Comparison of formalin, buffered formalin, and Bouin's fixation on the detection of human papillomavirus deoxyribonucleic acid from genital lesions

Detection of nucleic acid sequences homologous to human papillomavirus (HPV) relies primarily on their extraction from unfixed tissue. We detected HPV sequences in DNA extracted from paraffin-embedded tissue fixed in formalin (buffered and unbuffered) and Bouin's solution by dot blot hybridization. A detectable hybridization signal was noted in 32% of these fixed tissues which were chosen from cases where HPV DNA was detected in the unfixed tissue. When using a homologous 32P-labeled probe and a high stringency wash, the hybridization signal was lost if DNA was extracted after Bouin's fixation and diminished after formalin fixation, more so with unbuffered formalin. Similar differences in the hybridization signals among the different fixatives after high stringency wash were noted with in situ hybridization. Southern blot analysis showed that DNA extracted from tissues fixed in Bouin's was degraded and ranged in size from 100 to 500 base pairs as compared with 100 to 900 base pairs for DNA extracted from tissue fixed with unbuffered formalin. In contrast, no degradation was noted after fixation with buffered formalin. These results demonstrate that HPV sequences can be identified in DNA extracted from paraffin-embedded, fixed tissue. However, use of some fixatives may preclude identification of HPV type, by either dot blot or in situ hybridization.     

Effect of buffered formalin on amplification of DNA from paraffin wax embedded small biopsies using real-time PCR

BACKGROUND: The isolation of good quality DNA from routinely fixed and processed biopsy samples is crucial for the success of subsequent molecular analysis. AIMS: To compare the amount of beta actin DNA extracted from upper gastrointestinal tract biopsies fixed in buffered and unbuffered formalin. METHODS: Amounts of beta actin DNA extracted from forceps biopsies of the upper gastrointestinal tract fixed in unbuffered (n = 22) and buffered formalin (n = 16) were estimated by quantitative real-time polymerase chain reaction. RESULTS: The yield of beta actin DNA was significantly higher in biopsies fixed in buffered formalin than in those fixed in unbuffered formalin (median 2.8 x 10(4) and 5.3 x 10(2) DNA molecules, respectively; p < 0.005). Furthermore, fixation in buffered formalin led to a more reproducible DNA extraction, as indicated by the coefficient of variation (1.0 and 2.2, respectively). CONCLUSIONS: This study indicates that tissue samples should be fixed in buffered formalin to facilitate the use of molecular pathology analysis in routine biopsy material.     

Fixation temperature affects DNA integrity in the testis as measured by the TUNEL assay

Mature rat testes and liver were fixed with Bouin's fluid (BF) or modified Davidson's fixative (mDF) at room temperature (23 °C) or 4 °C, and DNA integrity was examined by the TUNEL assay. When testes were fixed in BF, TUNEL-stained cells were more prevalent than when fixation occurred in mDF. Independent of fixative, TUNEL-staining was higher when testes were fixed at room temperature relative to 4 °C. Significant effects were present for fixative and temperature of fixation, but not their interaction. Relative to BF, mDF also provided for lower TUNEL-staining in liver, but staining was not affected by fixation temperature. Since the TUNEL assay depends on the detection of fragmented DNA strands, harsh fixatives that induce breaks in the DNA can introduce substantial artifacts. Such potential artifacts are especially prevalent in a tissue such as testes with its ongoing division and differentiation activities. Therefore, the current findings lead the authors to conclude that fixation of mature testes in mDF at 4 °C minimizes generation of false TUNEL-positive cells.     

Fixation conditions for DNA and RNA in situ hybridization: a reassessment of molecular morphology dogma.

