2 (2007) 583–590

Physiology & Behavior 9

Integration of salivary biomarkers into developmental andbehaviorally-oriented research: Problems and solutions

for collecting specimens

Douglas A. Granger a,b,c,d,⁎, Katie T. Kivlighan e, Christine Fortunato a,c, Amanda G. Harmon a,b,Leah C. Hibel a,b, Eve B. Schwartz d, Guy-Lucien Whembolua a,b

a Behavioral Endocrinology Laboratory, The Pennsylvania State University, United Statesb Department of Biobehavioral Health, The Pennsylvania State University, United States

c Department of Biobehavioral Health, Human Development and Family Studies, The Pennsylvania State University, United Statesd Salimetrics LLC, State College, PA, United States

e Bloomberg School of Public Health, The Johns Hopkins University, United States

Received 23 August 2005; received in revised form 2 November 2006; accepted 2 May 2007

Abstract

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 theperception 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 sufficientsample volume for assay. In this report, we review the nature and circumstances surrounding some of these problems in the context ofdevelopmental science and then present alternatives that can be used by investigators to improve the next generation of studies. We expect ourfindings will ease the burden on research participants and assistants, reduce the rate of missing values in salivary data sets, and increase theprobability that salivary biomarkers will continue to be successfully integrated into developmental and behaviorally-oriented research.© 2007 Elsevier Inc. All rights reserved.

Keywords: Salivary cortisol; Testosterone; DHEA; Alpha-amylase; Saliva collection; Microsponge; Filter paper; Passive drool

1. Introduction

Saliva has been championed as a diagnostic fluid of thefuture [1]. Much of the attention saliva receives as a biologicalspecimen is due to the perception that the nature of samplecollection is quick, uncomplicated, and non-invasive [2]. Theliterature suggests that in most cases this perception matchesreality. Yet, in specific circumstances and populations, collect-ing saliva can be unexpectedly difficult, time consuming, andmay require considerable creativity (e.g., [3–5]) to gathersufficient sample volumes for assay. When sample collection is

⁎ Corresponding author. Behavioral Endocrinology Laboratory, Departmentof Biobehavioral Health, 315 Health and Human Development East, ThePennsylvania State University, University Park, PA 16802, United States.Tel.: +1 814 863 8402; fax: +1 814 863 7525.

E-mail address: dag11@psu.edu (D.A. Granger).

0031-9384/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.physbeh.2007.05.004

inadequate and assay protocols or laboratory technicians cannotaccommodate a partial sample, the missing data problemscreated can seriously compromise research, and hinderscreening and potential diagnostic agendas.

In this report, we review the nature of some of the uniqueproblems specific to the application of salivary biomarkers indevelopmental science and rigorously evaluate approaches tosaliva collection that have been used in an attempt to resolvesome of the difficulties with more traditional approaches. Wepresent alternatives that can be used by investigators, especiallyin special circumstances and with unique populations ofresearch participants, to improve the next generation of studies.We expect our findings will ease the burden on researchparticipants and research assistants, reduce the rates of missingvalues in salivary data, and consequentially increase theprobability of the successful integration of salivary biomarkersinto behaviorally-oriented research.

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2. Saliva collection: unexpected challenges

Contrary to popular belief, under certain circumstances, thecollection of saliva can be cumbersome, time consuming, andfrustrating for research participants and assistants. This appearsto be true for the very youngest and also the very oldest agegroup of research participants.

2.1. Early childhood

Saliva collection from full-term and preterm newborns, andinfants less than 3 months of age can be challenging [6]. Evenafter repeated or sustained collection efforts, the outcome isoften insufficient specimen volume (e.g., [7]). The problemappears to be that newborn infant's parotid glands have lowfluid production rates compared to older children and adults(e.g., [8]).

