One-step embedding method for maintaining orientation of pathological tissue specimens using agar thin films.

Pathological histology examination involves handling a variety of specimens that are cut according to regulations and placed in cassettes. Tissue fragments in the cassettes are then diagnosed after processing, embedding, thin sectioning, staining and other procedures using a processing machine. Maintaining tissue fragment order and orientation during these processes is important for accurate diagnosis. In this study, we present a method of maintaining tissue fragment order and orientation using a thin film of ultra-high-strength agar and evaluate its usefulness during tissue sectioning.Cassettes were prepared, each containing three pieces of porcine liver, and compared embedding time with and without agar thin films (ATFs). Embedding was performed by three medical laboratory scientists with different levels of experience.To enable one-step tissue sample embedding, ATFs were integrated with samples in the cassettes. This resulted in an average reduction of 6.22 s of embedding time per cassette compared with traditional embedding methods.Through the use of ATFs, tissue fragment order and orientation is maintained, and embedding process time shortened. Additionally, ATFs are easily prepared and stored in 10% neutral buffered formalin over extended periods, allowing for immediate use during sectioning. This method is ideal to implement in busy pathology laboratories.

The significance of the extent of tissue embedding for the detection of incidental prostate carcinoma on transurethral prostate resection material: the more, the better?

The aim of this study is to investigate the incidental prostate cancer (iPCa) detection rates of different embedding methods in a large, contemporary cohort of patients with bladder outlet obstruction (BOO) treated with transurethral surgery. We relied on an institutional tertiary-care database to identify BOO patients who underwent either transurethral loop resection or laser (Holmium:yttrium-aluminium garnet) enucleation of the prostate (HoLEP) between 01/2012 and 12/2019. Embedding methods differed with regard to the extent of the additional prostate tissue submitted following the first ten cassettes of primary embedding (cohort A: one [additional] cassette/10 g residual tissue vs. cohort B: complete embedding of the residual tissue). Detection rates of iPCa among the different embedding methods were compared. Subsequently, subgroup analyses by embedding protocol were repeated in HoLEP-treated patients only. In the overall cohort, the iPCa detection rate was 11% (46/420). In cohort A (n = 299), tissue embedding resulted in a median of 8 cassettes/patient (range 1-38) vs. a median of 15 (range 2-74) in cohort B (n = 121) (p < .001). The iPCa detection rate was 8% (23/299) and 19% (23/121) in cohort A vs. cohort B, respectively (p < .001). Virtual reduction of the number of tissue cassettes to ten cassettes resulted in a iPCa detection rate of 96% in both cohorts, missing one stage T1a/ISUP grade 1 carcinoma. Increasing the number of cassettes by two and eight cassettes, respectively, resulted in a detection rate of 100% in both cohorts without revealing high-grade carcinomas. Subgroup analyses in HoLEP patients confirmed these findings, demonstrated by a 100 vs. 96% iPCa detection rate following examination of the first ten cassettes, missing one case of T1a/ISUP 1. Examination of 8 additional cassettes resulted in a 100% detection rate. The extent of embedding of material obtained from transurethral prostate resection correlates with the iPCa detection rate. However, the submission of 10 cassettes appears to be a reasonable threshold to reduce resource utilization while maintaining secure cancer detection.

Application of Amplicon-Based Targeted NGS Technology for Diagnosis of Drug-Resistant Tuberculosis Using FFPE Specimens.

