Applied Nursing Research 19 (2006) 95 – 101

www.elsevier.com/locate/apnr
Clinical methods
Strategies for salivary cortisol collection and analysis in research
with children
Kirsten Hanrahan, MA, ARNPa,4, Ann Marie McCarthy, RN, PhD, PNP, FAANa,
Charmaine Kleiber, RN, PhD, FAANa, Susan Lutgendorf, PhDb,c,d, Eva Tsalikian, MDe
a
College of Nursing, University of Iowa, Iowa City, IA 52242, USA
b
Department of Psychology, University of Iowa, Iowa City, IA 52242, USA
c
Department of Obstetrics, University of Iowa, Iowa City, IA 52242, USA
d
Department of Gynecology, University of Iowa, Iowa City, IA 52242, USA
e
Department of Pediatrics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

Abstract Salivary cortisol has emerged in pediatric research as an easy-to-collect, relatively inexpensive,
biologic marker of stress. Cortisol is highly variable and is responsive to a wide range of factors that
should be considered when incorporating this measure into research with children. Strategies for
sample collection include: (1) standardizing the time for sample collection, including baseline
samples; (2) using consistent collection materials and methods; (3) controlling for certain drinks,
foods, medications, and diagnoses; and (4) establishing procedures and protocols. Other strategies
for laboratory analyses include: (1) selecting the appropriate assay and laboratory; (2) identifying
units of measure and norms; and (3) establishing quality controls. These strategies control extraneous
variables and produce reliable and valid salivary cortisol results.
D 2006 Elsevier Inc. All rights reserved.

1. Introduction in stress, coping, and health research with children

(Schmidt, 1997). However, collecting and measuring
Salivary cortisol has emerged in pediatric research as an
salivary cortisol in children present unique challenges.
easy-to-collect, relatively inexpensive, biologic marker of
The purpose of this article is to present strategies for the
stress. Cortisol can be used to assess responses to stressful
collection and analysis of salivary cortisol in research with
stimuli or to determine the effectiveness of interventions
children. A brief review of cortisol secretion and of factors
intended to reduce stress. However, cortisol is highly
impacting cortisol levels is conducted. Strategies used for
variable and is responsive to a wide range of factors that
collecting salivary cortisol in children and for laboratory
should be considered when incorporating this measure into
analyses are reported.
research with children. Researchers have developed meth-
ods for collecting salivary cortisol in children, and recent
laboratory techniques have made it possible to detect very 2. Cortisol secretion
small concentrations of cortisol in plasma and saliva. Two
Cortisol is secreted in response to increased stress in an
advantages that salivary cortisol has over plasma cortisol in
individual’s environment. Stress response involves the
pediatric research are that samples can be collected through
interaction of two systems: the sympathetic nervous system
relatively noninvasive techniques and that they can be timed
(SNS) and the hypothalamicpituitaryadrenal (HPA) axis.
without depending on the availability of a laboratory or
The SNS is associated with the release of norepinephrine
health care professional. Because of these advantages,
and epinephrine, which rapidly activate a bfight-or-flightQ
salivary cortisol has become a popular measure of cortisol
response. The HPA axis is activated more slowly, causing a
cascade of endocrine events (Carter & DeVries, 1999). The
* Corresponding author. Tel.: +1 319 363 6838. HPA axis is believed to play an important role in
E-mail address: kirsten-hanrahan@uiowa.edu (K. Hanrahan). physiological coping and can aid in mediating the effects
0897-1897/$ – see front matter D 2006 Elsevier Inc. All rights reserved.
doi:10.1016/j.apnr.2006.02.001
96 K. Hanrahan et al. / Applied Nursing Research 19 (2006) 95–101

of stress on health, mood, behavior, and stress-related
disease (King & Hegadoren, 2002; Sapolsky, 2000).