Saliva collection from older infants (3–18 months) has itsown unique set of challenges ranging from the children'sirregular sleep–wake cycles and frequent napping, to residuefrom liquids (formula, milk, juices) and food in infants' mouths,to young children's anxiety about strangers and compliancewith collection protocols. In a large-scale study (n=1193) of 6-and 15-month olds (The Family Life Project; FLP NICHD,PO1HD39667) we have documented the prevalence andcorrelates of these special circumstances. Briefly, the FLPemploys a developmental epidemiological design to studyfamilies that live in two of the four major US geographical areasof high child rural poverty—by design, low income (200%below the poverty line) and African American families wereover sampled. As part of the larger protocol, home visitorscollected and recorded field notes regarding saliva collectionproblems while collecting specimens before, 20 and 40 minafter infants participated in a series of tasks designed to elicitemotional reactivity. Content analyses [9] reveal that certainsaliva collection problems are more prevalent during earlyversus later infancy. During early infancy (5 to 10 months ofage,M=7.6 months), falling asleep and consuming breast milk,formula, and milk were the prevailing issues. Collecting salivafrom sleeping infants was often not possible or not permittedby primary caregivers, and resulted in “missingness” of salivaspecimens. The consumption of breast milk and formula hasthe potential to impact findings by contaminating specimenswith materials that cross-react (e.g., bovine hormones) withimmunoassays [10]. In contrast, during later infancy (14 to20 months of age, M=15.5 months), the prevalence of fallingasleep and milk product consumption decreased but corre-spondingly the consumption of other liquids (i.e., juices)increased. The consumption of acidic foods or beverages has thepotential to interfere with antibody binding in salivaryimmunoassays (e.g., [4]).

Equally important, the frequency of some of these problemsvaried by ethnicity and poverty status. In particular, Whiteinfants (n=688) and those from middle to upper class families(n=416) had a greater tendency to consume breast milk,formula, and milk (10.9% and 11.3%, respectively) thanAfrican American infants (n=500) or those from impoverished

or low-income (n=777) families (5.2% and 6.9%, respectively).Ethnicity and poverty status were also associated with infantnon-compliance to saliva collection protocols and resulted inhigh rates of “missingness.” Specifically, White infants andthose from middle to upper class families were more likely to benon-compliant than African Americans or those fromimpoverished or low-income families.

2.2. The oldest-old

On the opposite end of the developmental spectrum, col-lecting saliva samples from the oldest and most frail elderlyparticipants can also be unexpectedly difficult. Many facetsinvolved in the treatment and care of institutionalized elderly,such as disease states, cognitive status, immobility, fatigue,medications and hydration practices, may potentially affectthe ability to adequately assess salivary biomarkers. In a re-cent study of the oldest-old (n=116, 73% older than 85 years),as many as one third of all attempts to collect saliva failed toprovide a valid sample [11]. Xerostomia (dry mouth) was aparticular challenge affecting up to half of these nursing homeresidents. Indeed, of the 116 residents recruited at baseline,only 77 were able to provide valid samples and were includedin the follow-up. This disappointing completion rate occurreddespite a highly trained professional staff with extensivegeriatric nursing experience. Not surprisingly, sample collec-tion also took several times longer (10–15 min) than theliterature suggested (2 min). Hodgson et al. [11] investigatedwhether there were clinical differences between those able toprovide saliva samples and those who were non-compliant orprovided invalid samples and found no significant-meaningfuldifferences. Post-hoc analyses revealed that individuals tak-ing medications with diuretic properties (dehydration result-ing in limited saliva flow) were disproportionately highamong the group that presented the most challenge to salivacollection [11]. In retrospect, given that poly-pharmacy is thenorm rather than the exception among the oldest-old, andthere is a long list (n∼400) of over the counter and prescrip-tion medications with iatrogenic effects that include restric-tion of saliva flow [12], it is not surprising that collectingsufficient saliva volume can be challenging in this specialgeriatric population.

3. Consequences when saliva collection is problematic

A variety of potentially negative consequences occur whensaliva collection is ineffective. The most obvious is that missingvalues are created in data sets. Given that this phenomenon ispotentially associated with ethnicity, poverty, medication use,age, and perhaps patience and persistence of individual researchassistants, “missingness” is unlikely to be random. Non-randommissing data significantly complicates multivariate statisticalmodels of individual differences. For instance, this set ofproblems significantly minimized the sample size (n reducedfrom 116 to 77) in Hodgson et al.'s [11] study such that theywere unable to examine statistical models of moderation andmediation while controlling for all the necessary covariates. In

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short, missingness of samples restricts degrees of freedom andstatistical power.

