Written based on the NSH Webinar: Embedding Basics & Troubleshooting

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Written based on the NSH Webinar Jonesing For Johne’s - AFB Control Tissue

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Written by: Connie Wildeman, Director of Education, NSH

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Special thanks to Milestone Medical for supporting this episode. 

Written based on the article "Multiple levels of Gomori methenamine silver (GMS) stains do not improve diagnostic yield in esophageal biopsies" published in the Journal of Histotechnology

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Written by: Connie Wildeman, MPA, Director of Education at NSH

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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.

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.

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.

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.

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.

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.

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.

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.

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.

Written based on the NSH Webinar: Assessing Adhesion Slide Performance Across Histology Applications

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Written based on the NSH Laboratory Webinar- When Worlds Collide Through Organ Donation

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Written based on the NSH Laboratory Webinar Forget-Me-Not: Demystifying Dementia

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Written by: Jordan Terrell, HT(ASCP)cm

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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.

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Written by Antoinette EF Lona MSc., HTL(ASCP)cm 

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Written by: Ashley Stewart, Membership & Publications Manager

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Written based on the webinar Remembering Why—a Review of Patient Case Studies 

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Written by: Nicole Leon BS, HTL(ASCP)

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This blog was written based on Framework for Reporting Materials and Methods for Histology Assays webinar

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Blog is based on article in the June 2025 Journal of Histotechnology

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Based on an interview with Cristi Rigazio

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Written by: Lamar Jones, NSH President

Based on the Webinar By: Kei Kuroki, DVM, PhD, DACVP, of the University of Missouri

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Written by: Dr. Jules Elias, PhD

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Based on an Article By: Havnar, C., Holokai, L., Ichikawa, R., Chen, W., Scherl, A., & Shamir, E. R. (2024)

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Written By: Andrea Transou BS,HTL(ASCP), QIHC(ASCP)

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Written By: Jeniesha Russell, HT(ASCP)

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Written By: Toysha Mayer, DHSc, MBA, HT(ASCP)

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Written By: Khulood Ayad Majeed; College of Dentistry, University of Kirkuk

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Based on the Webinar By: Valerie Cortright, BA, HT(ASCP), HTL(ASCP), QIHC

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Written By: Cathay García Lauzurique, MHA, MSc, HTL(ASCP)

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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. 

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.

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. 

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.

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.

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.

Authors: Nicholas Pankow, BA; Gabriela De la Cruz, BS; Hannah Marie Atkins, PhD, DVM

In some diseases or conditions, it is challenging to leverage human tissues to determine patterns and resolve disease progression. Similarities between human and mouse biology make using murine models in pre-clinical studies possible. More specifically, mouse lungs are used to further analyze respiratory illnesses as they provide scalable models that can either be genetically manipulated to elicit human diseases or follow similar phenotypic outcomes. Mice are thus used in several different disease models that can be later translated to human conditions including asthma, COPD, toxicants, cystic fibrosis, as well as viral and bacterial infections. The standard histological embedding orientation of the lung provides an easy view of the main bronchus, alveoli, bronchioles, trachea, and related lymph nodes for distinct research inquiries. However, the standard histological orientation may not demonstrate the specific areas of interest to further investigate these conditions. By trimming the lung lobes in specific ways or altering the orientation during embedding, we can provide a specific focus to a study.  One alternative embedding strategy is the “max airway”, which focuses on showing the main bronchus anatomy on all lung lobes. The “morphometry” embedding strategy allows for multiple cross-sections of the left lung lobe and a cross-section of the main airway.  Another common method is the “lung sampling” strategy which provides a systematically chosen cross-section of the lungs. A “left lobe focus” strategy is used to review the bronchioles and alveolar areas. Finally, “whole lung” embedding can be useful for examining general lung morphology, adjacent mediastinal structures, and other organs. Using these different protocols, we can bring a desired focus to a study and have a more scalable experiment without using human tissue early in the process.

Authors: Jason (Jay) Innerhofer, PA(ASCP), M.H.S., Albuquerque, NM

The Paraform System eliminates the need to perform manual steps at embedding and great in terms of training, histology and turn around time.

