The choice of embedding media affects image quality, tissue R2* , and susceptibility behaviors in post-mortem brain MR microscopy at 7.0T.

PURPOSE: The quality and precision of post-mortem MRI microscopy may vary depending on the embedding medium used. To investigate this, our study evaluated the impact of 5 widely used media on: (1) image quality, (2) contrast of high spatial resolution gradient-echo (T1 and T2* -weighted) MR images, (3) effective transverse relaxation rate (R2* ), and (4) quantitative susceptibility measurements (QSM) of post-mortem brain specimens. METHODS: Five formaldehyde-fixed brain slices were scanned using 7.0T MRI in: (1) formaldehyde solution (formalin), (2) phosphate-buffered saline (PBS), (3) deuterium oxide (D2 O), (4) perfluoropolyether (Galden), and (5) agarose gel. SNR and contrast-to-noise ratii (SNR/CNR) were calculated for cortex/white matter (WM) and basal ganglia/WM regions. In addition, median R2* and QSM values were extracted from caudate nucleus, putamen, globus pallidus, WM, and cortical regions. RESULTS: PBS, Galden, and agarose returned higher SNR/CNR compared to formalin and D2 O. Formalin fixation, and its use as embedding medium for scanning, increased tissue R2* . Imaging with agarose, D2 O, and Galden returned lower R2* values than PBS (and formalin). No major QSM offsets were observed, although spatial variance was increased (with respect to R2* behaviors) for formalin and agarose. CONCLUSIONS: Embedding media affect gradient-echo image quality, R2* , and QSM in differing ways. In this study, PBS embedding was identified as the most stable experimental setup, although by a small margin. Agarose and Galden were preferred to formalin or D2 O embedding. Formalin significantly increased R2* causing noisier data and increased QSM variance.

A Novel Mohs Precision Tool.

BACKGROUND: Effective treatment by Mohs micrographic surgery requires preparation of high-quality slides. OBJECTIVE: To examine a novel tissue alignment device designed to address variability in tissue processing because of excessive sample trimming. MATERIALS AND METHODS: A device was designed to account for angular errors and unparalleled tissue embedding. A retrospective chart review was performed both with and without the use of the device over the course of a 4-year period (2012-2015). RESULTS: Between January 1, 2012, and June 10, 2014, before device implementation, mean number of stages per case was 1.65 (n = 3,680) and mean number of surgeries per day was 6.34 (n = 640). Between June 11, 2014, and October 02, 2015, with device implemented, the average number of stages per case between decreased to 1.58 (n = 2,562) and the number of daily surgeries increased to 7.05 (n = 358). This represents a significant decrease in number of stages per case by 0.07 stages (95% CI: -0.01 to -0.13, p = .02), as well as an increase in the number of cases per day by 0.71 cases (95% CI: 0.12-1.3, p < .01). CONCLUSION: Slide preparation using the novel alignment device may result in less tissue waste and more cases being performed daily.

[THE TECHNOLOGY "CELL BLOCK" IN CYTOLOGICAL PRACTICE].

The article presents summary information concerning application of "cell block" technology in cytological practice. The possibilities of implementation of various modern techniques (immune cytochemnical analysis. FISH, CISH, polymerase chain reaction) with application of "cell block" method are demonstrated. The original results of study of "cell block" technology made with gelatin, AgarCyto and Shadon Cyoblock set are presented. The diagnostic effectiveness of "cell block" technology and common cytological smear and also immune cytochemical analysis on samples of "cell block" technology and fluid cytology were compared. Actually application of "cell block" technology is necessary for ensuring preservation of cell elements for subsequent immune cytochemical and molecular genetic analysis.

Modified improvised pre-embedding method for core needle biopsies: A clinicopathologic study.

