Tissue Procurement Research Articles

Ethical Issues of Organ Transplantation

Organ Transplantation involves a surgical procedure where in a new organ replaces a deteriorating or damaged organ in the human body, Heart, lungs, Kidneys are examples of such organs. They are composed of tissues and cells that work together to perform specific essential functions in the human body. In the present scenario, the transplantation of many organs between well-matched human beings is marked with huge success. Heart and lung or heart-lung transplants, liver and pancreas (or pancreatic islets) transplants are also becoming more common, while kidney, cornea, bone marrow and skin transplants today are considered routine for certain conditions. According to Dr. Robert White, even a human head transplant, which should perhaps be better referred to as a body transplant, may be possible. However the balance between demand and supply is hugely distorted, marked simultaneously by high costs of some organ transplants. Consequently numerous questions come up today regarding procurement, distribution of scarce resources, and their coverage by community funds. The ethical and legal issues related to organ and tissue procurement and transplantation are often discussed in light of age old principles of autonomy, compassion, non-maleficence, free and informed consent, dignity, integrity and equality of human beings, justice, and the common good. "In the donation and transplantation of human organs, respect is to be given to the rights of the donor, the recipient and the common good of society”.

Diagnostic Evaluation of Solid Pancreatic Lesions: Endoscopic Ultrasound-Guided Fine Needle Aspiration Versus Percutaneous Ultrasound-Guided Core Needle Biopsy.

The main objective of the present study is to compare the safety, technical success and diagnostic yield of endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) versus ultrasound-guided percutaneous core-needle biopsy (US-CNB) in patients with solid pancreatic lesions. This is a retrospective study that involved all patients with a solid pancreatic lesion who underwent EUS-FNA or US-CNB between November 2019 and February 2021. Of all patients, 69 (84.1%) had inoperable malignancy, whereas 13 (15.9%) had chronic pancreatitis. Resectability status was ascertained by computed tomography. All core needle biopsies were performed by the same interventional radiologist via ultrasound guidance with an 18-gauge semi-automatic tru-cut needle. All EUS-FNA procedures were performed by the same gastroenterologist with a 27-gauge EUS-FNA needle. Technical success is defined as if the region of interest is reached and specimen taken from the pancreatic lesion. Diagnostic yield is defined as the procurement of sufficient tissue for pathological examination. Overall, 52 patients (mean age 58.5 ± 9.8years) who underwent EUS-FNA and 30 patients (60.1 ± 12.1years) who underwent US-CNB were included. Solid lesions were most commonly (61.5% in EUS-FNA and 50.0% in US-CNB groups) located in pancreatic head in both groups. Mean size of the lesions was comparable in both groups as well. The technical success was 100% in both groups. In 12 (14.6%) patients, pathology results revealed inadequate sampling (11 × in the EUS-FNA and 1 × in the US-CNB group). The diagnostic yield was significantly higher in US-CNB group than in EUS-FNA group (96.7% vs. 78.8%, respectively, p = 0.048). Of 11 patients in the EUS-FNA with inadequate sampling, pancreatic lesions were located in the pancreatic head in 7 (63.6%). No major complications were observed in neither of the groups. As a minor complication, one case of slight abdominal pain was detected in the EUS-FNA group. Based on the results of the present study, both US-CNB and EUS-FNA appeared safe; however, diagnostic yield in the US-CNB group was significantly higher.

Skin Bank Establishment in Treatment of Severe Burn Injuries: Overview and Experience with Skin Allografts at the Vienna Burn Center.

