A cost-effective and efficient ex vivo, ex situ human whole brain perfusion protocol for immunohistochemistry.

BACKGROUND: Chemical fixation of the brain can be executed through either the immersion method or the perfusion method. Perfusion fixation allows for better preservation of the brain tissue's ultrastructure, as it provides rapid and uniform delivery of the fixative to the tissue. Still, not all facilities have the expertise to perform perfusion fixation, with initial high cost and complexity of perfusion systems as the main factors limiting its widespread usage. NEW METHOD: Here we present our low-cost approach of whole brain ex situ perfusion fixation to overcome the aforementioned limitations. Our self-made perfusion system, constructed utilising commercially accessible and affordable medical resources alongside laboratory and everyday items, demonstrates the capability to generate superior histological stainings of brain tissue. The perfused tissue can be stored prior to proceeding with IHC for at least one year. RESULTS: Our method yielded high-quality results in histological stainings using both free-floating cryosections and paraffin-embedded tissue sections. The system is fully reusable and complies with the principles of sustainable management. COMPARISON WITH EXISTING METHODS: Our whole brain perfusion system has been assembled from simple components and is able to achieve a linear flow with a pressure of 70 mmHg corresponding to the perfusion pressure of the brain. CONCLUSIONS: Our ex situ method can be especially useful in research settings where expensive perfusion systems are not affordable or in any field with high time pressure, making it suitable for the field of forensic medicine or pathology in general.

Assessment of the Impact of Alternative Fixatives on HER2 Detection in Breast Cancer and Gastric Cancer Tumor Specimens.

The type of fixative used for preserving tumor specimens can significantly impact the performance of the immunohistochemistry and in situ hybridization assays used for assessing human epidermal growth factor receptor 2 (HER2) status. This study reports the prevalence of the use of alternative fixatives other than the guideline-recommended 10% neutral buffered formalin (NBF) during HER2 testing in a real-world setting. The effects of alternative fixatives [20% NBF and 10% unbuffered formalin (UBF) fixatives] on HER2 testing of breast cancer (BC) and gastric cancer (GC) cell lines and tissues are also assessed. Overall, 117,636 tumor samples received at a central laboratory from >8000 clinical trial sites across 60 countries were reviewed to determine the prevalence of alternative fixative usage. To investigate the impact of alternative fixatives, 27 cell lines (21 BC and 6 GC) and 76 tumor tissue samples (50 BC and 26 GC) were fixed in 10% NBF, 20% NBF, or 10% UBF, and evaluated for HER2 status by immunohistochemistry and in situ hybridization. Real-world data showed that 9195 (7.8%) tumor samples were preserved using an alternative fixative. In cell lines, overall percentage agreement, negative percentage agreement, and positive percentage agreement among the 3 fixatives were 100%. In tumor tissues, the agreement among 10% NBF, 20% NBF, and 10% UBF ranged between 94.7% and 96.6% for negative percentage agreement and 90.9% for overall percentage agreement compared with a range of 58.3% to 66.7% for positive percentage agreement. These results suggest that alternative fixatives may have the potential to convert HER2 status in tissues from positive to negative.

Evaluation of formaldehyde exposure among gross dissection after modified embalming solution and health assessment.

Mainly embalming fixative contains formaldehyde which is classified as a carcinogen. People who work with cadavers have been at higher risk of cancer after formaldehyde exposure. We have formulated a less-formalin fixative (contained 3.6% formaldehyde,23.8% ethanol, 15% glycerin, and 0.2% phenol in the water) for preserving cadavers. Therefore, the objective of the present study was to evaluate the level of atmospheric formaldehyde indoors and the breathing exposure of medical students during dissection classes. We also analyzed the pulmonary parameters and effects of formaldehyde. The levels of atmospheric formaldehyde indoors and personal breathing exposure were sampled during anatomy dissection classes (musculoskeletal system, respiratory system, and abdominopelvic organ system) using sorbent tubes with air sampling pumps. Samples were then analyzed using Gas Chromatography with Flame Ionization Detector (GC-FID). The mean level of formaldehyde indoor air among the three classes was 0.518 ± 0.156 ppm whereas the formaldehyde level in the personal breathing zone was 0.956±0.408 ppm, which exceeded the recommended exposure standards of international agencies, including NIOSH agency and PEL of Thailand legislation. The laboratory had high humidity, high room temperature, and poor air ventilation. There was a significant difference in FVC, FEV1, and PEF (p < 0.05) between the sexes of students. Comparison pulmonary parameters between students and instructors showed that all parameters of the pulmonary function test had no significant differences. General fatigue and burnings of eyes and nose associated with strong odor were the most common symptoms reported during the dissection classes. The modified embalming fixative was used less formalin with ethanol-glycerin mixture, and it was suitable for the study of medical students, with few side effects of respiratory problems. However, the modified exhaust ventilation with local table-exhaust ventilation and heating-ventilation-air conditioning system performance were urgent issues for reducing levels of formaldehyde indoor air in the dissection room.

