Leaching of Trace Elements from Biological Tissue by Formalin Fixation

In studies of trace elements in biological tissue, it is imperative that sample handling does not substantially change element concentrations. In many cases, fresh tissue is not available for study, but formalin-fixed tissue is. Formalin fixation has the potential to leach elements from the tissue, but few studies have been published in this area. The concentrations of 19 elements were determined by high-resolution inductively coupled plasma mass spectrometry in formalin in which human and rat brain samples had been stored for different time durations ranging from weeks up to several years. Additional analysis was carried out in fixed brain samples. There was substantial leaching of elements from the tissue into the formalin, and the leaching varied considerably between different elements. For example, formalin concentrations of As, Cd, Mg, Rb, and Sb increased more than 100-fold upon long-term (years) storage, while for Ni and Cr, the leaching was negligible. The degree of leaching was strongly time-dependent. In conclusion, formalin fixation and storage of biological tissue has the potential to leach substantial fractions of several trace elements from the tissue. The potential of leaching must be critically considered when using formalin-fixed biological tissue in trace metal analysis.     

福尔马林对固定标本DNA提取和扩增的影响

福尔马林被广泛应用于生物标本的长期保存。由于福尔马林可能影响标本DNA的质量,因此需要对福尔马林固定标本DNA的提取和扩增过程进行改进。影响从福尔马林保存标本中提取的DNA质量的主要因素包括福尔马林导致的DNA与蛋白质之间、蛋白质与蛋白质之间、DNA与DNA之间的交联,福尔马林溶液的化学成分、pH值及浓度,标本保存的时间和温度,标本保存部位等。本文总结了目前常用的对标本DNA提取和扩增过程的改进措施及其优点。     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed, paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Development of an optimal antigen retrieval protocol for immunohistochemistry of retinoblastoma protein (pRB) in formalin fixed,paraffin sections based on comparison of different methods

A novel protocol for antigen retrieval (AR) for immunohistochemistry (IHC) of retinoblastoma protein (pRB) in formalin fixed, paraffin embedded (FFPE) tissue sections was developed using 0.05% citraconic anhydride as the AR solution for heat treatment based on comparison of different methods. This new protocol has advantages including superior morphological preservation, greater reproducibility, and more intense staining after retrieval. Our study demonstrates the importance of comparing various AR protocols to obtain maximal IHC for standardization and for quantitative IHC.     

Comparison of Fixation Methods for Preservation of Morphology, RNAs, and Proteins From Paraffin-Embedded Human Cancer Cell-Implanted Mouse Models

Xenograft transplantation of human tumor cells into immunodeficient mice is an important method to clarify the roles of specific molecules or chemicals in vivo. Recently, this method has been reported as a definitive examination to identify tumor stem cells. In this study, the authors compared the morphology and the quality and quantity of ribonucleic acid (RNA) and protein in paraffin-embedded tissues of nude mice implanted with human uterine cervical cancer cells, followed by fixation with commonly used fixatives, including 4% paraformaldehyde (PFA), 10% neutral buffered formalin (NBF), 20% NBF, and 99% ethanol (EtOH). The quality of the isolated RNA from PFA- and NBF-fixed paraffin-embedded tissues was high, while EtOH-fixed tissues showed degradation of RNA. NBF-fixed tissues showed excellent quality of morphology, but EtOH-fixed tissues showed contraction of cells. Immunohistochemical results showed differences depending on fixations. The 99% EtOH-fixed samples showed decreases of Ki-67 and VEGF-A immunoreactivities, but improved cytokeratin immunoreactivity. This study indicated that formalin fixation is better than alcohol fixation for RNA preservation in paraffin-embedded cancer cell implantation models. Immunohistochemical results differed markedly depending on fixation materials and antibodies; therefore, suitable fixations are needed to quantify and compare the results of immunohistochemical staining on cancer cell implanted nude mice tissues.     

Molecular mechanisms of antigen retrieval: antigen retrieval reverses steric interference caused by formalin-induced cross-links

Abstract

The overwhelming majority of antibodies useful for formalin fixed, paraffin embedded (FFPE) tissues require antigen retrieval to reverse the effect of formalin fixation and re-establish immunoreactivity. How this reversal happens is poorly understood. We developed a new experimental model for studying the mechanism of formalin fixation and antigen retrieval. Epitope mapping studies on nine antibodies useful for FFPE tissues revealed that each consisted of a contiguous stretch of amino acids in the native protein (linear epitope). Small peptides representing the epitopes of antibodies to human epidermal growth factor receptor type (HER2), estrogen, and progesterone receptors were attached covalently to glass microscope slides in a peptide array. Most peptides retained immunoreactivity after formalin fixation. Immunoreactivity was completely abrogated for all peptides, however, if an irrelevant large protein was present during formalin-induced cross-linking. We hypothesize that cross-linking the irrelevant protein to the peptide epitopes sterically blocked antibodies from binding. Antigen retrieval dissociates irrelevant proteins and restores immunoreactivity. Because the epitopes for clinical antibodies require only primary protein structure, the fact that antigen retrieval probably denatures the secondary and tertiary structure of the protein is irrelevant. The same mechanism may occur in tissue samples subjected to formalin fixation and antigen retrieval.     

