## 2 Materials and Methods  
### Samples  
The sample size used in the current study was based on the available number of samples from the original project that had two arms: investigating serum and synovial fluid samples from client-owned dogs with OA secondary to naturally occurring degenerative (non-traumatic) cranial-cruciate ligament (CrCL) tears in one or both knees and controls [8; 9]. The presence of CrCL tears and OA changes in the OA group dogs had beenconfirmed intraoperatively by inspection of the joint via arthotomy or arthroscopy. The controls were otherwise healthy adult dogs with no orthopedic abnormalities and with both knees free of gross abnormalities upon evaluation immediately after euthanasia for reasons unrelated to the study. Evaluation of the knee joint in control dogs was via an opening of the knee joint and evaluation of the joint, including cartilage surfaces, ligaments, menisci, synovium, and joint capsule. Briefly, venous blood samples were collected, and serum was separated and saved in 0.5-1 mL aliquots in cryovials (Nalgene Cryogenic tubes, VWR International, Batavia, IL, USA) and preserved at \(-80\)\({}^{\circ}\)C until batch analysis. Synovial fluid samples had been collected for the original study using an aseptic technique from the knees of dogs with a cranial-cruciate ligament tear in the OA group under general anesthesia immediately prior to surgical intervention to treat knee instability (i.e., tibial plateau leveling osteotomy), and from healthy knees in the control group immediately after euthanasia. The synovial samples were also saved in 0.5 mL aliquots in cryovials and preserved at \(-80\)\({}^{\circ}\)C until batch analysis. After the completion of the original project, the unused aliquots of serum and synovial samples were maintained in \(-80\)\({}^{\circ}\)C storage for a minimum of five years.  
The available sample inventory was reviewed for OA and control samples with adequate volume left for analysis. For the OA group, the serum and synovial fluid samples were those obtained prior to any surgical intervention. If more than one aliquoted sample was available, the clearest was selected (i.e., none to minimal blood contaminated for synovial fluid and none to minimal hemolysis for serum samples). Only one serum sample per dog was selected. In the OA group, even if both knees were affected, only one sample from one knee per dog was included in the study. In the control group, both healthy knees of each dog had been sampled, but preferentially, only one sample from each dog was included.  
The age of samples at the time of the first spectral analysis was calculated based on the date the sample was obtained from the dog and the time when the sample was thawed and the first spectral analysis was performed. Samples from this initial storage period (i.e., analyzed within a year after being acquired) are referred to as \"short-term storage\" samples. The age of samples at the time of the second spectral analysis after being in storage for a minimum of 5 years was calculated based on the date of sample acquisition from the dog and the time when the sample was run for the second time. Samples from the longer storage period (>1 year) are referred to as \"long-term storage samples\". The interval between measurements for the short-term and long-term storage was calculated based on the dates the MIR spectra of each sample were obtained.