Quantitative assessment of optical clearing methods on formalin-fixed human lymphoid tissue.

Rising interest in three-dimensional volume imaging of biological tissues for diagnostic and research purposes, calls for appropriate optical clearing methods as an indispensable requirement for high-resolution imaging on a cellular level. In recent years, many clearing protocols have emerged, though most of them focus on murine central nervous tissue. Peripheral organs or tissues of human origin have only been investigated sparsely. Therefore, we tested eight established clearing methods (BABB, Ce3D, CUBIC, ECi, ChemScale, ChemScaleQQ5, SeeDB2 and PACT) on formaldehyde-fixed human tonsils. This application-oriented taxonomy can help researchers restrict the space of their survey on clearing techniques for lymphatic tissue as it provides information on each method in regard to its efficacy, clearing speed, preservation of fluorescence labelling, toxicity, expenditure and monetary costs. We found that all of the applied clearing protocols could render the sample tissues transparent. Ce3D and PACT achieved the highest degrees of tissue transparency. Since it requires less preparing and processing time and is lower in toxicity, we recommend Ce3D for the clearing of human lymphoid tissue.

External Quality Assessment Schemes for Biomarker Testing in Oncology: Comparison of Performance between Formalin-Fixed, Paraffin-Embedded-Tissue and Cell-Free Tumor DNA in Plasma.

Liquid biopsies have emerged as a useful addition to tissue biopsies in molecular pathology. Literature has shown lower laboratory performances when a new method of variant analysis is introduced. This study evaluated the differences in variant analysis between tissue and plasma samples after the introduction of liquid biopsy in molecular analysis. Data from a pilot external quality assessment scheme for the detection of molecular variants in plasma samples and from external quality assessment schemes for the detection of molecular variants in tissue samples were collected. Laboratory performance and error rates by sample were compared between matrices for variants present in both scheme types. Results showed lower overall performance [65.6% (n = 276) versus 89.2% (n = 1607)] and higher error rates [21.0% to 43.5% (n = 138) versus 8.7% to 16.7% (n = 234 to 689)] for the detection of variants in plasma compared to tissue, respectively. In the plasma samples, performance was decreased for variants with an allele frequency of 1% compared to 5% [56.5% (n = 138) versus 74.6% (n = 138)]. The implementation of liquid biopsy in the detection of circulating tumor DNA in plasma was associated with poor laboratory performance. It is important both to apply optimal detection methods and to extensively validate new methods for testing circulating tumor DNA before treatment decisions are made.

Feasibility and comparative analysis of Dirofilaria immitis microfilaria freezing and fixation for student instruction and assessment of clinical parasitology skills.