BACKGROUND: An optimal core needle biopsy (CNB) is expected to balance between tissue diagnosis, the accuracy of negative sampling, and concordance with reports from resected specimens to select the appropriate treatment. Though various techniques for CNBs are available, no guidelines exist for processing CNB, with practices varying from lab to lab for transport and processing. This prospective study aims to design a cost-effective, user-friendly pre-embedding method for CNBs to yield intact cores. OBJECTIVE: To compare the outcomes of CNBs by a conventional method with those processed by the modified pre-embedded processing protocol over 2 years. MATERIAL AND METHODS: Presurgical CNBs from SOL in various organs were subjected to the conventional free-floating method in formalin (control) for histopathology diagnosis. CNBs from the corresponding, freshly resected SOLs (test) were taken, inked with coloring inks if multiple, placed between two 2 × 2 cm polyurethane foam meshes fitted inside cassettes, fixed in formalin, and transported to the laboratory. The two CNB groups were coded and scored independently for intactness, tissue processing, ease of embedding, and ease of cutting sections. Data obtained were statistically analyzed. RESULTS: Test CNB cores were better processed, intact, linear, and aligned, compared to control CNBs. With four CNBs in one block, the number of blocks and sections were cut-down by one-fourth. CONCLUSION: CNBs processed using polyurethane foam and coloring inks were superior and economical against conventional free-floating CNBs. This technique can be practiced by surgeons at the bedside.

Sectionable cassette for embedding automation in surgical pathology.

Embedding automation can be a step ahead in histology processing development. Among advantages in replacing time-consuming manual embedding, the possibility of the final specimen orientation by the grossing person is very attractive for surgical pathology. There is not yet a satisfactory technological solution for 2 main problems in the design of a sectionable cassette for biopsy specimens and small specimens: maintaining the orientation of the sample at the end of grossing and substitute fine skill manual alignment of the sample at the surface of the block for microtomy. The technical note presents attempts to solve these problems in the design of sectionable cassette. The latest sectionable cassettes by Sakura Finetek for shave and core biopsy specimens are discussed in detail.

Application of large format tissue processing in the histology laboratory.

In clinical, research and veterinary laboratories of North America, large format histology has more recently been improved with newer equipment and better methodology. Large tissue specimens are frequently sliced in the grossing room and processed in multiple smaller, standard size tissue cassettes. Justifiably, submitting more blocks inherently lends itself to a greater confidence in the accuracy of the diagnosis, yet guidelines for tissue sampling often suggest taking fewer samples. For example, large tumor specimen protocols recommend taking one standard-sized tissue block for each cm diameter of tumor. However, cancers are the culmination of many complex changes in cell metabolism and often appear dissimilar at different tissue locations. As these changes have an uncertain behavior, many other tissue samples are often taken from areas that appear to have either a variable texture or color. Consequently, at microscopy, the complete tissue sample may need to be reassembled like a jigsaw puzzle as the stained sections are frequently presented over many slides. This problem has easily been overcome by using large format cassettes since the entire cross-section of the tissue sample can often be viewed on a single slide. Because these cassettes can effectively hold up to 10 times the volume of conventional standard size cassettes, they are a more efficient way of assessing large areas of tissue samples. This system is easily adapted for all tissue types and has become the established method for assessing large tissue samples in many laboratory settings.

Suitability of the CellientTM cell block method for diagnosing soft tissue and bone tumors.

