Nonspecific Staining Research Articles

SOX-10 and Melan-A Immunostaining in Areas of Focal Acantholytic Dyskeratosis and Epidermolytic Hyperkeratosis Within Dysplastic Nevi Biopsies: An Observational Study.

Focal acantholytic dyskeratosis (FAD) and epidermolytic hyperkeratosis (EHK) are common incidental epidermal histologic findings within dysplastic nevi biopsies. We evaluate whether areas of FAD and EHK within dysplastic nevi biopsies stain with immunostains used to characterize melanocytic neoplasms. In this case series, a natural language search of histopathology reports from our institution in the past year (2020-2021) identified dysplastic nevus biopsies with concurrent FAD and/or EHK. Tissue samples were examined for positive melanocytic immunostaining with SOX-10 and Melan-A in areas of FAD and EHK. Out of 32 biopsies, 20 of 26 FAD specimens (76.9%) and 2 of 6 EHK specimens (33.3%) showed unexpected suprabasal layer staining with a melanocytic marker that did not correspond to definitively identified melanocytes on the H&E-stained sections. The immunohistochemical staining of FAD and EHK was observed in 2 forms: nonspecific background staining or "true" staining (ie, seemed nuclear on SOX-10 or cytoplasmic on Melan-A). This pilot examination provides evidence that areas of incidental FAD within dysplastic nevi biopsies demonstrate unexpected suprabasal layer staining with melanocytic markers. When dermatopathologists evaluate melanocytic neoplasms with melanocytic markers, it is possible the presence of incidental FAD could lead to over diagnosing pagetoid scatter within these lesions. This study is a proof of concept with mild to moderately dysplastic nevi that do not typically incur the use of melanocytic stains; however, the implication of this unexpected staining pattern would be important when using melanocytic markers on borderline melanocytic neoplasms that have incidental FAD. Close correlation with H&E is imperative to prevent misinterpretation in these cases.

Synergistically Activated Aggregation-Induced Emission Probe for Precise In Situ Staining of Lipids in Atherosclerotic Plaques.

Visualizing the localization and distribution of lipids within arteries is crucial for studying atherosclerosis. However, existing lipid-specific probes face challenges such as strong hydrophobicity and nonspecific staining of lipophilic organelles or tissues, making them impractical for the precise identification of atherosclerotic plaques. To address this issue, we design a synergistically activated probe, Cbz-Lys-Lys-TPEB, which responds to cathepsin B (CTB) and H2O2 for the in situ generation of aggregation-induced emission luminogens (AIEgens). This enables specific staining of lipids within arteries and precise imaging of atherosclerotic plaques. The probe combines a tetraphenylethene building block with a hydrophilic peptide sequence (Cbz-Lys-Lys) and phenylboric acid module, providing excellent water solubility and fluorescence quenching in a molecular dispersion state. Upon interaction with H2O2 and CTB within plaques, the hydrophilic Cbz-Lys-Lys-TPEB probe is specifically cleaved and converted into hydrophobic AIEgens, leading to rapid aggregation and significant fluorescence enhancement. Interestingly, the in situ-liberated AIEgens display distinct lipid binding ability, effectively tracking the location and distribution of lipids in plaques. This synergistic target-activated AIEgen liberation strategy demonstrates significant feasibility for the reliable and accurate identification of atherosclerotic plaques, holding tremendous potential for clinical diagnosis and risk stratification of atherosclerosis.

Can multiplexing Immunohistochemistry for PD-L1, CD68, and CD3 improve scoring for “Keytruda®” Indication?

Abstract Introduction/Objective FDA-approved companion diagnostics immunohistochemistry scoring for PD-L1 is essential for Keytruda ® treatment in patients with NSCLC. The challenge with PD-L1 testing alone is false positive PD-L1 expression score due to alveolar macrophage infiltration. Multiplexing with PD-L1, CD68, and CD3 could help alleviate this issue. By viewing PD-L1 expression on tumor cells, CD68 expression on macrophages, and the presence of CD3+ T cells on a single slide, pathologists will be able to see the presence of an immune microenvironment (high PD-L1 expression with abundant macrophages) and the potential for response to other types of Immunotherapies (presence of T cells). Methods/Case Report Positive NSCLC FFPE blocks were sectioned and stained using Agilent Dako Omnis. Two combinations of multiplex staining were prepared for each block by staining PD-L1 (brown) alone first followed by CD3 and CD68. First slide: CD3(Vina Green), CD68(Magenta); Second slide: CD3(Magenta), CD68(Vina Green). To prevent wash out, dehydration and clearing occurred with two changes of 100% alcohol and three changes of xylene for manual cover slipping. Results (if a Case Study enter NA) Manual scoring of PD-L1 alone vs two different multiplex combinations was compared to Agilent Proscia digital AI scoring using a preliminary study set (9 positives, 2 negatives). CD68-green and CD3-magenta showed better discrimination than CD68-magenta and CD3-green at a glance. This could be due to the staining intensity and nonspecific background staining of CD68-magenta. Manual scoring was higher compared to digital scoring, which could be due to the human error of positive bias. Digital scoring was significantly lower for PD-L1 alone vs both multiplex stains. The digital scoring for the multiplex stains showed a higher scoring percentage by removing false positive macrophages. Conclusion Reflex scoring workflow is being developed to focus on difficult cases using the multiplex triple stain, i.e. clear negative and clear >50% with 3+ intensity. More cases will be collected and studied to further investigate the improved PD-L1 scoring with digital imaging assisted by artificial intelligence and to compare the multiplex color combinations.

