Tissue Processing Techniques Research Articles

Recent progress in optical clearing of eye tissues

The growing need for viewing the detailed 3D structures of various tissues and organs requires advanced tissue processing and imaging techniques. However, light scattering by tissues hinders detailed structural observations. To overcome this, the emerging technique of “tissue optical clearing” has been flourishing in recent decades, providing excellent opportunities for imaging deep, micro-scale structures of various organs, or even of the whole body. In recent years, advanced tissue clearing techniques have been optimized for specific tissues and organs. Among these tissues, the eye is unique owing to its delicate structure and pigmented retinal epithelial cells, calling for more work on making these tissues “transparent”. In this review, we searched Medline and Embase for studies published between January 2006 and August 2021 using the terms “tissue optical clearing”, “ophthalmology”, “eye”, and “optical clearing agents”, and we reviewed the publications on the optical clearing techniques of eye tissue from 2006 to the present, including both the clearing procedures and the subsequent analytical processes, thus gaining more insight into the application of tissue optical clearing in basic eye research. Furthermore, we discuss the future potential of optical clearing applications in clinical ophthalmology.

Personalising cancer medicine with prognostic markers.

Personalising cancer medicine with prognostic markers.

Microwave-Based Rapid Tissue Processing Technique: A Novel Aid in Histopathologic Laboratory.

Introduction:Conventional tissue processing takes a lot of time, but microwave processing method though rapid, its quality of tissue texture and staining properties is still questionable. Therefore, we conducted this study to find out the reliability of histological sections in the microwave method.Aim and Objectives:The aim is to assess whether microwave tissue processing, which is a rapid technique, is comparable to conventional tissue processing in terms of quality and reliability of histological sections.Materials and Methods:A total of 80 formalin-fixed tissue samples were taken and were divided into two pieces. One subjected to conventional tissue processing and the other subjected to microwave tissue processing. The slides were coded and evaluated by three independent observers using various parameters. All the data were subjected to Chi-square test.Results:Both the process did not differ in their efficiency in overall nuclear details, overall cytoplasmic detail, occurrence of artifact and tissue architecture, nuclear staining, cytoplasmic staining, and red blood cell staining. Although the other observers did not find any statistical significance, according to observer 3, conventional processing proved to have marginally significantly better epithelial connective tissue interface than microwave processing (P = 0.047).Conclusion:The microwave method of tissue processing is an economical procedure which reduces the obnoxious chemicals as well as the turnaround time.

Multicolor 3D Confocal Imaging of Thick Tissue Sections.

In multicellular organisms, most physiological and pathological processes involve an interplay between various cells and molecules that act both locally and systemically. To understand how these complex and dynamic processes occur in time and space, imaging techniques are key. Advances in tissue processing techniques and microscopy now allow us to probe these processes at a large scale and at the same time at a level of detail previously unachievable. Indeed, it is now possible to reliably quantify multiple protein expression levels at single-cell resolution in whole organs using three-dimensional fluorescence imaging techniques. Here we describe a method to prepare adult mouse bone tissue for multiplexed confocal imaging of thick tissue sections. Up to eight different fluorophores can be multiplexed using this technique and spectrally resolved using standard confocal microscopy. The optical clearing method described allows detection of these fluorophores up to a depth of >700μm in the far-red. Although the method was initially developed for bone tissue imaging, we have successfully applied it to several other tissue types.

Effect of different human tissue processing techniques on SARS-CoV-2 inactivation-review.