Depending on their extent, burn injuries require different treatment strategies. In cases of severe large-area trauma, the availability of vital skin for autografting is limited. Donor skin allografts are a well-established but rarely standardized option for temporary wound coverage. Ten patients were eligible for inclusion in this retrospective study. Overall, 202 donor skin grafts obtained from the in-house skin bank were applied in the Department of Plastic and Reconstructive and Aesthetic Surgery, Medical University of Vienna. Between 2017 and 2022, we analysed the results in patient treatment, the selection of skin donors, tissue procurement, tissue processing and storage of allografts, as well as the condition and morphology of the allografts before application. The average Abbreviated Burn Severity Index (ABSI) was 8.5 (range, 5-12), and the mean affected total body surface area (TBSA) was 46.1% (range, 20-80%). In total, allograft application was performed 14 times. In two cases, a total of eight allografts were removed due to local infection, accounting for 3.96% of skin grafts. Six patients survived the acute phase of treatment. Scanning electron microscope images and histology showed no signs of scaffold decomposition and intact tissue layers of the allografts. The skin banking program and the application of skin allografts at the Vienna Burn Center can be considered successful. In severe burn injuries, skin allografts provide time by serving as sufficient wound coverage after early necrosectomy. Having an in-house skin banking program at a dedicated burn centre is particularly advantageous since issues of availability and distribution can be minimized. Skin allografts provide a reliable treatment option in patients with extensive burn injuries.

Quality by Design: Development of Safe and Efficacious Full-Thickness Acellular Dermal Matrix Based on EuroGTPII Methodologies.

The activities of tissue establishments are constantly and rapidly evolving. The development of a new type of allograft, full-thickness acellular dermal matrix, with high mechanical properties to be used in tendon repair surgeries and abdominal wall reconstruction, has determined the need for quality by design process in order to assess evidence of quality, safety and efficacy. The EuroGTPII methodologies were specifically tailored to perform the risk assessment, identify and suggest tests in order to mitigate the potential risk consequences of a novel tissue preparation implementation. The new allograft and associated preparation processes were assessed using the EuroGTP methodologies and characterized to properly evaluate the novelty (Step 1), identify and quantify the potential risks and risk consequences (Step 2), and define the extent of pre-clinical and clinical assessments required to mitigate the risks identified in the assessment (Step 3). Four risk consequences associated with the preparation process were identified: (i) implant failure related with tissue procurement and the reagents used during the decellularization protocol; (ii) unwanted immunogenicity related with the processing; (iii) disease transmission linked with the processing, reagents used, reduction in the reliability of microbiology testing and the storage conditions; and (iv) toxicity related to the reagents used and handling of the tissue during clinical application. The outcome of the risk assessment was a low level of risk. Nevertheless, it determined the need for a series of risk mitigation strategies proposed to reduce each individual risk and to provide additional evidence of the safety and efficacy of full-thickness acellular dermal matrix grafts. EuroGTPII methodologies allow us to identify the risks and ensure the correct definition of pre-clinical assessments required to address and mitigate the potential risk consequences, before proceeding with clinical use of the new allografts in patients.

Composite tissue xenopreservation: Preliminary results of staged VCA in rat to mouse model.

The time between procurement and transplantation of composite tissues, especially regarding the limited donor pool, is a challenge effecting the outcomes of the transplantation. Current preservation techniques mainly include either cold preservation with a solution or machine perfusion using blood or certain oxygen-carrying solutions. However, none enables preservation beyond 24 h. Increasing this time to several days will provide better usage of the donor pool, safer transplantation of VCA with significant muscle content, and gives time to stabilize a patient before long surgical procedures. Herein, we described a novel strategy of xenopreservation (preservation via xenotransplantation) to preserve composite tissues for 7 days, followed by staged transplantation. We used two concordant species, female Sprague Dawley rats (n = 10) and female CF-1 mice (n = 10) in this study. Four of pair of animals are used for anatomical study. The groin flap of the rat was used as a xenograft and xenotransplanted to the neck area of the carrier mouse. Cyclosporine (CsA) was administered used as immunosuppressant. After 7 days of preservation on the mouse neck, xenotransplanted groin flap (called xenopreserved flap) was re-harvested, skin and vessels samples were collected for histopathological evaluation, and the xenopreserved flap was transplanted to the donor rat's opposite groin area. Anastomoses were performed between the flap's pedicle and the femoral vessels. Clinical observation regarding inflammation and tissue perfusion of the xenopreserved flap was monitored daily. Fifteen days after the second surgical procedure, the rats were euthanized, and skin and vessel samples were collected. Histologic evaluation, including inflammatory cell numbers, was performed. Wilcoxon test was used to compare the changes in inflammation severity and p < .05 was set for statistical significance. All xenopreserved groin flaps except one survived. Mean lymphocyte count before the second operation (at the end of the xenopreservation procedure) was 20,22 ± 0.44 and reduced to 13,14 ± 0.47 at the end of 15 days, and the difference was statistically significant (p < .05). This proof-of-concept study with preliminary results showed that xenotransplantation might be a novel strategy for preservation of VCA for a certain period of time. However, additional translational studies are needed to modulate the tissue changes following xenopreservation.

