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Higher synovial fluid white blood cell count in patients with knee osteoarthritis and metabolic syndrome
S84
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
general MMP activity was highest after 24 hours. Hyaluronidase treatment only interfered with MMP3 activity measurements but SF matrix effects were observed for all assays and need to be taken into account when quantifying biomarkers. 129 ITRAQ PROTEIN PROFILE ANALYSIS OF SYNOVIAL FLUID FROM OSTEOARTHRITIS PATIENTS WITH DIFFERENT AND REMARKABLE FEATURES OF CLINICAL SYMPTOM AND SYNOVITIS PATIENTS X. yuankun, Sr. y, S. Quan z, Z. Kaiwei z. y Peking Univ., Beijing, China; z GuiYang Colledge of Triditional Chinese Med., GuiYang, China Purpose: Osteoarthritis (OA) is a chronic musculoskeletal disorder that affect the joints with high prevalence rates. Clinical symptoms of OA are various and the experiences of OA patients for certain physical characteristics are different. Nevertheless, specific markers or certain specialty of molecular components in synovia fluid that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes of osteoarthritis remain largely unknown. Dissecting the protein aspects of osteoarthritis might significantly contribute to improving their early diagnosis and clinical management. Methods: The proteomic profiling of synovial fluid obtained from osteoarthritis and synovitis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. We compared quantitative and qualitative changes in proteomes of synovial fluid that occurred in 3 groups of OA patients with different and remarkable features (hot, cold, wet) of clinical symptom and 1 group of synovitis patients. 15 Patients diagnosed with OA were included in the study from 12 Nov. 2011 to 16 Oct. 2012, and 5 patients with synovitis
were diagnosed with synovitis excluding intra-articular infection, trauma and rheumatoid arthritis from 13 Nov. 2012 to 3 Jan. 2013. Results: In the iTRAQ analysis, 251 proteins were detected, and 246 of the 251 proteins were detected in all samples. Of all 251 proteins in Gene Ontology, 151 proteins with Gene Ontology functional annotation were detected in 3 Ontologies. We employed an iTRAQ approach followed by high resolution mass spectrometry to analyze the synovial fluid proteome from patients with osteoarthritis. Conclusions: We believe that the proteins detected in this study provide insights into our knowledge regarding the pathophysiology of osteoarthritis and contribute to better identification of biomarkers for early diagnosis and clinical management. 130 HIGHER SYNOVIAL FLUID WHITE BLOOD CELL COUNT IN PATIENTS WITH KNEE OSTEOARTHRITIS AND METABOLIC SYNDROME s, C. Orellana, J. Calvet, N. Navarro, M. García-Manrique, J. Grataco M. Larrosa. Hosp. Parc Taulí Univ.ri, Sabadell, Spain Purpose: Different studies have related knee osteoarthritis (OA) with a higher prevalence of classical cardiovascular risk factors and of metabolic syndrome. These patients would present with greater pain and disability levels and greater OA severity. Frequently these patients present with signs of synovial effusion. Objective: To evaluate the relationship between the synovial fluid white blood cell count and the classical cardiovascular risk factors, the metabolic syndrome and its individual components in patients with knee osteoarthritis and synovial effusion. Methods: Transversal study including consecutively women aged 50 to 80 years with symptomatic knee OA, Kellgren-Lawrence I-IV with significant synovial effusion. Demographic data and history of hypertension (HT), dyslipidemia, obesity or diabetes mellitus (DM) were collected. Blood samples were taken to determine basal glycemia, total, HDL- and LDL-cholesterol, insulinemia and glycated hemoglobin. Patients were evaluated for the presence of metabolic syndrome by strict NCEP-ATP-III criteria. All patients underwent joint