Good Quality Sections Research Articles

Durability Test of Microtome Blades with the High-Precision Tissue-Sectioning Machine

In this study, the durability of microtome blades, used for sectioning paraffin blocks, was evaluated with the goal of improving the quality of sections in pathology tests. First, for the durability test of microtome blades, a sectioning test device that realizes stable sectioning operations was developed. This device comprised precise stages supported by cross-roller guides, achieving sufficient rigidness. This device allowed automated repetitive sectioning and simultaneously measured the principal and thrust cutting forces. Samples embedding porcine kidney and rib tissues were used for the durability test. Two types of blades with different blade edge angles were used. Additionally, the rake face and cross-section of blades, as well as H&E-stained sections, were observed. In the durability test with porcine kidney tissue, good quality sections were obtained even after 100 times of sectioning with both microtome blades, showing sufficient durability. However, in sectioning porcine rib tissue, the microtome blade with a large blade edge angle produced good-quality sections in the initial phase of the durability test; however, defects such as overlapping of folds were observed after 100 times of sectioning. Meanwhile, the microtome blade with a small blade edge angle experienced blade damage from the early phase of the durability test, resulting in the production of unsuitable preparations for pathology tests. These results indicated that the microtome blade with a small blade edge angle lacked durability against hard tissues such as porcine ribs.

A feasible method for llama embryo paraffin embedding

Maternal recognition of pregnancy in llamas would be related to a still-unknown signal secreted by the embryo to extend the corpus luteum lifespan. In order to study llamas reproductive physiology and early pregnancy aspects, research on embryos between 8 and 12 days post-mating is essential. However, there is a lack of information on llama embryo paraffin embedding, a procedure that exhibits difficulties, mostly for embryo manipulation. This work presents an accessible, easy and rapid protocol for embedding llama blastocysts. Uterine flushings from four superovulated llamas were performed to recover embryos 8 days post-mating. Each embryo was fixed in 1 ml of 10 % neutral buffered formalin for 24 h, at 4 °C, and transferred to 70 % ethanol. Later, they were submitted to a series of baths: 500 µl PBS with 5 % bovine foetal serum solution (PBS+BFS) for 1 min; zona pellucida permeabilization with 500 µl PBS with 0.2 % Triton X-100 for 20 min; 500 µl PBS+BFS for 1 min; dehydration on baths with ascendent grades of ethanol (70 %, 96 %, and 100 % twice) for 10 min each; and bath of butanol for 5 min. Finally, embryos were embedded in a pellet of paraffin and afterwards in a paraffin block. Complete sections from 8 of 13 grade I and II embryos were obtained. In conclusion, by applying this method, good quality sections with preserved embryo morphology suitable for histological, histochemical or immunohistochemical approaches would be possible to acquire.

Evaluation of the efficacy of hematoxylin and eosin stain when xylene is completely replaced by turpentine or kerosene oil - A comparative study for oral tissues.

Microscopic examination of cells and tissues requires the preparation of very thin and good-quality sections mounted on glass slides and appropriately stained to demonstrate normal and abnormal structures. Before this step, the tissue must undergo preparatory treatment known as tissue processing. The various stages of tissue processing are dehydration, clearing, impregnation, and embedding, each with a particular duration for proper completion of the process. Xylene is the most frequently used clearing agent whose carcinogenic potential is well documented. Hence, attempts were made to substitute xylene with a biosafe clearing agent. The present study aimed to evaluate and compare the efficacy of hematoxylin and eosin stain (H and E stain) when xylene is completely replaced by turpentine or kerosene oil. A total number of 50 tissue samples were taken in the study, which included 40 study samples and 10 controls. All the samples were randomly separated into three groups and routine tissue processing and H and E staining were performed. The result was further subjected to statistical analysis by using Fisher's exact test. Group-1: Ten tissue samples were processed and H and E staining was done in xylene. Group-2: Twenty tissue samples were processed and H and E staining was done in turpentine oil. Group-3: Twenty tissue samples were processed and H and E staining was done in kerosene oil. Nuclear staining, cell morphology, and uniformity of staining were better in kerosene sections, while cytoplasmic and clarity of staining of turpentine sections were comparable with xylene sections. Turpentine and kerosene as clearing agents can be used in the future with certain modifications in their concentration and routine staining protocol.

