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.

Homemade Glove Retrieval Bag in Laparoscopic Complete Excision of Benign Ovarian Teratoma with Ovarian Tissue Preservation in Children: A Case Series.

STUDY OBJECTIVE: To present our experience of laparoscopic resection of pediatric benign ovarian teratomas with gonadal preservation, using a homemade glove retrieval bag. DESIGN, SETTING, PARTICIPANTS, INTERVENTIONS, AND MAIN OUTCOME MEASURES: Review of all girls with benign ovarian teratomas who were managed with laparoscopic ovarian-sparing surgery (OSS) at our hospital between January 2013 and December 2018. RESULTS: Eleven patients were included for analysis with a mean age of 6.1 years. Ten patients received elective surgery, whereas 1 patient received emergency surgery because of ovarian torsion. Main indication for OSS was the existence of a dissection plane between tumor margins and healthy ovarian tissue. Postoperative outcome and follow-up were uneventful with a median follow-up of 30.1 months (range; 12-60 months). CONCLUSION: Laparoscopic OSS can be safely performed for these tumors. Laparoscopic magnification with energy devices are excellent tools in such procedures. The homemade glove bag can be used to retrieve the tumor effectively in countries with limited resources.

Rapid tissue processing using a temperature-controlled collection device to preserve tumor biomarkers.

Precision tissue diagnostics rely on high quality input specimens so that assay results are not affected by artifact, but advances in collection and processing of tissue specimens have lagged behind innovations in diagnostic assay development. Therefore, we have designed and evaluated a novel surgical tissue collection device that maintains and monitors sample temperature and motion throughout transport so that the major preanalytical variable of tissue temperature can be controlled and measured. This device, in combination with an improved cold-hot tissue fixation protocol affords optimal biomarker preservation in less overall time, thereby simultaneously improving diagnostic quality and turnaround time. We collected 50 primary and metastatic liver tumors using a novel transport device. Tissue was fixed using a rapid cold-hot fixation protocol and immunohistochemical assays were used to assess the performance of the device, in comparison to control tissue preserved using standard clinical fixation protocol. Two pathologists evaluated the IHC studies in a blinded fashion to determine the immunophenotype of each tumor. The observed IHC staining intensities and the clinical impressions of the immunophenotypes did not differ between tissue collected with the novel device and control tissue, while improvements in processing time were achieved. The novel cold transport device and rapid fixation protocol can be successfully and safely combined and used to monitor specimen conditions, thus preserving the diagnostic utility of specimens and improving the overall turn-around time of the diagnostic process.

Tissue preservation through socket-shield technique and platelet-rich fibrin in immediate implant placement: A case report.

RATIONALE: In this report, a combination of socket-shield technique (SST) and platelet-rich fibrin (PRF) technique was used for immediate implant placement on a fractured central incisor. During the follow-up visit, cone beam computed tomography (CBCT) and clinical observation were used to evaluate the preservation outcome of peri-implant bone and gingiva. PATIENT CONCERNS: The patient was a 28-year-old healthy female patient who desired her fractured 21 to be replaced with an implant-supported single crown; the fractured 21 comprised a post-core crown with insufficient residual bone at the labial site. DIAGNOSIS: The root of 21 exhibited a complex root fracture; the labial portion of the alveolar ridge was thin (<1 mm) and partial ankylosis of the residual root was observed. INTERVENTIONS: Modified SST was applied to the labial portion of the residual root. The implant was placed immediately at the lingual site of the retained socket-shield root fragment; PRF was the placed in the gap between the root fragment and the implant. Final prosthodontic treatment was performed at 24 weeks after implant placement. OUTCOMES: Clinical examination and CBCT scanning at various follow-up visits time showed that the periodontal tissue was well- preserved. At 6 months after surgery, the average horizontal and vertical peri-implant bone resorption was 0.4 mm; a follow-up visit at 18 months post-loading indicated that peri-implant tissue was well preserved by the shield-technique and no significant peri-implant tissue resorption was displayed. LESSON SUBSECTIONS: In cases of anterior teeth with intact but insufficient residual alveolar ridge, the SST with PRF may be effective for preservation and maintenance of stable peri-implant tissue.

In-field collection and preservation of decomposing human tissues to facilitate rapid purification and STR typing.