Organ Donation and Transplantation: Contributions of Professionals on Interprofessional Work in Programs

Objective: This study aimed to identify the contributions for improvement in the interprofessional working relationship pointed out by professionals involved in organ and tissue procurement and transplantation programs. Method: exploratory study with a quanti-qualitative approach conducted over a period of 21 months, with the participation of health professionals invited to complete an online questionnaire on the Google form platform. After collection, the quantitative data were organized and analyzed descriptively, and the qualitative data were categorized and submitted to content analysis. Results: 130 health professionals participated in the study, 84% were female, of which 64% said they were white, between 41-59 years old, and 31% had worked for more than 15 years in the programs. The categories identified were: working conditions; appreciation of non-medical professionals; psychological support to professionals and funding for training of professionals pointed out as improvements in the work relationship in the programs. Conclusion: the study identified that working conditions and greater valorization of the interprofessional team allied to offering psychological support to professionals are necessary and important strategies, and may bring impacts on the satisfaction of professionals, on mental health, in addition to improvements in work processes, impacting transplantation rates.

Abstract 202: Perfused patient-derived tumor organoid models with autologous immune cells for preclinical drug development

Abstract The paradigm shift towards inclusion of primary patient samples in preclinical drug development bottlenecks at the tissue procurement step due to an on-going lack of ’research-ready’ tumor samples. Organoid technology was developed as a solution to this hurdle. Their ability to expand in culture makes them a renewable resource similar to cell lines while recapitulating key properties of the primary tissue, including tumor heterogeneity, structural organization and drug responses. As preclinical drug development continues to adopt organoid-based screening technology and immune-oncology agents continue to be advanced, there is high demand for more complex organoid-based models capable of more accurately predicting individual patient drug responses. Due to their development process, the vast majority of organoids do not contain immune cells, resulting in the need to utilize allogeneic immune cells for any immune cell involved interrogation of the models. Maintaining the immune cells for incorporation with the organoid model can overcome this hindrance. Additionally, perfused organoid models offer a complex, ex vivo model with the ability to replicate the mechanical forces imparted by blood flow and interstitial pressure. Compared to 2D and static 3D cultures, perfused 3D culture models show enhanced proliferative and migratory capacities and can be sustained as long-term cultures. In our previous studies, DNA methylation signatures in patient-derived breast microtumors grown in Kiyatec’s 3DKUBE™ perfusion bioreactor matched the primary tissues of derivation. Given the need for more complex biology when assessing immune-oncology agents, the physiological relevance in maintaining gene expression ex vivo, and the capability to sustain long-term viability, the impact of autologous immune cells and perfusion on the success of organotypic culture was investigated across a panel of primary patient tumors. Immune cells were profiled for status and activity and imaging and viability assays were used to evaluate static and perfused 3D organoid models for differences in viability and morphology. PCR was used to examine how mechanically transduced forces alter gene expression and DNA methylation signatures. Drug responses were measured using our KIYA-Predict™ platform, comparing organoid drug responses to primary tissue drug responses. Results of this study provide insight on alternative methods of organotypic culture and evidence for the utilization of more complex tissue modeling in preclinical drug development. Citation Format: Melissa Millard, Natalie Dance, Teresa M. DesRochers. Perfused patient-derived tumor organoid models with autologous immune cells for preclinical drug development [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 202.

Ex vivo, in situ perfusion protocol for human brain fixation compatible with microscopy, MRI techniques, and anatomical studies.

