HistoTalks: NSH Podcasts

Fixation on Histology: NSH Was Doing Distance Learning Before It Was Cool — Here's Why It Still Works Written by: Connie Wildeman, MPA, Director of Education at NSH To Read the Full Blog, Click Here

Title: Histological Whole Slide Scanning Reproducibility Study   Authors: Hannah Benton, BSa, Tomoe Shiomi, MS, HTL(ASCP)CM, CT(IAC)CM, Fatma Farooqi, BSc, HTL(ASCP), Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC and Luis Chiriboga, PhD, HT(ASCP)QIHC Abstract: Whole slide imaging (WSI) is an increasingly versatile method for capturing and sharing high-resolution digital images of stained histological slides. These images can be used for a variety of applications, including clinical diagnosis, pathology review, and image analysis. While many whole slide scanners exist with varying features tailored to different use cases, a critical factor across all platforms is the accuracy and reproducibility of the scanned images. To investigate scan consistency over time, a control slide was prepared using a tissue microarray stained with Hematoxylin and Eosin (H&E). Ink dots were applied to the slide to define a consistent scanning region. The slide was scanned 77 times over six months using an Aperio AT2 whole slide scanner at 40x magnification.Image analysis was performed using HALO software by Indica Labs. Both the entire scan area and individual tissue punches were analyzed to assess total stained area and stain intensity, quantified by optical density (OD) for both hematoxylin and eosin. A linear regression model was applied to data from all individual punches and the full scan region. Additionally, a two-way ANOVA was conducted to compare OD values of hematoxylin and eosin between the first 10 scans and the last 10 scans. Key findings were that hematoxylin showed a statistically significant decline in both stained areas and OD over time, while eosin demonstrated a statistically significant increase in stained area, but a decrease in OD. These results suggest potential degradation of staining quality or imaging consistency over time. Possible contributing factors include slide bleaching, light source variability, annotation region size, or other imaging conditions. These will be the focus of future investigations to better understand and control variability in longitudinal slide scanning studies.

Title: Employing Multi-Tissue Controls to Enhance Kidney Biopsy Protocol Education in a Program in Histotechnology Student Lab  Authors: Hyder Aljanabi, Damon Bendolph, Gabriella Casas, Yosan Embrafrash, Sara Hassan, Anastasja Kraft, Stephan Lloyd-Brown , Nida Mubeen, Minh Nguyen, Xena Orosco, Nicole Rivera, Moriam Sissoho, Tan Tang , Kaleena Ramirez, Toysha Mayer, Mark Bailey Abstract: In a Program in Histotechnology student laboratory, establishing a representative and clinical teaching laboratory environment is essential for preparing students to manage the complexities of diagnostic tissue processing. The objective of the project was to simulate real-world clinical procedures by integrating multi-tissue controls into student education competencies for kidney biopsy staining protocols. Students participated in the investigation, each receiving four pieces of formalin-fixed, paraffin-embedded (FFPE) tissue: kidney, liver, gastrointestinal tract (GI), and tonsil. The tissues served as controls to validate staining techniques commonly used in renal pathology. Students prepared tissue sections using a rotary microtome, sectioning tissue at four microns. In total, forty slides were prepared, with eighteen slides manually stained using specific histochemical methods. Stains included hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), periodic acid methenamine silver (PAMS), and the Gomori Trichrome technique. The results yielded identifiable cellular and structural features critical for diagnostic interpretation. A slide review was conducted, and acceptable representative slides were selected for digital imaging. In addition, the results demonstrated the four tissue types which may be approved to use as controls, due to the consistency of demonstrating staining characteristics and features required for evaluating kidney biopsy protocols. Upon technical validation, the use of multi-tissue controls contributed to educational and operational outcomes. Students gained quality assurance experience, and the experience reinforced special stain and laboratory operations competencies, teaching students how to conserve reagent use, and to reduce time and expense. Furthermore, the protocol introduced the application of digital pathology and quality assurance in a real-world lab setting. Our investigation supports the integration of multi-tissue controls in histotechnology education as a valuable tool for enhancing both learning and laboratory efficiency. Future studies are recommended to include additional tissue types, stains, and immunohistochemical markers, to further advance and expand histotechnology educational competencies.

Title: Comparative assessment of routine H&E and Mason's Trichrome Stain to Differentiate Normal from Infected Urinary Bladders in the Göttingen Minipig   Authors: Stephanie D. Rivera, MS, HT(ASCP); Anthony Romanello, BS; Ronnie Chamanza, BVSc, MSc, FRC Path, MRCVS, FIATP, Preclinical Sciences & Translational Safety, Johnson & Johnson Innovative Medicine, Spring House, Pa Abstract: The objective of this experiment was to evaluate the difference between normal and infected Göttingen minipig urinary bladder and determine the difficulty of microscopically evaluating infectious urinary bladder. The focus was on the histology of the tissue using routine Hematoxylin and Eosin(H&E) staining and the Masson Trichrome (MT) special stain to demonstrate the morphology of normal and infectious urinary bladder. To improve on the translation between preclinical and clinical studies, the Göttingen minipig model is appropriate to use for research for diseased human bladder treatment because the minipig anatomy and organ system are similar to humans. The procedure was designed to evaluate normal uninfected urinary bladder and bacteria infected (E. coli) urinary bladder by evaluating morphology/cellular changes associated with the resultant inflammatory response in the urinary bladder of the Ellegaard Gottingen Minipig. The H&E-stained urinary bladder tissue section and a Masson Trichrome stained urinary bladder section were used to evaluate control ‘normal' tissue vs infected tissue cellular differences. Optimal microscopic evaluation requires that the urinary bladders are properly fixed and processed. By using proper fixation and diligent histologic practices, all components of the urinary bladder are captured for proper histologic evaluation.