aspiration to confirm non-inflammatory synovial fluid without microcrystals and to perform a joint cell count. Results: One hundred and fourteen patients (94 women and 20 men) were analysed. The average age was of 68.5 and 64.7 years and the symptoms duration was 48.5 and 36 months in women and men, respectively. Percentages of each cardiovascular risk factors were for women and men: HTA 53.2% and 70%, dyslipidemia 51.1% and 55%, obesity 56.4% and 50%, DM 7.4% and 20%, and for the metabolic syndrome were of 38.3% and 45%, respectively, which meant greater frequencies than expected by age and gender in all cases, except for DM in which the percentage was the expected. Regarding joint cell count, women with metabolic syndrome had a higher number of synovial fluid white blood cells compared with women without metabolic syndrome (median 218.4 (155.3e299.1) and 111.7 (81.4e149.8), respectively (p ¼ 0.004). Furthermore, the joint white blood cell count increased as more individual components of the metabolic syndrome were present in female patients (p ¼ 0.024). However no differences were found in men. A history of DM was also associated with higher white blood cell in synovial effusions of women with knee OA (p ¼ 0.013), whereas only dyslipidemia was associated with joint white blood cell count in men (p ¼ 0.04). Conclusions: Higher white blood cell counts were found in synovial effusions of female patients with knee OA who had metabolic syndrome, and the number of cells increased as more individual components of the metabolic syndrome were present in patients. These facts
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
were not found in men, although the smaller sample size prevent from drawing clear conclusions. 131 ADIPONECTIN SERUM LEVELS AND DISEASE SEVERITY IN THE STR/ ORT MOUSE MODEL OF OSTEOARTHRITIS R. Giambelli, S.K. Bernini, A. Grotti, R. Cavagnoli, B. Barbetta, P. Garofalo, T. Zanelli, G. Caselli, L.C. Rovati. Rottapharm Biotech Srl, Monza, Italy Purpose: Adiponectin, an adipokine secreted from adipose tissue into the bloodstream, is involved in the modulation of different metabolic processes including glucose uptake and fatty acid oxidation. This adipokine acts via two receptors, AdipoR1 and AdipoR2, expressed mainly in the skeletal muscle and liver, respectively, but also in chondrocytes. Adiponectin signal transduction by AdipoR1 and AdipoR2 involves the activation of many signaling molecules like Peroxisome proliferatoractivated receptor gamma (PPARg) and 5' AMP-activated protein kinase (AMPK). Unlike other adipokines, whose production is increased with obesity, adiponectin serum levels decrease with increasing weight. The STR/Ort mouse is a model of spontaneous osteoarthritis (OA) that shows a human-like hyperlipidemic condition and is characterized by a very early pathology development. Furthermore, these animals have serum levels of adiponectin much lower than other mouse strains. It was suggested that systemic (plasma) and local (synovial fluid) adipokine levels would be associated with cartilage degeneration and synovial inflammation, linking obesity and adiposity with inflammation and pathology in OA patients. Moreover, a down-regulation of adiponectin receptors was recently reported in human OA chondrocytes. These observations indicate a possible protective role for adiponectin in the development of osteoarthritis. The purpose of our study was to establish a possible relationship between OA development and hypoadiponectinemia in this model. Methods: STR/Ort and CBA mice were recruited at 6 months (26 weeks) of age and euthanized. Serum was obtained from blood samples taken from vena cava at sacrifice, using Microvette® CB 300 capillary tubes (Sarstedt). Knee joints were collected, processed for histology and blindly scored according to both Mankin’s and OARSI methods. Adiponectin levels were evaluated from serum samples by a commercially