BACKGROUND: Detection of D. immitis microfilaria (mf) is an important diagnostic skill in veterinary medicine and is critical to Day 1 veterinarians and technicians. Finding a supply of blood containing mf to teach the technique and formalin's adverse environmental effects used in the diagnostic microscopic tests present a challenge. RESULTS: This study evaluated the use of cryopreserved and recently drawn mf-infected blood along with two fixative reagents, acetic acid or formalin for mf detection. The specific aims included determining if veterinary students could 1) detect cryopreserved mf added to fresh blood using routine diagnostic testing and 2) detect morphological differences in the mf. The 236 students were kept blind from the sample status. The ability of the students to identify mf and the mf morphology were compared for the samples and fixatives evaluated. The results demonstrate using a combination of cryopreservation and acetic acid for teaching microfilaria diagnostic techniques is fleasible; however, the quality of the mf morphology is less than optimal when compared to freshly acquired mf containing blood. Compared to reference values, the mf demonstrated a decrease in size with each additional variable evaluated. CONCLUSION: A majority (98.3%) of the 236 students correctly identified the presence of mf. Teaching laboratories could utilize cryopreserved mf-spiked donor blood in lieu of freshly collected mf-containing blood from a naturally or experimentally infected dog. Substitution of less hazardous chemicals for the fixative can be used. Finally, the change in size measurements provides a mechanism to ensure students can correctly measure mf as students are required to do verifiable measurements and cannot copy reference values from a text book since the cryopreservation and fixation methods cause the mf to measure smaller than textbook reference values.

Homologous and heterologous assessment of a novel biomaterial for venous patch.

OBJECTIVE: This study evaluated swine and bovine pulmonary visceral pleura (PVP) as a vascular patch. Venous patches are frequently used in surgery for repair or reconstruction of veins. Autologous patches are often limited by the number and dimension of donor tissue and can result in donor complications. Bovine pericardium is the most common heterologous patch used by vascular surgeons. Researchers, however, are continually seeking to improve heterologous and synthetic patches for improved outcome. METHODS: The PVP was peeled from swine and bovine lungs and cross-linked with glutaraldehyde. After sterilization and rinsing, the PVP patches were implanted in the jugular vein (10 × 35 mm) of pigs and dogs. Patency was evaluated by ultrasound, and animals were euthanized at 2 and 4 months. Neoendothelium and neomedia were evaluated by histologic analysis. RESULTS: The jugular vein patched by PVP in pigs and dogs remained patent at 2 and 4 months with no adhesions, inflammation, or aneurysm in the patches. The biomarkers of endothelial cells-factor VIII, platelet/endothelial cell adhesion molecule 1, and endothelial nitric oxide synthase-were detected in the neoendothelial cells. The expression of vascular smooth muscle cell (VSMC) α-actin was robust in the neomedia at 2 and 4 months. Neomedia composed of VSMCs developed to nearly double the thickness of adjacent jugular vein. The circumferential orientation of VSMCs in neomedia further increased in the 4-month group. CONCLUSIONS: The cross-linked swine and bovine PVP patch has a nonthrombogenic surface that maintains patency. The PVP patch may overcome the pitfall of compliance mismatch of synthetic patches. The proliferation of vascular cells assembled in the neoendothelium and neomedia in the patches may support long-term patency.

Safety assessment of antioxidants and color fixatives for the Korean population using dietary intake monitoring.

Food additives such as antioxidants and color fixatives are substances used in food intentionally for technical effect, such as decolorizing or intensifying the color of food. Based on the necessity of re-evaluating food additives for safety and to improve consumer perception, we conducted safety assessments for food additives according to the Risk Assessment Guidelines of the Korean Ministry of Food and Drug Safety. These safety assessments evaluated new risk information based on toxicology data and estimates of dietary intake exposures to food additives in comparison with the acceptable daily intake (ADI). Estimated daily intakes (EDI) of food additives were calculated using food consumption data for the Korean population derived from the Korea National Health and Nutrition Examination Survey and monitoring data based on the analysis of food additives in food products. Unlike contaminants, antioxidants and color fixatives are purposely added as food additives, and they are largely consumed in processed foods. Therefore, EDI was compared with ADI to investigate the likelihood of potentially hazardous effects in humans. The risk likelihoods of food additives, evaluated by comparing the EDI with the ADI, were less than 2% in the total population. Thus, exposure levels to antioxidants and color fixatives do not exceed the ADI. Based on the safety assessments conducted in this study, we estimate exposure to food additives to be within safe limits for all population groups.

Assessment of dynamic bioaccessibility of curcumin encapsulated in milled starch particle stabilized Pickering emulsions using TNO's gastrointestinal model.