Short tandem repeats (STR) are currently the gold standard in human identification for forensic casework purposes, and successful STR typing is dependent on sufficient quantity and quality DNA. In the aftermath of a mass disaster and some forensic cases, human remains are recovered for identification in various stages of decomposition, and ideally these remains are transported to a refrigerated facility in order to halt the decomposition process and preserve the integrity of DNA within the tissue. However, in situations where refrigeration is not available (e.g., after a mass disaster or in rural forensic casework), remains continue to be exposed to environmental insults after collection, causing further DNA damage and degradation. Therefore, successful STR typing is dependent on the time of collection and preservation of the DNA sample. This study aims to test two simple in-field collection and preservation methods for decomposing human tissues that are subsequently stored at room temperature for up to six months either in a tissue preservative solution (modified TENT buffer) or on an FTA® Elute Card. In addition, these collection and preservation methods were tested for their ability to facilitate more direct and faster processing of DNA from preserved tissues or DNA leached into the surrounding TENT preservative solution for STR typing. Pre-PCR methods tested in this study include a quick lysis of FTA® Elute Cards, silica-based purification (QIAquick®), enzyme-based extractions (PDQeX), and simple dilution of liquid preservative. The traditional DNA analysis pipeline, which includes DNA extraction and quantification, will be compared to an alternate direct PCR method, thereby allowing the elimination of these two time-consuming and costly steps. The results indicate that modified TENT preservative and FTA® Elute Cards both preserved DNA from relatively fresh tissue for up to six months at room temperature. However, mostly partial profiles were produced from decomposed tissues (day 6 - day 14 in this study) when stored for up to six months compared to when tissues were processed immediately following collection. Overall, the modified TENT preservative produced higher DNA concentrations and more successful STR results than FTA® Elute Cards. In addition, a rapid DNA extraction platform (PDQeX) generated the most successful STR typing results from the decomposed tissues stored in TENT for up to six months at room temperature. The direct PCR method used in this study generated comparable STR results to the traditional DNA analysis approach, warranting further investigation of direct PCR methods for forensic casework type samples.

Sterility and Safety Validation for Transport Packaging of Organs and Tissues.

The bags used in the transport of organs and tissues must be sterile, nontoxic, pyrogen free, and must serve as a barrier throughout their useful life. The goal of this study was to show the sterility, safety, and functionality of the bags subjected to irradiation, through validated procedures and techniques. The selected sterilization method was the use of gamma radiation. The sterilization dose was determined based on validated standards for the sterilization of medical products, ISO 11137-2: 2013 and ISO/TS 13004: 2013, using the Verification Dose Maximum method on samples belonging to 3 manufacturing lots. The ISO 10993-5: 2009 standard was used in the cytotoxicity tests, by means of extracts test and quantitative technique of MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. The tests to determine the expiration date of the kit were performed by ASTM F1980, accelerated aging, and ASTM D3078 to evaluate hermeticity. The irradiation dose validated to reach the required sterility safety level was 22.5 kGy. The constituent materials and the sterilization method do not generated cellular toxicity, and the product was not modified during the simulated time of 5 years. Sterilization by irradiation is a method that leaves no residue, does not harm the properties of the material because it is conducted in cold, and as the sterilizing agent, the energy absorbed by the product is highly penetrating and can be treated in its final packaging, with no risk of postcontamination. It is for this reason that it is prioritized over other methods of sterilization.

Clinically-Orientated Anatomy: Five exemplars to portray the concept

It has become almost a truism that, as for many biomedical sciences courses, gross anatomy tuition for healthcare curricula (including medicine and dentistry) should be integrated with clinical components to improve vocational relevance. Nevertheless, many fundamental questions remain to be answered relating to the content to be taught, who teaches the discipline, how the students react, and whether the students are prepared to integrate the clinical and biomedical components. We additionally need evidence of how the delivery of clinical content is influenced by technical developments such as medical imaging. This article documents some examples, or case scenarios, showing how interactions between professional anatomists and clinicians can be fostered, as well as providing illustrations of different teaching styles. From a review of the literature, as well as from our own experiences, we conclude that, for many branches of medicine, it is essential to have access to human bodies for both anatomical and clinical education and training and that postgraduate anatomical teaching remains important for a variety of specialities. We therefore support the notion that a close relationship between professional anatomists and surgeons can reinforce core anatomical knowledge by deepening the understanding of its clinical importance. Paradoxically, however, there is evidence that medical students do not believe that the teachers of anatomy should necessarily be clinically qualified. Furthermore, while students appreciate the value of using clinical examples, scenarios or case histories in anatomy teaching, they remain ambivalent about their use in assessments or examinations. This article also emphasises that anatomy is important both as a scientific and a clinical, translational discipline and argues that the discipline is crucial for appreciation of the human body, not just in disease, but also in health

No disponible

A Dry Method for Preserving Tear Protein Samples.

