Synovial Samples Research Articles

Shared lung and joint T cell repertoire in early rheumatoid arthritis driven by cigarette smoking

ObjectivesSmoking has been associated with an increased risk of developing rheumatoid arthritis (RA) in individuals carrying shared epitope (SE) HLA-DRB1 alleles. Yet, little is known about the regional and systemic...

IĸBζ as a Central Modulator of Inflammatory Arthritis Pathogenesis.

Current therapies targeting individual factors in inflammatory arthritis show variable efficacy, often requiring treatment with combinations of drugs, and are associated with undesirable side effects. NF-ĸB is critical for the production and function of most inflammatory cytokines. However, given its essential role in physiologic processes, targeting NF-ĸB is precarious. Hence, identifying pathways downstream of NF-ĸB that selectively govern the expression of inflammatory cytokines in inflammatory arthritis would be advantageous. We have previously identified IĸBζ as a unique inflammatory signature of NF-ĸB that controls the transcription of inflammatory cytokines only under pathologic conditions while sparing physiologic NF-ĸB signals. We generated mice harboring myeloid, lymphoid, and global deletion of Nfkbiz (the gene encoding IĸBζ). These models were subjected to serum transfer-induced arthritis. Additionally, pharmacologic inhibitors of IĸBζ were injected intraperitonially. Joint swelling, microcomputed tomography, immunohistochemistry, flow cytometry, and cytokine measurements were conducted using synovial tissue samples. Global deletion of Nfkbiz or depletion of neutrophils (vastly IĸBζ+ cells) reduced inflammatory synovial cells and increased anti-inflammatory and regenerative synovial cells, plummeted expression of inflammatory factors and ameliorated experimental mouse inflammatory arthritis. Further, expression of immune responsive gene-1, the enzyme responsible for itaconate production, was increased in synovial cells. Accordingly, the itaconate derivative dimethyl itaconate (DI) inhibited IĸBζ-mediated inflammatory factors. Further, in silico screen identified 8-hydroxyquinoline (HQ) as a putative inhibitor of IĸBζ not affecting physiologic NF-ĸB activity. Congruently, systemic administration of either DI or HQ inhibited joint swelling and damage. Our study positions IĸBζ as an inflammation-specific target for therapeutic consideration in rheumatoid arthritis because its inhibition spares the beneficial functions of NF-ĸB.

An Equine Protein Atlas Highlights Synovial Fluid Proteome Dynamics during Experimentally LPS-Induced Arthritis.

In human proteomics, substantial efforts are ongoing to leverage large collections of mass spectrometry (MS) fragment ion spectra into extensive spectral libraries (SL) as a resource for data independent acquisition (DIA) analysis. Currently, such initiatives in equine research are still missing. Here we present a large-scale equine SL, comprising 6394 canonical proteins and 89,329 unique peptides, based on data dependent acquisition analysis of 75 tissue and body fluid samples from horses. The SL enabled large-scale DIA-MS based quantification of the same samples to generate a quantitative equine protein distribution atlas to infer dominant proteins in different organs and body fluids. Data mining revealed 163 proteins uniquely identified in a specific type of tissue or body fluid, serving as a starting point to determine tissue-specific or tissue-type-specific proteins. We showcase the SL by highlighting proteome dynamics in equine synovial fluid samples during experimental lipopolysaccharide-induced arthritis. A fuzzy c-means cluster analysis pinpointed SERPINB1, ATRN, NGAL, LTF, MMP1, and LBP as putative biomarkers for joint inflammation. This SL provides an extendable resource for future equine studies employing DIA-MS.

