Preservation of histology by phenol-based fixative: mini review of recent findings / Preservación de la histología mediante un fijador basado en fenol: una mini revisión de los hallazgos recientes

SUMMARY: In surgical and anatomical training, use of cadaver remains the most ideal technique. Standard formaldehyde solution preserves cadaveric tissues for an extended period comparing to the unfixed tissues. However, it fails to retain the natural texture, color, and biomechanical features. Phenol based soft embalming methods were developed to maintain these properties, while simultaneously decreasing the biohazard risk. Soft embalming techniques have made the bodies more 'lifelike' and wellfitted for training. Though phenol fixation displays rewarding morphological maintenance, we have scanty evidences on the histological preservation. This mini review primarily discussed the latest reports regarding the effect of phenol-based fixation on the tissue histology. Published literatures revealed phenol-based fixation displayed comparable histological preservation to that ofgold standard paraformaldehyde-based solution. It was concluded that phenol-based solution is an excellent fixative used to preserve tissues for microscopic analysis.

RESUMEN: En el entrenamiento quirúrgico y anatómico, el uso de cadáveres sigue siendo la técnica más ideal. La solución estándar de formaldehído conserva los tejidos cadavéricos durante un período prolongado en comparación con los tejidos no fijados. Sin embargo, no conserva la textura, el color y las características biomecánicas naturales. Se desarrollaron métodos de embalsamamiento blando a base de fenol para mantener estas propiedades y, al mismo tiempo, disminuir el riesgo biológico. Las técnicas de embalsamamiento suaves han hecho que los cuerpos sean más "realistas" y estén mejor preparados para la enseñanza. A pesar que la fijación de fenol muestra un buen mantenimiento morfológico, existe evidencia escasa sobre la preservación histológica. Esta mini revisión se refirió principalmente a los últimos informes sobre el efecto de la fijación en base de fenol en la histología del tejido. La literatura publicada reveló que la fijación a base de fenol mostró una preservación histológica comparable a la de la solución a base de paraformaldehído. Se concluyó que la solución a base de fenol es un excelente fijador utilizado para preservar tejidos para análisis microscópico.

Cytomorphologic Analysis of Wet and Spray Fixation Methods in Oral Exfoliative Cytology

@#For a long time, oral exfoliative cytology (OEC) has been implemented as an effective preliminary diagnostic tool for pathological lesions and various methods for fixation of the cytology specimens have been studied. The present study was undertaken to compare the efficacy between the wet and spray type of fixation methods for Papanicolaou (PAP) stained oral cytosmears. The study comprised of 45 healthy subjects in the age group of 20-25 yrs. For each subject, two smears were collected from the buccal mucosa and subjected to wet and spray fixation methods respectively. Both the smears were stained using a commercial Rapid Pap Kit. Smears were observed microscopically and evaluated for cytomorphological features involving uniformity of staining, cellular morphology, nuclear morphology, cellular staining, nuclear staining and presence of impurities. Comparisons were made between the two methods of fixation and statistically analysed using McNemar non-parametric test. Cells were evenly distributed in wet-fixed smears (n=38, 95%) compared to spray fixed smears (n=19, 47.5%). Wet-fixed smears showed lesser impurities (n=13, 32.5%) than spray fixed smears (n=27, 67.5%). However, other parameters such as cytological and nuclear morphology, staining of cytoplasm and nucleus were found to be not significant when compared between the two methods of fixation (p<0.05). The study shows that wet-fixed smears have better cellular distribution and relatively fewer impurities when compared to the spray fixed smears. The method of wet-fixed smears may be used as an alternative to spray fixed smears. A larger sample size may be required for further validation.

A comparative study on the effect of fixation for guinea pigs eyeballs among three different fixation solution / 国际眼科杂志(Guoji Yanke Zazhi)