Title: Evaluating the Reverse Slide Embedding Method vs. Heat Extractor Embedding in the Mohs Laboratory: A Comparative Quality Review of 100 Cases Authors: Tashsa Cromedy, Heather Frye, Ochsner MD Anderson Cancer Center, St. Tammany Cancer Center A Campus of Ochsner Medical Center Abstract:  Overview Accurate tissue embedding is critical in Mohs micrographic surgery for complete margin assessment. This study evaluates the efficacy of a reverse slide embedding method compared to the conventional heat extractor technique. The goal was to determine which method yields fewer artifacts or discrepancies that may compromise histologic interpretation and margin assessment. Methods A total of 100 Mohs cases were retrospectively reviewed in a controlled laboratory setting. Two embedding techniques were compared: Reverse Slide Method: 50 cases were embedded by placing the tissue on a chilled slide before embedding, ensuring orientation preservation and minimizing heat exposure. Heat Extractor Method: 50 cases were embedded using the traditional heat extractor to flatten and orient tissue in the embedding medium. All slides were reviewed by a Mohs surgeon for processing artifacts, orientation challenges, and histologic discrepancies. Validation The Mohs surgeon identified a total of 17 artifact inconsistencies or discrepancies across all cases: 13 instances were associated with the heat extractor method. 4 instances occurred with the reverse slide method. These findings suggest that the reverse slide method may reduce artifacts and improve embedding accuracy compared to the heat extractor, offering potential benefits for tissue integrity and diagnostic confidence in the Mohs laboratory. Conclusion The reverse slide embedding method demonstrated a significant reduction in embedding-related artifacts compared to the heat extractor technique. These findings support its use in the Mohs laboratory to enhance tissue quality, reduce the risk of diagnostic errors, and improve patient outcomes. Further studies with larger sample sizes and multi-lab validations are recommended to confirm these results.  

Title: High-resolution histological preparation of Araneomorphae and Mygalomorphae chelicerae using a modified petrographic technique Authors: Damien Laudier, HTL(ASCP)QIHC, Laudier Histology Abstract: Producing quality histological preparations of spider chelicerae with articulated fangs and cheliceral teeth is exceptionally challenging, if not impossible, using conventional histology techniques. Typically, these structures are examined with topographic or radiographic imaging methods, such as scanning electron microcopy (SEM) and micro computed tomography (mIcro-CT). While both are very useful tools for morphological analysis, they're not capable of revealing the fine tissue structure and cellular details, that a histological section viewed under light microscopy can provide. This study describes a modified petrographic/hard tissue histology technique to prepare high-resolution histology sections, for qualitative and quantitative assessment of both cheliceral soft tissue and fang microstructure.

Title: Enhanced Collagen Detection in Liver Fibrosis: A Comparative Study of Picrosirius Red Staining With and Without Bouin's Pretreatment   Authors: Nate Rampy, BS, Amber Moser, BS, HTL(ASCP)cm, Hannah Benton, BS, Brad Bolon, DVM, MS, PhD, DACVP, DABT and Elizabeth A. Chlipala, BS, HTL(ASCP)QIHC, Premier Laboratory, LLC, Longmont, Colorado; GEMpath, Inc., Longmont, Colorado Abstract: The use of Bouin's solution as a post-fixation treatment, rather than a primary fixative, remains largely unexplored in Picrosirius Red (PSR) procedures for collagen detection. In this study, we compared the effectiveness of PSR staining in liver samples from mouse, rat, and human with and without Bouin's solution as a pretreatment step. Liver sections were fixed in 10% neutral buffered formalin, processed and embedded in paraffin before being sectioned at 4 microns and stained with PSR. Bouin's was applied prior to staining for 60 minutes at 70º C, not as a fixative, but as a mordant to enhance dye-tissue interactions. Stained slides were scanned at 20x with an Aperio AT2. Visual assessment and image analysis in bright field microscopy demonstrated that the slides pretreated with Bouin's had significantly improved collagen differentiation, with enhanced contrast. By comparison, slides stained without the Bouin's pretreatment showed weaker and less distinct collagen staining. Our findings suggest that Bouin's pretreatment significantly improves collagen staining contrast and differentiation. The use of Bouin's pretreatment may serve as a valuable revision to the standard histology protocol for PSR fibrosis evaluation as well as general collagen visualization.

Title: Wheat Germ Agglutinin (WGA) Staining Optimized for Image Analysis of Muscle Tissue Morphometry  Authors: Cheru, R. and Wolf, J.C., Experimental Pathology Laboratories, Inc., Sterling, Virginia Abstract: Wheat Germ Agglutinin (WGA) is a plant-derived lectin and fluorescent stain that binds to N-acetylglucosamine and sialic acid residues in tissues, making it a valuable histochemical tool for visualizing cell membranes and components of the extracellular matrix. In muscle tissue, WGA staining allows clear delineation of the laminin-labeled basal membrane outlining each myofiber, distinguishing it from the residual autofluorescence of the myofiber sarcoplasm. To support digital pathology applications, a WGA staining protocol was optimized for compatibility with image-based quantitative analysis. Formalin-fixed, paraffin-embedded muscle sections were stained with fluorescently labeled WGA, counterstained with DAPI for nuclear visualization, and mounted with antifade medium to preserve fluorescence. Image analysis of WGA-stained skeletal muscle was successfully performed by a pathologist using Image-Pro® Plus software, employing macros to assess myofiber size and count.