Cortisol, a glucocorticoid hormone, is the endproduct of
HPA axis activation in humans. The neurochemicals
involved in HPA axis stress response activation are
produced when a stressful event is experienced. There is
an estimated lapse of 15 to 30 minutes between a stress
event and the production and release of increased plasma
cortisol; an additional 2-minute lapse occurs before cortisol
increases in saliva (Gunnar & White, 2001). For example, if
a child experiences a stressful event, such as an immuni-
zation at 9:00 a.m., the child’s cortisol response will peak in
the serum after about 15 to 30 minutes (~9:20 a.m.) and in
the saliva approximately 2 minutes later (~9:22 a.m.).
Cortisol is normally secreted in short bursts, with 15 to
30 pulses over the course of 1 day (Jett et al., 1997; King &
Hegadoren, 2002). Like other hormones, cortisol has a
known circadian rhythm, fluctuating during the day in a
predictable cycle, and demonstrates diurnal (light and dark)
variations. Cycles in children’s cortisol levels are generally
established in early infancy (Groschl, Rauh, & Dorr, 2003;
Gunnar & White, 2001; Price, Close, & Fielding, 1983), but
fluctuations continue to occur. Cortisol levels generally peak
around 20 to 30 minutes after awakening, decrease to half
by midafternoon, and are lowest by midnight (King &
Hegadoren, 2002; Schmidt, 1997). Increased physical
activity levels (Kirschbaum & Hellhammer, 1994) and
eating (Gibson et al., 1999) may cause transient increases
that change cortisol levels. Changes in sleeping patterns,
waking times, and travel across time lines can alter the
diurnal rhythm of cortisol levels (Gunnar, Bruce, & Hick-
man, 2001; King & Hegadoren, 2002; Kudielka &
Kirschbaum, 2003). Variations related to individual patterns,
time of day, activity, sleeping, eating, certain medications,
certain illnesses, and use of salivary stimulants need to be
considered carefully by researchers using salivary cortisol as
a measure of stress and/or a research outcome.
Craske, Katz, Schwartz, & Zeltzer, 2000; Davis, Bruce, &
Gunnar, 2002; Davis, Donzella, Krueger, & Gunnar, 1999;
Dettling, Gunnar, & Donzella, 1999; Gunnar, Tout, de Haan,
Pierce, & Stansbury, 1997). Some factors may impact
individuals at different developmental stages in their lives,
causing fluctuations in cortisol levels. For example, in older
children, stage of puberty may be a factor (Kiess et al.,
1995); in older adolescents and adults, phase of the
menstrual cycle, hormone contraceptives, pregnancy, and
smoking may be factors that influence cortisol levels
(Kirschbaum & Hellhammer, 1994; Kirschbaum, Kudielka,
Gaab, Schommer, & Hellhammer, 1999). This means that
investigators must identify those factors that may be relevant
to the population of study.
A broad spectrum of diagnoses and medications (Table 1)
may also affect the functioning of the HPA axis and cortisol
levels (King & Hegadoren, 2002). Some diagnoses, such as
Cushing disease and Addison disease, overtly affect HPA
axis activity. Other diagnoses, such as personality and mood
disorders, have known variable effects on cortisol levels
(Kariyawasam, Zaw, & Handley, 2002; Luby et al., 2003;
Pajer, Gardner, Rubin, Perel, & Neal, 2001; Wedekind,
Bandelow, Broocks, Hajak, & Ruther, 2000; Weinstein et
al., 1999). In addition, medications that alter mood,
metabolism, or HPA axis activity should be considered
(Woodside, Winter, & Fisman, 1991). Of particular concern
in young children are the effects of steroids on HPA axis
activity. Steroids, which are increasingly used to treat
children for asthma and other diseases, have a direct effect
on HPA activation and cortisol level measurements.