available kit (Bertin Pharma, cat. A05187). Correlation between the OARSI score and adiponectin levels was examined using Spearman‘s rank correlation test. Results: Adiponectin serum levels in STR/Ort mice at 26 weeks of age were significantly lower compared to CBA mice at the same age (12.5mg/ ml ± 0.65 and 23.9mg/ml ± 0.98 respectively). We observed a significant negative correlation (p ¼ 0.007) between adiponectin levels and weight in STR/Ort mice but not in CBA mice. Among the 6 CBA mice tested, 10 of the 12 knees examined showed no signs of OA (mean OARSI score ¼ 0.3 and Mankin’s score ¼ 0.3). Conversely, in the 17 STR/Ort mice only 1 of 34 knees examined showed no signs of OA, while all the other knees showed high level of cartilage damage (mean OARSI score ¼ 14.1 and Mankin’s score ¼ 7.9), as revealed in all histopathological parameters evaluated. There was a negative correlation between adiponectin levels and disease severity (as measured by OARSI score), reaching statistical significance (p ¼ 0.04) when considering the less damaged knee. Conclusions: This study, while confirming that adiponectin serum levels in STR/Ort mice are significantly lower than in a normal strain like CBA at the same age, showed for the first time a negative correlation between adiponectin and disease severity in STR/Ort mice. These results suggest that decreased adiponectin may be associated with OA development and/or progression in this animal model. Spearman Coefficient of Correlation (Rho) between systemic levels of adiponectin and OARSI Score in the less damaged and more damaged knee joint in STR/Ort mice
Less damaged More damaged *p< 0.05 **p< 0.01
Grade
Stage
OARSI Score
0.28 0.15
0.62** 0.51**
0.50* 0.14
S85
132 A NOVEL SEROLOGICAL TYPE III COLLAGEN A DISINTEGRIN AND METALLOPROTEINASE WITH THROMBOSPONDIN MOTIFS (ADAMTS) DEGRADATION MARKER FOR ASSESSMENT OF THE EXTRACELLULAR MATRIX REMODELING IN PATIENTS WITH OSTEOARTHRITIS A.-C. Bay-Jensen y, K. Kjaer-Pedersen z, L. Arendt-Nielsen z, O. Simonsen x, Q. Zheng k, D.J. Leeming y, M.A. Karsdal y. y Nordic BioSci. A/S, Herlev, Denmark; z SMI, AAlborg Univ., AAlborg, Denmark; x AAlborg Hosp., AAlborg, Denmark; k Nordic BioSci. China, Beijing, China Purpose: ADAMTS’ (A Disintegrin And Metalloproteinase with Thrombospondin Motifs) play an important role in the turnover of cartilage through the generation of the two well-described neoepitopes TEGE and ARGS. Less described is the action of ADAMTS’ on other joint-related matrix proteins, such as type III collagen. Type III collagen in upregulated in both cartilage and synovium in OA, especially in the presence of pro-inflammatory cytokines. Thus the aim was to identify ADAMTS generated neo-epitope of type III collagen and to develop a competitive enzyme-linked immunosorbent assay (ELISA) for detection of such neo-epitope, C3A. Furthermore to test the association of the neo-epitope with OA clinical characteristic. Methods: Mass spectrometric (LC-MS/MS) analysis of purified type III collagen degraded by ADAMTSs revealed a type III collagen fragment generated by ADAM-TS-1, 4-, 8. Monoclonal antibodies were raised against this neo-epitope for ELISA development. Patient samples 216samples 216 patients with different degrees of knee pain (maximal pain during the last 24 hours rated on a visual analog scale [VAS]) (VAS 0-100) and 64 controls (VAS 0-9) were recruited. Patients were separated into 3 groups: VAS 10 to 39 (N ¼ 81), VAS 40 to 69 (N ¼ 70), and VAS 70 to 100 (N ¼ 65). WOMAC and Lequesne scores were collected. Patients were separated into 2 groups: low VAS pain 40. Results are shown as mean ± SEM. Differences between mean values were compared by non-parametric Mann-Whitney’s t-test for twotailed observations. Area Under the Receiver Operating Characteristic (AUROC) was calculated. Results: Technical performance: The human and rat sequence 486’GAPGFRGPAG’49 in the alpha 1 chain of