Alcohol Fixation Research Articles

Efficient Single Muscle Fiber Isolation from Alcohol-Fixed Adult Muscle following β-Galactosidase Staining for Satellite Cell Detection

Staining for β-galactosidase activity for whole tissues, sections, and cells is a common method to detect expression of β-galactosidase reporter transgene as well as senescence-dependent β-galactosidase activity. Choice of fixatives is a critical step for detection of β-galactosidase activity, subsequent immunostaining, and enzymatic digestion of tissue to dissociate cells. In this report, the authors examined several aldehyde and alcohol fixatives in mouse skeletal muscle tissues for their efficiency at improving detection of β-galactosidase activity as well as detection by immunostaining. In addition, fixatives were also analyzed for their efficiency for collagenase digestion to isolate single muscle fibers on postfixed β-galactosidase-stained whole skeletal muscle tissues. The results show that fixing cells with isopropanol yields the greatest reliability and intensity in both β-galactosidase staining as well as double staining for β-galactosidase activity and antibodies. In addition, isopropanol and ethanol, but not glutaraldehyde or paraformaldehyde, allow for the isolation of single muscle fibers from the diaphragm and tibialis anterior muscles following postfixed β-galactosidase staining. Using this method, it is possible to identify the amount of cells that occupy the satellite cell compartment in single muscle fibers prepared from any muscle tissues, including tibialis anterior muscle and diaphragm.

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.

Kitchen Microwave-Assisted Accelerated Method for Fixation and Processing of Oral Mucosal Biopsies: A Pilot Study

ABSTRACT Background and aim Conventional protocols of formalin fixation and processing add time and cost to histoprocessing. This study aims to evaluate an indigenously developed protocol for microwave histoprocessing and compare it with the routine protocol for histoprocessing. Materials and methods A total of 40 human buccal mucosal specimens were processed according to the microwave and the routine protocol. Sections obtained were coded and evaluated for the following: Section quality, staining quality, artifacts, cellular details in epithelium, nuclear detail and diagnostic ability. Parameters were graded as suboptimal, optimal and good, and results were statistically analyzed. Results The number of good and optimal specimens in both the protocols were almost similar for all parameters assessed with better results for cellular detail, nuclear detail, staining and sectioning quality seen in microwave-fixed samples. Artefacts were lesser in routine processing protocol. Conclusions Microwave alcohol fixation protocol provides good and faster fixation compared to routine fixation.

Burkitt's lymphoma: maximising the use of fine needle aspirates by long-term preservation for diagnosis and research

Fine needle aspirates from Burkitt's lymphoma and other tumours transferred directly into ThinPrep® PreservCyt® (Cytyc UK Ltd, Crawley, UK) buffered alcohol fixative retain their cellular and viral antigens and nucleic acids for many months at ambient temperatures. Despite the presence of blood and debris, cells dried onto slides from droplets and post-fixed in formalin, or sections of paraffin-embedded cell blocks from formalin post-fixed pellets, prove adequate for morphology, immunocytochemistry, in-situ hybridization and molecular biological analyses. Where there is lack of expertise in making thin smears or hospitals lack pathology laboratories and services, PreservCyt® provides an excellent medium for transport elsewhere for diagnosis and research.

The search for an optimal DNA, RNA, and protein detection by in situ hybridization, immunohistochemistry, and solution-based methods

Clinical trials and correlative laboratory research are increasingly reliant upon archived paraffin-embedded samples. Therefore, the proper processing of biological samples is an important step to sample preservation and for downstream analyses like the detection of a wide variety of targets including micro RNA, DNA and proteins. This paper analyzed the question whether routine fixation of cells and tissues in 10% buffered formalin is optimal for in situ and solution phase analyses by comparing this fixative to a variety of cross linking and alcohol (denaturing) fixatives. We examined the ability of nine commonly used fixative regimens to preserve cell morphology and DNA/RNA/protein quality for these applications. Epstein–Barr virus (EBV) and bovine papillomavirus (BPV)-infected tissues and cells were used as our model systems. Our evaluation showed that the optimal fixative in cell preparations for molecular hybridization techniques was “gentle” fixative with a cross-linker such as paraformaldehyde or a short incubation in 10% buffered formalin. The optimal fixatives for tissue were either paraformaldehyde or low concentration of formalin (5% of formalin). Methanol was the best of the non cross-linking fixatives for in situ hybridization and immunohistochemistry. For PCR-based detection of DNA or RNA, some denaturing fixatives like acetone and methanol as well as “gentle” cross-linking fixatives like paraformaldehyde out-performed other fixatives. Long term fixation was not proposed for DNA/RNA-based assays. The typical long-term fixation of cells and tissues in 10% buffered formalin is not optimal for combined analyses by in situ hybridization, immunohistochemistry, or – if one does not have unfixed tissues – solution phase PCR. Rather, we recommend short term less intense cross linking fixation if one wishes to use the same cells/tissue for in situ hybridization, immunohistochemistry, and solution phase PCR.

