Survey of NAACLS accredited histotechnology programs in the United States.

Histotechnology educational programs are accredited by the National Accrediting Agency for Clinical Laboratory Sciences (NAACLS) and currently number fewer than 50 in the United States which contributes to the shortages of laboratory personnel. A survey tool designed with REDCap software was distributed to all programs identified on the NAACLS website and consisted of three parts: a) program information, b) facility information, and c) challenges. Programs are located primarily in large urban centers where populations are most concentrated. The median class size was 6 which may explain the excellent student outcomes to include 96% graduation rates and 90.7% board of registry examination pass rates. Overall, programs had ample equipment, funding, and administrative support. Costs to attend the programs were relatively low (<$3,000 per semester) for over half of the programs. However, due to the small number of accredited education programs across the US, potential students do not often have access to an institution in their area. The programs indicated that the most common challenge was recruitment of adequate high-quality candidates which may explain, in part, the persistent shortage of personnel in the histology laboratory.

Neutrophil Activity and Extracellular Matrix Degradation: Drivers of Lung Tissue Destruction in Fatal COVID-19 Cases and Implications for Long COVID.

Pulmonary fibrosis, severe alveolitis, and the inability to restore alveolar epithelial architecture are primary causes of respiratory failure in fatal COVID-19 cases. However, the factors contributing to abnormal fibrosis in critically ill COVID-19 patients remain unclear. This study analyzed the histopathology of lung specimens from eight COVID-19 and six non-COVID-19 postmortems. We assessed the distribution and changes in extracellular matrix (ECM) proteins, including elastin and collagen, in lung alveoli through morphometric analyses. Our findings reveal the significant degradation of elastin fibers along the thin alveolar walls of the lung parenchyma, a process that precedes the onset of interstitial collagen deposition and widespread intra-alveolar fibrosis. Lungs with collapsed alveoli and organized fibrotic regions showed extensive fragmentation of elastin fibers, accompanied by alveolar epithelial cell death. Immunoblotting of lung autopsy tissue extracts confirmed elastin degradation. Importantly, we found that the loss of elastin was strongly correlated with the induction of neutrophil elastase (NE), a potent protease that degrades ECM. This study affirms the critical role of neutrophils and neutrophil enzymes in the pathogenesis of COVID-19. Consistently, we observed increased staining for peptidyl arginine deiminase, a marker for neutrophil extracellular trap release, and myeloperoxidase, an enzyme-generating reactive oxygen radical, indicating active neutrophil involvement in lung pathology. These findings place neutrophils and elastin degradation at the center of impaired alveolar function and argue that elastolysis and alveolitis trigger abnormal ECM repair and fibrosis in fatal COVID-19 cases. Importantly, this study has implications for severe COVID-19 complications, including long COVID and other chronic inflammatory and fibrotic disorders.

Histochemical and morphological evaluation of a glyoxal acid-free fixative.

The application of most chemical fixatives, such as formalin, in the anatomic pathology laboratory requires safety training and hazardous chemical monitoring due to the toxicity and health risks associated with their use. Consequently, the use of formalin has been banned in most applications in Europe; the primary exception is its use in the histology laboratory in lieu of a suitable and safer alternative. Glyoxal based solutions, several of which are available commercially, are the most promising alternative fixatives, because they are based on a mechanism of fixation similar to that of formalin. Unlike formalin, however, glyoxal based solutions do not dissociate from water and therefore do not require ventilation measures such as a fume hood. A primary barrier to the adoption of commercially available glyoxal based solutions is their low pH, which can produce undesirable morphological and antigenic tissue alterations; however, a recently available neutral pH glyoxal product (glyoxal acid free) (GAF) has been developed to mitigate the challenges of low pH. We compared the morphology and histochemistry among tissues fixed in 10% neutral buffered formalin, a commercially available acidic glyoxal product (Prefer), and GAF. Tissues fixed in formalin and Prefer exhibited similar morphology and staining properties; tissues fixed with 2% GAF exhibited deleterious effects.