The safety of the tissue transplant recipient is a top priority for tissue banks, and the emergence of the new coronavirus SARS-CoV-2 has raised significant concerns about the risks of releasing tissue for clinical use. In the present study, we conducted a literature review about the potential infectivity of SARS-CoV-2 in different biological tissues and the influence of various tissue processing and sterilization procedures on viral inactivation. The search revealed that SARS-CoV-2 binds to the human angiotensin-converting enzyme receptor to penetrate human cells. These receptors are present in skin cells, musculoskeletal tissue, amniotic membranes, cardiovascular tissue and ocular tissues, including the cornea. In general, we found that coronaviruses are stable at low temperatures, and inactivated upon exposure to extreme heat and pH. Notably, gamma irradiation, which has already been employed to inactivate SARS and MERS, could be useful for sterilizing skin, amnion and musculoskeletal tissues against SARS-CoV-2. We conclude that due to the limited information about the effects of physical and chemical tissue processing methods on viral neutralization, rigorous donor screening is still essential for tissue transplant recipient safety.

Rapid, Automated Tissue Processing Techniques for Small Biopsies Can be used to Produce Quality Sections for Diagnostic Reporting in Histopathology

Surgical histopathologists are currently working in a context of high demand and requirement for fast turnaround times to report small diagnostic biopsies. Use of automated rapid cycle processing could improve turnaround times for reporting small biopsies. There are few papers in the medical literature describing detailed assessment of staining quality in verification of automated rapid cycle processing methods. This study assessed quality of immunohistochemical and haematoxylin and eosin staining of 62 paired endometrial biopsy samples processed using standard overnight cycles compared with rapid cycles of either 2, 3 or 4 hours duration. We found that small biopsies adequately fixed in 10% neutral buffered formalin were of sufficient staining quality for use in diagnostic reporting if they were processed for 3 or 4 hours. Five samples in our 2-hour group were of insufficient staining quality for full microscopic evaluation and histopathological diagnosis. As such, the 2-hour method was rejected. Rapid cycles of three hours duration will be adopted in our laboratory for processing small samples. Prospective monitoring of report turnaround times will be needed to assess impact on future outcomes for patients.

Frozen-Section Tissue Processing in Mohs Surgery.

Mohs micrographic surgery (MMS) is the most reliable tissue-sparing technique in the management of cutaneous malignancies. Although the concept is simple, there is considerable variability in the mapping and processing techniques used by Mohs surgeons and histotechnicians. This review article aims to examine the frozen-section tissue processing techniques. Existing variations will be discussed and pearls offered to optimize the frozen processing technique. A PubMed search was performed for publications on methods of tissue processing in MMS. Our review highlights variations in debulking, embedding, processing adipose tissue, cartilage, and wedge resections. We offer pearls on how to avoid false-positive and false-negative margins and discuss advances in immunohistochemistry. Our article provides a how-to format on the different stages of tissue processing with pearls and techniques to optimize practice and improve accuracy.

Three-dimensional morphological analysis of placental terminal villi.

Transport of nutrients and waste between the maternal and fetal circulations during pregnancy takes place at the final branches of the placental villous trees. Therefore, and unsurprisingly, pregnancy complications have been related to the maldevelopment of terminal villi. However, a deep analysis of placental villous morphology has been limited by tissue processing and imaging techniques. In this proof-of-principle study, placental lobules were fixed by perfusion and small clumps of villi were stained, sectioned optically and reconstructed. Morphological and network analyses were suggested and demonstrated on samples of normal placentas. The results show that most parameters are almost constant within a placenta but that there exists an inter-individual variation. Network analysis suggests that the feto-placental capillary network has several paths within an individual villus, serving as an efficient transport system. Three-dimensional reconstruction from confocal laser scanning microscopy images is a potent technique able to quantify placental architecture and capture the significant irregularities in vessel diameter and membrane thickness. This approach has the potential to become a powerful tool to further our understanding of the differences in placental structure which may underlie pregnancy complications.

A novel in ovo model to study cancer metastasis using chicken embryos and GFP expressing cancer cells.

