Ethanol-fixed Tissues Research Articles

The Near-Infrared Spectroscopy of Ethanol-Fixed Tissues to Detect Illicit Treatments with Glucocorticoids in Bulls.

This study aimed to set up indirect, rapid methods involving near infrared (NIR) spectroscopy analysis, to detect illicit treatments with glucocorticoids in bull. The ethanol fixation method (EtOH) was applied to 7 different tissues obtained from 20 Friesian bulls, 12 of which were experimentally administered with dexamethasone as part of a growth-promoting protocol for 60 days and slaughtered 26 days after the end of the treatment. A perfect discrimination was obtained for the 7 sampled tissues, considering a full UV-Vis-NIR range (350 ÷ 2500 nm), for both false positive and negative animals. The validated true positive and negative errors were zero for the longissimus thoracis muscle, 10% for the skin-dermis, 15% for the fat, 25% for the thymus gland and the semitendinosus muscle, 30% for the sternomandibularis muscle and 35% for the skin-hair. A multiple test on the most accessible tissues, that is, the thymus gland, the sternomandibularis muscle and fat, can be used as an alternative to provide indications about animals that have been subjected to illicit treatments. In the short space of three days from the slaughter, NIR spectroscopy of ETOH fixed tissues, would allow at least cost the detection of a probable illicit which could eventually be reported to health authorities for specific investigation in the frame of official controls.

Using American Type Culture Collection Cell Lines to Evaluate Interlaboratory Variables for Estrogen Receptor and Human Epidermal Growth Factor Receptor 2 Immunostaining.

Most laboratories currently use patient tissues for validating immunohistochemical stains. To explore advantages of using cell lines with known antigenicity as a validation method. Five American Type Culture Collection (ATCC) cell lines with known negative, low positive, and moderate to strong estrogen receptor (ER) expression as well as negative, equivocal, and positive human epidermal growth factor receptor 2 (HER2) expression were cultured and made into cell blocks. One block from each cell line was fixed in formalin and another in ethanol before cell block preparation. Two sets of paired unstained slides from each block were sent to 10 different laboratories for HER2 and ER staining to be stained on runs from different days according to each laboratory's defined protocol. The 10 study participants evaluated 40 slides in a blinded fashion. For ER expression, all 80 interpretations for the ER strong and moderate positive cell lines had the target ER-positive result, and 74 of 80 ER-negative cell lines (92.5%) had agreement with the intended negative result. The ER low positive cell line showed varied but positive expression among all observers. The HER2 (3+)-positive cell lines yielded a target interpretation of 3+ in 65 of 80 interpretations (81.2%). For the HER2-negative cell line 69 of 78 interpretations (88.5%) were consistent with the target response (0 or 1+). No significant variation was observed between the ethanol- and non-ethanol-exposed cell lines, or between runs by the same laboratory. Variation from target results clustered within laboratories. This study indicates that variability between laboratories can be identified by using cell lines for quantitative or semiquantitative immunohistochemistry when using cultured cell lines of known antigenicity. These cell lines could potentially play a role in aiding anatomic pathology laboratories in validating immunohistochemistry tests for formalin- and ethanol-fixed tissues.

PCR, in-situ hybridization, and phylogenetic analysis suggest that 'big belly' disease in barramundi, Lates calcarifer (Bloch), is caused by a novel Vibrio species.

'Big belly' disease is a chronic, granulomatous bacterial enteritis and peritonitis, first reported in 3- to 4-week-old Asian seabass or barramundi, Lates calcarifer Bloch fry. Affected fry are emaciated and have a swollen abdomen, and the condition is referred to as 'skinny pot-belly' or 'big belly' disease. In this study, histopathological examinations of diseased fish from a batch of 2-month-old, 6- to 8-cm L.calcarifer fingerlings, kept in seawater recirculating aquaculture systems, showed pathology resembling 'big belly' disease. Ethanol-fixed tissues were tested positive using specific PCR primers based on 16S rRNA genes. In situ hybridization using dioxygenin-labelled positive PCR products on formalin-fixed paraffin-embedded tissues showed positive reactions with intralesional, clusters of the large, 'big belly' coccobacilli. A phylogenetic tree constructed based on analyses of these 16S rRNA gene PCR products from five positive fish suggests that the 'big belly' bacterium is most likely a novel Vibrio species.

Genetic Diversity and Geographic Distribution of Bat-borne Hantaviruses.