One-step copper deposition-induced signal amplification for multiplex bacterial infection diagnosis on a lateral flow immunoassay device

The lateral flow immunoassay (LFIA) is predominant in rapid diagnostic tests owing to its cost-effectiveness and operational simplicity. However, the conventional LFIA exhibits limited sensitivity and is susceptible to human variance for the result readout, impacting result interpretation. In this study, we introduced a novel one-step copper deposition-induced signal amplification lateral flow immunoassay (osa-LFIA) that markedly enhances the detection sensitivity for Staphylococcus aureus (protein A) and Pseudomonas aeruginosa (exotoxin A). Utilizing gold nanoparticles (AuNPs) as a catalyst, this approach employs ascorbic acid to reduce Cu2+ to Cu0, depositing on AuNPs at the test line and amplifying the signal. A user-friendly design features a three-dimensional paper structure incorporating pre-dried reagents, enabling a streamlined, efficient testing process. The osa-LFIA significantly lowers detection limits to 3 ng mL−1 for protein A and 10 ng mL−1 for exotoxin A, offering a tenfold improvement over conventional LFIA. Additionally, we developed a portable grayscale detection device, achieving less than 10% error in quantitative analysis compared to the data acquired and analyzed in the lab. This entire process, from detection to signal amplification, is completed in just 20 min. For the clinical trial, we utilized the osa-LFIA to test synovial fluid samples infected with Staphylococcus aureus. We also successfully detected different concentrations of the exotoxin A in parallel, with a recovery value of 96%–110%. Our findings demonstrate the osa-LFIA's potential as a rapid, highly sensitive, and simple-to-use diagnostic tool for detecting various pathogens, significantly advancing the field of rapid diagnostic testing.

Clinical performance evaluation of the BioFire Joint Infection Panel.

The BioFire Joint Infection (JI) Panel offers a significant advancement in the rapid diagnosis of joint infections by facilitating the simultaneous detection of multiple bacterial and fungal pathogens, as well as resistance markers, directly from synovial fluid samples. An article published in the Journal of Clinical Microbiology by Moran et al. (J Clin Microbiol 62:e00182-24, 2024, https://doi.org/10.1128/jcm.00182-24) presents both prospective and retrospective analyses of the panel's real-world clinical application. The study highlights the panel's benefits, such as its rapid turnaround time and ability to identify challenging pathogens, while also discussing its limitations, particularly in detecting certain off-panel organisms.

RNA-seq Based Transcriptome Analysis Reveals Role of Myoglobin in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease which manifests as joint destruction and bone erosion, could be caused by both genetic and environmental factors. Currently, the causes of RA are unknown, and targeted therapies are often associated with side effects and contraindications. The detection rate of RA in women is higher than men (3:1), however, there is still a lack of comprehensive understanding of the relationship between sex and RA. We hypothesized gender differences in RA prevalence and their associated mechanisms by performing genome-wide transcriptome analysis of synovial biopsy samples. The results indicated that myoglobin (MB) was differentially expressed between males and females, with higher expression in males than females in healthy populations, while the opposite was observed in RA patients. MB interacted with HLA class II histocompatibility antigen, DM beta (HLA-DMB) and the inflammatory factor interleukin 6 (IL-6) in the human synovial cell line MH7A.

A-281 Comparison of C-reactive Protein, Procalcitonin, and Presepsin in Blood and Synovial Fluid as Diagnostic Biomarkers for Septic Arthritis