Pickering emulsions stabilized by milled starch particles have been developed as a novel food-grade formulation to enhance the bioaccessibility of poorly soluble bioactive compounds (i.e., curcumin) by controlling the digestion of lipids in the human gastrointestinal (GI) tract. The dynamic bioaccessibilities of curcumin with and without encapsulation in the Pickering emulsion were evaluated using the dynamic TNO's gastrointestinal (TIM-1) model. For comparison, their digestion profiles were also studied using the in vitro pH-stat lipolysis model. With the combination of two in vitro models, the effect of the milled starch particle stabilized Pickering emulsions on the bioaccessibility of curcumin was fully revealed. There are large differences between the bioaccessibility values of curcumin samples obtained by these two models. Simulated small intestinal lipolysis in the pH-stat model revealed that the bioaccessibility of curcumin encapsulated in the Pickering emulsion was 27.6%, which was larger than 22.1% for free curcumin suspended in the bulk oil phase. The bioaccessibility of curcumin was 50.7% in the emulsion system and 7.8% in the bulk oil when using the TIM-1 model, which simulated the digestion conditions of the entire human GI tract. The digestion mechanism of the milled starch particle stabilized Pickering emulsions in the upper GI tract was well elucidated by the TIM-1 model. The gradual release and improved dissolution profile of the milled starch particle stabilized Pickering emulsions highlighted their potential as delivery systems for lipophilic bioactive compounds.

DNA metabarcoding from sample fixative as a quick and voucher-preserving biodiversity assessment method 1.

Metabarcoding is a powerful, increasingly popular tool for biodiversity assessment, but it still suffers from some drawbacks (specimen destruction, separation, and size sorting). In the present study, we tested a non-destructive protocol that excludes any sample sorting, where the ethanol used for sample preserving is filtered and DNA is extracted from the filter for subsequent DNA metabarcoding. When tested on macroinvertebrate mock communities, the method was widely successful but was unable to reliably detect mollusc taxa. Three different protocols (no treatment, shaking, and freezing) were successfully applied to increase DNA release to the fixative. The protocols resulted in similar success in taxa detection (6.8-7 taxa) but differences in read numbers assigned to taxa of interest (33.8%-93.7%). In comparison to conventional bulk sample metabarcoding of environmental samples, taxa with pronounced exoskeleton and small-bodied taxa were especially underrepresented in ethanol samples. For EPT (Ephemeroptera, Plecoptera, Trichoptera) taxa, which are important for determining stream ecological status, the methods detected 46 OTUs in common, with only 4 unique to the ethanol samples and 10 to the bulk samples. These results indicate that fixative-based metabarcoding is a non-destructive, time-saving alternative for biodiversity assessments focussing on taxa used for ecological status determination. However, for a comprehensive assessment on total invertebrate biodiversity, the method may not be sufficient, and conventional bulk sample metabarcoding should be applied.

Tannic Acid Solution: A Better Fixative Solution Than Formalin for Elastin and Collagen-Toxic and Morphological Assessment.

Formaldehyde is commonly used worldwide, even though it is classified as carcinogenic to humans by the International Agency for Research on Cancer. This has motivated intensive investigations of formaldehyde substitutes, and recently, some alternative solutions were found, which can potentially replace it. Previous research showed that tannic acid (TA) in glutaraldehyde solution has the ability to stabilize elastin and collagen. This provided a basis for the development of a new alcoholic fixative solution, particularly aimed at extracellular matrix components, with TA as a main component. Heart, brain, and intestinal samples were fixed by immersion in 10% regular formalin solution (RFS), 70% ethanol solution (ES), and tannic acid ethanolic solution (TAES). Next, tissue fragments were prepared for routine histology procedures. The toxicity of TA was analyzed using in silico tests for mutagenicity, as well as for cutaneous and respiratory toxicity. Analyses of photomicrographs demonstrated that all fixative solutions have the ability to preserve the fragments. The quantitative analyses showed that capability of TAES to preserve and stabilize elastin and collagen is superior to that of RFS and ES. We demonstrated that TA is not mutagenic, and it is less toxic for skin and respiratory tract. We therefore conclude that TAES can potentially represent a powerful and feasible alternative solution for fixing extracellular matrix of microscopic examination samples. Anat Rec, 301:1544-1550, 2018. © 2018 Wiley Periodicals, Inc.

