Fixation Fluid Research Articles

Role of Liquid-Based Cytology in the Endoscopic Diagnosis of Pancreatic Ductal Adenocarcinoma

Recently, endoscopic ultrasound-guided tissue acquisition (EUS-TA) has been widely used to diagnose pancreatic ductal adenocarcinoma (PDAC). The histological examination of core tissues acquired using novel biopsy needles is the primary diagnostic approach for patients with PDAC. However, in patients with early-stage PDAC, such as Stages 0 and I, EUS-TA can be challenging, and its diagnostic accuracy may be limited. This presents a clinical dilemma: The earlier that clinicians attempt to accurately diagnose PDAC, the more difficult it becomes to do so using EUS-TA. Liquid-based cytology (LBC) is a technique for preparing pathological specimens from liquefied cytology specimens by placing the collected material in a special fixative preservative fluid. LBC offers advantages, such as specimen optimization with reduced blood interference, a high cell-collection rate, and the simplicity of the procedure in the endoscopy room. The use of LBC may improve diagnostic accuracy, particularly for early-stage PDAC. Therefore, we emphasize that cytology remains a valuable tool for the endoscopic diagnosis of PDAC. In this review, we discuss the role of LBC in the endoscopic diagnosis of PDAC.

Integration of charging time, migration pathways and sealing analysis to understand hydrocarbon accumulation in complex fault blocks, the Pinghu Slope Belt of the Xihu Depression, East China Sea Basin

A large number of oil and gas fields have been successfully discovered in complex fault blocks worldwide. However, the role played by the fault-rock seal in the hanging wall traps is not fully understood due to the paucity of datasets. The trap examples of the Pinghu Slope Belt are used to analyze the migration and accumulation of hydrocarbons in the complex fault blocks, as well as the fault-sealing mechanism of the hanging wall traps. The burial history in typical wells, combined with the homogenization temperature of fluid inclusions, is used to determine the hydrocarbon charging time. Based on detailed 3D seismic interpretation, the paleo-structural features at critical moments and their control on hydrocarbon migration were discussed. Hydrocarbon accumulation characteristics were illustrated based on triangle juxtaposition diagrams. The result shows that there are two hydrocarbon charging episodes in the study area, respectively, 10.6–8.1 Ma and 2.8 Ma-present, with the latter being predominant. This phenomenon that the homogenization temperatures of inclusions are generally higher than the measured formation temperatures at the same depth is attributed to late fluid accumulation and not reaching thermal equilibrium with the reservoir fluids. The analysis of hydrocarbon generation history, trap fixation history and fluid inclusions indicate that the critical moment of the generation-migration-accumulation of most hydrocarbons ranges from 2.8 Ma to the present. Due to the difficulty in effectively recognizing the reflector (the top of the Santan Fm) and the nearly horizontal sediments above T20, the reflector T20 (5 Ma) near the top of the Santan Fm was chosen as an approximate proxy for this critical time (2.8 Ma). The paleo-structures of the Pinghu Slope Belt at 5 Ma are a gentle southeast-dipping slope. Based on the numerical simulation of three different fault scenarios (no faults, closed faults and open faults), wells A-H are located on the preferential migration pathways. Fault seal analysis shows that multiple juxtaposition types on the fault surface and moderate-potential to effective SGR values at the sandstone reservoirs in the middle and lower Pinghu Formation. Lateral and vertical reservoir compartmentalizations are presented in clean sand-sand juxtapositions with low-potential sealing (SGR<20%) in well A. The SGR-buoyancy pressure relationship from hanging wall traps and the global calibration plots from footwall traps have a consistent trend. The fault sealing mechanism of hanging wall traps is similar to that of the footwall traps. Therefore， those global calibration plots can be simultaneously applied to the fault seal evaluation of both hanging wall traps and footwall traps.