Next-generation sequencing (NGS) enables rapid identification of common and rare drug-resistant genetic variations from tuberculosis (TB) patients' sputum samples and MTB isolates. However, whether this technology is effective for formalin-fixed and paraffin-embedded (FFPE) tissues remains unclear. An amplicon-based targeted NGS sequencing panel was developed to predict susceptibility to 9 antituberculosis drugs, including 3 first-line drugs, by directly detecting FFPE tissues. A total of 178 tissue samples from TB patients who underwent phenotypic drug susceptibility test were retrospectively tested from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. Phenotypic drug susceptibility test results were used as the reference standard. We identified 22 high-quality mutations from 178 FFPE tissue samples, including 15 high+moderate+minimal confidence-level mutations associated with drug resistance (rpoB D435V, S450F/L; KatG S315T; inhA-fabG promoter c-15t; embB G406S, M306V; rpsL K43R, K88R, rrs a1401g, a514c; gyrA D94G/Y/A, A90V), 6 mutations not associated with resistance (rpoB D435Y, H445S, L430P, L452P; embB G406A/D), and one mutation site embB M306I defined as indeterminate. Compared to the phenotypic method, sensitivities (95% CI) for rifampicin, isoniazid, and ethambutol were 96% (79.65-99.90%), 93.55% (78.58-99.21%), and 71.43% (35.24-92.44%), respectively; while for second-line drugs, it varied from 23.53% (9.05-47.77%) for capreomycin to 86.84% (72.20-94.72%) for streptomycin. Specificities for all drugs were satisfactory (>94.51%). Therefore, important pathological FFPE tissue samples, despite partially degraded DNA, can be used as essential specimens for molecular diagnosis of drug resistant TB by amplicon-based targeted NGS technology. IMPORTANCE Amplicon-based targeted NGS technology focuses on a set of gene mutations of known or suspected associations with drug susceptibility in Mycobacterium tuberculosis (MTB). This method offers many benefits, such as low sequencing cost, easy customization, high throughput, shorter testing time and not culture dependent. Formalin-fixed and paraffin-embedded (FFPE) tissues are important pathological specimen in diagnosing tuberculous disease because they are noninfectious and provide excellent preservation of tissue morphology with low storage cost. However, the performance of amplicon-based targeted NGS method on FFPE samples has not been reported yet. Therefore, we evaluated the performance of this method using FFPE samples collected from January 2017 to October 2019 in the Department of Pathology, Beijing Chest Hospital, China. We demonstrate that the amplicon-based targeted NGS method performs excellent on FFPE samples, and it can be applied to pathological diagnosis of drug resistant tuberculosis.

Tissue processing and optimal visualization of cerebral infarcts following sub-acute focal ischemia in rats.

Transient cerebral ischemia followed by reperfusion in an infarcted brain comes with predictable acute and chronic morphological alterations in neuronal and non-neuronal cells. An accurate delineation of the cerebral infarct is not a simple task due to the complex shapes and indistinct borders of the infarction. Thus, an exact macroscopic histological approach for infarct volume estimation can lead to faster and more reliable preclinical research results. This study investigated the effect(s) of confounding factors such as fixation and tissue embedding on the quality of macroscopic visualization of focal cerebral ischemia by anti-microtubule-associated-protein-2 antibody (MAP2) with conventional Hematoxylin and Eosin (HE) staining serving as the control. The aim was to specify the most reliable macroscopic infarct size estimation method after sub-acute focal cerebral ischemia based on the qualitative investigation. Our results showed that the ischemic area on the MAP2-stained sections could be identified macroscopically on both cryo-preserved and paraffin-embedded sections from both immersion- and perfusion-fixed brains. The HE staining did not clearly depict an infarct area for macroscopic visualization. Therefore both immersion-fixed and perfused-fixed-MAP2 stained sections can be used reliably to quantify cerebral infarcts.

Tissue block staining and domestic adhesive tape yield qualified integral sections of adult mouse orbits and eyeballs.

The standard histological processing procedure, which produces excellent staining of sections for most tissues, fails to yield satisfactory results in adult mouse orbits or eyeballs. Here, we show that a protocol using tissue block staining and domestic adhesive tapes resulted in qualified integral serial cryo-sections of whole orbits or eyeballs, and the fine structures were well preserved. The histological processing protocol comprises paraformaldehyde fixation, ethylenediaminetetraacetic acid decalcification, tissue block staining with hematoxylin and eosin, embedding, adhesive tape aided sectioning, and water-soluble mounting. This protocol was proved to be the best in comparison with seven other related existing histological traditional or non-traditional processing methods, according to the staining slice quality. We observed a hundred percent success rate in sectioning, collection, and mounting with this method. The reproducibility tested on qualified section success rates and slice quality scores confirmed that the technique is reliable. The feasibility of the method to detect target molecules in orbits was verified by successful trial tests on block immunostaining and adhesive tape-aided sectioning. Application of this protocol in joints, brains, and so on,-the challenging integral sectioning tissues, also generated high-quality histological staining sections.

Ultrastructure of the Mitochondria-Associated Membranes in Human Tumor Specimens.

