco palmar mayor, fractura de dos o más huesos del carpo, luxaciones palmares irreductibles, fracturas articulares de radio distal y lesiones neurovasculares, los cuales requieren de múltiples procedimientos quirúrgicos para el manejo de tejidos blandos y musculoesqueléticos. Métodos: Se describe el reporte de un paciente con fractura de los huesos del carpo derecho, expuestos grados IIIB de Gustillo y Anderson por proyectil de arma de fuego, el cual fue tratado con artrodesis del carpo mediante injerto sintético, fijación externa y rotación de colgajo fasciocutáneo. Resultados: Paciente que fue sometido a tratamiento quirúrgico de la lesión del carpo consistente en fractura de todos los huesos del carpo, expuestos grados IIIB de Gustillo y Anderson por proyectil de arma de fuego quien fue sometido a tratamiento quirúrgico mediante artrodesis del carpo con injerto sintético, estabilización con fijador externo y cobertura con colgajo fasciocutáneo, obteniendo una evolución clínica mediante la escala de DASH con un score de 14.2 y Michigan Hand score de 70 puntos con un seguimiento de 12 meses. Conclusiones: El tratamiento con fijación externa más artrodesis del carpo con injerto sintético y cobertura cutánea con colgajo proporcionó un resultado funcional bueno para un paciente con lesión por proyectil de arma de fuego.

Background: The incidence of hand gunshot wounds in civil patients has increased in recent years; it causes long-term or permanent disability and has a personal, social and economic impact. Severe hand injuries include: involvement of the greater palmar arch, fracture of two or more carpal bones, irreducible palmar dislocations, articular fractures of the distal radius, and neurovascular injuries. All of these injuries require multiple surgical procedures for the management of soft and musculoskeletal tissues. Methods: We report the case of a patient with a Gustilo and Anderson grade IIIB open fracture of the right carpal bones caused by a gunshot projectile. He was treated with arthrodesis of the carpus using a synthetic graft, external fixation and rotation of a fasciocutaneous flap. Results: The patient underwent surgical treatment of the carpal injury that consisted of Gustilo and Anderson grade IIIB open fracture of all carpal bones caused by a gunshot projectile. Surgery included arthrodesis of the carpus with a synthetic graft, stabilization with an external fixator, and a fasciocutaneous graft as skin cover. The clinical course according to the DASH scale had a score of 14.2 and the Michigan Hand score was 70, with a 12-month follow-up. Conclusions: Treatment with external fixation plus arthrodesis of the carpus with a synthetic graft and skin cover using a flap provided a good functional result in a patient who sustained a gunshot wound.

A Comparative Study to Determine the Most Suitable Fixative for Immunocytochemical Staining of Cytospin Slides / 임상검사와정도관리

BACKGROUND: Fixation of cells is a critical procedure that can determine the success of immunocytochemical staining (ICC) of cytospin slides. In this study, we evaluated the efficacy of a number of fixatives to determine the ideal fixative for ICC of cytospin slides. METHODS: Sixteen patients with metastatic neoplasm in the body cavity were enrolled. Cytospin slides were prepared from each patient using 5 different fixatives (cold acetone, 95% ethanol, 1:1 methanol:ethanol, 3.7% formalin, and 3:1 methanol:acetone), and the suitability of each for use with Wright's stain was compared. For 4 of the samples, appropriate ICCs were performed using all 5 fixatives and the results were compared, while for 11 samples, only the first 3 fixatives were used for ICC. RESULTS: Using Wright stain, cold acetone, 95% ethanol, and 1:1 methanol:ethanol fixatives all showed similar efficacy when compared to the conventional methanol fixation method. However, the stain quality using 3.7% formalin or 3:1 methanol:acetone fixatives was poor due to deterioration of cell adhesion and distortion of cell morphology. Using ICC, cold acetone fixative showed stronger and more tumor-specific stainability than the 95% ethanol (decreased stainability in 6 stained slides, false positive staining of histiocytes/neutrophils in 4 stained slides, no staining of CD3 and terminal deoxynucleotidyl transferase [TdT]) and 1:1 methanol:ethanol fixatives (decreased stainability in 3 stained slides, false positive staining of histiocytes/neutrophils in 2 stained slides, no staining of CD3 and TdT). CONCLUSIONS: Cold acetone fixative was the most efficacious among the 5 fixatives tested in this study; therefore, it is the most appropriate fixative in the preparation of cytospin slides for ICC.