Alcohol Fixation of Induced Sputum Samples for Applications in Rural Communities

Sputum induction is a tool recommended for the assessment of airway inflammation and disease management. Currently, its use is limited because samples need to be processed within 3 h of induction (ie, while cells are viable); therefore, this procedure is unavailable to most clinicians. To develop a fixation method for induced sputum samples that allows for a delay in processing while maintaining sample integrity and not altering the standard processing method. Sputum samples were collected and split into three portions: a fresh sample processed using the routine method (within 3 h, using dithiothreitol); fixation in alcohol followed by delayed processing using the routine method (within 48 h to 72 h, using dithiothreitol); and fixation in formaldehyde followed by delayed processing using an alternative method (within 48 h to 72 h, using proteolysis). For each method, cytospins were prepared and differential cell counts were performed. Fixation in alcohol provides accurate measures of eosinophils and macrophages, but not neutrophils. Formaldehyde fixation provides accurate measures of neutrophils and macrophages, but not eosinophils. Alcohol fixation is a superior method for eosinophil quantification. It requires alteration of standardized methods for sputum sample processing and should be recommended for monitoring eosinophilic airway disease in settings where immediate processing of a sputum sample is not possible.

Silicon nitride as a versatile growth substrate for microspectroscopic imaging and mapping of individual cells

Herein is described a general sampling protocol that includes culture, differentiation and fixing of cells in their preferred morphology on the one sample substrate (Si(3)N(4)) to enable subsequent diverse modern microspectroscopic analyses. The protocol enables unprecedented correlated and complementary information on the intracellular biochemistry of metabolic processes, diseases and their treatment, which offers the opportunity to revolutionize our understanding of cell and tissue biology at a molecular level. The culture of adherent cells onto inexpensive Si(3)N(4) membranes allows microspectroscopic analyses across the electromagnetic spectrum, from hard X-ray fluorescence (both XRF and XANES), through to visible and fluorescence light microscopies, and infrared microspectroscopy without substrate interference. Adherent mammalian cell lines (3T3-L1 adipocytes and H9c2 cardiac myocytes) illustrate the in vitro application of these protocols. The cells adhered strongly to Si(3)N(4) membranes and visually displayed normal proliferative and phenotypic growth; more importantly, rapid alcohol fixation of cells did not affect their structural integrity for subsequent analyses.

Poor pregnancy outcome following assisted reproductive technology among women operated on for bilateral endometriomas

To determine the first line of infertility treatment for managing patients with unilateral or bilateral ovarian endometriomas. We evaluated pregnancy outcome in patients who had received ovarian surgery for unilateral (Group U, n = 47) or bilateral endometriomas (Group B, n = 38) and aspiration with or without alcohol fixation for unilateral (Group u, n = 37) or bilateral endometriomas (Group b, n = 22). Subsequently, 64 of these women, excluding 29 dropouts, underwent assisted reproductive technology. We compared the clinical pregnancy rates of the four groups. The cumulative pregnancy rate after operation of Group B (18%) was significantly lower than that of a cyst-free control group (n = 143; 44%) and Group U (43%). Group B had fewer oocytes fertilized during ART than did Group b (P < 0.005) and fewer blastocysts available for transfer (P < 0.005). The cumulative pregnancy rate of Group B was also lower than in Group b (P = 0.052). Pregnancy outcomes of Group B were not better than for Group b. Therefore, encouraging such women to move directly to ART might help avoid ovarian damage and improve their ability to achieve a pregnancy.

