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.

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.

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.

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.

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.

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.

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.

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.

Close-to-native ultrastructural preservation by high pressure freezing.

The objective of modern transmission electron microscopy (TEM) in life science is to observe biological structures in a state as close as possible to the living organism. TEM samples have to be thin and to be examined in vacuum; therefore only solid samples can be investigated. The most common and popular way to prepare samples for TEM is to subject them to chemical fixation, staining, dehydration, and embedding in a resin (all of these steps introduce considerable artifacts) before investigation. An alternative is to immobilize samples by cooling. High pressure freezing is so far the only approach to vitrify (water solidification without ice crystal formation) bulk biological samples of about 200 micrometer thick. This method leads to an improved ultrastructural preservation. After high pressure freezing, samples have to be subjected to follow-up procedure, such as freeze-substitution and embedding. The samples can also be sectioned into frozen hydrated sections and analyzed in a cryo-TEM. Also for immunocytochemistry, high pressure freezing is a good and practicable way.

Memento of the post-mortem.

When the Department of Pathology of the Radboud University Nijmegen Medical Centre in The Netherlands moved from its old to its new premises, the demolition of the marble post-mortem tables was impending, taking with it the Department's (physical) memory. To keep the importance of this memory and of the post-mortem itself on everyone's minds in the new building, the artist Piet Hein Eek was invited to incorporate these post-mortem tables into a work of art. This became a triptych: the three post-mortem tables were stood upright against the wall behind a mounted double sheet of glass, containing screen prints of enlarged microscopic images. The two emblems of pathology-post-mortem tables and double glass slides containing specimens-were thus united into a fascinating work of art: a true memento of the post-mortem, re-embedded in the contemporary world by its design.

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.

Evaluation of filler materials used for uniform load distribution at boundaries during structural biomechanical testing of whole vertebrae.

This study was designed to compare the compressive mechanical properties of filler materials, Wood's metal, dental stone, and polymethylmethacrylate (PMMA), which are widely used for performing structural testing of whole vertebrae. The effect of strain rate and specimen size on the mechanical properties of the filler materials was examined using standardized specimens and mechanical testing. Because Wood's metal can be reused after remelting, the effect of remelting on the mechanical properties was tested by comparing them before and after remelting. Finite element (FE) models were built to simulate the effect of filler material size and properties on the stiffness of vertebral body construct in compression. Modulus, yield strain, and yield strength were not different between batches (melt-remelt) of Wood's metal. Strain rate had no effect on the modulus of Wood's metal, however, Young's modulus decreased with increasing strain rate in dental stone whereas increased in PMMA. Both Wood's metal and dental stone were significantly stiffer than PMMA (12.7 +/- 1.8 GPa, 10.4 +/- 3.4 GPa, and 2.9 +/- 0.4 GPa, respectively). PMMA had greater yield strength than Wood's metal (62.9 +/- 8.7 MPa and 26.2 +/- 2.6 MPa). All materials exhibited size-dependent modulus values. The FE results indicated that filler materials, if not accounted for, could cause more than 9% variation in vertebral body stiffness. We conclude that Wood's metal is a superior moldable bonding material for biomechanical testing of whole bones, especially whole vertebrae, compared to the other candidate materials.

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.

Ultrahigh density microarrays of solid samples.

We present a sectioning and bonding technology to make ultrahigh density microarrays of solid samples, cutting edge matrix assembly (CEMA). Maximized array density is achieved by a scaffold-free, self-supporting construction with rectangular array features that are incrementally scalable. This platform technology facilitates arrays of >10,000 tissue features on a standard glass slide, inclusion of unique sample identifiers for improved manual or automated tracking, and oriented arraying of stratified or polarized samples.