Fixation and fixatives in histopathology: a review

If fixation is not adequate, the other processes that follow such as dehydration, clearing, infiltration, embedding, microtomy and staining, will also be inadequate. A poorly processed tissue will make it difficult for the Pathologist/Histoscientist to render a proper diagnosis. This article is aimed at elucidating more on fixatives and fixation process in histopathology. The information presented in this review was gathered primarily from an extensive literature search on PubMed, Scopus and Textbooks. Fixatives in histopathology can be grouped into simple and compound fixatives. Simple fixative is a solution or gas which contains only one active ingredient or that has a singlechemical solution. Examples include; Formaldehyde, Glutaraldehyde, Mercuric Chloride, Potassium Dichromate, Picric Acid, Osmium Tetroxide, Acetic Acid, Ethanol, Acetone and Chromic Acid. When two or more simple fixatives are combined in a solution, the resulting solution is called a compound fixative. This can be further divided into Microanatomical and cytological fixatives. Examples of a micro-anatomical fixatives include; 10% formal-saline, Heidenhain’s Susa, Boiun’s fluid, Formol Sublimate. Cytological fixatives are grouped into cytoplasmic and nuclear fixatives. Fixation terminates any ongoing biochemical reactions, and also increases the mechanical strength or stability of the treated tissues. To accomplish this, tissue samples are usually immersed immediately in a fixative fluid. The fixatives employed prevent autolysis and putrefaction from taking place. Fixatives are integral substances used in histopathology to forestall the actions of autolysis and putrefaction in tissue samples.

The effects of formalin fixation and fluid storage on stable isotopes in rodent hair

Abstract Stable isotopes are increasingly being used to unlock the wealth of information contained in specimens preserved in museum collections. However, preservation methods that employ formalin may confound ecological interpretations. To quantify the effects of formalin fixation and subsequent fluid storage in ethanol on the isotopic signatures of small mammal hair, we analyzed δ 13C and δ 15N values from specimens of seven rodent species that were sampled repeatedly both before and after varying lengths of formalin fixation (1–11 days) and ethanol storage (1–6 years). We supplemented these data with a 2-week fixation experiment using deer mice (Peromyscus maniculatus) in which no ethanol storage was employed. As expected, preservation in formalin and ethanol had no discernable effect on δ 15N values. In contrast, specimen δ 13C values decreased in a saturating fashion during formalin fixation and over subsequent years of fluid storage in ethanol. On the basis of models that we fit to these time series, we estimate the long-term effect of fixation and storage on δ 13C values to be −0.92‰ after 4 years. This biologically relevant shift in δ 13C values should be accounted for when inferring the diets of species from fluid-stored museum collections and when comparing across specimens with different preservation histories.

THE AMOUNT OF BALLOON FIXATION FLUID VOLUME EFFECTSS ON URINE LEAKAGE IN PERMANENT CATHETER INSTALLATION

THE AMOUNT OF BALLOON FIXATION FLUID VOLUME EFFECTSS ON URINE LEAKAGE IN PERMANENT CATHETER INSTALLATION&#x0D; &#x0D; Ririn Marwaningsih1, Sunarsih2, NikenAstuti 3&#x0D; &#x0D; 1Program studi D III, Sekolah Tinggi Ilmu Kesehatan Elisabeth Semarang &#x0D; Email : ririn.marwaningsih@yahoo.com&#x0D; 2RS Elisabeth Semarang Email : sunarsihutagalung1@gmail.com&#x0D; &#x0D; &#x0D; &#x0D; ABSTRACT&#x0D; Background: The appropriate volume of balloon fixation still becomes a matter of discussion in the permanent catheter installation. The providing of fixation fluid on varied catheter installation is still found practically. The incidence of urine leakage in patients with permanent catheters is still often found. The amount of fixation fluid volume used needs to be studied to provide input on how much the right fixation volume for the permanent catheter installation. The purpose of this study is to determine the role of the amount of catheter balloon fixation volume on the incidence of permanent catheter leakage.&#x0D; Methods: The design of this research is analytic observational with a cross-sectional approach. The research population was patients in private hospitals in Semarang who had permanent catheters installed. The sample uses total sampling in accordance with the inclusion and exclusion criteria by taking samples from 17-21 January 2019 as many as 53 respondents.&#x0D; Results: 33 (62.3%) respondents had 10 ml fixation volume permanent catheter (62.3%), 13 respondents (24.5%) had permanent 20 ml balloon fixation volume and 7 (13.2%) respondents had permanent 25 ml balloon fixation volume. There were 7 respondents (13.2%) who had urine leakage and 46 respondents (86.8%) did not. The Mann-Whitney test showed a p value of 0.047 (&lt;0.05).&#x0D; Conclusion: the volume of balloon fixation fluid has a role in the incidence of urine leakage in permanent catheter installation.&#x0D; Key words: volume of catheter balloon fixation fluid, catheter balloon, urine leakage, permanent catheter