Abstract Background Early detection and prompt treatment are crucial for managing septic arthritis (SA). However, diagnosing SA at an early stage can be challenging since the symptoms are non-specific, and there is no definitive diagnostic test available. This study aimed to investigate the potential of biomarkers as a reliable diagnostic tool for SA. Methods A prospective study was conducted to collect residual blood and synovial fluid samples from patients who underwent blood sampling and arthrocentesis for differential diagnosis due to suspected SA. The collected samples were analyzed for C-reactive protein (CRP), procalcitonin, and presepsin. The data obtained from the analysis were subjected to statistical analysis. Results From January to November 2023, 58 patients were enrolled. They comprised 24 men and 34 women with a median age of 71.1 (interquartile range, IQR 64.0-79.3) years. Twenty-one patients were diagnosed with SA and 37 with non-SA, including osteoarthritis (n=2), osteonecrosis (n=1), meniscus tear (n=1), abscess (n=1), contusion (n=2), and mechanical complications of implants (n=30). There were no significant differences in age or sex distribution between the two groups. The median blood and synovial fluid CRP levels were 4.24 and 1.85 mg/dL, respectively, in the SA group and 0.24 and 0.11 in the non-SA group. The median blood and synovial fluid procalcitonin levels were 0.0383 and 0.0403 ng/mL in the SA group and 0.0358 and 0.0488 in the non-SA group. The median blood and synovial fluid presepsin levels were 307 and 1389 pg/mL in the SA group and 192 and 959 in the non-SA group. Except for blood procalcitonin, all showed statistically significant differences between the SA and non-SA groups. In the ROC curve analysis, blood CRP displayed the highest area under the curve (AUC) of 0.795 with a 0.47 mg/dL cut-off value, 90.5% sensitivity, and 64.9% specificity. Synovial fluid CRP displayed similar results of 0.786 AUC, 0.30 mg/dL cut-off value, 85.0% sensitivity, and 71.4% specificity. Blood and synovial fluid procalcitonin had low AUCs of 0.532 and 0.659, respectively, and were insufficient to differentiate between SA and NS groups. The AUC (0.696 vs. 0.704), sensitivity (76.2% vs. 76.2%), and specificity (67.6% vs. 62.2%) of blood and synovial fluid presepsin were similar; however, synovial fluid showed a significantly higher cut-off value, 4.5 times that of blood (237 pg/mL vs. 1087 pg/mL). Conclusions Procalcitonin was not a reliable marker for distinguishing between SA and non-SA, regardless of whether it was measured in blood or synovial fluids. However, CRP and presepsin proved useful markers when measured in either blood or synovial fluid. In particular, when measuring presepsin in synovial fluid, an optimal cut-off value is needed.

Synovial vancomycin and meropenem concentrations in periprosthetic joint infection treated by single-stage revision combined with intra-articular infusion.

We aimed to determine the concentrations of synovial vancomycin and meropenem in patients treated by single-stage revision combined with intra-articular infusion following periprosthetic joint infection (PJI), thereby validating this drug delivery approach. We included 14 patients with PJI as noted in their medical records between November 2021 and August 2022, comprising eight hip and seven knee joint infections, with one patient experiencing bilateral knee infections. The patients underwent single-stage revision surgery, followed by intra-articular infusion of vancomycin and meropenem (50,000 µg/ml). Synovial fluid samples were collected to assess antibiotic concentrations using high-performance liquid chromatography. The peak concentrations of vancomycin and meropenem in the joint cavity were observed at one hour post-injection, with mean values of 14,933.9 µg/ml (SD 10,176.3) and 5,819.1 µg/ml (SD 6,029.8), respectively. The trough concentrations at 24 hours were 5,495.0 µg/ml (SD 2,360.5) for vancomycin and 186.4 µg/ml (SD 254.3) for meropenem. The half-life of vancomycin was 6 hours, while that of meropenem ranged between 2 and 3.5 hours. No significant adverse events related to the antibiotic administration were observed. This method can achieve sustained high antibiotic concentrations within the joint space, exceeding the reported minimum biofilm eradication concentration. Our study highlights the remarkable effectiveness of intra-articular antibiotic infusion in delivering high intra-articular concentrations of antibiotics. The method provided sustained high antibiotic concentrations within the joint cavity, and no severe side-effects were observed. These findings offer evidence to improve clinical treatment strategies. However, further validation is required through studies with larger sample sizes and higher levels of evidence.