More subtly, young children's behavioral reactivity tosample collection procedures has the potential to interferewith our ability to observe the relationships of interest. This isparticularly true when studying the effects of an acute event onintra-individual change in a stress-responsive salivary biomark-er (i.e., cortisol, alpha-amylase) over a short time interval. Inthis case, the intent of employing a “non-invasive”measure maybe moot because the specimen collection procedure iscontributing to a negative subjective experience held by theresearch participant. When this happens during the intervalbetween pre- and post-task salivary collections it has thepotential to confound a task-related change in the salivarybiomarker(s) assessed. Furthermore, this negative subjectiveexperience has the potential to effect subsequent saliva samplecollections when protocols call for repeated measures. Post-hocanalyses from the FLP revealed that a substantial portion of themissing data were from the 40-min post-stress task collectionpoint at both the 6- (n=64) and 15-month visit (n=80)compared to the baseline (n=0 and 16, respectively) and 20-min post-challenge (n=25 and 34, respectively) time points.

At another level, in the world of large project data collection(multi-site studies, program projects, and nationally represen-tative large-scale surveys) every second of time with studyparticipants is carefully scripted to maximize the amount ofinformation learned and minimize costs. In these instances,extra time with research participants to collect saliva specimensis directly linked to additional costs. Also, unexpected delays in,or unpredictable times for the completion of, saliva collectioncan create problems with a larger project's assessment schedule.This unpredictability can become a significant issue whenmulti-investigator projects are coordinating research agendasthat are “competing” for data collection time during a singleinterview session. In our experience, many large-scale, nationalsurveys have expressed interest but then elect not to includesalivary biomarkers in certain waves of data collection for thereasons described above.

4. All saliva collection devices do not perform the same: Acloser look

The most common strategy for saliva collection in studies ofadults involves the use of a cotton pledget (10×37 mm) toabsorb sample from the participant's mouth. Commerciallyavailable saliva collection devices often employ cotton absor-bent materials (e.g., Salivette, Sarstedt). Briefly, after 2–3 minin the oral cavity the cotton becomes saturated (appearingmatted or flattened) with saliva. It is then removed from thesubject's mouth, and saliva is expressed out of the cotton into acollection vial by centrifugation [13]. In most circumstances,this approach is convenient, simple, and has the capacity toyield 1.0 mL of whole saliva within a few minutes time. Thisoutcome, of course, depends on cooperative participants with asufficient saliva flow rate to make available enough specimento be absorbed. In studies of early child development (e.g.,[14,15], saliva collection has traditionally been collected using

sections of sterile braided cotton ropes (10×152 mm) availablethrough dental supply houses (e.g., Richmond, Charlotte, NC).The ropes are longer than actually needed to enable someone tohold onto the end of the braid and prevent children from chok-ing on or swallowing the material. Typically, only one end of therope is placed in the child's mouth. The saliva saturated end ofthe cotton rope is cut off with a pair of scissors, placed into thebarrel of a needleless syringe, and then compressed using theplunger to express saliva into a collection vial [14]. The cottonpledget from the Salivette device is occasionally used in studieswith children, but because of its short length it is a chokinghazard (American Academy of Pediatrics); therefore, the cottonpledget should not be used for children under age 6 years.

In situations or special populations of research participantswhere the volume of saliva available for collection is likely to besmall (e.g., newborns, frail elderly, or participants suffering withxerostomia), inherent features of the cotton absorbent materialscomplicate matters. Cotton absorbs fluid very efficiently andsmall liquid volume is quickly dispersed across a large surfacearea of the intertwined cotton fibers. When the surface area ofthe cotton used is large relative to the sample volume availableto be absorbed, the liquid can be so diffusely distributed in thefibers that, despite centrifugation or pressure, it is difficult torecover a sufficient test volume.