English

BACKGROUND Though histopathology is the gold standard investigation for diagnosing most benign and malignant lesions of bone, the decalcification process required for removing bone hardness makes it a time-consuming process. Sediment cytology is a rapid investigative procedure utilising the cells in the specimen containers after proper fixation, processing and staining. The objective of this study was to compare the diagnostic efficacy of sediment cytology of bone lesions with the histopathology. METHODS The study was conducted in 50 samples of bony tissues received in the histopathology lab during the study time. The fixative fluid containing specimens were first agitated for procuring the cells from the sample. The sediment obtained was processed and stained as a cytology sample and studied under microscope. Based on cytology findings they were classified into benign, malignant, inflammatory and inconclusive. The specimen was processed as routine histopathologic sample and final diagnosis was made from it. RESULTS Sediment cytology of malignant bone lesions showed a very high specificity (100 %) but only a moderate sensitivity of 80 %. Sediment cytology of benign bone lesions also showed a higher specificity of 100 % but only a moderate sensitivity of 84.6 %. CONCLUSIONS If adequately cellular, biopsy sediment cytology is a simple, inexpensive, rapid tool for the early diagnosis of bone lesions. Sediment cytology can act as a good complementary test to histopathology. KEYWORDS Bone Lesions, Cytology, Sediment Cytology, Histopathology

Different fixative solutions in the detection of mast cells in the canine and feline reproductive organs.

The aim of the study was to evaluate the usability of different fixative fluids in the detection of mast cells in ovaries and uteri of female dogs and cats. Samples were fixed in 4% formaldehyde, Carnoy's fluid, Mota's basic lead acetate and isotonic formaldehyde-acetic acid (IFAA). Mast cells (MCs) were detected by acidified toluidine blue staining and counted for various parts of the ovaries and uteri. In the ovaries of both species, the numbers of MCs were significantly (p < 0.05) higher in Carnoy than in formalin. No significant differences were found between Carnoy and Mota (tested only in cats). In the uterus, numbers of MCs were significantly (p < 0.05) higher in Carnoy, Mota and IFAA compared to formalin (canine endometrium, feline endometrium and feline myometrium), in Carnoy and Mota compared to formalin (canine myometrium) and in Mota compared to IFAA (feline myometrium). The majority of MCs were formalin-sensitive in the canine and feline uterus, in the canine ovary and in the feline cortex ovarii. In the feline medulla ovarii, the majority of MCs were formalin-resistant. No formalin-resistant MCs were detected in the feline tunica albuginea ovarii. Thus, using Mota's or Carnoy's fluid in the canine or feline female reproductive organs is recommended. This study improves methodology for all studies which clarify the role of MCs in the reproductive organs of the domestic and laboratory animals.

Cytological studies on Pyropia suborbiculata

In the present study, karyological observations on Pyropia suborbiculata (Bangiophycidae) at various stages of the life cycle were carried out. Living material of different life cycle stages of Py. suborbiculata were fixed in Carnoy’s fixative fluid and stored under the light conditions of 60–80 μmol photons m−2 s−1 to leach phycobilin pigments and then the samples were stained with Wittmann’s aceto-iron hematoxylin chloral hydrate and examined under a photomicroscope. The results indicated that vegetative cells, spermatangia, and carpogonia in gametophytic blade phase were haploids (n = 3), while zygotospores, vegetative cells, and conchosporangial branch cells in conchocelis phase were diploids (2n = 6). Meiosis occurred during the initial two-cell divisions of the germinating conchospores. Subsequently, the chromosome changed from diploid (2n = 6) to haploid (n = 3). When the blades released monospores for asexual reproduction, both the monospores and the cells of their germlings were haploid karyotypes (n = 3). In addition, when filamentous conchocelis cells at different developmental stages underwent mitosis, pairing of homologous chromosomes would occur in the nucleus during the prophase of division.