Exploring TFE3-Rearranged Renal Cell Carcinoma: A Case Study showing minimal expression of PAX-8

Abstract Introduction/Objective TFE3-rearranged renal cell carcinoma accounts for 1.6-4% of adult renal cell carcinomas (RCC). The histological characteristics of this entity can mimic many other renal cell neoplasms and definite diagnosis is based on immunohistochemical (IHC) and fluorescent in situ hybridization (FISH) analysis. Nonspecific IHC findings can lead to diagnostic challenges delaying confirmatory studies and underdiagnosis. Methods/Case Report A 28-year-old male with sickle cell disease and a right kidney multilocular cystic mass incidentally found during evaluation of splenomegaly underwent partial nephrectomy. The tumor showed papillary, alveolar and sheet-like growth patterns composed of eosinophilic to clear cells with pleomorphic nuclei, including multinucleate forms. Initial ancillary studies showed minimal neoplastic expression (less than 5%) of PAX-8. Repeat PAX-8 yielded similar results and laboratory controls were evaluated successfully. A full work-up showed positivity of AMACR (diffuse), CD10 (diffuse), Cathepsin K (patchy), CK7 (focal), CAIX (focal), and no significant expression of pancytokeratin, HMB-45, Melan-A and SMA. Fumarate hydratase was retained, and the tumor showed a non-specific staining pattern of S (2-succinyl)-cysteine. Controls were appropriate. A high suspicion for a TFE-rearranged RCC was maintained, and a final diagnosis was confirmed with FISH analysis demonstrating a TFE3 (Xp11.23) rearrangement. Results (if a Case Study enter NA) NA Conclusion Reports in the literature detail diffuse PAX-8 expression in TFE3-rearranged RCC. A high index of suspicion based on morphology in our case prompted FISH analysis leading to correct diagnosis. This case highlights the inconsistencies in the reported IHC profile of this entity. More studies are required to elaborate the presentations of this RCC subtype with numerous histologic mimics including the spectrum of immunostaining patterns to better assist patient care for this uncommon variant.

HcGAN: Harmonic conditional generative adversarial network for efficiently generating high-quality IHC images from H&E

Generating high quality histopathology images like immunohistochemistry (IHC) stained images is essential for precise diagnosis and the advancement of computer-aided diagnostic (CAD) systems. Producing IHC images in laboratory is quite expensive and time consuming. Recently, some attempts have been made based on artificial intelligence techniques (particularly, deep learning) to generate IHC images. Existing IHC stained image generation methods, still have a limited performance due to the complex structures and variations in cells shapes, potential nonspecific staining and variable antibody sensitivity. This paper proposes a novel technique known as harmonic conditional generative adversarial network (HcGAN) for generating high quality IHC-stained images. To generate the IHC images, the HcGAN model is fed with the widely available hematoxylin and eosin (H&E) images that contain cellular and morphological underlying structures of diverse cancer tissues. Such approach helps generate high quality IHC images mimicking the real ones that highlight the positive cells. The proposed HcGAN model is based generative adversarial learning with generator and discriminator networks. In HcGAN, harmonic convolution based on discrete cosine transform filter banks is employed in the generator and discriminator networks instead of the standard convolution in order to improve visual quality of the generated images and address the issue of overfitting. Our qualitative and quantitative results demonstrate that the proposed HcGAN achieved the highest performance over state-of-the-art methods using two publicly available datasets.

