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

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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.

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.

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:

Validation

The Mohs surgeon identified a total of 17 artifact inconsistencies or discrepancies across all cases:

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.

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.