Enhanced microscopic diagnosis of oesophageal candidiasis through additional cytospin analysis of the fixative of oesophageal biopsies

To confirm or to refute the diagnosis of candida oesophagitis as the most common infectious disease of the oesophagus is a standard diagnostic procedure in histopathology. The fungal hyphae colonise mainly the superficial layers of the oesophageal squamous mucosa. Tangentially cut sections of oesophageal biopsies in the paraffin block might lead to a false negative result concerning mycotic infection. The aim of this study was to investigate whether cytospin analysis of the formalin fixative in which the biopsies were stored and transported would be a tool to close the diagnostic gap.Oesophageal biopsies from 150 consecutive patients with the clinical diagnosis or question "candida" or "candida oesophagitis" have been investigated. The biopsies were routinely processed and stained with haematoxylin and eosin and periodic acid-Schiff reaction. In parallel, the fixative fluid, usually disposed of after use, was processed by using a cytospin centrifuge and prepared for cytological proof of fungal hyphae. The cytology slides were also stained with periodic acid-Schiff reaction. In this blind study, the pathologist investigating the results of one procedure was unaware of the results of the second procedure.Out of 89 positive cytology cases, 64 cases (71,9 %) also showed a positive histology result. In the remaining 25 cases (28,1 %), fungal hyphae were seen only after re-evaluation of the original histology slides (n = 6) or in further serial sections using the complete tissue in the block (n = 5). In 14 cases, no hyphae could be detected histologically. Only in one of the 61 cytospin-negative cases was candida seen in histology.Our results show that diagnosing oesophageal candidiasis can be improved by more than one quarter using the formalin fixative for cytospin cytology.

Brain perfusion fixation in male pigs using a safer closed system

Tissue fixation methods are well established for rodents, but not for large animals. We present a simple technique for in situ brain perfusion fixation in a male porcine model, using cervical vessels for inflow and outflow and achieving a closed system. Thirty-four pigs, aged 4.7 ± 0.6 months and weighing 60.7 ± 10.9 kg, were anaesthetised and mechanically ventilated. The ipsilateral common carotid artery and external jugular vein were dissected and constituted the inflow and outflow access, respectively. The brains were perfused and fixed in situ with heparinised saline followed by buffered formaldehyde. Then, specimens (brain, cerebellum and brainstem) were extracted and processed for histology. Fixative fluid leakage was avoided, achieving a closed system. This technique minimises the exposure to toxic chemicals such as formaldehyde and associated hazards (inherent toxicity, eye irritation), thereby increasing operators' safety. Perfusion was performed with a peristaltic pump for 20-30 minutes at an optimum rate of 0.20 l/min and required only 5 litres of the fixative. The specimens were sufficiently hardened to be extracted. High-quality tissues were available for histology analysis. This technique offers a user-friendly closed system for brain perfusion fixation which can be adapted for other tissues of the head, face and neck.

Assessment of alternatives to environmental toxic formalin for DNA conservation in biological specimens.

One essential step of museum and clinical specimen preservation is immersion in a fixative fluid to prevent degradation. Formalin is the most largely used fixative, but its benefit is balanced with its toxic and carcinogenic status. Moreover, because formalin-fixation impairs nucleic acids recovery and quality, current museum wet collections and formalin-fixed, paraffin-embedded clinical samples do not represent optimal tanks of molecular information. Our study has been developed to compare formalin to two alternative fixatives (RCL2® and ethanol) in a context of molecular exploitation. Based on a unique protocol, we created mammalian fixed collections, simulated the impact of time on preservation using an artificial ageing treatment and followed the evolution of specimens' DNA quality. DNA extraction yield, purity, visual integrity and qualitative and quantitative ability to amplify the Cox1 gene were assessed. Our results show that both RCL2 and ethanol exhibit better performances than formalin. They do not impair DNA extraction yield, and more importantly, DNA alteration is delayed over the preservation step. The use of RCL2 or ethanol as fixative in biological collections may insure a better exploitation of the genetic resources they propose.

