Fixation Fluid Research Articles

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

AbstractPurpose 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 reactionMethods 170 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 evaluatedResults 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 processConclusion A standardized protocol for sampling and handling vitreous biopsies, fixing in PreservCyt® and processing by the Cellient® 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

PTH-033 A Systematic Approach to Standard Endoscopic Sampling of Bile Duct Strictures is Highly Accurate in the Diagnosis of Biliary Strictures

IntroductionAccurate diagnosis of bile duct strictures as malignant or benign is imperative for optimal patient management, but is frequently difficult. Histological and cytological samples can be obtained at ERCP. Various...

A new and standardised method to sample and analyse vitreous biopsies

AbstractPurpose To investigate and prepare a universal protocol for sampling and analysing vitreous material. Vitreous biopsies are difficult to handle because of the paucity of cells, the gelatinous formation and structure of the vitreous, the low frequency of biopsies and the burden of long‐during, delicate handwork by the technician.Methods After a standardised 23gauge vitrectomy, 50 consecutive vitreous samples were analysed with the Cellient® tissue processor (Hologic). This machine is a fully automatic processor from a specified container with PreservCyt® (fixative fluid) with cells to paraffin. Cytology was compared with fixatives Cytolyt® ( contains a mucolyticum) and Preservcyt®.Results In 96% (48 of 50 cases) sufficient material was found for diagnosis. Cytolyt® wash was necessary in 15% of cases to prevent clotting of the delicate tubes in the Cellient®, this procedure causes a loss of cellular material. Immuno‐histovhemical stainings were equal in quality with both preservatives. Labour hours of processing by technician was diminished by 4, compared with former, not‐standardised techniques.Conclusion A standardised protocol for sampling and handling viteous biopsies by a 23G vitrectomy, fixing in PreservCyt® and processing by the Cellient® gives a superior result in morphology, number of cells, possibility of immuno‐histochemical stainings and technician labour hours.

Is an alcoholic fixative fluid used for manual liquid-based cytology accurate to perform HPV tests?

Is an alcoholic fixative fluid used for manual liquid-based cytology accurate to perform HPV tests? Christian Garbar1, Corinne Mascaux1, Philippe De Graeve2, Philippe Delvenne31Department of Biopathology, Institute Jean Godinot, Reims Cedex, France; 2Centre de Pathologie des Coteaux, Toulouse, France; 3Department of Pathology, University of Liege, Tour de Pathologie, Domaine Universitaire du Sart Tilman, Liège, BelgiumAbstract: In Europe, the alternative centrifuge method of liquid-based cytology is widely used in cervical screening. Turbitec® (Labonord SAS, Templemars, France) is a centrifuge method of liquid-based cytology using an alcoholic fixative fluid, Easyfix® (Labonord). It is now well accepted that the association of liquid-based cytology and human papillomavirus test is indissociable of cervical screening. The aim of this work was to demonstrate that Easyfix alcoholic fluid is reliable to perform Hybrid Capture® 2 (QIAGEN SAS, Courtaboeuf, France). In this study, 75 patients with colposcopy for cervical lesions served as gold standard. A sample was collected, at random, for Easyfix fixative cytological fluid and for Digene Cervical Sampler (QIAGEN). The results of Hybrid Capture 2 (with relative light unit >1) showed no statistical difference, a positive Spearman’s correlation (r = 0.82, P < 0.0001), and a kappa value of 0.87 (excellent agreement) between the two fluids. It was concluded that Easyfix is accurate to use in human papillomavirus tests with Hybrid Capture 2.Keywords: human papillomavirus, hybrid capture 2, Turbitec®, cervix cytology, liquid-based cytology

Cancer Biomarkers: Closer to Delivering on their Promise

Taguchi etal. describe, in this issue of Cancer Cell, a quantitative comparative biomarker discovery approach that integrates animal lung cancer models with validation in well-controlled human clinical study sets. This approach overcomes many of the major barriers that have held back the field of cancer biomarkers in the past.

Complications Associated with Posterior Lumbar Surgery

Complications associated with posterior lumbar surgeries include pseudarthrosis, loss of fixation, instrumentation malposition, and cerebrospinal fluid leakage. An understanding of the associated risk factors and their incidences can help to reduce the likelihood of complications. Adhering to meticulous operative technique is also essential. Once complications occur, establishing the diagnosis and tailoring treatment to each individual patient can help to reduce the risk for progression of symptoms and can limit additional complications.