Conventional transmission electron microscopy is an essential tool to understand the structure-function relationships and play a vital role in biological research. Mitochondria-associated membranes are linked with cancer processes in a fundamental manner. A conventional transmission electron microscopy method for preparing specimens in clinical and research settings for the study-analysis of the mitochondria-associated membranes in human tumors is presented. The sample processing includes chemical fixation by immersion, dehydration, embedding, polymerization, sectioning, and staining.

Optimization of protocols for pre-embedding immunogold electron microscopy of neurons in cell cultures and brains.

Immunogold labeling allows localization of proteins at the electron microscopy (EM) level of resolution, and quantification of signals. The present paper summarizes methodological issues and experiences gained from studies on the distribution of synaptic and other neuron-specific proteins in cell cultures and brain tissues via a pre-embedding method. An optimal protocol includes careful determination of a fixation condition for any particular antibody, a well-planned tissue processing procedure, and a strict evaluation of the credibility of the labeling. Here, tips and caveats on different steps of the sample preparation protocol are illustrated with examples. A good starting condition for EM-compatible fixation and permeabilization is 4% paraformaldehyde in PBS for 30 min at room temperature, followed by 30 min incubation with 0.1% saponin. An optimal condition can then be readjusted for each particular antibody. Each lot of the secondary antibody (conjugated with a 1.4 nm small gold particle) needs to be evaluated against known standards for labeling efficiency. Silver enhancement is required to make the small gold visible, and quality of the silver-enhanced signals can be affected by subsequent steps of osmium tetroxide treatment, uranyl acetate en bloc staining, and by detergent or ethanol used to clean the diamond knife for cutting thin sections. Most importantly, verification of signals requires understanding of the protein of interest in order to validate for correct localization of antibodies at expected epitopes on particular organelles, and quantification of signals needs to take into consideration the penetration gradient of reagents and clumping of secondary antibodies.

Comparative liquid chromatography/tandem mass spectrometry lipidomics analysis of macaque heart tissue flash-frozen or embedded in optimal cutting temperature polymer (OCT): Practical considerations.

RATIONALE: Biobanks of patient tissues have emerged as essential resources in biomedical research. Optimal cutting temperature compound (OCT) blends have shown to provide stability to the embedded tissue and are compatible with spectroscopic methods, such as infrared (IR) and Raman spectroscopy. Data derived from omics-methods are only useful if tissue damage caused by storage in OCT blends is minimal and well understood. In this context, we investigated the suitability of OCT storage for heart tissue destined for liquid chromatography/tandem mass spectrometry (LC/MS/MS) lipidomic studies. METHODS: To determine the compatibility of OCT storage with LC/MS/MS lipidomics studies. The lipid profiles of macaque heart tissue snap-frozen in liquid nitrogen or stored in an OCT blend were evaluated. RESULTS: We have evaluated a lipid extraction protocol suitable for OCT-embedded tissue that is compatible with LC/MS/MS. We annotated and evaluated the profiles of 306 lipid species from tissues stored in OCT or liquid nitrogen. For most of the lipid species (95.4%), the profiles were independent of the storage conditions. However, 4.6% of the lipid species; mainly plasmalogens, were affected by the storage method. CONCLUSIONS: This study shows that OCT storage is compatible with LC-MS/MS lipidomics of heart tissue, facilitating the use of biobanked tissue samples for future studies.

Ex Vivo Whole-Mount Imaging of Leukocyte Migration to the Bone Marrow.

The bone marrow is the major hematopoietic organ, consisting of distinct microenvironmental niches for the production of hematopoietic cells. Advanced visualizing methods are required to define and better understand the interactions between stromal and hematopoietic cells. In this chapter, we describe an ex vivo whole-mount imaging technique of the bone marrow, which allows for a fast, high-quality, and three-dimensional visualization of different bone marrow components. We provide a guide for conducting adoptive transfer experiments of fluorescently labeled leukocytes and visualizing their location in the bone marrow with respect to the bone marrow vasculature. This method presents a quick, easy, and inexpensive approach to image the bone marrow in three dimensions.

Protein-Retention Expansion Microscopy (ExM): Scalable and Convenient Super-Resolution Microscopy.

