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Clinical Therapeutics/Volume ], Number ], 2015
Salivary Cortisol Results Obtainable Within Minutes of Sample
Collection Correspond With Traditional Immunoassays
Elizabeth A. Shirtcliff, PhD
1
; Robert L. Buck, PhD
2
; Mary J. Laughlin, PhD
2
;
Thomas Hart, MS
3
; Craig R. Cole, PhD
2
; and Paul D. Slowey, PhD
2
1
Department of Human Development and Family Studies, Iowa State University, Ames, Iowa;
2
Oasis Diagnostics, Vancouver, Washington; and
3
Middleton Research, Middleton, Wisconsin
ABSTRACT
Purpose: Cortisol is frequently assayed as a stress-
responsive biomarker which changes over the course
of minutes to meet the demands of a person’s social
context. Salivary cortisol is often used as a non-
invasive sampling method that possesses important
health implications. A critical barrier to psychobio-
logical research that involves salivary cortisol is a time
delay of days to months before cortisol results are
obtained via immunoassay, long after the person is no
longer proximate to the social context in which they
provided the sample. The present study was designed
to address this critical barrier through creation of a
lateral flow test (LFT) cortisol device capable of
measuring salivary cortisol within minutes of sample
collection. The LFT is frequently used within com-
mercial point-of-care settings to obtain rapid answers
to the presence/absence of a biomarker. The present
study extends the LFT into the research domain by
presenting performance characteristics of a quantita-
tive LFT that measures salivary cortisol within 20
minutes of sample collection.
Methods: Saliva samples from 29 adults (15 men)
were obtained in the morning and afternoon by using
Passive Drool and then the Super SAL Extra Collec-
tion Device (hereafter Super SAL) and later assayed
with LFT and a commercially available enzyme
immunoassay.
Findings: Results indicate the LFT correlated well
with these collection methods (R¼0.872 with Super
SAL, R¼0.739 with Passive Drool, Po0.0001)
and at comparable levels to correspondence of Super
SAL with Passive Drool (R¼0.798, Po0.0001)
which were measured with the same assay.
Implications: These results open an exciting new
possibility to integrate this technologic advance into
stress research, including knowing and potentially
changing the person’s social context in a time-
sensitive manner. Methodological improvements such
as this have the possibility of refining conceptual
models of stress reactivity and regulation. (Clin Ther.
2015;]:]]]–]]])&2015 Elsevier HS Journals, Inc. All
rights reserved.
Key words: collection methods, cortisol, immuno-
assay, lateral flow technology, stress regulation.
INTRODUCTION
Stress is a leading cause of morbidity and mortality in the
United States.
1
Asmallsetofbiomarkersprovide
information about chronic and acute stressors.
2
Cortisol
is putatively the most frequently investigated stress
biomarker
3
because cortisol is linked with many
physiologic processes such as neural development and
cell death,
4
immune function,
5
learning and memory,
6
sleep,
7
metabolism and fat distribution,
8
growth and
development,
9
reproduction,
10
and aging.
11
The pow-
erful role of cortisol in shaping health outcomes is
illustrated by its clinical value in treatments that range
from mild rashes in over-the-counter creams to life-saving
efforts.
12
Cortisol is the end product of the hypothalamic-
pituitary-adrenal (HPA) axis. After appraisal of the
social context in limbic and paralimbic neural circui-
tries,
13,14
the hypothalamus releases corticotropic
releasing hormone that initiates a hormonal cascade
that culminates with adrenocorticotropic hormone
that stimulates the release of cortisol from the adrenal
cortex and into the blood.
15
After 15 minutes from
Accepted for publication February 12, 2015.
http://dx.doi.org/10.1016/j.clinthera.2015.02.014
0149-2918/$ - see front matter
&2015 Elsevier HS Journals, Inc. All rights reser ved.
]2015 1
the onset of a stressor, cortisol concentrations peak.
