Salivary Collection for Periodontitis Biomarkers: A Review of Pre-analytical Variables and Recommendations for Standardization
Keywords:
periodontitis; salivary biomarkers; saliva collection; specimen handling; standardizationAbstract
Background: Periodontitis is a prevalent inflammatory disease affecting tooth-supporting structures. While clinical examination remains the diagnostic gold standard, saliva is an appealing medium for detecting biomarkers that may aid in screening and monitoring. However, differences in collection, handling, and storage can markedly influence biomarker stability and detection.
Objective: To summarize current evidence on how salivary collection and preanalytical factors affect biomarker integrity in periodontitis.
Methods: A narrative review was conducted of English-language studies published between January 2020 and July 2025, identified through PubMed, Scopus, and Web of Science. Studies were included if they measured salivary biomarkers relevant to periodontitis and reported collection or handling protocols. Data on collection methods, sample source, timing, participant preparation, and storage were synthesized qualitatively.
Results: Unstimulated whole saliva collected by passive drooling or spitting was the most common method due to its simplicity and ability to preserve biomarker integrity. Stimulated saliva altered protein profiles but offered more stable circadian patterns. Cottonbased absorbent devices reduced analyte recovery, while glandspecific sampling was rarely used. Biomarker levels varied with sampling time and participant preparation. Proper handling—immediate centrifugation, aliquoting, and ultralow temperature storage—was critical, as warmer storage conditions and repeated freeze–thaw cycles accelerated biomarker degradation.
Conclusion: Methodological heterogeneity in salivary biomarker studies limits reproducibility. Standardizing unstimulated collection protocols, controlling pre-collection variables, and adopting consistent handling and storage practices are essential to advance the clinical utility of salivary biomarkers in periodontitis.
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1. Papapanou PN, Sanz M, Buduneli N, et al. Periodontitis: Consensus report of workgroup 2 of the 2017 World Workshop on the Classification of Periodontal and Peri‐Implant Diseases and Conditions. J Periodontol. 2018;89(Suppl 1):S173-82. DOI: 10.1002/JPER.17-0721.
2. Tonetti MS, Greenwell H, Kornman KS. Staging and grading of periodontitis: Framework and proposal of a new classification and case definition. J Periodontol. 2018;89(Suppl 1):S159-72. DOI: 10.1002/JPER.18-0006.
3. Fidyawati D, Masulili SLC, Iskandar HB, et al. Clinical and Radiographic Parameters for Early Periodontitis Diagnosis: A Comparative Study. Dent J. 2024;12(12):407. DOI: 10.3390/dj12120407.
4. Ebersole JL, Hasturk H, Huber M, et al. Realizing the clinical utility of saliva for monitoring oral diseases. Periodontol 2000. 2024;95(1):203-19. DOI: 10.1111/prd.12581.
5. Ng TK, Udeh‐Momoh C, Lim MA, et al. Guidelines for the standardization of pre‐analytical variables for salivary biomarker studies in Alzheimer's disease research: An updated review and consensus of the Salivary Biomarkers for Dementia Research Working Group. Alzheimers Dement. 2025;21(2):e14420. DOI: 10.1002/alz.14420.
6. Kim HJ, Park DH, Han SH, et al. Optimal storage time and temperature of human oral samples to minimize microbiome changes: A scoping review. Jpn Dent Sci Rev. 2024;60:220-31. DOI: 10.1016/j.jdsr.2024.05.001.
7. Jasim H, Ghafouri B, Ernberg M. The diurnal pattern of cytokines, chemokines and growth factors in human saliva—a pilot study. Front Dent Med. 2024;5:1420081. DOI: 10.3389/fdmed.2024.1420081.
8. Albagieh H, Alshehri AZ, Alduraywishi AS, et al. Evaluation of Salivary Diagnostics: Applications, Benefits, Challenges, and Future Prospects in Dental and Systemic Disease Detection. Cureus. 2025;17(1):e77520. DOI: 10.7759/cureus.77520.
9. Sachelarie L, Stefanescu CL, Murineanu RM, et al. Role of Salivary Biomarkers IL-1β and MMP-8 in Early Detection and Staging of Periodontal Disease. Medicina. 2025;61(4):760. DOI: 10.3390/medicina61040760.
10. Relvas M, Mendes-Frias A, Gonçalves M, et al. Salivary IL-1β, IL-6, and IL-10 Are Key Biomarkers of Periodontitis Severity. Int J Mol Sci. 2024;25(15):8401. DOI: 10.3390/ijms25158401.