Comparative Study of Microwave Oven-Assisted Tissue Processing with Isopropanol and Mineral Oil Mixture and Propylene Glycol Methyl Ether on Morphological Quality of Tissue Sections

Background: 
 Over several years, xylene has been traditionally utilised as the clearing agent of choice in tissue-processing due to effectiveness in rapidly clearing tissue, facilitating the paraffin infiltration process. However, xylene use adversely impacts the health of personnel with long term exposure due to toxicity. In order to overcome these effects and replace it with a safer alternative agent, the present study aims to compare quality of tissue sections processed using an isopropanol and mineral oil mixture and propylene glycol methyl ether (PGME) as xylene substitutes.
 
 Methods: 
 Rat skeletal muscle tissue samples (n=20) were prepared for each processing protocol with xylene substitutes. Tissue specimens were processed according to the proposed microwave protocol. The clearing steps were performed using isopropanol and mineral oil mixture, and PGME, replacing xylene. From each paraffin-embedded block, one section of 4-5µm thickness tissue was obtained and conventionally-stained with Haematoxylin and Eosin (H&E). The histological sections were microscopically assessed and scored by a pathologist. A qualitative analysis was performed with the results obtained.
 
 Results: 
 The overall score obtained for xylene processed tissue was 100% with a score of 2 for all the 3 parameters assessed. However, the outcome for tissue processed with isopropanol and mineral oil mixture was 28.6% unsatisfactory, 28.6% satisfactory and 42.8% good. In PGME-treated tissues, 14.3% were unsatisfactory sections, 71.4% were satisfactory and 14.3% produced good quality sections. Overall, tissues processed using both substitutes exhibited sufficient staining quality in terms of the aforementioned parameters as compared to xylene-processed tissues, though significant difference in scores were observed.
 
 Conclusion:
 Despite several challenges faced in the study, isopropanol and mineral oil mixture and PGME can be suggested as alternative clearing agents to xylene, provided having access to a more sophisticated microwave oven with precise temperature control for complete tissue-processing.

How Ground Penetrating Radar helps to understand the Nhecolândia lakes landscape in the Brazilian Pantanal wetland

Abstract The Pantanal wetland is an active sedimentary basin representing a relevant depositional setting for alluvial sedimentation studies. However, sedimentation homogeneity and the lack of outcrops makes sedimentary analysis more difficult. The Lower Nhecolândia is located at the Southern edge of the Taquari river megafan, whose genetic origin has been disputed as fluvial or eolian deposition. GPR analysis was used to characterize the subsurface stratigraphy and understand the region’s geomorphic evolution. The 100 MHz GPR provided continuous good quality sections up to a depth of 8 m. Two continuous reflections are disconformities that bound three depositional sequences characterized by distinct radar facies. The lower facies presents an upper erosional truncation followed by reflections presenting ∼1.5 m deep channelized forms and concave-up low amplitude reflections. The intermediate facies (∼4 m thick) presents a base with erosional truncation followed by concave-upward forms, ∼10 m wide, 1–3 m deep, separated by 1–2 m, and offlapping geometry. The upper facies has a flat base and thickness of 2–4 m, with parallel reflections; it shows a strong correlation between the radar facies and the forms preserved in the landscape, suggesting that channelized fluvial streams did not form them. The results obtained indicate that GPR use in the Pantanal is an important method to elucidate its geologic evolution.

Vibratome Sectioning of Plant Materials for (Immuno)cytochemical Staining.

A vibrating microtome is widely used to produce good-quality sections of plant organs or tissues. This method allows for an improved preservation of antigenicity and structure and is compatible with most (immuno)cytochemical staining procedures.

Comparison of four softening agents used on formalin-fixed paraffin-embedded nail biopsies with inflammatory disease

ABSTRACTObtaining high-quality sections of the nail plate poses a significant challenge to histopathology technicians world over. Nail is a heavily keratotic hard tissue that tends to split or tear while sectioning when processed and embedded in a routine manner. Many agents such as phenol, alcohol, and thioglycolate have been tried for the purpose of softening a variety of experimental materials. However, there is no clear consensus on any single agent. The study was conducted with the aim of evaluating and comparing the role of various compounds as softening agents for nail biopsies with inflammatory disease. Thirty paraffin-embedded nail biopsies were subjected to four softening agents: distilled water (DIH20), 30% potassium hydroxide (KOH), hair removal cream, and fabric conditioner. The ease of sectioning, the incidence of juddering (i.e. ‘venetian blind’ effect), and the shattering of tissue were recorded. Hematoxylin and eosin-stained sections were examined microscopically. Sectioning was very easy after using fabric conditioner, with good quality sections, and hair removal cream produced comparable results. The incidence of juddered, shattered sections after using hair removal cream was considerably higher (63.33%) compared to fabric conditioner-treated nails (16.67%). Microtomy of nail biopsies was found to be easiest after using 30% KOH with moderate section quality. DIH2O could neither allow easier sectioning nor obtain good sections for interpretation. Fabric conditioner and hair removal cream proved to be the effective keratin softeners, while 30% KOH worked effectively when the nail plate alone was submitted for histological examination.