16–18
As a lipid-soluble hormone, cortisol easily crosses
through cellular membranes which allows it to travel
directly to cell nuclei to change gene expression,
19,20
especially in the brain where it is responsible for
terminating the stress response
4
via negative
feedback.
21,22
Cortisol also acts throughout the body
where it influences physiology over seconds, minutes,
hours, and days.
23
Salivary measurement of small
steroids such as cortisol take advantage of the fact
that free cortisol is lipid soluble; this biologically
active fraction of total cortisol passes through the
acinar cells to enter saliva via passive diffusion in
proportion to cortisol’s entrance into cell nuclei.
24
Measuring cortisol in saliva has opened a window of
opportunity to conduct stress-related research that
involves many repeated measures
25
or applications
with vulnerable populations
26,27
or in unique set-
tings.
28,29
Salivary measurement has even indicated
unique diagnostic and treatment information about
cortisol-related diseases such as Addison syndrome or
Cushing disease which previously required much more
invasive measurements throughout treatment.
30–33
First-generation salivary cortisol assays relied on radio-
labeled cortisol antibodies in radioimmunoassays that
were highly sensitive and specific. This sensitivity was
enhanced with the next generation of assays that
quantified cortisol concentrations via optical density
measurements in commercially available enzyme (EIAs)
or luminescence immunoassays (LIAs) that required
much lower sample volumes to be effective. These
particular methods rely on the degree of color change
of bound horseradish peroxidase to estimate hormone
concentrations relative to a standard curve. The range
of sensitivities with the use of these assays provided a
limit of detection in the range of picogram per milliliter,
a necessary prerequisite for salivary steroid hormone
detection whereby concentrations are low.
A critical barrier in the stress field is that current
immunoassay methods do not provide reportable
results for cortisol for days to months after collection.
Recent studies are beginning to explore the feasibility
of point-of-care measurement of salivary cortisol,
34–37
typically using lateral flow test (LFT) assays to deliver
qualitative or quantitive test results within minutes of
sample collection.
38
The present study describes the
first in a new generation of assays for measuring
salivary hormones such as cortisol by using an LFT
device (Oasis Diagnostic Corporation, Vancouver, WA).
The first US patent that used the term lateral flow was
filed in 1988 and was awarded in 1990,
39
although
several companies had patented elements of that
concept earlier. The first successful commercial use
of an LFT was the EPT urine human chorionic
gonadotropin hormone dipstick pregnancy test. LFT
has been widely used in industry since the late 1980s
and early 1990s, primarily to target the commercial
needs for point-of-care assays.
40
In similar fashion to
EIA and LIA, LFT relies on the detection of an emitted
signal (in this case fluorescence) to provide a fully
quantitative cortisol readout, but, unlike EIA and LIA
or even technologies such as mass spectrometry or
microarrays, LFTs are not widely used in the academic
community. The reasons for this may be related to
education, intellectual property considerations, and a
lack of LFT suppliers that cater to the specific needs
and recommendations of researchers.
The cortisol LFT technology is a unique proprietary
format that improves on several related patented
technologies.
41–43
We designed this LFT specifically
for the research community to provide major advan-
tages over currently available methods for saliva and
include the following: (1) the assay takes minutes from
sample collection to end results; (2) the assay and
reading unit (Litebox Image Analysis Module,
[LIAM]) are portable, allowing real-time cortisol
assessment to be performed in a variety of point-of-
care and nontraditional settings; and (3) the assay
retains many of the advantages of available methods
(eg, high sensitivity and quantitative output), so few
sacrifices in assay quality are required for real-time,
point-of-care cortisol measurement. The present study
describes the methodology and performance charac-
teristics of real-time LFT cortisol, including compar-
ison of this technology with another commercially
available non-LFT assay. The goal was to determine
correspondence and, by extension, viability of obtain-
ing real-time cortisol scores within the near future.
