Fixation In Buffered Formalin Research Articles

The particularities of connective fibers from the wall of varicose veins extirpated by cryostripping.

The varicose vein affects more than 30% of the general population. Significantly increased rates were noticed in women and older population. From the histopathological point of view, venous arterialization, smooth muscle cell hypertrophy, and hyperplasia are the main changes noticed in varicose vein disease. Some of the main therapeutic methods used in the management of varicose disease are injection sclerotherapy, conservative, surgical, saphenous vein inversion and removal, high saphenous ligation, ambulatory phlebectomy, transilluminated powered phlebectomy, endovascular management, cryostripping. The aim of this study was to evaluate the morphology of connective fibers from the wall of the varicose veins extirpated by cryostripping. The study included 109 samples taken by cryostripping method. Hematoxylin-Eosin, Masson's trichrome, Silver and Orcein staining were applied. The assessment of fibers was made according to score values between 0 and 3. It was found no major structural differences in terms of alterations of collagen fibers induced by the applied surgical procedure. It was noticed duplications and multiplications of the internal elastic lamina, as diffuse and nodular forms. Depletion of elastic fibers at the media was a lesion identified in most of the specimens. The depletion of reticulin fibers correlates with the accumulation of collagen fibers, which partially or completely replace the network in the media and intima. No correlation was found between changes in the reticulin network and the time between prelevation and buffered formalin fixation, the maximum time investigated being five days. The Orcein staining in the venous vessel evaluation panel may be a useful investigation.

A comparison of fixation and immunofluorescence protocols for successful reproducibility and improved signal in human left ventricle cardiac tissue.

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.

Comparison of 10% Buffered Formalin Fixation and Carnoy's Solution in Revealing the Axillary Lymph Node Counts in Modified Radical Mastectomy Specimens

Comparison of 10% Buffered Formalin Fixation and Carnoy's Solution in Revealing the Axillary Lymph Node Counts in Modified Radical Mastectomy Specimens

Volume of 4% neutral buffered formaldehyde affects the results of HER2 gene detected by fluorescence in situ hybridization in primary invasive breast cancer

To investigate the effects of the volume of 4% neutral phosphate buffered formalin fixative solution on the detection of human epidermal growth factor receptor 2 (HER2) gene by fluorescence in situ hybridization (FISH) in primary invasive breast cancer. Tissue samples were collected from 109 patients with primary invasive breast cancer admitted in Zhongshan Boai Hospital from June 2014 to October 2016. The ratios of 4% phosphate buffered formalin fixative solution to sample volume samples were 3:1, 6:1, 9:1, 10:1, 15:1, 20:1 or 25:1 (groups A, B, C, D, E, F and G), respectively. Paraffin sections were made after 15 h of fixation. The amplification of HER2 gene was detected by FISH. The gene amplification results of HER2 were observed and compared in different groups. Fluorescence microscope showed that the tissue contour in groups A, B and C was vague, cell debris appeared, and the probe was positioned poorly; while the tissue contour was clear and complete in groups D, E, F and G and the probe was positioned accurately. The positive rate of HER2 was gradually increased from group A to D(χ2=8.601, P<0.01), and that remained stable at 24.77% in groups D to G. The positive rate of gene amplification in groups D, E, F and G was significantly higher than that in groups A, B and C (all P<0.05). When using FISH to detect HER2 gene in samples of primary breast invasive carcinoma, the volume of fixative solution should be at least 10 times of the sample volume to obtain accurate and stable results.

First detection and genotyping of Giardia intestinalis in stool samples collected from children in Ghazni Province, eastern Afghanistan and evaluation of the PCR assay in formalin-fixed specimens