Cancer metastasis is the leading cause of cancer-related mortality worldwide. To date, several in vitro methodologies have been developed to understand the mechanisms of cancer metastasis and to screen various therapeutic agents against it. Nevertheless, mimicking an in vivo microenvironment in vitro is not possible; while in vivo experiments are complex, expensive and bound with several regulatory requirements. Herein, we report a novel in ovo model that relies on chicken embryo to investigate cancer cell invasion and metastasis to various organs of the body. In this model, we directly injected green fluorescent protein (GFP) expressing cancer cells to the heart of chicken embryo at 3 days of incubation, then monitored cell migration to various organs. To this end, we used a simple tissue processing technique to achieve rapid imaging and quantification of invasive cells. We were able to clearly observe the migration of GFP expressing cancer cells into various organs of chicken embryo. Organ specific variation in cell migration was also observed. Our new slide pressing based tissue processing technique improved the detectability of migrated cells. We herein demonstrate that the use of GFP expressing cancer cells allows easy detection and quantification of migrated cancer cells in the chicken embryo model, which minimizes the time and effort required in this types of studies compared to conventional histopathological analysis. In conclusion, our investigation provides a new cancer metastasis model that can be further improved to include more complex aspects, such as the use of multiple cell lines and anti-metastatic agents, thus opening new horizons in cancer biology and pharmaceutical research.

Imaging the placental glycocalyx with transmission electron microscopy

There is a significant glycocalyx present at the maternal-fetal interface of the human placenta, with increasing evidence to suggest it has an important role in placental function.Glycocalyx is adversely affected by traditional tissue processing and fixation techniques. Using transmission electron microscopy, we present methodologies for reliably imaging and measuring glycocalyx of both the syncytiotrophoblast and fetal capillary endothelium in term healthy placentae.These techniques can be used to study the role of the placental glycocalyx in both health and disease, including pre-eclampsia.

Abstract: Fat Embolism in Gluteal Lipoinjection: Not Only a Matter of “Where” but Also of “What”

PURPOSE: Gluteal fat grafting became extremely popular during the last five years according to ASPS statistics.1 Parallel, there have been deaths reports following this procedure, usually associated with gluteal vessels damage and macroscopic fat embolism (MAFE).2 Some necropsies have failed to find macroscopic fat tissue in lungs or tore gluteal veins, but found oil in lung parenchyma in the microscopic evaluation, which has been called microscopic fat embolism (MIFE).3 Fat embolism is known to occur even without lipoinjection, in patients subjected only to liposuction.4 But all papers published so far about safety in gluteal fat transplantation focus only on MAFE pathophysiology, outlining buttocks areas, machinery, and techniques that are more prone to provoke a vessel wall damage. To date, there is no initiative discussing measures to avoid MIFE, therefore not contemplating all fatal cases that occur. METHODS and MATERIALS: MIFE pathophysiology, the interplay between vascular wall physiology and liposuction components, taking into account the effects of surgical metabolic response on vascular reactivity are discussed and correlated to clinical current practices. RESULTS: Fat injections into the muscular tissue are related to fat embolism not only because the presence of large vessels within its mass that can be damaged and suction the fat graft through the tearing but also because muscles have a higher overall vascularity and concentration of vessels compared to other tissues that allow more absorption of the free oil normally contained in the lipoaspirate through its lipophilic walls. CONCLUSION: Initiatives to develop fat tissue harvesting and processing machinery and techniques that avoid the presence of free oil in the graft material are pivotal to ensure maximal safety in autologous fat graft procedures.

OR50 Donor specific HLA antibody formation following distal tibial allograft and subsequent graft resorption: A case report