METHODS
Participants
All procedures were approved by the institutional
review board at a research university, and all participants
provided informed consent. Participants were excluded if
they were taking oral steroids, had eaten or consumed
alcohol within 1 hour, or were 436 years of age. After
exclusion, the final sample size was N ¼29.
Clinical Therapeutics
2 Volume ]Number ]
Procedures
Participants provided four saliva samples, two in
the morning before 11:30 AM (mean =8:54 AM;
range =6:11–11:27 AM) and two in the afternoon/
evening after 1 PM (mean =4:46 PM; range =1:29–
11:11 PM). These rough times were selected to obtain
high and low cortisol concentrations anticipated by
the diurnal rhythm of the hormone. A total of three
participants provided morning or afternoon/evening
samples only, leaving a total of 55 samples. Order of
sampling was not counterbalanced to avoid carry-over
effects. First, participants provided an unstimulated
saliva sample via Passive Drool by expectorating
directly into a 2-mL polypropylene cryovial (mean
[SD] =3.43 [3.07] minutes for collection times).
Immediately after the Passive Drool sample and
questionnaire (10.53 [15.97] minutes later), partici-
pants provided a second saliva sample by using the
Super SAL Extra Collection Device (Oasis Diagnostic
Corporation), hereafter termed Super SAL. This de-
vice serves as the first stage of LFT cortisol collection
because it contains an absorbent pad that collects up
to 2 mL of saliva (2.63 [1.52] minutes for collection
times). The absorbent pad filters many extraneous
substances from saliva which is necessary with LFT,
because these types of assays are sensitive to viscosity
and sample quality. Once the sample volume ad-
equacy indicator on Super SAL indicated that ample
volume was collected (by changing from light green to
blue), participants removed the collection device,
inserted it into the compression tube, and expressed
the saliva into a cryovial by using force on the handle.
The compression tube contains a proprietary filtration
medium, which removes additional extraneous mate-
rials and unwanted particulates that can compromise
the LFT. In this way, the Super SAL provided purified
saliva for the LFT and a confirmation specimen for
later testing. Samples were immediately frozen at
–801C and shipped on dry ice to Oasis Diagnostic
Corporation for assay by LFT. Residual samples were
then shipped on dry ice to Middletown Research
(Middletown, WI) for independent confirmation by
using the EIA method.
Methods
IBL Cortisol Assay
Both Passive Drool and Super SAL specimens were
assayed with a solid-phase enzyme-linked immuno-
sorbant assay by using reagents with the same lot and
expiration date purchased from IBL International
(Hamburg, Germany; www.IBL-International.com).
The IBL kit uses 50 uL of saliva with an expected
range of 0.015 to 5 mg/dL cortisol and reported
correspondence with IBL cortisol luminescence assay
of R=0.96. Intra- and inter-assay CVs are, on
average, 4.9% and 8.2%, respectively.
LFT Cortisol System
Saliva from Super SAL was assayed with the LFT;
Passive Drool was not used, given viscosity concerns.
Given the novelty of this assay, nonproprietary details
are provided. The basic platform is a positive read small
molecule competitive quantitative LTF strip with several
components. The first major component is a test
housing that incorporates the two LFT strips. A sample
pad located directly below the housing sample well acts
as a reservoir to distribute saliva to a conjugate pad.
The conjugate pad then accepts the saliva as a fluid
medium for hydration of the dried conjugate. The
VerOFy LFT point-of-care cortisol system then uses
two europium fluorescent particle-based conjugates that
react with free and conjugate bound cortisol in the
saliva sample (hereafter termed fluid because the prop-
erties of saliva are purposely altered to accommodate
the LFT). A nitrocellulose membrane then accepts
reacted conjugate from the conjugate pad. The nitro-
cellulose membrane then binds the reaction products to
the two capture areas that each contain two capture
bands that are immobilized onto the nitrocellulose
membrane. The first pair of bands consists of immobi-
lized bovine serum albumin-cortisol. The second pair of
bands is a combination of binding molecules that
scavenge any conjugate that does not bind to the bovine
serum albumin-cortisol bands. An absorption pad then
accepts fluid from the nitrocellulose membrane, provid-
ing the capillary engine to continuously pull fluid
through the strip.
