Chapter 41

Saliva and Gingival Crevicular Fluid (GCF) Collection

for Biomarker Screening
Petros Papagerakis, Li Zheng, Doohak Kim, Raed Said, Amber A. Ehlert,
Kevin K. M. Chung, and Silvana Papagerakis

Abstract
Assaying different biological markers (biomarkers) is commonly used to monitor health status and aid in the
diagnosis of diseases. With the recent advances in highly sensitive protein assays, whole saliva (WS) and
gingival crevicular fluid (GCF) appear to be fluids that may contain important biomarkers with various
applications in dentistry and medicine. Herein, we describe the process of GCF and WS sample collection
and preparation for assaying clinically relevant biomarkers in clinical screening trials. Analysis of biomarkers
in WS and GCF represents an easy and practical approach for the diagnosis and screening of different
pathological conditions particularly in epidemiological surveys.

Key words Biomarkers, Saliva, Gingival crevicular fluid, Screening, Diagnostics

1 Introduction

Biological markers (biomarkers) are used to objectively measure

normal biological processes, pathogenic processes, or responses to
a therapeutic intervention. In addition, biomarker screening can be
used to detect a person’s susceptibility to a specific disease or
disease process. Biomarkers have been traditionally analyzed in
body fluids such as blood, urine, and cerebrospinal fluid. With the
recent advances in highly sensitive protein assays, whole saliva
(WS) and gingival crevicular fluid (GCF) appear to be potential
fluids that may contain important biomarkers with various applica-
tions in dentistry and medicine [1–4]. One main advantage of
biomarker assays is their ability to detect changes before clinical
signs or symptoms become obvious. For example, analysis of
dentin- and cementum-specific biomarkers can be utilized to
detect orthodontic root resorption before radiographic signs are
noticed [5, 6].

Petros Papagerakis (ed.), Odontogenesis: Methods and Protocols, Methods in Molecular Biology, vol. 1922,
https://doi.org/10.1007/978-1-4939-9012-2_41, © Springer Science+Business Media, LLC, part of Springer Nature 2019

549
550 Petros Papagerakis et al.

Whole saliva is the fluid that is found in the oral cavity. It

contains secretions from the major and minor salivary glands as
well as non-salivary components. These non-salivary components
include gingival crevicular fluid, nasal and bronchial secretions,
serum and blood derivatives from wounds, desquamated epithelial
linings, food components, and microorganisms that reside in the
oral cavity [7]. Gland-specific saliva can be collected to diagnose
pathology specific to one of the major salivary glands, whereas WS
is collected to reflect systemic conditions. Indeed, several types of
inflammatory biomarkers associated with both oral diseases and
systemic diseases have been detected in WS, such as interleukins
1β, 6, and 8 (IL-1β, IL-6, and IL-8), tumor necrosis factor-α
(TNF-α), and matrix metalloproteinases 8 and 9 (MMP-8
and MMP-9) [8–12]. Moreover, an increasing number of specific
molecular markers for different diseases, such as oral and breast
cancer, cardiovascular diseases, and human immunodeficiency virus
(HIV), are being identified [12–15]. Hence, salivary proteomics
has been a growing topic of research due to saliva’s ease of collec-
tion and its ability to be a diagnostic tool for systemic diseases.
Saliva can be collected with or without stimulation [16]. Stimulated
and unstimulated saliva produce different flow rates and have dif-
ferent protein compositions and pH [17]. Therefore, it is impor-
tant to use only one type of collection technique when performing
studies. Salivary flow rate can be affected by the degree of hydration
of the subject and olfactory stimulation and also has a circadian
rhythm with peak flow in the afternoon [16]. Therefore, it is
important to control these variables when collecting saliva samples.
Gingival crevicular fluid was first recognized in 1899 as a fluid
that emerges between the surface of the tooth and the epithelial
integument [18]. In healthy sites, GCF production is thought to be
a serum transudate due to the passage of fluid from the surrounding
capillaries into the sulcus [19]. In inflamed or mechanically stimu-
lated sites, GCF production is an inflammatory exudate thought to
act as a protective mechanism to flush away bacteria and transport
antibacterial substances [20]. Gingival crevicular fluid contains
many substances such as blood serum and plasma components,
periodontal epithelium, inflammatory and immune cells, enzymes,
cytokines, interleukins, and products of tissue breakdown [5]. In
healthy sites, the protein concentration is similar to interstitial fluid,
whereas in inflamed sites, GCF protein concentration is similar to
that of serum [21]. Gingival crevicular fluid is an excellent source
for assaying specific dental biomarkers of oral conditions due to its
ability to be site specific (e.g., assaying for abnormalities in dental
tissues in one tooth).
Several techniques have been used to collect GCF [22–24].
The most common and practical method entails the use of absor-
bent filter paper strips to collect the GCF samples. The strip can be
inserted just at the entrance of the crevice, to the base of the pocket,
 Biomarkers in Saliva and GCF 551