Singlet oxygen detection in vivo is hindered by nonspecific SOSG staining

Singlet oxygen is considered an important cell damaging agent due to its propensity to react with organic compounds. This drives the interest in developing methods for determination of 1O2. Simplicity of application and high sensitivity makes fluorescent probes a popular choice for in vivo 1O2 detection. Despite its proclaimed cell-impermeability, the commercially available Singlet Oxygen Sensor Green (SOSG) is widely applied to support assertions of 1O2 involvement in cell and tissue damage. Our investigation, however, demonstrate that different microbial species and cancer cells become fluorescent when exposed to SOSG under conditions which exclude generation of 1O2. Cells, permeabilized with chlorhexidine or by heat exposure under anaerobic conditions, exhibited SOSG fluorescence. Permeabilized cells could be stained with SOSG even 24 h post-permeabilization. Since SOSG is cell impermeable, the main factor that led to fluorescent staining was plasma membrane damage. Spectral analyses of different batches of SOSG revealed that SOSG endoperoxide (SOSG-EP) did not increase even after prolonged storage under the recommended conditions. The commercial preparations of SOSG, however, were not SOSG-EP free, which can produce erroneous results when SOSG staining is used as a proof of singlet oxygen production in vivo.

A versatile approach to quality control of protein-based receptor layers by reversible, nonspecific staining for multiplex SPRi immunosensing

SPRi protein arrays for rapid, label-free immunodetection offer an attractive approach for high throughput biosensing and analysis of molecular interactions. However, their routine use for quantitative analysis requires adequate reproducibility and homogeneity of receptor assemblies manufactured ex-situ using microdispensing techniques. There is also an emerging need to develop versatile and non-destructive methods for the determination of protein surface density for quality control of the immobilization process. Here, we report on a simple, one-step method of surface proteins visualization by their reversible, non-specific staining with anionic dyes: Ponceau S (PS), Amido Black (AB) or Coomassie Brilliant Blue G (CBB-G). Using rabbit IgG antibody and transferrin as model receptors, we demonstrate the possibility of protein surface density determination in a broad range up to ∼ 3 ng∙mm−2 (for a 2D monolayer) and up to at least 12.5 ng∙mm−2 (for a 3D hydrogel-coated substrate). The ranges of dynamic response for the developed methods cover the typical surface densities required for biosensing and studies of the binding kinetics. It was demonstrated that each of the investigated dyes manifests advantages in specified applications. While CBB-G shows the highest sensitivity of SPRi response, AB requires the gentlest labeling conditions, and PS shows the highest association rate and ease of washing out, even from protein layers of high density. The feasibility of the developed method for the quality control of SPRi microarray was confirmed. The advantages of the new, dye-based, non-specific labeling method over immunolabeling in terms of simplicity of implementation and versatility have also been highlighted.

Utility of C4d Immunohistochemistry in the Diagnosis of Esophageal Pemphigus Vulgaris.

Aims. To assess the utility of C4d immunohistochemistry for esophageal pemphigus vulgaris. Methods and results. We searched for patients with a history of esophageal pemphigus vulgaris who had esophageal biopsies for routine hematoxylin and eosin (H&E) staining. A total of 8 biopsies from 7 patients were available. We also identified 18 non-pemphigus esophageal biopsies for controls. C4d immunohistochemistry was performed on each biopsy. Five of 6 (83%) biopsies with classic pemphigus vulgaris histologic findings were positive for intercellular staining at the basal layer. The negative biopsy was in a patient that had recently received high-dose corticosteroid treatment for a flare. Two biopsies with atypical histologic features for pemphigus vulgaris had negative C4d staining but positive direct immunofluorescence (DIF) studies. Various nonspecific C4d staining patterns were observed in the controls, but none showed the intercellular staining pattern that was observed in pemphigus vulgaris. Conclusions. Suprabasal clefting with acantholysis and "tombstone effect" are described histologic features of pemphigus vulgaris on H&E. However, procedural artifact may mimic these findings. Currently, the gold standard for pemphigus vulgaris is DIF, which is not always available because it cannot routinely be performed on formalin-fixed paraffin embedded tissue. Our study shows that C4d immunohistochemistry may be a useful adjunct in evaluating esophageal pemphigus vulgaris.

Development of a deep-learning model tailored for HER2 detection in breast cancer to aid pathologists in interpreting HER2-low cases.