type III collagen generated by ADAMTS -1, -4, -8 was identified by LC-MS amongst several epitopes. The native reactivity of this Mab was high against both human serum (90% inhibition). The lower limit of detection (LDL) for the assay was 0.76ng/mL. Dilution recovery was within 100 ± 15% for human serum (mean 90 and 71%) and human EDTA and heparin plasma (mean 96 and 86% respectively). The inter- and intra-assay variation was below 10%. ADAMTS-4 seems to generate more of the fragment compared to ADAM-TS -1 and -8. No cross-reactivity was seen to the elongated synthetic peptide sequence that extended one amino acid at the neo-epitope site. Association with OA: Serum C3A levels was significantly associated with the pain recorded by VAS (b ¼ -4.4, p ¼ 0.0082), WOMAC (b ¼ 0.6, p ¼ 0.0073) and Lequesne (0.4, p ¼ 0.0011) after adjustment for age, BMI and gender. There no were association between C3A and WOMAC function or KL grade. Patients with low pain and KL 1 had significantly higher levels of C3A than patients with low pain levels and KL 3 (fig. 1). The AUC for separating the two groups (1 vs. 3) was 0.69 (p<0.05), whereas it was 0.71 (ns) for the demographics. The full model provided and AUC of 0.75 (p<0.05). There were no difference in the C3A levels in patients mild to severe structural OA all with high pain (category 4 to 6). Conclusions: We developed a novel biomarker measuring the human type III collagen neo-epitopes derived from the activity of ADAMTS'. We tested C3A in a cross-sectional OA study and found that serum levels of C3A was inversely correlated to pain measures indicating that this could be a biomarker of symptomatic OA. Furthermore we found that patients with a VAS pain score of less than 40 mm and a mild degree (OA sub-category 1) could be separated from those with a severe OA (OA sub-category 3) with an AUC of 75%. C3A may be a biomarker for identification of those with minimum symptomatic knee OA, but with early and potentially progressing OA.
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
general MMP activity was highest after 24 hours. Hyaluronidase treatment only interfered with MMP3 activity measurements but SF matrix effects were observed for all assays and need to be taken into account when quantifying biomarkers. 129 ITRAQ PROTEIN PROFILE ANALYSIS OF SYNOVIAL FLUID FROM OSTEOARTHRITIS PATIENTS WITH DIFFERENT AND REMARKABLE FEATURES OF CLINICAL SYMPTOM AND SYNOVITIS PATIENTS X. yuankun, Sr. y, S. Quan z, Z. Kaiwei z. y Peking Univ., Beijing, China; z GuiYang Colledge of Triditional Chinese Med., GuiYang, China Purpose: Osteoarthritis (OA) is a chronic musculoskeletal disorder that affect the joints with high prevalence rates. Clinical symptoms of OA are various and the experiences of OA patients for certain physical characteristics are different. Nevertheless, specific markers or certain specialty of molecular components in synovia fluid that might help in early diagnosis, monitoring disease progression and in improving therapeutic outcomes of osteoarthritis remain largely unknown. Dissecting the protein aspects of osteoarthritis might significantly contribute to improving their early diagnosis and clinical management. Methods: The proteomic profiling of synovial fluid obtained from osteoarthritis and synovitis patients was carried out by using iTRAQ labeling followed by high resolution mass spectrometry analysis. We compared quantitative and qualitative changes in proteomes of synovial fluid that occurred in 3 groups of OA patients with different and remarkable features (hot, cold, wet) of clinical symptom and 1 group of synovitis patients. 15 Patients diagnosed with OA were included in the study from 12 Nov. 2011 to 16 Oct. 2012, and 5 patients with synovitis