Authors: Lilly Guevara, Kristie Wolfe-Steele, Brian Johnson

While procedures exist in AP laboratories to protect patient samples and prevent loss through specimen tracking, misplaced cassettes between grossing and tissue processing are still problematic. Significant time may be spent locating a misplaced cassette in this part of the workflow. Pathologists Bio-Medical (PBM) Pathgroup implemented a new system to address this need for preprocessing sample tracking in their AP workflow. PBM worked to create their own solution for tracking AP samples. This included the use of barcodes, scanners, and high-resolution cameras to track samples throughout the lab. It was a time consuming and costly effort pursued over the course of years. However, visibility in the pre-processing workflow was still problematic. In some instances, cassettes scanned at grossing were not used at all, or a cassette was inadvertently returned to the specimen container. In order to truly advance patient safety, improve quality, and optimize lean workflow, this needed to be addressed. In March 2023, PBM implemented a system that automated data capture for cassettes at preprocessing. This resulted in a reduction in time associated with reconciling misplaced/missing cassettes from grossing to embedding: from as long as a few days to as short as a couple hours. Another benefit of the new system was tracking cassettes by processor retort. This is especially important for laboratories that have multiple dual retort processors supporting various tissue types. In the past, lab staff spent significant time solving processing quality issues and identifying which cassettes were loaded on which processor(s). The new system software enables full reporting of this information. This system has provided a quick method to investigate processing errors with various specimen types. These changes created a more efficient laboratory, reduced stress for staff, and enhanced turnaround times.

Authors: From University of Texas M.D. Anderson Cancer Center Center, Houston, TX- Fatimah Ansar; Carla Arredondo; Tran Huynh; Victoria Jones; Jennifer Le; Duong Nguyen; Andres Ronquillo-Erazo; Minnu Varghese; Kaleena Ramirez; Toysha Mayer; Mark A. Bailey

Identifying the presence of fungi through special histotechniques is a critical diagnostic histopathology test for patients who may have acquired a fungal infection localized in the lungs. The purpose of this study was to determine if the mushroom species Agaricus bisporus, generally known as the Button Mushroom and, commonly used for cooking, may be used a special stain control slide. Is the Button Mushroom a reliable fungal control to use when performing a Grocott Methenamine Silver technique test?  The investigators hypothesize the Agaricus bisporus mushroom is a good substitute control for result verification  of the GMS staining technique and readily available compared to tissue controls. The button mushrooms were purchased from a local grocer in Houston, TX, subsequently grossed into eight 1.0x1.0x0.4cm representative sections and submitted for routine tissue processing, embedding, sectioning and stained using the H&E and Grocott Methenamine methods. Inclusive, for the test a commercially available human lung tissue control (+) for fungi was compared to the Button Mushroom results and to ensure the GMS technique produced the appropriate result. The processed button mushrooms GMS stain did not yield a definitive result to conclude the outcome of the staining method. The stained GMS slides demonstrated an indistinguishable contrast between the morphology of the mushroom and the presence of fungi; however, the H&E demonstrated the morphology well, however, further studies are necessary to confirm the presence of fungi.   The use of alternative controls are dependent on the specific clinical; research goals or experimental conditions to provide appropriate and relevant results. The limitations of the research was, not together with, the  GMS to perform a PAS stain on the button mushrooms. Subsequently, we will perform the PAS technique on the button mushrooms and test a different alternative source to establish a viable control for the substitution of human tissue (+) for fungi.  

Authors:  Dr. Tanuja Shet, Dr. Aditi Rathi 

Introduction: HER2/neu gene amplification on Fluorescence in-situ hybridization (FISH) is defined as average HER2neu gene signals > 4 and HER2:CEP17 ratio of > 2, in breast cancer. However, with tumor heterogeneity interpretation of these cut-offs is challenging and some cases test as borderline. We attempted a study analyzing Gene protein assay (GPA) which combines HER2/neu immunohistochemistry (IHC), and D-DISH (dual color dual in-situ hybridization) assay to help resolve this issue.   

Methods:  A total of 31 cases reported as HER2/neu amplified with intermixed tumor heterogeneity on FISH were taken for this study. The original HER2/neu count was between 4 to 6 and ratio near 2 in most cases. GPA was done using 4B5 HER2/neu IHC and Roche/Ventana D-DISH kit on Ventana Ultra machine by doing IHC first followed by D-DISH.

Result:  On GPA, 3/31 cases were reported as non-amplified, and the rest were amplified. Advantage of GPA was that it helped count D-DISH as per membrane staining and identified more tumor heterogeneity in contrast to FISH in six cases. The results of the remaining cases were the same.