or until minimum resistance is felt [25]. The advantages of this

technique include being quick and easy to use, being able to apply it
to individual sites, and causing minimal trauma when used cor-
rectly. The amount of GCF collected may be measured by various
means; however, the most common and accurate method used
today is the electronic method. This method measures the amount
of GCF collected on Periopaper® by using an electronic transducer
(Periotron®, Winnipeg, Manitoba). The electronic method is based
on the fact that the flow of electronic current is affected by the
wetness of the paper strip and thus provides a digital readout [26].
Recent advances in proteomic technologies have facilitated the
comprehensive profiling of protein expression in body fluids and
tissues [27]. Applying these proteomic approaches on the readily
accessible saliva samples from patients can aid in early prediction of
the disease status. Indeed, mass spectrometry-based proteomic
approaches have been recently used to identify characteristic saliva
biomarkers for multiple diseases such as Sjögren’s syndrome and
gastric cancer [28, 29].

2 Materials

2.1 Whole Saliva Materials needed:

Sampling and
1. 14 mL polypropylene 17
100 mm Falcon tube.
Preparation
2. Disposable funnel.
3. Wet ice in Styrofoam cup.
4. Distilled water.
5. Aprotinin (1 mg/mL in water).
6. Phenylmethanesulfonyl fluoride (PMSF) (200 mM in MeOH).
7. Labeling machine/label maker for cataloguing tubes (optional;
can use fine-tipped Sharpie marker).
8. Pipettors (for 20 μL and 1000 μL).
9. Pipettor tips (for 20–200 μL and 1000 μL).
10. 1.5 mL microfuge tubes.

2.2 Gingival Materials needed:

Crevicular Fluid
1. Periopaper® gingival fluid collection strips (OraFlow,
Sampling and
Smithtown, NY).
Preparation
2. Clock/timer able to record the seconds.
3. Labtop cooler or dry ice container to place tubes containing
samples.
4. Clinical exam kit with cotton forceps.
5. Cheek retractors and bite block.
552 Petros Papagerakis et al.

6. 2
2 gauze squares.
7. Cotton rolls.
8. Permanent fine-tip marker or label maker.
9. 1.5 mL microfuge tubes.
10. 12
75 mm polystyrene culture tube with caps (Fisher Scien-
tific, Pittsburgh, PA).
11. Pipettors (for 20, 100, 200, and 1000 μL).
12. Pipettor tips (for 20–200 μL and 1000 μL).
13. GCF extraction buffer (24.5 mL phosphate-buffered saline
(pH 7.4), 125 μL phenylmethylsulfonylfluoride (PMSF)
(200 mM in MeOH), 250 μL aprotinin (1 mg/mL in water),
and 83.5 μL of 30% human serum albumin.

2.3 Protein Profiling 1. Dithiothreitol (DTT).

2. Iodoacetamide.
3. Acetone.
4. 2% sodium dodecyl sulfate (SDS).
5. Tris-HCl (pH 8).
6. Qubit fluorometer.
7. 10% Bis-Tris protein mini-gel.
8. MOPS buffer system.
9. Automated in-gel digestion system.
10. Ammonium bicarbonate.
11. Acetonitrile.
12. Trypsin.
13. Formic acid.
14. Nano LC-MS/MS with an HPLC system interfaced to a mass
spectrometer.