Effects of Meloidogyne incognita on Nitrogen Fixation in Soybean

The effects of a North Carolina population of Meloidogyne incognita on N₂ fixation on root-knot-susceptible ''Lee 68'' and moderately resistant ''Forrest'' soybean were evaluated 50, 75, I00, and 135 days after inoculation with nematodes. Nematodes stimulated N₂ fixation in Lee 68 by 50 days and in Forrest by 75 days. At all other intervals, N₂ fixation was either depressed or unaffected by nematodes. Additional observations indicate that the susceptibility of Lee 68 is associated with greater rates of penetration by larvae and more favorable responses of host tissues to nematodes than occur in Forrest. With time, however, the histological reactions of both hosts became less favorable for nematode development. Resistant or hypersensitive responses became common in Forrest by 75 days but not in Lee 68 until 90 days after inoculation. This population of M. incognita may stimulate N₂ fixation at a specific time interval and depress it at others; therefore, disease of susceptible soybeans caused by this nematode is probably not primarily due to a net loss of fixed nitrogen but to pathogenicity similar to that which occurs on nonlegume hosts.     

Combined effects of formalin fixation and tissue processing on immunorecognition

Abstract

It is accepted that aldehyde-based fixation of cells can affect immunodetection of antigens; however, the effects of tissue processing on immunodetection have not been analyzed systematically. We investigated the effects of aldehyde-based fixation and the various cumulative steps of tissue processing on immunohistochemical detection of specific antigens. DU145 (prostate) and SKOV3 (ovarian) cancer cell lines were cultured as monolayers on microscope slides. Immunohistochemical detection of Ki67/MIB-1 and proliferating cell nuclear antigen (PCNA) was evaluated after various fixation times in 10% neutral buffered formalin and after each of the several cumulative steps of tissue processing. The effect of antigen retrieval (AR) was evaluated concomitantly as an additional variable. Our results indicate that in addition to fixation, each of the tissue processing steps has effects on immunorecognition of the epitopes recognized by these antibodies. Extensive dehydration through ethanols to absolute ethanol had only modest effects, except for the detection of Ki67/MIB-1 in SKOV-3 cells where the effect was stronger. In general, however, establishment of a hydrophobic environment by xylene resulted in the greatest decrease in immunorecognition. AR compensated for most, but not all, of the losses in staining following fixation and exposure to xylene; however, AR gave consistent results for most steps of tissue processing, which suggests that AR also should be used for staining PCNA. The cellular variations that were observed indicate that the effects of fixation and other steps of tissue processing may depend on how antigens are packaged by specific cells.     

Immunohistochemical Double Staining of Microwave Enhanced and Nonenhanced Nuclear and Cytoplasmic Antigens

Many of the antigens commonly investigated in histopathology can be enhanced by microwave pretreatment (MWPT) of formalin fixed, paraffin embedded tissue sections. We developed a double labeling method using microwave heating to detect otherwise undetectable nuclear antigens combined.with immunohisto-chemistry (IHC) of cytoplasmic or membranous antigens that do not benefit from MWPT. We used the same primary antibody solutions used in single antibody IHC. The staining technique is based on the alkaline phosphatase anti-alkaline phosphatase (APAAP) and the labeled avidin-biotin (LSAB) methods. Four different protocols were tested, each modifying the sequence of MWPT, APAAP and LSAB staining. In this study Ki67, estrogen receptor, progesterone receptor, c-neu, CD68 and desmin primary antibodies were used in routinely formalin fixed, paraffin embedded tissues of 50 tumor specimens. MWPT followed by LSAB for microwave enhanced antigens and APAAP for antigens that cannot be enhanced by MWPT gave the best double staining results. This method improves characterization of tumor cell features from paraffin embedded tissue and should aid analysis of tumor differentiation, receptor status and nuclear proteins in the single cells in archival tissues.     

