Tissues In Formalin Research Articles

Assessing the Quality of Long-Term Stored Tissues in Formalin and in Paraffin-Embedded Blocks for Histopathological Analysis.

Introduction:Formalin is the most commonly used fixative which enables for long-term storage of specimens and preserves morphologic features allowing the microscopic evaluation for future research analysis. Archival collections of the tissue serve as a reliable tool for diagnostic research purpose. They have an important role in on-going patient care, allows for evaluation of recurrent cases for diagnostic purpose and rare case specimens can also be used as an educational tool as well as for further biomedical research purposes. However, studies assessing quality and their usefulness for such purposes are scanty. Hence, the present study is aimed at evaluating and comparing the tissue changes after long-term storage in formalin as well as in paraffin-embedded blocks.Methodology:Three study groups include specimens stored in formalin for a minimum of 5 years (long-term fixed tissue) and their corresponding paraffin-embedded old tissue blocks along with freshly fixed tissues taken as controls which were subjected to routine histopathological procedures and were assessed for macroscopic and microscopic evaluation.Chi-square test and Z-proportion tests were considered for statistical analysis.Results:Prolonged storage of the tissues in formalin showed variation in color and consistency, difficulty in cutting during grossing with inadequate sectioning characters, loss of tissue integrity and architecture, and inadequate nuclear and cytoplasmic details.Conclusion:On histological analysis, prolonged formalin-stored specimens showed deleterious effects than archival blocks. Hence, it can be proposed that tissues are better preserved in paraffin blocks rather than in formalin for further biomedical research purposes.

Single acquisition label-free histology-like imaging with dual-contrast photoacoustic remote sensing microscopy.

.Significance: Histopathological analysis of tissues is an essential tool for grading, staging, diagnosing, and resecting cancers and other malignancies. Current histopathological imaging techniques require substantial sample processing, prior to staining with hematoxylin and eosin (H&E) dyes, to highlight nuclear and cellular morphology. Sample preparation and staining is resource intensive and introduces potential for variability during sample preparation.Aim: We present a method for direct label-free histopathological assessment of formalin-fixed paraffin-embedded tissue blocks and thin tissue sections using a dual-contrast photoacoustic remote sensing (PARS) microscopy system.Approach: To emulate the nuclear and cellular contrast of H&E staining, we leverage unique properties of the PARS system. Here, the ultraviolet excitation PARS microscope takes advantage of DNA’s unique optical absorption to provide nuclear contrast analogous to hematoxylin staining of cell nuclei. Concurrently, the optical scattering contrast of the PARS detection system is leveraged to provide bulk tissue contrast reminiscent of eosin staining of cell membranes.Results: We demonstrate the efficacy of this technique by imaging human breast tissue and human skin tissues in formalin-fixed paraffin-embedded tissue blocks and frozen sections, respectively. Salient nuclear and extranuclear features such as cancerous cells, glands and ducts, adipocytes, and stromal structures such as collagen are captured.Conclusions: The presented dual-contrast PARS microscope enables label-free visualization of tissues with contrast and quality comparable to the current gold standard for histopathological analysis. Thus, the proposed system is well positioned to augment existing histopathological workflows, providing histological imaging directly on unstained tissue blocks and sections.

Impact of Preanalytical Factors on the Measurement of Tumor Tissue Biomarkers Using Immunohistochemistry.

Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute's Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12-24 hr to avoid confounding effects of these preanalytical factors on IHC.

Detection of microorganisms on formalin-fixed and stored pathology tissues: A microbiological study

Background:Formalin is widely used to fix histological preparations and as preservatives in embalming solutions and is an age-long practice in medical laboratories. It is generally accepted that the risk of contracting infections is relatively high among medical laboratory workers and pathologists. Recent studies have, however, suggested that formalin does not effectively inactivate all kinds of microbes in formalin-fixed tissue (FFT). Long time preserved tissues in formalin may develop growth of microbes on the surface of the formalin.Aims and Objectives:The purpose of the study is to determine the growth of microorganisms on the surface of FFTs.Materials and Methods:Fifty-one containers of 10% formalin with fixed tissues and undiscarded formalin solution not containing tissues of years 2013–2015 (17 in each year) were selected, and samples for inoculation onto the cysteine lactose electrolyte deficient agar plates were taken from the surface of the FFT using sterile cotton tips. The growth of the colonies was checked for after 48 h.Results:Out of 51 samples from 2013 to 2015, 17 had shown growth of microbial colonies. Six out of 17 samples of 2013, 7 out of 17 of 2014 and 4 out of 17 samples of 2015 had colonies of microbes on agar plates. Gram-negative bacilli, Bacillus subtilis and micrococci were mostly found.Conclusion:There were viable microbes on the surfaces of formalin solution containing pathology tissue. Since cross-contamination by microbes may occur during regrossing or processing, protocols to decrease cross-contamination should be instituted.