Poster Title: CelLockTM and Axlab: A mutual symbiotic relationship resulting in increased patient care quality. Authors: Clifford M Chapman ( Medi-Sci Consultants), Karla Escobar (Axlab -US), Timm Piper (Axlab –US) Abstract: An ever increasing number of tiny specimens are received in histology laboratories every day. Fine needle aspirates, needle biopsies, gastrointestinal and skin biopsies, along with research specimens such as organelles: all pose challenges in receiving, processing and preparing stained slides from such specimens. Axlab is a Danish based company founded in 1993 which specializes in finding novel solutions for pathology laboratories. Axlab recently announced the release of their AS-410M automated sectioning equipment. Receiving FDA clearance in 2024, this equipment has been successfully implemented worldwide, resulting in enhanced section quality and increased workflow efficiency. CelLockTM is an innovative standardized method for collecting individual cells and small tissue fragments for subsequent routine, immunohistochemical and molecular pathology diagnostic and investigative techniques. The CelLock method, which utilizes a novel product CelLGelTM , results in the collection and retention of 99.9% of the original specimen within a paraffin embedded cell-block. In addition, the specimen is precisely located within the paraffin block, close to the surface, and is identified by a marker to cue when sections should be taken. Axlab is currently investigating the use of CelLock and CelLGel in the preparation of cell blocks which can be precisely sectioned on their automated sectioning equipment. The initial results are presented in this poster.

Poster Title: The High Cost of Understaffing: A Case Study in Surgical Pathology Consequences   Authors: Emily Nangano, MS, PA(ASCP)cm; Gillian Bass; Rob Terranova Abstract: Laboratories are the diagnostic backbone of healthcare, yet staffing decisions are often driven by budget constraints rather than operational needs. This case study examines the real-world consequences of delayed staffing action within the anatomic pathology department at a large academic medical center. Faced with a predicted shortfall in grossing coverage due to reduced resident support and unchanged PA staffing levels, institutional leadership opted against proactive hiring. As a result, grossing FTEs fell from 6.5 to 3.5, and histology staffing experienced a drop to 3 technicians from the usual 9 due to attrition and burnout. This staffing collapse led to turnaround time delays of up to 6–8 weeks and forced the lab to outsource specimen processing. Over the following seven months, the institution spent nearly $4 million on reference lab services. Staff morale declined sharply, clinician trust eroded, and senior PAs and histotechs resigned. Even after additional staff were hired, it took more than a year to stabilize operations. This poster presents supporting data, including FTE changes, outsourcing costs, and turnaround time impacts. It also explores how temporary, qualified locum tenens staffing solutions—such as Pathologists' Assistants and histotechnologists, and cytologists—can help bridge coverage gaps and prevent costly disruptions. Ultimately, this case underscores the critical importance of timely, proactive staffing strategies. The hidden costs of under-resourcing the laboratory go beyond dollars—they affect staff well-being, institutional reputation, and patient care outcomes.

Fixation on Histology: The Hunt for the Perfect Slide Written based on the NSH Webinar: Assessing Adhesion Slide Performance Across Histology Applications To Read the Blog, Click Here. 

Fixation on Histology: From Professional to Patient, A Med Tech's Organ Donation Journey Written based on the NSH Laboratory Webinar- When Worlds Collide Through Organ Donation To Read the Blog, Click Here. 

Fixation on Histology: Dementia Demystifying Written based on the NSH Laboratory Webinar Forget-Me-Not: Demystifying Dementia To Read the Blog, Click Here

Fixation on Histology: So You Want To Be A Manager? Written by: Jordan Terrell, HT(ASCP)cm To Read the Blog, Click Here

Fixation on Histology: Meet Nicole Leon, NSH's 2025 Histotechnologist of the Year   To read the blog, click here

Fixation on Histology: How Digital Image Analysis Strengthens H&E Staining Quality Control Based on the article Utilizing image analysis by optical density to evaluate changes in hematoxylin and eosin staining quality after reagent overuse” published in the Journal of Histotechnology. To read the full blog, click here. 

Fixation on Histology: U.S. Cancer Research at a Breaking Point: NIH's Grant Race Tightens Sharply Written by Antoinette EF Lona MSc., HTL(ASCP)cm  To Read the Full Article Click Here

Fixation on Histology: The Devil You Know: Common Reasons Change Doesn't Happen in the Lab To Read the Full Blog, Click Here

Fixation on Histology: Are You Making Time to Invest in Your Career? Written by: Ashley Stewart, Membership & Publications Manager To Read the Full Blog, Click Here.

Fixation on Histology: Remembering the Person Behind the Pieces of Tissue Written based on the webinar Remembering Why—a Review of Patient Case Studies  To Read the Full Blog Click Here

Fixation on Histology: CAP Made Competency Assessment a Tiny Bit Less… Confusing (Yes, really!) Written by: Nicole Leon BS, HTL(ASCP) To read the full blog, click here. 

Fixation on Histology: The Role of Images in Research Reproducibility This blog was written based on Framework for Reporting Materials and Methods for Histology Assays webinar To Read the Full Blog, Click Here.

Fixation on Histology: Enhanced Method for PGP 9.5 Immunohistochemical Labeling in Small Fiber Neuropathy Blog is based on article in the June 2025 Journal of Histotechnology To read the full blog, click here. 

Fixation on Histology: The Lena Spencer Scholarship Fund You can read the full blog here. 