Managing chocolate cysts before OPU for in vitro fertilization: prophylaxis for better outcome

OBJECTIVE: Ovarian endometriosis occurs in 50% of women with endometriosis, and the need to treat it prior to IVF is controversial. The objective of this study was to evaluate the impact of aspiration and alcohol fixation of chocolate cysts prior to IVF treatment on the outcome.

Immunohistochemical detection of estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression in breast carcinomas

Fine-needle aspiration (FNA) is a rapid and accurate procedure for the detection of breast carcinomas. The evaluation of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression by immunohistochemistry (IHC) is performed routinely on formalin-fixed, paraffin-embedded needle-core (NC) or excision tissue block (TB) preparations, according to the American Society of Clinical Oncology/College of American Pathologist guidelines. In this retrospective study, the authors compared expression levels of ER, PR, and HER2 in ethanol-fixed BC FNA cell block (CB) samples with expression levels in formalin-fixed NC and TB samples. Forty-one breast carcinoma CB samples with concurrent or subsequent NC and TB samples were identified. Patients who had received neoadjuvant or adjuvant chemotherapy were excluded. CB samples initially were fixed in 50% ethanol (4-12 hours), and this was followed by formalin fixation (minimum, 6 hours). NC samples were placed promptly in formalin for a minimum of 6 hours. Within 4 to 8 hours, TB samples were fixed in formalin for 6 to 48 hours. Fluorescence in situ hybridization (FISH) results were also compared. IHC for ER on alcohol-fixed CB samples had good correlation with NC and TB samples. PR results on TB samples had excellent agreement with NC samples. A higher discordance rate wais observed when PR results were compared between CB samples and NC samples. HER2 detection on ethanol-fixed CB samples resulted in a higher rate of positive and equivocal staining than NC or TB samples. HER2 IHC on TB samples demonstrated better correlation with FISH results than CB or NC samples. Alcohol fixation did not affect ER results in breast carcinoma, but it may alter tumor cell PR antigenicity. The authors concluded that CB samples could be used to triage patients for tamoxifen therapy, but they are not reliable for the assessment of HER2 status; therefore, CB results should be correlated with results from NC or TB samples.

Histology without formalin?

Because formalin is toxic, carcinogenic, and a poor preserver of nucleic acids, for more than 20 years, there have been numerous attempts to find a substitute, with as many different alternative fixatives, none totally successful. With a fast penetration, formaldehyde is a slow and reversible fixative that requires 24 to 48 hours to completely bind to tissue; thus, any surgical specimen arriving to the laboratory between 8 AM and 4 PM and processed conventionally for the slides to be ready the following day will be only between 30% and 66% bound and even less fixed when the dehydration starts, resulting in an additional and also incomplete alcoholic fixation. This causes infiltration problems and can affect subsequent tests, especially immunohistochemistry. Formaldehyde fixation is tissue thickness independent between 16 microm and 4 mm but is faster at above room temperature, so the fixation of specimens with less than 24 hours in formalin can be improved if the fixing stations in the conventional tissue processors are set at 40 degrees C. If the safety measures are improved to offer a work environment with a time weighted average level of 0.4 ppm, and the contact with formalin is reduced to a minimum by discouraging its neutralization and limiting the recycling practice to filtering methods, formalin could remain as the routine fixative, with modified methacarn for those specimens requiring nucleic acids studies. This is a preferred solution than having to validate all the standard and special procedures, including those US Food and Drug Administration approved, if formalin is replaced by another fixative without its advantages. To the question posed in the title of this article, the answer is "Yes, it can be done, but that is neither likely nor worth it!"

Predicting the viability of liver allografts procured from non-heart-beating donors: The role of histopathology

Predicting the viability of liver allografts procured from non-heart-beating donors: The role of histopathology

Comparison of Carnoy’s Solution and 96% Ethyl Alcohol Fixation in Bloody Pap Smears

To compare 2 methods of fixation in bloody Pap smears with Carnoy's solution and 96% ethyl alcohol.After observation of contact bleeding, 2 samples were prepared from cervical cells with conventional Pap smear. One sample was fixed in 96% ethyl alcohol and another sample was fixed in Carnoy's solution.Of 450 slides, 410 were selected for study. In study of cell adequacy, diagnosis of squamous cells and glandular cells was better in Carnoy's-fixed slides. Blood contamination of slides was reduced in Carnoy's-fixed slides (13.85% vs. 49.51%), and clearance of slides was increased in Carnoy's-fixed slides. Diagnosis of inflammatory cells and pathogenic microorganisms in was increased in Carnoy's-fixed slides, but no difference was seen in diagnosis of epithelial cell and glandular cell abnormalities.Carnoy's solution can be used as an effective fixative in bloody smears in conventional Pap tests.