Gross anatomy study on isolated formalin-fixed anatomical preparations

The anatomic dissection has always had a crucial role in teaching gross Anatomy in medical academic courses. Nowadays the easy availability of several programs of virtual dissection together with the difficulties in finding useful human bodies as well as the complicated procedures of preparation, make anatomic dissection practice barely available in our Universities. The use of dissected preparations obtained from isolated organs or well-defined anatomical regions rather than the use of whole cadavers may be useful in many instances to overcome the difficulties described above. In particular, the long-established use of formalin-fixed specimens may still represent an easy and useful support to teach gross anatomy. However, due to its toxicity and cancerogenic activity, formalin use as a fixative has been recently strongly restricted, making both dissection as well as prosection of formalin-fixed specimens difficult to be performed. The E. Morelli’s Museum, located in the C. Forlanini Hospital of Rome, contain a collection of anatomical preparations dating back to the half of past century, which represents not only a historical but also a scientific and didactic patrimony. This collection includes over one thousand of anatomical preparations, the majority of which are fixed and stored in unsealed glass vase. Furthermore, at present most preparations, many of which are of admirable workmanship, are abandoned. Therefore, on behalf of Sapienza University of Rome, during the last months we are engaged in the recovery and safeguard of these specimens. The procedures we are using include systematic cataloguing of all specimens, adding with photograph and comments as well as several recovery treatments, tailored to the singular specimen condition (change or recovery of split vases, complete or partial change of fixative fluid, improvement of the presentation of anatomical preparation, etc.). All vases were finally filled up with fixative and sealed with a synthetic resin, which polymerizes under water. This procedure of airtight closure of vases prevents any dispersion of formalin gas in the air, making anatomical preparations available for educational as well as museum purposes. Our final goal is the recovery, restoration and cataloguing of these preparations in order to reevaluate the Anatomic Museum center as the foundation of Medical school training.

An improved anatomical MRI technique with suppression of fixative fluid artifacts for the investigation of human postmortem brain phantoms.

Phantoms are often used to assess MR system stability in multicenter studies. Postmortem brain phantoms best replicate human brain anatomy, allowing for a combined assessment of the MR system and software chain for data analysis. However, a wash-out of fixative fluid affecting T1 values and thus T1-weighted sequences such as magnetization-prepared 180 degrees radiofrequency pulses and rapid gradient-echo (MP-RAGE) has been reported for brain phantoms, hampering their immediate use. The purpose of this study was the creation of anatomical data that provide the characteristics of conventional data while avoiding this artifact. Two brain phantoms were scanned at several time points, acquiring conventional MP-RAGE data and quantitative T1 and proton density (PD) maps. Assuming a suitable cutoff value T1cut , synthetic MP-RAGE data were created from these maps, being T1-weighted for T1 > T1cut to reduce fluid signal in the sulci, but PD-weighted for T1 < T1cut for artifact suppression. A time-dependent artifact was observed in the T1 but not in the PD maps. The temporal stability of the synthetic data was greatly improved as compared to the conventional data. The proposed method enables anatomical imaging of postmortem brain phantoms, avoiding artifacts induced by the wash-out of fixative fluid, and thus achieving high signal stability shortly after fixation. Magn Reson Med 77:1115-1123, 2017. © 2016 International Society for Magnetic Resonance in Medicine.

The added value of undiluted vitreous biopsy samples processed by the Cellient® tissue processor (Hologic) in unsolved uveitis

PurposeIn this prospective study, the added value of undiluted vitreous biopsy samples in the diagnosis of unsolved uveitis was evaluated. Vitreous biopsies are difficult to handle because of the paucity of cells and the gelatinous structure of the vitreous. Histopathological analysis of the vitreous is useful in challenging cases to differentiate uveitis from lymphoma or infection and to define the type of cellular reaction.Methods97 consecutive undiluted vitreous samples were isolated in patients with unsolved intermediate or posterior uveitis. A 1.5–2.5 cc sample was taken through a single 23G or 27G port using the EVA vitrectomy platform (DORC) with a twin duty cycle high speed cutter. The samples were analysed with the Cellient® tissue processor (Hologic). This machine is a fully automated processor starting from a specified container with PreservCyt® (fixative fluid) with cells to paraffin. Routine histochemical and immunostainings were evaluated.ResultsIn 94.8% of the cases, sufficient material was found to provide an added value in the diagnostic workup. In 34%, a Cytolyt® mucolytic wash was necessary to prevent clotting of the tubes in the Cellient® tissue processor due to the viscosity of the sample. In 7% the diagnosis was an acute inflammation (presence of granulocytes), in 42% chronic active inflammation (presence of T‐lymphocytes), in 36% low‐grade inflammation (presence of CD68 cells, with &lt;5% T‐lymphocytes); and in 9% a malignant process (lymphoma). In 5% no diagnosis was found. In the chronic active inflammation group 39% was a granulomatous inflammatory process.ConclusionsThis standardized protocol for sampling and handling undiluted vitreous biopsies gives a superior result in morphology, number of cells, and possibility of immuno‐histochemical stainings. The diagnosis can be established or confirmed in 94.8% of cases.