Fixation on Histology: Veteran Histologist Cristi Rigazio Recognized for Her Dedicated Leadership Based on an interview with Cristi Rigazio Click to Read The Full Blog Here

President's Message: May 2025 Written by: Lamar Jones, NSH President

Fixation on Histology Blog: The Vital Role Histotechnologists Play in Veterinary Medicine Based on the Webinar By: Kei Kuroki, DVM, PhD, DACVP, of the University of Missouri Click to Read this Post

Fixation on Histology Blog: The Man Behind the Award: Dr. Jules Elias and the Power of Pursuing Passion Written by: Dr. Jules Elias, PhD Click to Read this Post

Fixation on Histology Blog: Enhancing Organoid Research with Histogel-Based Embedding Techniques Based on an Article By: Havnar, C., Holokai, L., Ichikawa, R., Chen, W., Scherl, A., & Shamir, E. R. (2024) Click to read this post.

Fixation on Histology Blog: Phlebotomy to Research: A Journey Through Histotechnology Written By: Andrea Transou BS,HTL(ASCP), QIHC(ASCP) Click to read this post.

Fixation on Histology Blog: My Histology Journey - Jeniesha Russell, HT(ASCP) Written By: Jeniesha Russell, HT(ASCP) Click to read this post.

Fixation on Histology Blog: My Histology Journey - Toysha Mayer Written By: Toysha Mayer, DHSc, MBA, HT(ASCP) Click to read this post.

Fixation on Histology Blog: Mastering the Art of Immunohistochemistry: Essential Techniques for Reliable Diagnostic Results Written By: Khulood Ayad Majeed; College of Dentistry, University of Kirkuk Click to read this post.

Fixation on Histology Blog: Where Does Tissue Contamination Happen Most? Based on the Webinar By: Valerie Cortright, BA, HT(ASCP), HTL(ASCP), QIHC Click to read this post.

Fixation on Histology Blog: 10 Game-Changing Leadership Lessons Written By: Cathay García Lauzurique, MHA, MSc, HTL(ASCP) Click to read this post.

Audio reading from the July 2023 NSH Fixation on Histology Blog, Hot Dog as an Alternative Source for Control Tissue. Read entire article.

P08-Diagnostic Cytopathology Cell Block Preparation Methods- Anna Patterson FIBMS MSc CSci Diagnostic Cytology Scheme Coordinator Helen Naylor MSc Diagnostic Cytology Technical Specialist Cell blocks from Cytopathology samples have always had value in the diagnostic process as a complement to the traditional Cytopathology stains – Papanicolaou and Romanowsky.  This has become more important to provide material for Immunocytochemistry to refine malignant diagnosis, and more recently, for the use of molecular testing to aid in the choice of tailored chemotherapy regimens.  If this information can be obtained from Cytopathology samples, which are less invasive than biopsy samples, the patient will benefit. A variety of preparation methods are available for the preparation of cell blocks from cells from Diagnostic Cytopathology samples.  The most popular methods will be discussed and how they can be used to optimise the quality of cell preservation if used correctly. Information garnered from the results of the recently introduced UK NEQAS CPT Diagnostic Cytopathology Cell block scheme will be presented and how this information can be circulated to laboratories experiencing difficulties with their preparation methods as an advisory service. This will include. • Understanding the clinical importance and diagnostic purpose of correct procedures in Diagnostic Cytopathology Cell Block preparation. • Identifying and determining factors affecting best practice and quality in Diagnostic Cytopathology Cell Block practice and how to resolve them. • Identifying and understanding the causes of artefacts experienced in Diagnostic Cytopathology Cell Block preparation methods and how to eliminate and prevent them. P11- Participating In A Digital Diagnostic Cytopathology Interpretive Proficiency Testing (iEQA) Scheme -Helen Naylor, Anna Patterson, Chantell Hodgson, Ashley Makela The iLabXCell Digital Diagnostic Cytopathology Interpretive Proficiency Testing Scheme or iEQA is facilitated by UK NEQAS Cellular Pathology Technique (CPT), and is designed to promote quality, excellence, and education for all involved in screening and reporting of Diagnostic Cytopathology. It is open to medical and non-medical staff, trainees, advanced practitioners, and staff associated with Cytopathology. iEQA provides superior, outstanding, and representative case examples for individuals to examine in a remote setting and submit an opinion via the user-friendly digital platform.  Offering 2 circulations annually it provides • Easy access for registration  • Ability to choose specimen types  • Advanced slide viewing • Clinical details to assist diagnoses • Categorisation of images using benign or malignant  Flexibility Remote learning allows participants to continue learning alongside their peers, in an environment accommodating their needs. It allows the flexibility of international participants to access the platform at any time, convenient to them. Remote learning via the iEQA platform, offers the flexibility for users to pick up learning where they left it – anywhere, anytime, from any location. Self-paced learning Traditional learning has long failed to acknowledge the individualised nature of learning, opting instead for a generalised approach that may not be optimised for everyone. One of the many benefits of this iEQA is providing participants more independence to: • Spend more time on cases/case types they find difficult • Revisit the images as often as they need  Environmental benefits Digitising slides has led to a decrease in physical resources previously necessary for iEQA to function. Everything participants need is accessible on-line, eliminating the historical environmental impact and the time taken for circulations to be distributed and completed.   Future developments An on-line educational image library, containing images of Diagnostic Cytopathology cases of urines, serous fluids, respiratory and head and neck cases used in previous circulations for educational and training purposes.         