To illustrate this phenomenon, Harmon et al. [16] usedbraided cotton rope (Richmond, Charlotte, NC) or the cottonpledget from the Salivette device to absorb saliva in volumes of0.25, 0.50, 1.0, and 1.5 mL. The different saliva volumes werepipette into cryovials and the end of the absorbent materialswere held in the oral fluid for 1 min. After 1 min, all the liquid ineach sample was completely absorbed. Next, followingstandard procedures used in the field, the saturated end of thecotton rope was cut off, placed into the barrel of a 5-cm3

needleless syringe, saliva was expressed into a 2-mL cryovial(e.g., [14]), or the Salivette pledget was centrifuged [13]. Onaverage, the percent volume recoveries for the Salivette cottonpledget were 38.30%, 59.40%, 73.93%, and 82.58%, for initialsample volumes of 0.25, 0.50, 1.0, and 1.5 mL, respectively.The comparable percent recoveries were 15.45%, 31.50%,64.20%, and 74.42% for the cotton rope. Fig. 1 presents theaverage percent recoveries (and standard errors) of salivarecovered from cotton rope when much smaller volumes areavailable to be absorbed (range 25 μL to 400 μL). The findingsreveal that virtually no liquid volume is recovered using cottonabsorbent materials until the volume of sample available to beabsorbed exceeds 200 μL. These findings are consistent withthose of Herrington et al. [7] who reported a loss of 54% of thesamples collected in preterm infants due to lack of adequateamounts of saliva collected with cotton plugs.

Cotton absorbent materials have other well documentedrestrictions that also limit their use for saliva collection.Previous studies (e.g., [17]) show that samples collected usingthis approach can be accurately assayed for only a handful ofsalivary markers, such as cortisol, cotinine, and most recentlyalpha-amylase [18]. This list is short given the variety ofsalivary assays now available and efforts underway to developadditional assays in the future. The process of absorbing and

Fig. 1. Mean (standard error) percent volume recovered for saliva usingmicrosponge (hydrocellulose) and “Traditional Cotton Rope” sample collectionmethods [16].

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filtering saliva through cotton causes “interference” in immu-noassays for many salivary biomarkers. This phenomenonoccurs for salivary testosterone, sIgA, dehydroepiandrosterone,estradiol, progesterone, 17 OH Progesterone, and androstene-dione (e.g., [17,18,19]).

In summary, the use of cotton-based absorbent materials tocollect saliva can be problematic when the expected volume ofsaliva available to be absorbed is small, and can negativelyaffect the validity of many salivary assays when the saliva isfiltered through the cotton. We speculate that the interferencecaused by filtering saliva through cotton is due to either the(1) concentration of saliva due to the capture and retention ofwater molecules by the cotton, (2) release of material from thecotton (plant hormone-like substances) that cross-reacts ornonspecifically interfere with immunoassays, or (3) bindingand retention of molecules (e.g., sIgA) of interest by the cottonfibers. Regardless of the mechanism, investigators need to beaware of these potential problems when considering the use ofcotton absorbent materials to collect saliva samples (e.g.,[19]).

5. Solutions to problems with saliva collection

In an attempt to solve the problems that cotton absorbentmaterials create for saliva collection in studies of early child-hood, several possibilities have been explored. In this section,we first review attempts to stimulate saliva flow to increase thevolume of sample available to be absorbed as well as the re-duction of sample test volume requirements in immunoassays.Then we evaluate alternative sample collection strategies andcharacterize their relative advantages and disadvantages.