The recent discovery that multiple species of shrews and moles (order Eulipotyphla, families Soricidae and Talpidae) from Europe, Asia, Africa and/or North America harbour genetically distinct viruses belonging to the family Hantaviridae (order Bunyavirales) has prompted a further exploration of their host diversification. In analysing thousands of frozen, RNAlater-preserved and ethanol-fixed tissues from bats (order Chiroptera) by reverse transcription polymerase chain reaction (RT-PCR), ten hantaviruses have been detected to date in bat species belonging to the suborder Yinpterochiroptera (families Hipposideridae, Pteropodidae and Rhinolophidae) and the suborder Yangochiroptera (families Emballonuriade, Nycteridae and Vespertilionidae). Of these, six hantaviruses are from Asia (Xuân Son virus and Dakrông virus in Vietnam; Láibin virus in China and Myanmar; Huángpí virus and Lóngquán virus in China; and Quezon virus in the Philippines); three are from Africa (Mouyassué virus in Côte d'Ivoire and Ethiopia; Magboi virus in Sierra Leone; and Makokou virus in Gabon); and one from Europe (Brno virus in the Czech Republic). Molecular identification of many more bat-borne hantaviruses is expected. However, thus far, none of these newfound viruses has been isolated in cell culture and it is unclear if they cause infection or disease in humans. Future research must focus on myriad unanswered questions about the genetic diversity and geographic distribution, as well as the pathogenic potential, of bat-borne viruses of the family Hantaviridae.

Genetic variation of mitochondrial genes among Echinococcus multilocularis isolates collected in western China

BackgroundAlveolar echinococcosis (AE) is a life-threatening human disease caused by Echinococcus multilocularis transmitted between rodents and dogs/foxes in the Northern Hemisphere. The study aims to identify the genetic variation of the parasite in AE patients from China.MethodsE. multilocularis isolates were collected from wild small mammals (n = 6) and AE patients (n = 56) from western China. Genomic DNA was extracted from different tissue samples including paraffin tissue blocks, ethanol fixed tissues and frozen tissues surgically removed. Two mitochondrial gene fragments (526 bp for cob and 474 bp for nad2) of E. multilocularis were amplified and sequenced.ResultsThe parasite fragment sequences of cob fragments from AE patients showed two haplotypes, and nad2 gene fragment sequences had four haplotypes. The gene sequences from Microtus sp. were 100% identical to the sequences of some isolates from AE patients. These haplotypes were distributed in both Qinghai and Xinjiang provinces. Alignment analysis with the sequences from the GenBank databases showed five genotypes including three Asian genotypes, one from Europe and one from North America.ConclusionsMost AE patients harbored the Asian genotype 1 which may be an indication of its relative frequency in the definitive hosts and the environment or of its pathogenicity to humans, which calls for further research.

Hantaviruses: Rediscovery and new beginnings

Virus and host gene phylogenies, indicating that antigenically distinct hantaviruses (family Bunyaviridae, genus Hantavirus) segregate into clades, which parallel the molecular evolution of rodents belonging to the Murinae, Arvicolinae, Neotominae and Sigmodontinae subfamilies, suggested co-divergence of hantaviruses and their rodent reservoirs. Lately, this concept has been vigorously contested in favor of preferential host switching and local host-specific adaptation. To gain insights into the host range, spatial and temporal distribution, genetic diversity and evolutionary origins of hantaviruses, we employed reverse transcription-polymerase chain reaction to analyze frozen, RNAlater®-preserved and ethanol-fixed tissues from 1546 shrews (9 genera and 47 species), 281 moles (8 genera and 10 species) and 520 bats (26 genera and 53 species), collected in Europe, Asia, Africa and North America during 1980–2012. Thus far, we have identified 24 novel hantaviruses in shrews, moles and bats. That these newfound hantaviruses are geographically widespread and genetically more diverse than those harbored by rodents suggests that the evolutionary history of hantaviruses is far more complex than previously conjectured. Phylogenetic analyses indicate four distinct clades, with the most divergent comprising hantaviruses harbored by the European mole and insectivorous bats, with evidence for both co-divergence and host switching. Future studies will provide new knowledge about the transmission dynamics and pathogenic potential of these newly discovered, still-orphan, non-rodent-borne hantaviruses.