Over 50% of breast cancer cases are "Human epidermal growth factor receptor 2 (HER2) low breast cancer (BC)", characterized by HER2 immunohistochemistry (IHC) scores of 1+ or 2+ alongside no amplification on fluorescence in situ hybridization (FISH) testing. The development of new anti-HER2 antibody-drug conjugates (ADCs) for treating HER2-low breast cancers illustrates the importance of accurately assessing HER2 status, particularly HER2-low breast cancer. In this study we evaluated the performance of a deep-learning (DL) model for the assessment of HER2, including an assessment of the causes of discordances of HER2-Null between a pathologist and the DL model. We specifically focussed on aligning the DL model rules with the ASCO/CAP guidelines, including stained cells' staining intensity and completeness of membrane staining. We trained a DL model on a multicentric cohort of breast cancer cases with HER2-IHC scores (n = 299). The model was validated on two independent multicentric validation cohorts (n = 369 and n = 92), with all cases reviewed by three senior breast pathologists. All cases underwent a thorough review by three senior breast pathologists, with the ground truth determined by a majority consensus on the final HER2 score among the pathologists. In total, 760 breast cancer cases were utilized throughout the training and validation phases of the study. The model's concordance with the ground truth (ICC = 0.77 [0.68-0.83]; Fisher P = 1.32e-10) is higher than the average agreement among the three senior pathologists (ICC = 0.45 [0.17-0.65]; Fisher P = 2e-3). In the two validation cohorts, the DL model identifies 95% [93% - 98%] and 97% [91% - 100%] of HER2-low and HER2-positive tumours, respectively. Discordant results were characterized by morphological features such as extended fibrosis, a high number of tumour-infiltrating lymphocytes, and necrosis, whilst some artefacts such as nonspecific background cytoplasmic stain in the cytoplasm of tumour cells also cause discrepancy. Deep learning can support pathologists' interpretation of difficult HER2-low cases. Morphological variables and some specific artefacts can cause discrepant HER2-scores between the pathologist and the DL model.

P-322 Development of a CD138-based histopathological scoring system to diagnose chronic endometritis

Abstract Study question Does a standardized histopathological classification system lead to a robust interobserver agreement to diagnose chronic endometritis (CE) and can an accurate computation algorithm be developed? Summary answer The proposed scoring system has a particularly good to excellent interobserver agreement among pathologists. A machine-learning algorithm outperformed pathologists in the clinically most relevant 2-tier-system. What is known already Plasma cell (PC) presence in the stromal endometrium has been proposed as a marker for CE, yet no standardized classification system that considers the amount or distribution of PCs exists. Also, interobserver agreement has been insufficiently reported in the previous publications on the topic. Without a robust histopathological score, the potential clinical impact of CE on ART treatment outcomes cannot be investigated. This issue is strongly reflected in the heterogeneity of the current diagnostic and therapeutic approaches, specifically for patients experiencing repeated implantation failure (RIF) or recurrent pregnancy loss (RPL). Study design, size, duration A 5-tier classification system based on CD138-positive PCs (Roche B-A38/automated Ventana system) was established: class0 (no PCs), classIPC (isolated PCs), class1 (³1 cluster of 5-19 PCs/0.25mm2), class2 (³1 cluster of 20-49 PCs/0.25mm2), class3 (³1 cluster of 50 or more PCs/0.25mm2). A test set of 78 endometrial biopsies (collected over two years) was selected by a certified pathologist who sorted >1000 biopsies to ensure the full spectrum of PC counts/distribution was accounted for. Participants/materials, setting, methods The 3DHistech-platform digitized the slides (pixel size 0.2738x0.2738mm) and a scoring session was set up in the Pathomation software suite. Six pathologists scored independently after training. Overall agreement was expressed by Fleiss’ Kappa values. Pairwise interobserver agreement was calculated using Cohen’s Kappa statistics. A supervised machine-learning algorithm was developed in QuPath(V0.4.2) and considered a seventh observer in the interobserver agreement calculations. Main results and the role of chance The CD138-based semiquantitative scoring system showed particularly good to excellent interobserver agreement among all observers. Overall agreement values were 0.722 for the 5-tier system and 0.858 for the 2-tier system (0+IPC versus 1 + 2+3, which was considered the clinically most relevant classification, i.e. CE absent versus diagnosed). Pairwise interobserver agreement was 0.871-0.951. Intra-class correlations varied from 0.727 to 0.839 in the 5-tier system and 0.893 to 0.965 in the 2-tier system. All findings were statistically significant (p < 0.001). The supervised machine-learning algorithm was able to compete with human pathologist observers and outperformed them in the 2-tier system. The proposed classification system provides a framework for assessing the presence and severity of endometrial PC infiltration and can guide further research. Limitations, reasons for caution Inconsistent staining intensity, non-specific epithelial staining and background interference are common problems that might limit interlaboratory use of the algorithm. Furthermore, the algorithm has not yet been associated to patient/clinical parameters. Accurate analysis of an endometrial biopsy might still be insufficient to provide a formal diagnosis of CE. Wider implications of the findings To unravel the association between PCs in endometrial biopsies (as a proxy for the diagnosis of CE) and the reproductive outcomes following ART, a standardized methodology is of crucial importance for future studies. Higher accuracy, efficieny and automation are advantages when using computer-aided diagnostics for CE. Trial registration number BUN 143202042810

Identification of HER2 ultra-low based on an artificial intelligence (AI)-powered HER2 subcellular quantification from HER2 immunohistochemistry images.

