Quantitation of the cellular content of saliva and buccal swab samples

Abstract

Buccal swabs and saliva are the two most common oral sampling methods used for medical research. Often, these samples are used interchangeably, despite previous evidence that both contain buccal cells and blood leukocytes in different proportions. For some research, such as epigenetic studies, the cell types contributing to the analysis are highly relevant. We collected such samples from twelve children and twenty adults and, using Papanicolaou staining, measured the proportions of epithelial cells and leukocytes through microscopy. To our knowledge, no studies have compared cellular heterogeneity in buccal swab and saliva samples from adults and children. We confirmed that buccal swabs contained a higher proportion of epithelial cells than saliva and that children have a greater proportion of such cells in saliva compared to adults. At this level of resolution, buccal swabs and saliva contained similar epithelial cell subtypes. Gingivitis in children was associated with a higher proportion of leukocytes in saliva samples but not in buccal swabs. Compared to more detailed and costly methods such as flow cytometry or deconvolution methods used in epigenomic analysis, the procedure described here can serve as a simple and low-cost method to characterize buccal and saliva samples. Microscopy provides a low-cost tool to alert researchers to the presence of oral inflammation which may affect a subset of their samples. This knowledge might be highly relevant to their specific research questions, may assist with sample selection and thus might be crucial information despite the ability of data deconvolution methods to correct for cellular heterogeneity.

Distinctions between sex and time in patterns of DNA methylation across puberty

“…Finally, we compared our results for overlap with previously reported methylation quantitative trait loci (mQTLs) in buccal cells 31 , which contain a similar composition of epithelial cells as saliva 32 . We assessed the overlap of our significant sites that surpassed biological threshold with significant mQTLs within a distance of 1 Mb in a recent study of buccal cells in a sample of males and females (mean age 7.5 years) 31 .…”

DRD4 methylation as a potential biomarker for physical aggression: An epigenome‐wide, cross‐tissue investigation

“…Of note, the Smith et al study examined saliva, not buccal swabs. However, these two DNA sources typically contain the same mixture of cell‐types (i.e., buccal epithelial cells and leukocytes), although proportions of buccal epithelial cells tend to be higher in buccal swabs (Theda et al, ). Furthermore, our sensitivity analyses for cell‐type heterogeneity further supported the comparability of DRD4 DNAm patterns between buccal swabs and saliva (see Supporting Information Methods).…”

Effect of short-term prescription opioids on DNA methylation of the OPRM1 promoter

“…A peculiar challenge we faced was that the site of sample collection was also the site of surgery. Saliva has a highly heterogeneous cellular makeup and is estimated to constitute about ~45% epithelial cells, and about ~55% leukocytes from circulating blood [40]. The main goal of the study was to detect the effect of short-term and comparatively low-dose opioids, while accounting for the larger perturbation caused by surgery.…”

“…The reference-free method estimated four major cell types. Although the saliva certainly has more than just 4 types of cells [40], the classification into 4 broad groups likely reflects the limitation in the in-silico approach to resolve finer differences between cellular subtypes. Cell 1 most likely represented the granulocyte population (chiefly neutrophils), which constitutes the most abundant leukocyte subtype in circulating blood, and responsible for innate immunity and acute inflammatory response.…”