Background The association between donor specific Human Leukocyte Antigen (HLA) antibody formation and small bone allograft resorption has not been studied. We present the case of a patient treated for glenoid bone loss with a distal tibial allograft who formed donor specific HLA antibodies against the allograft and had subsequent graft resorption. Methods The patient received a right glenoid reconstruction using distal tibia allograft with Bankart repair in June 2015. X-ray and CT-Scans were performed pre-and post-surgery at standard checkpoints. Patient blood and serum samples were collected pre-and-post-surgery for HLA typing and HLA antibody testing. Results Pre-operative CT scans revealed large Hill-Sachs and bony Bankart lesions, and pre-operative HLA antibody testing revealed no HLA antibodies. The 2-week post-operative radiograph revealed proper graft fixation, and the patient had good range of motion 6-weeks post-surgery. However, 6-week post-operative HLA antibody testing revealed development of donor specific HLA antibodies directed at HLA-A2 antigen as well as several antigens belonging to the A2 crossreactive group (A24, A68, A69, B57 and B58), cPRA of 69%. Eplet analysis showed reactivity to 62GE and 151AHV eplets. At 3-months post-surgery, the patient was weak in internal rotation, and the HLA-A2 antibodies continued to be detected at 5-months post-surgery. Shortly after the antibody peak, the 6-month post-operative CT arthrogram revealed significant graft resorption. Interestingly, the donor specific HLA-A2 antibodies were undetectable 6 months after resorption was complete. Discussion This case report shows that small bone allografts can lead to HLA sensitization. The temporal correlation between donor specific HLA antibody formation and clinical findings strongly suggest an association between HLA antibody formation and graft resorption. It is unclear what impact HLA antibody formation may have on future regrafting in this patient. This case shows a clear association between the formation of donor specific HLA antibodies and graft resorption, which suggests a link between the two events. Our future studies will address the frequency of HLA antibody formation, and investigate whether tissue processing techniques could be improved to reduce bone allograft antigenicity.

Evaluation of Preparation Techniques for Optimal Hepcidin Detection in Human Hepatocyte Culture

Approximately 40% of adults aged 65 and older are affected by Chronic Kidney Disease (CKD). Anemia is an associated symptom seen in many patients diagnosed with CKD, promoting elevated cardiovascular risk, decreased quality of life, and increased morbidity and mortality in affected populations. Hepcidin‐25 (hepcidin) is a key iron regulating hormone secreted by hepatocytes to regulate and maintain body iron homeostasis and dietary iron absorption. Hepcidin does this by stifling iron release from macrophages, preventing the normal recycling of iron in the body needed to support erythropoiesis. Elevated levels of hepcidin have been observed in both adults and children suffering from CKD, while its pathological mechanism of action is still relatively unstudied. There is a growing need to further the understanding of hepcidin's role in CDK and other iron related pathologies. Currently there is no standardized methodology for hepcidin evaluation studies on relevant human cell lines, and there is a growing need for studies to be done in this field to assist biomedical researchers in the translation of clinically relevant lab findings from the benchtop to the bedside. Hep G2 is a perpetual human hepatic cancer cell line confirmed to produce hepcidin and a commonly used lineage when studying hepatic function in vitro. While it is confirmed that Hep G2 cells produce hepcidin when cultured, there is currently no standardized procedure for detection of this peptide in vitro from these, or any other human derived cell lines. The expanding clinical and biomedical implications of hepcidin has prompted the need to determine an optimal method for detection of hepcidin in vitro. In this study we use of Hep G2 cells as a model for hepcidin peptide detection and various tissue processing techniques to prepare the cells hepcidin quantification via ELISA.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Adipose tissue: New tissue processing technique for adipose tissues.

Adipose tissue: New tissue processing technique for adipose tissues.

Rapid tissue processing technique: A novel method using methyl salicylate.

Introduction:Tissue processing involves transition of the biopsy tissue in graded concentration of various chemicals to make the tissue amiable for sectioning. The entire process takes 2–3 working days before a microscopic slide is ready for diagnosis. In order to shorten the turnaround time, rapid tissue processing method using methyl salicylate was developed.Aim:The aim of this study is to develop a rapid tissue processing technique using methyl salicylate as a clearing agent and to compare it with routine tissue processing technique.Materials and Methods:A total of 70 tissue specimens were cut into two equal halves. One each was processed by routine processing technique (RoPT) and rapid processing technique (RaPT). Tissue specimens were measured before and after processing. Quality of staining and cellular-level shrinkage were observed and scored for specimens. Statistical analysis using Welch's unequal variances t-test was performed. Costs of chemicals in both the techniques were compared to see the cost-effectiveness of RaPT.Results and Conclusion:Outcomes of both the processing techniques were comparable with statistically not significant P values for all the parameters. Hence, the results of RaPT technique are satisfactory, and the use of this technique may prove beneficial to pathology laboratories.