· AIM:To compare the effect of fixation and select the optimal fixation solution and condition for PAS staining of guinea pig eyes among three different fixation solution:4％ paraformaldehyde solution,4％ glutaraldehyde solution and Davidson solution.· METHODS:Totally 30 healthy guinea pigs were divided into 6 groups:Group Ⅰ-Ⅱ were fixed in 4％ paraformaldehyde solution and 4％ glutaraldehyde solution for 24h,respectively;Group Ⅲ-Ⅴ were fixed in Davidson solution for 3,6 and 24h,respectively;and Group Ⅵ were fixed in Davidson solution for 3h and then transferred into 10％ neutral formaldehyde solution for 48h.All groups were sectioned by routine section method and undergone PAS staining,and then observed by light microscope.· RESULTS:It was found that the group which was fixed by Davidson solution for 3h,remained the most complete structure for PAS staining (Group Ⅲ).While the effect of fixation for the group which was transferred into 10％ neutral formaldehyde solution for preserving for 48h after fixing in Davidson solution for 3h was also acceptable for PAS staining (Group Ⅵ).The retinal cells remained clear and in order for both groups which was mentioned above.· CONCLUSION:The best fixation condition for PAS staining for eyes of guinea pigs is fixation in Davidson solution for 3h among these fixation conditions,while it is also suitable to transfer the eyes into neutral formaldehyde solution after fixing in Davidson solution for 3h for preserving for long periods,which is not severely reduced the effect of fixation for PAS staining.

Comparison of Fixation Effects of Three Different Complex Fixatives on Mouse Lens Tissue / 中国医科大学学报

Objective To compare the effects of three kinds of fixative solutions on paraffin section of mouse lens tissue and optimize the fixing?method of paraffin section in mouse lens tissue. Methods Three kinds of conventional fixatives were selected for the test ,including the conven?tional Davison’s solution,modified Davison’s solution and 10%neutral buffered formalin. Mice eyeball tissues were fixed with three different fixa?tives,embedded,sliced and then stained with HE method. The paraffin slices were observed under the light microscope. Results The structures of lens and retina fixed in conventional Davison ’s fixative solutions were clear and intact ,and the cells were arranged regularly and compactly. There was no eyeball distortion,contraction and retinal detachment in the eyeballs fixed in modified and conventional Davison’s fixative solution. However,the ones fixed in 10%neutral buffered formalin showed eyeball distortion and contraction,space and spherules. Conclusion The mice lens slides made from tissues fixed by conventional Davison ’s fixative solution are better than fixed by modified Davison ’s fixative solution and the 10%neutral buffered formalin fixed ones.

Sediment cytology in diagnostic evaluation of oral neoplasms.

Aim: To evaluate the role of sediment cytology of biopsy specimen fixatives in early diagnosis of oral neoplasms. Materials and Methods: Cytological smears were prepared by centrifuging the fixatives in which the biopsy specimens were received. The smears were analyzed and the cytological results were compared with histological diagnosis. Results: Of 20 lesions studied by sediment cytology, 8 were labeled as benign, 9 as malignant and 3 cases as inconclusive. Final histopathological diagnosis labeled 12 lesions as malignant and 8 lesions as benign. Comparing the cytological diagnosis with histological sections, 17 out of 20 cases were concordant. The overall diagnostic accuracy of 85% was achieved. Conclusion: Biopsy sediment cytology is a good complimentary method to histopathology in the study of oral biopsy material.

Comparison on rat and mice intestine slides’ quality fixed with three different fixatives and embeded by paraffin / 中国比较医学杂志

Objective To compare the rat and mice intestine slides quality fixed with three different fixatives and embeded by paraffin.Methods Three kinds of conventional fixatives were selected for the test, they are 10% neutral buffered formalin,Bouin’s solution and modified Davidson’ s solution.SD rat’ s and KM mice’ s intestinal tissues were fixed with the three different fixatives, then embeded, sliced and stained with HE as routine rut.To observe the slides quality under microscope.Results Sheding and necrosis of intestine villus epithelia were observed in slides made from formalin fixed intestines, which is speculated due to the fixations.The phenomenon was not observed in Bouin's solution and modified Davidson’ s solution fixed tissues.And the tissue structure were more fine and clear in them.Conclusions The rat and mice intestine slides made from tissues fixed by Bouin's solution and modified Davidson's solution are better than from the 10%neutral buffered formalin fixed ones.

Quick Golgi method: Modified for high clarity and better neuronal anatomy.

The Golgi methods have long been used to study the neuronal soma, axons, dendritic arborization and spines. The major concerns of the Golgi method have been its unpredictable nature (inconsistency of impregnation of the stain), time consumed, tissue hardening and clear background, resulting in several modifications to improve the cellular visualization. In the present work we describe a modification of the rapid-Golgi method that takes the benefit of perfusion fixation (with rapid-Golgi solution) then post-fixation in the same fixative for 36 h followed by 36 h impregnation in aqueous AgNO3 followed by vibratomy. This modification is simpler, faster and inexpensive, provides a consistent staining of neurons with good resolution of neuronal soma, dendritic arborization as well as spines with much reduced formation of silver chromate crystals and background in just 3 days.