Increase of KLK7, cytokeratin 5/6, and elafin expression in head and neck squamous cell carcinoma compared with lung squamous cell carcinoma.

Squamous cell carcinoma is the most common primary tumor in the head and neck epithelium and is the second most common primary tumor type in the lung. Although morphologically indistinguishable from each other with hematoxylin and eosin stain on histology, the tumors have different protein expression profiles. Using 24 formalin-fixed paraffin embedded squamous cell carcinomas of the lung and 24 squamous cell carcinomas in the head and neck, protein expression for cytokeratin 5/6, kallikrein 7, and elafin was evaluated by immunohistochemistry. All three proteins were found to evidence higher expression in head and neck squamous cell carcinoma as compared with that of squamous cell carcinoma of the lung. The differences in expression may help clinical differentiation between primary tumors of the lung from metastatic tumors to the lung from the oral/laryngeal cavities.

The presence of H. pylori in laparoscopic sleeve gastrectomy specimens is associated with increased mucosal thickness, presence of secondary follicles, increased chronic inflammation, and intestinal metaplasia.

Helicobacter pylori is putatively present in over half of the global human population and is recognized as a carcinogenic agent that increases the likelihood of infected patients developing gastric adenocarcinoma or gastric lymphoma. Although there are several means for testing for H. pylori, the gold standard remains the invasive histologic evaluation. The current most popular form of bariatric surgery is the laparoscopic sleeve gastrectomy (LSG) and is the only bariatric surgery which supplies a specimen for histologic evaluation. While non-invasive testing is effective in diagnosing and monitoring H. pylori infection, histological examination of biopsies and resections is the only way to grade chronic inflammation and evaluate specimens for additional pathologies such as intestinal metaplasia. The investigators evaluated 203 sequential LSG specimens collected from a major metropolitan hospital over the period of one year. Specimens were processed to paraffin, stained with hematoxylin and eosin, alcian blue, and immunohistochemistry to determine the presence of H. pylori, chronic inflammation, presence of secondary lymphoid follicles in the mucosa, mucosal thickness, and presence of intestinal metaplasia. Statistical analyses demonstrated a significant positive correlation among all factors examined. The overall positivity rate of H. pylori in LSG specimens was 18.2% but ranged from 6.9-23.8% depending on whether the treating clinician performed routine pre-surgical endoscopy. The presence of H. pylori was associated with a higher average chronic inflammation grade, intestinal metaplasia, thicker mucosa, and presence of lymphoid follicles with germinal centers in the mucosa.

Mast Cell Concentrations in Pancreatic Disease Processes.

Mast cells enumeration has been performed using various histologic staining techniques with the goal of elucidating the influence mast cells exert on pathologic processes. In this study, 77 human pancreatic tissues evidencing morphologically normal pancreas, benign fibrotic changes, endocrine tumors, and adenocarcinoma were evaluated using Wright stain and immunohistochemistry markers for tryptase and CD117. Mast cell counts were similar with tryptase and CD117 but were both significantly higher than counts obtained with the Wright stain. Furthermore, all analyses demonstrated that endocrine tumors and morphologically normal pancreatic tissues had significantly lower mast cell counts as compared with benign fibrosis and adenocarcinoma suggesting that the highly fibrotic nature of both pancreatitis and adenocarcinoma are related to increased mast cell concentrations.

A comparison of staining methods for Helicobacter pylori in laparoscopic vertical sleeve gastrectomy resections.