Increased prevalence of attention deficit hyperactivity
disorder (ADHD) and the use of medications for treatment
may also affect HPA activity and cortisol levels. Kariyawa-
sam et al. report that children with ADHD or oppositional
defiant disorder (ODD) have significantly lower salivary
cortisol levels compared to healthy controls. This finding

3. Individual factors impacting cortisol levels Table 1

Medications and diagnoses that may affect cortisol levels
Cortisol levels in children have the same circadian Drugs that may Drugs that may Drugs that may Diagnoses that
patterns as adults and show similar variability related to influence increase decrease may alter
individual factors (Kiess et al., 1995; Kudielka, Buske- cortisol levels cortisol levels cortisol levels cortisol levels
Kirschbaum, Hellhammer, & Kirschbaum, 2004). Large Herbal Amphetamine Androgen Cushing
variations in baseline levels and the response magnitude of products disease
cortisol levels exist within and between individuals (Bruce, Substances of Estrogen Barbiturates Addison
abuse disease
Davis, & Gunnar, 2002; Gunnar, Brodersen, Krueger, & Corticosteroids Ethyl alcohol Dexamethasone Depression
Rigatuso, 1996; Smyth et al., 1997). Factors that may impact Sex hormones Lithium Levadopa Panic disorders
cortisol variability in an individual include genetic differ- Antidepressants Methadone Phenytoin Abdominal
ences (allelic variations), age, weight, pubertal stage, and sex obesity
differences (Bartels, de Geus, Kirschbaum, Sluyter, & Drugs that alter Nicotine Stuttering
mood
Boomsma, 2003; Groschl et al., 2003; Gunnar & White, Caffeine Spironolactone Schizophrenia
2001; Heuser et al., 2000; Kiess et al., 1995; Kudielka et al., Personality
2004; Price et al., 1983). Individual characteristics, such as disorder
temperament, coping style, social competency, and pain sen- ADHD
sitivity, can further modify cortisol responsiveness (Chen, Data from Kariyawasam et al., 2002; King & Hegadoren, 2002.
 K. Hanrahan et al. / Applied Nursing Research 19 (2006) 95–101
suggests a dysfunction of the HPA axis in these children,
which may be a reflection of underarousal, elevated
threshold for stress, or low sensitivity in the HPA axis.
Lower cortisol levels in the ADHD/ODD group were
restricted to the subgroup not on stimulant medications,
whereas the subgroup on stimulant medications was
comparable to healthy controls. New medications are
emerging for the treatment of ADHD; the effects of these
medications on the HPA system and cortisol levels are
variable and not fully understood.
Food products may cause variability in salivary cortisol
levels for two reasons: (1) they can alter the oral
environment, and (2) they can alter HPA activation. Some
drinks and foods lower salivary pH, which may affect
cortisol assays and produce false high or low levels
(Schwartz, Granger, Susman, Gunnar, & Laird, 1998).
Other drinks or food products may alter HPA activation
because of hormones or stimulants contained in them. For
example, milk products may contain endogenous hormones
that falsely increase salivary cortisol levels. A variety of
foods and drinks contain caffeine, a stimulant that may
increase HPA axis activity. Meals high in protein may cause
a transient increase in cortisol (Gibson et al., 1999).
4. Strategies for collecting salivary cortisol in children
As the literature suggests, a number of factors need to be
addressed before incorporating measurements of salivary
cortisol into pediatric research. The research team must
establish a consistent collection process that includes
controlling for factors that might interfere with accurate
measurements of cortisol in saliva. Strategies for sample
collection include: (1) standardizing the time for sample col-
lection, including baseline samples; (2) using consistent col-
lection materials and methods; (3) controlling for certain
drinks, foods, medications, and diagnoses; and (4) establish-
ing procedures and protocols. Other strategies for the
laboratory analyses include: (1) selecting the appropriate
assay and laboratory; (2) identifying units of measure and
norms; and (3) establishing quality controls.
4.1. Standardize the time for sample collection including
baseline samples
To control for the effects of circadian and diurnal rhythms,
the time of day samples are collected should be standardized.
Ideally, samples are collected at a similar time of day for all
subjects. However, this is not always possible in a clinical
research study involving children. For example, in a study of
children having intravenous catheters placed for a variety of
diagnostic tests, the time of the procedure is determined by
clinic flow, and standardization for the study may not be
feasible. To determine salivary cortisol response, the times
must vary with the stressor of interest (in this case intravenous
start time). As in this case, statistical manipulation of results
can control for effects related to the time of day samples are
collected when standardized times are not feasible.