We present a method for human brain fixation based on simultaneous perfusion of 4% paraformaldehyde through carotids after a flush with saline. The left carotid cannula is used to perfuse the body with 10% formalin, to allow further use of the body for anatomical research or teaching. The aim of our method is to develop a vascular fixation protocol for the human brain, by adapting protocols that are commonly used in experimental animal studies. We show that a variety of histological procedures can be carried out (cyto- and myeloarchitectonics, histochemistry, immunohistochemistry, intracellular cell injection, and electron microscopy). In addition, ex vivo, ex situ high-resolution MRI (9.4T) can be obtained in the same specimens. This procedure resulted in similar morphological features to those obtained by intravascular perfusion in experimental animals, provided that the postmortem interval was under 10 h for several of the techniques used and under 4 h in the case of intracellular injections and electron microscopy. The use of intravascular fixation of the brain inside the skull provides a fixed whole human brain, perfectly fitted to the skull, with negligible deformation compared to conventional techniques. Given this characteristic of ex vivo, in situ fixation, this procedure can probably be considered the most suitable one available for ex vivo MRI scans of the brain. We describe the compatibility of the method proposed for intravascular fixation of the human brain and fixation of the donor's body for anatomical purposes. Thus, body donor programs can provide human brain tissue, while the remainder of the body can also be fixed for anatomical studies. Therefore, this method of human brain fixation through the carotid system optimizes the procurement of human brain tissue, allowing a greater understanding of human neurological diseases, while benefiting anatomy departments by making the remainder of the body available for teaching purposes.

Abstract P2-12-01: A comprehensive assessment of researcher needs to increase biospecimen donations for breast cancer research: A Patients and Researchers Together (PART) Program

Abstract A comprehensive assessment of researcher needs to increase biospecimen donations for breast cancer research: A Patients and Researchers Together (PART) Program Introduction: Tissue-based breast cancer research can help translate scientific discoveries to improve treatments or reduce cancer risk. Tissue donation programs require a partnership between patients being asked to donate tissue and researchers using tissue for translational investigation. Lineberger Comprehensive Cancer Center (LCCC) has established a program called Patients and Researchers Together (PART) to increase the number of patients that donate tissue for research. PART believes patients will be more willing to donate tissue if they are informed about the research process and its impact and are valued as partners of LCCC. The PART program is evaluating the tissue donation process to positively impact efficiencies. Methods: PART leaders conducted interviews to assess current and future needs of LCCC cancer care providers involved in tissue-based research. A total of 13 breast cancer care providers at LCCC with different lengths of research experience were interviewed. These included 4 surgeons, 4 clinical study coordinators, 2 pathologists, 2 medical oncologists and 1 member of the Tissue Procurement Facility (TPF) staff. Interviewees identified key themes, provided insight into barriers and facilitators, and recommended resources to enhance the tissue-based research process. Results: Interviewees noted the high need for fresh tissue, which requires real-time coordination between clinicians, clinical study coordinators, surgeons, pathologists and researchers. Interviewees identified numerous barriers to the tissue donation process that hinder patient recruitment. These included: lack of knowledge about tissue-based research, time commitment required by an additional procedure, loss of privacy, lack of trust and knowledge about how the tissue will be used and access to cancer care (transportation). Interviewees also identified facilitators for recruiting patients to donate tissue for research, including: tailored communication between the patient and their cancer care team, a high interest in medical research, plain language and understandable educational materials and input of a trusted care team member at the appropriate time. Interviewees identified the need for additional tissue collection support and provided recommendations to improve the tissue donation process. Recommendations included: 1) increase the quantity of tissue available for research, 2) increase the racial and treatment history diversity of patients involved in tissue donation research, 3) increase diversity in the biological types of tissue donations (i.e., blood, stool, tumor), and 4) ensure that the available tissue is optimal for use in research. In addition, interviewees identified resources to enhance the tissue-based research process. One suggested resource is a team of coordinators tasked with managing the day-to-day operations of the tissue collection process and serving as a point of contact throughout. Ideally, the designated team would facilitate communication throughout the tissue collection process from the identification of a patient to the tissue’s use in research. Other recommendations included plain language patient educational materials, internal website improvements to enhance communication between researchers, and a written standard operating procedure guide shared with clinical and research team members. Finally, interviewees noted the importance of communication between tissue collection process stakeholders, highlighting the disparity in knowledge regarding best practices between new and more experienced researchers. Findings can inform the development of educational materials and other strategies to increase patient participation in tissue-based breast cancer research. Citation Format: Hayley Morris, Missy Van Lokeren, Patty Spears, Jennifer A. Potter, Vernal Branch, Patient And Researchers Together Team, Charles M. Perou, Lisa Carey. A comprehensive assessment of researcher needs to increase biospecimen donations for breast cancer research: A Patients and Researchers Together (PART) Program [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P2-12-01.