Effects of three different preservation methods on the mechanical properties of human and bovine cortical bone

Background and purpose In the development of new strategies for fracture fixation, new methods have to be tested biomechanically under in vitro conditions before clinical trials can be performed. The gold standard for laboratory evaluations is fresh-frozen specimen. As the availability of fresh-frozen specimens is limited and since their use bears infectious risks, specimens treated with various chemical embalming fluids are also used. These preservation methods may alter the mechanical properties of the specimens used. Therefore, the aims of the present study were to determine the effects of three different preservation methods (formalin fixation (FO), Thiel-fixation (TH), and alcohol–glycerine fixation (AG)) on the elastic and postyield mechanical properties of cortical bone and to compare these properties to those of fresh-frozen (FF) specimens. Materials and methods Cylindrical cortical specimens (diameter 3 mm, length 60 mm) were obtained from human femurs ( n = 48) and bovine tibiae ( n = 40). Before specimen immersion in different fixation fluids, bone mineral density (BMD) as well as the initial Young's modulus was determined. The Young's modulus was determined in a nondestructive bending test, and measurements were repeated after 6 months of immersion in fixative solution. Subsequent to the nondestructive test, a destructive 3-point bending test was conducted to assess the postyield and fracture properties. Results The BMD as well as the initial Young's modulus showed no significant differences between the four test groups. After 6 months in fixative solution, the Young's modulus was significantly lowered in human Thiel specimens and only showed minor changes in formalin- and alcohol–glycerine-treated specimens. The plastic energy absorption of human and bovine specimens was altered significantly. Formalin as well as alcohol–glycerine fixation yielded a significant decrease in plastic energy absorption, whereas Thiel fixation significantly increased the plastic energy absorption. Discussion/conclusion Because of the significantly altered plastic mechanical properties of cortical bone, the use fresh-frozen bone specimens is recommended in biomechanical studies investigating failure loads of orthopaedic implants. The use of embalmed specimens should be restricted to pilot tests.

Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement

The mechanical properties of soil (cohesion, friction, stiffness and permeability) are important parameters for engineering constructions and ecosystems in sedimentary environments. BioGrout is an in situ soil strengthening technique involving microbial-induced carbonate precipitation (MICP). This process involves hydrolysis of urea by bacteria containing the enzyme urease in the presence of dissolved calcium ions, resulting in calcium carbonate precipitation. In order to control the BioGrout process for engineering applications, it is necessary to improve understanding of the relevant phenomena and develop efficiencies to enable up-scaling of the technology to suit commercial applications. Control of a homogeneous distribution of bacterial activity in a sand bed is considered crucial in order to prevent clogging during injection and provide homogeneous reinforcement results. This paper describes a methodology to distribute and fix bacteria (with their enzyme activity) relatively homogeneously in a sand bed, before supplying cementation reagents. The methodology is based on a two-phase injection procedure: a bacterial suspension is injected into the sand body, immediately followed by a fixation fluid (i.e. a solution with high salt content). It is proposed that bacteria are retarded by adsorption and filtration processes and are permanently adsorbed to the sand grains when overtaken by the fixation fluid. The presented experimental approach for optimizing bacterial fixation in porous media can be used as a tool to design the treatment protocol for engineering applications in practice.

Validation of T2* Technique in Ex-Vivo Myocardial Tissue.

