## 3 Results  
There were 52 serum and 51 synovial fluid samples available from the 70 dogs originally in the OA group, while 49 serum and 52 synovial fluid samples were available from the 50 dogs in the control group. In the control group, one dog did not have adequate serum samples available. Two of the control dogs did not have adequate synovial fluid samples; therefore, the additional three samples were selected from the contralateral knees of three of the control dogs that had adequate samples from both knees. In the OA group, all 70 dogs had radiographic evidence of OA changes (i.e., joint effusion, intra-articular and peri-articular osteophylosis). The knee OA changes were present unilaterally in 63 (30 right, 33 left) and bilaterally in seven dogs. In cases with bilateral OA secondary to CrCL tears, only the more clinically affected site that was operated on was sampled. The median (IQR) of time in a frozen state before the first run (short-term storage) and the second run (long-term storage) were 1.1 (1.1) and 6.8 (1.1) years, respectively. The time interval between the first and second measurement was 5.7 (0.03) years. At first, MSCA was used to evaluate whether there could be any significant difference in the spectral profiles of the samples after short- or long-term storage, and serum samples were considered. When the overall variability in the preprocessed spectral profiles was partitioned according to the ANOVA scheme, it was found that only 1.61% of the total variability among the serum spectra was ascribable to the effect of aging (between-samples differences accounted for 77.23% of the total variance, while the remaining part is residual variation, associated with random error). The variability observed between the two time points was not impacted by the type of sample (OA versus control). Although the relatively low amount of spectral variance was associated with the differences between short-term and long-term storage, permutation testing indicated that these differences were statistically significant (\(p<0.0001\)). Principal component analysis (PCA) of the effect matrix resulting from the ANOVA decomposition indicated that long-term stored serum samples were characterized, on average, by a lower intensity of most of the peaks. The same analysis was then conducted on the synovial fluid samples. Also, in this case, when the overall variability in the preprocessed spectral profiles was partitioned according to the ANOVA scheme, it was found that the largest part of the spectral variance was due to between-samples differences (85.54%), with only 2.98% of the total variability among the spectra corresponding to the effect of aging. Permutation testing indicated that the effect of sample ageing on the spectral signature, though relatively small, could still be deemed statistically significant (\(p<0.0001\)). Interpretation of the observed difference in synovial-fluid spectra by PCA of the corresponding effect matrix confirmed that long-term storage results, on average, in less intense absorption peaks. Based on the outcomes of this exploratory analysis, which indicated that sample ageing could result in small but significant differences in the spectral signature, in a second stage of the study we wanted to verify if the analysis of long-term stored samples could still provide reliable predictions and, if so, whether models built on the two set of samples (short- and long-term stored) resulted in the same set of putative markers. First, the spectra collected on the serum samples were considered. The quality of the predictive models was evaluated by considering the figures of merit calculated on the outer loop of the rDCV procedures, which correspond to the classification performances on samples not used for model building or optimization. Moreover, since rDCV uses resampling, this allows for the estimation, for each figure of merit, of not only a single value but also a confidence interval (in particular, in the remainder of the text, 95% confidence level will always be considered).