1115 Background: HER2-targeted antibody-drug conjugates (ADCs) can effectively target tumor cells even in HER2-low breast cancers (1+ and 2+/no amp). We developed an AI-based whole-slide image (WSI) analyzer for immunohistochemistry (IHC)-stained slides, exploring a cutoff differentiating between true HER2 negative versus HER2 ultra-low. Methods: An AI-based IHC WSI analyzer was developed using 6,188 WSIs stained by 19 types of IHC including HER2 and from various cancers, annotated by 153 pathologists. The model predicts cells as tumor cells or non-tumor cells and generates HER2 expression continuous score (HER2ecs, 0-100%), quantifying IHC staining intensity of membrane, nucleus, cytoplasm, and membrane+cytoplasm separately for each cell. A total of 401 HER2 IHC WSIs were acquired from Samsung Medical Center (n=275), Kyung Hee University Hospital (n=78), and Commercial Biobank (n=48), scored as 0 (n=347) and 1+ (n=54) by pathologists. Results: In total, 67,169,114 tumor cells and 119,113,886 non-tumor cells from 401 WSIs were successfully evaluated. Comparing HER2 score 0 versus 1+ by pathologists, mean HER2ecs of nucleus (1.7% vs 4.1%), cytoplasm (6.5% vs 12.9%), and membrane (3.6% vs 8.2%) were comparable. In HER2 score 0 WSIs, membrane HER2ecs of tumor cell was 3.6 ± 7.8% (mean ± standard deviation), while that of non-tumor cell was 0.9 ± 2.9% (p <0.001 vs. tumor cell), suggesting, even in HER2 score 0 cases, tumor cell exhibits HER2 expression beyond non-specific HER2 staining intensity. Based on our internal validation dataset, membrane-specific HER2ecs of 6.0% was the optimal threshold discriminating HER2 no-staining cells versus HER2 incomplete and faint/barely perceptible cells (AI-HER2-weak) per ASCO/CAP guideline. Among HER2 score 0 by pathologists, when applying this cutoff, the proportion of WSIs showing AI-HER2-weak or higher membrane-specific HER2ecs in over 10% of all tumor cells was 23.6% (82/347). Interestingly, that of WSIs among HER2 score 1+ by pathologists was 51.9% (28/54), which is comparable to 52.3%, objective response rate of trastuzumab deruxtecan in HER2-low patients in DESTINY-Breast04. Conclusions: AI-powered subcellular-level analysis enables identification of 23.6% HER2 ultra-low WSIs among HER2 score 0 by pathologist, which is defined by AI-HER2-weak or higher membrane-specific HER2 expression ≥ 10%. Clinical validation of the AI-powered HER2 ultra-low definition is warranted.

Quantum Dot-Fab' Conjugates as Compact Immunolabels for Microtubule Imaging and Cell Classification.

Antibodies and their conjugates of fluorescent labels are widely applied in life sciences research and clinical pathology. Among diverse label types, compact quantum dots (QDs) provide advantages of multispectral multiplexing, bright signals in the deep red and infrared, and low steric hindrance. However, QD-antibody conjugates have random orientation of the antigen-binding domain which may interfere with labeling and are large (20-30 nm) and heterogeneous, which limits penetration into biospecimens. Here, we develop conjugates of compact QDs and Fab' antibody fragments as primary immunolabels. Fab' fragments are conjugated site-specifically through sulfhydryl groups distal to antigen-binding domains, and the multivalent conjugates have small and homogeneous sizes (∼12 nm) near those of full-sized antibodies. Their performance as immunolabels for intracellular antigens is evaluated quantitatively by metrics of microtubule labeling density and connectivity in fixed cells and for cytological identification in fixed brain specimens, comparing results with probes based on spectrally-matched dyes. QD-Fab' conjugates outperformed QD conjugates of full-sized antibodies and could be imaged with bright signals with 1-photon and 2-photon excitation. The results demonstrate a requirement for smaller bioaffinity agents and site-specific orientation for the success of nanomaterial-based labels to enhance penetration in biospecimens and minimize nonspecific staining.