Natural history study and tissue procurement of neuroendocrine neoplasms (NENs).

TPS659 Background: Neuroendocrine neoplasms (NENs) are a rare spectrum of malignant neoplasms originating from neuroendocrine cells, most commonly affecting the gastrointestinal tract, pancreas, and lungs. Tumors may vary from low grade neuroendocrine tumors (NET) to high grade neuroendocrine carcinomas (NEC). The annual occurrence of NENs is increasing worldwide and currently the incidence in the US is about 6 cases per 100,000 people per year. A variety of therapeutic options are available for advanced NENs, however, when to apply a given option, what combination therapeutic approach should be used, how long treatment should be continued is unclear and controversial. Moreover, unlike common cancers, pre-clinical models for NENs are sorely limited. Without robust pre-clinical models our understanding of tumor pathophysiology and novel drug development can be challenging. Methods: A prospective study to evaluate the natural history and sample acquisition of NENs comprehensively and longitudinally. Patients ≥ 18 years old with histological confirmation of NENs, biochemical evidence of NENs, or by imaging studies of NENs are eligible. Participants are invited to NIH for biannual evaluations or as clinically indicated. Patients complete individual medical history, family history, and laboratory assessments including blood, saliva, and tumor for RNA/DNA analysis. A tumor analysis via a 500+ gene panel (Illumina TruSight Oncology 500 Panel) is performed for comprehensive genomic and epigenomic analysis. When feasible, fresh tissue is collected to develop pre-clinical models for drug testing. If clinically indicated, anatomic and functional imaging is performed. We aim to 1) create a repository of biological samples and conduct future investigations to understand the basic biology of NENs with the goal to develop novel treatment approaches, biomarkers of treatment response, and new prognostic and diagnostic models, 2) acquire clinical data so that the overall genomic, proteomic, and metabolomic alternations can be correlated to clinical parameters, 3) establish organoid cultures, cell line models, and xenograft models corresponding to NENs of various grades for drug screening. This protocol will evaluate the natural history of NENs, allow tissue acquisition, and study this heterogenous group of neoplasms with unique tumor biology, and current clinical management to stabilize correlations with clinical outcomes and develop novel therapies. Clinical trial information: NCT05237934 .

The Impact of Donor Preparation for Tissue Procurement on Postmortem Vitreous Isopropanol Concentration.

Volatile chemicals can be relevant in the determination of the cause and manner of death by forensic pathologists. Isopropanol is a secondary alcohol that is occasionally seen on postmortem toxicology testing. A series of 11 forensic autopsy cases was previously reported in which the presence of isopropanol in the vitreous humor was suspected to be due to postmortem contamination from the body preparation process for tissue procurement.In collaboration with a tissue procurement agency, donor vitreous humor was collected from one eye before body preparation for procurement and from the other eye postpreparation. The specimens underwent testing for volatile substances by headspace gas chromatography.Of the 50 cases, 8 (16%) showed statistically significant changes in the prepreparation and postpreparation isopropanol concentrations. Postpreparation isopropanol concentrations ranged from 5 to 104 mg/dL (median, 18 mg/dL). Seven of the 8 cases had undetectable prepreparation isopropanol, whereas the remaining case had a detectable prepreparation isopropanol.In conclusion, surface contamination of the decedent's body with chemicals used in body preparation can lead to the passive absorption into the body, resulting in the presence of isopropanol in postmortem toxicology samples. Forensic pathologists need to be aware of this when interpreting postmortem samples after tissue procurement.

ENDOTHELIAL GLYCOCALYX SHEDDING IN INTRA-ABDOMINAL SEPSIS: A FEASIBILITY STUDY.