were diagnosed with synovitis excluding intra-articular infection, trauma and rheumatoid arthritis from 13 Nov. 2012 to 3 Jan. 2013. Results: In the iTRAQ analysis, 251 proteins were detected, and 246 of the 251 proteins were detected in all samples. Of all 251 proteins in Gene Ontology, 151 proteins with Gene Ontology functional annotation were detected in 3 Ontologies. We employed an iTRAQ approach followed by high resolution mass spectrometry to analyze the synovial fluid proteome from patients with osteoarthritis. Conclusions: We believe that the proteins detected in this study provide insights into our knowledge regarding the pathophysiology of osteoarthritis and contribute to better identification of biomarkers for early diagnosis and clinical management. 130 HIGHER SYNOVIAL FLUID WHITE BLOOD CELL COUNT IN PATIENTS WITH KNEE OSTEOARTHRITIS AND METABOLIC SYNDROME s, C. Orellana, J. Calvet, N. Navarro, M. García-Manrique, J. Grataco M. Larrosa. Hosp. Parc Taulí Univ.ri, Sabadell, Spain Purpose: Different studies have related knee osteoarthritis (OA) with a higher prevalence of classical cardiovascular risk factors and of metabolic syndrome. These patients would present with greater pain and disability levels and greater OA severity. Frequently these patients present with signs of synovial effusion. Objective: To evaluate the relationship between the synovial fluid white blood cell count and the classical cardiovascular risk factors, the metabolic syndrome and its individual components in patients with knee osteoarthritis and synovial effusion. Methods: Transversal study including consecutively women aged 50 to 80 years with symptomatic knee OA, Kellgren-Lawrence I-IV with significant synovial effusion. Demographic data and history of hypertension (HT), dyslipidemia, obesity or diabetes mellitus (DM) were collected. Blood samples were taken to determine basal glycemia, total, HDL- and LDL-cholesterol, insulinemia and glycated hemoglobin. Patients were evaluated for the presence of metabolic syndrome by strict NCEP-ATP-III criteria. All patients underwent joint aspiration to confirm non-inflammatory synovial fluid without microcrystals and to perform a joint cell count. Results: One hundred and fourteen patients (94 women and 20 men) were analysed. The average age was of 68.5 and 64.7 years and the symptoms duration was 48.5 and 36 months in women and men, respectively. Percentages of each cardiovascular risk factors were for women and men: HTA 53.2% and 70%, dyslipidemia 51.1% and 55%, obesity 56.4% and 50%, DM 7.4% and 20%, and for the metabolic syndrome were of 38.3% and 45%, respectively, which meant greater frequencies than expected by age and gender in all cases, except for DM in which the percentage was the expected. Regarding joint cell count, women with metabolic syndrome had a higher number of synovial fluid white blood cells compared with women without metabolic syndrome (median 218.4 (155.3e299.1) and 111.7 (81.4e149.8), respectively (p ¼ 0.004). Furthermore, the joint white blood cell count increased as more individual components of the metabolic syndrome were present in female patients (p ¼ 0.024). However no differences were found in men. A history of DM was also associated with higher white blood cell in synovial effusions of women with knee OA (p ¼ 0.013), whereas only dyslipidemia was associated with joint white blood cell count in men (p ¼ 0.04). Conclusions: Higher white blood cell counts were found in synovial effusions of female patients with knee OA who had metabolic syndrome, and the number of cells increased as more individual components of the metabolic syndrome were present in patients. These facts
Abstracts / Osteoarthritis and Cartilage 24 (2016) S63eS534