It is estimated that faecal-orally transmitted diseases are common in Afghanistan, as a consequence of poor hygienic standards of life and widespread contamination of water and food with both human and animal faeces. However, there is little information in the literature concerning infections caused by intestinal parasites in the Afghan population. In this study, we report the occurrence of Giardia intestinalis assemblages (A and B) in formalin-fixed stool samples collected from 245 Afghan schoolchildren living in Ghazni Province in eastern Afghanistan. Detection of the parasite’s DNA and genotyping was performed using real-time PCR, specific to the β-giardin gene of G. intestinalis. Positive results were recorded in 52 (21.2%) samples. Genotyping was successful in 39 faecal samples and showed the predominance of assemblage B of G. intestinalis in this population (15 assemblage A and 24 assemblage B). Co-infection with both genotypes A and B was detected in four samples. Additionally, we evaluated the effect of 10% buffered formalin fixative on the detection of G. intestinalis DNA using real-time PCR and nested PCR characterised by different lengths of PCR products (74 and 479 bp, respectively). The human faeces containing the Giardia cysts were tested for 16 weeks. Amplification of G. intestinalis DNA with real-time PCR was possible up to 6 weeks of preservation of stool sample in formalin, compared to only 2 weeks with nested PCR. This suggests that real-time PCR is a more suitable tool in cases where stool samples have to be kept in formalin for longer periods of time.

Presentation of Neurolytic Effect of 10% Lidocaine after Perineural Ultrasound Guided Injection of a Canine Sciatic Nerve: A Pilot Study.

BackgroundPhenol and alcohol have been used to ablate nerves to treat pain but are not specific for nerves and can damage surrounding soft tissue. Lidocaine at concentrations > 8% injected intrathecal in the animal model has been shown to be neurotoxic. Tests the hypothesis that 10% lidocaine is neurolytic after a peri-neural blockade in an ex vivo experiment on the canine sciatic nerve.MethodsUnder ultrasound, one canine sciatic nerve was injected peri-neurally with 10 cc saline and another with 10 cc of 10% lidocaine. After 20 minutes, the sciatic nerve was dissected with gross inspection. A 3 cm segment was excised and preserved in 10% buffered formalin fixative solution. Both samples underwent progressive dehydration and infusion of paraffin after which they were placed on paraffin blocks. The sections were cut at 4 µm and stained with hemoxylin and eosin. Microscopic review was performed by a pathologist from Henry Ford Hospital who was blinded to which experimental group each sample was in.ResultsThe lidocaine injected nerve demonstrated loss of gross architecture on visual inspection while the saline injected nerve did not. No gross changes were seen in the surrounding soft tissue seen in either group. The lidocaine injected sample showed basophilic degeneration with marked cytoplasmic vacuolation in the nerve fibers with separation of individual fibers and endoneurial edema. The saline injected sample showed normal neural tissue.ConclusionsTen percent lidocaine causes rapid neurolytic changes with ultrasound guided peri-neural injection. The study was limited by only a single nerve being tested with acute exposure.

Inflammatory Bowel Disease: Anatomical and Histopathological Study

Worldwide, the frequency of chronic inflammatory bowel disease (CIBD), including ulcerative colitis (UC) and Crohn's disease (CD) increased rapidly through the last century making their histopathological diagnosis daily task for all pathologists. The accurate histopathological diagnosis of UC and CD requires an intimate correlations between the clinical, laboratory and endoscopic findings in conjunction to the suitable obtained biopsy material, as there are several diagnostic confusions between CIBD and non CIBD forms. The aim of this study was to focus and analyze certain practically selected histopathological features for the diagnosis of UC and CD including crypt architectural abnormalities, inflammation and an associated dysplasia or adenocarcinoma. This present study was performed on 100 specimens of tissue samples taken from small and large bowel including 3 ileocecal resections which were done for suspected an IBD. All the tissue samples received fixed in 10% neutral buffered formalin fixative solution, and paraffin-embedded tissue sections were prepared and examined microscopically for the histopathological features which distinguishing CIBD from non CIBD colitis. This study revealed 95 cases CIBD, among them 55 cases CD and 40 cases were UC, whereas the remaining 5 cases were non CIBD colitis. This study concluded that the diagnosis of IBD needs interdepartmental correlations to avoid under- or over-diagnosis. As well as the accurate discrimination of CIBD from non CIBD forms, differentiate UC from CD, and to identify any dysplastic lesions which is the backbone for therapeutic and prognostic strategies.

Optimizing breast cancer diagnosis in Kenya: Importance of standardization of technical methodologies for comparative breast cancer data.

