## 3 Results  
The concentrations of BTM on a salivary matrix of 20 subjects were evaluated after the identification and exclusion of n 3 subjects with aberrant values reported by the statistical evaluation. The assessment for suspected Tukey right-sided outliers was used as low values below the limit of quantification were previously excluded. The distribution of the data showed a parametric distribution only for the P1 NP data; therefore, the calculation of the biological variability parameters for PTHrP and for TRACP 5b were calculated. The results are reported in Table 1. Particularly, it was verified that the salivary concentrations of P1 NP, TRAcP 5b and PTHrP were higher than the LoD of the analytical method. The Boxplot of the distribution of the values of the BTM evaluated shows the statistical summary of the concentration of the individual determinations and confirms the absence of any outside values included (Figure 1).  
The analytical variability (CVA) of BTM assays, was determined by calculating the between-run imprecision, in accordance with the procedure described in Clinical and Laboratory Standards Institute (CLSI) document EP15-A2 for P1 NP, TRACP 5b and PTHrP were 5.1%, respectively (average concentration 2.0 \(\upmu\)g/L); 4.5% (mean concentration 1.50 U/L) and 9% (mean concentration 0.52 ng/mL).  
Estimates of CVA and components of biological variation for all subjects and derived individuality index (II, RCV) of salivary BTM assays are shown in Table 2. Mean biological within-subject variance (CVI) was higher for PTHrP (23.0%) and minimal for P1 NP (5.6%) while the biological variance between subjects (CVG) was higher for TRAcP 5b (35.0%). The within-subject biological variability (CVI) and between-subject BV (CVG) estimates were not calculated for sex (male and female) and on age subgroups for all measurands because the number of subjects included in the study did not allow for a correct layering. The individual mean value and the absolute range of values for each individual included in the study were reported in the multiple-variable graphs. The graphs of P1 NP and TRACP 5b confirmed the high individuality of P1 NP and TRACP 5b. In fact, the concentrations of the analytes in the single subject did not cover the entire range of distribution of the values (Figure 2). The RCV was maximum for PTHrP (68.4%) and minimum for P1 NP (21.2%) indicating that the significance of the change in concentrations between the two measurements was different for the BTM evaluated for laboratory test interpretation. P1 NP and TRACP 5b, based on the values obtained for the biological variability, showed high individuality indicating that for the interpretation of the results of these biomarkers the comparison with any reference intervals calculated on the saliva was of little use. For the evaluation of the individuality index (II), the cut-off reported in the literature of 0.60 was considered suitable.  
\begin{table}
\begin{tabular}{l l l l l} \hline
**Variable** & **P1 NP (\(\mu\)g/L)** & **TRACP-5b (U/L)** & **PTHrP (ng/mL)** \\ \hline Lowest value & 1.20 & 1.00 & 0.33 \\ Highest value & 3.50 & 2.90 & 0.90 \\ Arithmetic mean & 2.44 & 1.62 & 0.60 \\ Median & 2.40 & 1.40 & 0.57 \\ Standard deviation & 0.44 & 0.56 & 0.12 \\ D’Agostino-Pearson test for & accept Normality & reject Normality & reject Normality \\ Normal distribution & (\(p\) = 0.7126) & (\(p\) = 0.0001) & (\(p\) = 0.0412) \\ Suspected outliers Tukey & None & None & None \\ right-sided (alpha-level 0.05) & & & & \\ \hline \end{tabular}
\end{table}
Table 1: Distribution of the obtained values: P1 NP exhibits normal distribution while TRACP 5b and PTHrP are not normally distributed.  
Figure 1: Boxplot of the distribution of the values of the BTM of the concentration of the individuality determination: there are not outside values that may be included.