Neutral buffered formalin (NBF) (4% neutral buffered formaldehyde) has been advocated by most investigators as the primary fixative of choice for in situ hybridization (ISH), and specific anecdotal cautions interdicting the use of precipitating fixatives, which otherwise may offer certain advantages such as superior nuclear detail, are common. Few systematic studies addressing ISH fixation conditions have been published. We reasoned that heavy metals present in some precipitating fixatives may compromise duplex formation during ISH. Cell lines containing known viral gene content (CaSki, 200 to 600 human papilloma virus 16 copies/cell, and SiHa, 1 to 2 human papilloma virus 16 copies/cell) and two negative cell lines (K562 and MOLT 4) were expanded to >10(10) and pellets fixed in NBF, zinc formalin, B5, and Bouin's and Hollande's solutions, and subjected to DNA ISH using biotinylated genomic probes. Ten tissue biopsies fixed in both Hollande's and NBF solutions were also evaluated for human papilloma virus content using DNA ISH. Additionally, 17 cases of Hodgkin's disease fixed in B5 and formalin were compared for Epstein-Barr encoded RNA detection using RNA ISH with fluorescein isothiocyanate-labeled oligonucleotides. Catalyzed reporter deposition combined with Streptavidin-Nanogold staining and silver acetate autometallography (Catalyzed reporter deposition-Ng-autometallography ISH) and a conventional indirect alkaline phosphatase method were used for detection for both DNA and RNA. Contaminating heavy metals entrapped in fixed tissues were removed by two exposures to Lugol's iodine. Results for both DNA and RNA ISH comparing B5 and NBF fixatives were virtually identical. Hollande's, Bouin's, B5, and zinc formalin fixed tissue showed results indistinguishable from NBF fixed tissue in DNA ISH. Precipitating fixatives such as B5 and Hollande's solution may be used for DNA and RNA ISH under appropriate conditions.     

Buffered formalin is the superior fixative for the detection of HPV DNA by in situ hybridization analysis.

In situ hybridization is used commonly for detection of human papillomavirus (HPV) DNA. There is little information, however, on whether the detection of HPV DNA by in situ hybridization can be affected by the way in which the tissue is fixed. To address this question, the authors compared the hybridization signal using this technique under low stringency conditions for several genital condylomata containing HPV 6 or 11 that were randomly subdivided and fixed in various fixatives for 16 hours. In all cases, the largest proportion of cells with koilocytotic atypia that had detectable HPV DNA was in buffered formalin-fixed tissue (80%), followed by tissue fixed in unbuffered formalin (70%), Hartman's solution (40%), and Bouin's solution (10%). After a high stringency wash, the greatest decrease in the overall hybridization signal was with tissue fixed in Bouin's solution; a minimal decrease was noted with tissue fixed in buffered formalin. Fixation in Bouin's solution for 2 hours gave in situ hybridization results comparable with buffered formalin fixation but with poorer cytologic detail. It is concluded that, of the fixatives studied, buffered formalin is superior for the detection of HPV DNA by in situ hybridization analysis.     

Evaluation of RNA and DNA extraction from liquid‐based cytology specimens

Molecular diagnosis using DNA and RNA derived from malignant tumors and molecular biological tools such as the quantitative polymerase‐chain‐reaction (qPCR) is a commonly used technique in clinical pathology. In this report, we compared the qualitative extraction of RNA and DNA from cancer cells fixed using several liquid‐based cytology (LBC) kits. Ten to 1,000 cells from the T24 urinary bladder cancer cell line and SKG‐II cervical cancer cell line were fixed with 55% methanol and three different methanol‐based LBC solutions. The mRNA levels of CD44 in T24 cells and E7 in SKG‐II cells and DNA levels of p53 in T24 cells and E7 in SKG‐II cells were analyzed by qPCR. mRNA and DNA extracted from T24 and/or SKG‐II cells fixed with methanol‐based LBC solutions were efficiently detected, but to differing degrees, by qPCR. mRNA, and DNA from cells fixed with a formaldehyde‐containing fixative liquid were detected at significantly low copy numbers by qPCR. Our results demonstrate that LBC systems are powerful tools for cytopathology and immunocytochemistry applications. However, the appropriate fixative must be selected for cell preservation when a small number of LBC samples is used for molecular testing, particularly in RNA‐based molecular analyses. Diagn. Cytopathol. 2016;44:833–840. © 2016 Wiley Periodicals, Inc.     