BackgroundThe T2* technique has been validated as a non-invasive measurement of myocardial iron load. However, calibration of the technique against myocardial tissue remains to be determined. Since such calibration is easiest to perform using post-mortem tissue, it is useful to know the effects of storage of the tissue in formalin, and in particular whether iron leaks from the heart during storage, and whether fixation alters the myocardial T2* significantly.MethodA heart was donated after death of a patient with sideroblastic anemia who had been poorly compliant with deferoxamine treatment and died in heart failure. The pre-mortem myocardial T2* was 5.2 ms indicating severe myocardial iron loading. The majority of the heart was assigned to a calibration study of myocardial iron, but 2 samples from the heart were available for this study.Sample 1 was an apical short-axis slice of the left ventricle. This was preserved in formalin for 3 days prior to measurement of the initial post-mortem myocardial T2* which was 5.2 ms (Siemens Sonata 1.5T MR scanner). The T2* of the slice was then measured weekly for 4 weeks; then 2 weekly for 8 weeks; and finally 4 weekly.Sample 2 was a 7.35 g piece of apical myocardium which was stored in 280ml formaldehyde. A 1ml sample of formaldehyde solution was taken for iron analysis from sample 2 initially and at 2 days, and then weekly for 4 weeks; two-weekly for 6 weeks; and then 4-weekly for 12 weeks. The iron concentration in the formalin was measured using Inductive Coupled Plasma with Mass Spectometry (ICPMS).ResultsThe results from sample 1 are shown as myocardial T2* against days elapsed. The T2* rose from a pre-mortem and post-mortem (equivalent values) of 5.2ms to a mean of 6ms over the first 20 days, and after this was stable (figure 1).The results from sample 2, show an increase from 16.6 microgram to 74.9 microgram over the initial 3–4 weeks after which little significant additional accumulation occurred (figure 2). Estimation of the total iron content of the myocardial sample (prior to destructive iron measurement at the end of the study) suggested that the total iron leaked into the formalin was 0.44% of the iron in the myocardial sample.ConclusionThere was a small increase in myocardial T2* over the first 20 days of formalin fixation, but little significant change after that. This was associated with a loss of approximately 75 micrograms of iron into the formalin fixation solution, which was estimated as only 0.44% of the iron in the myocardial sample. This suggests that formalin fixation has a small but measurable effect on myocardial T2* over time, and this is not explained by loss of iron into the fixation fluid. [Display omitted] [Display omitted]

Cotton Block Method

To describe the cotton block method, an easy, inexpensive, 1-step method of obtaining a cell block after fine needle aspiration biopsy.Before connection to a 10-mL syringe, the plastic hub of a 22-23-gauge needle is filled with the 3-4-mm, woolly tip of a cotton bud. Aspiration is performed as described elsewhere. After smear preparation, the material remaining in the needle and the material retained in the cotton wool mesh are immediately fixed by aspiration ofa fixative fluid (70% alcoholic formaldehyde-acetic acid). After fixation, the cotton tip is removed and routinely processed for paraffin embedding, and the sections are stained by routine methods used in cytopathology.The results demonstrate that the amount and quality of material obtained in the cotton wool tip is similar to that in the traditional cell block obtained from the pellet after centrifugation of aspirated fluid.The method is easy to perform and cost effective and is a rapid way to prepare cell blocks of high quality, allowing special staining techniques and improving cytohistologic correlation.

Changes in trabecular bone structure in rickets and osteomalacia: the potential of a medico‐historical collection

AbstractIn this study lumbar vertebral bodies with and without disturbed mineralization from a medico‐historical collection of the 19th century (housed in the Federal Museum for Pathological Anatomy, Vienna) were investigated, using non‐invasive radiological and invasive histological techniques. Macerated, or partly dissected, or fixation fluid conservated skeletal preparations from 61 individuals with known age and gender were examined. The specimens of 37 individuals had gross pathologic alterations from rickets or osteomalacia. The specimens from 24 individuals without Known bone disease served as age‐matched controls. Bone structure and mineralization in all specimens were evaluated by non‐invasive techniques (conventional radiographs and computed tomography). Thirteen selected specimens from both groups were also analysed by quantitative computed tomography (qCT) and dual‐energy X‐ray absorptiometry (DEXA). In these cases invasive techniques (light and scanning electron microscopy [backscattered electron (BSE)‐ and secondary electron (SE)‐mode] on surface‐stained and carbon‐sputtered undecalcified histological ground sections) were applied. Corresponding microradiographs were also used for computer‐assisted morphometry. The clinically established method of determination of bone mineral densities (BMD) by qCT and DEXA failed in these historical specimens, but all other non‐invasive and invasive techniques were successfully applied and yielded results corresponding to radiological changes and histomorphometric parameters. In the rachitic vertebrae it was not volume densities (bone volume per tissue volume = BV/TV), but numbers of trabeculae (trabecular number = Tb.N.) which were increased, compared to the age‐matched vertebral bodies without bone disease. In osteomalacic vertebrae the volume densities (BV/TV) were decreased in the whole vertebral body, while Tb.N. were only decreased in the middle third of the vertebral body. Accordingly, surface densities (SV) in non‐adults, and also specific surfaces (S/V) in adults were increased, suggesting increased bone resorption. Depending on the preparation technique used in the present study, resorption lacunae and characteristic unmineralized osteoid seams could be demonstrated. In conclusion, it might be useful to establish such techniques and standards for the diagnosis of disturbed mineralization in clinical patients. Copyright © 2003 John Wiley & Sons, Ltd.

