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.     

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.     

Assessment of fixatives, fixation, and tissue processing on morphology and RNA integrity

Molecular characterization of morphologic change requires exquisite tissue morphology and RNA preservation; however, traditional fixatives usually result in fragmented RNA. To optimize molecular analyses on fixed tissues, we assessed morphologic and RNA integrity in rat liver when sections were fixed in 70% neutral-buffered formalin, modified Davidson's II, 70% ethanol, UMFIX, modified Carnoy's, modified methacarn, Bouin's, phosphate-buffered saline, or 30% sucrose. Each sample was subjected to standard or microwave fixation and standard or microwave processing, and sections were evaluated microscopically. RNA was extracted and assessed for preservation of quality and quantity. Modified methacarn, 70% ethanol, and modified Carnoy's solution each resulted in tissue morphology representing a reasonable alternative to formalin. Modified methacarn and UMFIX best preserved RNA quality. Neither microwave fixation nor processing affected RNA integrity relative to standard methods, although morphology was modestly improved. We conclude that modified methacarn, 70% ethanol, and modified Carnoy's solution provided acceptable preservation of tissue morphology and RNA quality using both standard and microwave fixation and processing methods. Of these three fixatives, modified methacarn provided the best results and can be considered a fixative of choice where tissue morphology and RNA integrity are being assessed in the same specimens.     

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.     

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.     

Immersion fixation methods for glycol methacrylate-embedded testes

Routine processing of testicular tissue through 10% neutral buffered formalin (NBF) into paraffin produces severe cellular shrinkage which obscures most of the morphologic detail. The following studies were performed to compare different combinations of immersion fixatives and embedding media for optimal cellular detail in the final histologic sections. We examined sections of testes from rats, mice, and rabbits fixed in either NBF, Bouin's, Zenker's, or Helly's fixatives, and embedded in either standard paraffin or glycol methacrylate. The results were similar in all 3 species. In paraffin, Bouin's or Helly's fixatives produced the fewest artifacts, while in glycol methacrylate, NBF-fixed tissue showed the greatest intracellular detail and preservation. The merits and limitations of each method are discussed for the rat, with exceptions for the other species noted where appropriate. The use of glycol methacrylate as the support medium for sectioning makes high-quality tissue sections available from formalin-fixed testes.     

Immunohistochemical demonstration of c-erbB-2 oncoprotein in gastric adenocarcinoma: comparison of cryostat and paraffin wax sections and effect of fixation.

AIMS--To investigate the effects of fixation on the immunohistochemical demonstration of c-erbB-2 oncoprotein using paraffin wax and cryostat sections; to compare c-erbB-2 expression in non-neoplastic and neoplastic gastric tissues. METHODS--Adjacent blocks of tumour and non-neoplastic tissue from four gastrectomy specimens were put into a panel of 10 fixatives including acetone, B5, Bouin's fluid, Carnoy's fluid, buffered formalin, formol dichromate, zinc formalin, 4% paraformaldehyde, periodate-lysine-paraformaldehyde (PLP) and periodate-lysine-paraformaldehyde-dichromate (PLPD) before embedding in paraffin wax for sectioning. Similar tissue blocks were snap frozen and cryostat sections were postfixed in these fixatives, either alone or in combination, before immunostaining. RESULTS--In paraffin wax embedded sections the best fixative was PLP, and in frozen tissues the best results were obtained after fixation of cryostat sections in buffered formalin followed by cold methanol and acetone. Applying these fixatives to samples from a further 16 gastrectomy specimens, strong membrane staining of c-erbB-2 protein was found in the tumour in eight of 16 cases (50%) using paraffin wax sections, and staining was stronger in the better differentiated carcinomas. For frozen tissues, positive membrane staining was found in all gastric adenocarcinomas, but differential staining intensity associated with tumour differentiation could not be detected. CONCLUSIONS--These results indicate that fixation and paraffin wax embedding affect the results of immunohistochemical demonstration of c-erbB-2 in gastric cancer. The choice of fixative is critical in the demonstration and evaluation of c-erbB-2 protein expression by immunohistochemistry in gastric carcinomas. Staining results also vary depending on whether frozen or paraffin wax embedded tissues are studied.     