Immunocytochemistry for hepatocyte nuclear factor‐1β (HNF‐1β): A marker for ovarian clear cell carcinoma

Recent microarray studies have shown that the expression of hepatocyte nuclear factor-1beta (HNF-1beta) was significantly up-regulated in clear cell carcinoma (CCC) of the ovary. HNF-1beta may be a useful marker for CCC in peritoneal fluid cytology. We designed an experimental study using three CCC cell lines to evaluate the influence of alcohol fixation or air drying on immunocytochemistry for HNF-1beta. Each cell line was cultured on chamber slides or transplanted into the abdominal cavity of nude mice, then the slides or ascites smears of nude mice were immunostained with or without microwave-mediated epitope retrieval. Immunoreactivity with HNF-1beta, which was either alcohol-fixed or air-dried, was noticeably improved after microwave heating. In contrast, two serous adenocarcinoma cell lines never showed immunoreactivity. Based on these results, 21 archival Papanicolaou-stained slides of peritoneal fluid (5 CCCs, 12 serous, 2 mucinous, and 2 endometrioid adenocarcinomas) were decolorized and immunostained under heating pretreatment. Five of 5 CCCs were distinctively positive for HNF-1beta, whereas all non-CCC ovarian cancers were negative for this protein. The present study clearly demonstrated that heating epitope retrieval improved the immunoreactivity for a nuclear protein in alcohol-fixed or air-dried cytology specimens. HNF-1beta is likely to be helpful for the diagnosis of CCC in the peritoneal fluid.

Primary Intraocular Lymphoma: Improving the Diagnostic Procedure

To analyze the clinical features of primary intraocular lymphoma (PIOL) and to describe cytochemical and immunocytochemical findings of the vitreous specimens as well as the reasons for delayed diagnosis of PIOL. Prospective noncomparative study. Eleven patients referred to the uveitis or medical retina units, Department of Ophthalmology, University of Helsinki, were diagnosed as having PIOL between 2000 and 2005. The median follow-up of the patients was 32 months. Clinical features and diagnostic workup of uveitis were described. Twelve vitrectomies were performed on 9 patients. The first 5 biopsies were fixed in an equal volume of 50% alcohol. The specimens of the next 7 vitrectomies were handled without alcohol, and tissue culture medium was added to the samples. Clinical features of PIOL, intervals from ocular symptoms and from first ophthalmological examination to diagnosis, and the role of a proper handling of the vitreous sample in the diagnosis of PIOL. Six females (54%) and 5 males (46%) (median age, 61 years) were included. Ten patients had ocular symptoms for 1 to 30 months (median, 8) before the first contact with an ophthalmologist. Uveitis was bilateral in 9 patients. Vitreitis was seen in all patients, and it was severe in 8. Fundus lesions dominated in 3 patients. Six patients lost useful vision in one eye before the diagnosis of PIOL. Cytologic and immunohistochemical stainings prepared of the unfixed vitreous specimens showed PIOL in 6 patients. The samples fixed in alcohol were nondiagnostic in 4 patients, and in them, verification of diagnosis was based on brain biopsy (3) or cerebrospinal fluid (1) findings. Seven patients died due to primary nervous system lymphoma. Diagnosis of PIOL is difficult but can be improved. Severe bilateral vitreitis in an elderly patient is a characteristic finding of PIOL. Alcohol fixation may jeopardize the identification of PIOL cells in the vitreous sample. Optimal handling of the vitreous specimens and examination of the slides by an experienced cytopathologist are critical in the diagnostic workup of PIOL.

Preservation of Tissue Urate Crystals With the Use of a Rapid Tissue-Processing System

The recognition of crystals in routinely processed hematoxylin and eosin-stained tissue sections is a problem familiar to all histologists and histopathologists. Unless special care is taken to fix specimens containing urate crystals with alcohol fixatives, followed by special processing, the crystals are dissolved, and examination of histologic sections of the suspected crystals with polarized light is unrewarding. With the development of a microwave-assisted, rapid throughput tissue-processing system, combined with routine use of an alcohol-based molecular-friendly fixative, we are now able to detect urate crystals in routinely processed tissue sections, including those previously fixed in formalin. Two representative cases are illustrated. (The J Histotechnol 29:17, 2006)Submitted: July 5, 2004; accepted with revisions December 23, 2005

Alcoholic fixation of blood to surgical instruments—a possible factor in the surgical transmission of CJD?