A-158 Multi-site verification and performance of body fluids in-between Horiba Medical Yumizen h2500 with Sysmex XN-10 automated hematology analyzers and manual microscopy for morphology assessment

Abstract Background The analysis of body fluids plays an important role in the diagnosis and management of a wide variety of conditions. As standards for method validation and laboratory developed tests evolve, the body fluids analysis is receiving increased attention from both laboratories and regulatory bodies. We present the evaluated result of the validation and clinical performance accuracy of HORIBA Medical Yumizen H2500 analyzers as compared to SYSMEX XN-10 analyzers and to manual slide microscopy examination as a reference method for differential analysis for body fluids samples. Methods Depending on each laboratory collected procedures, synovial samples were collected with Sodium Heparin or K2EDTA or without anticoagulant and the other samples were collected with K2EDTA or without anticoagulant. As recommended, most (94%) of the synovial fluids were treated with the hyaluronidase. For the comparison of Yumizen H2500 versus XN-10 analyzers, the study was performed in 2 US sites and 1 French site collecting 390 samples totally: 138 serous fluids (Ascites, Pleural, Peritoneal, Pericardial), 169 synovial fluids and 83 CSF fluids. Samples were analyzed as soon as possible after collection (with a limit of 24 hours for serous and synovial fluids and 4 hours for CSF) and within 2 hours between both instruments. For the comparison of Yumizen H2500 versus reference method (manual microscopy), the study was performed in 2 US sites collecting 58 samples: 34 serous fluids (Ascites, Pleural, Peritoneal, Pericardial), 12 synovial fluids and 12 CSF fluids. Two qualified morphology examiners made the manual microscopy analysis and the average of the results of the 2 slide readings was used for the analysis. Results Considering all types of body fluids, the H2500/XN results are very well correlated for BFWBC (0.969), BFRBC (0.999), BFMN% (0.912), BFMN# (0.89), BFPN% (0.912), BFPN# (0.97). Similarly, the statistical analysis per body fluids type shows very good correlation. For serous type, all results are higher than 0.876, for synovial type all results are higher than 0.879 and for cerebrospinal fluids type, all results are higher than 0.788. Among all samples, less than 8 samples (<2%) were out of the 95% interval. The comparison Yumizen H2500 versus manual microscopy, all the correlation results (r²) are very good: 0,9705 for Polynuclear and 0,9742 for Mononuclear. The study demonstrated better results of Yumizen H2500 versus manual microscopy than versus XN-10 for BFPN% (0.9705 versus 0.912) and BFMN% (0.9742 versus 0.912). Among 58 samples, only 4 (6.9%) were out of the 95% interval for BFPN% and BFMN%. Conclusions The Yumizen H2500 results for body fluid WBC differentiation (Polynuclear and Mononuclear) are very well correlated with the results from manual slide microscopy evaluation. The comparison of the results analyzed on Yumizen H2500 and on XN does not show any significant difference.

Imaging Mass Cytometry in Psoriatic Disease Reveals Immune Profile Heterogeneity in Skin and Synovial Tissue

Imaging Mass Cytometry (IMC) is a technology that enables comprehensive analysis of cellular phenotypes at the tissue level. We performed a multi-parameter characterization of structural and immune cell populations in psoriatic skin and synovial tissue samples aimed at characterizing immune cell differences in patients with psoriasis, psoriatic arthritis (PsA).A panel of 33 antibodies was used to stain selected immune and structural cell populations. IMC data were segmented into single cells based on combinations of antibody stains. Single cells were then clustered into cell categories based on pre-specified markers. The spatial relationships of different cell populations were assessed using neighborhood analysis.Among all cell types in the skin and synovium, lymphoid cells accounted for the most prevalent cell type. T cells and macrophages were the most prevalent immune cell type in the synovium and B cells and NK cells were also identified. Neighborhood analysis showed high correlation between synovial T cells, B cells, macrophages, dendritic cells and neutrophils suggesting spatial organization.Innate and adaptive immune cells can be reliably identified using IMC in skin and synovium. Inter-patient heterogeneity exists in tissue cell populations. IMC provides opportunities for exploring in depth underlying immunological mechanisms driving psoriasis and PsA.

The Significance of Intracellular Versus Extracellular Calcium Pyrophosphate Crystals in Diagnosing Calcium Pyrophosphate Crystal Arthritis.