P06-Understanding the Quality of your Electron Microscopy Provider in this Era of Outsourcing of Services- Tracey de Haro MSc, FIBMS, UK NEQAS CPT TEM Scheme Coordinator Specialist Scientific Lead for Electron Microscopy University Hospitals Of Leicester NHS Trust, UK  Background Electron Microscopy (EM) remains vital to the diagnostic repertoire for the diagnosis of pathologies. Surveys of diagnostic TEM units in the UK were carried out in 2012, in 2019 (unpublished) and is currently being repeated. These surveys showed that a large amount of EM is now outsourced to units away from the originating trust. Whether UK or globally, when pathology departments are looking for a supplier of diagnostic EM services, the only questions they ask of the EM units are “how much does it cost” and “what is your turnaround time?” Are these the only relevant questions to ask? Considerations The following relevant issues should be considered alongside cost and speed; The technical quality of an electron microscopy service; Can the EM unit produce good quality sections and images that maximizes the chances of observing relevant pathologies? Data from UK NEQAS CPT show that only 50% of EM units participating in the diagnostic TEM scheme achieve excellent scores of 9 or 10 out of 102 not achieving excellent quality could potentially compromise a diagnosis. The knowledge the staff have of ultrastructural pathology • Do the staff assessing your samples know what to look for? Having proof of EM staff's knowledge in ultrastructural pathology is essential when relying on them to provide relevant images and a considered report on features seen or not seen. Questions to Ask Your EM Provider To ensure that the EM service you are sending your samples to is ‘fit for purpose', you should not only consider the speed and cost of the service but also; • Ensure that your potential provider is accredited to ISO 15189 standards. • Ask for evidence of participation in an EQA scheme specifically for technical TEM. • Ask for TEM EQA results over the past 12 months and ensure they are consistently achieving excellent marks. • Do the EM staff participate in regular knowledge and competence competency review specifically for TEM and ultrastructural assessment? • How much experience of ultrastructural pathology do the members of staff examining your samples have and do they have any qualifications in this area? • Get endorsements from other users of the EM service to evidence the quality of work offered. Summary Access to EM services remains vital across the globe, but in the UK is increasingly being outsourced to units remote from the originating trust. In this case, the pathologist is reliant on the images and ultrastructural report being accurate to inform diagnosis. To ensure accuracy, the EM unit, whether they be UK based or part of our international community, all should participate in a quality EQA scheme and all staff should be experienced and have access to training to ensure they are educated to a high level in ultrastructural pathology. Without this, the referring trust cannot be guaranteed the service they are paying for is fit for purpose. P15-Understanding the Quality of your Electron Microscopy Provider in this Era of Outsourcing of Services: How does a Technical EQA Scheme Add Value? -Tracey de Haro MSc, FIBMS, UK NEQAS CPT TEM Scheme Coordinator Specialist Scientific Lead for Electron Microscopy University Hospitals Of Leicester NHS Trust, UK Background Benefit of Participation in an EQA Scheme  Summary The Transmission Electron Microscopy (TEM) Scheme was developed in 2018 as a means of assessing the quality of the final output of diagnostic TEM Units against a defined assessment standard. The aim of this EQA scheme is to allow EM units to have their work regularly assessed and measured against other TEM units in an anonymous and supportive manner. This ensures that results are reliable and comparable no matter where they are performed. Education in technical aspects of the TEM process is also offered as a knowledge and competence exercise as part of each EQA assessment. EQY Scheme Organization TEM scheme Participants are asked to submit 4 digital images from each of 2 contrasted TEM cases. There are 6 EQA assessment runs per year. Specific tissue types for each case are requested for each assessment run. Renal cases are requested for each run as case 1 and muscle or nerve are requested in rotation for case 2. However alternative tissue types can be submitted for either case if participants do not examine those tissue types. Details of technical fixation, processing and imaging for each case are required to be submitted as part of Data Entry. This allows generation of ‘best method' reports for high achieving EQA scores to be issued to Participants. Each image is anonymously assessed against the defined assessment  criteria by a pair of expert peer assessors. Each assessor will award a score out of 5 giving a total score for each image out of 10. Participants receive the following for each run; • EM Individualized report detailing the scores awarded for each image, along with information about where marks were lost and why • EM best method report detailing methods that achieved high scores • EM image report giving examples of images that achieved a range of scores from the maximum score to low scoring images, along with why those scores were allocated  Summary Participation in a technical TEM EQA scheme provides factual information that can also be used to advertise and promote an EM unit as providing a proven quality service.  Whilst being participating in an educational exercise within each EQA assessment, allows individual and laboratory knowledge and competence to be assess and demonstrated. Evidencing your EM unit provides a high quality, gold standard service is vital when diagnostic electron microscopy is often being outsourced to external EM units. Cost of service and turn round times for results are often asked, but quality of work for diagnostic results should also be considered. Consistency of technical quality is important to ensure pathologists receive high quality diagnostic information. This mitigates against misinterpretation from poor quality images, assuring a safe, accurate diagnosis. This is good news for the participating EM units, good news for the diagnostic process and ultimately, good news for patients.  

Research Requires Flexibility: Protease-Free Permeabilization Expands FISH Tissue Applications.-Andrelie Branicky, Shared Laboratory Resources, Lerner Research Institute, Cleveland Clinic, Cleveland, OH Fluorescence in situ hybridization (FISH) visualizes the presence of a specific DNA or RNA sequence in a tissue sample or cell. This method, particularly the mRNA version, detects gene expression when protein might not be present or IHC is impossible. FISH combined with immunohistochemistry enables spatial transcriptomics, which provides significantly more information about the tissue microenvironment. Formalin-fixed paraffin-embedded (FFPE) tissues are the standard for tissue preservation in the clinical world. Most commercial mRNA probe and amplification systems are built around the model of FFPE tissue that can withstand harsh protease permeabilization. In the research world, tissues are fixed in different fixatives for varying times; all at the discretion of the investigator instead of an organization like the CLIA.  Given the wide range of tissue preparations, the HCR automated FISH-ISH protease-free program provides the flexibility to combine  FISH and fluorescent immunohistochemistry on tissue fixed in a variety of ways such as: 10% NBF, Histochoice (a glyoxal-based fixative), and methanol/acetic acid, with only minor changes to the basic protocol. Additionally, the lack of harsh protease pre-treatment maintains tissue integrity and morphology for staining and imaging. 