3 Methods

3.1 Whole Saliva Carry out all procedures at room temperature unless otherwise
(WS) Collection, specified.
Processing, Storage,
and Preparation
3.1.1 Whole Saliva 1. Prepare tubes for specimen collection:
Collection (a) Label tube with subject information.
(b) Fill Styrofoam cup with wet ice, and place Falcon tube in
cup surrounded by the ice.
(c) Place a disposable funnel into the Falcon tube.
 Biomarkers in Saliva and GCF 553

2. Subjects should abstain from brushing their teeth, chewing

gum, eating, or drinking for at least 1.5 h before the visit (see
Note 1).
3. During the collection period, the subject shall be seated
straight up with eyes open and head tilted slightly forward.
4. The subject will hold the Styrofoam cup with the Falcon tube
inside on wet ice during the collection process.
5. The subject will be instructed to minimize orofacial move-
ments to minimize influence on salivary flow (see Note 2).
6. Give the subject distilled drinking water, and ask that they rinse
their mouth out well for 1 min. The subject can then expecto-
rate or swallow the water. Wait 90 s before beginning collection
process. Subject can swallow saliva normally during this time.
7. When starting collection, invite the subject to swallow any
remaining saliva but to abstain from swallowing for the remain-
der of the collection process. The subject is asked to allow the
saliva to accumulate on the floor of the mouth until enough
saliva has pooled, so that they are able to tilt their head forward
to allow the saliva to drip into the funnel.
8. Collection is complete once 3 mL of whole saliva is collected.
This usually takes 10–15 min. Leave the sample on ice, and
perform the processing and storage procedure as soon as pos-
sible (see Note 3). With all the samples still on ice, the protease
inhibitor cocktail containing 80 μM of aprotinin, 4 mM of
bestatin hydrochloride, 1.4 mM of N-trans-epoxysuccinyl-L-
leucine 4-guanidinobutylamide (E-64), 2 mM of leupeptin,
1.5 mM of pepstatin A, and 104 mM of 4-2-aminoethylbenze-
nesulfonyl fluoride hydrochloride (AEBSF) (Sigma-Aldrich,
catalog # P8340, St. Louis, MO) is to be added immediately
chairside to the cell-free WS at a 1:100 dilution to inhibit the
degradation of the proteins in the sample. After vortexing for
30 s, the sample is divided into 500 μL aliquots and stored at
80  C until further analysis.

3.1.2 WS Sample 1. Measure the total volume of saliva within the Falcon tube.
Processing and Storage 2. Per 2 mL of saliva, add the following to inhibit protein degra-
dation (see Note 4):
(a) 20 μL aprotinin (1:100 dilution of 1 mg/mL stock; stored
at 20  C, thaw before use)
(b) 10 μL PMSF (1:200 dilution of 200 mM stock; stored at
20  C but does not freeze)
3. Mix tube well by inverting end over end 10–15 times to mix
completely; then divide into 500 μL aliquots in 6 pre-labeled
1.5 mL microfuge tubes.
554 Petros Papagerakis et al.

4. Transfer tubes into storage boxes and label box accordingly.

5. Make a log sheet with a grid to match each storage box. This
greatly facilitates future sample location and retrieval.
6. Store box in 80  C freezer.

3.1.3 WS Sample 1. When ready for analysis, thaw tube on wet ice.
Preparation 2. Once samples are fully thawed, homogenize samples for 5 s
with an ultrasonic tissue homogenizer (at setting 5). This will
break up the mucous portion of the whole saliva and will make
collection of the supernatant much easier (see Note 5).
3. Centrifuge the samples for 15 min at ~10,000 g at 4  C to
remove insoluble material and obtain a cell-free supernatant.

3.2 Gingival Gingival crevicular fluid (GCF) contains many components that can
Crevicular Fluid (GCF) be used as diagnostic aids. GCF samples are taken from the mesial
Sample Collection, or distal aspect of teeth in humans. The sampling is performed after
Processing, Storage, completing a plaque index score and in order to avoid mechanical
and Preparation irritation and/or bleeding from PD measurements with the peri-
odontal probe. Attention must be given such that the sample is
relatively free of plaque and there is no contamination by saliva and
blood. Carry out all procedures at room temperature unless other-
wise specified.