BACKGROUND: The diagnosis of tumors of soft tissue and bone (STB) heavily relies on histological biopsies, whereas cytology is not widely used. CellientTM cell blocks often contain small tissue fragments. In addition to Hematoxylin and Eosin (H&E) interpretation of histological features, immunohistochemistry (IHC) can be applied after optimization of protocols. The objective of this retrospective study was to see whether this cytological technique allowed us to make a precise diagnosis of STB tumors. METHODS: Our study cohort consisted of 20 consecutive STB tumors, 9 fine-needle aspiration (FNAC) samples, and 11 endoscopic ultrasonography (EUS) FNACs and included 8 primary tumors and 12 recurrences or metastases of known STB tumors. RESULTS: In all 20 cases, H&E stained sections revealed that diagnostically relevant histological and cytological features could be examined properly. In the group of 8 primary tumors, IHC performed on CellientTM material provided clinically important information in all cases. For instance, gastrointestinal stromal tumor (GIST) was positive for CD117 and DOG-1 and a PEComa showed positive IHC for actin, desmin, and HMB-45. In the group of 12 secondary tumors, SATB2 was visualized in metastatic osteosarcoma, whereas expression of S-100 was present in 2 secondary chondrosarcomas. Metastatic chordoma could be confirmed by brachyury expression. Two metastatic alveolar rhabdomyosarcomas were myf4 positive, a metastasis of a gynecologic leiomyosarcoma was positive for actin and estrogen receptor (ER) and a recurrent dermatofibrosarcoma protuberans expressed CD34. CONCLUSION: In the proper clinical context, including clinical presentation with imaging studies, the CellientTM cell block technique has great potential for the diagnosis of STB tumors.

DNA and RNA isolated from tissues processed by microwave-accelerated apparatus MFX-800-3 are suitable for subsequent PCR and Q-RT-PCR amplification.

Over the past decade, methods of molecular biology have appeared in diagnostic pathology and are routinely applied on formalin-fixed, paraffin-embedded histological samples, processed via conventional embedding methods. Due to its reagent- and cost-effectiveness, embedding techniques that utilize microwave acceleration in one or more steps of histoprocessing are increasingly used by numerous laboratories. The demand arises that tissues processed this way should also be suitable for the requirements of molecular pathology. In this study, both conventionally embedded and MFX-800-3 machine-processed tissue samples from the same source were used for isolation of DNA and RNA and for performing PCR and real-time PCR. PCR amplification of the beta-globin gene, as well as the real-time PCR amplification of the ABL mRNA was successful in all cases. Our conclusion is that samples processed by the vacuum assisted automatic microwave histoprocessor MFX-800-3 are perfectly applicable for DNA and RNA isolation and provide appropriate templates for further PCR and realtime PCR studies.

Creating tissue microarrays by cutting-edge matrix assembly.

Tissue microarrays have become widely adopted for effective parallel in situ analysis of hundreds of tissues placed onto single slides. Traditionally, tissue core punches are transferred into predrilled holes within a scaffold block of paraffin or other material, and sectioned transversely by a microtome to generate array sections. While core-based arraying has greatly advanced tissue analyses, some of the limitations include restricted feature sizes and numbers, variable core depths of unpredictable tissue quality and inability to array thin-walled, stratified tissue samples such as intestines, vessels or skin. Overcoming these limitations, the authors have developed a practical arraying method that combines serial cutting and edge-to-edge bonding of samples to assemble a scaffold-free array matrix -- cutting-edge matrix assembly. Using cutting-edge matrix assembly, the authors have successfully placed more than 10,000 individual tissue pieces on a single histology glass slide. The potential biomedical utility and ongoing efforts to further develop the assembly technology and analysis of high-density cutting-edge matrix assembly tissue microarrays is discussed.

Whole-mount in situ hybridization to assess advancement of development and embryo morphology.

Whole-mount in situ hybridization (WISH) is widely used to visualize the site and dynamics of gene expression during embryonic development. Various methods of probe labeling and hybridization detection are available nowadays. Meanwhile the technique was adapted to be used on many different species and has evolved from a manual to a larger scale and automated procedure. Standardized automated protocols improve the chance to compare different experimental settings reliably. The high resolution of this method is ideally suited for examination of manipulated (e.g., cloned) embryos often displaying subtle changes only. Embedding and sectioning of in situ hybridized specimen further enhance the detailed examination of their gene expression and morphology.

Inexpensive holder for hand-trimming resin-embedded tissues.