5.1. Stimulating saliva flow

The literature is full of creative approaches to increasesample volumes by stimulating saliva flow using a variety ofsubstances, such as various chewing gums [20,21], powderedsugar and drink mix crystals (e.g., [14,22]), and cotton swabsimpregnated with citric acid. While some recent modificationsof this basic approach (e.g., soaking cotton ropes in drink mix

and then dehydrating them) have been used effectively (e.g.,[3,23]), many materials used to stimulate saliva flow have thepotential, if not used minimally and consistently, to compromisesample integrity and negatively influence salivary assay perfor-mance. Schwartz et al. [4] showed that powdered drink mixcrystals used to stimulate saliva flow in most studies of earlychild development prior to 1998 increased saliva acidity. WhenpH drops below 4 (lower pH values indicate higher acidity), theantibody–antigen reaction necessary for accurate measurementof salivary biomarkers by immunoassay is compromised. Theresult is artificially high estimates, that do not dilute in parallelto the standard curve (i.e., indicating interference is present;[24]). Even if a small amount of material is used to stimulatesaliva flow, problems with pH can be created because thesmaller the sample volume collected the more concentrated thematerial will be in the sample assayed. In an effort to reduce thenegative consequences of these effects, sample pH can bescreened prior to or during the immunoassay process.

The concentrations of some biomarkers (e.g., sIgA) can alsobe subtly influenced by salivary flow rate (e.g., [25]).Stimulating saliva flow may require that concentration pervolume units need to be adjusted for flow rate (volume/min). Inour collective experience, many new investigators are caughtunaware by this fact and may not record flow rate. Anytime theoption of stimulating saliva flow is exercised, care should betaken to ensure that there are no iatrogenic effects on theperformance of the specific assays to be used prior to datacollection (e.g., [4,26]).

In direct response to concerns about the collection of suf-ficient sample volume, and in an attempt to prevent the need forusing materials to stimulate saliva flow, the immunodiagnos-tic community has redesigned assays specifically for use withsaliva and substantially reduced test volume requirements andbuffers than minimize problems associated with variability insample pH (e.g., Diagnostic Systems Laboratories, Webster,TX; IBL-Hamburg, Hamburg, Germany; Salimetrics, StateCollege, PA; Diagnostic Systems). Ten years ago the test vol-ume requirements for salivary cortisol, cotinine, and testoster-one ranged from 0.2 to 1.0 mL. Relatively high sample testvolumes were required because salivary assay protocols weresimple modifications of immunoassays design for use withserum (e.g., [27,28]). Given the low levels of most markers insaliva, these modifications almost always required increasingsaliva test volumes many times higher (5–10 fold) over thosespecified for use with serum. Specialized salivary immunoassayprotocols are now commercially available and often onlyrequire 25–50-μL test volumes (1/8th of an eye dropper drop).These protocols have eliminated much of the need to stimulatesaliva flow prior to sample collection in most circumstances.Despite these improvements, however, the collection of eventhese small volumes can at times still prove to be daunting—asnoted above.

5.2. “Passive” drool

The technical advances that eliminated large test volumesdo not address the limits of cotton pledgets with respect to

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interference in salivary immunoassays. That situation has beenaddressed in our own research by abandoning the cottonabsorbent material when objectives involve the assay of multiplesalivary markers. Instead, we have participants donate “wholesaliva” as follows: subjects are asked to imagine they are chewingtheir most favorite food, to close their eyes and imagine theyare eating that food item, and to slowly and gently move theirjaws as if chewing that food. The process of moving the jawas if chewing generates saliva, this saliva is then allowed to poolbriefly under the tongue before being gently “drooled” through ashort common plastic drinking straw into a collection vial.This procedure is capable of generating large sample volumes (1–5 mL) within 3–5 min.

Whole saliva collected by passive drool has the followingdistinct advantages, (1) enables a large sample volume to becollected, (2) minimizes the influence of substances used tocollect or stimulate saliva flow on immunoassays, (3) pro-duces a sample that can be assayed for multiple markers, and(4) allows unused sample to be frozen in an archive for futureassay without concern that there would be interference withthose assay protocols. Of course this procedure requires acompetent, compliant, aware (and awake), and capable re-search participant. While effective with children over 6 yearsof age, and correspondingly most adults, this alternative doeslittle to resolve the special circumstances with very youngchildren, any research participant who is sleeping, and the frailelderly. In those situations, it is still essential to have sometype of material that can be used to absorb saliva from thesubjects' mouths.

