Quantifying blood leakage into the oral mucosa and its effects on the measurement of cortisol, dehydroepiandrosterone, and testosterone in saliva Katie T. Kivlighan, a Douglas A. Granger, a, * Eve B. Schwartz, b Vincent Nelson, b Mary Curran, b and Elizabeth A. Shirtcliff c a Behavioral Endocrinology Laboratory, Department of Biobehavioral Health, The Pennsylvania State University, University Park, PA 16802, USA b Salimetrics LLC, State College, PA 16804-0395, USA c Department of Psychology, University of Wisconsin, Madison, WI 53706-1696, USA Received 18 November 2003; revised 21 January 2004; accepted 22 January 2004 Available online 21 April 2004 Abstract 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, HemastixR 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. D 2004 Elsevier Inc. All rights reserved. Keywords: Blood contamination in saliva; Cortisol; Testosterone; Dehydroepiandrosterone Introduction Monitoring steroid hormones in saliva rather than in urine, blood spot, or serum specimens has several well- documented advantages for biobehavioral researchers (see Kirschbaum et al., 1992; Malamud and Tabak, 1993). Yet, as the number of studies assessing salivary hormones has increased, so has our understanding that special circum- stances involving the presence of exogenous cross-reacting substances in the mouth (i.e., infant formula, breast milk; Magnano et al., 1989), as well as characteristics of the process by which saliva flow is stimulated (i.e., citric acid containing powders, chewing gum; Schwartz et al., 1998) and collected (i.e., cotton-based absorbent material; Shirtcl- iff et al., 2001), have potential to influence assay validity. Ten years ago, the literature warned that when blood is present in the oral mucosa, quantitative estimates of hor- mones measured in saliva may be compromised (Malamud and Tabak, 1993). Given the rising popularity of the use of salivary biomarkers, it is somewhat surprising that little empirical research has specified the impact of this potential confound on measurement validity. In this study, we attemp- ted to fill this important knowledge gap. Blood and its components can leak into the oral mucosa as a result of microinjuries such as burns, abrasions, or cuts 0018-506X/$ - see front matter D 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.yhbeh.2004.01.006 * Corresponding author. Behavioral Endocrinology Laboratory, De- partment of Biobehavioral Health, The Pennsylvania State University, 315 E. Henderson Building, University Park, PA 16802. Fax: +1-814-863-7525. E-mail address: DAG11@PSU.EDU (D.A. Granger). www.elsevier.com/locate/yhbeh Hormones and Behavior 46 (2004) 39 – 46