11. Boynes SG, Sofiyeva N, Saw T, et al. Assessment of salivary matrix metalloproteinase (MMP8) and activated salivary matrix metalloproteinase (aMMP8) in periodontitis patients: a systematic review and meta-analysis. Front Oral Health. 2025;6:1444399. DOI: 10.3389/froh.2025.1444399.
12. Zalewska EA, Lawicka R, Grygorczuk P, et al. Importance of Metalloproteinase 8 (MMP-8) in the Diagnosis of Periodontitis. Int J Mol Sci. 2024;25(5):2721. DOI: 10.3390/ijms25052721.
13. Rakic M, Calciolari E, Grant MM, et al. Host Markers of Periodontal Diseases: Meta‐Analysis of Diagnostic Accuracy Studies. J Clin Periodontol. 2025;(Suppl 29):155-81. DOI: 10.1111/jcpe.14167.
14. Ali Alftaikhah SA, Issrani R, Alnasser M, et al. Salivary biomarkers in periodontitis: a scoping review. Cureus. 2023;15(12):e50207. DOI: 10.7759/cureus.50207.
15. Ebersole JL, Kirakodu SS, Zhang XD, et al. Salivary features of periodontitis and gingivitis in type 2 diabetes mellitus. Sci Rep. 2024;14(1):30649. DOI: 10.1038/s41598-024-77434-2.
16. Dolińska E, Wiśniewski P, Pietruska M. Periodontal Molecular Diagnostics: State of Knowledge and Future Prospects for Clinical Application. Int J Mol Sci. 2024;25(23):12624. DOI: 10.3390/ijms252312624.
17. Sukhera J. Narrative reviews: flexible, rigorous, and practical. J Grad Med Educ. 2022;14(4):414-7. DOI: 10.4300/JGME-D-22-00480.1.
18. Foratori-Junior GA, Ventura TMO, Grizzo LT, et al. Is there a difference in the proteomic profile of stimulated and unstimulated saliva samples from pregnant women with/without obesity and periodontitis? Cells. 2023;12(10):1389. DOI: 10.3390/cells12101389.
19. Agnello L, Giglio RV, Del Ben F, et al. Evaluation of core Biomarkers of Alzheimer's disease in saliva and plasma measured by chemiluminescent enzyme immunoassays on a fully automated platform. Sci Rep. 2024;14(1):16084. DOI: 10.1038/s41598-024-66923-z.
20. Zhao X, Chen X, Lu Y, et al. Saliva metabolomics: a non-invasive frontier for diagnosing and managing oral diseases. J Transl Med. 2025;23(1):582. DOI: 10.1186/s12967-025-06587-z.
21. Yeh CK, Christodoulides NJ, Floriano PN, et al. Current development of saliva/oral fluid-based diagnostics. Tex Dent J. 2010;127(7):651-61. PMCID: PMC3742318.
22. Li Y, Ou Y, Fan K, et al. Salivary diagnostics: Opportunities and challenges. Theranostics. 2024;14(18):6969-90. DOI: 10.7150/thno.100600.
23. López A, Estebala P, García-Ruíz E, et al. Biomarkers in Saliva to Assess the State of Periodontal Disease. In: Aitken-Saavedra J, Rojas-Alcayaga G, Braz-Silva PH. Salivary Analysis - Biomarkers, Clinical Applications, and Emerging Technologies [Working Title]. London: IntechOpen; 2025. DOI: 10.5772/intechopen.1010390.
24. Murray JM, Stone JE, Abbott SM, et al. A Protocol to Determine Circadian Phase by At‐Home Salivary Dim Light Melatonin Onset Assessment. J Pineal Res. 2024;76(5):e12994. DOI: 10.1111/jpi.12994.
25. Mortazavi H, Yousefi-Koma A-A, Yousefi-Koma H. Extensive comparison of salivary collection, transportation, preparation, and storage methods: a systematic review. BMC Oral Health. 2024;24(1):168. DOI: 10.1186/s12903-024-03902-w.
26. Teles FR, Chandrasekaran G, Martin L, et al. Salivary and serum inflammatory biomarkers during periodontitis progression and after treatment. J Clin Periodontol. 2024;51(12):1619-31. DOI: 10.1111/jcpe.14048.
27. Maar S, Czuni L, Hassve JK, et al. Technical considerations regarding saliva sample collection to achieve comparable protein identification and detection via one-and two-dimensional gel electrophoresis among humans. Heliyon. 2024;10(24):e40752. DOI: 10.1016/j.heliyon.2024.e40752.
28. Amadeu JK, Lemes AL, Schussel JL, et al. Effect of storage time and temperature on salivary total antioxidant capacity, total oxidant status, and oxidant stress index. Acta Stomatol Croat. 2019;53(2):119-24. DOI: 10.15644/asc53/2/3.
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