Tears covering the ocular surface are important biofluids containing thousands of molecules, including proteins, lipids, metabolites, nucleic acids, and electrolytes. Tears are valuable resources for biomarker research of ocular and even systemic diseases. For application in biomarker studies, tear samples should ideally be stored using a simple, lowcost, and efficient method along with the patient's medical records. For this purpose, we developed a novel Schirmer's strip-based dry method that allows for storage of tear samples in vacuum bags at room temperature. Using this method, tear protein patterns can also be preserved. Liquid chromatography-mass spectrometry/mass spectrometry analysis of proteins recovered by the dry method and traditional wet method showed no significant difference. Some tissue/organ-enriched proteins were identified in tear samples, thus tears might be a good window for monitoring changes of these tissues or organs. This dry method facilitates sample transportation and enables the storage of tear samples on a large scale, increasing the availability of samples for studying disease biomarkers in tears.

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen.

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

How does closed system vitrification of human oocytes affect the clinical outcome? A prospective, observational, cohort, noninferiority trial in an oocyte donation program.

OBJECTIVE: To evaluate whether is possible to vitrify oocytes in an aseptic (hermetically closed) fashion and maintain clinical results comparable with those of fresh oocytes. DESIGN: Prospective, observational, cohort, noninferiority trial. SETTING: Private in vitro fertilization center. PATIENT(S): One hundred eighty-four recipients of donated vitrified oocytes. INTERVENTION(S): Closed system vitrification. MAIN OUTCOME MEASURE(S): Pregnancy rate per cycle and clinical pregnancy rate per cycle. RESULT(S): No statistically significant differences were observed between two groups regarding the pregnancy rate per cycle (63.1% vs. 60.9%) or the clinical pregnancy rate per cycle (55.4% vs. 58.7%). Biochemical pregnancy rate was statistically significantly higher in the fresh group (7.6% vs. 2.2%). The mean number of embryos transferred was similar (2.0 ± 0.0 vs. 1.97 ± 0.3). Concerning embryologic data, there were no statistically significant differences regarding the fertilization, cleavage, top quality day-3 embryo, or blastocyst rates, whereas the top quality blastocyst rate on day 5 was statistically significantly higher in the fresh oocyte group (31.7% vs. 26.1%). CONCLUSION(S): Aseptically (in a closed system) vitrified oocytes show similar clinical efficiency compared with their sibling fresh oocytes.

A Mobile Extracorporeal Extremity Salvage System for Replantation and Transplantation.

BACKGROUND: Traumatic amputation is the second leading cause of limb loss in the United States. The preferred treatment is salvage and replantation of the amputated limb, whenever possible, and allotransplantation is a novel procedure whereby healthy limbs are procured from deceased organ donors and transplanted into the amputee recipient. A major restriction for both procedures is the irrecoverable muscle damage occurring due to ischemia. We investigated the feasibility of using a novel lightweight, mobile perfusion device specifically designed to perfuse amputated porcine limbs with an acellular perfusion solution to delay ischemic muscle damage prior to transplantation or replantation. METHODS: Bilateral hind limbs of Yorkshire pigs were amputated; one of the limbs was preserved by perfusion in the mobile perfusion device, and the other by storage in ice slurry for 12 hours. RESULTS: Five sets of bilateral limbs were preserved as described previously. A defined pressure of 30 mm Hg was reliably maintained in the arterial system without loss of flow. Comparison of the perfusate composition before and after limb passage revealed significant differences. Muscle biopsies showed a consistent progression of clusters of hypoxic cells in the control limbs with time. Similar changes could not be observed in the perfused tissue. CONCLUSIONS: We have designed and built a small, mobile perfusion device that is operational and that more closely mimics the normal physiological environment when compared with the current standard of preservation in ice slurry. This project may have far-reaching implications for the treatment of limb loss through replantation and transplantation.

[Midterm Outcomes of Root Replacement Using Homograft for Aortic Valve Infective Endocarditis].

From August 2003 to June 2013, 9 patients with aortic valve endocarditis underwent aortic root replacement using homografts which were harvested and preserved in our institute. The median patient age was 62 years (range 46~84) and 5 patients were men. Four cases were prosthetic valve infections. The in-hospital mortality was 0%. In 8 of 9 cases were evaluated on midterm outcomes. At a median of 52 months (range 19~156), overall survival was 100%, freedom from cardiovascular events was 87.5%. The peak aortic pressure gradient was 9.04 ± 4.2 mmHg. Aortic regurgitation was less than 2 of 4 in all cases.