While developing a new protein removal test for the quality control of surgical instrument cleaning, it was noted that alcohol firmly binds blood to stainless steel. Creutzfeldt–Jacob disease is one of the transmissible spongiform encephalopathies (TSE) that has been transmitted between humans and chimpanzees by electroencephalogram electrodes, previously ‘sterilized’ using ethanol and formaldehyde. Although ethanol has a bactericidal action, it also binds protein to metal. Prion proteins found in TSE are thought to be the causal agents of spongiform disease and it is likely that these proteins are also bound to the stainless steel of surgical instruments by alcohols. Where spongiform disease is a possibility, alcohol, and probably formaldehyde, should not be used to decontaminate neurosurgical instruments.

Bemisia tabaci

<i>Bemisia tabaci</i>

Can PAP staining of air dried cervical scrape smears replace the traditional wet fixed smears?

An alternative to ‘air drying’ artifacts seen in improperly wet fixed cervico-vaginal smears would be total elimination of fixation in alcohol and allowing the smears to dry in air. The primary objective of this study was to evaluate and compare the cell morphology in rehydrated air-dried smears (ADS) and the conventional wet fixed smears (WFS) and also to study the effect of postponing rehydration of smears. Three hundred and fifty cervical scrape smears were prepared from material collected from 10O patients. First smear from every patient (100) was wet fixed in 95% ethanol. The remaining smears (250) were air-dried and were divided in two groups. Group 1 included 100 ADS that were stained by Papanicolaou staining within three to four hours of collection. Group II included 150 ADS stained after 24 (50), 48 (50) and 72 (50) hours, to see the effect of postponing rehydration of ADS. Rehydration of ADS was done in normal saline for 20-30 seconds, followed by post fixation in 5% glacial acetic acid in 95% ethanol Using a scoring method the cytomorphology seen in ADS was compared with that of WFS for effect on background blood and mucus, nuclear chromatin, nucleolus, cytoplasmic details, overall ease of interpretation of the smear and degree of air drying artifact. The cytomorphology in ADS stained within 3-4 hours was comparable if not superior to that seen in WFS, as shown by the p values that were highly significant for inter-observer agreement and also comparable with the p values of WFS for all the parameters. In Group II cases the details of nuclear chromatin, nucleolus, differentiation of cytoplasm and restoration of its transparency were gradually and significantly lost with postponement of rehydration. However p value for overall ease of interpretation of cytomorphology was significant except for the smears rehydrated at 72 hours. Overall the smears showed no significant drying artifact even when rehydrated at 72 hours. Additional advantages like, reduction in the demand of alcohol and ease of preparing and transport of smears, can promote collection of Pap smears in more number of women especially in the rural areas.

Pretreatment for Ovarian Endometrial Cyst before in vitro Fertilization

Assisted reproductive technology is a widely accepted treatment for infertile women with endometriosis. The presence of an ovarian endometrial cyst reduces the quality of oocytes, while surgical resection of endometrioma may reduce the ovarian reserve for ovarian stimulation by exogenous gonadotropins. To determine what pretreatment should be performed for ovarian endometrial cyst before IVF-ET, we analyzed IVF outcomes with or without pretreatment in patients with endometrioma. Infertile women with endometrioma who underwent IVF-ET were divided into 3 groups, including patients who had received laparotomy or laparoscopy, patients for whom the endometrioma content had been aspirated and treated with or without alcohol fixation, and patients who did not undergo pretreatment. The number of retrieved oocytes, rate of mature oocytes, and fertilization rate were compared among groups. The results showed that pretreatment for endometrioma reduces the number of retrieved oocytes. Although oocyte quality as a rate of mature oocytes was not affected by the presence of an ovarian endometrial cyst, the fertilization rate was improved by cyst aspiration. We propose that surgical pretreatment is not necessary for ovarian endometrial cyst before IVF-ET, but cyst aspiration may be beneficial after several failed attempts of IVF.