Acute and chronic calcium pyrophosphate (CPP) crystal arthritis is characterized by the presence of synovial CPP crystals within a clinically inflamed joint. CPP crystals may be situated intracellularly or extracellularly; however, the clinical significance of their location remains understudied. The objective of this retrospective cohort study was to assess the relevance of the CPP crystal location in diagnosing acute/chronic CPP crystal arthritis. Data were collected from Waikato District Health Board to identify a study population with synovial fluid samples positive for CPP crystals. The cohort was stratified into 2 groups based on crystal location: intracellular and extracellular. The proportions of acute/chronic CPP crystal arthritis cases were compared between these groups. Acute/chronic CPP crystal arthritis was diagnosed when synovial CPP crystals were present, with objective evidence of joint inflammation and no other alternative diagnosis. Further analysis was made with respect to demographics, other laboratory results, and cartilage calcification. This study included 134 patients: 108 with intracellular CPP crystals and 26 with extracellular CPP crystals. Acute/chronic CPP crystal arthritis was diagnosed in 85% of cases in the intracellular and 50% in the extracellular group (P < 0.001). Following exclusion of septic arthritis cases, acute/chronic CPP crystal arthritis was diagnosed in 97% of patients in the intracellular group and in 62% of those in the extracellular group (P < 0.001). The presence of intracellular CPP crystals is more strongly associated with acute/chronic CPP crystal arthritis than with extracellular CPP crystals alone.

Do Intra-articular Metal Ion Levels Predict Adverse Local Tissue Reaction in Revision Total Hip Arthroplasty for Mechanically Assisted Crevice Corrosion?

BackgroundMechanically assisted crevice corrosion is a complication that may occur in vivo at modular metal interfaces following metal-on-polyethylene total hip arthroplasty (THA). Metal ions released in vivo may be associated with adverse local tissue reactions (ALTRs). While there is no definitive value, high serum ion levels are implicated as contributors to ALTR, and various screening levels have been recommended. The purpose of this investigation was to evaluate the relationship between synovial fluid (SF) cobalt and chromium ion levels and the risk of developing ALTR. MethodsThis was a retrospective cohort study of 552 patients who underwent 621 metal-on-polyethylene primary THAs. A total of 69 patients underwent revision THA due to symptomatic primary failure with elevated serum metal ions levels. There were 28 who had preoperative serum and intraoperative SF chromium and cobalt samples. Patient demographics, surgical, and laboratory data were collected. Descriptive statistics, Mann-Whitney U, analysis of variance tests, and linear regression analyses were performed. ResultsThere were 40.6% of revisions that had preoperative serum and intraoperative SF samples. The mean time to revision was 5.7 (range, 3.8 to 7.6) years. Mean SF cobalt and chromium levels were 870.9mcg/L (range, 1.1 to 8,300.0) and 573.5mcg/L (range, 1.3 to 10,000.0). Mean serum and SF cobalt-chromium ratios were 4.0 (range, 0.9 to 7.1) and 6.4 (range, 0 to 15.1), respectively. Elevated serum cobalt levels were predictive of ALTR (P = 0.002), SF levels were not. Analysis of preoperative serum to SF cobalt-chromium ratios showed poor correlation (R2 = 0.05). ConclusionsThere was no correlation between SF ion levels and ALTR. Also, serum and intra-articular ion levels did not correlate. The SF levels did not provide additional value over serum levels for diagnosis or prognosis of mechanically assisted crevice corrosion. Further studies are needed to better understand the relationship between serum and SF ion levels and its relationship to ALTR.

Streptococcus dysgalactiae Septic Arthritis of a Pre-Weaned Holstein Bull Calf - a Case Report

Septic arthritis is a joint inflammation caused by an infectious agent. Septic arthritis takes a long time to heal, and as such it may have a significant economic impact. A one-month-old Friesian Holstein calf was lame and had swelling in the left carpal joint. Synovial fluid samples from the left carpal joint were used for bacterial identification, which use API Strept 20 (bioMérieux, Inc – USA). The identification results showed that septic arthritis was caused by Streptococcus dysgalactiae subspecies dysgalactiae,. Streptococcus dysgalactiae subspecies dysgalactiae play a significant role even though it plays a major role in mastitis in dairy cows.