97
The number of samples obtained must also be estab-
lished. More samples provide more information on individ-
ual fluctuations. However, the number of samples obtained
in a study may be limited by the availability of funds and the
clinical situation. For intervention studies, the number of
samples should include both measurements of baseline
levels for the child and the child’s response to stressful
events or interventions. Baseline samples provide a measure
of the child’s normal cortisol levels during a typical day.
Researchers have just begun to establish reference values for
salivary cortisol in healthy children (Groschl et al., 2003).
Although definitive normal values are not well established,
this does not preclude the use of salivary cortisol in the
research of children. The individual child acting as his or her
own control in experimental situations may be the best
reference value (Schmidt, 1997).
4.2. Use consistent collection materials and methods
The materials and techniques used to collect samples
may influence the accuracy of testing. Steroid hormones,
such as cortisol, have varied affinity for plastic and may
bind to the walls of collection tubes. Some laboratories have
developed special low-affinity plastic collection tubes for
steroid measures (IBL, Hamburg, n.d.). Use of one
consistent collection device for cortisol sampling is high-
ly recommended.
A common way of obtaining saliva for analysis in
adults involves the use of the commercially available
Salivette (Sarstedt Aktiengesellschaft and Company;
Kirschbaum & Hellhammer, 1994). The Salivette looks
like a 2-in. cotton dental roll and is packaged in a plastic
test-tube-like container. Participants are instructed to chew
or mouth the Salivette for approximately 30 to 45 seconds
(Sarstedt, n.d.). Although tasteless, the Salivette has a
consistency that some children find unpalatable. Because
this approach may not be appropriate for infants and young
children, other ways of obtaining saliva from children have
been developed. One alternative process for collecting
saliva in children involves asking the child to spit (also
referred to as passive drool) through a short straw into a
plastic collection tube (Granger, Schwartz, Booth, &
Arentz, 1999; Schwartz et al., 1998). Other options for
saliva collection in younger children or infant populations
include using a syringe or absorbent cotton swabs
(Herrington, Olomu, & Geller, 2004; Joyce, Keck, &
Gerkensmeyer, 2001; Schwartz et al., 1998).
Another strategy to assure the collection of adequate
samples is to use salivary stimulants, such as flavored drink
crystals or sugarless gum. This strategy is particularly
attractive to young children who like flavors. However,
salivary stimulants should be used with caution. Although
the rate of salivary flow does not affect the cortisol level
(Kirschbaum, n.d.-a), salivary stimulants may alter salivary
pH, causing an elevation in the cortisol assay (Schwartz
et al., 1998). Sensitivity to pH may be determined by the
cortisol assay selected. Using only salivary stimulants that
98 K. Hanrahan et al. / Applied Nursing Research 19 (2006) 95–101
have been standardized for cortisol testing with a specific
assay is advisable. Allowing at least 3 minutes between
chewing gum (or other stimulants) and collecting salivary
samples is also recommended (Granger et al., 1999).
4.3. Control for certain drinks, foods, medications,
and diagnoses
Certain foods, medications, and diagnoses known to
affect salivary cortisol levels should be documented, and
exclusion criteria should be determined. For example, in
studies with young children, food or drinks that may be
potentially problematic are milk products and those that
contain caffeine or that alter salivary pH. To control for
alterations in salivary cortisol related to food and drinks,
study participants may be asked to refrain from eating or
drinking for 30 minutes prior to sample collection. A child
who has eaten b 30 minutes prior to sampling may be asked
to rinse his or her mouth and to wait 3 to 5 minutes (long
enough to re-establish the natural oral environment and a
salivary pH of about 6.4–7.4) before providing a sample
(IBL, Hamburg, n.d.). Caffeine intake on the day of sample
collection may be recorded for later consideration in
statistical analysis. Exclusion criteria for medications and
diagnoses need to be clearly established for the study
population. For example, children taking oral steroids in the
30 days prior to collecting the sample may be excluded.
However, children on topical and inhaled steroids may be
included because systemic absorption of these is limited.
Children with ADHD may be included, and statistical
analysis may be used to determine if differences with the
general population exist.