were not found in men, although the smaller sample size prevent from drawing clear conclusions. 131 ADIPONECTIN SERUM LEVELS AND DISEASE SEVERITY IN THE STR/ ORT MOUSE MODEL OF OSTEOARTHRITIS R. Giambelli, S.K. Bernini, A. Grotti, R. Cavagnoli, B. Barbetta, P. Garofalo, T. Zanelli, G. Caselli, L.C. Rovati. Rottapharm Biotech Srl, Monza, Italy Purpose: Adiponectin, an adipokine secreted from adipose tissue into the bloodstream, is involved in the modulation of different metabolic processes including glucose uptake and fatty acid oxidation. This adipokine acts via two receptors, AdipoR1 and AdipoR2, expressed mainly in the skeletal muscle and liver, respectively, but also in chondrocytes. Adiponectin signal transduction by AdipoR1 and AdipoR2 involves the activation of many signaling molecules like Peroxisome proliferatoractivated receptor gamma (PPARg) and 5' AMP-activated protein kinase (AMPK). Unlike other adipokines, whose production is increased with obesity, adiponectin serum levels decrease with increasing weight. The STR/Ort mouse is a model of spontaneous osteoarthritis (OA) that shows a human-like hyperlipidemic condition and is characterized by a very early pathology development. Furthermore, these animals have serum levels of adiponectin much lower than other mouse strains. It was suggested that systemic (plasma) and local (synovial fluid) adipokine levels would be associated with cartilage degeneration and synovial inflammation, linking obesity and adiposity with inflammation and pathology in OA patients. Moreover, a down-regulation of adiponectin receptors was recently reported in human OA chondrocytes. These observations indicate a possible protective role for adiponectin in the development of osteoarthritis. The purpose of our study was to establish a possible relationship between OA development and hypoadiponectinemia in this model. Methods: STR/Ort and CBA mice were recruited at 6 months (26 weeks) of age and euthanized. Serum was obtained from blood samples taken from vena cava at sacrifice, using Microvette® CB 300 capillary tubes (Sarstedt). Knee joints were collected, processed for histology and blindly scored according to both Mankin’s and OARSI methods. Adiponectin levels were evaluated from serum samples by a commercially available kit (Bertin Pharma, cat. A05187). Correlation between the OARSI score and adiponectin levels was examined using Spearman‘s rank correlation test. Results: Adiponectin serum levels in STR/Ort mice at 26 weeks of age were significantly lower compared to CBA mice at the same age (12.5mg/ ml ± 0.65 and 23.9mg/ml ± 0.98 respectively). We observed a significant negative correlation (p ¼ 0.007) between adiponectin levels and weight in STR/Ort mice but not in CBA mice. Among the 6 CBA mice tested, 10 of the 12 knees examined showed no signs of OA (mean OARSI score ¼ 0.3 and Mankin’s score ¼ 0.3). Conversely, in the 17 STR/Ort mice only 1 of 34 knees examined showed no signs of OA, while all the other knees showed high level of cartilage damage (mean OARSI score ¼ 14.1 and Mankin’s score ¼ 7.9), as revealed in all histopathological parameters evaluated. There was a negative correlation between adiponectin levels and disease severity (as measured by OARSI score), reaching statistical significance (p ¼ 0.04) when considering the less damaged knee. Conclusions: This study, while confirming that adiponectin serum levels in STR/Ort mice are significantly lower than in a normal strain like CBA at the same age, showed for the first time a negative correlation between adiponectin and disease severity in STR/Ort mice. These results suggest that decreased adiponectin may be associated with OA development and/or progression in this animal model. Spearman Coefficient of Correlation (Rho) between systemic levels of adiponectin and OARSI Score in the less damaged and more damaged knee joint in STR/Ort mice
Less damaged More damaged *p< 0.05 **p< 0.01
Grade
Stage
OARSI Score
0.28 0.15
0.62** 0.51**
0.50* 0.14
S85