Septic Arthritis Modeling Using Sonographic Fusion with Attention and Selective Transformation: a Preliminary Study.

Conventionally diagnosing septic arthritis relies on detecting the causal pathogens in samples of synovial fluid, synovium, or blood. However, isolating these pathogens through cultures takes several days, thus delaying both diagnosis and treatment. Establishing a quantitative classification model from ultrasound images for rapid septic arthritis diagnosis is mandatory. For the study, a database composed of 342 images of non-septic arthritis and 168 images of septic arthritis produced by grayscale (GS) and power Doppler (PD) ultrasound was constructed. In the proposed architecture of fusion with attention and selective transformation (FAST), both groups of images were combined in a vision transformer (ViT) with the convolutional block attention module, which incorporates spatial, modality, and channel features. Fivefold cross-validation was applied to evaluate the generalized ability. The FAST architecture achieved the accuracy, sensitivity, specificity, and area under the curve (AUC) of 86.33%, 80.66%, 90.25%, and 0.92, respectively. These performances were higher than using conventional ViT (82.14%) and significantly better than using one modality alone (GS 73.88%, PD 72.02%), with the p-value being less than 0.01. Through the integration of multi-modality and the extraction of multiple channel features, the established model provided promising accuracy and AUC in septic arthritis classification. The end-to-end learning of ultrasound features can provide both rapid and objective assessment suggestions for future clinic use.

Histological synovitis score in juvenile idiopathic arthritis and other pediatric synovial inflammatory conditions

ObjectiveThe Krenn’s scoring is a system for histopathological grading of synovial inflammation, and in adults, useful in etiological grouping. The score has only rarely been used in pediatric samples. We aimed to assess the performance of the score in juvenile idiopathic arthritis and other pediatric synovial inflammatory processes in categorization of the disease and in finding characteristic pathological features. DesignWe collected an unselected series of pediatric (age < 16 years) routine synovial biopsy samples to represent normal synovium and inflammatory conditions. The final diagnosis based on clinical follow-up was determined and classified as normal synovium, and different groups according to etiology. Total of 142 patients were analyzed. According to the score, case was classified to normal, low- or high-grade synovitis. ResultsThe synovitis scores in clinically normal synovium were low with 48 % of cases with scores of low-grade synovitis. In structural joint disorders scores varied from normal to low grade synovitis with occasional cases of high grade synovitis. In transient/reactive arthritis scores showed increase, majority clustering to low grade synovitis. In JIA and in bacterial synovitis the scores were higher than in the other groups high grade synovitis being the dominant grade. Extended oligoarthritis showed higher score than persistent oligoarthritis. ROC analysis indicated that JIA could be differentiated from other conditions. ConclusionsThe Krenn’s synovitis score is useful in the etiological classification of pediatric synovial samples, high Krenn’s score suggesting JIA. Observed differences between the subcategories of oligoarthritis may be useful in subclassifying these types of JIA.

Prevalence and factors associated to calcium pyrophosphate arthritis in patients with gout

Aim: To ascertain the prevalence of calcium pyrophosphate arthritis (CPPA) at diagnosis and during follow-up of patients with gout. Methods: Inception cohort of patients with gout prospectively recruited and followed-up from 1994–2023. Gout-case was defined as crystal-proved tophus or arthritis, or the presence of tophus plus double contour with ultrasonography. CPPA was defined as the presence of intra-leukocyte calcium pyrophosphate (CPP) crystals in synovial fluid (SF) and neat chondrocalcinosis in plain radiographs. Age, gender, time from onset of symptoms, number of flares, joint distribution, previous and prescribed treatments, colchicine prophylaxis, comorbidities, alcohol intake, use of diuretics, renal function, and previous vascular disease were available for analysis. Results: A total of 1,544 patients with gout, with an average of 4-year follow-up, were available for analysis. CPPA was observed in 127/1,544 cases (8.2%). In 37/1,544 patients (2.4%) CPP and monosodium urate (MSU) crystals were observed in the same SF sample at gout diagnosis, and 90/1,544 (5.8%) showed CPP crystals apart from the diagnosis of gout. CPPA-gout cases had more flares per year, but no more frequent polyarticular distribution at baseline compared to non-CPPA-gout. CPPA-gout cases were older at baseline and showed lower renal function. Women, patients using diuretics, patients with hypertension, and those with previous vascular events showed CPPA more frequently. Multivariate analysis showed that only age and use of diuretics were independently associated with CPPA, as other variables apparently associated were dependent on aging. Interestingly, an analysis of the prevalence in the three decades available showed an increased CPPA diagnosis through time, probably associated with increased awareness of the association. Conclusions: (1) CPPA is not infrequent in patients with gout; (2) it is associated with aging and diuretic use; (3) awareness of this association may increase the rate of diagnosis.