Effects of several commonly used fixatives on DNA and total nuclear protein analysis by flow cytometry

Five commonly used fixatives (AZF, B-5, Bouin's, formalin, and Zenker's) were evaluated for their effect on the flow cytometric analysis of DNA and total nuclear protein (TNP) in solid tumors. Data were obtained with the use of colonic adenocarcinoma, squamous carcinoma of the lung, mammary adenocarcinoma, and spleen with a plasma cell leukemic infiltrate. The parameters examined were G0-G1 DNA staining intensity, %G0-G1, percent coefficient of variation (%CV), percent debris, and TNP staining intensity. The results showed that variations in the fixation of solid tumor significantly affected flow cytometric-derived parameters. In this study, paraffin-embedded tissue (PET) fixed in 10% (v/v) neutral buffered formalin (NBF) produced the best results, with a %CV below 4.7, whereas fixatives such as Zenker's and B-5 produced poor %CVs (above 6.0) or uninterpretable TNP and light scatter data. These data suggest that a portion of all tissue samples be fixed in NBF to allow for subsequent analysis by fixative-sensitive assays such as DNA in situ hybridization and flow cytometry.     

PCR amplification from paraffin-embedded tissues. Effects of fixative and fixation time.

The polymerase chain reaction (PCR) DNA amplification method is a powerful new tool for the retrospective analysis of paraffin-embedded tissue (PET). The technique has afforded the sensitive and specific detection of nucleic acid sequences associated with genetic and infectious diseases. However, PET processing conditions vary in their suitability for amplification. The authors have examined the effects of 11 fixatives at three fixation times. The effect of fixation was measured by the ability of the DNA in a treated tissue to serve as a template for the amplification of DNA fragments that ranged from 110 to 1,327 base pairs in length. Specimens fixed in acetone or 10% buffered neutral formalin were found to be best suited for subsequent analysis by PCR. A second group of fixatives, including Zamboni's, Clarke's, paraformaldehyde, formalin-alcohol-acetic acid, and methacarn, compromised amplification efficiency. Tissues treated with Carnoy's, Zenker's, or Bouin's, respectively, were even less desirable for amplification analysis.     

Comparison of formalin and Bouin's reagent for fixation of coagulase negative staphylococcal biofilm.

Methodological modifications, particularly the use of different fixatives, may account for discrepancies between studies of the relation between virulence and biofilm production in vitro by isolates of coagulase negative staphylococci. The efficacy of formalin and Bouin's reagent for fixing coagulase negative staphylococcal biofilms in a microtitre tray assay was compared. The optical density of stained adherent growth by three strains was reduced by an average of 20% following fixation with 10% formaldehyde compared with Bouin's reagent. This difference seemed to be mainly because of increased background staining and blackening of the biofilm when Bouin's reagent was used. Formalin fixation was also effective at identifying early and late biofilm production in adherence growth kinetic experiments with 10 coagulase negative staphylococcal clinical isolates.     

Modified formalin and methanol fixation methods for molecular biological and morphological analyses

Several simplified fixation methods were examined to determine their suitability for both molecular biological analyses and morphological study. Fixation with 10% v/v formalin alone at 4°C and containing 5 mmol/L ethylenediamine-N, N, N', N'-tetraacetic acld (EDTA) at room temperature preserved significantly more high-molecular-weight DNA than 10% v/v formalin fixation at room temperature. The morphological differences between tissues fixed using these modified formalin fixation methods and conventional 10% v/v formalin fixation were negligible. Of the dehydration fixatives tested, 100% methanol did not cause regional differences due to artificial tissue shrinkage and the morphology of sections prepared by methanol fixation was preserved consistently better than that of acetone- or ethanol-fixed sections. All three dehydration fixatives preserved relatively higher-molecular-weight DNA and RNA, compared with formalin. Cold formalin, formalin containing EDTA at room temperature and 100% methanol are recommended as standard and additional fixatives routine clinic pathological laboratory use.     