4.4. Establish procedures and protocols
To ensure that samples are collected in a consistent
manner, protocols for research team members should be
developed. An example of a procedure for sample collection
is the bchew-and-spitQ method (Table 2). For some studies,
parents may need to obtain salivary samples at home.
Because cortisol concentrations in unfrozen samples remain
stable for at least 5–7 days, these samples can be returned
by standard mail (Aardal & Holm, 1995; Clements &
Parker, 1998). Examples of handouts developed to assure
consistent collection procedure at home are provided
(Tables 3 and 4). Questions should be designed for the
target audience (e.g., older children should also be asked
about smoking and illicit drug use).
In some cases, cortisol samples must be shipped from the
clinical site to the laboratory for analysis. Specific proce-
dures for handling, shipping, and storing samples should be
developed. Salivary samples can be stored in a 208C
freezer until they are ready to be shipped to the laboratory.
When shipping samples, allowing a thaw cycle does not
appreciably alter the specimens, although evaporation may
occur (Herrington et al., 2004). Thawing may be avoided,
but not guaranteed, by shipping samples on dry ice.
Refreezing thawed samples should be avoided, as evidence
Table 2
Salivary sample collection procedure: Chew-and-spit method
Purpose
To obtain saliva for cortisol levels at appropriate times
(Time 1 clinic = preprocedure cortisol; Time 2 clinic = cortisol
collected 20 – 30 minutes after intravenous procedure)
Steps
(1) Give child one piece of original-flavored Trident sugarless gum to
chew on until the mouth starts watering.
(2) Have child remove the gum from the mouth and discard it.
Wait for 3 minutes for saliva to clear.
(3) Open the vial.
(4) Place straw into the vial.
(5) Instruct child to direct saliva through the tube into the vial.
(6) If no saliva appears in the vial, have the child gently blow saliva out
of the straw.
(7) Fill the vial one thirds full.
(8) Close the vial tightly.
(9) Mark take-home labels with time samples collected in the clinic
(Time 1 home matches Time 1 clinic, and Time 2 home matches
Time 2 clinic).
(10) Document on the Biological Sample Tracking Record.
(11) Send for storage refrigeration.
Precautions
! Avoid eating or drinking caffeinated products for 2 hours before
sampling.
! Avoid eating or drinking dairy products for 15 minutes before
sampling.
! Steroids, including dexamethasone, prednisone, and others,
may interact with the analysis process, causing falsely elevated levels.
Do NOT collect sample if the child has been on oral steroids within
the last month.
! Use research questions to screen for food and drugs that interfere with
levels and track samples. Note on questions but DO collect sample.
! If unable to collect Time 2 clinic sample 15 to 35 minutes after
intravenous stick, then discard sample Time 2 clinic sample and
do not collect home samples.
! Samples should be refrigerated within 24 hours and sent to a freezer
within 72 hours.
! If the child is unable to participate in the collection method, use the
Salivette method.
suggests that it may result in significantly lower cortisol
levels (Aardal & Holm, 1995).
5. Strategies for laboratory analyses
5.1. Select the appropriate assay and laboratory
The reliability of the measurement of cortisol levels is
determined by the assay and the laboratory selected.
Researchers should first review the various types of assays
available, identify the assay most appropriate for their
needs, and then identify a laboratory that is able to carry out
the appropriate analysis. Initially, commercial assays
intended for serum testing were adapted to saliva for
noninvasive testing. More recently, highly sensitive assays
specific to saliva analysis have become available using
chromatographic procedures and immunologic methods.
Assays may differ in the use of radioactive materials,
 K. Hanrahan et al. / Applied Nursing Research 19 (2006) 95–101
Table 3
Instructions for home sample salivary cortisol collection
Collecting cortisol samples
Cortisol is a hormone that increases when you are stressed. We can
measure cortisol in saliva (spit) samples. We need saliva samples taken
from your child on a bnormalQ day to compare with samples taken in
the clinic. Try to pick a typical day. Weekends often work best
because of the time samples need to be collected.
The time the samples are collected is important. We have marked on
the sample bag times for you to collect samples. Try to collect samples
as close to the times specified as possible. These are the same times
we collected samples in the clinic.