Estudio comparativo entre soluciones conservadoras con y sin formol en placenta humana / Comparative study between conservative solutions with and without formaldehyde in human placenta

Las técnicas de fijación y conservación anatómica son realizadas habitualmente con soluciones que contienen formol, dado su bajo costo. Estas tienen varias desventajas como el olor irritante, rigidez, cambios de coloración de las estructuras, así como toxicidad con potencial cancerígeno, teratogénico y mutagénico para quien lo manipula. Por esto, es deseable utilizar soluciones sin formol. El objetivo de este trabajo fue comparar 2 métodos de conservación cadavérica, uno con formol (solución de Montevideo) y otro sin formol (método de Prives) utilizando la placenta humana como órgano experimental, evaluando sus parámetros macroscópicos. Se utilizaron 46 placentas humanas de partos normales y gestación a término. Las placentas fueron separadas en dos grupos (n=22 y n=24 respectivamente). El primer grupo de placentas fue perfundido con una solución con formol y el segundo grupo en una solución sin formol. Luego ambos grupos fueron sumergidos y mantenidos en sus soluciones respectivas por dos semanas y posteriormente retiradas dejándolas al aire a temperatura ambiente por 2 semanas más. Se analizaron las variables cuantitativas de peso y diámetro en cada una de las piezas, así como las variables cualitativas de consistencia, color, olor y crecimiento de micro/macro organismos. La recopilación de datos fue realizada previo al lavado, a los 14, 21 y 28 días. Los resultados mostraron que las placentas conservadas con el método de Prives presentaron mejor conservación en relación a su diámetro, consistencia, color y menor olor irritante en relación a las placentas tratadas con solución con formol. En ningún caso hubo crecimiento de micro o macroorganismos. En conclusión, emplear soluciones alternativas que sustituyan ventajosamente al formol como la fórmula de Prives conservan mejor las características macroscópicas de las placentas sin generar un olor irritante, deteniendo el proceso de descomposición.

The fixation and conservation techniques of anatomic material are commonly performed with solutions containing formaldehyde, given its low cost. These have several disadvantages such as the irritating odor, stiffness, discoloration of the structures, toxicity, carcinogenic, teratogenic and mutagenic risk for those who are exposed. Therefore it is desirable to use solutions without formaldehyde. The aim of this study was to compare 2 methods of anatomical conservation, one with formalin (Montevideo's solution) and one without formalin (Prives method) using the human placenta as an experimental organ model evaluating its macroscopic parameters. We used 46 human placentas from normal deliveries and term pregnancy. The placentas were separated into two groups (n=22 and n=24 respectively). The first group of placentas was perfused with formaldehyde solution and the second group in a solution without formaldehyde. Then both groups were immersed and maintained in their respective solutions for two weeks and then withdrawn leaving the air at room temperature for 2 weeks. Quantitative variables were analyzed for weight and diameter of each piece, and qualitative variables as consistency, color, odor and growth of micro/macro organisms were evaluated. Data collection was performed prior to washing at 14, 21 and 28 days. The results showed that conserved placentas with Prives method showed better conservation in relation to its diameter, consistency, color and less irritating odor in relation to placentas treated with formalin solution. In no case was growth of micro or macro organism. In conclusion, using advantageously at alternative solutions to formaldehyde, as the formula of Prives method, better preserved macroscopic characteristics of placentas without generating an irritating smell, stopping the decomposition process.

Research of the best way to the double-labeling immunofluorescence staining / 中国医师杂志

Objective Detailed descriptions of the double-labeling immunofluorescence staining for fluorescence microscopy provides an ideal sample.Methods Rat liver frozen sections were used fixative were 95％ alcohol,95％ formaldehyde and acetone,frozen sections,with anti-CSE,Ki-67 polyclonal antibody and incubated with FITC,Cy3 fluorescence-labeled secondary fluorescently labeled secondary antibody staining,observed under a fluorescence microscope.Results Acetone fixed group visible in the proliferative phase (S phase) cells showed a red fluorescent nucleus,cytoplasmic green fluorescence.Conclusion The impact of double labeling immunofluorescence the effect of sample links and many factors,including the two most important factors are 2 and coordination with the primary antibody and select the appropriate fixative.

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.