Authors: Philip Seifert, HTL(ASCP), Xinyao Hu, Bianai Fan and Darlene A. Dartt, PhD, Schepens Eye Research Institute of Mass Eye and Ear, Boston, MA

The mPrep/S capsules (Microscopy Innovations) are customized pipette tips with screen inserts designed for various electron microscopy applications. Capsule processing methods were developed for mouse ocular and mineralized bone for histology and transmission electron microscopy (TEM) applications and compared to manual and carousel processed methods. The novel methods tested enable processing larger sized tissues (~3 mm diameter) including whole, anterior or posterior segments of mouse eyes and optic nerves within a single capsule that use 75% less reagents and time. Capsule processed tissue embedded in Glycol Methacrylate, methyl methacrylate/butyl methacrylate and EMBED812 epoxy resin were sectioned for histological staining including H&E, toluidine blue, osmium-paraphenylenediamine, Von Kossa and TEM imaged. Stained slide scanned micrographs and TEM images from capsule processed samples were measured and compared to conventional processed using a carousel processor or manual processing.  There were no detectable differences in tissue preservation, staining, optic nerve axon density or processing related artifacts in any of the tissue processed into the resins.  Use of capsules with pipette propelled mixing delivers accelerated diffusion of reagents and infiltration of embedding media into specimens that decreases both processing volume and time compared to manual or carousel processing.  A variety of embedding media resins may be used with capsules for histology and electron microscopy tissue processing. 

Authors: Elizabeth Druffel, HT ASCP, Mayo Clinic, Rochester, MN; Dr. Joaquin Garcia, MD, Mayo Clinic, Rochester MN

Authors: Robert Lott, HTL(ASCP), Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Joshua Greenlee, MBA IASSC, HTL(ASCP)cm, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Melissa Alexander, MD PhD, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Jodi Barrientos, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; David Ferber, MD, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Beth Sheppard, MBA, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Shalini Singh, MD, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Teri Saylor, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC; Shanya Scott, Anatomical Pathology Patient Interest Association, Raleigh, NC; Erico von Bueren, MD PhD MOR, Anatomical Pathology Patient Interest Association (APPIA), Raleigh, NC

Authors: Deborah M. Holzapfel, PhD, Francesca Mazzoni, PhD, Jack Coleman, PhD

Authors: Matthew Lunetta BS HT(ASCP)cm, Cameron Felty DO, Megan Grivois BS HT(ASCP)cm, Chris Hansen HT(ASCP), Chris Jackson MD, and Peter Seery BS PA(ASCP)cm , Liz Rizzo PA(ASCP)cm LSSBB 

Authors: Joseph Vereen, MS, Annexon Biosciences, Brisbane, CA
Alessia Tassoni, PhD, Annexon Biosciences, Brisbane, CA
Larry Mattheakis, PhD, Annexon Biosciences, Brisbane, CA
Ellen Cahir-McFarland ,PhD, Annexon Biosciences, Brisbane, CA
Ted Yednock, PhD, Annexon Biosciences, Brisbane, CA
Yaisa Andrews-Zwilling, PhD, Annexon Biosciences, Brisbane, CA

In this episode, Pamela Vizcarra discusses how she started out as a lab assistant and was able to receive on-the-job training to become a histology supervisor in a research lab doing clinical trials. 

In this episode, Peter Socki explains how his love of crime shows as a child lead to him becoming a histotechnologist working in a medical examiner's office.

In this episode, Sean Moynihan discusses how he applies his degree in biology with a focus on nuclear medicine to what he does on a day-to-day basis working in a dermatopathology outreach lab.

In this episode, Katelin Murphy discusses how she became interested in histology through a recommendation by her high school guidance counselor and now works in a veterinary pathology lab. 

In this episode, Jennifer Betustak explains how she went from being a phlebotomists to becoming a Histology Supervisor.

In this episode, we speak with Janet Tunnicliffe about her experience in British Columbia working as a histotech and she gives thoughtful advice to those considering Histotechnology as a career.  

Published:

Kelli C. Kiekens, Gabriella Romano, Dominique Galvez, Ricky Cordova, John Heusinkveld, Kenneth Hatch, William Drake, Zaynah Kmeid, Jennifer K. Barton"Re-engineering a Falloposcope Imaging System for Clinical Use", Translational biophotonics, Volume2, Issue4, November 2020, e202000011.

Authors: Damien Laudier, Laudier Histology