Immunohistochemical assays in prostatic biopsies processed in Bouin's fixative

AIMS: To investigate the problems involved in undertaking immunohistochemistry (IHC) and nuclear morphometry using Bouin's fixed prostate biopsies. METHODS: Archival Bouin's fixed and formalin fixed, paraffin wax embedded prostatic biopsies were immunostained for three nuclear biomarkers (minichromosome maintenance protein 2 (MCM-2), p27, and Ki-67), one membrane localised biomarker (C-erb-B2), CD34, and alpha methylacyl-CoA racemase (AMACR). The quality of IHC staining was compared between tissues prepared separately in both fixatives. Feulgen staining was also performed on Bouin's fixed tissues to check its suitability for nuclear morphometry. RESULTS: MCM-2 staining was completely negative in Bouin's fixed tissues, whereas p27 showed more background and excess cytoplasmic staining in Bouin's fixed versus formalin fixed tissues. C-erb-B2 showed non-specific, strong luminal cell staining in the Bouin's fixed tissue. Feulgen staining was also very weak in Bouin's fixed tissue. However, Ki-67, AMACR, and CD34 worked equally well in Bouin's and formalin fixed tissues. CONCLUSIONS: Bouin's fixed tissues may be unsuitable when subsequent IHC and morphometry are contemplated. An awareness of which antibodies are suitable for use in Bouin's fixed biopsies is essential.     

Morphology of the fetal rat testis preserved in different fixatives

Histopathological examination of the testes of exposed fetuses and neonates is important in assessing the developmental effects of environmental toxins, including sex hormone modulators. Modified Davidson's fluid (mDF) has been suggested as a superior substitute for Bouin's fluid for fixation of adult animal testes. We compared the morphology of fetal rat testes stained with hematoxylin and eosin (H&E) or immunochemically after fixation in 10% neutral buffered formalin (NBF), Bouin's fluid, or mDF. Fixation in mDF resulted in more sharply defined nuclear detail and better preservation of cellular cytoplasm on H&E-stained sections of rat testes on gestation day 19. Use of Bouin's fluid did not allow satisfactory detection of apoptotic cells by fluorescent terminal deoxynucleotide transferase-mediated deoxy-UTP nick labeling. Staining with the immunoperoxidase system and the conventional chromogen diaminobenzidine tetrahydrochloride to visualize 5-bromo-2-deoxyuridine-positive cells demonstrated that the number of positive nuclei and intensity of staining were similar with all 3 fixatives. Immunostaining for cytoskeletal protein vimentin was more intense and provided better details of the Sertoli cell cytoplasm with formalin fixation than with mDF. Our study demonstrates that fixation in mDF provided better morphologic detail in the fetal rat testis compared with 10% NBF and Bouin's fluid and illustrates the importance of establishing the correct fixation conditions for each immunostaining protocol.     

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.     

Optically clear nuclei in papillary carcinoma of the thyroid: demonstration of one of the fixation artifacts and its practical usefulness

To examine whether or not optically clear nuclei are one of the fixation artifacts in papillary carcinoma of the thyroid. Experiments using an optimal immersion fixation method by making thin-sliced specimens, 5 x 2 x 2 mm in size were adopted. Fixatives tested were 10% formalin, 10% buffered formalin, mixture of alcohol and formalin (4: 1), 95% alcohol, 4% phosphate-buffered paraformaldehyde, Carnoy's fixative, Bouin's fixative and various concentrations of formalin. Tested thyroid lesions in total were: cases with papillary carcinoma (38); follicular carcinoma (one); follicular adenoma (15); adenomatous goiter (11); Hashimoto's thyroiditis (two); Grave's disease (five); and normal thyroid tissue around tumor (five). Routinely processed papillary carcinoma showed a high occurrence (64.6%) of the optically clear nuclei contrasted by a low occurrence (almost 0%) in other lesions. However, this rate in papillary carcinoma decreased (< 35.3%) in all experimental groups irrespective of the choice of fixatives. Frequent occurrence (61.9-66.6%) of the optically clear nuclei was reproduced in papillary carcinoma fixed in a higher concentration of formalin (40-60%) in the experimental groups. These results demonstrated that the optically clear nuclei are one of the fixation artifacts, although still useful as a diagnostic criterion.     

Effects of fixation on polymerase chain reaction detection of Mycobacterium leprae.

The effects of standard fixatives (10% neutral buffered formalin, ethanol and mercury based) on the detection of Mycobacterium leprae DNA by the polymerase chain reaction (PCR) were studied. Mercury-based fixatives (Zenker's and Carnoy-Lebrun's fluids) strongly inhibited PCR amplification of M. leprae DNA. Ten percent neutral buffered formalin was inhibitory, but significant inhibition was observed only when fixation times exceeded 24 h. Ethanol-based fixatives provided the best medium for holding specimens for subsequent PCR with both free bacilli and skin biopsy specimens containing M. leprae. The M. leprae-specific, 360-bp region of the 18-kDa protein gene could be amplified from paraffin-embedded sections of formalin-fixed skin biopsy specimens from patients with either multibacillary or paucibacillary infections when proper fixation conditions were used. Results of the study demonstrate that tissues properly fixed with two standard fixatives (10% neutral buffered formalin and 50 or 70% ethanol) can be analyzed by PCR for the presence of M. leprae with no loss in specificity and only minimal diminution in sensitivity compared with the specificities and sensitivities obtained by use of freshly prepared, unfixed specimens.     