An objective diagnosis of gout and calcium pyrophosphate deposition disease with machine learning of Raman spectra acquired in a point-of-care setting.

Raman spectroscopy is proposed as a next-generation method for the identification of monosodium urate (MSU) and calcium pyrophosphate (CPP) crystals in synovial fluid. As the interpretation of Raman spectra requires specific expertise, the method is not directly applicable for clinicians. We developed an approach to demonstrate that the identification process can be automated with the use of machine learning techniques. The developed system is tested in a point-of-care-setting at our outpatient rheumatology department. We collected synovial fluid samples from 446 patients with various rheumatic diseases from three centra. We analyzed all samples with our Raman spectroscope and used 246 samples for training and 200 samples for validation. Trained observers classified every Raman spectrum as MSU, CPP or else. We designed two one-against-all classifiers, one for MSU and one for CPP. These classifiers consisted of a principal component analysis model followed by a support vector machine. The accuracy for classification of CPP using the 2023 ACR/EULAR CPPD classification criteria was 96.0% (95% CI 92.3-98.3), while the accuracy for classification of MSU with using the 2015 ACR/EULAR gout classification criteria was 92.5% (95% CI 87.9-95.7). Overall, the accuracy for classification of pathological crystals was 88.0% (95% CI 82.7-92.2). The model was able to discriminate between pathologic crystals, artifacts, and other particles such as microplastics. We here demonstrate that potentially complex Raman spectra from clinical patient samples can be successfully classified by a machine learning approach, resulting in an objective diagnosis independent of the opinion of the medical examiner.

Association of Cartilage, Synovial Fluid and Membrane Pathological Findings in Serbian Mountain Horses Without Signs of Lameness

Abstract Osteoarthritis (OA) in horses often affects the metacarpal/metatarsophalangeal (MCP/MTP) joints and its detection in the early stages is crucial for effective management. I t was hypothesized that the extent of cartilage damage positively correlates with synovial membrane (SM) and synovial fluid (SF) pathological findings in the MCP/MTP joints of Serbian mountain horses that transported heavy loads but did not show signs of lameness. The study was conducted on 32 MCP/MTP joints of eight horses between seven to 12 years of age. Horse limbs were transferred from the abattoir to the necropsy room and SF was sampled. Its appearance, total nucleated cell count (TNCC), mononuclear cell count (MNC), total proteins (TP), and haptoglobin (Hp) were determined. Samples of SM were collected from the dorsal palmar/plantar pouch for histology. A macroscopic examination of gross condylar pathology of the third metacarpal/metatarsal bone was performed with Indian Ink staining. Scoring was done based on Osteoarthritis Research Society International recommendations. SF was clear, pale yellow, and mostly fairly viscous. Half of the samples had TNCC above, and all had TP within the reference range. Hp values were below the reference range and were omitted from further analyses. TNCC correlated with MNC (ρs=0.81, P&lt;0.001), microscopic (ρs=0.62, P=0.003) and macroscopic scores (ρs=0.47, P=0.008). In addition, MNC correlated with macroscopic scores (ρs=0.40, P=0.03). All pathological findings were mild and their correlation indicated that these processes are interrelated and that could be ascribed to early OA.