Abstract PO5-26-09: Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67

Abstract Background and Aims Ki-67 is a well-established biomarker of proliferation in breast cancer (BC). However, its value in treatment decision making is hampered by a lack of analytical reproducibility. Regular participation in external quality assessment (EQA) substantially improves inter-laboratory concordance. Pre-requisites in establishing a fit-for-purpose EQA for Ki-67 are a well-validated testing substrate and a reproducible method of assessing Ki-67 scores in participants’ submitted materials. We report here on the results of work using cell line controls analysed by digital image analysis (DIA) as a first step towards providing such an EQA. Materials and Methods A formalin-fixed paraffin embedded (FFPE) cell line microarray (CLMA) was designed and produced in conjunction with Array Sciences LLC (Sausalito, USA). It was comprised of cores taken from a pure population of Sf9 caterpillar cells, which have been shown to be completely unreactive with most commercial antibodies to human Ki-67, together with cores of Sf9 cells mixed with four different human BC cell lines. These were BT-20 (85% BC cells), ZR-75-1 (75%), BT-474 (65%) and BT-483 (55%). Sections from the CLMA were mounted onto glass microscope slides together with sections from a FFPE tonsil sample and two BC samples; one BC showed high (~30%, BC-high) and the second, low proliferation (~5%, BC-low). In both BC samples proliferating cancer cells were distributed homogeneously throughout the the block. Unstained sections were distributed to laboratories participating in the Scheme’s Ki-67 BC programme; after routine IHC-staining for Ki-67 returned slides were centrally visually assessed for stain quality and subjected to DIA using an in-house application developed on Visiopharm software (Visiopharm A/S, Hoersholm, Denmark). Results Slides were returned by 37 laboratories. Analysis of Ki-67 scores obtained on the Sf9 core identified two distinct groups. The first (n = 27) were negative or showed low Ki-67 scores (mean = 1.1%, 95% CIs: 0.2-1.9%), the second (n = 10) displayed a step-change in scores (mean = 49.9%, 95% CIs: 33.2-66.7%); the means of the two groups were significantly different (P< 0.0001). When Ki-67 scores for each of the tissue samples were dichotomized into similar groups, the means of those groups differed significantly for the two BC samples (P< 0.001), but not for tonsil. Quality scores generated by visual assessment did not differ significantly between the two groups. However, when slides bearing Sf9 cores demonstrating aberrant Ki-67 scores were visually examined nuclear staining was clearly visible, and non-specific nuclear staining could also be identified in the matched BC tissue samples, but not in the tonsil sections. Correlation of Ki-67 scores between the four BC cell line cores and each of the BC tissue samples was examined using Pearson’s correlation statistics. The r statistic range was 0.57-0.71 in comparisons between BC cell line cores and the BC-high sample, and 0.53-0.70 for those with BC-low; in each case BT-483 showed the highest correlation score and BT-20 the lowest. A similar analysis was undertaken between the tonsil and the two BC tissues. The r statistic for correlation between tonsil and BC-high scores was 0.72; it was 0.64 for those between tonsil and BC-low. Conclusions By using a pure population of Sf9 cells we have developed a sensitive indicator of non-specific nuclear staining in IHC preparations stained for Ki-67 which identifies the presence of the artefact quantifiably. Cores made from Sf9/BC cell line mixtures (especially BT-483) produce Ki-67 scores which correlate with those obtained in breast cancer samples at a similar level to those achieved between tonsil and BC samples; this is true for both high and the low proliferation ranges. Cell line mixtures can be adjusted to show Ki-67 scores in the clinically relevant ranges, and they do not show the inherent biological variations seen in tissue controls such as tonsil. Citation Format: Andrew Dodson, Fitim Berisha, Dawn Wilkingson, Lila Zabaglo, Suzanne Parry. Digital image analysis and a novel set of cell line samples as aids in the development of a quantitative external quality assessment programme for Ki-67 [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO5-26-09.

Consistency Analysis of Programmed Death Ligand 1 Expression in Non–Small Cell Lung Cancer Between Pleural Effusion and Matched Primary Lung Cancer Tissues by Immunohistochemical Double Staining

In clinical practice, programmed death ligand 1 (PD-L1) detection is prone to nonspecific staining due to the complex cellular composition of pleural effusion smears. In this study, diaminobenzidine (DAB) and 3-amino-9-ethylcarbazole (AEC) immunohistochemistry double staining was performed to investigate PD-L1 expression in tumor cells from malignant pleural effusion (MPE). MPE was considered as a metastasis in non–small cell lung cancer patients; thus, the heterogeneity between metastatic and primary lung cancer was revealed as well. Ninety paired specimens of MPE cell blocks and matched primary lung cancer tissues from non–small cell lung cancer patients were subjected to PD-L1 and thyroid transcription factor-1(TTF-1)/p63 immunohistochemistry double staining. Two experienced pathologists independently evaluated PD-L1 expression using 3 cutoffs (1%, 10%, and 50%). PD-L1 expression in MPE was strongly correlated with that in matched primary lung cancer tissues (R = 0.813; P < .001). Using a 4-tier scale (cutoffs: 1%, 10%, and 50%), the concordance was 71.1% (Cohen’s κ = .534). Using a 2-tier scale, the concordance was 75.6% (1%, Cohen’s κ = 0.53), 78.9% (10%, Cohen’s κ = 0.574), and 95.6% (50%, Cohen’s κ = 0.754). The rates of PD-L1 positivity in MPE (56.7%) were higher than that in lung tissues (32.2%). All 27 discordant cases had higher scores in MPE. The double-staining method provided superior identification of PD-L1-positive tumor cells on a background with nonspecific staining. In conclusion, PD-L1 expression was moderately concordant between metastatic MPE cell blocks and matched primary lung carcinoma tissues, with variability related to tumor heterogeneity. MPE should be considered to detect PD-L1 when histological specimens are unattainable, especially when PD-L1 expression is >50%. PD-L1 positivity rates were higher in MPE. Double staining can improve PD-L1 detection by reducing false-negative/positive results.