PrPSc detection in formalin-fixed paraffin-embedded tissue by ELISA

BackgroundFormalin-fixed paraffin-embedded tissue is regularly employed in the diagnosis of transmissible spongiform encephalopathies (TSE) by immunohistochemistry (IHC), the standard by which all other TSE diagnostic protocols are judged. While IHC affords advantages over diagnostic approaches that typically utilize fresh or frozen tissue, such as Western blot and ELISA, the process of fixing, staining, and analyzing individual sections by hand does not allow for rapid or high throughput screening. However, preservation of tissues in formalin is not dependent upon the availability of refrigeration.FindingsFormalin-fixed paraffin-embedded tissues from TSE transmission studies of scrapie in sheep, chronic wasting disease in white-tailed deer or transmissible mink encephalopathy in cattle were cut at 5 μm thickness. Samples containing the tissue equivalent of as little as one 5 μm section can be used to readily discriminate positive from negative samples.ConclusionsThis approach cannot replace IHC but may be used along with IHC as both a more rapid and readily high throughput screen where fresh or frozen tissues are not available or impractical.

Effects of isopropanol storage time on histochemical and immunohistochemical stains in lung tissue

Objectives: Prolonged storage of tissues in formalin is known to result in progressively increasing cross-linking of proteins and subsequent difficulties in histochemical and immunohistochemical staining. One solution to this problem is to fix the tissues in formalin and then store them in alcohol. Our institution uses isopropanol for this purpose. However, little is known on how isopropanol storage may affect the quality of subsequent staining of sections taken from these tissues.Methods: To test this hypothesis, lung tissue samples from a transplant donor lung were fixed for 24 hours in 10% neutral buffered formalin and then stored in 70% isopropanol at 4°C for 0 days, 1 week, 2 weeks, 4 weeks, 8 weeks, 12 weeks, and 24 weeks. All lung tissue samples were stained for hematoxylin and eosin, Masson’s trichrome, periodic acid Schiff, CD31, CD68, and smooth muscle actin. Staining intensities were scored (1 = weak; 2 = moderate; and 3 = intense) and the inter-observer variability was evaluated.Results and discussion: There were no significant differences in the staining intensities of hematoxylin and eosin, Masson’s trichrome, periodic acid Schiff, CD31, CD68, and smooth muscle actin in the lung tissue samples with increasing storage time in isopropanol. Thus, the tissues that are stored in isopropanol at 4°C can yield good quality staining for research and clinical investigations.

A study of the effect of formalin preservation on normal and cancerous breast tissues using small angle X-ray scattering (SAXS)

Small angle X-ray scattering (SAXS) has the ability to provide information on a molecular and supra-molecular scale from biological tissue specimens. It has been postulated that this information will be useful in providing histopathological diagnoses for certain diseases of the breast. In this category, we include cancer, a major health problem for a number of populations around the world. So far studies in our group have been made using flash-frozen tissue samples. This limits the range and ease of use of the technique. If we were able to obtain the same information from preserved tissues then a more extensive use of SAXS diagnosis would be possible. Here we report on the first investigations into this possibility. In the research reported in this paper, 84 human breast biopsies including cancer and normal tissues were obtained from human patients. Small angle scatter data were collected at station 2.1 of the SRS at the Daresbury Laboratory, UK using a beam size of 0.25 mm 2 at the sample and a wavelength of 1.54 Å. The sample to detector distance was 2000 mm. The results verify that there is a quantifiable difference between the scatter curves from flash-frozen cancer and normal breast tissue in the range of scatter vector Q between 0.4 and 0.7 nm −1. After preserving the tissues in formalin, the difference between the normal and cancerous tissues is less marked. The preservation of the tissue in formalin can essentially mask the effects that disease would have on the tissue supra-molecular structure rendering the preserved specimens of less useful for this histopathology technique.

Microwave irradiation as a form of fixation for dental pulp

This study investigated developments in microwave energy fixation and the general applicability of microwave fixation to studies of dental pulp. Rat mandibles with incisors were dissected out and immersed in various solutions before and after exposure to microwave energy. Histological examination showed no combination with microwave fixation to be equal in quality to control tissues fixed in formalin. Inferior but satisfactory results could be achieved by immersion of tissues in formalin after microwave irradiation, a step thought to protect the microwave fixed tissue from subsequent damage caused by decalcification. Good fixation of tissues outside the pulp was achieved using microwave energy. Further investigation is required to perfect microwave fixation of pulpal tissue.

Effects of products of autolysis of tissue and urine on the viability, morphology, and antigenicity of Leptospira interrogans serovar pomona.