A simple and inexpensive mount for securely holding resin-embedded tissues for trimming prior to sectioning for light or electron microscopy is described. The unit is composed of a base plate from which a 12.7-mm drill chuck protrudes. A tissue block is clamped into a microtome chuck and that is mounted in the drill chuck. A standard dissecting microscope is placed over the unit and used to magnify the block for trimming. Total cost of materials is estimated to be $20-40 USD.

Postembedding immunocytochemistry of large sections of brain tissue: an improved flat-embedding technique.

A method for osmicating, dehydrating, and flat-embedding large slabs of brain tissue in epoxy resin is presented. This permits the production of semithin sections for postembedding immunocytochemistry that are far larger than can be obtained with other embedding approaches. Vibratomed slabs, 50-200 microns thick and as large as 6 x 8 mm are embedded in a 'soft' Araldite epoxy. The slabs are laminated onto the flat surface of a pre-cast epoxy slide. After polymerization, the tissue can be studied on the slide as a whole mount to view osmicated fiber tracts, or in experimental tract-tracing studies, to locate retrogradely labeled cells before semithin sections are cut. The rigidity of the epoxy slide ensures that the slabs remain flat and are easily removed and mounted for resectioning. Semithin sections are cut using 8 mm wide glass knives or a 6 mm wide diamond knife and are mounted singly or in serial pairs and immunostained using conventional etching and immunoperoxidase techniques. The relative softness of the epoxy permits dozens of semithin sections to be cut from large blocks without appreciably degrading a glass knife edge. After further polymerization the embedment is also compatible with electron microscopy.

A standardized method for brain-cutting suitable for both stereology and MRI-brain co-registration.

We have developed an agar-embedding method for brain-slicing that minimizes the geometrical distortions which arise from handling and slicing the fixed postmortem brain. To facilitate postmortem brain-magnetic resonance imaging (MRI) co-registration, each hemisphere is processed separately. We embed the fixed brain hemisphere with reference markers in agar. The block containing the brain and markers is sliced at a fixed interval using a rotary slicer. Each slice is photographed with a high-resolution digital camera. The digital images are realigned as a 3-dimensional volume via a control point-based registration method for multi-slice registration. The realigned multiple slices of the reconstructed postmortem hemisphere are then co-registered to corresponding slices of an in vivo reference MRI-volume. We illustrate these postmortem MRI-brain co-registration methods to correlate in vivo T2-weighted MRI hyperintensities in gray and white matter with underlying pathology. For design-based stereology, the volume of interest (VOI) is defined using reproducible anatomical boundaries. This method is suitable for stereologic measures of structures ranging from defined nuclei to whole brain.

New strategies for embedding, orientation and sectioning of small brain specimens enable direct correlation to MR-images, brain atlases, or use of unbiased stereology.

We present a newly developed brain slicing machine and technique for tissue embedding, which enable orientation of fresh or fixed brain tissue from small laboratory animals, in any given position, and subsequent tissue sectioning into slabs with an optional thickness between 0.5 and 20 mm. The oriented tissue slabs may be analysed directly, or processed further on a cryostat or vibratome, into thin stainable histological sections, and aligned to MR-images or brain atlases, depending on the reference used for the initial orientation. Additionally, we describe a new embedding medium (HistOmer) which is an alginate cold polymer ready for instant use after mixing with water. HistOmer allows accurate positioning of the tissue during embedding, and at the same time supports and protects the embedded tissue during sectioning. HistOmer is, therefore, described comprehensively and compared with other commonly used embedding media. This novel slicing technique may also, as illustrated, be used to perform isotropic random orientation of the embedded tissue, before sectioning into tissue slabs of the same thickness. The technique thereby fulfills the requirements for optimal tissue sampling and preparation needed for an unbiased stereologic analysis.

Cryofixation, cryosubstitution, cryoembedding for ultrastructural, immunocytochemical and microanalytical studies.