The second major component is LIAM which is a
fluorescent LFT cassette reader with onboard capa-
bility to analyze and transfer cortisol data via blue-
tooth connection. After the LFT strips are exposed to
the sample, the VerOFy test housing is inserted into
the LIAM reader because the test housing is specifi-
cally engineered to expose the nitrocellulose mem-
brane on the LFT strip to exciting UV light in a precise
orientation to ensure reproducible image analysis. The
LIAM uploads test information, including lot-to-lot
calibration curve parameters by reading a QR code on
E.A. Shirtcliff et al.
]2015 3
the housing. The LIAM is also equipped with a digital
temperature sensor that allows kinetic temperature
corrections to be made within the operating range of
181Cto301C because LFT is known to be sensitive to
temperature. Ultimately, the LIAM will generate an
algorithm to perform the quantitation to report
cortisol scores in units of picogram per milliliter with
data transferred to a computer or smartphone. At
present, results are reported as test-to-reference ratio
(T/R) in which low ratio values indicate cortisol near
zero and higher ratio scores indicate greater binding in
the secondary capture zone relative to the primary
capture zone on the strip. That is, binding of cortisol
to the primary zone is displaced and captured by the
secondary band when cortisol is high (Figure 1).
Statistical Analysis
First, we examined performance specifications of
the assay by using ttests and the CV. Correspondence
across assay type was calculated as Pearson bivariate
correlations and then Fisher R-to-Ztransformation to
determine whether the magnitude of one correlation
was significantly larger than another. Finally, a series
of linear regressions relied on the R
2
to elucidate how
much variance in LFT or Passive Drool, respectively,
were accounted for by the other assays. We then
added collection time or high-versus-low concentra-
tion, respectively, to the linear regression to determine
whether these explained or changed the correspond-
ence across assays.
RESULTS
Performance Specifications
Time Course
To determine the time course of the LFT, that is the
optimal time to read the cortisol scores after sample
collection, saliva samples were run through our
collection and purification system and read at
5-minute intervals. The rise in cortisol concentrations
as the sample flows down across the nitrocellulose
membrane and binds to cortisol is shown in Figure 2.
Stability appeared to be achieved at 20 minutes after
collection. To probe further, a series of ttests were
calculated in which samples assayed before a time
threshold were compared with samples assayed after a
time threshold. Samples assayed at 10 minutes, (t¼
4.7; Po0.002) and 15 minutes (t¼2.8; Po0.025)
were significantly lower than the samples assayed
later. The sample assayed at 20 minutes (t¼1.91;
P¼0.10) and 25 minutes (t¼1.35; P¼0.22) and
30 minutes (t¼0.97; P¼0.36) were not significantly
different from one another, suggesting stability in con-
centration was achieved at 20 minutes. Hereafter,
all LFT is reported according to a read time of
20 minutes.
Variation
Performance specifications for LFT cortisol were
good. Of the possible 55 LFT devices, 4 failed quality
control (QC) specifications (7.2%) on the basis of area
of the strip or image brightness.
Figure 1. Illustration of the LIAM reader and test
housing in which the VerOFy LFT strips
are integrated, as well as the Super SAL
Extra Collection Device. LFT ¼lateral
flow test; LIAM ¼Litebox Image Ana-
lysis Module.
2.5
2
1.5
1
0.5
0
15
10
Time (in Minutes) before Reading LFT Cortisol
T/R Ratio for Cortisol
15 20 25 30 35 40
Replicate 1
Replicate 2
Mean
Figure 2. Time course of LFT cortisol suggests
the optimal read time is 20 minutes
after collection, but stable reads are
achieved earlier. LFT ¼lateral flow
test; T/R ¼test-to-reference.
