HistoTalks: NSH Podcasts

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.

Alternative Strategies For Analyzing Pre-Clinical Mouse Lungs

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.

BENEFITS of Tissue-Tek Paraform Sectionable Cassette System

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.

Leveraging preprocessor scanning to improve safety, quality, and lean workflow in the anatomic pathology lab.

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.

The Button Mushroom (Agaricus bisporus): An Alternative Control Source for the Grocott Methenamine Silver Technique

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.  

 Gene Protein Assay(GPA): Unveiling Tumor Heterogeneity in HER2/neu Positive Breast Cancer

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.

Novel Use of Pipette Based Capsules for Glycol Methacrylate, Methyl/Butyl Methacrylate and Epoxy Resin Processing with Mouse Tissue That Reduce Processing Solution Volume and Time.  

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. 

Join us for our AwardsCast series as we have short conversations with the 2023 NSH Award & Scholarship Recipients. In this episode, we talk to Colleen Forster of the University of Minnesota, the recipient of the Immunohistochemistry Education Scholarship, where we discuss how every day in a core research lab is different and keeps her learning new things as well as her plans for learning more about the hot new area of spatial omnics.