The effect of the products of autolysis on Leptospira interrogans serovar pomona was investigated in bovine kidney tissues and urine inoculated with this organism. No viable leptospires were found at 24 h or subsequently after inoculation of kidney tissues or urine. Leptospires and their soluble antigens were rapidly destroyed in tissues stored at 20 degrees C and could not be detected by the fluorescent-antibody test and the enzyme-linked immunosorbent assay. On the other hand, leptospires were detectable by dark-field microscopy and fluorescent-antibody test 38 days after addition to urine. The rate of destruction proceeded less rapidly in tissues stored at 4 degrees C. Under the conditions of this study, soluble leptospiral antigen levels decreased less rapidly than did the number of detectable leptospires. Similarly, breakdown of leptospires, demonstrated by dark-field microscopy and fluorescent-antibody test, progressed during the first 5 days after inoculation of urine, when levels of soluble leptospiral antigen remained constant. Although the number of intact leptospires was markedly reduced after freezing of tissues at -20 degrees C, soluble leptospiral antigen levels remained unaltered. Neither intact leptospires nor their soluble antigens were detected after preservation of tissues in Formalin. However, formolization of urine delayed the destruction of leptospires and increased the levels of soluble leptospiral antigen during at least the first 5 days after inoculation. These results indicate that assays such as the enzyme-linked immunosorbent assay should be useful for the detection of soluble leptospiral antigens in urine and in autolyzing tissues such as those taken from a bovine fetus aborted as a result of leptospiral infection.

Studies of trace-metal levels in human tissues--VI. Concerning the estimation of lead levels in human lung and vertebra with particular reference to formalin fixation.

The difficulties arising from the study of lead levels in lung and vertebra raise a number of matters relevant to all studies of trace element concentrations in human tissues. First, the fixation of tissues in formalin, prior to trace element analysis, should be avoided because of the likelihood of loss by leaching. Secondly, meaningful comparisons among different studies are difficult, if not impossible, to make unless standard conditions for the weight basis of expression of the results are used in preparing the tissues. Thirdly, if results are expressed as median metal concentrations, the mean concentration should also be given. Fourthly, authors must indicate the steps they have taken to ensure both the precision and accuracy of their data. Fifthly, it is desirable that there should be more consistency between different studies in the age groups used to show the relation (if any) between age and tissue metal levels. Sixthly, the value of a data set involving trace-metal levels in human tissues is enhanced if information on the tissue donors includes their smoking history. The contribution of cigarette smoking to renal cadmium levels is now well established.

Demonstration of amyloid with Mesitol WLS-Congo Red: application of a textile auxiliary to histochemistry.

Previous histochemical investigations demonstrated similarities in the binding of Congo Red and other direct cotton dyes by amyloid and cellulose. It seemed therefore of interest to determine whether or not the cellulose-like reactivity of amyloid extends also to dye solutions containing an anionic reserving agent. These reagents are used in the dyeing of wool-cellulose (Halbwolle) fabrics to prevent binding of direct cotton dyes by proteins. Mesitol WLS-Congo Red solutions stained amyloid selectively; other tissue structures, except some hyaline deposits in arterioles, remained unstained. The cause of this non-specific reaction could not be determined with certainty. Therefore, the alkaline Congo Red method is recommended for histochemical identification of amyloid. However, the Mesitol WLS-Congo Red technic was very useful for demonstration of amyloid after prolonged storage of tissues in formalin; amyloid in such material showed little or no reactivity with the alkaline Congo Red or the Sirius dye methods. This pilot study indicates that anionic reserving agents can be effectively employed under conditions of histochemical technics.

Observation on the remarkable stability of transmissible mink encephalopathy virus

Transmissible mink encephalopathy (TME) virus retained biological activity after prolonged storage in formalin (about three and a half years) and in paraffin tissue blocks used for histologic preparations (about six and a half years). Thus, TME tissues in formalin and in paraffin blocks should be handled as contaminated materials.

DISTRIBUTION OF CHOLINESTERASE IN THE LARVAE OF HYPODERMA BOVIS AND HYPODERMA LINEATUM (DIPTERA: OESTRIDAE) AND ITS INHIBITION BY SYSTEMIC INSECTICIDES