3.2.1 GCF Collection and 1. Label microfuge tubes accordingly prior to taking samples with
Storage subject info, time point/date, study name/initials, and
sample site.
2. Cheek retractors and a bite block are placed, and the area
around each sample site is isolated using cotton rolls (see
Note 6). The area is dried with gauze and a quick blast of air
from the air/water syringe making sure not to direct any air
flow into the gingival sulcus.
3. Using cotton forceps, hold the orange nylon portion of the
Periopaper® so that the white cellulose portion of the methyl-
cellulose strip can be carefully inserted into the gingival crevice
until a slight resistance is felt (Fig. 1). Each GCF strip will
remain in position for a total of 60 seconds (see Note 7) before
immediate removal (see Notes 8 and 9).
4. Following GCF collection, each strip is placed into its
corresponding labeled microfuge tube (Fig. 2) and is immedi-
ately placed onto dry ice for transport to the laboratory and
storage in a 80  C freezer until analysis (see Notes 10
and 11).

3.2.2 GCF Sample Proteins within the harvested crevicular fluid are extracted from the
Preparation GCF strips using an elution method adapted from Giannobile et al.
[26], involving a series of washes and centrifugations. The elution
 Biomarkers in Saliva and GCF 555

Fig. 1 Placement of methylcellulose strips into gingival sulcus. Using cotton

forceps, hold the orange nylon portion of the Periopaper® so that the white
cellulose portion of the methylcellulose strip can be carefully inserted into the
gingival crevice approximately 1 mm or until slight resistance is felt. Optimally
each GCF strip will remain in position for a total of 60 s before removal (The GCF
strip can be removed after 60 s or less (10–30 s), depending of the GCF flow
variables). Because they are site specific, GCF collection studies are very useful
in measuring the levels of dental biomarkers for a particular tooth

Fig. 2 Example of the GCF amount collected in one case. The volume can be
determined using an electronic measuring device, the Periotron® (Winnipeg,
Manitoba), which measures the electrical current flow of the wetted strips
556 Petros Papagerakis et al.

Fig. 3 Securing GCF strip to the side of the culture tube. After adding GCF elution
buffer, the strip is secured at the top of a 12
75 mm polystyrene culture tube
using a cap to hold the orange portion of the strip in place. Ensure that the white
portion of the strip lays flat against the wall of the tube

buffer used for GCF protein extraction is to be made fresh and kept
on wet ice throughout the entire extraction process to inhibit
protease activity (see Note 12).
Procedure:
1. The GCF strips are transported on wet ice from the 80  C
freezer, thawed at room temperature, and kept on wet ice
during the entire procedure.
2. A total of 11 μL of extraction buffer is pipetted directly onto
the cellulose (white) portion of each Periopaper strip. We use
11 μL because there is approximately 1 μL of elution buffer
that will remain on the strip even after centrifugation—leaving
10 μL of washed fluid at the bottom of the tube. The strip is
then secured at the top of a 12
75 mm polystyrene culture
tube using a cap to hold the orange portion of the strip in place.
Ensure that the white portion of the strip is laying flat against
the wall of the tube (Fig. 3).
3. The tubes are then centrifuged at 2000 rpm for 5 min at 4  C.
This process is repeated four additional times to yield 50 μL
total volume.
4. This entire product of 50 μL is then transferred to a sterile
1.5 mL microfuge tube. Each tube is labeled as previously
described, to contain the necessary information.
5. The strips are then soaked in 60 μL of elution buffer and
centrifuged at 2000 rpm for 5 min at 4  C as a final wash to
collect any remaining protein. Approximately 50 μL can be
 Biomarkers in Saliva and GCF 557

recovered from this wash and is transferred to the previously

collected 50 μL to yield a total value of 100 μL. The tubes are
then placed in a 80  C freezer until ready for analysis (see
Notes 13–15).

3.3 Protein Profiling Protein profiling of both WS and GCF samples can be conducted
for WS and GCF by custom protein extraction, sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE), robotic in-gel
digestion with trypsin, and liquid chromatography-mass spectrom-
etry (LC-MS/MS), followed by database searching and reporting
for protein identification.

3.3.1 Protein Extraction 1. Thaw samples on ice.

2. Remove 500 μL of each sample, and clarify it by centrifugation
at 10,000 g for 10 min.
3. Reduce the sample with 10 mM dithiothreitol at 25  C for
30 min.
4. Alkylate the sample with 15 mM iodoacetamide at 25  C for
45 min (see Note 16).
5. Concentrate the proteins by adding 4
volumes of 20  C
acetone, and incubate overnight at 20  C.
6. Collect precipitated proteins by centrifuging for 10 min at
5000
g and 4  C.
7. Wash the protein pellets twice with 20  C acetone (see
Note 17).

