Journal of Histochemistry & Cytochemistry

Gold standard for immunohistochemical analyses is the manual assessment by two specialist pathologists. This process is time-consuming, highly dependent on the respective evaluator and often difficult to reproduce. The use of image analysis software, such as ImageJ, QuPath, or CellProfiler, which employ machine learning and/or deep learning mechanisms to perform biomarker analyses, offers a potential solution to these problems. The objective of our study is to evaluate whether digital assessment using the open-source software QuPath is comparable to manual evaluation and to examine the inter-evaluator variability between the two manual evaluators and two software-based evaluations. Six tissue microarrays (TMAs) were constructed for a cohort of 309 patients with primary oral squamous cell carcinoma (OSCC). The tumor tissue and corresponding non-lesional squamous epithelial mucosa specimen were immunohistochemically stained for the biomarkers Ki67, as a nuclear marker; the epidermal growth factor receptor (EGF-R), as a membranous marker; and the major histocompatibility complex class I (MHC-I) heavy chain (HC) expressed on the membrane and in the cytoplasm. The staining pattern was analyzed by two experienced, independent manual evaluators and by QuPath. The percentage of positive cells, for Ki67, and the histoscore (H-score) based on the percentage of positive cells and their staining intensity, for EGF-R and MHC-I, were determined as final values. The results yielded high to excellent spearman correlation coefficients for all three biomarkers (p<0.001) in lesional and non-lesional tissues. The Bland–Altman plots demonstrated a high degree of agreement between manual and software-based analysis, as well as inter-evaluator variability demonstrating a high comparability of the evaluation methods. However, a prerequisite for a proper software-based analysis is an accurate, time-consuming annotation of the single specimen, which requires users with a comprehensive understanding of histology and extensive training in QuPath. Once these requirements are met, the software-based analysis offers advantages for large-scale biomarker studies due to objective and reproducible comparability of the stainings leading to a greater accuracy as well as the reuse of established conditions across similar analyses without requiring further operator input.

The Wilms’ Tumor protein WT1 is a zinc-finger transcription factor with crucial roles in organogenesis, cell differentiation, tissue homeostasis, and oncogenesis. While its expression has been extensively studied in various tissues, its presence in the nervous system, particularly in peripheral glial cells, remains largely unexplored. In this study, we examined WT1 expression in the Schwann cells of mechanosensory corpuscles, nerve bundles, and free nerve endings (FNEs) within human penile tissues. Using single and double immunohistology, we analyzed WT1 coexpression with Schwann cell markers (S100, nestin, SOX10) and its association with axonal (neurofilaments, neuron-specific enolase, tyrosine hydroxylase) and perineurial/endoneurial markers (Glut-1, α-SMA, CD34). We found consistent WT1 cytoplasmic expression in the Schwann cells of Pacinian, Meissner, Krause, genital, Golgi-Mazzoni, and Ruffini-like corpuscles, with variable staining intensity. Confocal microscopy revealed WT1 colocalized with nestin but not S100, suggesting involvement in cytoskeletal organization. In addition, we documented WT1 in myelinating Schwann cells of nerve bundles, with distinct staining patterns in Cajal bands and Schmidt–Lanterman incisures, as well as in non-myelinating Schwann cells of FNEs. This is the first study to describe WT1 expression in sensory corpuscles, implicating it in Schwann cell development, maintenance, or plasticity, with potential relevance for peripheral nerve biology, pathology, and mechanosensation.

Immunocytochemistry (IHC) and immunofluorescence (IF) offer crucial diagnostic insights in clinical settings. Most IF assays are performed using fluorophore-conjugated antibodies, but these fluorophores can be subject to issues, such as photobleaching and autofluorescence, that result in lower signal-to-noise ratios (SNR). Gold nanoparticles provide greater signal stability and scatter light well, making them easily separable from the background of biological tissues and improving SNR. In this study, we sought to determine the labeling efficiency, signal quality, and image artifacts of 2.2, 10, and 40 nm diameter gold nanoparticle probes conjugated to antibodies in IF applications on fixed, permeabilized cells in comparison to traditional fluorophores. Overall, micrographs of nanoparticle labels had higher SNR due to lower background signal, and punctate appearances as compared with the continuously distributed signal of the immunofluorescent label. Signal-to-noise ratios varied with nanoparticle diameter, and signal fidelity was worse for keratin versus epithelial growth factor receptor (EGFR). Labeling of EGFR was successful using both extracellular and intracellular epitopes, while poor labeling of keratin 19 with 10 nm diameter nanoparticles was improved by pretreatment with heat and sonication, suggesting hindrance of nanoparticle labels within the fixed, permeabilized cell.

Chemokine-like receptor 1 (CMKLR-1) is a G-protein-coupled receptor that functions as the binding site of chemerin. It induces chemotaxis in various immune cells, and it has recently been linked to vascular remodeling in inflammation. In this study, we investigated CMKLR-1 expression in human aorta samples, focusing on its distribution across different cell types and its potential association with clinical and histomorphological data, particularly concerning aortic dissection and aneurysms. We analyzed 62 aorta samples taken from patients undergoing dissection surgery (n = 27) or aneurysm/dilatation (n = 35) using immunohistochemical CMKLR-1 staining. CMKLR-1 expression was primarily observed in the smooth muscle cells (SMCs) of the aortic media layer, and band-like coloring appeared in the unstained center section. CMKLR-1 positivity in the inner and outer parts of the media layer was observed in only a few cases. One-third of the vasa vasorum exhibited staining. Staining in lymphocytes, macrophages, and endothelia was rare. No significant differences in CMKLR-1 expression were found between the dissection and aneurysm cases, and the clinical or histomorphological data. Although CMKLR-1 expression did not distinguish between the studied conditions, its presence in the aortic media, especially in SMCs, is noteworthy.

PC12 cells are a cell line originating from rat adrenal medullary chromaffin (AMC) cells. They extend a neurite-like structure in response to nerve growth factor (NGF). Thus, unstimulated and NGF-stimulated PC12 cells are used as models for AMC cells and sympathetic ganglion cells, respectively. However, how closely unstimulated and stimulated PC12 cells resemble AMC cells and sympathetic neurons, respectively, has not been elucidated sufficiently. We explored these issues by using biochemical and immunocytochemical methods. AMC cells and PC12 cells selectively expressed uncoupling protein 3 (UCP3) and uncoupling protein 4 (UCP4), respectively, and glucocorticoid activity inhibited UCP4 expression in PC12 cells. PC12 cells expressed extremely low levels of chromaffin granule–associated proteins, whereas the amount of synaptophysin, a synaptic vesicle–associated protein, was much higher than that in the adrenal medulla. Similar to AMC cells, the muscarinic receptor type 1 was located at the cell periphery in unstimulated PC12 cells, and its expression was markedly enhanced by NGF. Furthermore, NGF stimulation abolished the expression of GABA signaling molecules in PC12 cells. The results suggest that the properties of unstimulated PC12 cells are between those of AMC cells and sympathetic ganglion cells and GABA signaling is intrinsic to AMC cells.