The added value of undiluted vitreous biopsy samples processed by the Cellient® tissue processor (Hologic) in unsolved uveitis

PurposeIn this prospective study, the added value of undiluted vitreous biopsy samples in the diagnosis of unsolved uveitis was evaluated. Vitreous biopsies are difficult to handle because of the paucity of cells and the gelatinous structure of the vitreous. Histopathological analysis of the vitreous is useful in challenging cases to differentiate uveitis from lymphoma or infection and to define the type of cellular reaction.Methods97 consecutive undiluted vitreous samples were isolated in patients with unsolved intermediate or posterior uveitis. A 1.5‐2.5cc sample was taken through a single 23G or 27G port using the EVA vitrectomy platform (DORC) with a twin duty cycle high speed cutter. The samples were analysed with the Cellient® tissue processor (Hologic). This machine is a fully automated processor starting from a specified container with PreservCyt® (fixative fluid) with cells to paraffin. Routine histochemical and immunostainings were evaluated.ResultsIn 94.8% of the cases, sufficient material was found to provide an added value in the diagnostic workup. In 34%, a Cytolyt® mucolytic wash was necessary to prevent clotting of the tubes in the Cellient® tissue processor due to the viscosity of the sample. In 7% the diagnosis was an acute inflammation (presence of granulocytes), in 42% chronic active inflammation (presence of T‐lymphocytes), in 36% low‐grade inflammation (presence of CD68 cells, with &lt;5% T‐lymphocytes); and in 9% a malignant process (lymphoma). In 5% no diagnosis was found. In the chronic active inflammation group 39% was a granulomatous inflammatory process.ConclusionsThis standardized protocol for sampling and handling undiluted vitreous biopsies gives a superior result in morphology, number of cells, and possibility of immuno‐histochemical stainings. The diagnosis can be established or confirmed in 94.8% of cases.

Pengaruh Hidroksiquinolin Pada Pembuat Preparat Kromosom Akar dan Kalus Bawang Putih (Allium sativum L.)

The effective method in making chromosome prepararation was able to count the number of chromosome easier, especially for analysis the ploidy level in plants. Garlic ( Allium sativum L.) is one of the plants that the chromosome is difficult to be counted because their chromosomes are long and difficult to spread during the squashing. This research was to find out the effect of hydroxiquinoline on making chromosome preparation of root and callus in garlic. The steps of making chromosome preparation were fixation in Farmer’s fluid, put in hydroxiquinoline for an hour, hydrolysis in 1N HCl for two minutes at 60°C, put in Carnoy for 30 minutes, and stained in 1% aceto orcein for 20 minutes. The treatment were gift hydroxiquinoline for an hour after put in fixative fluid and the control were making preparation without gift hydroxiquinoline. The result of this research suggest that the method of making preparation by treatment with hydroxiquinoline for an hour after fixation produced the chromosomes that the stain were sharp and contrast, and also it produced well spread chromosomes that were clearly defined of both callus and root tip in garlic.