132 A NOVEL SEROLOGICAL TYPE III COLLAGEN A DISINTEGRIN AND METALLOPROTEINASE WITH THROMBOSPONDIN MOTIFS (ADAMTS) DEGRADATION MARKER FOR ASSESSMENT OF THE EXTRACELLULAR MATRIX REMODELING IN PATIENTS WITH OSTEOARTHRITIS A.-C. Bay-Jensen y, K. Kjaer-Pedersen z, L. Arendt-Nielsen z, O. Simonsen x, Q. Zheng k, D.J. Leeming y, M.A. Karsdal y. y Nordic BioSci. A/S, Herlev, Denmark; z SMI, AAlborg Univ., AAlborg, Denmark; x AAlborg Hosp., AAlborg, Denmark; k Nordic BioSci. China, Beijing, China Purpose: ADAMTS’ (A Disintegrin And Metalloproteinase with Thrombospondin Motifs) play an important role in the turnover of cartilage through the generation of the two well-described neoepitopes TEGE and ARGS. Less described is the action of ADAMTS’ on other joint-related matrix proteins, such as type III collagen. Type III collagen in upregulated in both cartilage and synovium in OA, especially in the presence of pro-inflammatory cytokines. Thus the aim was to identify ADAMTS generated neo-epitope of type III collagen and to develop a competitive enzyme-linked immunosorbent assay (ELISA) for detection of such neo-epitope, C3A. Furthermore to test the association of the neo-epitope with OA clinical characteristic. Methods: Mass spectrometric (LC-MS/MS) analysis of purified type III collagen degraded by ADAMTSs revealed a type III collagen fragment generated by ADAM-TS-1, 4-, 8. Monoclonal antibodies were raised against this neo-epitope for ELISA development. Patient samples 216samples 216 patients with different degrees of knee pain (maximal pain during the last 24 hours rated on a visual analog scale [VAS]) (VAS 0-100) and 64 controls (VAS 0-9) were recruited. Patients were separated into 3 groups: VAS 10 to 39 (N ¼ 81), VAS 40 to 69 (N ¼ 70), and VAS 70 to 100 (N ¼ 65). WOMAC and Lequesne scores were collected. Patients were separated into 2 groups: low VAS pain 40. Results are shown as mean ± SEM. Differences between mean values were compared by non-parametric Mann-Whitney’s t-test for twotailed observations. Area Under the Receiver Operating Characteristic (AUROC) was calculated. Results: Technical performance: The human and rat sequence 486’GAPGFRGPAG’49 in the alpha 1 chain of type III collagen generated by ADAMTS -1, -4, -8 was identified by LC-MS amongst several epitopes. The native reactivity of this Mab was high against both human serum (90% inhibition). The lower limit of detection (LDL) for the assay was 0.76ng/mL. Dilution recovery was within 100 ± 15% for human serum (mean 90 and 71%) and human EDTA and heparin plasma (mean 96 and 86% respectively). The inter- and intra-assay variation was below 10%. ADAMTS-4 seems to generate more of the fragment compared to ADAM-TS -1 and -8. No cross-reactivity was seen to the elongated synthetic peptide sequence that extended one amino acid at the neo-epitope site. Association with OA: Serum C3A levels was significantly associated with the pain recorded by VAS (b ¼ -4.4, p ¼ 0.0082), WOMAC (b ¼ 0.6, p ¼ 0.0073) and Lequesne (0.4, p ¼ 0.0011) after adjustment for age, BMI and gender. There no were association between C3A and WOMAC function or KL grade. Patients with low pain and KL 1 had significantly higher levels of C3A than patients with low pain levels and KL 3 (fig. 1). The AUC for separating the two groups (1 vs. 3) was 0.69 (p<0.05), whereas it was 0.71 (ns) for the demographics. The full model provided and AUC of 0.75 (p<0.05). There were no difference in the C3A levels in patients mild to severe structural OA all with high pain (category 4 to 6). Conclusions: We developed a novel biomarker measuring the human type III collagen neo-epitopes derived from the activity of ADAMTS'. We tested C3A in a cross-sectional OA study and found that serum levels of C3A was inversely correlated to pain measures indicating that this could be a biomarker of symptomatic OA. Furthermore we found that patients with a VAS pain score of less than 40 mm and a mild degree (OA sub-category 1) could be separated from those with a severe OA (OA sub-category 3) with an AUC of 75%. C3A may be a biomarker for identification of those with minimum symptomatic knee OA, but with early and potentially progressing OA.