Clinical Therapeutics
4 Volume ]Number ]
Strips were selected apriorifor quality control on
the basis of total area of the strip and image brightness.
All strips were developed with a saliva pool purified
with Super SAL and run five times by using strips from
the same lot (CORT140528CB-2). The interassay CV
was excellent, with 6.99% variation across the five
replicates. The interassay CV for low QC strips was not
acceptable, given the low area of the strip and poor
light quality (49.15%), indicating the importance of
high QC. The reading for one replicate run to illustrate
the calculation of the T/R is shown in Figure 3.
To determine the lower limit of detection, the
precision of read was measured with a purified saliva
pool that contained low cortisol in T/R units on the
basis of five bright readings. A calculated %CV was
determined to be 7% (ie, SD of 0.032), the lowest
concentration of cortisol that can be distinguished
from zero is 0.513 T/R (2 SDs from zero T/R). With
the use of our current algorithm as a reference, this
corresponds to 0.91 ng/mL (ie, 2.51 nmol/L), which is
well within the expected range of salivary cortisol
with the use of the IBL EIA kit.
Participant Characteristics
For the human saliva component, participants in-
cluded 32 adults (15 men) between the ages of 18 and
36 years (27.5 [4.704] years). Participant ethnicities
included white (65.6%), Hispanic (18.8%), African
American (3.1%), Asian or PacificIslander(3.1%),and
other (6.3%). For all of the saliva samples collected,
only 1 sample of 51 (2%) fell below the lower limit of
detection of 0.91 ng/mL (ie, 2.51 nmol/L).
Correspondence with EIA
Correspondence across assay types is based on 55
samples for the immunoassays and 51 samples for
LFT cortisol. Bivariate correlations of LFT with other
methods were highly significant (R¼0.872 with
Super SAL; R¼0.739 with Passive Drool; both,
Po0.0001). With the use of Fisher R-to-Ztrans-
formation, we found that LFT indicated a trend for a
better correlation with Super SAL than with Passive
Drool (Z¼1.93, P¼0.054). The intercorrelation of
Passive Drool with Super SAL was highly significant
(R¼0.798 with Super SAL, Po0.0001). With the
use of Fisher R-to-Ztransformation, we determined
that the correlations of Passive Drool with Super SAL
or LFT were comparable in magnitude (Z¼0.24, P¼
0.47). These intercorrelations are shown in Figure 4.
A series of linear regressions were then run in
which Passive Drool and then Super SAL predicted
LFT cortisol to further determine whether Super SAL
or Passive Drool was best related to LFT. Passive
Drool significantly predicted LFT cortisol (β¼0.74,
Po0.0001), explaining 55% of the variance in LFT
Example LIAMTM output used to
generate T/R Ratio
Density (pixels)
Distance (pixels)
0 100
120
100
80
60
40
20
200 300 400 500 600 700
Figure 3. Example LIAM output used to generate
T/R ratio. Like other assays, initial
results are reported in optical density
and then later converted to concentra-
tion values. LIAM ¼Litebox Image
Analysis Module; T/R ¼test-to-
reference.
2LFT T/R
Ssal
1.8
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
1.6
1.4
1.2
1
0.8
0.6
0.4
0 0.5 1 1.5
SuperSal Cotisol
Passive Drool Cortisol n
g
/mL
LFT Cortisol T/R Ratio
2 2.5 3
Figure 4. Intercorrelations between techniques
for measuring cortisol are high. Both
Passive Drool and Super SAL cortisol
are obtained with a commercially
available assay from IBL; LFT cortisol
was obtained within 20 minutes of
assay start. LFT ¼lateral flow test;
Ssal, Super SAL; T/R ¼test-to-
reference.
E.A. Shirtcliff et al.
