Detection of Novel Gingival Crevicular Fluid Biomarkers Using Multiplex Bead Immunoassays

Objectives: Multiplex Bead Immunoassays (Luminex) allows for the concomitant examination of a large numbers of host-derived analytes in gingival crevicular fluid (GCF). Kits targeting biomarkers of potential relevance to periodontal disease pathogenesis have become recently available, including markers of: angiogenesis, bone turnover, cancer, oxidative stress, skin biology, CD8+ T cell activity, and extracellular matrix degradation. Thus, the objectives of the study were to: 1) measure levels of 41 host-derived biomarkers in GCF; 2) identify which analytes are differentially expressed in periodontally healthy and periodontitis sites.
Methods: We collected GCF samples from the mesiobuccal sites of up to 32 teeth per subject from 19 periodontally healthy and 15 periodontitis subjects. We pooled 8 samples from 3 distinct categories of sites: 1) shallow sites from healthy subjects (SH; n=19), 2) shallow sites from periodontitis subjects (SP; n=15) and 3) deep sites from periodontitis subjects (DP; n=15). Samples from each category were eluted separately and analyzed using 8 different Luminex kits. We determined significance of differences between groups using nonparametric ANCOVA adjusting for age, gender and ethnicity and adjusted for multiple comparisons using False Discovery Rate (FDR) set at 1%. We also ran logistic regression to estimate the area under the ROC curve (AUC) and determine the diagnostic accuracy of the different biomarkers.
Results: Of the 41 analytes only MMP-9 could not be quantified due to values above the standard curve. GCF perforin, Growth Differentiation Factor 15 (GDF-15) and Tartrate-resistant acid phosphatase type 5 (TRAP5) were statistically significantly higher in DP compared to SH sites. The AUC for the GCF biomarkers that best discriminated between DP and SH were: 0.88, 0.86 and 0.85 for GDF-15, TRAP5 and YKL-40, respectively.
Conclusions: All analytes examined were detectable in GCF. Biomarkers previously associated with cancer metastasis (GDF-15, TRAP5 and YKL-40) and the cytolytic mediator perforin (derived from CD8+ T cells) might be associated with periodontitis.