Abstract 4862: Whole genome amplification allows for testing of valuable specimens by array comparative genomic hybridization

Abstract Many cancer clinical specimens are obtained in minute quantities and in varying states of degradation; for example, when they are formalin or ethanol fixed. In addition, many investigators seek tests on multiple genomic analytes and frequently insufficient DNA remains for all inquiries after the initial priority testing. To accommodate all clinical scientific inquiries, the Sigma GenomePlex whole genome amplification (WGA) protocol has been evaluated as a means to produce sufficient genomic material for array comparative genomic hybridization (aCGH) downstream testing. However previous studies, including our own, have shown that testing degraded samples with WGA coupled to oligo CGH arrays resulted in decreased data quality. We contend this could be due to unequal genomic representation in the WGA product. Our objective was to assess the importance and efficacy of using correctly matched reference DNA and to compensate for the WGA amplification bias, potentially improving aCGH results. We obtained DNA from ethanol-fixed prostate tissues in which both tumor and normal tissue compartments were separately isolated by laser capture microdissection or by directed needle-core sampling from formalin-fixed paraffin embedded blocks. We sought to optimize the Sigma WGA protocol by testing the following parameters: source of reference DNA (commercial versus matched normal), WGA cycle number, and the use of size-exclusion columns post-WGA. Quality control measures for the purified WGA product included electrophoresis (gel and Agilent Bioanalyzer), and a multiplex PCR to determine the extent of DNA genomic representation across the WGA product. ChromaSpin columns were used to reduce the number of smaller WGA amplicons prior to aCGH. WGA products were assayed with Agilent 8×60K CGH microarrays and chromosomal aberrations and data quality were assessed using BioDiscovery Nexus software. Data will be presented comparing the effectiveness of using sheared reference DNA as opposed to matched normal DNA, the use of QC methods prior to hybridization, any benefit in removing small WGA DNA products, and varying the WGA cycle number. We continue to evaluate how these modifications can bring improvement to WGA when coupled to aCGH, because there is promise that this method could expand the genomic testing opportunities for valuable and rare clinical specimens. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4862. doi:10.1158/1538-7445.AM2011-4862

Resurrection of DNA Function In Vivo from an Extinct Genome

There is a burgeoning repository of information available from ancient DNA that can be used to understand how genomes have evolved and to determine the genetic features that defined a particular species. To assess the functional consequences of changes to a genome, a variety of methods are needed to examine extinct DNA function. We isolated a transcriptional enhancer element from the genome of an extinct marsupial, the Tasmanian tiger (Thylacinus cynocephalus or thylacine), obtained from 100 year-old ethanol-fixed tissues from museum collections. We then examined the function of the enhancer in vivo. Using a transgenic approach, it was possible to resurrect DNA function in transgenic mice. The results demonstrate that the thylacine Col2A1 enhancer directed chondrocyte-specific expression in this extinct mammalian species in the same way as its orthologue does in mice. While other studies have examined extinct coding DNA function in vitro, this is the first example of the restoration of extinct non-coding DNA and examination of its function in vivo. Our method using transgenesis can be used to explore the function of regulatory and protein-coding sequences obtained from any extinct species in an in vivo model system, providing important insights into gene evolution and diversity.

Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies

Convergence of Heat Shock Protein 90 with Ubiquitin in Filamentous α-Synuclein Inclusions of α-Synucleinopathies

Single-cell immunohistochemical mutation load assay (SCIMLA) using human paraffin-embedded tissues.

It would be advantageous to measure mutation load in situ in order to determine the relationship between a high mutation load and increased risk for cancer or other diseases and to evaluate sources of possible mutagen exposure. Previously, in situ mutation detection assays have been plagued with multiple rounds of amplification and high rates of false-positives and false-negatives. The single cell immunohistochemical mutation load assay (SCIMLA) was developed to measure somatic mutation frequency, pattern, and spectrum in normal tissues with a single round of amplification. The P53 gene was utilized as a mutation reporter because of the unusual property that missense mutations often cause P53 protein to accumulate in the cell, allowing the mutant proteins to be detected by immunohistochemical staining. Alternative reporter genes with stabilized mutant proteins may be envisioned. Single cells that stain positively for P53 protein overabundance (red cells) were microdissected from ethanol-fixed and paraffin-embedded tissues. A novel stimulated-PCR (S-PCR) protocol permitted successful amplification of a 1.8-kb segment of the P53 gene (i.e., exons 5-9) in 87% of single mammary cells. Subsequent sequence analysis demonstrated that 35% of the amplified red-stained epithelial cells from normal breast tissue have missense mutations at evolutionarily conserved amino acids. Jackpot mutations, presumably due to clonal expansion, were common. False-positive missense mutations at conserved residues were observed in 3% of the clear cells (i.e., without red stain), presumably due to DNA polymerase error in early PCR cycles. The allele dropout rate was measured at 40% of the amplified cells. SCIMLA is applicable to a variety of tissues, utilizes a single amplification of an endogenous gene, displays mutant cells in situ, and may be adapted to other species.