5.3. Filter paper

At least four reports suggest that collecting saliva on filterpaper is a viable option for use with newborns or the oldest-old[8,29,30]. Only two provide sufficient detail to evaluateprocedural steps, and only one affords sufficient informationon assay validation. First, Dombrowski et al. [8] used Whatmangrade 42 filter papers cut in 2.54×9.0-cm strips. They collectedsample from newborns by gently placing the filter paper on theanterior portion of the tongue and holding it in place until“sufficient” saliva was obtained. One centimeter was then cutfrom the saturated end of the paper (they assumed it contained0.1 mL of saliva), and placed into a 12×75 borosilicate glasstube with 1.0 mL of absolute ethanol. After mixing, the paperwas removed and discarded. The ethanol (containing thesalivary biomarkers of interest) is evaporated, and thecompletely dry sample was reconstituted in 0.5-mL buffer andthen assayed.

To our surprise, the salivary cortisol levels reported byDombrowski seemed to read very high. Out of curiosity weevaluated the procedure. First, we examined the assumption thatthe filter paper would absorb sample consistently betweenindividuals, then we evaluated if there was a difference in thelevel of cortisol estimated using the filter paper versus passivedrool (control) procedures. Nine adults were asked to rinsetheir mouths with water and wait 10 min before donating1.5 mL of saliva via passive drool (as above). Then, within

seconds they placed a Whatman grade 42 filter paper(2.54×9 cm) on their tongue for 20 s. Immediately thereafter,the volume of sample absorbed was estimated by weight, g/mL(cm3). On average, the paper absorbed 59 μL of saliva in 20 s.Despite standardizing the time of exposure in the mouth, therewas considerable range in the volume of saliva collected (52 μLor 13% below the mean, to 72 μL or 23% above the mean)between individuals.

Next, we followed the ethanol extraction proceduredescribed by Dombrowski et al. [8] and assayed the 9 matchedfilter paper and passive drool collected samples for cortisol. Onaverage, saliva collected using filter paper returned at least 5-fold higher (M=1.78 μg/dL; range 1.58 to 1.99 μg/dL) cortisolestimates than the passive drool condition (M=.26 μg/dL; range.16 to .55 μg/dL), t (8)=29.80, pb .0001. Individual differencesin the levels of salivary cortisol measured using the twocollection conditions were not conserved, r (7)= .43, ns. Theseresults were disappointing, and confirm our suspicions aboutthe very high cortisol values reported in samples collected usingfilter paper [8].

Most recently, Neu et al. [31] described a filter paper col-lection procedure for use with newborns. The protocol differsfrom that of Dombrowski et al. [8] in that they used a differentextraction method and a commercially available cortisol assayspecifically designed for use with saliva. They report the col-lection protocol in sufficient detail as to be repeated from theirpaper, and provide extensive validation data. The protocol ap-pears to have acceptable recovery of whole saliva from thefilter paper, and acceptable linearity of dilution (×2 and ×4 butnot below). They report saliva collection was very efficient andeffective with only 2% of the samples lost due to inadequatewetting of the filters. These findings are encouraging, and theuse of this alternative collection approach seems promising.

In summary, the amount of saliva absorbed by filter paperwill depend on (1) the amount of saliva available, (2) timethe filter paper is left in the mouth, (3) where in the mouththe paper is placed, and (4) individual differences in thecomposition of the saliva. With respect to the latter, in ourexperience, saliva samples can be very viscous or stringy, andothers can contain substantial particulate matter. Given thesesources of variability, it would seem very challenging todetermine a precise quantitative (concentration per volume)metric for salivary markers collected with this method as wecannot determine an accurate estimate of the volume absorbedwhen the paper is placed in the subject's mouth. Nevertheless,in circumstances when it is not possible to gather samplesby other means, filter paper may represent a viable option.Following the protocol presented by Neu et al. [31], thismethod's limitations appear minor compared to the alternativeof no sample at all.