The diagnostic value of tissue button technique for specimen accusation during endobronchial ultrasound-guided transbronchial fine-needle aspiration.

The quality of tissue acquisition during endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a major determinant of the diagnostic yield of the procedure. In the tissue button (TB) technique, the retrieved cellular specimen is fixed in ethanol and subsequently scraped from slide using surgical blade into formaldehyde and processed like ordinary tissue biopsy thus potentially increasing its diagnostic value. To retrospectively evaluate the diagnostic yield of a TB technique in patients undergoing EBUS-TBNA for various malignant and benign conditions. The diagnostic yield of specimen obtained by two methods (TB and traditional cell-block technique) performed during the same procedure are outlined in 46 patients who underwent EBUS-TBNA (median age = 65, range 19-85 years). Overall, in both malignant and benign conditions, TB resulted in clear diagnostic material in 43/46 (93.4%) patients. Specifically, TB provided clear histological diagnosis of malignancy (either primary lung cancer or metastases from extra-thoracic cancer) in 30/46 (65.2%) patients and granulomatous inflammation in 11/46 (23.9%) of patients. Only in two patients TB did not provide diagnostic material. The newly introduced TB technique provides valuable histological diagnostic material during EBUS-TBNA both malignant and benign conditions. Given its simplicity and its high diagnostic yield, TB should be considered to be used as one of the preferred specimen acquisition modalities during EBUS-TBNA specimen processing. Direct comparison to alternative tissue processing techniques during EBUS-TBNA should be explored in further randomized prospective studies.

Tissue processing techniques for fabrication of covered stents for small-diameter vascular intervention.

Pericardial tissue is a widely used biomaterial for cardiovascular implants, such as covered stents. The use of glutaraldehyde (GA) has become the method of choice for pericardial tissue fixation, making it immunologically acceptable in humans. However, GA-treated tissue is prone to several problems, such as degeneration and calcification that may lead to long-term failure. Here, we studied two alternative tissue processing techniques: fixative-free decellularization and limited exposure to GA. We've shown that both methods achieve better mechanical properties and promote better host acceptance, tissue remodeling and long-term durability. Since the availability of autologous tissue for transplantation is limited, these methods should be adopted for other types of cardiovascular devices, such as bioprosthetic valves, ultimately achieving better long-term results for patients.

Comparison of multiple techniques for endobronchial ultrasound-transbronchial needle aspiration specimen preparation in a single institution experience.

The optimal method for specimen preparation of endobronchial ultrasound-transbronchial needle aspiration (EBUS-TBNA) is still controversial. This study aims to compare several techniques available for EBUS-TBNA specimen acquisition and processing, in order to identify the best performing technique. We retrospectively reviewed the data of 199 consecutive patients [male, 73%; median age, 64 years (IQR: 52-74 years)] undergoing EBUS-TBNA at our institution from 2012 through 2014 for diagnosis of hilar-mediastinal lymph node enlargement suspect of neoplastic (n=139) or granulomatous (n=60) disease. All procedures were performed by two experienced bronchoscopists, under conscious sedation and local anaesthesia, using 21/22-Gauge (G) needle, without rapid on-site evaluation (ROSE). Five specimen-processing techniques were used: cytology slides in 42 cases (21%); cell-block in 25 (13%); core-tissue in 60 (30%); combination of cytology slides and core-tissue in 51 (26%); combination of cytology slides and cell-block in 21 (10%). To assess the diagnostic accuracy of each tissue-processing technique we compared the EBUS-TBNA results to those obtained with surgical lymphadenectomy, or 1-year follow-up in non-operated patients. Diagnostic yield, accuracy and area under the curve (AUC) were as follows. Cytology slides: 81%, 80%, 0.90; cell-block: 48%, 33%, 0.67; core-tissue: 87%, 99%, 0.96; cytology slides + core-tissue: 80%, 100%, 1.00; cytology slides + cell-block: 86%, 100%, 1.00. Cytology slides and core-tissue method showed non-significantly different diagnostic yield (P=0.435) and AUC (P=0.152). In our single-institution experience, cytology slides and core-tissue preparations demonstrated high and similar diagnostic performance. Cytology slides combination with core-tissue or cell-block showed the highest performance, however these combination methods were more resource-consuming.