Caffeine and milk products interfere with testing. We ask that your child
not have caffeine on the morning of sample collection. Milk should
be avoided for 30 minutes before sampling. Steroids can interfere
with testing, too. If your child is on steroids now and was not present
at the clinic appointment, please call the project director or site
coordinator listed.
Directions
(1) Have the child rinse the mouth with water 30 minutes before
sampling time.
(2) Do not allow the child to eat or drink 30 minutes before
collecting samples.
(3) Give your child one piece of original-flavored Trident gum to chew
on until the mouth starts watering. (If you do not have the supplied
gum, you can try to collect saliva without a gum).
(4) Have the child remove the gum from the mouth and discard it. Wait
for 3 to 5 minutes for gum to clear from the saliva.
(5) Open the collection tube and place the straw inside.
(6) Instruct the child to direct saliva through the tube into the vial.
(7) If no saliva appears in the vial, have the child gently blow saliva out
of the straw.
(8) Fill the tube one thirds full. Close the tube tightly.
(9) Put the tube back into the bag and seal.
(10) Write on the bag the time the sample was collected.
(11) Refrigerate samples until ready for mailing.
Cortisol sample checklist
q Choose a typical day.
q No caffeine before sampling.
q No milk products within 30 minutes before sampling.
q Use only the gum supplied as stimulant for saliva. Wait 3 to 5 minutes
for gum to clear from the saliva.
q Collect samples within 30 minutes of the time designated on the bag.
q Fill the tubes one thirds full.
q Make sure to mark on the bags the times you collected the samples.
q Refrigerate samples until they are ready for mailing.
q Place both samples in the mailing bag.
q Send by US mail. Postage has been paid. No special handling is
needed.
sensitivity of the method, and equipment needs. Nonisotopic
immunoassays for salivary cortisol have made testing more
readily available. These commercially available kits use
markers that compete to bind to cortisol receptor sites. The
criteria to consider in selecting the appropriate assay
include: sensitivity, lower detection level, intra-assay and
interassay variations, availability of controls, degree of
automation, and availability of equipment and laboratory
facilities (IBL, Hamburg, n.d.). Consultation with experts
familiar with the assays and performance criteria helps in
identifying an appropriate assay for researchers’ needs.
Researchers without facilities or preparation to carry out
99
Table 4
Cortisol home questions
We appreciate your time and commitment to collecting home cortisol
samples. It is important to have complete information about the
samples you collected even if the brulesQ were not followed. Please
answer the following questions about the day that the samples were
collected.
(1) Was today a typical day at home?
q Yes
q No (explain) _______________________________
(2) Was your child healthy and feeling well today?
q Yes
q No (explain) _______________________________
(3) Did your child participate in any vigorous physical activity today
before the samples were collected (e.g., soccer practice, swimming)?
q Yes (explain) __________________ At what time? ________ a.m./p.m.
q No
(4) Did your child have an emotional event today before sampling (such
as fighting with sibling, prolonged crying for more than 10 minutes)?
q Yes (explain) __________________ At what time? ________ a.m./p.m.
q No
(5) Did your child eat or drink anything with caffeine today before sampling?
q Yes (explain) __________________ At what time? ________ a.m./p.m.
q No
(6) Did your child use the gum supplied just prior to sampling?
q Yes
q No, no gum used
q No, used something else (explain) _____________
(7) Did your child have anything to eat in the 30 minutes prior to sampling?
q Yes (explain) __________________ At what time? ________ a.m./p.m.
q No
(8) Did your child have milk products in the 30 minutes prior to sampling?
q Yes (explain) __________________ At what time? ________ a.m./p.m.
q No
(9) List the medications your child is taking. If there are no medications,
please indicate.
(10) Is there anything else we should know?
their own laboratory work must identify a laboratory
wherein they could perform these procedures. The criteria
for selecting a laboratory in which to do a specific assay
should include: availability, quality performance measures
by the laboratory, experience, and cost.