Expansion microscopy (ExM) is a method to expand biological specimens ~fourfold in each dimension by embedding in a hyper-swellable gel material. The expansion is uniform across observable length scales, enabling imaging of structures previously too small to resolve. ExM is compatible with any microscope and does not require expensive materials or specialized software, offering effectively sub-diffraction-limited imaging capabilities to labs that are not equipped to use traditional super-resolution imaging methods. Expanded specimens are ~99% water, resulting in strongly reduced optical scattering and enabling imaging of sub-diffraction-limited structures throughout specimens up to several hundred microns in (pre-expansion) thickness.

Acupoint catgut embedding for the treatment of peptic ulcers: A protocol for systematic review and meta analysis.

BACKGROUND: Peptic ulcer (PU) is a common clinical disease of the digestive system, which can occur in all ages, gastric and duodenal ulcers are the most commonly seen PUs in clinical practice. The main manifestations are chronic and periodic rhythmic upper abdominal pain, accompanied by indigestion symptoms such as pantothenic acid, belching, and nausea. Serious complications such as bleeding, perforation, obstruction and canceration are easy to occur, endangering the life safety of patients. There are many ways to treat PU in clinic, and acupoint catgut embedding therapy has its unique advantages. Hence, our systematic review aims to evaluate the efficacy and safety of acupoint embedding therapy in the treatment of PU and to provide a reliable basis for physician. METHODS: We will search electronic databases including PubMed, Embase, Cochrane Library, China National Knowledge Infrastructure (CNKI), Wanfang Database (WF), China Biomedical Literature Database (CBM), and China Scientific Journals Database (VIP) from establishment to April 2021, and will manually searched the list of medical journals as a supplement. Two authors will screen the studies independently, as well as extract data information, and assess methodological quality through the Cochrane risk of bias (ROB) tool. The Stata software (Version 16.0) software will be used for statistical analysis. RESULTS: By evaluating the current status of acupoint catgut embedding for Peptic ulcer disease, this study would prove the effectiveness and safety of acupoint embedding therapy, and will be published in a peer-reviewed journal. CONCLUSION: This systematic review will provide a credible evidence-based for acupoint catgut embedding in the treatment of peptic ulcer. INPLASY REGISTRATION NUMBER: INPLASY202130097.

In-laboratory breast specimen radiography reduces tissue block utilization and improves turnaround time of pathologic examination.

BACKGROUND: This study was performed to determine whether in-laboratory specimen radiography reduces turnaround time or block utilization in surgical pathology. METHODS: Specimens processed during a 48-day trial of an in-lab cabinet radiography device (Faxitron) were compared to a control group of specimens imaged in the mammography suite during a prior 1-year period, and to a second group of specimens not undergoing imaging of any type. RESULTS: Cases imaged in the mammography suite had longer turnaround time than cases not requiring imaging (by 1.15 days for core biopsies, and 1.73 days for mastectomies; p < 0.0001). In contrast, cases imaged in-lab had turnaround time that was no longer than unimaged cases (p > 0.05 for core biopsies, lumpectomies and mastectomies). Mastectomies imaged in-lab required submission of fewer blocks than controls not undergoing any imaging (mean reduction of 10.6 blocks). CONCLUSIONS: Availability of in-lab radiography resulted in clinically meaningful improvements in turnaround time and economically meaningful reductions in block utilization.

A flat embedding method for transmission electron microscopy reveals an unknown mechanism of tetracycline.

Transmission electron microscopy of cell sample sections is a popular technique in microbiology. Currently, ultrathin sectioning is done on resin-embedded cell pellets, which consumes milli- to deciliters of culture and results in sections of randomly orientated cells. This is problematic for rod-shaped bacteria and often precludes large-scale quantification of morphological phenotypes due to the lack of sufficient numbers of longitudinally cut cells. Here we report a flat embedding method that enables observation of thousands of longitudinally cut cells per single section and only requires microliter culture volumes. We successfully applied this technique to Bacillus subtilis, Escherichia coli, Mycobacterium bovis, and Acholeplasma laidlawii. To assess the potential of the technique to quantify morphological phenotypes, we monitored antibiotic-induced changes in B. subtilis cells. Surprisingly, we found that the ribosome inhibitor tetracycline causes membrane deformations. Further investigations showed that tetracycline disturbs membrane organization and localization of the peripheral membrane proteins MinD, MinC, and MreB. These observations are not the result of ribosome inhibition but constitute a secondary antibacterial activity of tetracycline that so far has defied discovery.

