Eve B. Schwartz

Use of salivary biomarkers in biobehavioral research: cotton-based sample collection methods can interfere with salivary immunoassay results

In a series of studies, we evaluated the susceptibility of immunoassays for saliva biomarkers to interference effects caused by cotton materials used to absorb saliva during sample collection. Salivary assay results for testosterone, DHEA, progesterone, and estradiol are artificially high, and for sIgA artificially low, when samples are collected using cotton absorbent materials. In contrast, results for salivary cortisol, DHEA-S, and cotinine are not affected by the use of cotton collection methods. The order of individual results from samples collected using cotton versus no-cotton methods for certain markers is not conserved, suggesting that for some biomarkers this collection method can be a significant source of unsystematic error. It was shown, using DHEA as an example, that the cotton interference effect is of sufficient magnitude to attenuate the association between serum and saliva levels. Awareness of this issue is critical to ensure measurement validity in future studies and analyses of archived samples collected using cotton materials.

Integration of salivary biomarkers into developmental and behaviorally-oriented research: Problems and solutions for collecting specimens

Saliva has been championed as a diagnostic fluid of the future. Much of the attention that saliva receives as a biological specimen is due to the perception that the nature of sample collection is quick, uncomplicated, and non-invasive. In most cases, this perception matches reality; however, in some special circumstances and populations collecting saliva can be unexpectedly difficult, time consuming, and may not yield sufficient sample volume for assay. In this report, we review the nature and circumstances surrounding some of these problems in the context of developmental science and then present alternatives that can be used by investigators to improve the next generation of studies. We expect our findings will ease the burden on research participants and assistants, reduce the rate of missing values in salivary data sets, and increase the probability that salivary biomarkers will continue to be successfully integrated into developmental and behaviorally-oriented research.

Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration, Hemastix reagent strips to detect hemoglobin, and an immunoassay for transferrin. The presence of blood in saliva immediately after microinjury was confirmed by all methods. Hemoglobin and transferrin levels remained elevated over baseline for at least 30 min. Levels of salivary testosterone increased over baseline and remained elevated for 30 min in response to microinjury. Microinjury induced change in salivary testosterone was more closely associated with the change in transferrin than hemoglobin levels or discoloration ratings. On average, levels of salivary dehydroepiandrosterone (DHEA) did not increase in response to microinjury. However, individual differences in microinjury induced change in DHEA were associated with discoloration ratings. Salivary cortisol levels, on average, were neither responsive to microinjury, nor were individual differences in cortisol change associated with blood contamination measures. Neither diurnal nor gender-related differences in baseline hormone levels predicted the impact of blood leakage on quantitative salivary measurements. The findings suggest ecologically valid minor-to-moderate level microinjuries to the oral cavity have negligible effects on the measurement of salivary cortisol, but may be important to quantify and control when assessing other hormones especially testosterone.

Assessing dehydroepiandrosterone in saliva: a simple radioimmunoassay for use in studies of children, adolescents and adults

While salivary assays for some hormones are widely used, the availability of assays for salivary DHEA is limited. By adapting a commercially available radioimmunoassay serum kit, we developed a reliable, efficient and sensitive measure of DHEA in saliva that does not require separation or extraction. The minimum detection limit was 4.0 pg/ml. Intra-assay coefficients of variation (CV%) were on average 4.05, and inter-assay CVs averaged 9.70. Method accuracy, determined by spike recovery, and linearity, determined by serial dilution, averaged 99.55 and 92.03%. Levels in matched serum and saliva samples showed strong linear relationships for adult males and females. Specific guidelines are developed for sample collection, storage, and preparation procedures. Reference ranges for salivary DHEA levels are provided for 64 children ages 8-11, 96 adolescents ages 12-17 and 48 adults ages 30-45. Salivary DHEA levels are shown to reflect developmental, gender and diurnal differences.