The Centriole-Centrosome Complex is Affected by Microgravity During Cell Division and in Cilia of Sea Urchin Embryos: Results from Space Flight Experiments

Sea urchins have been used for over a century as a remarkable animal model system in which to study cell, molecular, and developmental biology. The studies presented here have used sea urchin eggs and embryos for pioneering experiments to explore the effects of microgravity on the cytoskeleton during a space flight on the space shuttle Endeavor. The culture conditions followed those described previously utilizing the Aquatic Research Facility (ARF) to fertilize and culture eggs and embryos up to the pluteus stage under controlled temperature (12°C) and fixation conditions. To achieve a final fixation with 0.5% glutaraldehyde and 4μM taxol, concentrated fixation fluid was injected at preselected time points to preserve microtubules, centrioles, centrosomes, microfilaments, mitochondria, and cell membranes.The analysis of the results revealed that the centriole-centrosome complex during cell division and cilia formation showed alterations in samples that had been exposed to microgravity while control cells cultured in a centrifuge at lg in space and those cultured on ground appeared normal.

The Human Patella: A Comparison of Three Preparation Methods

consuming and serial sections were difficult to acquire with the paraffin tape-transfer technique. We also investigated procedures to reduce processing time by drilling shallow holes in the anterior surface to improve the penetration of fixative and decalcification fluids. The processing time was significantly reduced from 50 to 32 days. (The J Histotechnol 21:151, 1998)

Healing of a severe microarterial trauma: An experimental study

A severe microarterial trauma (arteriotomy/intimectomy) was introduced to the central arteries of rabbit ears. Twenty-one patent vessels, 3 at each interval of 2 hours, 1, 3, 7, 14, 30, and 90 days after reperfusion were taken for light and scanning electron microscopy studies. These vessels remained patent at all time intervals. A physiological "in vivo" perfusion technique with fixation fluid of normal temperature (37 degrees C), pressure (120 mm Hg) and osmolarity (300 mOsm) was used to ensure fixed relaxed vessels. The endothelial, internal elastic, and part of the medial layers of vessel walls were removed following intimectomy. The inner surface of the intimectomy region was irregular and partly covered with thrombotic materials between 2 hours and 3 days postreperfusion. Endothelialization of the traumatized region was first observed at 3 days and was completed between 7 and 14 days. Intimal hyperplasia developed during this period and had not diminished in thickness by 30 or 90 days. The trauma and healing process of the arteriotomy/intimectomy are similar to the healing of vascular anastomoses and virtually identical to the clinical trauma of vessel avulsion, but neointima formation is more pronounced and engages vessel walls far outside the intimectomy site.

Immunohistochemical localization of bovine placental retinol-binding protein.

An immunogold staining method was used in combination with epipolarization microscopic detection to demonstrate the presence of bovine placental retinol-binding protein in bovine extraembryonic membranes. Amnion, chorion and allantois were fixed in Bouin fixation fluid and embedded in polyethylene glycol 1500. Sections (5 mm) were cut and transferred onto Digene silanated slides and immunostained using rabbit antiserum raised against bovine placental retinol-binding protein followed by goat anti-rabbit IgG labeled with 1 nm gold. Gold particles after silver enhancement were viewed and photographed under epipolarization microscopy. Epithelial cells of all three membranes (i.e. amniotic ectoderm, chorionic trophectoderm, and allantoic endoderm) were immunoreactive, while mesodermal cells, collagen, and blood cells were not. These data, together with our previous observation that these three placental membranes synthesize and secrete retinol-binding protein, indicate that epithelial cells lining the amnion, chorion and allantois are the major sources of this protein. The presence of retinol-binding protein in placental membranes and their fluids may be indicative of an important role for retinol in placental differentiation and development.

Microwave fixation of water-cooled insect tissues for immunohistochemistry

In this paper a new technique for microwave-accelerated tissue fixation and washing is described. The temperature of the irradiated specimens is controlled by means of a specially designed water-cooling device. Two types of fixation fluids, one of them containing rather concentrated picric acid, and a washing procedure were tested empirically on dissected nervous tissue of the Colorado potato beetle, Leptinotarsa decemlineata (Say). Entire beetles were processed in a similar way. It was shown that specimens could be irradiated as long as was required for good fixation and washing results, without the accumulation of excessive heat. No differences in morphology and immunoreactivity were observed when compared with standard immersion-fixed controls. A considerable reduction in processing time was achieved.