A method to prepare large resin sections for counting myelinated axons in rodent CNS and PNS structures.

We present a method that allows preparing histological sections from large blocks of nervous tissue embedded in epoxy resin. Resin-embedding provides excellent resolution especially for the myelin-rich white matter and is often being used for visualizing the myelinated axons in peripheral nerves. However, because of the limited penetration of the reagents, only very small tissue specimens can be processed in this way. Here, we describe a method that enables to embed large specimens and their sectioning on a standard sliding microtome. To process the large specimens, modifications in several steps of the processing technique had to be made. In this paper we demonstrate, that with this technique 1-3 µm thick transversal sections can be prepared from the resin-embedded specimens as large as rat brain hemisphere. Such a large section allows simultaneously: 1.) overviewing and delineating the gross anatomical structures, and 2.) observing the subcellular details at the highest possible optical magnifications. Such a large section with excellent resolution allows application of unbiased stereological methods and reliable quantification of very small objects within the area of interest.

Removal of optimal cutting temperature (O.C.T.) compound from embedded tissue for MALDI imaging of lipids.

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) is a common molecular imaging modality used to characterise the abundance and spatial distribution of lipids in situ. There are several technical challenges predominantly involving sample pre-treatment and preparation which have complicated the analysis of clinical tissues by MALDI-MSI. Firstly, the common embedding of samples in optimal cutting temperature (O.C.T.), which contains high concentrations of polyethylene glycol (PEG) polymers, causes analyte signal suppression during mass spectrometry (MS) by competing for available ions during ionisation. This suppressive effect has constrained the application of MALDI-MSI for the molecular mapping of clinical tissues. Secondly, the complexity of the mass spectra is obtained by the formation of multiple adduct ions. The process of analyte ion formation during MALDI can generate multiple m/z peaks from a single lipid species due to the presence of alkali salts in tissues, resulting in the suppression of protonated adduct formation and the generation of multiple near isobaric ions which produce overlapping spatial distributions. Presented is a method to simultaneously remove O.C.T. and endogenous salts. This approach was applied to lipid imaging in order to prevent analyte suppression, simplify data interpretation, and improve sensitivity by promoting lipid protonation and reducing the formation of alkali adducts.

Effect of acupoint catgut embedding in chronic fatigue syndrome patients: A protocol for systematic review and meta-analysis.

BACKGROUND: Chronic fatigue syndrome (CFS) is a relatively complex and disabling illness with a substantial economic burden and functional impairment. Until now, many CFS patients lack appropriate healthcare. Acupoint catgut embedding is an effective and emerging alternative therapy for CFE. With this research, we endeavor to investigate the effect and safety of ACE for CFS. METHODS: Eight databases will be searched from inception to December 2020: PubMed, EMBASE, The Cochrane Library, Web of Science, China National Knowledge Infrastructure, Chinese Biomedical Literature Database, Chong-Qing VIP database, and Wan-fang database. We regard studies as eligible for inclusion if they were RCTs done in CFS patients, compare acupoint catgut embedding to another treatment strategy, and report fatigue changes at the end of the intervention period. Two independent reviewers complete the study selection, data extraction, and the risk of bias assessment. We assess pooled data using a random-effects model through Revman software (v.5.3) and Stata (version 15.0). ETHICS AND DISSEMINATION: Ethics approval is not required because the individual patient data will not be involved, with no privacy concerns. This systematic review and meta-analysis will provide a reference for CFS patients and clinicians on the non-drug interventions. We will publish and disseminate the results of this review in a peer-reviewed journal or relevant conference. OSF REGISTRATION NUMBER: 10.17605/OSF.IO/7SHD9 (https://osf.io/7shd9).

Irreversible electroporation ablation against subcutaneously implanted VX2 tumors in rabbits: findings of shearwave ultrasound elastography.