139 Background: An analysis of 322 cases referred to Aga Khan University, Nairobi, revealed 56% estrogen receptor (ER) positive tumors and 35% prevalence of triple-negative breast cancer (TNBC). Findings were retrospective and limited by inability to control pre-analytical variables that could potentially impact results. Methods: As part of an ongoing prospective study assessing prevalence of TNBC in the three major ethnic groups in Kenya, we gathered a multidisciplinary team from 10 collaborating health facilities around Kenya for an educational workshop. The objectives were to assess baseline capabilities and pre-analytic variables at each center, identify gaps and provide hands-on training in order to ensure accuracy and validity of ER/PR/HER2 prevalence data gathered as part of the study. Results: See table. Breast cancer biopsies ranged from one to 20 per month per center. Diagnosis was predominantly by FNA and ER/PR/HER2 was not routinely performed. Buffered formalin fixative and standardized CAP reporting format was employed only at one center. A survey 3 months following the workshop demonstrated increase in diagnostic core biopsiesby 90%, and uniform use of buffered formalin fixative, and adoption of synoptic reporting. 66 prospective cases of breast cancer from the 10 institutions with patients from different ethnic backgrounds have been subsequently collected and IHC data will be presented. Conclusions: Much has been made of the difference in prevalence of TNBC in Africa as compared to North America, yet little attention has been paid to differences in diagnostic methodologies and basic tissue handling techniques that can potentially alter results. Despite limitations of resources, educational workshops make it possible to improve the practice of breast cancer diagnosis, and thereby enable accurate comparative analysis between breast cancers in the developing and the developed world. [Table: see text]

Abstract 1265: Tissue is alive: Preserving phosphoproteins and tissue morphology in clinical trial samples

Abstract An urgent clinical goal is to identify subpopulations of cancer patients that may respond to targeted kinase inhibitors and/or their phosphorylated substrates. Consequently, phosphorylated signal pathway proteins have emerged as a critical class of analytes for therapeutic stratification. Technologies now exist that can measure protein phosphorylations and map cell signaling pathways in a single core-needle biopsy, thus relying on the inhibition of any kinase/phosphatase activity within the sample immediately following excision. Unfortunately applying these technologies to clinical samples has been hindered because phosphoprotein epitopes are not adequately preserved by formalin fixation and paraffin embedding, while freezing tissue samples may not be feasible in multi-center clinical trial sites and cannot adequately preserve morphology. To facilitate clinical trial molecular profiling, where immediate snap-freezing of tumor biopsies is not feasible, we have created a novel, one-step, room temperature preservative that stabilizes proteins/phosphoproteins equivalent to snap-freezing and tissue/cell morphology equivalent to neutral buffered formalin fixation. In addition, our preservative solution simultaneously fixes and decalcifies bony tissue, thus permitting molecular profiling of bony tissues that was never before possible. We have utilized our Biomarker and Histology Preservative (BHP-Cell and/or BHP-Tissue) in a breast cancer multi-site clinical trial (US Oncology 05-074/GSK LPT109096) and a clinical research multiple myeloma trial. BHP has been validated to a) function as a transport medium while preserving histomorphology, b) maintain full antigenicity for clinical immunohistochemistry (such as Ki-67, ER, PR, Her2, p63, and phosphorylated epitopes), c) preserve phosphoprotein epitopes for cell signaling pathway profiling by reverse phase protein microarray (RPMA), d) be compatible with frozen sections or paraffin embedding, and e) obviate the need for additional decalcification. Preservation of tissue morphology has been demonstrated in 25 mouse, feline, and human tissues with enhanced immunohistochemical properties for &amp;gt;20 antigens using standard IHC protocols. RPMA data from LPT10906 shows differentially deranged signaling networks in the pre-treatment biopsies for patients that did not have a pathologic complete response compared to responders. Our multiple myeloma trial quantifies cell signaling pathway expression pre/post kinase inhibitor treatment, in an ex vivo treatment model, on both myeloma and bone marrow cells, preserved in BHP. We are currently evaluating the integrity of nucleic acid preservation in parallel with proteins to provide a universal one step biospecimen tissue fixative for clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1265. doi:1538-7445.AM2012-1265

Influence of Preservation in Two Kinds of Formaldehyde Solutions on the Fracture Characteristics of Bovine Femoral Compact Bones