]2015 5
cortisol (F[1,49] ¼58.9, Po0.0001). When Super
SAL was added as a predictor, an additional 22% of
the variance in LFT cortisol was explained (F[1,48] ¼
44.49, Po0.0001), suggesting Super SAL is related
to LFT cortisol beyond the correlation with Passive
Drool. More specifically, although Super SAL pre-
dicted LFT cortisol independently of Passive Drool
(β¼0.79, Po0.0001), Passive Drool no longer
contributed independently to the prediction of LFT
cortisol (β¼0.10, P¼0.40). Conversely, we found
that Super SAL alone explained 76% of the variance
in LFT cortisol, (F[1,49] ¼155.82, Po0.0001), but
Passive Drool did not add further in the prediction of
LFT cortisol, explaining 0.4% additional variance in
LFT cortisol (F[1,48] ¼0.73, P¼0.4). These findings
suggest that LFT cortisol is related to other methods
of measuring cortisol and may be best related to
Super SAL cortisol that shares a collection device
methodology.
Given that Passive Drool may be considered the
gold standard, we then determined which method was
best correlated with Passive Drool cortisol. We con-
ducted a parallel series of linear regressions to see
whether Super SAL or LFT best correlated with
Passive Drool. Beyond the 55% of the variance in
Passive Drool cortisol explained by LFT cortisol,
Super SAL explained an additional 11% of the
variance in Passive Drool (F[1,48] ¼15.29, Po
0.0001), and only Super SAL remained an independ-
ent predictor of Passive Drool for Super SAL (β¼
0.68, Po0.0001) and for LFT (β¼0.15, P¼0.4).
LFT cortisol did not explain the variance in Passive
Drool cortisol beyond Super SAL (R
2
¼0.5%,
F[1,48] ¼0.73, P¼0.4). Findings suggest Passive
Drool cortisol is significantly related to LFT cortisol,
but Super SAL explains additional variance in corti-
sol beyond the LFT method perhaps related to the fact
that both Passive Drool and Super SAL were assayed
with the same IBL assay. Taken together, all three
methods are highly intercorrelated, but Super SAL
may be uniquely related to the other technologies,
likely through shared collection devices (ie, LFT) or
assay techniques (ie, Passive Drool).
To determine whether intercorrelations signifi-
cantly differed between morning and afternoon/eve-
ning collection times (correspondence of LFT with
Super SAL was R¼0.857 in the morning and R¼
0.719 in the afternoon/evening; correspondence of
LFT with Passive Drool was R¼0.567 in the
morning and R¼0.729 in the afternoon/evening; all
Ps o0.05), we conducted a series of linear regressions
in which the main effect of assay type and time
(morning or afternoon/evening) were included in step
1, and the interaction between assay type and time of
day was added in step 2. With Passive Drool as the
outcome, we did not find that the intercorrelation with
LFT differed by time of day (F[1,47] ¼0.54, P¼
0.47) nor did we find that the intercorrelation with
Super SAL differed by time of day (F[1,51] ¼1.0, P
¼0.32). Similarly, we did not find that the intercor-
relation of LFT with Super SAL differed by time of
day (F[1,47] ¼0.03, P¼0.87). Findings suggest that
the intercorrelations across assay type did not system-
atically change across the day.
Parallel analyses contrasted specifically with low-
versus-high cortisol concentrations across each assay
type by using a median split. We did not find that the
intercorrelation of Passive Drool with LFT (F[1,47] ¼
0.29, P¼0.59) or Passive Drool with Super SAL
(F[1,51] ¼2.24, P¼0.14) or LFT with Super SAL
(F[1,47] ¼0.73, P¼0.4) differed by low-versus-high
concentrations, respectively. In summary, these three
assay types are highly intercorrelated, and the magni-
tude of their association does not systematically vary
by time of day or concentration.
DISCUSSION
The biomarker cortisol is frequently investigated in
psychobiological research, with hundreds of studies
across the past few decades suggesting its utility. A
critical barrier in the stress field is that current
immunoassay methods do not provide reportable
results for cortisol for days to months after collection.