5.4. Hydrocellulose microsponge

Microsponge devices are used during ocular surgery towick tears away from the eye and keep the area under opera-tion dry. They typically have a short plastic applicator shaft(0.4×5.2 mm) that serves as a handle. Affixed to the end of the

Table 1Means (standard error) and correlation coefficients comparing microsponge(hydrocellulose) and passive drool saliva collection methods for five commonsalivary biomarkers

Microsponge Control R

DHEA (pg/mL) 96.82 65.54⁎ .655(41.12) (13.95)

Cortisol (μg/dL) 0.132 0.132 .937⁎⁎⁎

(0.19) (0.20)α-amylase (U/mL) 151.07 153.48 .998⁎⁎⁎

(34.22) (35.03)Testosterone (pg/mL) 77.12 138.24⁎⁎ .307

(18.86) (29.52)Cotinine (ng/mL) 188.78 182.17 .996⁎⁎⁎

(20.26) (20.35)

Note: ⁎pb .05, ⁎⁎pb .01, ⁎⁎⁎pb .001; all Ns=10, except N=5 for dehydroe-piandrosterone (DHEA).

Fig. 2. Microsponge (hydrocellulose) sample collection approach absorbssufficient saliva volume for assay within 20–30 s, and its maximum volume isachieved within 60 s.

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handle is a small (0.7×1.8) arrowhead shaped sponge. Giventhese devices are used to collect very small volumes of liquid(tears) we considered the microsponge as having high potentialto resolve some of our problems with saliva sampling. The firstconcern was whether the device would absorb enough sampleand yield sufficient saliva sample test volumes for assay. To testthis issue hydrocellulose microsponges (DB OpthalamicSystems, Walton, MA) were placed under the tongue for 15,30, 45, 60, 75, 90, 105, and 120 s. Fig. 2 reveals the average(standard error) of sample volume (μL) recovered. There was acurvilinear relationship with the microsponge reaching itsmaximum absorbed volume of approximately 300 μL within1 min. Thus, the minimum sample test volume for a typicalsalivary assay (25–50 μL) was achieved within 20–30 s.

Next, we examined sample recovery under conditionsdesigned to represent very small volume availability (e.g.,newborns, frail elderly, xerostomia). We used the microspongeto absorb saliva in known volumes of 25, 50, 100, 200 and400 μL. As above, different saliva volumes were pipetted intocryovials and the end of the sponge was held in the liquid for1 min. Each microsponge was then placed into a 2-mL cryovial,centrifuged to remove the liquid in the microsponge, and thenvolume absorbed was determined by weight. Fig. 1 presents theaverage percent of saliva available that was recovered from thesponge. As can be seen, in Fig. 1, the findings confirm that thepercent recovery from the microsponge is an improvement overthe cotton pledget. On average, the recovery was 40% from thesponge and 19% from the cotton swab, t (4)=4.40, pb .05. Thepercent recovery for both the sponge and the cotton pledgetincreased as the volume of sample to be absorbed increased.However, the sponge clearly out-performed the cotton pledgetin terms of percent recovery when sample volumes were below100 μL. Our conclusion is that the sponge yields an adequatetest volume given a much smaller sample available to becollected than does the cotton pledget method (see also [16]).

We next turn to feedback from investigators employing themicrosponge under saliva collection circumstances that haveproven challenging using the cotton pledget [9]. Informalobservations and feedback from FLP home visitors reveal that

the small size of the microsponge often allowed collection ofsufficient saliva volumes from sleeping infants without wakingthem. Infants were often willing to allow the sponge to beplaced in their mouths after they refused the cotton pledget.Anecdotal reports imply that the number of infants willing tomouth the microsponge increased if the mother was theinvolved in conducting the saliva collection and if there aredistraction techniques used concurrently with the collection. Inboth the 6- and 15-month waves of assessment, we determinedthat a 1-min collection period (total time in mouth — minutedoes not have to be continuous) and the use of 2 sponges percollection maximizes success of collecting sufficient volume.Using this method, the FLP investigators have been able tocollect sample volumes sufficient for duplicate testing ofsalivary cortisol (N100 μL) in more than 90% of the samples[9].