Better choice of fixatives provides better histological details of the alveolar-capillary interface

AIMS: To evaluate lung disease, pulmonary tissues should be fixed by inflation. However, many histological sections prepared after inflation fixation show wire-like alveolar septa with capillary collapse. We investigated the reason for this artefact. METHODS: To evaluate the effect of fixatives, we used the following commercially available solutions: regular 10% neutral buffered formalin (NBF), 20% NBF, 10% and 5% formalin prepared by diluting the 20% NBF, modified formalin solution as a substitute for 10% NBF, and 10% formalin prepared by diluting the 100% formalin without any buffers. RESULTS: The osmolarity of the fixative was found to be responsible for the collapse artefact. Ten per cent formalin, prepared by diluting 100% formalin, the commercially available substitute for 10% NBF, and 5% formalin prepared by diluting 20% NBF, yielded the best pulmonary tissue morphology, including that of the alveolar-capillary interface. CONCLUSIONS: Pulmonary physicians and pulmonary pathologists should use a suitable fixative solution for obtaining a better pulmonary architecture as well as to preserve the tissue block in optimal condition for future assessment of pulmonary diseases.     

Variable fixation of staphylococcal slime by different histochemical fixatives

A variety of histochemical fixatives were used to compare the fixation of bacterial films produced by a standard slime-producing strain ofStaphylococcus epidermidis on plastic tissue culture plates. Some reagents were completely ineffective in fixing the slime layer, whereas others gave variable results. The best alternative to the fixative of the reference method, the potentially explosive Bouin's reagent, was air drying.     

Human intestinal mucosal mast cells: evaluation of fixation and staining techniques

The staining properties of tissue mast cells are influenced by the method of fixation. Differences in fixation and staining techniques may explain the contradictory results in the published reports on the number of human mucosal mast cells (MMC) in the gastrointestinal mucosa in health and disease. We have examined the influence of fixatives on the staining properties of human MMC in operative biopsy specimens of human jejunum. Specimens were divided into pieces, each of which was fixed in one of the following fixatives: Carnoy's, basic lead acetate (BLA), Baker's, Bouin's, isotonic formol-acetic-acid (IFAA), 10% neutral buffered formalin, formol sublimate, and formol saline. Thereafter, tissues were paraffin-embedded and 5 micron sections were cut and stained with either astra-blue/safranin pH 0.3, or toluidine blue pH 0.5. Counts of the number of MMC/mm2 were obtained for each fixation method. The results show a critical influence of the fixative on the number of mast cells identified after staining. For example with astra-blue/safranin the mean MMC/mm2 count was 40 in formol-saline-fixed specimens, and 268 in Carnoy's-fixed specimens. In biopsies fixed with formalin-based fixatives, mast cells were more readily stained with toluidine blue. It is recommended that Carnoy's or BLA be used as the fixative for any light microscopic study of human MMC.     

Intracellular myoglobin--a specific marker for skeletal muscle differentiation in soft tissue sarcomas. An immunoperoxidase study.

Intracellular myoglobin represents an excellent marker for specific characterization of normal (adult and fetal) and malignant skeletal muscle cells in paraffin sections. With an immunoperoxidase indirect sandwich technique for detection of intracellular myoglobin, positive staining was observed in 13 of 17 rhabdomyosarcoma specimens including 5 of 7 of the alveolar type, 5 of 5 of the embryonal type, and 3 of 5 of the pleomorphic type. Initial fixation in Zenker's-acetic acid solution gave optimal staining, but satisfactory results were obtained with fixation in formalin, Bouin's, and B5 solutions. Other types of sarcomas (13 cases) and other types of tumors (24 cases) that sometimes mimic rhabdomyosarcoma on histologic examination gave negative results. The immunoperoxidase method affords a sensitive and specific method for identifying rhabdomyoblasts in tissue on the basis of intracellular myoglobin and is of use in distinguishing rhabdomyosarcomas from other sarcomas and from malignant tumors of other types.