Effects of Enhanced UV-B Radiation on Nitrogen Fixation in Arctic Ecosystems

Recent global climate models predict a further significant loss of ozone in the next decades, with up to 50% depletion of the ozone layer over large parts of the Arctic resulting in an increase in ultraviolet-B radiation (UV-B) (280–315 nm) reaching the surface of the Earth. The percentage of total annual ecosystem N input due to biological nitrogen fixation by cyanobacteria might be as high as 80% and the contribution to total annual N uptake by plants up to 20%. A possible reduction of nitrogen fixation raises serious concerns about already nutrient impoverished plant communities. This review shows that nitrogen fixation by moss-associated cyanobacteria in arctic vegetation was dramatically reduced after six years of exposure to enhanced UV-B radiation. In subarctic vegetation, nitrogen fixation activity of moss-associated cyanobacteria was not affected by 6 years of enhanced UV-B radiation. However, a 50% increase of summer precipitation resulted in a 5- to 6-fold increase in activity. Long-term effects of UV-B radiation on nitrogen fixation activity have been examined only in two lichens, giving contrasting results. Peltigera aphthosa (L.) Willd., having external cephalodia, experienced a significant reduction, whereas Peltigera didactyla (With.) J.R. Laudon, having cyanobacteria in the photobiont layer below the upper cortex, did not experience any changes due to radiation regimes. The difference is probably related to the location of the cyanobacteria. While the Nostoc cells are protected by the fungal, melanized upper cortex in P. didactyla, they are exposed and unprotected in P. aphthosa, and their own synthesis of UV-B absorbing compounds appears to be low. Under certain environmental conditions, an increasing UV-B radiation will dramatically affect nitrogen fixation in arctic tundra vegetation, which in turn may have severe influence on the nitrogen budget in these environments. Further long-term studies are necessary to conclude if these effects are temporal and how concurrent climatic changes will influence the nitrogen balance of the ecosystem.     

Standardization in immunohistochemistry: the role of antigen retrieval in molecular morphology

Molecular morphology seeks to integrate the traditional morphologic criteria of surgical pathology with immunohistochemical and in situ hybridization techniques that allow demonstration of a variety of molecules, proteins, RNA and DNA in a tissue section. While immunohistochemistry has proven to be successful for demonstrating lineage related biomarkers of value for diagnosis and classification of tumors, concerns have been raised periodically about validation of reagents, overall reproducibility of the staining method, and interpretation of results. These concerns have been heightened by the burgeoning interest in prognostic markers, where the question extends beyond a relatively simple positive or negative result to an absolute need for quantification of the staining result; not only is it positive, but how much is there? In this presentation at the Annual Meeting of the Biological Stain Commission in June, 2005, I advocate a total test approach that requires systematic attention to pre-analytic, analytic, and post-analytic issues. The approach encompasses all aspects of test performance from specimen acquisition, through fixation, antigen retrieval, processing, staining, interpretation, and reporting of results. A similar systematic approach also may be adopted for in situ hybridization methods, which have performance requirements that in many ways parallel immunohistochemistry.     

Capillarity in Rat Skeletal Muscle: Effects of Rapid Freezing versus Perfusion Fixation

We determined the effects of rapid freezing and perfusion fixation on fiber geometry and capillarity in rat skeletal muscle. Fiber areas were significantly decreased, and capillary densities significantly increased, in perfusion-fixed versus quick-frozen muscle. Significant differences in capillary-to-fiber ratios were not observed, suggesting that differences in fiber geometry, not the methods of quantifying capillaries, accounted for the differences in capillary density. We conclude that estimates of fiber geometry, capillarity, and diffusive gas conductances obtained from perfusion-fixed muscles are subject to significant error due to shrinkage.     

Diagnostic value of clot examination for malignant cells in serous effusions

Objective:  To assess the diagnostic value of clot examination for satisfactory processing and confirmation of malignancy in serous effusions in routine cytological evaluation and compare the results with those of conventional smear and cell block preparations.
Methodology:  Body cavity fluids ( n  = 600) received in our laboratory were processed according to a pre-designed protocol for the study as follows: Day1: on receipt of the specimen, smears were made and a cell block was prepared from the sediment. Day2: after overnight sample storage of the remaining specimen at 2–8 °C all fluids were examined for the presence of a clot at the bottom of the container. Fluids in which clot had formed were fixed in formalin. The clot was then placed on a lens paper, wrapped and processed routinely. Diagnostic yields were compared.
Results:  In this study, we included 600 cases of serous fluids from pleural, pericardial and peritoneal effusions. In 73% ( n  = 437) of samples, clot formation was seen, while in 27%, ( n  = 163) no clot had formed. Routine smear and cell block preparations showed malignant cells in 9.6% ( n  = 42). However, with the addition of the clot preparation, the number of cases in which atypical/malignant cells were seen increased from 42 to 85 (19.4%), with a P  < 0.001. Special stains and immunohistochemistry (IHC) were also performed on clot preparations in 10 difficult cases.
Conclusion:  Clot preparation from body cavity fluids on the second day can be used as an adjunct to smear and routine cell block preparation to improve the accuracy and yield of the cytological diagnosis and may also be of great help for special studies such as IHC staining.     