Failure to obtain time-sensitive information is espe-
cially problematic for this hormone because cortisol
constantly changes to allow the person to efficiently
encode and filter salient environmental cues.
44
With
the use of LFT and a portable LIAM we were able to
assay cortisol reliably within 20 minutes with strong
assay performance specifications and correspondence
with other methods.
Real-time cortisol measurement has the potential to
advance the field of stress physiology. Real-time
technology with other psychobiological measures
(eg, blood pressure, heart rate, or skin conductance)
indicate the tremendous effect that real-time results
can have on the stress field, including permitting
biofeedback studies,
46
clinical trials to improve
Clinical Therapeutics
6 Volume ]Number ]
relaxation,
47
and enhancing cardiac diagnosis and
clinical outcomes.
48
We set out to confront the time-
sensitivity barrier with the HPA axis because these
systems can be dissociated.
49
We believe the field is
poised to consider new HPA biomarkers, given the
powerful effect of advances in cortisol technology (eg,
hair cortisol
45
).
Obtaining salivary cortisol scores within 20 mi-
nutes of sample collection is advantageous because
this time course matches well with the time course of
peak cortisol reactivity after an acute stressor and
precedes the time in which behavioral effects of
cortisol are anticipated
23
or before negative feedback
emerges.
50
Practically speaking, this technology may
be useful for ensuring that the person is no longer
stressed by laboratory arrival before initiating a
targeted stressor. More interestingly, this techno-
logy may open the possibility of identifying stress
responders while still within the laboratory. Success-
ful laboratory stressors frequently trigger a stress
response in 50% to 70% of participants.
13,17
Even
for robust laboratory challenges, it is problematic
when a large percentage of persons are nonrespond-
ers. Some persons may appear as nonresponders when
they appraise the challenge, but successfully regulate
responsivity before crossing the relatively high stress
threshold of the HPA axis.
51
Conversely, it may be
maladaptive for some persons to appear as
nonresponders.
52
In this case, failure to find an HPA
stress response signifies that the person was
hyporesponsive to stress, with a low capacity to
mount a stress response even in situations that call
for it. This lack of malleability in the HPA axis is
problematic because such persons would not be able
to recalibrate their physiologic functioning to meet the
demands of a changing environment, increasing the
person’s risk of stress-related diseases
53
and
problems.
54
Early identification of nonresponders
within 20 minutes is an important step toward
distinguishing between these types of nonresponders
and further advancing the understanding of psycho-
pathologic risk.
The time course of 20 minutes of delay is typical of
LFT,
38
with shorter read times for qualitative results
and longer read times for quantitative results.
36
We
recognize that shorter delays may be desirable while
still maintaining quantitative results as for the VerOFy
system. Future experiments will determine whether
earlier readings on the LIAM produce cortisol scores
that can be reliably extrapolated to inform the
person’sfinal cortisol reading. It is possible at
present that each sample could be read multiple
times, beginning as early as 5 minutes after
collection and then finalized by 20 minutes after
collection. In the research setting, for example, this
possibility would allow stressors to be modified
during the challenge after a nonresponder is
identified so that the experimenter could enhance the
acute challenge. A handful of studies has explored
how modifications to social evaluative threat
51,55–59
or uncontrollability
57,60,61
can be experimentally
adjusted to influence rates of cortisol responsivity.
The next step is to implement such modifications with
information about the person’s current physiology
before initiation of a modification.
Correspondence of LFT cortisol with Passive Drool
and Super SAL cortisol scores obtained with the IBL
commercial assay was good with correlations near or
above R¼0.80. Future analyses will determine
whether similar correspondence is obtained with mass
spectrometry and other commercially available assays.