Fluid preservation: a comprehensive reference

Introduction Acknowledgements Part I. Fluid Preservation Techniques and Collections Chapter 1. History of Fluid Preservation Fluid Preservation in the Ancient World History of Ethyl Alcohol Origin of the Name Alcohol Glass Containers The Discovery of Preservation of Specimens in Ethyl Alcohol Early Instructions for Preserving Specimens in Fluids Later Instructions for Preserving Specimens in Fluids Fluid Preserved Collections Other Fluid Preservatives Summary Chapter 2. Fixation Fixation Formaldehyde Origin of the Names Formaldehyde, Formol, and Formalin Commercial Formaldehyde Formaldehyde as a Fixative and Preservative Fixative pH Range Narcotization Fixatives for Botanical Specimens Temperature, Time, and Rates of Penetration of Fixatives The Penetration-Fixation Paradox Lipids and Fixation Formaldehyde and Field Work Paraformaldehyde Post-Formaldehyde Fixation Washing Unwanted Effects of Formaldehyde Gluteraldehyde Aldehyde Safety Alternative and Proprietary Fixatives Chapter 3. Preservation Preservation without Fixation Freezing Transfer between Fluids Preservative Quality Old Fluid Preservatives Botanical Use of Fluid Preservation Glycerin Isopropyl Alcohol Fluid Preservation for DNA Extraction Evaporation Clearing and Staining Anatomical and Histological Fluid Preparations Mounting Specimens Inside Containers Glycol, Phenol, and Phenoxetol as Preservatives Novec Fluid Mineral Oil Universal Fixatives Criteria for Evaluating Alternative Fixative and Preservative Fluids Chapter 4. Effects of Fixatives and Preservatives on Specimens Changes in Body Dimensions and Biomass Changes in Color Solvent Extraction in Fixatives and Preservatives Chapter 5. Managing Fluid Preserved Collections Identification of Fluid Preservatives Checking Fluid Concentration Re-Use of Old Alcohol pH of Preservative Solutions Preparing Fixatives and Preservatives Containers and Seals Alternatives to Glass Containers Labeling The Storage Environment Topping Up and Replacing Preservatives Why do Closures Fail? Bacterial and Fungal Growth in Fluid Collections-Detection and Remediation Rehydration of Fluid Preserved Specimens Moving Collections Exhibition of Fluid Preserved Specimens Dealing with Old Containers and Old Specimens Repair of Damaged Fluid Preserved Specimens Health and Safety Fire Prevention Formaldehyde Safety Chapter 6. Fluid Preserved Collections as Cultural Patrimony Why Preserve Specimens in Fluid? The Fluid Preserved Human Fond Memories of Fluid Preservation Fluid Preservation in Visual Art Fluid Preservation in Literature Fluid Preservation in Film Fluid Preservation in Popular Culture The Aesthetics of Fluid Preservation Part II. Literature in this Field Chapter 7. Literature Cited. Chapter 8. Literature Reviewed but Not Cited Part III. Reference Tables Table 1. Fluid preservation techniques. Table 2. Timeline of milestones in published fluid preservation techniques. Table 3. Tissue matrix types. Table 4. Proprietary fixatives (based on manufacturer's MSDS, advertisements, and published analyses). Table 5. Narcotizing agents. Table 6. Disinfectant mechanisms of some preservatives (based in part on Volk and Wheeler 1984). Table 7. Summary of factors that affect the long-term usefulness of fluid preserved specimens (after Simmons 2002). Table 8. Timeline of the known introduction of chemicals in fluid preservation. Table 9. Anatomical fixation and preservation techniques. Table 10. Clearing and staining techniques. Table 11. Disinfectant mechanisms of some preservatives (based in part on van Dam 2003). Table 12. Criteria for identifying alternative preservative fluids (based in part on van Dam 2003). Table 13. Summary of fixative and preservative induced changes in invertebrates. Table 14. Summary of fixative and preservative induced changes in invertebrates by taxonomic group. Table 15. Summary of fixative and preservative induced changes in vertebrates by taxonomic group. Table 16. Summary of fixative and preservative induced changes in vertebrates by taxonomic group. Table 17. Proprietary preservatives (based on manufacturer's MSDS, advertisements, and published analyses). Table 18. Summary of published fluid concentration and pH testing of fluid preserved collections. Table 19. Characteristics of containers for fluid preserved specimens (based on Simmons 2002). Table 20. Oxygen permeablility of container materials. Table 21. Published recommendations for label substrates and inks. Table 22. Rehydration techniques for fluid preserved specimens. Table 23. Treatments and practices that are not recommended for fluid preserved specimens. Appendix Index About the Author

A new and standardized method to sample and analyse vitreous biopsies in unsolved uveitis by the Cellient® automated cell block system