3.3.2 Protein 1. Suspend the pellets in 55 μL of 2% SDS and 50 mM Tris-HCl.

Quantification, SDS-PAGE, 2. Quantify the protein yield using a Qubit fluorometry assay.
and Automated In-Gel
3. Process 10 μg of each sample by 5 cm SDS-PAGE using a 10%
Digestion
Bis-Tris protein mini-gel and the MOPS buffer system.
4. Excise the mobility region into 10 equally sized bands for
in-gel digestion.
5. Process each band by using a robotic in-gel digestion system
with the following protocol:
(a) Washing with 25 mM ammonium bicarbonate followed
by acetonitrile
(b) Reduction with 10 mM dithiothreitol at 60  C followed
by alkylation with 50 mM iodoacetamide at RT
(c) Digestion with trypsin at 37  C for 4 h
(d) Quenching with formic acid (the supernatant can be ana-
lyzed directly without further processing)
558 Petros Papagerakis et al.

3.3.3 Mass Spectrometry Analyze the tryptic digest by nano LC/MS/MS with an HPLC
and Data Processing system interfaced to a mass spectrometer (we used a nano LC-MS/
MS with a Waters NanoAcquity HPLC system interfaced to a
ThermoFisher Q-Exactive).
Procedure:
1. Load the peptides on a trapping column, and elute them over a
75 μm analytical column at 350 nL/min.
2. Pack both columns with suitable resin (see Note 18).
3. Operate the mass spectrometer in data-dependent mode, with
the Orbitrap operating at 60,000 FWHM and 17,500 FWHM
for MS and MS/MS, respectively.
4. Select the 15 most abundant ions for MS/MS.
5. Using a copy of the resulting Mascot file, search the data with
the following parameters:
Enzyme: Trypsin
Database: Swiss-Prot human (concatenated forward and reverse
plus common potential contaminants)
Fixed modification: Carbamidomethyl (C)
Variable modifications: Oxidation (M), acetyl (N-term), pyro-
Glu (N-term Q), deamidation (N/Q)
Mass values: Monoisotopic
Peptide mass tolerance: 10 ppm
Fragment mass tolerance: 0.02 Da
Max missed cleavages: 2
6. Parse the Mascot DAT files into the scaffold algorithm for
validation, filtering, and creation of a nonredundant list per
sample (see Notes 19 and 20).

4 Notes

1. Due to diurnal variation of analytes in saliva, subjects should

always have their visits scheduled at a given time of day.
2. The subject should not swallow and should not speak during
the collection process.
3. To familiarize the subject with this method, it is wise to run a
1–2 min trial collection prior to the actual collection period.
4. Another protease inhibitor cocktail containing 80 μM of apro-
tinin, 4 mM of bestatin hydrochloride, 1.4 mM of N-trans-
epoxysuccinyl-L-leucine 4-guanidinobutylamide (E-64), 2 mM
of leupeptin, 1.5 mM of pepstatin A, and 104 mM of 4-2-
aminoethylbenzenesulfonyl fluoride hydrochloride (AEBSF)
 Biomarkers in Saliva and GCF 559

(Sigma-Aldrich, catalog # P8340, St. Louis, MO) can be used.

