Search bioRxiv⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.1101/2020.03.17.994210

Validating a semi-quantitative method for assessment of degree of methylene blue staining in sentinel lymph nodes

Ram, A. S.·Oblak, M. L.·Matuszewska, K.·Singh, A.·Petrik, J.
View original source ↗Export metadata ↓

Abstract

Purpose To develop a digital algorithm and validate a semi-quantitative scoring method for surface methylene blue (MB) staining in whole lymph nodes (LN).Methods Lymph nodes from canine models undergoing sentinel lymph node (SLN) mapping were prospectively assessed ex vivo and photographed. Two blinded observers evaluated all images and assigned a semi-quantitative score based on surface staining (0 – no blue stain, 1 – 1-50% stained, 2 – 51-100% stained). A standard reference for degree of blue staining was based on signal-to-background ratios using computer-based imaging software with an output measurement of percentage of staining of the LN. Agreement between observers was assessed using the Kappa coefficient.Results 124 lymph nodes were included and demonstrated strong agreement (K = 0.8007, p < 0.0001) between results of semi-quantitative scoring and image analysis. Also, strong interobserver and intraobserver agreement was observed for the scoring system (K = 0.8051, p < 0.0001 and K = 0.9493, p < 0.0001, respectively).Discussion Agreement between the observer-based scoring system and imaging software illustrates a validated method in assessing MB staining, without the need for analysis software. The use of a semi-quantitative scoring system shows promise for a simple, objective assessment of MB staining in surgery and for future study. Lymph nodes can have variable surface colour, which can make assessment of blue staining challenging for novice observers in certain cases. This study describes a digital algorithm for quantitative analysis of blue staining in LN thereby providing a novel and objective reporting mechanism in scientific research involving SLN mapping.Competing Interest StatementThe authors have declared no competing interest.View Full Text

Explore related subjects

pathologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Ram, A. S., Oblak, M. L., Matuszewska, K., Singh, A., Petrik, J.. 2020-03-24. Validating a semi-quantitative method for assessment of degree of methylene blue staining in sentinel lymph nodes. https://doi.org/10.1101/2020.03.17.994210

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Relative expression of THY1 and PDGFRA in equine endometrosis in relation to severity, histopathological features and endometrial transcripts

Equine endometrosis is a degenerative uterine disease characterized by periglandular fibrosis and represents a major cause of subfertility in mares. While stromal cell activation results in pathological tissue remodeling, the involvement of cell-surface integrators (THY1) and growth factor receptors (PDGFRA) was not researched. This study evaluated the relative mRNA expression of THY1 and PDGFRA across Kenney and Doig (KD) endometrosis categories and specific histopathological features. Additionally, transcriptomic correlation profiles between target genes and markers of myofibroblast differentiation (ACTA2), cellular proliferation (MKI67), and steroid hormone receptors (ESR1, PGR) were analyzed. Endometrial samples (n = 47) were quantified using RT-qPCR. Relative mRNA expression of THY1 differed significantly across KD categories (p = 0.014), exhibiting a transient upregulation in category IIA compared with category I (p = 0.009) and category IIB (p = 0.002). Furthermore, THY1 relative mRNA expression was significantly elevated in tissues with glandular basal lamina disruption (p = 0.022). Conversely, PDGFRA relative mRNA expression remained stable across disease categories (p > 0.05). THY1 relative mRNA expression correlated very strongly with ACTA2 ({rho} = 0.875, p < 0.001) and strongly with ESR1 ({rho} = 0.652, p < 0.001). PDGFRA relative mRNA expression exhibited a very strong positive correlation with ESR1 ({rho} = 0.864, p < 0.001) and a strong positive correlation with MKI67 ({rho} = 0.674, p < 0.001). These results indicate that THY1 transcription is transiently induced during early stromal remodeling and basal lamina loss, whereas PDGFRA maintains constitutive transcript stability. Observed co-expression profiles reflect statistical co-variation among stromal remodeling, proliferative, and steroid-responsive cell populations in bulk tissue.

pathology↗
bioRxiv2026

sPLA2 inhibitor-loaded nanoparticles mitigate spontaneous osteoarthritis in guinea pigs

Osteoarthritis (OA) is a prevalent degenerative joint disease lacking effective disease-modifying therapies. Secreted phospholipase A2 (sPLA2), a key upstream regulator of inflammatory lipid mediators, has been implicated in OA pathogenesis, but its therapeutic potential in age-related OA remains unclear. Here, we evaluated the role of sPLA2 and the efficacy of nanoparticle-mediated sPLA2 inhibition in a spontaneous OA model using female Dunkin-Hartley (DH) guinea pigs. We first characterized OA progression and observed age-dependent increases in cartilage degeneration, synovitis, oxidative stress, and sPLA2 expression and enzymatic activity. We then developed sPLA2 inhibitor (thioetheramide-PC)-loaded nanoparticles (sPLA2i-NPs) and demonstrated their superior ability, compared to free inhibitor, to prevent IL-1{beta}-induced cartilage degeneration in guinea pig cartilage explants. In vivo, intra-articular administration of sPLA2i-NPs from 3 months of age significantly attenuated cartilage destruction, synovial inflammation, and osteophyte formation in 7- and 10-month-old guinea pigs. These structural improvements were accompanied by normalization of anabolic and catabolic gene expression and a marked reduction in synovial fluid sPLA2 activity. Importantly, longitudinal gait analysis revealed that sPLA2i-NP treatment preserved limb loading, propulsion, and locomotor performance, demonstrating functional recovery. No systemic toxicity was observed following long-term treatment. Collectively, our findings identify sPLA2 as a key driver of spontaneous OA and establish nanoparticle-mediated sPLA2 inhibition as a promising disease-modifying strategy for age-related OA.

pathology↗
bioRxiv2026

Midlife nonlinearities across scale in Biondi body amyloid formation in the human choroid plexus

Human choroid plexus epithelial cells (CPECs), which produce cerebrospinal fluid and form the blood-cerebrospinal fluid barrier, accumulate discrete intracellular amyloid inclusions called Biondi bodies (BBs). Using systematic manual and machine learning approaches on the choroid plexus glomus, we determined the percentages of CPECs containing thioflavin S-stained BBs as well as BB load and spatial distributions in whole slide images from 77 cases across the human lifespan. On average, BB prevalence was near zero until age 40, rose steeply in the 5th and 6th decades, and exceeded 50% after age 70. As prevalence plateaued, BB load continued to rise with age. Accelerated BB accumulation was seen in two cases of diabetic ketoacidosis, including a 16-year-old, while an 85-year-old with the relatively rare APOE2/E2 genotype had fewer BBs than expected. BB prevalence in the choroid plexus glomus was not associated with Alzheimer's disease, but was lower in females than males. Notably, spatial clustering of BBs was universal across cases while nearest-neighbor analyses suggested cell-to-cell, prion-like BB spread that can account for the nonlinearities seen at tissue and population scales. The abrupt, nonlinear BB accumulation during midlife coincides with or precedes other common aging-associated neuropathologies of the human brain, consistent with broad BB and choroid plexus contributions to brain aging and disease.

pathology↗