A novel panel of short mononucleotide repeats linked to informative polymorphisms enabling effective high volume low cost discrimination between mismatch repair deficient and proficient tumours.

Somatic mutations in mononucleotide repeats are commonly used to assess the mismatch repair status of tumours. Current tests focus on repeats with a length above 15bp, which tend to be somatically more unstable than shorter ones. These longer repeats also have a substantially higher PCR error rate, and tests that use capillary electrophoresis for fragment size analysis often require expert interpretation. In this communication, we present a panel of 17 short repeats (length 7-12bp) for sequence-based microsatellite instability (MSI) testing. Using a simple scoring procedure that incorporates the allelic distribution of the mutant repeats, and analysis of two cohort of tumours totalling 209 samples, we show that this panel is able to discriminate between MMR proficient and deficient tumours, even when constitutional DNA is not available. In the training cohort, the method achieved 100% concordance with fragment analysis, while in the testing cohort, 4 discordant samples were observed (corresponding to 97% concordance). Of these, 2 showed discrepancies between fragment analysis and immunohistochemistry and one was reclassified after re-testing using fragment analysis. These results indicate that our approach offers the option of a reliable, scalable routine test for MSI.

Validation of a New Low-Cost, Methanol-Based Fixative for Cervical Cytology and Human Papillomavirus Detection.

OBJECTIVE: To test the performance of a new fixative for pap smear collection for liquid-based cervical cytology, CellPreserv® and compare it with the commercially available, PreservCyt® used in the diagnosis and detection of human papillomavirus (HPV). METHODS: Seven hundred twenty five women participated in this study after signing an informed consent. The specimens were collected using a traditional device, agitated in PBS, and equally divided in both fixatives. The slides were prepared routinely, stained by Papanicolaou, examined blindly by 2 cytologists, and reviewed by one cytopathologist. To search for HPV, 1,000 µL from each fixative was taken and processed by polymerase chain reaction. RESULTS: Considering the adequacy of samples, both fixatives had similar results - 0.33 and 0.32% of the cases unsatisfactory for PreservCyt® and CellPreserv®, respectively. Considering the 701 satisfactory cases and comparing the new fixative to the traditional fixative, there was 99.3% concordance between both. The results regarding the HPV detection was 100% concordant between the 2 fixatives. CONCLUSION: The new methanol-based fixative, CellPreserv®, is cheaper and equally efficient for treating cervical cancer screening and for HPV detection, and can be safely used by the health system prevailing in low-income countries.

Adapted methods for scanning electron microscopy (SEM) in assessment of human sperm morphology.

Infertility is a widespread problem, and in some cases, the routine basic semen analysis is not sufficient to detect the cause of male infertility. The use of the scanning electron microscope (SEM) could provide a detailed insight into spermatozoa morphology, but it requires specific sample preparation techniques. The purpose of this study was to select, adjust, and optimize a method for the preparation of spermatozoa samples prior to SEM analysis, and to establish the protocol required for its use in clinical practice. We examined sperm samples of 50 men. The samples were fixed with modified iso-osmolar aldehyde solution followed by osmium post-fixation. In the first method, dehydration of the cells and subsequent critical point drying (CPD) were performed on a coverslip. In the second method, the samples were dehydrated in centrifuge tubes; hexamethyldisilazane (HMDS) was used as a drying agent instead of CPD, and the samples were air-dried. The third procedure was based on a membrane filter. The samples were dehydrated and dried with HMDS in a Gooch crucible, continuously, without centrifugation or redispersion of the sample. Our results showed that the fixation with modified iso-osmolar aldehyde solution followed by osmium post-fixation, and combined with dehydration and CPD on a coverslip, is the most convenient procedure for SEM sample preparation. In the case of small-size samples or low sperm concentration, dehydration and drying with HMDS on the membrane filter enabled the best reliability, repeatability, and comparability of the results. The presented procedures are suitable for routine use, and they can be applied to confirm as well as to correct a diagnosis.