Secondary reagents for immunohistochemical research of rat brain

BACKGROUND: The critical factor when working with immunohistochemistry in laboratory animals is to select appropriate secondary antibodies, that allow clear and specific visualization of tissue antigens. Many reliable secondary reagents are currently not available for purchase, which determines the high relevance of replacing them with other detection systems.AIM: To verify the effectiveness of available secondary reagents for immunohistochemical research of the rat brain.METHODS: Brain samples from Wistar and SHR rats (n=4) were used for the study. Iba-1, GFAP and vimentin immunohistochemistry was carried out using various polymer-based detection systems, namely UltraVision Quanto, N-Histofine Simple stain MAX PO ® and UnoVue Rabbit HRP.RESULTS: All three studied polymer systems demonstrated visualisation of target proteins in brain tissues and cells corresponding to the general understanding of structures containing Iba-1, GFAP and vimentin. The UnoVue Rabbit HRP and Quanto Detection System HRP kits showed good and similarly specific immunohistochemical reaction. However, the UnoVue Rabbit HRP kit was less sensitive compared to the Quanto Detection System HRP. The reaction with N-Histofine® Simple Stain MAX PO was not optimal due to the presence of non-specific background staining that was not present with other reagents.CONCLUSION: Two secondary antibody kits from the three studied showed optimal efficiency of immunohistochemical reaction and minimal background staining. N-Histofine® Simple Stain MAX PO is not suitable for immunohistochemical research of rat brain tissue.

PEGylation of NIR Cd0.3Pb0.7S aqueous quantum dots for stabilization and reduction of nonspecific binding to cells

Cd0.3Pb0.7S (CdPbS) aqueous quantum dots (AQDs) made with 3-mercaptoproprionic acid (MPA) as a ligand have the advantages of emitting near-infrared light, well above 800 nm, that completely circumvents interference from tissue autofluorescence and have significant amounts of ligands for bioconjugation. However, retaining the right amount of MPA became a challenge when using CdPbS AQDs for bioimaging because retaining too much MPA could lead to significant nonspecific staining in cell imaging while insufficient MPA could cause AQDs instability in biological systems. Here we examined PEGylation (i.e. chemically linking amine-functionalized polyethylene glycol (PEG)) to modify MPA on the AQDs surface to improve AQDs stability and reduce nonspecific staining. In addition, for conjugation with antibodies, a bifunctional PEG with a carboxyl functionality was used to permit chemical linkage of a PEG to an antibody on the other end. It was found that performing PEGylation at the thiol concentration where the zeta potential becomes saturated stabilized the CdPbS AQDs suspension and reduced nonspecific binding to cells. Furthermore, with the bifunctional PEG, the CdPbS AQDs were conjugated with antibodies and the AQD-Ab conjugates were shown to stain cancer cells specifically against normal cells with a signal-to-noise ratio of 8.

Nonspecificity of Immunohistochemistry for Mycobacteria Species Using a Rabbit Polyclonal Antibody.

Recent publications have featured immunohistochemistry (IHC) as a sensitive tool for detecting Mycobacterium tuberculosis and nontuberculous mycobacteria, but performance is limited to cases suspected to have mycobacterial infection. To examine cross-reactivity of a polyclonal antimycobacterial antibody with various types of pathogens, tissues, and inflammatory patterns. Surgical pathology files during a period of 6 years were searched, and 40 cases representing a variety of pathogens, tissue types, and inflammatory responses were retrieved. Cases were stained with a rabbit polyclonal antimycobacterial antibody (Biocare Medical, Pacheco, California). The cases and associated histochemical stains, culture, and molecular results were reviewed by 3 pathologists. All 8 cases of mycobacterial infection previously diagnosed by other methods were positive for mycobacteria by IHC. In addition, multiple bacterial and fungal organisms and 1 case of Leishmania amastigotes were also immunoreactive with the mycobacterial IHC. Although highly sensitive for mycobacteria, the polyclonal antibody shows significant cross-reactivity with other organisms. This is a sensitive but nonspecific stain that can be used as an alternative confirmation method for mycobacteria, but attention should be paid to inflammatory reaction and organism morphology when IHC is positive to avoid misdiagnosis.