Background: The endothelial glycocalyx layer (EGL) is a complex meshwork of glycosaminoglycans and proteoglycans that protect the vascular endothelium. Cleavage or shedding of EGL-specific biomarkers, such as hyaluronic acid (HA) and syndecan-1 (SDC-1, CD138) in plasma, have been shown to be associated with poor clinical outcomes. However, it is unclear whether levels of circulating EGL biomarkers are representative of the EGL injury within the tissues. The objective of the present feasibility study was to describe a pathway for plasma and tissue procurement to quantify EGL components in a cohort of surgical patients with intra-abdominal sepsis. We sought to compare differences between tissue and plasma EGL biomarkers and to determine whether EGL shedding within the circulation and/or tissues correlated with clinical outcomes. Methods: This was a prospective, observational, single-center feasibility study of adult patients (N = 15) with intra-abdominal sepsis, conducted under an approved institutional review boards. Blood and resected tissue (pathologic specimen and unaffected peritoneum) samples were collected from consented subjects at the time of operation and 24-48 hours after surgery. Endothelial glycocalyx layer biomarkers (i.e., HA and SDC-1) were quantified in both tissue and plasma samples using a CD138 stain and ELISA kit, respectively. Pairwise comparisons were made between plasma and tissue levels. In addition, we tested the relationships between measured EGL biomarkers and clinical status and patient outcomes. Results: Fifteen patients with intra-abdominal sepsis were enrolled in the study. Elevations in EGL-specific circulating biomarkers (HA, SDC-1) were positively correlated with postoperative SOFA scores and weakly associated with resuscitative volumes at 24 hours. Syndecan-1 levels from resected pathologic tissue significantly correlated with SOFA scores at all time points ( R = 0.69 and P < 0.0001) and positively correlated with resuscitation volumes at 24 hours ( R = 0.41 and P = 0.15 for t = 24 hours). Tissue and circulating HA and SDC-1 positively correlated with SOFA >6. Conclusions: Elevations in both circulating and tissue EGL biomarkers were positively correlated with postoperative SOFA scores at 24 hours, with resected pathologic tissue EGL levels displaying significant correlations with SOFA scores at all time points. Tissue and circulating EGL biomarkers were positively correlated at higher SOFA scores (SOFA > 6) and could be used as indicators of resuscitative needs within 24 hours of surgery. The present study demonstrates the feasibility of tissue and plasma procurement in the operating room, although larger studies are needed to evaluate the predictive value of these EGL biomarkers for patients with intra-abdominal sepsis.

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

Performance of Biopsy Tools in Procurement of Lung Tissue in Robot-Assisted Peripheral Navigation: A Comparison

Introduction: Robot-assisted navigation bronchoscopy (RANB) has been gaining traction as a new technology for minimally invasive biopsies of peripheral pulmonary lesions (PPLs). Cryobiopsy is an established method of procuring satisfactory lung tissues and can be safely paired with RANB. While some studies have evaluated the diagnostic accuracy and yield of this procedure, there is limited data on the utility of various biopsy tools, the sequence of use and differences in tissue characteristics based on the sampling techniques. Therefore, this study aims to examine the real-life performance of needle, forceps and cryoprobe when utilized in succession with RANB in the biopsy of PPLs, and to evaluate the specific types of tissue samples obtained from each instrument. Methods: In a single-center retrospective study, 50 patients presenting 52 PPLs underwent biopsies sequentially using fine-needle aspiration (FNA), forceps, and cryoprobe. All procedures were performed via cone-beam CT-assisted RANB. Performance metrics, such as diagnostic yield, sensitivity, and specificity, were determined by classifying malignancy as true positives and explicit benign findings as true negatives. Tissue sizes and subtypes were based on pathology description and compared with Student’s t test and χ², respectively. Results: Cryobiopsy, when performed sequentially after FNA then forceps, was able to retrieve more alveolar tissue (p = 0.0098) among diagnosed lesions and showed higher diagnostic yield (p = 0.001) in PPL biopsy with RANB platform. Cryobiopsy was also able to obtain larger sample size (p = 0.0087). No difference was observed in the diagnosis of malignancy between forceps and cryobiopsy (p = 0.8877). Conclusion: The integration of RANB and sequential biopsies, utilizing FNA, forceps, and cryoprobe, can efficiently diagnose PPLs and procure alveolar tissues. Further research based on histopathological subtypes is required to assess its prognostic significance.