An evaluation of tyramide signal amplification and archived fixed and frozen tissue in microarray gene expression analysis

Archival formalin-fixed, paraffin-embedded and ethanol-fixed tissues represent a potentially invaluable resource for gene expression analysis, as they are the most widely available material for studies of human disease. Little data are available evaluating whether RNA obtained from fixed (archival) tissues could produce reliable and reproducible microarray expression data. Here we compare the use of RNA isolated from human archival tissues fixed in ethanol and formalin to frozen tissue in cDNA microarray experiments. Since an additional factor that can limit the utility of archival tissue is the often small quantities available, we also evaluate the use of the tyramide signal amplification method (TSA), which allows the use of small amounts of RNA. Detailed analysis indicates that TSA provides a consistent and reproducible signal amplification method for cDNA microarray analysis, across both arrays and the genes tested. Analysis of this method also highlights the importance of performing non-linear channel normalization and dye switching. Furthermore, archived, fixed specimens can perform well, but not surprisingly, produce more variable results than frozen tissues. Consistent results are more easily obtainable using ethanol-fixed tissues, whereas formalin-fixed tissue does not typically provide a useful substrate for cDNA synthesis and labeling.

Immunohistochemical demonstration of S-phase cells by anti-bromodeoxyuridine monoclonal antibody in cattle tissues

Bromodeoxyuridine (BrdU), a non-radioactive thymidine analogue, was administered to 15 cattle at a dosage of 1-10 mg/kg intravenously or intraperitoneally to demonstrate S-phase cells in the tissues. The organs and tissues were fixed in 10% neutral buffered formalin or in 70% ethanol, sectioned, denatured with hydrochloric acid, and treated with monoclonal antibody against BrdU. Immunohistochemical methods were used to "visualize" BrdU-labelled nuclei. BrdU-positive cells were satisfactorily demonstrated in both formalin- and ethanol-fixed tissues of animals given doses of 2 mg/kg or over, by either route of administration. Large numbers of BrdU-positive cells indicative of active cell production were found in the basal region of the stratified squamous epithelium, the neck between gastric pits and gastric glands in the abomasum, and the crypts of Lieberkühn of the small and large intestines. Moderate numbers of positive cells were observed amongst inflammatory cells in cases of nephritis and in granulation tissue. Numerous positive cells were detected in leukaemia cells. The study showed that BrdU can be used to measure proliferative S-phase cells in cattle, as in human beings, mice and rats.

Correlation of fixation, HCL hydrolysis and proteolytic digestion in bromodeoxyuridine immunohistochemistry of human stomach labeled by extracorporial perfusion system.

Bromodeoxyuridine (BrdU) immunohistochemistry has recently been introduced for the visualization of DNA-synthesizing nuclei. To determine the optimal conditions for detecting BrdU-incorporated nuclear DNA in formalin- or ethanol-fixed, paraffin-embedded human gastric tissues, we tested several different pretreatment procedures of hydrolysis with HCl and enzymatic digestion with actinase before the immunohistochemical staining. Three cases of surgically resected stomachs were perfused with artificial blood containing BrdU and fixed in formalin, ethanol or with their combination for different durations. The results of BrdU-immunohistochemical stainings were checked and the labeling indices were compared. As for hydrolysis, treatment with 2N HCl at 25°C for 90min was sufficient to detect the immunoreactivity in the stomach irrespective of fixative conditions tested. However, to obtain the most suitable stainings an additional enzymatic digestion was needed, and the optimal condition with actinase was different according to the fixation time in formalin- and/or ethanol-fixed tissues. There are also a few differences in the demonstrability of nuclear BrdU among various kinds of cells, i.e. normal epithelial cells, immunoblasts in the lymphoid follicle and cancer cells. Our method could extend the range of application of BrdU immunohistochemistry for cell kinetic studies using human surgically resected organs.

Effects of fixation time and enzymatic digestion on immunohistochemical demonstration of bromodeoxyuridine in formalin-fixed, paraffin-embedded tissue.

We studied the effects of fixation time and enzymatic digestion on immunohistochemical staining for bromodeoxyuridine (BUdR) in excised rat and human gastrointestinal tissues and human brain tumors which had been fixed in formalin after intravenous administration of BUdR shortly before biopsy of tissue. In formalin-fixed rat gastrointestinal tissues not treated with proteinase, the reaction products were insufficient to identify BUdR-positive cells. Results similar to those in ethanol-fixed tissue were obtained when formalin-fixed tissue sections were treated with protease, pepsin, or trypsin. The longer the material had been fixed in formalin, the longer the incubation in proteinase required to identify BUdR-labeled nuclei. The BUdR labeling indices of formalin-fixed human brain tumor specimens treated with protease were comparable to those of ethanol-fixed tissues. Sufficient BUdR staining was obtained even in tissues fixed in formalin for prolonged periods. Therefore, the BUdR labeling index can be determined retrospectively in clinical materials stored in formalin.