New Design for Fixation of Surgically Obtained Lung Specimens

A new design for lung fixation at a constant transpulmonary pressure is described. The head pressure of the apparatus can easily be set in a range from 15 to 95 cm H2O and maintained at a constant level through the recirculation of the fixative fluid. Moreover, the dimensions of the device are smaller than those previously reported. To analyze the efficiency of the equipment the estimated total post-fixing lung volume (TLV) in 12 surgically obtained lung specimens was calculated and compared with helium single-breath total lung capacity measurements (TLCsb). The TLV/TLCsb ratio was 102 +/- 5%. In addition, TLV was related to predicted TLC values (TLCpred) from different reference equations. The TLV/TLCpred ratios ranged from 89 to 99%. Estimated volumes of formalin fixed lungs did not show significant differences to those obtained by TLCsb measurements (p = 0.49). Consequently, it can be concluded that the fixation procedure performed with this device does not produce changes in air space dimensions of surgically obtained lung specimens. Moreover, the variability in TLV/TLC ratios depends upon either the technique of TLC measurement or the set of predicted values used.

Quantitative Pathology Today — A Technical View

This paper gives an overview of the different techniques which are currently used in quantitative pathology. Morphometry is used to denote the interactive quantitative analysis of single cells or tissue sections by means of an eyepiece graticule, a projection microscope or a graphic tablet. It is a simple, inexpensive, relatively fast technique, and allows the quantitation of both cell and architectural (tissue) properties. Standard cell and tissue preparations can be employed for such investigations. This requires (minimal) standardization of the cell and tissue handling process. The degree of acidity (pH) of the fixation fluid, such as "neutral" (or buffered) formalin is probably the most important and easy to measure factor, and should be kept between 6 and 8 in order to obtain reproducible nuclear area measurements. Another important factor for reproducible results is the magnification in relation to the size of the particles measured. Careful selection of relevant areas, cells and nuclei done by a skilled pathologist, is often essential, as well as quality control of the measuring process. In static cytometry, a relatively popular application is the measurement of the DNA content of single cells in slides. Although having the advantage of optical control, the measuring technique of the transmission systems is, at the present state of development, laborious. This restricts the measurable number of cells to one hundred or only a few hundred at maximum. As a result, the reproducibility rate, as well as the capacity to detect small differences is only moderate.(ABSTRACT TRUNCATED AT 250 WORDS)

Postmortem recognition and specimen fixation in early congenital and experimental limb deformities.

A method is recommended that facilitates the postmortem assessment of early stages of congenital or experimental paralytic and postural limb deformities, which otherwise are difficult to demonstrate on small specimens. One of the early signs detectable is change in the range of passive movements in the limb, and for its fine and objective assessment it is recommended that limb weight and gravity be used. Any disturbance of the original in vivo shape and position of the extremity during fixation of the postmortem specimen is prevented by suspension and free floating in the fixation fluid.

Fluorescent compounds as retrograde tracers compared with horseradish peroxidase (HRP). II. A parametric study in the peripheral motor system of the cat

Fluorescent compounds which are currently used as retrograde tracers were tested in the cat peripheral motor system and compared with horseradish peroxidase (HRP). The tracers were either injected into forelimb muscles or applied to the proximal end of transected forelimb nerves. The remaining muscles of the limb had been carefully denervated. Following intramuscular injection all fluorescent compounds labeled spinal cord motoneurons, the DAPI compounds labeled endothelial cells in addition. In the nerve application mode tracer positive motoneurons were only observed when propidium iodide (PI) and the DAPI compounds were used, whereas bisbenzimide (BB), nuclear yellow (NY) and primuline did not label any cells. The fluorescence of BB labeled motoneurons was predominantly located in the cytoplasma. NY positive motoneurons showed a different localization of the fluorescent label between the different neurons of the same motornucleus: in some neurons it was exclusively located in the nucleus, in others predominantly in the cytoplasma, in the majority in both compartments. The intracellular distribution of the BB and the NY label was independent of the pH of the fixation fluid. The fluorescent tracers labeled the motoneuronal cell columns in their complete rostrocaudal extent and in a position identical to the one obtained with HRP. However, some substances (PI, fast blue) labeled less motoneurons of a motornucleus than did HRP, none of the fluorescent tracers labeled more. The results are discussed under several aspects: use of the investigated fluorescent compounds as single tracers; use of several tracers in the same animal to map collateral projections of one and the same neuron; use of several tracers in the same animal to establish the topographical relation between several independent neuronal populations.