BACKGROUND: Helicobacter pylori is an important public health concern due to its status as a carcinogenic bacterium. Well adapted to the acidic environment of the human stomach, the variety of strains and virulence factors of the organism when interacting with the host immune system creates an individualistic response. Although estimates suggest that approximately half of the global population is infected with H pylori, the majority of infected persons remain asymptomatic while harboring an increased risk of intestinal metaplasia and gastric cancers. Therefore, appropriate diagnostic testing protocols are imperative. METHODS: This study compared labeling methodologies, including Wright stain, alcian yellow toluidine blue (AYTB), and immunohistochemistry (IHC) on formalin-fixed paraffin-embedded stomach resections from sleeve gastrectomy patients, to detect H pylori infection. RESULTS: Although all 3 labeling methods evidenced similar specificity in H pylori detection, the IHC method was significantly more sensitive. However, the IHC cost per test was approximately 5-fold higher than that of the Wright or AYTB stains, and the technical time required per test was at least 6-fold that of Wright or AYTB. CONCLUSION: Despite the higher cost per test, IHC is the most sensitive and preferred method for determination of H pylori infection.

Immunohistochemical evaluation of hormones secreted by pancreatic endocrine tumors.

The endocrine component of the pancreas is located primarily in the islets of Langerhans, but is also found as single cells among the acinar cells and duct epithelium. It currently is thought that endocrine tumors of the pancreas (PETs) arise from pluripotent stem cells located within the ductal epithelium rather than from existing endocrine cells. Islet cell components include alpha, beta, PP, delta and epsilon cells, which secrete glucagon, insulin, pancreatic polypeptide, somatostatin and ghrelin, respectively. We investigated immunohistochemical labeling of 24 formalin fixed paraffin embedded PETs to identify which hormones were produced most frequently. Glucagon was the most frequently secreted hormone (83%) in PETS followed by insulin, ghrelin, pancreatic polypeptide and somatostatin.

Histologic examination of canned cat food.

Cat food production is a billion-dollar industry in the United States, with most pet owners trusting pet food companies to provide their pets with complete nutrition. Moist or canned cat food is healthier than dry kibble for cats due to its higher water content promoting healthy kidney function, but ingredient labels on canned cat food are lengthy with ambiguous terminology including 'animal by-products.' Forty canned cat food samples were collected from grocery stores and were processed using routine histologic methods. Hematoxylin and eosin-stained tissue sections were evaluated microscopically to determine the cat food content. Many brands and flavors were composed of well-preserved skeletal muscles admixed with various animal organs, which closely approximates nutritional components found in natural feline prey. However, several samples demonstrated marked degenerative changes suggesting a delay in food processing and potential decrease in nutrient content. Four samples contained cuts consisting of skeletal muscle only with no organ meat. Surprisingly, 10 samples contained fungal spores and 15 demonstrated refractile particulate matter. A cost analysis demonstrated that although the overall quality of canned cat food increases as the average cost per ounce increases, low-cost high-quality canned cat food is available.

Glyoxal fixation: an immunohistochemistry assay evaluation.

Analysis of surgical pathology specimens by histological techniques including immunohistochemistry (IHC) assays is a mainstay of disease diagnosis in humans. Neutral buffered formalin (NBF) is currently the primary fixative used, but its use is not without risks due to toxicity and carcinogenicity. Several glyoxal-based fixatives have been commercially produced, are considered safer alternatives to NBF, and produce histochemical staining results comparable to that of tissues fixed in NBF. However, previous studies evaluating IHC assay results in tissues fixed in NBF and glyoxal solutions have indicated mixed results. This study demonstrated that while tissues fixed in NBF were slightly superior to tissues fixed in glyoxal solutions among the 34 antibodies assayed with IHC, all fixative solutions produced results compatible for use in an anatomic pathology laboratory.

Application of dyes to cytology cell blocks and biopsy tissues before processing enhances specimen visualization during embedding and microtomy.

Cytology specimens and biopsy tissues are frequently small and pale, making them difficult to visualize grossly in paraffin. Ten dyes were assayed on small tissues to determine if specimen discernibility could be increased during the embedding and microtomy steps in the histological process. The ideal dye should not remain visible in a tissue section microscopically after subsequent staining and must not interfere with immunohistochemistry (IHC) assays. This study found that Harris hematoxylin and 1% aq. toluidine blue solution were the best labelers for gross tissue visualization and did not adversely affect post-processing staining and IHC assays.