Morphology of Rat Testis Preserved in Three Different Fixatives / 华中科技大学学报（医学）（英德文版）

Histopathological examination of testes is important in assessing spermatogenesis and testicular function.Modified Davidson's fluid (mDF) has been proposed as a superior substitute for Bouin's fluid (BF) for fixation of adult animal testes.Besides,4％ paraformaldehyde (PFA) has been commonly used to fix testes with convenience.We compared the morphology of the rat testis fixed in 4％ PFA,mDF,or BF using hematoxylin and eosin (HE)-stained sections.Fixation in 4％ PFA resulted in obvious tissue shrinkage artifacts,especially between seminiferous epithelium cells.Shrinkage artifacts were also observed in the central area of the testes fixed in BF.Use of mDF did not cause shrinkage artifacts between seminiferous tubules,though a small amount can be observed in seminiferous tubules between germ cells.Clarity of nuclear detail in testes fixed in mDF and BF is better compared to 4％ PFA.Our study demonstrated that fixation in mDF provided better morphologic details in the rat testis as compared with 4％ PFA and BF.

The detectable time limit of intact STR profile for human tissues in unbuffered formalin / 中国法医学杂志

Objective To evaluate the STR profiling availability of formalin-fixed tissues and the detectable time limit of intact STR profile.Methods The different human tissues were fixed with 10-fold diluted commercial 40%formalin fixative for different duration under 15～20℃,and then DNA was extracted using the method of QIAamp~(R)DNA and IQ~(TM) DNA System.The extracted DNA was quantified with QuantifilerTM kit and amplified by both AmpFSTR identifiler kit and AmpFSTR MiniFiler kit.The STR profile was analyzed by GeneMapper ID v3.2 on 3100-Avant.Resuls The STR profiling availability of formalin-fixed tissues was relevant to the formalin fixing duration mainly.as well as the type of tissues and the template concentration and protocol of DNA extracting.The optimal ranges of template concentration is 1～3ng/μL and the QIAamp extracting method was preferable.There are differences in the degradation rate between various types of tissues in the unbuffered formalin fixative,and the lung tissue showed the slowest rate and liver and intestine tissues the fastest.Intact miniSTR profile of all the tissues detected could be obtained within 15 days duration of formalin fixing while intact STR profile could be obtained within 4 days.Conclusion The major factor that impact the availability of STR profiling of formalin-fixed tissues is the fixing duration in unbuffered formalin,as well as the type of tissues,method of extraction,concentration of PCR template and the kinds of STR loci.

Optimization of Wet Fixation Methods for AFM Imaging of Human Fibroblast Cells

We investigated the effect by the chemical fixative on human fibroblast cells (HFCs) in order to make nano-scale images using by the atomic force microscopy (AFM). The cell fixation needed to be optimized as prerequisite step for the preparation before analysis. AFM imaging after optimal wet fixation can provide practical, simple and fast technique for scanning living cells. In this study, AFM images - topography and amplitude - and the optic images of HFCs which were fixed with phosphate buffered saline (PBS), 2:1 ethanol:acetic acid, 4% glutaraldehyde and 37% formaldehyde were compared respectively. The final effect by washing with PBS or distilled water (D.W.) was examined after 4% glutaraldehyde fixation. To determine the optimal fixation method for HFCs, we performed quantitative and qualitative analysis by the height profile, the presence of artifacts and the morphology of well-conserved fibroblastic topography image by AFM. From AFM image which showed fibroblastic cellular morphology and differential height value of cytoplasm (670+/-47 nm, n=10) and nucleus (847+/-32 nm, n=10) in HFCs, we proposed that wet fixation by 4% glutaraldehyde, followed by final washing with PBS, could be the most suitable preparation for AFM imaging of HFCs, which enable us to approach easily on living cells with the least shrinkage.

Usefulness of Cytologic Study of Intraoperative Suction Fluid in Brain Tumors / 대한세포병리학회지

In diagnosing a brain tumor, it is essential to obtain samples from many areas of the tumor. Although there are reports about the suitability of material obtained by cavitron ultrasonic surgical aspirator(CUSA), there is a paucity of reports regarding conventional intraoperative suction. This study was performed to evaluate the usefulness of the suction fluid and the effect of different hemolytic fixatives. Intraoperative suction fluid was obtained from 2 pituitary adenomas and 2 choroid plexus carcinomas. In two cases of mixed astro-oligodendroglioma, one of glioblastoma multiforme and 3 of meningioma, the fluid was collected by CUSA. Each sample was divided into four bottles for the different fixatives such as 0.1N HCl, 10% acetic acid, 95% alcohol, and no additive. All cases were evaluated by the both cytologic smear and cell block preparations, and were reviewed with concomitant histologic diagnosis. The result showed a good correlation between the cytologic study and the histologic diagnosis and 95% alcohol was found to be superior to other fixatives in cell preservation.