The Occurrence of Amphizoic Amebae in Domestic Animals

SYNOPSIS Random examination of domestic animals revealed the frequent presence of free-living amebae in their bodies. In diseased or dead cows, dogs, pigs, rabbits, pigeons, and turkeys 15 strains of amebae were found, belonging to the genera Acanthamoeba (A. polyphaga), Hartmannella (H. vermiformis) , and Vahlkampfia (V. avara, V. enterica, V. inornata). , They were usually accompanied by other infectious agents in different parts of the host bodies. Pathogenicity of 3 isolates could not be demonstrated by inoculation of laboratory animals.
Some features of the isolates differed from those previously known for members of these genera. These strains may be considered amphizoic amebae according to Page (1974).     

Hardware for In-Flight Fixation of Plants in Microgravity

NASA-approved hardware was designed for an experiment involving in-flight fixation of 4.8-day-old seedlings of Brassica perviri-dis. The hardware is inexpensive, reliable, and simple to construct. This hardware aboard flight 61-C of space shuttle Columbia functioned perfectly, and required less than 0.5 hr of crew time for training. Analyses of cellular ultrastructure indicated excellent ultrastructural preservation.     

经后路一期病灶清除、自体植骨融合、内固定治疗胸腰椎结核疗效观察

目的:研究经后路一期病灶清除、自体植骨融合、内固定治疗胸腰椎结核的疗效.方法:选择胸腰椎结核的患者作为研究对象,随机分为给予自体植骨融合的观察组和椎间融合器植骨融合的对照组,观察手术相关指标、椎体功能相关指标、活动状态和生活质量.结果:观察组手术时间、术中出血量、术后引流量、术后卧床时间、Cobb角均明显少于对照组;治疗后椎间隙高度、椎体融合率、KPS评分和生活质量优良率明显高于对照组.结论:经后路一期病灶清除、自体植骨融合、内固定治疗能够改善治疗效果、提高生活质量,具有积极的临床价值.     

Effects of long-term fixation on histological quality of undecalcified murine bones embedded in methylmethacrylate

While long-term fixation and storage of specimens is common and useful for many research projects, it is particularly important for space flight investigations where samples may not be returned to Earth for several months (International Space Station) or years (manned mission to Mars). We examined two critical challenges of space flight experimentation: the effect of long-term fixation on the quality of mouse bone preservation and the preservation of antigens and enzymes for both histochemical and immunohistochemical analyses, and how the animal/sample processing affects the preservation. We show that long-term fixation minimally affects standard histological staining, but that enzyme histochemistry and immunolabeling are greatly compromised. Further, we demonstrate that whole animal preservation is not as suitable as whole leg or stripped leg preservation for long-term fixation and all histological analyses. Overall, we recommend whole leg processing for long-term storage of bone specimens in fixative prior to embedding in plastic for histological examination.     

Immunohistochemistry on Resin Sections: A Comparison of Resin Embedding Techniques for Small Mucosal Biopsies

We have modified resin embedding methods to provide optimal information from en-doscopic biopsies. Mucosal biopsies were fixed either in buffered formalin and processed for embedding in Araldite or in acetone containing protease inhibitors and embedded in glycol meth-acrylate (GMA). GMA embedding generated an im-munophenotypic profile similar to that obtained in frozen sections while yielding far superior morphology and greater numbers of sections from small biopsies. The phenotypic markers included those for T cells, macrophages, mast cells, eosin-ophils and neutrophils. We have also demonstrated collagens, cell adhesion molecules and integrin molecules. Sections of similar quality were obtained with Araldite but the repertoire of antibodies was restricted to those which can be applied to formalin fixed, paraffin embedded tissues. We suggest that for optimal results, small biopsies to be subjected to immunochemistry are fixed in acetone at -20 C with the inclusion of protease inhibitors and embedded in GUIA with careful temperature control.     

Effects of preservation on pigmentation and length measurements in larval lampreys

The effects of two methods of preservation (fixation and storage in 10% formalin, and fixation in 10% formalin followed by storage in 95% alcohol) on pigmentation and morphometric features used for identification of larval Ichthyomyzon lampreys were analysed. Both short‐term (3 weeks) and long‐term (6 months) studies were conducted using digital analysis of images of fresh and preserved lampreys. Six standard morphometric lengths and 10 areas of pigmentation were analysed. All measurements were significantly affected by preservation. Preservative type affected pigmentation and morphometric characteristics differently, and characters were affected to different degrees. Multiple measurements over time showed that almost all changes occurred within 3 weeks of preservation. Regression equations allowed for accurate correction of preservation effects on morphometric measurements, but the effects on pigmentation levels were less predictable. Effects of preservation on larval lampreys need to be considered when comparing fresh and preserved specimens because they influence critical identification features.