Optimal fixation for total preanalytic phase evaluation in pathology laboratories. A comprehensive study including immunohistochemistry,DNA, and mRNA assays

The purpose of this study was to set the optimal preanalytical fixation protocol to enhance analytical and postanalytical phase accuracy and consistency. Twenty‐five normal colorectal tissues were fixed using various formalin concentrations, pHs, and fixation periods. All specimens were embedded in paraffin and 4 μm sections were used for immunohistochemistry of Ki‐67, and extraction and amplification of DNA and RNA. The Ki‐67 labeling index and the successful gene amplification rate for DNA and mRNA were evaluated and compared among variously fixed tissue samples. Ki‐67 positivity was enhanced by low pH and short fixation time, and was influenced by the type of antibody, but not by the staining (with or without using an autostainer) method. DNA amplification by PCR was strongly influenced by pH of formalin. cDNA amplification could be accomplished only with the shortest PCR fragment of 142 bp, and longer fixation times impaired the amplification. These data suggest that multiple different factors influence immunohistochemical results and gene amplification using DNA and mRNA. We recommend, based on data from this comprehensive analysis, a 10% neutral buffered formalin and fixation times of no longer than 1 week to produce consistent immunohistochemical slides and DNA amplification within 500 bp in pathology laboratories.     

Formalin-fixed paraffin-embedded clinical tissues show spurious copy number changes in array-CGH profiles

Formalin-fixed paraffin-embedded (FFPE) archival clinical specimens are invaluable in discovery of prognostic and therapeutic targets for diseases such as cancer. However, the suitability of FFPE-derived genetic material for array-based comparative genomic hybridization (array-CGH) studies is underexplored. In this study, genetic profiles of matched FFPE and fresh-frozen specimens were examined to investigate DNA integrity differences between these sample types and determine the impact this may have on genetic profiles. Genomic DNA was extracted from three patient-matched FFPE and fresh-frozen clinical tissue samples. T47D breast cancer control cells were also grown in culture and processed to yield a fresh T47D sample, a fresh-frozen T47D sample and a FFPE T47D sample. DNA was extracted from all the samples; array-CGH conducted and genetic profiles of matched samples were then compared. A loss of high molecular weight DNA was observed in the FFPE clinical tissues and FFPE T47D samples. A dramatic increase in absolute number of genetic alterations was observed in all FFPE tissues relative to matched fresh-frozen counterparts. In future, alternative fixation and tissue-processing procedures, and/or new DNA extraction and CGH profiling protocols, may be implemented, enabling identification of changes involved in disease progression using stored clinical specimens.     

Modeling formalin fixation and histological processing with ribonuclease A: effects of ethanol dehydration on reversal of formaldehyde cross-links

Understanding the chemistry of protein modification by formaldehyde fixation and subsequent tissue processing is central to developing improved methods for antigen retrieval in immunohistochemistry and for recovering proteins from formalin-fixed, paraffin-embedded (FFPE) tissues for proteomic analysis. Our initial studies of single proteins, such as bovine pancreatic ribonuclease A (RNase A), in 10% buffered formalin solution revealed that upon removal of excess formaldehyde, monomeric RNase A exhibiting normal immunoreactivity could be recovered by heating at 60 degrees C for 30 min at pH 4. We next studied tissue surrogates, which are gelatin-like plugs of fixed proteins that have sufficient physical integrity to be processed using normal tissue histology. Following histological processing, proteins could be extracted from the tissue surrogates by combining heat, detergent, and a protein denaturant. However, gel electrophoresis revealed that the surrogate extracts contained a mixture of monomeric and multimeric proteins. This suggested that during the subsequent steps of tissue processing protein-formaldehyde adducts undergo further modifications that are not observed in aqueous proteins. As a first step toward understanding these additional modifications we have performed a comparative evaluation of RNase A following fixation in buffered formaldehyde alone and after subsequent dehydration in 100% ethanol by combining gel electrophoresis, chemical modification, and circular dichroism spectroscopic studies. Our results reveal that ethanol-induced rearrangement of the conformation of fixed RNase A leads to protein aggregation through the formation of large geometrically compatible hydrophobic beta-sheets that are likely stabilized by formaldehyde cross-links, hydrogen bonds, and van der Waals interactions. It requires substantial energy to reverse the formaldehyde cross-links within these sheets and regenerate protein monomers free of formaldehyde modifications. Accordingly, the ethanol-dehydration step in tissue histology may be important in confounding the successful recovery of proteins from FFPE tissues for immunohistochemical and proteomic analysis.     