Presumed consent in organ donation – limitations of consent model regarding transplantology in Poland

Purpose: Organ transplantation is a treatment for patients with end-stage organ failure limited by the number of organs. The aims of this article are: to compare the relevance of the indicated systems for the number of donated organs, to analyze the status of organ donation, to identify current issues and possible measures in order to increase the number of donations from deceased donors in Poland. Design/methodology/approach: The paper refers to theories from behavioral economics: status quo bias, human tendency to procrastinate, aversion to changes. It also analyzes statistical data of donated organs and the number of objections raised in Poland. Findings: There is a widespread public approval for organ donation in Poland, however most people do not state their decisions. Countries with an opt-out system have a higher rate of organ donations. In Poland in 2020 less than 0,1% of population objected to donate organs, yet 12% of potential donors were disqualified due to record in the Central Register of Objections or family’s statement. In 2016 only 20% of population was aware that presumed consent is legally binding. Moreover, 75% have never talked with their relatives about donating their organs. Research limitations/implications: The article relies on general data from statistical databases and population-based survey studies, which do not include detailed information on the subject described, and thus do not provide data for reliable in-depth statistical analysis. Due to unavailability of numeric data on the reasons for abandoning the organ procurement, the article relied on the available general statistical classification. The authors find an qualitative research to be worth considering in the studied area in order to identify profound causes of the problem and to find a target group for future experimental research. Practical implications: As majority of Polish population is pro-donation the presumed consent for organ donation is preferable to increase the number of donors. Social implications: Profound ignorance of the legislations and broad unawareness of the family's preference is the foundation of public fear and uncertainty towards organ donation. Originality/value: As the social factor appears to be the limiting factor for organ procurement it is fundamental to educate the society about transplantation in a relevant way in order to increase the number of donations in Poland. Keywords: Organ Transplantation, Tissue and Organ Procurement, Presumed Consent. Category of the paper: Research paper/General review.

Human Schwann Cells in vitro I. Nerve Tissue Processing, Pre-degeneration, Isolation, and Culturing of Primary Cells.

This paper presents versatile protocols to prepare primary human Schwann cell (hSC) cultures from mature peripheral nervous system tissues, including fascicles from long spinal nerves, nerve roots, and ganglia. This protocol starts with a description of nerve tissue procurement, handling, and dissection to obtain tissue sections suitable for hSC isolation and culturing. A description follows on how to disintegrate the nerve tissue by delayed enzymatic dissociation, plate the initial cell suspensions on a two-dimensional substrate, and culture the primary hSCs. Each section contains detailed procedures, technical notes, and background information to aid investigators in understanding and managing all steps. Some general recommendations are made to optimize the recovery, growth, and purity of the hSC cultures irrespective of the tissue source. These recommendations include: (1) pre-culturing epineurium- and perineurium-free nerve fascicles under conditions of adherence or suspension depending on the size of the explants to facilitate the release of proliferative, in vitro-activated hSCs; (2) plating the initial cell suspensions as individual droplets on a laminin-coated substrate to expedite cell adhesion and thereby increase the recovery of viable cells; and (3) culturing the fascicles (pre-degeneration step) and the cells derived therefrom in mitogen- and serum-supplemented medium to accelerate hSC dedifferentiation and promote mitogenesis before and after tissue dissociation, respectively. The hSC cultures obtained as suggested in this protocol are suitable for assorted basic and translational research applications. With the appropriate adaptations, donor-relevant hSC cultures can be prepared using fresh or postmortem tissue biospecimens of a wide range of types and sizes.

Factors influencing decision making on donating organs for transplantation in the opinion of the respondents - study using the CAWI method

Introduction: Organ transplantation is a procedure of transferring living cells, tissues or organs within one organism or between two organisms. Despite the great achievements of transplantology and the legal basis regulating organ and tissue procurement and transplantation, there are still difficulties in overcoming social and psychological resistance related to this method of treatment and related to granting consent for organ donation. The aim of the study was to determine the knowledge of the factors influencing the decision to transplant organs among the respondents. Material and methods: The study was conducted using the CAWI method, where invitations were sent to a randomly selected group of respondents on the basis of specialized mailing lists, the research tool was a self-designed questionnaire. Participation in the study was anonymous and voluntary. Results and conclusions: Respondents have knowledge about transplantation. 52.4% of the respondents would agree that all organs should be taken from them after death in order to save the lives of other people, the remaining group was against it. The acceptance of organ donation may have an impact on the development of transplantology, especially thanks to its understanding by families who have to make decisions in this regard.