Choice of Internal Fixatives for the Intertrochanteric Fractures of the Femur in the Elderly

PURPOSE: To prepare the appropriate guideline in choosing the internal fixatives for the intertrochanteric fractures of the femur in the elderly. MATERIALS AND METHODS: We reviewed 95 cases of intertrochanteric fractures of the femur from January, 1999 to December, 2003. We fixed the fracture with Proximal Femoral Nail in 37 cases (PFN group), Dynamic Hip Screw in 56 (DHS group), Dynamic Condylar Screw in 2 cases (DHS group). We reviewed operation time, blood loss during operation, changes in neck-shaft angle and sliding of lag screw. RESULTS: There were no significant differences in the parameters between the 2 groups in stable fracture. In unstable fractures, operation time in PFN group and DHS group revealed 103.9 and 128.2 minutes respectively; mean amount of blood loss during operation revealed 523.2 and 573.1 ml respectively. Mean changes in the neck-shaft angle at final follow-up in PFN group and DHS group revealed 4.6 degrees and 4.1 degrees; sliding of lag screw averaged 3.4 and 6.5 mm respectively. Among the DHS group, cases of additional fixation with trochanteric supporting plate revealed 3.1 degrees of changes in neck-shaft angle and 4.2 mm of lag screw sliding. CONCLUSION: In cases of stable fractures, any fixative might suffice. In cases of unstable fractures, there were no significant differences in results of treatment between these two groups, however, PFN group revealed shoter operation time and less blood loss during operation. It seemed to be necessary to apply additional fixation with trochanteric supporting plate when using DHS in unstable cases.

Comparison of various procedures in the processing of binucleated-lymphocytes on to glass slide to detect chromosomal breakage.

Binucleated lymphocytes can be screened for micronuclei to assess chromosomal damage. There are various procedures to get slides containing binucleated lymphocytes, that are different in harvesting, fixation, and slide preparation methods. Screening binucleated lymphocytes to find a micronucleus needs at least 800 cells with intact cytoplasm. This study aimed to analyze the various procedures and simplified procedures to know which procedure gave the most abundant binucleated lymphocytes with intact cytoplasm and best staining properties for the purpose of micronucleus scoring. Seven heparinized blood samples were obtained from the Dept. of Obstetrics and gynecology, Faculty of medicine, University of Indonesia, Jakarta. The 7 blood samples were subjected to 17 procedures different in harvesting (with or without washing), slide preparation (smear and spot method, and using a cytocentrifuge), and fixation methods (methanol for 1 minute, methanol brief, methanol/glacial acetic acid 3:1 or 9:1). Our results showed that fixatives containing glacial acetic acid are not suitable for micronucleus test. To generate binucleated lymphocytes with intact cytoplasm as much as possible, the procedure should be conducted without washing steps. Methanol fixation either briefly or 1 minute is preferable, and for the ease of screening cytocentrifuge preparation, followed by spot method is preferable.

Detection of Helicobacter pylori in the Gastric Mucous Layer in Pediatric Patients

BACKGROUND: Helicobacter pylori is present mainly in the gastric mucous layer. However, the mucous layer, along with the bacteria, is lost during conventional tissue processing in which formalin is used for fixation. The purpose of this study is to ascertain - if the mucous layer is preserved by using Carnoy solution as a fixative - whether the detection rate of H. pylori is increased in pediatric patients. METHODS: Five pieces of gastric mucosal tissue were obtained from the gastric antrum and the body of one hundred pediatric patients. One of the specimens was fixed with formalin. Another specimen was fixed with Carnoy solution. The tissue sections were stained with hematoxylin-eosin and immunohistochemically stained for H. pylori. For reference, a rapid urease test was performed on the remaining three specimens. RESULTS: In the formalin-fixed tissue, the detection rate of H. pylori was 13% in the gastric antrum and 12% in the body (overall 16%). In the Carnoy solution-fixed tissue, the mucous layer was preserved and the detection rate of H. pylori was 23% in the antrum and 27% in the body (overall 28%). The positive rate of the rapid urease test was 26% in the antrum and 28% in the body (overall 29%). CONCLUSIONS: When the number of H. pylori is small in the gastric mucosa, the bacteria may not be detected by conventional histologic methods. In that case, the detection rate of H. pylori may be increased by using Carnoy solution, rather than formalin, as a tissue fixative.