Hidradenitis suppurativa lesions are associated with decreased collagen, decreased elastin, and increased neovascularization.

Hidradenitis suppurativa (HS) is a dermatological condition characterized by ruptured hair follicles inducing a localized, but massive chronic inflammatory response. Analysis of 104 HS lesions in formalin-fixed paraffin-embedded tissues identified the most common histologic features found associated with HS were migrating epithelial sheets, acute and chronic inflammation, perivascular chronic inflammation in regions adjacent to active lesions, and granulation tissue. Additionally, using a modified Masson's trichrome stain principally for collagen, Verhoeff van Gieson elastic stain, and CD34 immunohistochemistry assay for endothelium, lesional areas in the dermis with chronic inflammation exhibited a significant decrease in collagen and elastin, and an increase in neovascularization.

Dynamic Leukocyte Populations Are Associated With Early- and Late-stage Lesions in Hidradenitis Suppurativa.

Hidradenitis suppurativa (HS) is a chronic inflammatory skin condition typically targeting the axillary and anogenital regions of the body. The massive inflammatory cell infiltrate produced in this cryptogenic condition has led investigators in the attempt to link particular inflammatory cell fractions and cytokines to disease development, and ultimately to disease treatment. This study qualitatively and quantitatively analyzes the white blood cell fractions of macrophages, B-lymphocytes, T-lymphocytes, plasma cells, and granulocytes in 104 HS lesions on formalin-fixed paraffin-embedded tissues using immunohistochemistry (IHC). Four dermis-associated epithelial categories were investigated from persons with HS: 15 unaffected HS skin (US), 19 distended but unruptured follicle epithelium (UF), 62 migrating stratified squamous epithelium (MSSE) from ruptured follicles, and 35 degraded migrating epithelial sheets (DMES). In addition, 27 control skin (CS) from persons without HS were evaluated. Analysis of cell counts indicated that non-migratory dermal epithelium (CS, US, and UF) stimulated very little inflammatory response. However, contrary to previous studies which indicated macrophages to be the chief inflammatory cell in HS, this study showed that plasma cells were the primary cell type present in early-stage HS lesions (MSSE), whereas granulocytes were the major cell population seen in late-stage HS lesions (DMES).

Glyoxal: a proposed substitute for formalin in H&E and special stains.

Neutral-buffered formalin (NBF) has been used as the primary fixative in anatomic pathology laboratories for decades. Although it yields excellent morphologic and staining results, NBF poses significant health hazards requiring tissue to be grossed under a grossing/chemical fume hood. Glyoxal fixatives offer far less toxic alternatives and do not necessitate use of a grossing hood. Using freshly extracted canine and feline testes, ovaries, and uteri, the effects of glyoxal and NBF fixation were compared. While NBF is still considered the gold standard, some glyoxal fixatives perform as well as NBF in regards to morphology, H&E staining properties, and histochemical staining properties.

Comparison of bleaching protocols utilizing hematoxylin and eosin stain and immunohistochemical proliferation marker MCM3 in pigmented melanomas.

Melanomas represent the malignant transformation of melanocytes, cells found primarily in the skin to protect epithelium and underlying connective tissues from harmful effects of ultraviolet light. Melanomas vary greatly in morphology and may continue to produce melanin markedly, mildly, or not at all. Performing and evaluating hematoxylin and eosin stains as well as immunohistochemical stains on pigmented melanomas has been a long-standing challenge due to the obscuring pigment. Protocols for removing melanin to reveal cellular morphology have been used successfully for years, but coupling these protocols with stains for immunohistochemistry represents an added challenge. In this study, the investigators evaluated results of various melanin bleaching protocols on tissue morphology, completeness of melanin removal, and immunohistochemistry staining quality. It was found that 1% formamide in 3% H2O2 under bright light without heating outperformed other tested protocols.