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, 11 (12), e0166976

Reliability of Potential Pain Biomarkers in the Saliva of Healthy Subjects: Inter-Individual Differences and Intersession Variability


Reliability of Potential Pain Biomarkers in the Saliva of Healthy Subjects: Inter-Individual Differences and Intersession Variability

Eva M Sobas et al. PLoS One.


Aim: Salivary cortisol, α-amylase (sAA), secretory IgA (sIgA), testosterone, and soluble fraction of receptor II of TNFα (sTNFαRII) could serve as objective pain measures, but the normal variability of these potential biomarkers is unknown.

Patients & methods: Saliva was collected with the passive secretion method from 34, pain-free subjects in two single samples at least 24 hours apart. Biomarker variation and intersession reliability were assessed with the intraclass correlation coefficient (ICC). Also, we calculated the within-subject standard deviation (Sw) and the reproducibility (2.77 × Sw) of intersession measures.

Results: Salivary cortisol, sAA, sIgA, testosterone, and sTNFαRII yielded the following ICCs: 0.53, 0.003, 0.88, 0.42 and 0.83, respectively. We found no statistically significant systematic differences between sessions in any biomarker except for testosterone, which showed a decrease on the second day (p<0.001). The reproducibility for salivary cortisol, sAA, sIgA, testosterone, and sTNFαRII were 0.46 ng/ml, 12.88 U/ml, 11.7 μg/ml, 14.54 pg/ml and 18.29 pg/ml, respectively. Cortisol, testosterone and TNFαRII measurement variability showed a positive correlation with the magnitude (p<0.002), but no relationship was found for sAA and sIgA.

Conclusions: Salivary sIgA and sTNFαRII show a remarkable good reproducibility and, therefore, could be useful as pain biomarkers. When using the passive secretion method, intersession variations in salivary sIgA of more than 11.7 μg/ml may reflect true biomarker change. In the case of sTNFαRII this will depend of the magnitude. The estimates herein provided should help investigators and clinicians differentiate actual biomarker modification from measurement variability.

Conflict of interest statement

Dr. Roberto Reinoso is employed by Vision I+D. This does not alter our adherence to PLOS ONE policies on sharing data and materials.


Fig 1
Fig 1
Bland-Altman graphs showing the intersession reproducibility for each biomarker: (A) sAA, (B) sIgA, (C) Testosterone, (D) Cortisol, and (E) sTNFαRII. The solid lines represent the upper and the lower LoA (limits of agreement): crude 95% LoA are depicted in A and B, and the 95% back transformed LoA after the 10 base log-transformation are shown as functions of the mean of the two measurements in C, D y E (X denotes the corresponding biomarker mean). Dashed line represents the mean difference value between 2nd and 1st salivary collections, and shaded area the magnitude between this mean difference value and zero.

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Grant support

Vision I+D is a spin-off of University of Valladolid (our University). It provided the salary of Dr. Roberto Reinoso. Esteve Laboratories provided the salary of Sebastian Videla and had contributed with an unrestricted grant for the consumables used in this research. Neither Vision I+D nor Esteve had played any role in the design of this study. Roberto Reinoso and Sebastian Videla did not play any role in the study design, data collection and analysis. Sebastian Videla has contributed as external advisor, also participating in the preparation of the manuscript and Roberto Reinoso has analyzed some biomarkers, participating also in the preparation of the manuscript.