20 μL of the cocktail (1:100 dilution of a 1 mg/mL stock;
stored at 20  C, thaw before use) can be added to 2 mL of
saliva. The cocktail must be added immediately chairside to the
cell-free WS at a dilution of 1:100 to inhibit the degradation of
the proteins in the sample. After vortexing for 30 s, the sample
can then be divided into aliquots and stored at 80  C until
further analysis.
5. Be sure to have samples on ice as the homogenization process
will heat the sample up.
6. If present, any supragingival plaque is to be gently removed
prior to sampling.
7. Collection times for GCF samples can vary in the literature.
While the majority of studies reported 30 s, 60 s and 3 min have
been also found in the literature. The reason for this variation
depends on the fluid-flow dynamics of the host. Healthy hosts
have a slower GCF rate, while unhealthy hosts (i.e., periodontal
disease) have faster rates. It is important to note that the
amount of time used for collection should remain constant
throughout a study and constant from patient to patient.
8. Manipulation of the sulcus prior to sampling may create bleed-
ing. It is advised to not manipulate the sulcus prior to taking
GCF samples. If bleeding occurs at the site prior to sampling, it
must be rinsed and cleared away prior to taking the sample.
Blood appearing on the strip can affect the microbiologic
testing results.
9. Multiple samples can be collected at once by sequentially plac-
ing the strips in different sites. This will save time from sam-
pling each site individually and waiting 60 seconds each. For
example, to collect four samples from four different sites (60 s
collection time), a strip can be placed at 0, 15, 30, and 45 s at
site number 1, 2, 3, and 4, respectively. At 60, 75, 90, and
105 s, strips 1, 2, 3, and 4 can be removed, respectively (Fig. 4).
10. Assess the paper strip for over saturation of saliva. If saliva
saturation should occur, wait 90 s and another sample of
GCF may be taken. Retaking of a second sample may occur if
there is a failure to acquire a quality sample on the first attempt.
Examples of this might be that the strip did not fully engage
into the sulcus. If more than one sample in the same site is
necessary, wait 90 s prior to taking another GCF sample. This
allows the GCF flow to return to the sulcus. The same techni-
ques should be utilized on second sampling.
11. It is recommended that since the strips are stored without
stabilizers or protease inhibitors, subsequent analysis is
560 Petros Papagerakis et al.

Fig. 4 Placement of four methylcellulose strips in an orthodontic patient. In this

case, the GCF strips were placed in the sulcus at the mesiobuccal line angles for
a total of 60 s for each maxillary incisor. Four samples here are sampled at once
using the following sequence: at 0-, 15-, 30-, and 45-s intervals, a strip will be
placed at site number 1, 2, 3, and 4, respectively. Another sample was then
retaken at the distobuccal line angles after waiting 90 s between samplings to
allow for GCF flow to return to the sulcus

performed as soon as possible to minimize potential protein

degradation.
12. The elution buffer is best made fresh prior to GCF elution. It is
recommended to make the elution buffer within 24 h of use to
ensure proper elution of the GCF solution. Smaller batches are
made at a time to ensure fresh elution buffer is used. Do not
freeze-thaw the elution buffer for any reason prior to use.
13. GCF elution is best done by eluting one patient at a time, with
all of their time points eluted during the same washing interval.
While eluting one time point in the centrifuge, a second time
point can be eluted by washing at the same time, etc. This
ensures that if for any reason there is a variation in the elution
buffer, the elution buffer composition itself will remain con-
stant for each subject throughout all their time points.
14. Ideally, all patients should be eluted at the same time. How-
ever, due to time constraints, one may wish to do only a few
patients at a time. Minimize the variation in time of elutions for
each patient, and one operator should perform the elution
process to ensure there is no variation in the elution technique.
15. Label each centrifuge tube with tape and a permanent marker
to prevent labels from rubbing off during the elution process,
which may get wet while on ice. Aliquot the final solution
based on the number of protein assay kits you will be running
 Biomarkers in Saliva and GCF 561

(i.e., if 200 μL is needed per ELISA kit, aliquot into 200 μL

amounts) to minimize the freeze-thaw cycles of each solution.
16. It is recommended to incubate the samples in the dark after
alkylation.
17. We also recommend air-drying the pellets to remove residual
acetone.
18. We recommend packing with Jupiter Proteo resin
(Phenomenex).
19. Filter the data using 1% protein and peptide FDR and with at
least two unique peptides required per protein.
20. An example of detailed protein identification data can be found
in an Excel workbook accompanying this chapter, where we
identified the proteins from saliva samples acquired from 10 cli-
ents (numbered: 22640-22649) and divided into 2 groups: RA
group (224640-44) and CO group (22645-49). The Excel file
contains four worksheets:
– Two protein report worksheets that contain the full list of
proteins identified (including known contaminants and
reverse hits) and their molecular weight and spectral counts
(SpC) with a summary of the data
– An NSAF Calc. sheet that contains the conversion to spec-
tral abundance factor (SAF) and subsequent normalized
spectral abundance factor (NSAF). This was based on the
following equation:

NSAF ¼ ðSpC=MW Þ=ΣðSpC=MW ÞN

where SpC is spectral counts, MW is protein MW in kDa,
and N is total number of proteins.
– T-test sheet that contains SpC and NSAF values, the average
of the NSAF values for the two groups RA and CO, and also
a T-test performed using the NSAF data.

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