Possible use of corneal lenticule in surgery of corneal diseases (literature review)

In this review, we analyzed the domestic and foreign literature on the use of corneal lenticula obtained by keratorefractive surgery using the SMILE technology (SMall Incision Lenticula Extraction). Research is being actively carried out on the use of a lenticular tissue for refractive purposes: for the correction of hyperopia (LIKE – Lenticular Intrastromal Keratoplasty), for the correction of presbyopia (PEARL – PrEsbyopic Allogenic Refractive Lenticule). A significant amount of works are devoted to the use of lenticular tissue for the treatment of keratectasias of various origins. For example, a number of authors for the treatment of keratoconus suggest implantation of a lenticule into the recipient’s corneal pocket formed by a femtolaser (SLAK – Stromal lenticule addition keratoplasty). Clinical cases of combined treatment are described: implantation of a lenticule and corneal intrastromal segments for the treatment of corneal pellucid degeneration. A large number of works are devoted to the use of lenticules for tectonic coverage of ulcerative defects, marginal thinning in Mooren’s ulcer. Several clinical cases of the use of a corneal lenticule to cover a deep corneal defect in recurrent pterygium are described. This review also included articles on the storage and decellularization of corneal lenticules. The analyzed articles show a wide area of application of the corneal lenticule; however, more research is required in each of the areas of application, and it is also necessary to solve the problem of procurement and storage of lenticular tissue.

Protocol for the Systematic Fixation, Circuit-Based Sampling, and Qualitative and Quantitative Neuropathological Analysis of Human Brain Tissue.

Human brain tissue has long been a critical resource for neuroanatomy and neuropathology, but with the advent of advanced imaging and molecular sequencing techniques, it has become possible to use human brain tissue to study, in great detail, the structural, molecular, and even functional underpinnings of human brain disease. In the century following the first description of Alzheimer's disease (AD), numerous technological advances applied to human tissue have enabled novel diagnostic approaches using diverse physical and molecular biomarkers, and many drug therapies have been tested in clinical trials (Schachter and Davis, Dialogues Clin Neurosci 2:91-100, 2000). The methods for brain procurement and tissue stabilization have remained somewhat consistently focused on formalin fixation and freezing. Although these methods have enabled research protocols of multiple modalities, new, more advanced technologies demand improved methodologies for the procurement, characterization, stabilization, and preparation of both normal and diseased human brain tissues. Here, we describe our current protocols for the procurement and characterization of fixed brain tissue, to enable systematic and precisely targeted diagnoses, and describe the novel, quantitative molecular, and neuroanatomical studies that broadly expand the use of formalin-fixed, paraffin-embedded (FFPE) tissue that will further our understanding of the mechanisms underlying human neuropathologies.

Anatomical barriers against SARS-CoV-2 neuroinvasion at vulnerable interfaces visualized in deceased COVID-19 patients

Can SARS-CoV-2 hitchhike on the olfactory projection and take a direct and short route from the nose into the brain? We reasoned that the neurotropic or neuroinvasive capacity of the virus, if it exists, should be most easily detectable in individuals who died in an acute phase of the infection. Here, we applied a postmortem bedside surgical procedure for the rapid procurement of tissue, blood, and cerebrospinal fluid samples from deceased COVID-19 patients infected with the Delta, Omicron BA.1, or Omicron BA.2 variants. Confocal imaging of sections stained with fluorescence RNAscope and immunohistochemistry afforded the light-microscopic visualization of extracellular SARS-CoV-2 virions in tissues. We failed to find evidence for viral invasion of the parenchyma of the olfactory bulb and the frontal lobe of the brain. Instead, we identified anatomical barriers at vulnerable interfaces, exemplified by perineurial olfactory nerve fibroblasts enwrapping olfactory axon fascicles in the lamina propria of the olfactory mucosa.