Tissue Sampling and Processing for Histopathology Evaluation.

Histological procedures aim at providing good-quality sections that can be used for a light microscopic evaluation of tissue. These are applicable to identify either spontaneous or diseases-induced changes. Routinely, tissues are fixed with neutral formalin 10%, embedded in paraffin, and manually sectioned with a microtome to obtain 4-5 μm thick paraffin sections. Dewaxed sections are then stained with HE&S (hematoxylin-eosin and saffron) or can be used for other purposes (special stains, immunohistochemistry, in situ hybridization, etc.). During this processing, many steps and procedures are critical to ensure standard and interpretable sections. This chapter provides key recommendations to efficiently achieve this objective.

Respiratory rate estimation during triage of children in hospitals

Accurate assessment of a child’s health is critical for appropriate allocation of medical resources and timely delivery of healthcare in Emergency Departments. The accurate measurement of vital signs is a key step in the determination of the severity of illness and respiratory rate is currently the most difficult vital sign to measure accurately. Several previous studies have attempted to extract respiratory rate from photoplethysmogram (PPG) recordings. However, the majority have been conducted in controlled settings using PPG recordings from healthy subjects. In many studies, manual selection of clean sections of PPG recordings was undertaken before assessing the accuracy of the signal processing algorithms developed. Such selection procedures are not appropriate in clinical settings. A major limitation of AR modelling, previously applied to respiratory rate estimation, is an appropriate selection of model order. This study developed a novel algorithm that automatically estimates respiratory rate from a median spectrum constructed applying multiple AR models to processed PPG segments acquired with pulse oximetry using a finger probe. Good-quality sections were identified using a dynamic template-matching technique to assess PPG signal quality. The algorithm was validated on 205 children presenting to the Emergency Department at the John Radcliffe Hospital, Oxford, UK, with reference respiratory rates up to 50 breaths per minute estimated by paediatric nurses. At the time of writing, the authors are not aware of any other study that has validated respiratory rate estimation using data collected from over 200 children in hospitals during routine triage.

A ‘Waterfall’ Transfer-based Workflow for Improved Quality of Tissue Microarray Construction and Processing in Breast Cancer Research

A major focus in cancer research is the identification of biomarkers for early diagnosis, therapy prediction and prognosis. Hereby, validation of target proteins on clinical samples is of high importance. Tissue microarrays (TMAs) represent an essential advancement for high-throughput analysis by assembling large numbers of tissue cores with high efficacy and comparability. However, limitations along TMA construction and processing exist. In our presented study, we had to overcome several obstacles in the construction and processing of high-density breast cancer TMAs to ensure good quality sections for further research. Exemplarily, 406 breast tissue cores from formalin-fixed and paraffin embedded samples of 245 patients were placed onto three recipient paraffin blocks. Sectioning was performed using a rotary microtome with a "waterfall" automated transfer system. Sections were stained by immunohistochemistry and immunofluorescence for nine proteins. The number and quality of cores after sectioning and staining was counted manually for each marker. In total, 97.1 % of all cores were available after sectioning, while further 96 % of the remaining cores were evaluable after staining. Thereby, normal tissue cores were more often lost compared to tumor tissue cores. Our workflow provides a robust method for manufacturing high-density breast cancer TMAs for subsequent IHC or IF staining without significant sample loss.