Cryofixation, cryosubstitution and cryoembedding are a set of low-temperature methods for immunocytochemical and microanalytical ultrastructural studies. This review covers the theoretical and practical aspects of these cryomethods, simple, low-cost, safe devices that provide reproducible results and a summary of recent results. Sections prepared by these three cryomethods can be used to determine elemental composition, molecular composition, functions and 3-D ultrastructure. The information obtained can be treated by multivariate statistical methods. Thus, each cellular compartment can be identified by its morphology, molecular and elemental composition and function and changes in these data during physiological and pathological processes can be monitored.

Tissue carriers for processing fragile bio-materials in immuno-electron microscopy.

The preparation and use of tissue carriers for processing fragile biological materials from postfixation up to embedding in electron microscopy is described. Trimmed small pieces of vibratome sections can be very practical and safely transported in these carriers throughout osmification, dehydration for long times, and infiltration up to embedding in epoxy resins. This small device is especially suited for post-embedding immunogold electron microscopy.

A new antiroll device for cryostat wax sectioning.

A new antiroll device has been developed to replace the antiroll guide plate for cryostat wax sectioning. With this device, a continuous ribbon of 3-4 microns sections can be obtained. The sections are flat, uncreased, and compression is reduced to a minimum.

Comparative analysis of duodenal biopsy tissue orientation supported on vegetable matrix versus filter paper.

BACKGROUND: Biopsy tissue may get disoriented if filter paper is used as a supporting medium. Use of vegetable matrix (cucumber) as a supporting medium may obviate the need of lifting the tissue while making blocks and thus avoid disorientation that occurs during this step. OBJECTIVE: To compare the orientation of duodenal biopsy tissue supported on vegetable matrix (cucumber) and on filter paper. METHODS: Over one year, 40 patients (20 with large-volume diarrhea, 20 with dyspepsia) were included in the study. Two pairs of duodenal biopsy tissues were obtained during gastroscopy; one pair was placed on filter paper, the other on vegetable matrix. Tissue and vegetable matrix were embedded together while making blocks, whereas the tissue had to be lifted off in case of filter paper. Sections were stained and assessed for crypt-villous alignment, parallel orientation of crypts and presence of visible muscularis mucosae with the help of a scoring system. RESULTS: Compared to biopsy tissue supported on filter paper, vegetable matrix-supported tissues were better oriented. Scores were rated as bad, good and very good in 8, 11 and 21 vegetable matrix-mounted tissues and in 21, 11 and 8 filter paper-mounted tissues, respectively. CONCLUSION: Duodenal biopsy tissue supported on vegetable matrix (cucumber) is better oriented than that on filter paper.

Polydimethylsiloxane embedded mouse aorta ex vivo perfusion model: proof-of-concept study focusing on atherosclerosis.

Existing mouse artery ex vivo perfusion models have utilized arteries such as carotid, uterine, and mesenteric arteries, but not the aorta. However, the aorta is the principal vessel analyzed for atherosclerosis studies in vivo. We have devised a mouse aorta ex vivo perfusion model that can bridge this gap. Aortas from apoE((-/-)) mice are embedded in a transparent, gas-permeable, and elastic polymer matrix [polydimethylsiloxane (PDMS)] and artificially perfused with cell culture medium under cell culture conditions. After 24 h of artificial ex vivo perfusion, no evidence of cellular apoptosis is detected. Utilizing a standard confocal microscope, it is possible to image specific receptor targeting of cells in atherosclerotic plaques during 24 h. Imaging motion artifacts are minimal due to the polymer matrix embedding. Re-embedding of the aorta enables tissue sectioning and immuno-histochemical analysis. The ex vivo data are validated by comparison with in vivo experiments. This model can save animal lives via production of multiple endpoints in a single experiment, is easy to apply, and enables straightforward comparability with pre-existing atherosclerosis in vivo data. It is suited to investigate atherosclerotic disease in particular and vascular biology in general.