Effect of formalin fixation on pcr amplification of DNA isolated from healthy autopsy tissues

The aim of this study is to investigate the effects of formalin fixation on the degradation of DNA molecules in five different healthy tissues exempted during the autopsy, as well as the selection of the method that is most suitable for the DNA isolation. Heart muscle, liver, brain, lung and kidney tissue obtained from the healthy people who suddenly died from a violent death were used. The parts of tissue were fixed in 10% phosphate-buffered formalin as well as in 4% unbuffered formalin at room temperature. Morphology of tissue was studied using H&E staining. The DNA was isolated 6?h, 1–7 days (every 24?h), 10, 14, 28 days and 2 months after fixation using two different methods: extraction with phenol-chloroform-isoamyl alcohol as well as with PureLink Genomic DNA Kit. Yield and purity of the DNA samples were measured spectrophotometrically at 260?nm and 280?nm. The PCR amplifications of the glycerol-3-phosphate dehydrogenase 1 (GPD1, 150 bp), ß actin (ACTB, 262 bp) and ribosomal protein L4 (RPL4, 407 bp) genes were performed to evaluate the degree of DNA fragmentation. The RPL4 gene was amplified up to 72?h, ACTB gene up to 14 days and GPD1 gene up to 28 days from tissue fixed in phosphate-buffered formalin using phenol-chloroform-isoamylalcohol protocol for DNA isolation. Liver and kidney gave better results of PCR amplification, but statistical significance between tissues was not found. Preserving period, fixative and DNA extracting method are important factors for successful PCR amplification. The healthy tissue, fixed in phosphate-formalin up to 28 days, can be useful source in molecular studies. Tissues fixed in unbuffered formalin are suitable for molecular analysis up to 7 days.     

The effect of fixation and trypsinization on the immunohistochemical demonstration of intracellular immunoglobulin in paraffin embedded material

Using an indirect labelled immunoperoxidase technique the influence of fixation time on the antigenicity of intracellular immunoglobulin in lymphoid tissue fixed in buffered formalin has been investigated. Within a fixation period of 96 hours a decrease of 15% of stainable immunoglobulin containing cells was found, for every 24 hours the fixation time was prolonged. By comparing sections from tissue fixed in buffered formalin and selected fixatives (Lillie's AAF, Bouin's fluid, Clarke's fluid and 96% ethanol 1% acetic acid (E--A) processed at 4 degrees C and at 25 degrees C) an increased number of stained immunoglobulin containing cells was found in tissue fixed in Lillie's AAF, Bouin's fluid, Clarke's fluid and E--A processed at 4 degrees C. No difference was found between tissue fixed in buffered formalin and E--A processed at 25 degrees C. In addition the effect of pretreatment of the sections with trypsin on the number of stainable immunoglobulin containing cells was investigated. Trypsinization of sections from formalin fixed material increased the number of stainable cells substantially. No essential effect was seen on tissue fixed in Lillie's AAF and Bouin's fluid. In contrast trypsin treatment of sections from tissue fixed in Clarke's fluid and E--A completely destroyed the tissue. No differences were observed between different immunoglobulin classes examined as regards the effect of fixation time, fixatives and trypsinization.     