Undecalcified Bone Preparation for Histology, Histomorphometry and Fluorochrome Analysis

Undecalcified bone histology demonstrates the micro-architecture of bone. It shows both the mineralised and cellular components of bone, which provides vital information on bone turnover or bone formation and resorption. This has tremendous importance in a variety of clinical and research applications. It yields beautiful images1 and allows for techniques such as fluorochrome assessment and histomorphometry2. Fluorochrome analysis is a technique where fluorescent dyes that bind to calcium are injected at a particular time point, which allows for quantification of the amount of mineralisation at that given time. Histomorphometry is a process of bone quantification at the microscopic level. Performing undecalcified bone histology is technically challenging, particularly with large size specimens. It requires variations in technique from those used in standard paraffin embedded histology. This video illustrates the process of producing good quality sections and demonstrates the technical difficulties and methods with which to overcome them. Specimen preparation, fixation and processing are achieved with a manner similar to other soft tissues, however due to the density and lower permeability of bone considerably longer fixation and processing times are required, often taking several weeks. Embedding is achieved using a supporting medium with similar or equal hardness and density to the bone such as methacrylate- based resins, but unlike paraffin infiltration and embedding, this is an irreversible step. Sectioning can be achieved by grinding which produces a thicker section, which is optimal for studies such as fluorochrome analysis. This is best achieved using a diamond blade on a macrotome. Alternatively, thinner sections can be produced for light microscopy and this is achieved using a sledge microtome with a very sharp blade. The sledge microtome provides the additional strength and stability required for large, hard blocks. Resin embedded sections can be stained with a variety of stains, which are demonstrated.

A comparison of different embalming fluids on the quality of histological preservation in human cadavers

There are significant problems in obtaining normal human material for histology for teaching or research purposes. This study shows that tissue from cadavers embalmed for teaching can be used for routine histology. Twelve cadavers embalmed with four different formalin-containing embalming fluids were used (n = 3 per fluid): (1) formalin mix (10% formalin); (2) Dunedin mix (an alcohol-based fluid containing phenol); (3) Michigan mix (a water-based fluid); and (4) phenoxyethanol mix (an alcohol-based fluid containing phenoxyethanol). Tissue blocks of liver, heart, kidney, skin and skeletal muscle were taken from each cadaver, paraffin embedded, sectioned and stained with haematoxylin and eosin (H & E), Periodic Acid Schiff (PAS), or Mallory trichrome (Malt). Each section was assigned an overall score based on the histological quality of the cellular components of the tissue. Sections were scored from 1 to 3 (1 = poor, 2 = satisfactory, 3 = good). Satisfactory sections were obtained from all cadavers except those embalmed with the Dunedin mix. The Michigan and phenoxyethanol fluids resulted in consistently good quality sections. No significant differences in tissue morphology were observed between the different stains. The clearest morphology was observed in the skin and skeletal muscle sections, and in tissues embalmed with fluids which do not contain phenol.

Processing rodent embryonic and early postnatal tissue for in situ hybridization with radiolabelled oligonucleotides

Publisher Summary The expression pattern of a gene during development is a valuable piece of information. Reliable and presentable data require good quality sections from accurately aged animals. Practice and care are necessary to dissect, manipulate, and prepare the delicate tissues from these stages without damage or distortion. This chapter describes steps and hints useful for preparing embryonic and early postnatal (P0-P20) tissue for the in situ hybridization (ISH) protocol with radiolabeled oligonucleotides. Following on from the genome sequencing projects, it is believed that this particular method with radiolabeled oligonucleotides is ideally suited for large-scale mapping of gene expression during development. The X-ray film method permits a large number of gene expression patterns to be simultaneously compared. Even at the level of X-ray film analysis, quite detailed information on changes in gene expression can be gleaned. For small embryos whole-mount ISH with digoxigenin (DIG) riboprobes is more appropriate.

Departmental audit in surgical anatomical pathology

Internal auditing of performance by pathology providers is a necessary component of total quality management. In this study a peer review of 10% of departmental surgical anatomical pathology accessions received over a seven month period was performed. A number of critical performance parameters were analysed including turn-around times, accuracy of reports and technical proficiency. The results demonstrated an approximate 2% significant error rate in macroscopic and microscopic descriptions, technically good quality sections and stains and generally satisfactory turn-around times. The value and costing of such an audit and changes initiated by the audit are discussed.

Nuclear Bubbling: An Overlooked Artifact

AbstractSporadic appearance of a nuclear artifact had hampered accurate microscopic evaluation of lymph node and bone marrow biopsies. Although troubleshooting included specimen handling, fixation and staining, all changes in methodology failed to produce consistently good quality sections. Improved morphologic detail was attained only after blocks were recut and sections were completely dried before heat-affixation. Complete drying of sections before heat-affixation was the only factor that consistently prevented the artifact. (The J Histotechnol 13:135, 1990).