The Development of a Cocktail of Microglia and GFAP For Easy Diagnosis - Anisha Bhasin B.S, Sarah Holguin, MBA. B.S, Joe Vargas, M.S Microglia and GFAP are distinct neural markers, typically used separately to diagnose the degree of neurological infection and injury. Microglia, a glial cell, is used in the immune response of the central nervous system. GFAP is an astrocyte marker; astrocytes provide structural support and make up the blood-brain barrier. Using the two in conjugation with one another would prove to be an efficient diagnostic tool. A cocktail was constructed with optimal titration to observe the two markers in unison. In clinical usage, it will provide an efficient diagnosis of chronic inflammatory conditions of the central nervous system. The staining was conducted in IHC and fluorescence to compare morphology and count. Due to anatomical similarities, there tends to be morphological confusion between microglia and GFAP. However, when stained in conjunction with one another, notable differences can allow for easy distinction. This is why a cocktail run with a dual staining technique would be a superior diagnostic tool in comparison to testing the two markers independently.

Study tools for the histotechnologist (HTL) Histotechnician (HT) certification exam- Amber Moser1, BS, Hannah Benton1, BS, Taylor Wallace1, BS, and Elizabeth A. Chlipala1, BS, HTL(ASCP)QIHC Premier Labs, Longmont, CO Histology laboratories have seen an increase in workplace shortages since the COVID-19 pandemic and an increase in noncertified applicants to fill open positions. The traditional route for histology certification is the completion of a histology education series with an accredited institution; however, there are many other routes to qualify for certification. With declines in histology program graduate numbers, and the change in work force after the COVID-19 pandemic, it is expected that nontechnical positions will continue to be filled by noncertified individuals. This may lead to an increase in alternative routes of certification, specifically through laboratory experience. These tools provide additional resources for individuals studying for the HT/HTL certification exams. They are catered to individuals without access to an accredited educational program. Information has been organized in ways that focus on areas that may be particularly difficult, information heavy or exam relevant. They utilize multiple learning modalities, specifically written, spoken, and visual information retrieval methods, with opportunities for auditory and group learning. They are also intended to be cost effective. Materials include laminated study templates, laminated scratch paper, flashcards, a slideshow for visual tissue assessment, and a general study topic list. Questions are both simple and complex, and representative of exam questions. It should be noted, however, that this is not a comprehensive study guide and is intended to be used in addition to an online study program or exam preparation course and textbook resources. These tools are incomplete guides which can be added to and personalized.  

Somagen Method of Alcohol Reagent Tracking (SMART) Optimization to Realize Workflow Efficiencies within the Laboratory-Anne de Wolf, BSc MLT Somagen Diagnostics, Edmonton, Alberta, Canada; Christine Chiu MLT Somagen Diagnostics, Toronto, Ontario, Canada; Annie Vallee Somagen Diagnostics, Montreal, Quebec, Canada; James Tang, BSc MLT Somagen Diagnostics, Edmonton, Alberta, Canada Tissue processing in recent decades has changed without subsequent changes to reagent maintenance. Reagent maintenance protocols have been ‘Grandfathered down' without consideration for the reagent quality and how this is related to tissue types and block numbers processed. The concentration of the last absolute alcohol station needs to be >98 % to effectively remove water from tissue before clearing and paraffin infiltration.1, 2 Using the Eagle Eye Digital hydrometer can provide reagent quality information which could also provide potential downstream efficiencies to laboratory workflow and cost savings in labour and reagents.

Method for FFPE Organ-Chip -Lindsay Parmelee HTL(ASCP) 1,2,3, Stephanie Pei Tung Yiu PhD 1,2,3,4, Chi Ngai Chan PhD 1,2,3, Sizun Jiang PhD 1,2,31 Center for Virology and Vaccine Research 2 Beth Israel Deaconess Medical Center 3 Harvard Medical School 4 Wyss Institute Organoids are currently being developed for applications in biomedicine such as drug development and disease research. With many organoid models emerging on the market there is growing need to develop methods to adapt organoids to the established work flows and assays that are the foundations of modern research.  A method was developed for processing, embedding, and sectioning Organ-Chips for FFPE workflows. Organ-Chips were treated with Histogel and trimmed to fit standard sized cassettes. Followed by processing and embedding as usual before proceeding to the microtome. The resulting slides have successfully been stained with hematoxylin and eosin and immunohistochemistry, as well as shown promising results for use on multiplexing platforms. Although the process is laborious and requires continued refinement the slides should be sufficient for highplex proteomics and transcriptomics methods and can be used in addition to FFPE tissues for research into immunology, oncology, and virology.