High quality of DNA retrieved for Southern blot hybridization from microwave-fixed, paraffin-embedded liver tissues

To overcome the degradation problem encountered in DNA extracted from formalin-fixed, paraffin-embedded tissue blocks, several methods of tissue fixation were examined in order to improve the quality of the DNA recovered for use in nucleic acid analysis. The fixation methods included formalin fixation alone, alcohol fixation alone, and microwave fixation with tissues immersed in phosphate-buffered saline (PBS), alcohol, or formalin. Unfixed fresh frozen tissue served as the control. Using hepatitis B virus (HBV) DNA sequences and the type I human procollagen gene as markers and liver tissue as a target, microwave fixation, with formalin omitted, not only preserved the DNA very well, but also the labile viral antigen. Both high molecular weight-integrated and free-form HBV DNAs were well preserved, and suitable for polymerase chain reaction and Southern blot analysis. The restriction enzyme fragment pattern of DNA recovered from these paraffin blocks was identical to that of unfixed fresh frozen tissue. Microwave fixation also preserved the labile preS2 epitope of the hepatitis B surface antigen (HBsAg) considerably better than formalin. These results suggest that microwave fixation is superior to routine formalin fixation for the preservation of excellent quality of genomic and viral DNAs for nucleic acid hybridization analysis. Alcohol, often used for nucleic acid purification, was also a good fixative for preserving DNA and the antigenicity of the labile antigen, especially when carried out in combination with microwave fixation.     

The AMeX method: a multipurpose tissue-processing and paraffin-embedding method. Extraction of protein and application to immunoblotting.

The authors have previously reported a new fixation and paraffin-embedding method (the AMeX method), which preserves many antigens as well as high molecular-weight DNA and RNA that are normally destroyed by the routine formalin fixation and paraffin-embedding process. In the present study, the authors analyzed the preservation of protein suitable for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting in tissue fixed by the AMeX method. The method used for extraction of protein from AMeX-processed tissue sections after deparaffinization was the same as that for extraction from fresh tissues. The total amount of protein extracted from 50-mg (wet weight) AMeX-processed mouse liver tissue was the same as that from fresh tissue. The electrophoretic mobility and staining intensity of protein on SDS-polyacrylamide gel, and the immunoblotting pattern and staining intensity with several antibodies, were identical for both AMeX-processed and fresh tissue. Degradation of protein was minimal for storage periods of 2 years in paraffin block. The authors also showed that pellets of cultured cells can be processed by this method for immunologic analysis. This new fixation and paraffin-embedding method is a useful tool for obtaining information on correlations between morphologic features and immunochemical and molecular biological data.     

Southern blot analysis of DNA extracted from formal-saline fixed and paraffin wax embedded tissue

Model experiments were designed to assess whether DNA could be recovered from formol-saline fixed peripheral blood lymphocytes and tonsil tissue for use in Southern blot gene analysis. Lymphocytes were fixed for 30 min and tonsil for 6 and 24 h, then paraffin embedded. High molecular weight DNA was extracted by prolonged digestion (2-7 days) with proteinase K or protease XXIV in the presence of 1 per cent sodium dodecyl sulphate. Restriction, transfer and hydridization were possible without modification of standard procedures. Multiple copy sequences were demonstrated using Mspl and Bst Nl restriction and hybridization for the Y chromosome (pHY 2.1 probe), single copy genes using EcoRI and BamHl restriction for the T-cell receptor beta chain (T beta probe), and Bgl II and Hind III for the immunoglobulin heavy chain (JH probe). Identical banding to unfixed tissue was achieved except when 24 h fixed extracts were used. With these, demonstration of the 24 KB Bam Hl/T beta and 9.2 KB Hind III/JH bands was not obtained. These findings suggest that as the fixation time is extended, alterations to DNA will limit the available range of restriction enzyme/probe combinations. However, with careful choice of these the extraction of DNA from formalin fixed and paraffin embedded pathological tissue for Southern blotting should be profitable.     