5.2. Identify units of measure and norms
Once a laboratory has been identified, researchers need
to clarify the units in which the cortisol results will be
reported and to identify conversion formulas for comparing
values, if needed. Some laboratories report results in
nanomoles per liter; however, salivary cortisol is typically
reported in the literature as micrograms per deciliter, which
results in a narrower range of values. Formulas for the
Table 5
Conversion formulas
Reported units Factor Converted units
nmol/l 0.03625 Ag/dl
ng/ml 2.76 nmol/l
Ag/dl 27.6 nmol/l
Data from IBL, Hamburg, 2004; Jay Clinical Services, 2001.
100 K. Hanrahan et al. / Applied Nursing Research 19 (2006) 95–101
Table 6
Creating control specimens for measuring salivary cortisol reliability
(adapted from Gunnar & White, 2001)
Purpose
Demonstrate consistent cortisol levels among control samples to verify
the reliability of procedures for processing.
Procedure
!Collect a large pool of saliva.
!Mix well in a laboratory-used automated mixer.
!Aliquot to tubes.
!Freeze and ship according to study procedures.
!Blind the laboratory to which specimens are controls.
!Set the level of CV.
conversion of values between nanomoles per liter and
micrograms per deciliter are presented in Table 5.
Next, it is important to establish a normal range of values
to identify outliers. Studies with bnormalQ children (often a
control group) (Bruce et al., 2002; Davis et al., 1999;
Groschl et al., 2003; Gunnar, Bruce, et al., 2001; Gunnar,
Morison, Chisholm, & Schuder, 2001; Gunnar & Vazquez,
2001; Kiess et al., 1995) suggest that values b 0.01 Ag/dl
(c0 nmol/l) or N 1.0 Ag/dl (c28 nmol/l) may be outliers.
Another approach to identify outliers is to use statistical
analysis (e.g., N 2 SD from the mean) for a specific data set.
Each outlier should be reviewed to see if there is a clinical
explanation for outliers, such as food or medications.
5.3. Establish quality controls
Quality controls for laboratory measures of cortisol are
established by a coefficient of variance (CV). A CV is a
measure of variance from the mean and is calculated by
dividing the standard deviation by the mean for each sample
and multiplying by 100%. The value should be b 12 to 15%
(Kirschbaum, n.d.-b). The laboratory should routinely
monitor and report their CV levels for intra-assay reliability
(variation within each assay batch) and interassay reliability
(variation between batches).
Reliability procedures for the specific data set should
also be established using duplicate analysis, control speci-
mens, and random specimens. A duplicate analysis involves
repeating the analysis on the specimen and comparing the
two values. Because the analyses are from the same
specimen, the expectation is that the values will be the
same or very similar. The CV on duplicate analyses should
be below 10% (IBL, Hamburg, n.d.). Control specimens can
be created and included in each batch of study samples sent
to the laboratory. The procedures for creating control
specimens are described in Table 6. The assumption is that
cortisol levels should be similar for each control specimen.
The CV for the control samples should not be N 12 to 15%
(Kirschbaum, n.d.-a). Finally, random specimens can be
inserted into batches to establish a range of values. Random
specimens are obtained from different people at various
times during the day under variable conditions; therefore,
there should be a wide range of cortisol levels. The CV for
random samples should be N 12%, as variability is expected.
6. Conclusions
In summary, salivary cortisol levels are increasingly used
in pediatric research as a measure of stress. Although
cortisol is an appropriate biologic indicator of stress, to
ensure that the results are reliable, investigators need to
consider a wide range of factors when incorporating this
measure into research. Strategies for assuring consistent and
reliable sample collection and laboratory analyses of
salivary cortisol levels increase the validity of results. Data
from pediatric studies can contribute to our understanding of
children’s responses to stressful events and the ability to
determine effective interventions for children.
Acknowledgment
This study is funded by RO1 grant NR05269-01A2 from
the National Institute for Nursing Research. The authors
gratefully acknowledge Dr. Megan Gunnar and Bonny
Donzella of the University of Minnesota, and Dr. Clemens
Kirshbaum of Dusseldorf University for their generous
advisement and contributions to this project. We appreciate
the children and families who were eager to help others
through participation in this research.
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