Much of the degradation in correspondence was
introduced before LFT, but this was necessary, given
that LFT is sensitive to sample viscosity and partic-
ulates. With traditional assays, particulates in saliva
are reduced by the first freeze-thaw cycle, centrifuga-
tion, and (in some cases) dilution or extraction. With
the LFT, none of these initial steps are required to
avoid the time delay in protocols that these steps
necessitate (ranging from 10 minutes to 2 days).
Indeed, costly laboratory equipment such as ultracold
freezers or centrifuges are not needed, which opens a
possibility of sample collection and assay in a range of
settings without access to refrigeration or reliable
shipping.
62,63
The current methodology requires the
portable fluorescence LIAM reader (whose cost is on
par with an inexpensive laptop computer) to obtain
results but no further large equipment. This technol-
ogy thus reduces cost by eliminating the need for
costly laboratory equipment, shipping, and storage.
Moreover, cortisol is currently analyzed at substantial
cost per sample, regardless of its utility. This practical
burden is large, given that stress protocols fail in 30%
to 50% of participants.
13,17,64
Real-time analytical
information about this stress biomarker has the ability
to exert a powerful effect on the stress field. Although
final costs are not yet determined, we anticipate that
the cost of VerOFy cortisol will be substantially
E.A. Shirtcliff et al.
]2015 7
cheaper than current laboratory-based methods that
are typically $12 to $25 for external laboratories.
The present study has several limitations. First, we
recognize that correspondence of LFT with tradi-
tional assays is not perfect, and further validation
may be desirable on a study-by-study basis. There-
fore, the collection device was designed to allow for a
confirmation specimen to be obtained and shipped to
a laboratory, if desired. Second, we recognize the
inherent variability in results and ongoing calibration
of the algorithm to convert LTF cortisol to stand-
ardized units, and, consequently, we recommend that
the device is used for research purposes at this time.
Indeed, we believe that a major advantage of this
technology is that it is being created through collab-
oration between researchers and LFT experts to
create a device that best accommodates the needs
and desires of the research community. This diverges
from the traditional applications of LFT. Nonethe-
less, we acknowledge that at present the technology is
not yet likely to be applicable to diagnostic or treat-
ment adherence domains, such as for Addison syn-
drome or Cushing disease, in which laboratory- or
clinic-based approaches are more widely ac-
cepted.
32,65
Third, time-course data require having
the portable LIAM on site. This was not necessary to
obtain correspondence data in the present study;
nevertheless, planned studies will obtain cortisol data
in real time across different sites. Finally, future
directions include extension of this technology to
other biomarkers frequently investigated in saliva.
Regardless of these limitations, we believe that meas-
urement of this time-varying stress biomarker can be
enhanced through targeted exploration of its time-
sensitive nature. The advantage of obtaining cortisol
values within 20 minutes of saliva sample collection
represents an exciting new methodological direction
with the potential to contribute to conceptually
effective research directions.
ACKNOWLEDGMENTS
We thank the Stress Physiology Innovative Team
(SPIT laboratory) for all their help as well as the
employees at Oasis Diagnostics and Middleton
Research.
CONFLICTS OF INTEREST
This research was supported by a phase I (R43AT
006634) and phase II (R44 AT006634) Small Business
Innovative Research Award to Dr. Slowey. Dr. Shirtcl-
iff does not have a financial stake in the product
and sits as a volunteer on the board of advisors of
Oasis Diagnostics. Drs. Buck, Laughlin, Cole, and
Slowey are owners or employees of Oasis Diagnostics,
the company developing the VerOFy technology.
Dr. Shirtcliff collected all samples and conducted all
statistical analyses to ensure the analyses were inde-
pendent of the Oasis team. The authors have indicated
that they have no other conflicts of interest regarding
the content of this article.
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Address correspondence to: Elizabeth Shirtcliff, PhD, Iowa State University,
4380 Palmer, Suite 2330, Ames, Iowa 50011-4380. E-mail: birdie@
iastate.edu
Clinical Therapeutics
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