Ultra-rapid microwave-stimulated tissue processing with a modified protocol incorporating microwave fixation

AIMS: To develop an ultra-rapid microwave (MW)-stimulated histoprocessing protocol that incorporates MW fixation and produces consistent, high quality sections. METHODS: A range of fresh autopsy tissues was divided into three groups composed of equal numbers of small and large tissue blocks. Group 1 tissues were fixed for 8 hours in 4% buffered formaldehyde and processed in a conventional tissue processor through a 15-hour cycle. Group 2 tissues were processed in a MW histoprocessor according to the manufacturer's recommended protocol of irradiation in a proprietary reagent before embedding in paraffin. Group 3 tissues were processed with our protocol that incorporated the addition of MW fixation in 4% buffered formaldehyde and MW irradiation in isopropyl alcohol after irradiation in the proprietary reagent. RESULTS: The manufacturer's protocol resulted in 'grey' and 'wet look' artefacts in large tissue blocks. These artefacts did not occur in our protocol which produced sections that were indistinguishable from those obtained with conventional 23-hour processing. Histochemical and immunohistochemical stains were also indistinguishable and the tissue blocks cut very smoothly. CONCLUSIONS: The incorporation of an additional step to ensure adequate tissue fixation and one to ensure optimal dehydration and clearing resulted in a MW histoprocessing protocol that produced consistent results for both large and small tissue blocks. Paraffin-impregnated blocks can be produced very rapidly from fresh small and large tissue blocks in 45 and 100 minutes, respectively.     

The effect of immersion formaldehyde fixation on human placental weight

The prevalence of the human immunodeficiency and hepatitis viruses has led to considerable concern by health care workers about safer means of examining surgical pathological specimens. The human placenta needs to be examined when there are complications during pregnancy, labor, and delivery; when the fetus is born with apparent problems; and when the delivered placenta is abnormal. Placentas are routinely immersion fixed with neutral buffered 3.7% to 4.0% formaldehyde solution before examination and without obtaining a fresh weight. This study was undertaken to determine if there was a significant change in weight between a fresh and fixed placenta. The results show a 7.67% increase in placental weight after formaldehyde fixation for 24 hours. Thus, the practice of formaldehyde fixation prior to weighing and examination can be continued and still allow for accurate estimation of fresh placental weight.     

A microscopic survey on the efficiency of well-known routine chemical fixatives on cryosections

This study was designed to analyze and compare tissue preservation efficiency of acetone (AC), formaldehyde (FA) and paraformaldehyde (PFA) on cryosections. Brain, kidney, heart and liver tissue of adult Balb/c mice were fixed with either FA or PFA prior to cryosectioning, or fixed with AC alone immediately after cryosectioning. Hematoxylin and eosin staining showed that AC is a poor fixative in preserving the general tissue and cellular organization. PFA, and to a lesser extent FA, produced significantly better results. Another set of cryosections were further analyzed to test the properties of those fixatives to preserve proteins from specific cell structures. Cytokeratin filaments, F-actin filaments and nuclei were immunolabeled and examined using confocal microscopy. Results demonstrated that, overall, PFA is the best fixative tested. However, FA fixation gave poor results in preserving neuronal tissues. Immunofluorescence confirmed the inefficiency of AC fixation, after which no specific labelling of cytokeratin filaments was detectable. Nevertheless, actin filaments were detectable on AC-fixed samples, a finding that was supported by the quantification of fluorescein-phalloidin binding to F-actin. Overall, the data suggest that AC fixation is unacceptable for preservation of most samples, whereas FA and PFA fixation should be chosen according to the tissues and proteins to be studied.     