GPR55 is expressed in glutamate neurons and functionally modulates drug taking and seeking in rats and mice

G protein-coupled receptor 55 (GPR55) has been thought to be a putative cannabinoid receptor. However, little is known about its functional role in cannabinoid action and substance use disorders. Here we report that GPR55 is predominantly found in glutamate neurons in the brain, and its activation reduces self-administration of cocaine and nicotine in rats and mice. Using RNAscope in situ hybridization, GPR55 mRNA was identified in cortical vesicular glutamate transporter 1 (VgluT1)-positive and subcortical VgluT2-positive glutamate neurons, with no detection in midbrain dopamine (DA) neurons. Immunohistochemistry detected a GPR55-like signal in both wildtype and GPR55-knockout mice, suggesting non-specific staining. However, analysis using a fluorescent CB1/GPR55 ligand (T1117) in CB1-knockout mice confirmed GPR55 binding in glutamate neurons, not in midbrain DA neurons. Systemic administration of the GPR55 agonist O-1602 didnt impact ∆9-THC-induced analgesia, hypothermia and catalepsy, but significantly mitigated cocaine-enhanced brain-stimulation reward caused by optogenetic activation of midbrain DA neurons. O-1602 alone failed to alter extracellar DA, but elevated extracellular glutamate, in the nucleus accumbens. In addition, O-1602 also demonstrated inhibitory effects on cocaine or nicotine self-administration under low fixed-ratio and/or progressive-ratio reinforcement schedules in rats and wildtype mice, with no such effects observed in GPR55-knockout mice. Together, these findings suggest that GPR55 activation may functionally modulate drug-taking and drug-seeking behavior possibly via a glutamate-dependent mechanism, and therefore, GPR55 deserves further study as a new therapeutic target for treating substance use disorders.

Visualizing the invisible: novel approaches to visualizing bacterial proteins and host-pathogen interactions.

Host-pathogen interactions play a critical role in infectious diseases, and understanding the underlying mechanisms is vital for developing effective therapeutic strategies. The visualization and characterization of bacterial proteins within host cells is key to unraveling the dynamics of these interactions. Various protein labeling strategies have emerged as powerful tools for studying host-pathogen interactions, enabling the tracking, localization, and functional analysis of bacterial proteins in real-time. However, the labeling and localization of Salmonella secreted type III secretion system (T3SS) effectors in host cells poses technical challenges. Conventional methods disrupt effector stoichiometry and often result in non-specific staining. Bulky fluorescent protein fusions interfere with effector secretion, while other tagging systems such as 4Cys-FLaSH/Split-GFP suffer from low labeling specificity and a poor signal-to-noise ratio. Recent advances in state-of-the-art techniques have augmented the existing toolkit for monitoring the translocation and dynamics of bacterial effectors. This comprehensive review delves into the bacterial protein labeling strategies and their application in imaging host-pathogen interactions. Lastly, we explore the obstacles faced and potential pathways forward in the realm of protein labeling strategies for visualizing interactions between hosts and pathogens.

The use of fluorescence lifetime imaging (FLIM) for in situ microbial detection in complex mineral substrates.

The utility of fluorescence lifetime imaging microscopy (FLIM) for identifying bacteria in complex mineral matrices was investigated. Baseline signals from unlabelled Bacillus subtilis and Euglena gracilis, and Bacillus subtilis labelled with SYTO 9 were obtained using two-photon excitation at 730, 750 and 800nm, identifying characteristic lifetimes of photosynthetic pigments, unpigmented cellular autofluorescence, and SYTO 9. Labelled and unlabelled B. subtilis were seeded onto marble and gypsum samples containing endolithic photosynthetic cyanobacteria and the ability to distinguish cells from mineral autofluorescence and nonspecific dye staining was examined in parallel with ordinary multichannel confocal imaging. It was found that FLIM enabled discrimination of SYTO 9 labelled cells from background, but that the lifetime of SYTO 9 was shorter in cells on minerals than in pure culture under our conditions. Photosynthetic microorganisms were easily observed using both FLIM and confocal. Unlabelled, nonpigmented bacteria showed weak signals that were difficult to distinguish from background when minerals were present, though cellular autofluorescence consistent with NAD(P)H could be seen in pure cultures, and phasor analysis permitted detection on rocks. Gypsum and marble samples showed similar autofluorescence profiles, with little autofluorescence in the yellow-to-red range. Lifetime or time-gated imaging may prove a useful tool for environmental microbiology. LAY DESCRIPTION: The standard method of bacterial enumeration is to label the cells with a fluorescent dye and count them under high-power fluorescence microscopy. However, this can be difficult when the cells are embedded in soil and rock due to fluorescence from the surrounding minerals and dye binding to ambiguous features of the substrate. The use of fluorescence lifetime imaging (FLIM) can disambiguate these signals and allow for improved detection of bacteria in environmental samples.