For the evaluation of the physical properties of bones, specimens are often either deep-frozen or chemically fixed with reagents such as formaldehyde (formalin) or ethanol for antisepsis and sterilization. However, formalin contains formic acid, which dissolves bone minerals such as Ca and P. To suppress this bone mineral elution, we tested neutral buffered formalin as a fixative. In this study, we investigated the influence of formalin and the neutral buffered formalin fixation on the fracture characteristics of bovine femoral compact bones over a relatively long-term preservation period. In both the formalin and the neutral buffered formalin fixation, bone minerals migrated rapidly into the fixatives. Thus, some aqueous solutions such as a normal saline solution may also dissolve bone minerals. In the neutral buffered formalin fixation, fine calcium phosphate grains precipitated on the surface of the soft tissues of blood vessels in the Haversian canal, as observed by scanning electron microscopy. An element analysis with energy-dispersive X-ray spectroscopy also demonstrated the presence of Ca and P. Thus, the precipitated grains are assumed to be hydroxyapatite. In this study we evaluated the influence of formalin preservation, which reduces the fracture toughness of bovine femoral compact bones, and concluded that due to the formation of chemical bridges from the reaction of formaldehyde with the collagen fibers in the bones, the collagen fibers are cured and hardened, resulting in a reduction in the fracture toughness of the bovine femoral compact bones.

ウシ大腿皮質骨の破壊特性に及ぼすホルマリン保存液の影響

To evaluate the physical properties of a bone, a specimen is often deep-frozen or chemically fixed with reagents such as formaldehyde solution (formalin) or ethanol for antisepsis and sterilization. However, formalin contains formic acid and dissolves bone minerals such as Ca and P into the fixative. To suppress bone mineral solution, we used neutral buffered formalin as the fixative solution. In this study, we investigated the effect of formalin and of neutral buffered formalin fixation on the fracture characteristics of bovine femoral compact bone during relatively long-term preservation. With both formalin and neutral buffered formalin fixation, bone mineral migrates rapidly into the fixative solution. Thus, formic acid is not solely responsible for the dissolution of bone minerals, but some aqueous solutions such as saline can also dissolve bone mineral. With neutral buffered formalin fixation, small calcium phosphate grains precipitate at the surface of blood vessels in the Haversian canal, as observed by scanning electron microscopy. An energy-dispersive spectroscopy also demonstrates the presence of Ca and P. Thus, the precipitated grains are assumed to be hydroxyapatite. In this study we evaluate the effect of formalin preservation, which is to reduce the fracture toughness of bovine femoral compact bone, and to form chemical bridges by the reaction of formaldehyde with the collagen fiber of bone. The collagen fiber was cured and hardened, resulting in a reduction in the fracture toughness of bovine femoral compact bone.

Effect of buffered formalin fixation on DNA preservation for subsequent real-time PCR analysis

Effect of buffered formalin fixation on DNA preservation for subsequent real-time PCR analysis

Studies on the haemotoxicity of chloramphenicol succinate in the Dunkin Hartley guinea pig

In man, chloramphenicol (CAP), induces two major haemotoxic effects. First, a reversible, dose-related reticulocytopenia and anaemia developing during treatment. Second, a non-dose-related aplastic anaemia (AA), developing weeks after treatment, is often irreversible and fatal. In previous studies, we developed a mouse model of the reversible reticulocytopenia/anaemia using CAP succinate (CAPS); attempts to induce AA in the mouse with CAPS were unsuccessful; in the rat, CAPS induced only minimal haemotoxicity. We therefore wished to investigate haematological changes caused by CAPS in a third rodent, particularly in relation to the induction of significant 'late stage' bone marrow depression (AA). Female guinea pigs were gavaged with CAPS in three experiments. In a dose ranging study, CAPS (at 2500 and 3500 mg/kg) was administered daily for 9 days, and blood examined at 1 day post dosing. CAPS induced increased erythrocyte values (an apparent haemoconcentration effect), and reduced reticulocytes and femoral marrow nucleated cell counts (FNCC). In a second experiment, CAPS was given at 333, 666 and 1000 mg/kg (13 days); haematological changes were compared with results from the initial study, with evidence of dose-related effects. In a final experiment, CAPS was administered (825 mg/kg, 16 days) and blood studied at 1, 12, 28 and 63 days post dosing. At day 1, erythrocyte values were decreased (NS), and reticulocytes and FNCC were reduced; the marrow was hypocellular with erythroid depletion. At 12 and 28 days, values returned towards the normal range. At 63 days, parameters were normal. Thus, CAPS (825 mg/kg for 16 days) induced changes comparable to the reversible bone marrow depression seen in man; but there was no evidence of 'late stage' (i.e. at 63 days) marrow depression, as would be seen in a developing or overt marrow aplasia (AA). The guinea pig (like the mouse) is a model for the early events, but is not a good model for CAP-induced AA in man.