AbstractLarvae of Hypoderma bovis (L.) and H. lineatum (De Vill.) when incubated with the substrate acetylthiocholine iodide showed the presence of cholinesterase throughout the central nervous system and sense organs, in gonads, oenocytes, Malpighian tubes, anlagen of adult structures, part of the posterior midintestine, and at the junctions of the muscles of the body wall. Incubation with the substrate 5-bromoindoxyl acetate showed all the aforementioned and in addition demonstrated the presence of aliesterase (organophosphorus-sensitive esterase) in the cell cytoplasm of all the midintestinal epithelium, the ring gland, tracheal epithelium, salivary duct epithelium, and in the perineurium of the nerve sheath. Aromesterase (organophosphorus-resistant esterase) was particularly abundant in the epithelium of the midintestine. Lipase was prominent in the lipid droplets of the fat body cells and in similar droplets in the haemocytes.When the larvae or their tissues, prior to incubation with the substrates, were treated with any one of the three organophosphorus insecticides coumaphos (Co-Ral), trichlorfon (Neguvon, Dipterex), or Ruelene, the cholinesterase was inhibited and so was much of the aliesterase, but not the aromesterase or lipase. Fixation of the larval tissues in formalin did not result in loss of enzyme activity, but on the other hand was not necessary for good histochemical demonstration of the enzymes.It is concluded that these insecticides by their anticholinesterase action kill the first-instar cattle grubs before the latter can migrate to the lumbar region of the bovine host.

Uptake of Benzpyrene by a Chromosomal Phospholipid

THE uptake of 3,4-benzpyrene by cells has been of interest to many workers, particularly in view of the carcinogenic properties of this substance. Moreover, it is a fluorescent compound and has been used as a fluorochrome for lipids into which it will dissolve from a hydrotropic solution ; caffeine is usually used for dispersing it finely in water, since alone benzpyrene appears to be almost completely hydrophobic. Berg1 has used this method in a detailed and extensive study of the distribution of lipids in a wide range of normal and cancerous animal cells and in the root tips of Allium cepa. In order that the lipids should not be affected by his preparatory procedures, he fixed the tissues in formalin which had been stored over powdered chalk ; chalk seems to have the same effect as calcium chloride, which is frequently added with the intention of maintaining a high concentration of calcium ions to immobilize lipids2. This method of fixation seems to be much favoured by histologists. The tissue was sectioned with a freezing microtome, and the sections were then stained with benzpyrene. After a very careful investigation, Berg was forced to conclude that in animal and plant nuclei lipids are not present except in the nuclear membrane, and that biochemical demonstrations of these compounds in extracted nuclei were due to contamination by the cytoplasm. Indeed, he found that the nucleus was the one region in cells that never showed uptake of benzpyrene.

Frozen Sections from Bouin-Fixed Material in Histopathology

It has become almost a dogma that, for obtaining satisfactory frozen sections, fixation of the tissues in formalin is necessary; formalin is exclusively recommended for this purpose in the textbooks of microscopical and pathological technic.1 It has, however, several important disadvantages, which become the more acutely felt when fixation in more than one fixative is impossible, as happens so often in surgical pathology. When, after the preparation of the frozen sections, embedding in paraffin is necessary, formalin-fixed tissues show a marked shrinkage, especially when rapid embedding methods (e. g. dioxane) are used. This shrinkage can only be prevented by the time-consuming hardening of the blocks in K2Cr2O7. The trichrome stains, the phosphotungstic-hematoxylin stain and the azan method, which are slowly superseding the hematoxylin-eosin and van Gieson stains in histopathology, give only mediocre or inferior results after formalin fixation, unless the sections are refixed.

Avitaminosis. XVIII. Peripheral Nerves in Vitamin B1 Deficiency as Observed by Polarized Light.

Although the literature is full of reports on nerve degeneration in avian polyneuritis and B deficiency in the rat and other mammals, the most recent contributions in this field reveal conflicting evidence. In order to circumvent uncertainties in the interpretation of Marchi preparations and any possible artefacts that may be introduced during fixation and staining, Sutton, Setterfield and Krauss have recently applied the polarized light technique for the study of degeneration of myelinated nerves in vitamin A deficiency. This technique was found by Setterfield and Baird to have advantages over the Marchi method of staining with osmic acid, since it does not depend upon fixation and staining but on the chemical structure of the myelin sheath; moreover, it is more rapid, more sensitive and constant. The polarized light technique, however, as employed by Sutton and associates requires previous fixation of tissues in formalin, which procedure may introduce artefacts. We have applied the technique of Sutton and co-workers in studies of vitamin B deficiency and have checked the formalin procedure by elimination of all fixatives as well as all staining operations. Small pieces of spinal cord, trigeminal, sciatic and optic nerves from avitaminotic and litter mate controls on the same plane of nutrition, were fixed in 95% alcohol in liquid air, and dehydrated in vacuo, at −32°C., by the Altmann-Gersh-freezing-drying technique, as modified by Scott and Williams. Frozen sections were made and compared under polarized light with those fixed in 10% formalin. Twelve groups of rats were used for this comparative study. The pathological animals of 9 groups were on diets deficient only in vitamin B1 and had varying degrees of polyneuritis, ranging from slight to marked paralysis.