Assessing Adhesion Slide Performance Across Histology Applications -Colin Brewer, CellPath, Newtown, Wales; Rachel Finn, HTL, StatLab, McKinney, TX; Neil Haine, PhD, CellPath, Newtown, Wales; Arielle Hobson, StatLab, McKinney, TX; Moritz Kamphenkel, Knittel Glass, Braunschweig, Germany; Ronja-Melinda Komoll, Ph.D, Knittel Glass, Braunschweig, Germany; Racheal Moore, HT, StatLab, McKinney, TX; Nicole Romer, Knittel Glass, Braunschweig, Germany; Edeltraud Schikora, Knittel Glass, Braunschweig, Germany; Stefan Welsch, Knittel Glass, Braunschweig, Germany; Susan Willis, CellPath, Newtown, Wales Adhesion slides are widely preferred for IHC to aid in securing tissue sections to the slide and prevent reworks that could potentially postpone a patient diagnosis and drive-up costs in the lab. The cost of reworking a failed IHC slide due to poor tissue adhesion is estimated to be ~$80 per slide, considering the reagent cost and workload administration.1 Adhesion slides reinforce tissue adherence and integrity, minimizing the need to recut and restain the sample to ensure proper tissue morphological characteristics. Adhesion slides may also be used for H&E stains and special stains for added adhesion, but could retain excess reagent, or background staining, on the slide. In this study the differences in contact angle and in tissue adherence during microtomy were analyzed,  investigation was done on whether different adhesion slides exhibit similar levels of background staining during histological staining procedures, and evaluation and comparison of the tissue adhesion properties of adhesion slide brands across different tissue types and applications was performed.  After wide-ranging testing of adhesion slide characteristics, this study exhibited that not all adhesion slides are created equal. While water bath behavior showed to not be a relevant factor, there was considerable variation in background staining and tissue adhesion between slides. The results of this study suggested that it is important to determine what the needs are for your laboratory based on the types of staining done and tissue types used. It is also important to test adhesion slides to find the right slide or slides for your laboratory applications.

The Use of Immunohistochemistry vs. qPCR to Detect BRAFV600E in Thyroid Cancer- Kannitha Chek HTL (ASCP), Nicholas Hoo-Fatt, MS, HTL (ASCP), DP, Samantha Diamond, M.D., Haresh Mani, M.D., Myong Ho “Lucy” Nam, M.D. Introduction: This study aimed to validate the effectiveness of the Ventana anti-BRAFV600E antibody in detecting the BRAFV600E mutation in thyroid cancer using immunohistochemistry (IHC) as an initial screening test. The BRAFV600E mutation is a common genetic alteration in thyroid cancer, particularly papillary thyroid carcinoma (PTC), and detection is crucial for prognosis and treatment decisions. Methods: The research examined 12 thyroid cancer cases (11 PTC and 1 anaplastic thyroid carcinoma) and 12 non-cancerous thyroid cases. This study was conducted by using both the IHC BRAFV600E antibody as well as genetic testing methods including Next-Generation Sequencing (NGS) and the Biocartis Idylla rapid qPCR module for BRAF mutation. Results: The antibody successfully identified the BRAFV600E mutation in all cancer cases, aligning with results from other genetic testing methods, and as expected, non-cancerous cases showed no mutation-specific staining. The study highlighted important considerations in interpreting IHC results, such as the presence of non-specific brown staining due to colloid containing brown pigments in negative cases. It also emphasized the importance of proper tissue representation, as demonstrated by one case that initially lacked staining. While the research provides strong evidence for the accuracy of the Ventana anti-BRAFV600E antibody, it also identified limitations. The study lacked PTC cases negative for the BRAFV600E mutation, which would have provided a more comprehensive validation. Conclusion: The researchers suggest further investigation with a broader range of samples, including more anaplastic thyroid carcinoma cases and follicular variant of PTC, to explore potential correlations between tumor heterogeneity and staining intensity. This validation study contributes to improving thyroid cancer diagnosis and treatment by authenticating the reliability IHC methods in detecting a key genetic mutation. It also highlights the importance of continued research to optimize diagnostic methods in thyroid cancer.

Whole NHP Brain Microtomy Technique- S Wessel, MS, HTL (ASCP),AskBio, Discovery 1, Morrisville, North Carolina T Marais, AskBio France, Paris Brain Institute (ICM), Paris, France M Martini, PhD, PhD, AskBio, Discovery 1, Morrisville, North Carolina, M Faridounnia, PhD, MSc, AskBio, Discovery 1, Morrisville, North Carolina, D Gregorowicz, HTL (ASCP),AskBio, Discovery 1, Morrisville, North Carolina, L Haertel, HT (ASCP), AskBio, Discovery 1, Morrisville, North Carolina M Hamilton, HTL (ASCP), AskBio, Discovery 1, Morrisville, North Carolina, M Vallucci, AskBio France, Paris Brain Institute (ICM), Paris, France ,S Alves, PhD, MSc, AskBio France, Paris Brain Institute (ICM), Paris, France, J Mysore, BVSc, MVSc, PhD, DACVP, AskBio, Discovery 1, Morrisville, North Carolina Preserving the integrity and conformation of capsid proteins, enzymes, and antigens while working with large tissue sections presents a “big” challenge in histology laboratories. With our Whole NHP (Non-Human Primate) Brain microtomy technique, we maintained a frozen NHP brain as a single piece while coronally sectioning through the entire cerebral hemispheres followed by cerebellum/brain stem. The brain remained frozen on a sliding microtome by utilizing a freezing stage and molds filled with powdered dry ice. As the brain was sectioned at 40um the molds were removed to uncover additional tissue for cutting until the tissue had been entirely sectioned. Serial pictures and an organized series of well plates were used to preserve anatomical order. By sectioning the brain as a whole and maintaining control over anatomical context we achieved results that may enhance the accuracy and thoroughness of histopathological examinations. Furthermore, serial sectioning of the Paraformaldehyde-fixed frozen brain preserved the antigenicity for various immunohistochemical assays. We believe this technique offered certain advantages over the routine paraffin-embedded sectioning method, especially for the biodistribution studies practiced during the developmental stage of gene therapies. 