Histologic validation of vacuum sealed, formalin-free tissue preservation, and transport system.

We describe five validation trials of new vacuum sealing technologies that change the approach to the preanalytic "front end" of specimen transport, handling, and processing and illustrate their adaptation and integration into existing Lean laboratory operations with reduction in formalin use and personnel exposure to this toxic and potentially carcinogenic fixative. These trials provide histologic assessment by numerous pathologists of tissues processed in this new paradigm and define the financial advantages of applying this technology to the postanalytic or "back end" process of tissue storage. We conclude that the TisssueSAFE and SealSAFE vacuum sealing systems are both promising technologies for preserving fresh human specimens that can promote a safer environment by markedly reducing formalin use in operating room theaters and can minimize formalin use by laboratories.

Towards the lean lab: the industry challenge.

In anatomic pathology, the current state encompassing the pre-analytic processes of tissue collection, handling, examination, preparation, processing, and storage are largely uncontrolled, inconsistently performed, and/or not standardized according to the sound scientific data. Pre-analytic defects result in nearly three-quarters of the problems in laboratory diagnostics. This is evident in quality surveys from well-respected institutions that document high miss rates in the required basics of information related to patient and tissue identity, let alone parameters documenting quality aspects related to the surgical specimen and its preservation. This talk will describe the historical approach to tissue processing and identify gaps from worldwide observations in current laboratory practices. It will also offer potential methodological and technological solutions and process improvements that laboratories may consider in serving the ultimate users of pathology information: the clinician and the patient. It illustrates the need for scientifically validated specimen guidelines and a performance based, standardized and documented "chain of custody" of the pre-analytical steps from the patient's body through fixation. For thought leaders and professional standard setters, opportunities for optimizing molecular studies exist in specimen collection, transfer, grossing, fixation, and decalcification protocols. In this evolving era of molecular profiling and personalized therapeutic decision-making, a well-reasoned and coordinated focus on pre-analytic processes that optimizes specimens for subsequent testing will result in: Improved specimen quality for molecular testing Improved accuracy of diagnostic and molecular test results Reduced Turnaroundtimes for same-day diagnosis Enhanced satisfaction of clinicians and patients.

Tools to assess tissue quality.

Biospecimen science has recognized the importance of tissue quality for accurate molecular and biomarker analysis and efforts are made to standardize tissue procurement, processing and storage conditions of tissue samples. At the same time the field has emphasized the lack of standardization of processes between different laboratories, the variability inherent in the analytical phase and the lack of control over the pre-analytical phase of tissue processing. The problem extends back into tissue samples in biorepositories, which are often decades old and where documentation about tissue processing might not be available. This review highlights pre-analytical variations in tissue handling, processing, fixation and storage and emphasizes the effects of these variables on nucleic acids and proteins in harvested tissue. Finally current tools for quality control regarding molecular or biomarker analysis are summarized and discussed.

A review of room temperature storage of biospecimen tissue and nucleic acids for anatomic pathology laboratories and biorepositories.

UNLABELLED: Frozen biospecimens are crucial for translational research and contain well-preserved nucleic acids and protein. However, the risks of freezer failure as well as space, cost, and environmental concerns of frozen biospecimens are substantial. OBJECTIVE: The purpose of the study was to review the current status of room temperature biospecimen storage. METHODS: We searched Pubmed and vendor websites to identify relevant information. RESULTS: Formalin-fixed paraffin embedded (FFPE) tissues have great value but their use is limited by cross-linking and fragmentation of nucleic acids, as well as loss of enzymatic activity. Stabilization solutions can now robustly preserve fresh tissue for up to 7days at room temperature. For longer term storage, commercial vendors of chemical matrices claim real time stability of nucleic acids of over 2 years and their accelerated aging studies to date suggest stability for 12years for RNA and 60years for DNA. However, anatomic pathology biorepositories store mostly frozen tissue rather than nucleic acids. Small quantities of tissue can be directly placed on some chemical matrices to stabilize DNA, however RNA and proteins are not preserved. Current lyophilization approaches can preserve histomorphology, DNA, RNA, and proteins though RNA shows moderate degradation after 1-2years. Formalin-free fixatives show improved but varying abilities to preserve nucleic acids and face validation as well as cost barriers in replacing FFPE specimens. The paraffin embedding process can degrade RNA. CONCLUSION: Development of robust long-term room temperature biospecimen tissue storage technology can potentially reduce costs for the biomedical community in the face of growing targeted therapy needs and decreasing budgets.

