Assessment of Pain Biomarkers in Orthopedic Patients Undergoing Total Knee Arthroplasty

Brief name: Peripheral immune pain biomarker assessment in knee osteoarthritis Description: This is an observational study in which no therapeutic intervention is delivered. Participants are observed cross-sectionally with respect to pain, function, psychological measures (PROMs), and peripheral immune biomarkers (blood and synovial fluid samples). The study planned to enrol 3 groups of participants: 1) 20 patients awaiting total knee arthroplasty (TKA) including 20 painful knees with moderate to severe osteoarthritis (OA), 2) 20 controls, the healthy contralateral pain-free knee of these 20 patients awaiting TKA, and 3) 20 different healthy controls for venous blood measurements (described under "comparator"). This will result in 40 participants, and 60 knees in total: 20 painful knees with osteoarthritis that is awaiting TKA, 20 pain-free contralateral control knees of the same patients, 20 pain-free control knees of different patients. Preoperatively, at the pre-admission clinic 2-4 weeks before surgery, enrolled TKA-awaiting patients (n= 20) will receive routine venous blood sampling for elective surgery preparations at Noarlunga Hospital. At that same blood collection moment, blood will be collected for this study. Perioperatively, during the elective TKA surgery, synovial fluid will be collected from the painful knee receiving TKA surgery. Similarly, the contralateral pain-free knee will be punctuated for synovial fluid when the patient is still under narcosis. What is involved for participants: Participants will undergo a single study visit (healthy controls) or two study-related time points embedded within routine clinical care (TKA patients). Venous blood sampling: - Patients awaiting total knee arthroplasty (TKA): one venous blood sample (30 mL) collected preoperatively during the routine pre-admission clinic visit (2–4 weeks before surgery). Synovial fluid sampling (TKA patients only): - During the elective TKA procedure, synovial fluid (30 mL) is aspirated from the painful knee immediately after surgical exposure. - While the patient remains under general anaesthesia, synovial fluid (30 mL) is also aspirated from the contralateral pain-free knee. All sampling procedures are performed by credentialed clinical staff as part of, or in conjunction with, routine care. What is being observed / measured: - Peripheral immune responses measured in peripheral blood mononuclear cells and synovial fluid mononuclear cells, including interleukin-1 beta release following ex vivo Toll-like receptor 2 and 4 stimulation (painCELL assay). - Immune cell phenotyping using hyperspectral imaging of unstimulated blood and synovial fluid samples (painHS assay). - Clinical pain severity assessed using the Numeric Rating Scale (NRS). - Psychological status assessed using validated questionnaires: Hospital Anxiety and Depression Scale (HADS; approximately 5–10 minutes) Pain Catastrophizing Scale (PCS; approximately 5 minutes) - Functional outcomes assessed using: Oxford Knee Score (OKS) Knee Injury and Osteoarthritis Outcome Score (KOOS) Mode and setting of data collection: All assessments and sample collection occur face-to-face at Noarlunga Hospital/ Flinders Medical Centre in a hospital outpatient or operating theatre setting. Duration of observation: Each participant is observed cross-sectionally. Data collection occurs at a single time point for healthy controls, and over a short perioperative window for TKA patients (preoperative clinic visit and day of surgery). There is no longitudinal follow-up. Personalisation or adaptation: No intervention, dose adjustment, or personalisation applies, as this is an observational study. Intervention fidelity or adherence: Not applicable, as no intervention is delivered.