Encapsulated Cell Therapies in Sheep as a Translational Model - Trisha M. Fabijanic, B.S., University of Arizona, Tucson, AZ, Amy C. Kelly, Ph.D, University of Arizona, Tucson, AZ, Eliza H. Johnson, University of Arizona, Tucson, AZ, Delaney A. Drew, B.S., University of Arizona, Tucson, AZ, Demetri A. Vlachos, B.S., University of Arizona, Tucson, AZ, Charles W. Putnam, M.D., Ph.D., University of Arizona, Tucson, AZ, Sean W. Limesand, Ph.D., University of Arizona, Tucson, AZ, Klearchos K. Papas, Ph.D., University of Arizona, Tucson, AZ Introduction: Cell therapy has become increasingly studied for therapeutic use in drug delivery and regenerative medicine. Devices intended for implantation of cells require construction of membranes that can safely contain transplanted cells, provide a barrier to the host immune system and an outer vascularizing membrane to induce close vessel formation. Sheep make an excellent model because of their nearly comparable size, physiology, and disease profile with humans, as well as their docility, capacity for repeated blood draws, and ability to tolerate numerous implants during a single operation. In this study, sheep were implanted with devices containing allogeneic primary fetal sheep or xenogeneic Rat-2 fibroblasts to determine cell survival and immune response. Methods: Primary fetal sheep fibroblasts were isolated and cultured in RPMI. Rat-2 fibroblasts (ATCC) were cultured in DMEM. Cells were loaded into devices at different densities and transplanted subcutaneously. Sheep underwent allogeneic or xenogeneic transplantation with empty control and perforated devices transplanted as well. Devices were explanted at days 3, 7, 21 and 38 to evaluate host responses, vascularization, and cell survival through histological evaluation. Results: Sheep fibroblasts survived at different densities, confirming alloprotection with good vascularization and absence of a major foreign body response or extensive inflammation around the implants. Devices overloaded at the highest densities exhibited some host immune responses. Perforated devices exhibited a greater immune response that infiltrated the device. A robust immune response was seen around xenogeneic transplants with no encapsulated cells surviving. Conclusion: Sheep make a valuable translational animal model in cell therapy research. The results of this study concluded alloprotection in immunoisolating devices, allorejection in purposely perforated devices, and host immune response to xenogeneic transplantation, representing a feasible model for translational development of cell therapies. Future work will include continued allogeneic studies while avoiding xenogeneic rejection by incorporating immunomodulatory technologies.

Cellular localization of the pink bollworm Cry1Ac Bt protein receptor PgCad1 in cultured insect cells Authors: Melanie Miranda (BS, HTL- ASCP, University of Arizona-Comparative Pathology Core Laboratory, Tucson, AZ) Jeff Fabrick (PhD, United States Department of Agriculture, Phoenix, AZ) Transgenic cotton genetically engineered to produce insecticidal proteins from the bacterium Bacillus thuringiensis (Bt) are used to manage insect pests, including the pink bollworm (Pectinophora gossypiella), a globally invasive pest of cotton. However, the evolution of resistance to Bt Cry proteins (Cry1Ac and Cry2Ab) by the pink bollworm in parts of Asia has reduced the effectiveness of Bt cotton. Several mutations in the midgut cadherin gene PgCad1 are genetically linked with the resistance of the Cry1Ac Bt toxin, which can result in disrupted cellular trafficking of the cadherin receptor to the surface of the midgut membrane and decrease toxin binding. Here, we established specialized insect histology and immunohistochemistry (IHC) protocols for reliable localization the PgCad1 receptor in cultured Tni insect cell lines. Such protocols may be useful for in situ localization of wild type PgCad1 and mutant variants in midgut cells of the pink bollworm.    

2023 NSH co-presenters Gabriela De la Cruz and Lauren Ralph give great advice on selecting a topic and share why they decided to co-present at the 2023 NSH Convention. 

NSH member Ariel (Ari) Liberda discusses her decision to present at the 2023 NSH Convention.  She shares her journey and advice to anyone who is considering taking the leap!

Making the FNA Count Author: Jeffery Rinker The hospital at Sanford Bemidji Medical Center has 119 beds and performs an average of 150 FNAs each year. Our histologists do both histology and cytology, with FNAs comprising the majority of our cytology. Since 2020, our non-diagnostic rates on FNA samples have fluctuated. In 2022, our rate was as high as 28%. Compared to the non-diagnostic rate of 10% outlined in The Bethesda System for Reporting Thyroid Cytopathology, our results were over two times the accepted rate. To bring non-diagnostic rates down at our facility, a look at processing from beginning to end.  Radiology was the first place to start an overview of the process. During observation, we discovered that multiple passes of the needle contributed the bloodier samples, which affected the specimens processed later. To counter this, we reduced the number of smears and increased the amount of specimen deposited into CytoLyt (ThinPrep). Next, the cytology department process was examined. During this examination, it was discovered the CytoLyt (Thinprep) specimen was not being processed because of lack of specimen. When it was processed the slides were being dried to long leading to artifact on the slide. By increasing the sample size in radiology and using a slide dryer to regulate the drying, the lab was able to produce a more consistent result.  Following these changes, the lab projected to meet or exceed the goal of less than 10% non-diagnostic slide results by the end of 2023.