The effect of time, temperature and storage device on umbilical cord blood gas and lactate measurement: a randomized controlled trial.

OBJECTIVE: Umbilical cord blood gas analysis has a significant and growing role in early neonatal assessment. Factors often delay analysis of cord blood allowing values to change. Consequently, this study evaluates the impact of time, temperature and method of storage on umbilical blood gas and lactate analyses. METHODS: Umbilical cord segments from 80 singleton deliveries were randomized to: cords at room temperature (CR), cords stored on ice (CI), syringes at room temperature (SR) or syringes stored on ice (SI). Analysis occurred every 15 minutes for one-hour. Mixed model analysis of variance allowing for repeated measures was utilized. RESULTS: Cord arterial pH deteriorated in CR, CI, and SI within 15 minutes (p ≤ 0.001), with SR stable until 60 minutes (p = 0.002). Arterial pCO(2) remained stable in SR and CI, increased in SI (p = 0.002; 45 minutes) and decreased in CR (p < 0.001; 45 minutes). Arterial base excess deteriorated in CR and SI (p ≤ 0.009; 15 minutes), SR (p < 0.001; 30 minutes), and CI (p < 0.001; 45 minutes). Arterial lactate levels increased within 15 minutes in all groups (p < 0.001). CONCLUSIONS: Cord blood gas values change rapidly after delivery. Smallest changes were seen in SR group. Data suggest that analyses should be conducted as soon as possible after delivery.

Preservación de órganos / Organ preservation

El mantenimiento de la viabilidad de los órganos desde su extracción hasta el trasplante es un factor crucial para la adecuada función y la supervivencia del injerto. En los últimos años, este proceso se ha convertido en un verdadero reto, ya que, como consecuencia de la escasez de donantes, se utilizan con mayor frecuencia donantes con criterios expandidos, en los que la funcionalidad del órgano está más afectada. El daño de los órganos ocurre principalmente como resultado de la lesión por isquemiareperfusión, en relación con las lesiones derivadas de la propia preservación. Para minimizar este daño se usan diferentes técnicas de preservación de los órganos, cuyo objetivo es optimizar la función del órgano una vez que se restablezca la perfusión. Por su extremada simpleza, la conservación en frío estática es el método de preservación más utilizado, ya que presenta una serie de ventajas, como su disponibilidad, casi universal, y su facilidad de transporte. Sin embargo, es cuestionable si este método es capaz de prevenir el deterioro de la calidad de los órganos del grupo de donantes con criterios expandidos. En este artículo se describen pormenorizadamente los métodos de preservación actuales, con especial énfasis en la utilización de las máquinas de perfusión continua (AU)

Maintaining organ viability from extraction to transplantation is crucial to ensure the function and survival of the graft. In recent years, maintaining organ viability has become more challenging because the shortage of donors has led to broader criteria for donor acceptability and consequently to organs with greater compromise. Organ damage occurs primarily as a result of ischemia-reperfusion injury, which is associated to additional damage from the preservation process. To minimize this damage, different techniques of organ preservation are used with the aim of optimizing organ function once perfusion is restored. Static cold storage is the most commonly used method of preservation because it is extremely simple, nearly universally available, and easy to transport. However, static cold storage may be unable to prevent the deterioration of the quality of organs from donors included under the broader criteria. In this article, we describe current preservation techniques; we place special emphasis on continuous machine perfusion (AU)

Preservation of porcine hepatocytes in three-dimensional bioreactor at room temperature using epigallocatechin-3-gallate.

A bioartificial liver (BAL) assist system employing a three-dimensional (3D) bioreactor has been studied as a temporary support in liver failure. In the present study, a novel preservation method of primary cultured porcine hepatocytes in monolayer and 3D culture systems was studied. Epigallocatechin-3-gallate (EGCG), which has recently been found to have various bioactivities, was selected as a key compound for hepatocyte preservation. Hepatocytes isolated from porcine liver using the collagenase perfusion method were pre-cultured for 6 days, preserved at room temperature in the presence of EGCG at various concentrations for 4 days, and post-cultured in normal medium for another 6 days. In the monolayer culture, only albumin production rate was fully recovered after preservation when EGCG concentration was high (0.25mg/mL). In contrast, albumin production and ammonium metabolism in the 3D bioreactor under the same condition recovered to 72+/-16% and 98+/-32%, respectively, of levels before preservation. These results indicate that hepatocytes can be preserved in the presence of 0.25mg/mL of EGCG at room temperature, especially in a 3D culture system, which is promising technology for BAL preparation.