Biomechanical properties of bovine tendon xenografts treated with a modern processing method

Xenograft tendons have been used in few human studies, with variable results. With the advent of novel tissue processing techniques, which may mitigate against an immune-mediated rejection response without adversely affecting mechanical properties, there may now be a clinical role for xenograft tendons, particularly in knee ligament reconstruction. We hypothesize that ‘BioCleanse®’ processed bovine extensor digitorum medialis (EDM) tendons exhibit favorable time-zero pre-implantation biomechanical characteristics when compared to both unprocessed bovine EDM tendons and BioCleanse® processed human cadaveric allograft tibialis anterior tendons.In this in vitro case controlled laboratory study, three groups of tendons underwent a 5-stage static loading test protocol: 15 BioCleanse® bovine (BCB), 15 fresh frozen unprocessed bovine (FFB), and 12 BioCleanse® human allograft (BCA) tendons. Cross-sectional area of the grafts was measured using an alginate molding technique, and tendons were mounted within an Instron® 5565 Materials Testing System using cryogenic clamps.BCB tendons displayed a higher ultimate tensile stress (p<0.05), with equivalent ultimate failure load, creep, and modulus of elasticity when compared to the FFB tendons (p>0.05). BCB tendons had an equivalent cross-sectional area to the BCA tendons (p>0.05) whilst exhibiting a greater failure load, ultimate tensile stress, less creep and a higher modulus of elasticity (p<0.05).The BioCleanse® process did not adversely affect the time-zero biomechanical properties of bovine xenograft EDM tendons. BioCleanse® processed bovine xenograft EDM tendons exhibited superior biomechanical characteristics when compared with BioCleanse® processed allograft tibialis anterior tendons. These findings support further investigation of xenograft tendons in orthopedic soft tissue reconstructive surgery.

Comparison of tissue processing methods for microvascular visualization in axolotls

The vascular system, the pipeline for oxygen and nutrient delivery to tissues, is essential for vertebrate development, growth, injury repair, and regeneration. With their capacity to regenerate entire appendages throughout their lifespan, axolotls are an unparalleled model for vertebrate regeneration, but they lack many of the molecular tools that facilitate vascular imaging in other animal models. The determination of vascular metrics requires high quality image data for the discrimination of vessels from background tissue. Quantification of the vasculature using perfused, cleared specimens is well-established in mammalian systems, but has not been widely employed in amphibians. The objective of this study was to optimize tissue preparation methods for the visualization of the microvascular network in axolotls, providing a basis for the quantification of regenerative angiogenesis. To accomplish this aim, we performed intracardiac perfusion of pigment-based contrast agents and evaluated aqueous and non-aqueous clearing techniques. The methods were verified by comparing the quality of the vascular images and the observable vascular density across treatment groups. Simple and inexpensive, these tissue processing techniques will be of use in studies assessing vascular growth and remodeling within the context of regeneration. Advantages of this method include:•Higher contrast of the vasculature within the 3D context of the surrounding tissue•Enhanced detection of microvasculature facilitating vascular quantification•Compatibility with other labeling techniques