Degradomics defines proteolysis information flow from human knee osteoarthritis cartilage to matched synovial fluid and the contributions of secreted proteases ADAMTS5, MMP13 and CMA1 to articular cartilage breakdown

ObjectivesProteolytic cartilage extracellular matrix breakdown is a major mechanism of articular cartilage loss in osteoarthritis (OA) pathogenesis. We sought to determine the overlap of proteolytic peptides in matched knee OA cartilage and synovial fluid on a proteome-wide scale to increase the prospective biomarker repertoire and to attribute proteolytic cleavages to specific secreted proteases. DesignMatched human knee OA cartilage and synovial fluid (n=5) were analyzed by N-terminomics using Terminal Amine Isotopic Labeling of Substrates (TAILS), comprising labeling and enrichment of protein N-termini, high-resolution mass spectrometry and positional peptide mapping. Donor non-OA articular cartilage was digested with CMA1, MMP13 or ADAMTS5, and TAILS was used to identify cleavage sites, which were matched against cartilage and synovial fluid degradomes. ResultsOf over 20,000 cleaved peptides in the combined OA cartilage and synovial fluid degradomes, 677 peptides, originating from 153 proteins, were present in all cartilage and synovial fluid samples. CMA1, MMP13 and ADAMTS5 digestion of cartilage identified numerous cleavage sites for each protease and distinct cleavage site preferences. Peptides resulting from the activities of these proteases were detected in OA cartilage and synovial fluid. ConclusionsProteolytic fragments from both cartilage and circulating proteins are detectable by synovial fluid degradomics. CMA1, MMP13 and ADAMTS5 activity profiles in cartilage are distinct from each other and the previously determined HtrA1 profile. This work expands the proteolytic biomarker space for OA investigation, suggests that multiple, diverse proteases contribute to cartilage destruction, and demonstrates that their specific contributions can each be defined by multiple biomarkers.

Quantification of macrophage activity in knee synovial tissue using [18F]FEPPA positron emission tomography

ObjectiveInflammation in knee osteoarthritis (OA) is mediated primarily by synovial tissue macrophages, but non-invasive measurement of macrophage activity in vivo is a challenge. Activated macrophages markedly increase expression of the translocator protein (TSPO). In the context of other diseases TSPO-PET using the [18F]FEPPA tracer (binding to TSPO) has been reported to be an effective method for imaging macrophages in vivo. The goal of this study was to validate the use of [18F]FEPPA PET radiotracer to accurately measure macrophage activation in knee synovial tissue. DesignTen participants with late-stage OA scheduled for knee replacement surgery were recruited. Prior to surgery, 5 of the participants underwent a clinical [18F]FEPPA PET/MRI scan and the standard uptake value (SUV) in the suprapatellar synovial region was calculated. Suprapatellar synovial tissue samples were collected from all participants at the time of surgery and were imaged ex vivo with [18F]FEPPA autoradiography. Tracer uptake was compared to TSPO antibody immunostaining in serial tissue sections. The correlation between the [18F]FEPPA uptake and gold standard TSPO fluorescent intensity was measured. RESULTSThe autoradiography [18F]FEPPA signal in the synovial lining was correlated with the TSPO signal in the. lining macrophages measured by immunohistochemistry (r = 0.81, p = 0.0040, CI [0.37, 0.95]). Similarly, PET/MRI scans demonstrated similar correlation between SUV in synovial tissues in vivo and in situ TSPO signal in lining macrophages {r = 0.90, p = 0.083, CI [0.086, 0.99]) Conclusion[18F]FEPPA uptake in knee synovial tissue is strongly correlated to the expression of TSPO in synovial lining macrophages, suggesting that clinical [18F]FEPPA PET/MRI may be a valid measure of true macrophage activity in synovial tissues in knee OA. [18F]FEPPA may be an effective tool to assess macrophage activation both ex vivo and in vivo, and should be investigated further to assess its performance and measurement properties in different clinical contexts of knee OA including disease states and responses to treatment.