AbstractPurpose In this prospective study a universal protocol for sampling and analyzing vitreous material was investigated. Vitreous biopsies are difficult to handle because of the paucity of cells and the gelatinous structure of the vitreous. Histopathological analysis of the vitreous is useful in difficult uveitis cases to differentiate uveitis from lymphoma or infection and to define the type of cellular reaction.Methods After isolation of vitreous content by a single port biopsy or by a standardized 23G vitrectomy, 170 consecutive vitreous samples were analyzed with the Cellient® tissue processor (Hologic). This machine is a fully automated processor starting from a specified container with PreservCyt® (fixative fluid) with cells to paraffin. Cytology was compared with fixatives Cytolyt® (contains a mucolytic agent) and Preservcyt®. Routine histochemical and immunostainings were evaluated.Results In 79 cases (46%) a single port biopsy and in 91 cases (54%) a tree port pars plana vitrectomy was performed. In 94% of the cases, sufficient material was found for establishing the diagnosis. In 18%, a Cytolyt® wash was necessary to prevent clotting of the tubes in the Cellient® due to the viscosity of the sample. In 21% the diagnosis was an acute inflammation (presence of granulocytes), in 32% chronic active inflammation (presence of T‐lymphocytes), in 35% low‐grade inflammation (presence of CD68 cells, without T‐lymphocytes); and in 5% a malignant process. In 9% a granulomatous inflammation was detected, in 5% glial tissue (floaters). In 6% no diagnosis was found.Conclusion A standardized protocol for sampling and handling vitreous biopsies, fixing in PreservCyt® and processing by the Cellient® system gives a superior result in morphology, number of cells, and possibility of immuno‐histochemical stainings. The diagnosis can be established or confirmed in more than 90% of cases.

A new and standardized method to sample and analyse vitreous samples by the Cellient automated cell block system.

In this prospective study, a universal protocol for sampling and analysing vitreous material was investigated. Vitreous biopsies are difficult to handle because of the paucity of cells and the gelatinous structure of the vitreous. Histopathological analysis of the vitreous is useful in difficult uveitis cases to differentiate uveitis from lymphoma or infection and to define the type of cellular reaction. Hundred consecutive vitreous samples were analysed with the Cellient tissue processor (Hologic). This machine is a fully automated processor starting from a specified container with PreservCyt (fixative fluid) with cells to paraffin. Cytology was compared with fixatives Cytolyt (contains a mucolyticum) and PreservCyt. Routine histochemical and immunostainings were evaluated. In 92% of the cases, sufficient material was found for diagnosis. In 14%, a Cytolyt wash was necessary to prevent clotting of the tubes in the Cellient due to the viscosity of the sample. In 23%, the diagnosis was an acute inflammation (presence of granulocytes); in 33%, chronic active inflammation (presence of T lymphocytes); in 33%, low-grade inflammation (presence of CD68 cells, without T lymphocytes); and in 3%, a malignant process. A standardized protocol for sampling and handling vitreous biopsies, fixing in PreservCyt and processing by the Cellient gives a satisfactory result in morphology, number of cells and possibility of immuno-histochemical stainings. The diagnosis can be established or confirmed in more than 90% of cases.

Brucella abortus Induces Collagen Deposition and MMP-9 Down-Modulation in Hepatic Stellate Cells via TGF-β1 Production

In patients with active brucellosis, the liver is frequently affected by histopathologic lesions, such as granulomas, inflammatory infiltrations, and parenchymal necrosis. Herein, we examine some potential mechanisms of liver damage in brucellosis. We demonstrate that Brucella abortus infection inhibits matrix metalloproteinase-9 (MMP-9) secretion and induces collagen deposition and tissue inhibitor of matrix metalloproteinase-1 secretion induced by hepatic stellate cells (LX-2). These phenomena depend on transforming growth factor-β1 induction. In contrast, supernatants from B. abortus-infected hepatocytes and monocytes induce MMP-9 secretion and inhibit collagen deposition in hepatic stellate cells. Yet, if LX-2 cells are infected with B. abortus, the capacity of supernatants from B. abortus-infected hepatocytes and monocytes to induce MMP-9 secretion and inhibit collagen deposition is abrogated. These results indicate that depending on the balance between interacting cells and cytokines of the surrounding milieu, the response of LX-2 cells could be turned into an inflammatory or fibrogenic phenotype. Livers from mice infected with B. abortus displayed a fibrogenic phenotype with patches of collagen deposition and transforming growth factor-β1 induction. This study provides potential mechanisms of liver immune response induced by B. abortus-infected hepatic stellate cells. In addition, these results demonstrate that the cross talk of these cells with hepatocytes and macrophages implements a series of interactions that may contribute to explaining some of mechanisms of liver damage observed in human brucellosis.