PURPOSE: This study aimed to evaluate stiffness changes of rabbit subcutaneous VX2 tumors before and after irreversible electroporation (IRE) ablationby shearwave ultrasound elastography (SWE). METHODS: IRE was performed on 20 subcutaneously implanted VX2 tumors in rabbits (R-SIVX2). Tumor stiffness was measured by SWE at different time points (before IRE,120minutes after IRE,7 days after IRE and 14 days after IRE). RESULTS: Before IRE, the mean stiffness (Emean) of tumors was (10.45 ± 1.07) KPa. 120 minutes after I RE, the Emean of tumors obviously rose to (70.53 ± 9.87) KPa. 7 days after IRE, the Emean of tumors decreased to (40.22 ± 9.01) KPa. 14 days after IRE, the Emean of tumors was (15.17 ± 1.00) KPa. A clear boundary was observed between the ablation area and the normal tissues in the pathological results. CONCLUSIONS: The stiffness of the VX2 tumors experienced a first rise process and tend to be normal in the procedure of IRE. SWE could provide tissue stiffness information of different IRE ablation period as a non-invasive method.

Acupoint catgut embedding therapy for functional constipation: A protocol for systematic review and meta-analysis.

BACKGROUND: This review will assess current evidence related to the effectiveness and safety of acupoint catgut embedding therapy for functional constipation (FC) and provide efficacy assessments for clinical applications. METHODS: We will search the following databases for relevant trials: PubMed, EMBASE OVID, Cumulative Index of Nursing and Allied Health Literature, OVID MEDLINE, Web of Science, the Cochrane Central Register of Controlled Trials, Cochrane library, and Scopus. We will also search the following Chinese databases for trials published in the Chinese literature: China National Knowledge Infrastructure Database (CNKI), Chinese Scientific Journals Database, Wan Fang Database, Chinese Biomedicine and other resources from inception to December 2020. Only randomized controlled trials comparing acupoint catgut embedding versus acupuncture or sham acupuncture or placebo or other therapies will be included. The outcomes involved mean spontaneous bowel movements, complete spontaneous bowel movements, the Bristol Stool Form Scale, the Cleveland Clinic Score, Patient Assessment of Constipation symptom and so on. The risk of bias assessment and quality of evidence for outcomes will be appraised using the Cochrane Risk of Bias Tool and the Grading of Recommendations, Assessment, Development and Evaluation guidelines. RevMan 5.3 software will be employed for the meta-analysis. RESULTS: This work will compare and arrange the comparative efficacy of acupoint catgut embedding with different treatments for FC by summarizing the current evidences. CONCLUSION: The results of this meta-analysis may help doctors determine the best treatments for patients to manage FC. ETHICS AND DISSEMINATION: This is a protocol with no patient recruitment and personal information collection, approval by the ethics committee is not required. OSF REGISTRATION NUMBER: DOI 10.17605/OSF.IO/XTKE2.

Acupoint catgut embedding for the treatment of sciatica: A protocol for a systematic review.

BACKGROUND: Sciatica is a common and frequent peripheral neuropathic pain disease, which causes a great burden on peoples life. Recently, acupoint catgut embedding (ACE) has been widely applied for treating sciatica in China, however, there is no enough evidence to prove the efficiency and safety of ACE for sciatica. Our study aims to evaluate the efficiency and safety of ACE for sciatica. METHODS AND ANALYSIS: Searches of the Cochrane Library, PubMed, Springer Medline, EMBASE, China National Knowledge Infrastructure (CNKI), Wan-Fang Data (WANFANG), Chinese Biomedical Literature Database (CBM), and Chinese Scientific Journal Database (VIP databases) will be performed from inception to November 2020. The main outcomes are the pain intensity and the whole efficiency assessment. The secondary outcomes will include Oswestry Disability Index (ODI), life quality, physical examination, and adverse events. Two reviewers will separately conduct the study selection, data extraction and study quality assessments. RevMan 5.3 software will be used for meta-analysis. RESULTS: This study will provide an evidence-based review of acupoint catgut embedding therapy for sciatica according to the pain intensity, the whole efficiency assessment, life quality, DOI index and adverse events. CONCLUSIONS: This systematic review will present the current evidence for acupoint catgut embedding therapy for sciatica. ETHICS AND DISSEMINATION: Ethical approval is unnecessary as this protocol is only for systematic review and does not involve privacy data. The findings of this study will be disseminated electronically through a peer-review publication or presented at a relevant conference. TRIAL REGISTRATION NUMBER: INPLASY2020110087.