Influence of different fixation protocols on the preservation and dimensions of cardiac tissue

Recent extensive progress in invasive cardiac procedures has triggered a wave of dozens of heart morphometric anatomical studies that are carried out largely using autopsied samples fixed in formaldehyde solution prior to observations and measurements. In reality, very little is known about changes in heart tissue dimensions during fixation. The aim of this study was therefore to investigate how fixation affects the dimensions of cardiac tissue, and if different types and concentrations of reagents affect this phenomenon. A total of 40 pig heart samples were investigated, and seven different measuring sites were permanently marked in every heart prior to fixation. Four study groups (n = 10 each) were assembled that differed only in concentration and the type of fixative: (i) 2% formaldehyde solution; (ii) 4% formaldehyde solution (formalin); (iii) 10% formaldehyde solution; (iv) alcoholic formalin. The samples were measured before and after fixation at the following time points: 24 h, 72 h and 168 h. It was found that different fixatives significantly affected different parameters. Almost all of the heart dimensions that were measured stabilized after 24 h; later changes were statistically insignificant in the point‐to‐point comparison. Change in the length of the interatrial septum surface was not altered significantly in any of the fixatives after 24 h of preservation. It was found that 10% formaldehyde increased the thickness of muscular tissue only after 24 h; this thickening was reduced after 72 h and was insignificant at 168 h. Other heart parameters in this group do not present significant changes over the entire fixation time duration. In conclusion, the 10% formaldehyde phosphate‐buffered solution appeared to be the best fixative among the fixatives that were studied for cardiac morphometric purposes; this solution caused the smallest changes in tissue dimensions. Measurements should be obtained at least after 1 week of preservation when most parameters exhibit the smallest changes compared with the non‐preserved samples.     

四种固定液对肾上腺组织冰冻切片固定效果的比较

目的　以肾上腺组织为例观察比较丙酮、4%多聚甲醛、AAF和95%乙醇四种固定液的固定效果,为临床做出更好的快速冰冻切片,及时、准确的做出病理诊断提供实验基础。 方法　取人正常肾上腺组织,冰冻切片后分别用四种固定液进行固定,然后行HE染色,观察各组的组织结构、细胞核染色、细胞质染色和核质对比度情况。 结果　经冷丙酮固定的肾上腺组织切片,镜下组织结构清晰,高倍镜下观察：细胞核形态清晰完整,核染色和胞质染色清晰对比度好;经4%多聚甲醛固定的切片,镜下组织结构不清,细胞核形态较清晰,核染色和胞质染色对比度差;经AAF液固定的组织切片,镜下组织结构尚清晰,细胞核部分溶解,核染色均一,胞质染色较清晰;经95%乙醇固定的组织,组织结构尚清晰,核溶解普遍,胞质染色不清晰。 结论　四种固定液都可用于固定肾上腺组织及其它细胞成分较多的病理肿瘤组织,但冷丙酮效果最好。     

Immunohistochemical identification of mast cells in formaldehyde-fixed tissue using monoclonal antibodies specific for tryptase

An avidin-biotin enhanced immunoperoxidase procedure using monoclonal antibodies (AA1, AA3, and AA5) prepared against human mast cell tryptase resulted in intense staining of mast cells in paraffin-embedded tissue. The distribution of mast cells observed was similar to that seen when adjacent serial sections were stained using a standard procedure with toluidine blue, though the immunoperoxidase technique permitted the identification of significantly more mast cells. With monoclonal antibody AA1, immunostaining was entirely specific for mast cell granules, and there was negligible background staining in a range of tissues including lung, tonsil, colon, gastric mucosa, skin, and pituitary. There was no staining of antibody on basophils or on any other normal blood leukocyte. The technique was effective with tissue fixed in either Carnoy's or neutral buffered formalin, though the internal mast cell structure was better preserved with formaldehyde fixation. The immunoperoxidase staining procedure with monoclonal antibody AA1 is a highly specific and sensitive means for the detection of mast cells in routinely processed tissues.