Comparison of Bouin Solution and Buffered Formalin Fixation on the Detection Rate by In Situ Hybridization of Human Papillomavirus DNA in Genital Tract Lesions

Abstract In situ hybridization is a commonly used technique for the detection of nucleic acid sequences but its efficacy can be affected by many parameters. Human papillomavirus (HPV) infection of the genital tract is a useful model for determining how the various parameters may effect the hybridization signal. In this study, the rate of detection of HPV DNA was compared for 294 genital tract lesions fixed in Bouin solution and 306 lesions fixed in buffered formalin. The detection rate in low grade vulvar and cervical intraepithelial lesions fixed in buffered formalin was 96% and 91%, which compared to 83% and 76% , respectively, of tissues fixed in Bouin solution. The detection rate was 73% and 70% for vulvar and cervical high grade intraepithelial lesions fixed in buffered formalin, respectively, versus 50% and 47% for the comparable tissues fixed in Bouin solution. Thus, the decrease in the detection rate was greater for both the vulvar and cervical high grade lesions compared to the low grade lesions ...

Comparison of Bouin Solution and Buffered Formalin Fixation on the Detection Rate by In Situ Hybridization of Human Papillomavirus DNA in Genital Tract Lesions

Comparison of Bouin Solution and Buffered Formalin Fixation on the Detection Rate by In Situ Hybridization of Human Papillomavirus DNA in Genital Tract Lesions

A histological comparison of phase-partition fixation with fixation in aqueous solutions.

In phase-partition fixation, tissue is immersed in a non-aqueous solvent at equilibrium with an aqueous solution of a fixing agent to minimize osmotic effects. Preservation of morphology afforded by phase-partition fixation using formalin and glutaraldehyde and several organic solvents was compared to aqueous 10% neutral buffered formalin fixation for five tissues. It was shown that phase-partition fixation can provide excellent fixation for light microscopy if the proper combinations of fixatives and solvents are used.

A Simple Method for Histochemical Demonstration of Viral Inclusion Bodies in Cell Monolayers in Microtitration Plates

The present paper describes a Bouin's-formalin fixation and Giemsa staining procedure for demonstrating viral cytoplasmic inclusion bodies in cellular monolayers in microtitration plates. Monolayers are fixed in Bouin's fixative for 15 min followed by 10% neutral buffered formalin fixation overnight. After fixation, the monolayers are stained with 4% (v/v) Giemsa stain. The method is superior to the separate use of formalin, methanol or Bouin's fixation-staining methods and compares favorably to immunocytochemical techniques for sensitivity.

THE DEMONSTRATION AND DISTRIBUTION OF WATER SOLUBLE BLOOD GROUP O (H) ANTIGEN IN TISSUE SECTIONS USING A FLUORESCEIN LABELLED EXTRACT OF ULEX EUROPEUS SEED.

A fluorescein labelled extract of Ulex europeus seed was found to be satisfactory for demonstrating water soluble H antigen in formalin fixed paraffin embedded tissue sections. Commercially available anti-A typing serum was labelled with fluorescein and used to demonstrate water soluble A antigen in formalin fixed paraffin embedded tissues. The reactivity of A and H antigen after neutral buffered formalin fixation was superior to that observed after Zenker's, Helly's or Bouin's fixation; reactivity in paraffin sections of formalin fixed tissues were equal to that of fresh frozen sections. Autolysis did not prevent the demonstration of blood group antigen A or H in fixed paraffin embedded tissues but was associated with some diffusion of the antigens. The distribution of H antigen in formalin fixed paraffin embedded tissue section for patients with type A, B and O blood also was studied. In secretors blood group substances were widely distributed in the epithelial mucins of the body whereas in nonsecretors the antigens are largely confined to the deep portion of the pyloric mucosa of the stomach and Brunner's glands. The distribution of A and H antigen within a gland, such as Brunner's glands, was not homogeneous. That is, some acini contained A antigen but no H antigen; others contained H antigen but no A antigen: a few contained both A and H antigen. The significance of this mosaic distribution which previously has not been described was discussed.