Economic, human and environmental health benefits of replacing formaldehyde in the preservation of corpses.

Formaldehyde has been prominent in preserving biological tissues since the nineteenth century. Despite being admittedly harmful to health and to the environment, it is still widely used. The Morphology Department of the University of Brasília - Brazil, applied the rethink, reduce, reuse, recycle and responsibility methodology to their activities in an effort to protect the health of laboratory workers and users, save resources and reduce damage to the environment. Here we evaluate the results obtained a decade after the implementation of this proposal (2005-2015). Formaldehyde was replaced by alcohol and glycerol solutions in corpse conservation. Over five thousand dollars in public funds that would have been destined to buying preserving substances were saved annually, and over a hundred thousand liters of water that would have been contaminated and thrown into the sewage system were spared. The environment used to implement the study was improved and anatomical parts kept for study had their lifespan extended. It is noteworthy that such simple adjustments could cause pronounced changes in laboratory activities. We would avoid contaminating billions of liters of water and it would be possible to save millions if similar practices were implemented in all educational institutions having similar routines.

Cytocentrifuged biopsy fixative preparation: A simple cost-effective technique facilitating microscopic diagnosis of lumen-dwelling intestinal parasites.

OBJECTIVE: Direct microscopic visualization is the most specific method for detecting intestinal parasites and is commonly achieved by stool examination or mucosal biopsy. However, postfixation, the intestinal biopsy fragment is often curled, and the entire surface of the biopsied mucosa is seldom viewed microscopically. Tissue processing further distorts morphology of the organisms and causes diagnostic difficulties. Examining multiple sections for parasite detection is time-consuming and often requires aid of special stains and/or immunohistochemistry. To overcome these disadvantages, we hypothesized that the fixative in which biopsies are transferred may provide a valid representation of the biopsied mucosal surface and therefore aid in the identification of mucosal surface parasites. MATERIALS AND METHODS: Formalin in which biopsies were transferred was retained, stored at 4°C and processed with a cytocentrifuge. Totally, 120 consequent duodenal biopsy fixatives were processed in this way and the cytocentrifuged smears visualized after May-Grunwald-Giemsa staining. Findings of these smears were correlated with their corresponding formalin fixed paraffin embedded tissue sections. RESULTS: Cytocentrifuged formalin preparations were found to be representative of the mucosal surface contents. Giardia trophozoites were visualized in 10/120 preparations with distinct morphological characteristics which were seldom appreciable in tissue sections, eliminating the need for special stains. Furthermore, two of the corresponding histology sections did not demonstrate the parasites despite step sections, while in one case few parasites could be identified in the step sections. CONCLUSIONS: Cytocentrifuged fixative preparation is a simple and cost-effective technique which can be routinely employed for intestinal parasite characterization.

Assessment of alternatives to environmental toxic formalin for DNA conservation in biological specimens.

One essential step of museum and clinical specimen preservation is immersion in a fixative fluid to prevent degradation. Formalin is the most largely used fixative, but its benefit is balanced with its toxic and carcinogenic status. Moreover, because formalin-fixation impairs nucleic acids recovery and quality, current museum wet collections and formalin-fixed, paraffin-embedded clinical samples do not represent optimal tanks of molecular information. Our study has been developed to compare formalin to two alternative fixatives (RCL2® and ethanol) in a context of molecular exploitation. Based on a unique protocol, we created mammalian fixed collections, simulated the impact of time on preservation using an artificial ageing treatment and followed the evolution of specimens' DNA quality. DNA extraction yield, purity, visual integrity and qualitative and quantitative ability to amplify the Cox1 gene were assessed. Our results show that both RCL2 and ethanol exhibit better performances than formalin. They do not impair DNA extraction yield, and more importantly, DNA alteration is delayed over the preservation step. The use of RCL2 or ethanol as fixative in biological collections may insure a better exploitation of the genetic resources they propose.

Assessment of PAXgene Fixation on Preservation of Morphology and Nucleic Acids in Microdissected Retina Tissue.