The influence of protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and ethanol fixation.

The effect of three proteases--trypsin, pepsin, and pronase--on the immunohistochemical staining of keratins with a broad-spectrum monoclonal antibody was investigated in paraffin sections of formalin and ethanol-fixed tissues by means of the peroxidase-antiperoxidase method. Both the length of exposure to the fixative and the duration of proteolysis were varied over a wide range. Ethanol-fixed tissues showed excellent preservation of the antigenicity of keratins, and no appreciable differences in immunostaining related to the length of fixation were found. The use of proteolytic enzymes did not improve these results; on the contrary, it caused rapid tissue disintegration. Formalin-fixed epithelial tissues stained weakly or failed to stain unless they were treated with a proteolytic enzyme. The optimal length of proteolysis varied with the degree of fixation; tissues that were fixed for long periods of time in formalin required longer exposure to a proteolytic enzyme and were more resistant to digestion than were tissues that were fixed briefly. No significant advantage of one protease over another was found in this study. We conclude that a proteolytic step must precede immunostaining for keratins if the tissue is fixed in formalin, but that the digestion period must be adjusted according to the length of exposure to the fixative. The superiority of alcohol over formalin fixation for the preservation of the antigenicity of keratins is confirmed by this study.

Prolonged incubation time in immunohistochemistry: effects on fluorescence staining of immunoglobulins and epithelial components in ethanol- and formaldehyde-fixed paraffin-embedded tissues.

By prolonging the incubation time from 30 min to 20 hr at room temperature, fluorochrome conjugates may be applied at about ten times higher dilution and yet produce specific immunofluorescence staining of enhanced intensity. This modification of the direct method is important for reagent economy and, in addition, affords improved staining features for all antigens tested in formaldehyde-fixed tissues. It is particularly valuable when paired staining is used to characterize lymphoproliferative B-cell processes in pathological routine material; a clear-cut distinction between polyclonal and monoclonal expression of cytoplasmic immunoglobulin (Ig) is obtained, and cells giving rise to a false staining pattern are easily pinpointed. It is likewise advantageous to use prolonged incubation with conjugate for the localization of Ig-producing and Ig-bearing B cells in saline-extracted ethanol-fixed tissues, and the same holds true for Ig and C3 in immune-complex deposits. Also IgE on the surface of mast cells in tissues from atopic subjects is visualized distinctly with this modification. However, the localization or epithelial components is not consistently improved in ethanol-fixed tissues when the incubation time is prolonged; secretory products such as lactoferrin, lysozyme, amylase, and secretory component (SC) are not always immobilized sufficiently by ethanol fixation to avoid diffusion artifacts and a substantial loss from the cytoplasm. Differences in intracellular storage probably contribute to the variable antigen stability.

Comparison of conventional and immunofluorescent techniques for the detection of Entamoeba histolytica in rectal biopsies

Two comparative studies of six staining techniques in detection of Entamoeba histolytica in rectal biopsies were performed. The techniques evaluated were direct and indirect fluorescent antibody technique, and four conventional stains: PAS, H & E, Trichrome, and PTAH). On ethanol-fixed tissue, the direct fluorescent antibody technique was the most sensitive, detecting amoebas in 84% of rectal biopsies from 19 aspirate-positive dysentery patients. PAS staining was specific, almost as sensitive, and amoebas could be rapidly detected. The other three conventional methods, however, detected amoebas in only 53–58% of the aspirate-positive patients and required considerable efforts to find the amoebas. The indirect fluorescent antibody technique performed on ethanol-fixed tissues was comparable to the direct technique. The same technique used on formalin-fixed tissues had high background staining, but amoebas could still be detected more efficiently than with conventional staining. Following 20 hr or more treatment with metronidazole, no patient had amoebas detectable in their rectal biopsies by any technique used. Patients with amoebic dysentery and necrotic ulcers on proctoscopy had amoebas detected more frequently in rectal biopsy than did patients with mild disease as determined by proctoscopy. Entamoeba histolytica in rectal biopsies can be detected most easily in ethanol-fixed tissue stained with either a fluorescent antibody or with PAS. If amoebas are not detected in the rectal biopsy, amoebic colitis cannot be excluded.