Integrating the measurement of salivary α-amylase into studies of child health, development, and social relationships

To advance our understanding of how biological and behavioral processes interact to determine risk or resilience, theorists suggest that social developmental models will need to include multiple measurements of stress-related biological processes. Identified in the early 1990s as a surrogate marker of the sympathetic nervous system component of the stress response, salivary-amylase has not been employed to test biosocial models of stress vulnerability in the context of child development until now. In this report, we describe a standard assay that behavioral scientists can use to improve the next generation of studies and specific recommendations about sample collection, preparation, and storage are presented. More importantly, four studies are presented with mother–infant dyads (N= 86), preschoolers (N= 54), children (N = 54), and adolescents (N = 29) to illustrate individual differences in stress-related change in α-amylase levels, that patterns of α-amylase stress reactivity distinctly differ from those measured by salivary cortisol, and associations between individual differences in α-amylase and social relationships, health, negative affectivity, cognitive/academic/behavior problems, and cardiovascular reactivity. We conclude that the integration of measurements of the adrenergic component of the locus ceruleus/autonomic (sympathetic) nervous system, as indexed by salivary α-amylase, into the study of biosocial relationships may extend our understanding of child health and development to new limits.

Assessing Estradiol in Biobehavioral Studies Using Saliva and Blood Spots: Simple Radioimmunoassay Protocols, Reliability, and Comparative Validity

We developed simple, reliable, and highly sensitive assay modifications of commercially available radioimmunoassay kits to measure estradiol in saliva and blood spot specimens. The saliva assay has average intra- and interassay coefficients of variation (CV) of 6.45 and 9.01%, with average analytical and serial dilution recoveries 100.65 and 89.25%. The blood spot assay has average intra- and interassay CVs of 7.57 and 8.22%, with analytical and serial dilution recoveries of 80.50 and 108.50%. The analytical sensitivity ranges of the saliva (0.25-7.50 pg/ml) and blood spot (2. 00-375 pg/ml) assays are sufficient to determine levels in the majority of pre- and postpubertal males and females. Blood spot assay results are correlated with serum estradiol levels for adult males, r (17) = 0.73, and females, r (18) = 0.96. In contrast, the serum-saliva correlation is only modest for adult females, r (14) = 0.60, and not significant for adult males. Substitution of blood spot assay results for serum values underestimates the known serum estradiol-behavior correlation by only 3.45%, whereas substitution of saliva assay results for serum values underestimates the association by 37.55%. The findings have important implications for the use and potential misuse of noninvasive measures of estradiol in studies of health and human development.

Oxytocin is not a valid biomarker when measured in saliva by immunoassay.

The integration of oxytocin (OT) into behavioral science seems to hold considerable promise for advancing our understanding of human health and development but methodological issues restrict the measurement of OT in large studies, in everyday social settings, or when repeated sampling is required. Measuring OT in saliva could overcome many of these limitations. In this paper, we rigorously evaluate the feasibility of doing so. A series of experiments leads to the conclusion that saliva does not contain oxytocin in measurable amounts, and that OT is not a valid salivary biomarker when measured by currently available immunological methods. Levels of immuno-reactive OT in saliva are primarily due to non-specific interference with antibody-antigen binding. We can state with a high degree of certainty that measurement of OT in saliva does not yield meaningful indices of individual differences or intra-individual change.

Blood contamination and the measurement of salivary progesterone and estradiol.

The impact of blood leakage due to microinjury to the oral cavity on the measurement of salivary reproductive hormones was examined. Saliva samples were collected before, immediately after, and then every 15 min for 1 h following vigorous tooth brushing. Blood in saliva was quantified by visual inspection of discoloration and an immunoassay for transferrin. Levels of progesterone increased, and levels of estradiol decreased, in saliva after microinjury. These changes were present only immediately after microinjury. The findings have implications for the use of salivary assays in biobehavioral research, short-term dynamic investigations, pharmacokinetic analyses, and studies of chronobiological changes in progesterone and estradiol levels.