Our previous studies show that the process of absorbing andfiltering saliva through cotton causes “interference” in immu-noassays for many salivary biomarkers [17]. Table 1 presentsmeans and standard deviations comparing samples collectedusing the sponge and passive drool methods for a representativesample of the more commonly assayed salivary biomarkers.This comparison reveals that the sponge may be appropriate foruse with samples to be assayed for cortisol, cotinine, and alpha-amylase but not for other markers such as dehydroepiandros-terone and testosterone.

In summary, the microsponge method is capable ofabsorbing adequate sample quickly when conditions limit theamount of saliva available to be sampled. Similar to filter papermethods, the microsponge is small and has the potential to beused to gather sample when research participants might besleeping without awakening them. Given how quickly it cangather sufficient sample, young children seem to tolerate it intheir mouths better than the traditional cotton pledget method.However, the sponge is not without limitations. Its small sizemeans that investigators should take steps to be sure the spongeis not ingested. The device does fit inside a standard “chokehazard tube” but can be easily modified in the field to resolvethis issue. More importantly, the range of salivary biomarkers

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that can be assayed from samples collected using the sponge isnot all inclusive. As always, it is recommended that pilot studiesare conducted to confirm that the collection method does notinterfere with the measurement of the markers of interest usingthe exact assay protocol to be used for the project.

6. General conclusion

Taken together, our observations underscore that thecollection of saliva in the very young and the oldest-oldresearch participants can be unexpectedly challenging. Tradi-tional saliva sampling techniques that involve the use of cottonmaterials to absorb sample have limitations that restrict theirutility in these special circumstances and unique populations.The nature of this problem involves the very small amountsof saliva available to be collected, and the poor recovery ofsmall volumes of saliva once absorbed into cotton material.In our experience, attempts to resolve this problem by usingsubstances during sample collection to stimulate saliva flowhave the potential to create additional problems (e.g., [4]).

The alternative sample collection approaches we evaluated(i.e., passive drool, filter paper, and microsponges) had veryspecific advantages and disadvantages. Passive drool enabledthe measurement of multiple salivary biomarkers withoutconcern of interference caused by material employed tostimulate or absorb the sample. The technique, however,requires a cooperative and capable research participant. Filterpaper appears to be useful to collect sufficient sample for assaywhen small volumes of saliva are available (e.g., neonates). Theuse of this method to quantify concentration per volume indicesof salivary biomarkers has, so far, yielded mixed results. Themost recent findings [31] are particularly encouraging for themeasurement of salivary cortisol. Additional exploration of thisalternative collection approach seems worthwhile. In particular,it would be valuable to know whether the filter paper works aswell for other salivary markers (e.g., testosterone, cotinine,alpha-amylase) as it does for cortisol.

The microsponge method absorbed sufficient saliva volumefor assay within a very short time period, it was possible to usethe device to collect a sample from sleeping infants withoutwaking them, and young children tolerated the device in theirmouth for an adequate amount of time. To ensure adequatesample volume was collected, we found it worthwhile using twosponges simultaneously. The utility of the microspongeapproach to saliva collection is limited by the fact that it causesinterference in immunoassays for some salivary markers.

In conclusion, although there are many advantages affordedby measuring biological markers in saliva, this report adds to theliterature documenting many subtle issues that must be carefullycontrolled to ensure measurement validity [4,17,26,32]. To thebest of our knowledge, there is not a “one size fits all solution” tosampling saliva in biobehavioral research. The pros and cons ofsample collection methods will need to be balanced in relation tothe special needs and practical constraints of each individualresearch agenda. We expect these findings will help ease theburden on research participants and research assistants, reducethe rate of missing values in salivary data sets, and increase the

probability of the successful integration of salivary biomarkersinto behaviorally-oriented research.

Acknowledgements

This research was supported in part by the BehavioralEndocrinology Laboratory and the Child Youth and FamiliesConsortium at The Pennsylvania State University as well as theFamily Life Project funded by the National Institute of ChildHealth and Development (PO1HD39667-01A1). Thanks aredue to the Family Life Project Investigators; student assistants,Claire Kang, Kathryn Sawruk, Jeff Marguin, Olga Rumyanst-seva, Rachel Anolik, Reneca Green, Lauren Davis and ErinKelly; and Mary Curran for biotechnical support with assays.

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