AIM: To compare the effect of the two fixatives on tissue morphology and utility to obtain good quality nucleic acids for molecular analysis from micro-dissected retinal samples. METHODS: Enucleated specimens from New Zealand white rabbits were fixed in formalin or PAXgene fixative according to standard protocols, and then processed and embedded in paraffin for sectioning. Sections were stained with hematoxylin and eosin to assess the structural integrity of retina. Retinal tissue on slides was micro-dissected. DNA/RNA were extracted and assessed for preservation of the quality and quantity of the retinal tissue. RESULTS: The retinal morphology was well preserved with both PAXgene and formalin fixation. The RNA yield obtained using both fixation methods was similar, but RNA from PAXgene fixed paraffin embedded (PFPE) samples had better purity than that from formalin fixed paraffin embedded (FFPE) samples. There was a twofold greater yield of DNA in PFPE compared to FFPE samples but with similar purity. Quantitative reverse transcription PCR analyses showed that the mean cycle threshold values for beta-actin, beta-microglobin, Opsin 1-sw, Rhodopsin, and 18S RNA of the PFPE group were significantly lower than those of the FFPE group (p < 0.01). Greater than 10-fold greater levels of gene expression were detected in PFPE relative to FFPE for the above genes. CONCLUSION: PAXgene fixed tissue retinal morphology is comparable to FFPE tissue. PAXgene may be a good alternative to formalin, providing good tissue morphology and ability to isolate high quality nucleic acids from micro-dissected paraffin embedded retinal samples.

Health Technology Assessment: introducing a vacuum-based preservation system for biological materials in the anatomic pathology workflow.

INTRODUCTION: The objective of this work is to assess the implementation of a newly introduced medical equipment technology for the vacuum-based preservation of biological materials within an Anatomic Pathology service. METHODS: The approach selected for the analysis is the Health Technology Assessment (HTA ), a comprehensive evaluation method based on relevant scientific evidence and designed to support healthcare decision makers in purchasing, replacing or disposing of technologies. The analysis focused on specific domains such as Technology, Organization, Safety and Economy. RESULTS: The study proves that the use of such technology ensures the biological specimen to be suitably preserved (up to 72 hours), both reducing the amount of fixative being employed in the diagnostic process (30% to 55%) and resulting, in the particular context under examination, in savings of 93%. DISCUSSION: The HTA reported no significant drawbacks related to the use of the technology being examined. Nonetheless, the workflow for managing the transfer of biological materials from the Operating Room to the Anatomic Pathology department needs to be redefined - in terms of handling, processing, storage and disposal. Other elements concerned the monitoring of storage temperature, fresh tissue handling and especially fixative amount reduction, which positively impacts on the operators' safety with regard to chemical hazards.

The importance of tissue handling of surgically removed breast cancer for an accurate assessment of the Ki-67 index.

AIM: Insufficient attention for the Ki-67 immunohistochemistry has been given to the importance of tissue handling for surgical breast cancer specimens. We sought to investigate the effect of fixation status on the Ki-67. METHODS: We examined the effect of fixative, time to and duration of fixation using surgical specimens, and finally, compared the paired Ki-67 index in the tumour between core needle and surgical specimen. RESULTS: The Ki-67 was significantly higher when 10% neutral buffered formalin was used (p=0.0276). Insufficient fixation caused a drastic reduction in the Ki-67 index (p=0.0177), but not significant in oestrogen receptor (ER) and human epidermal growth factor receptor 2 (HER2). Sixteen hours delayed time to fixation also caused a reduction of the Ki-67 (p=0.0284), but not significant in ER. Prolonged fixation significantly led to a gradual reduction in the Ki-67 in a time-dependent manner, but not in both ER and HER2. Finally, cutting the tumour before fixation improved fixation status and consequently caused an increased level of the Ki-67 index (p=0.0181), which resulted in a strong correlation of the Ki-67 between core needle and surgical specimen (r=0.8595). CONCLUSIONS: Tissue handling of surgical specimen is critical for assessing the Ki-67 compared with ER and HER2. We should pay more attention to tissue fixation status for the standard assessment of the Ki-67 index.