Periodontology 2000, Vol. 70, 2016, 65–79 © 2015 John Wiley & Sons A/S.
ey & Sons A/S. Published by John Wiley & Sons Ltd
Printed in Singapore. All rights reserved
Clinical and technical
considerations in the analysis of
gingival crevicular fluid
R E B E C C A R. W A S S A L L & P H I L I P M. P R E S H A W
Gingival crevicular fluid is an inflammatory exudate
that flows from the gingival sulcus or periodontal
pocket. Volumes are typically low (microliter quanti-
ties), and generally increase with increasing inflam-
mation in the gingival and periodontal tissues.
Gingival crevicular fluid is a complex mixture of sub-
stances derived from serum and from locally gener-
ated mediators of inflammation and tissue turnover/
breakdown. It also contains cellular components,
such as neutrophils and bacterial components.
Gingival crevicular fluid plays a protective role in
host–bacteria interactions, via a physical protective
effect (dilution of bacteria and their products, plus
outflow of fluid) as well as transportation of antibac-
terial substances into the pocket (1, 22). The quantity
of gingival crevicular fluid flowing from a site is influ-
enced not only by the degree of inflammation in the
tissues but also by the extent of ulceration of the sul-
cular/pocket epithelium (1). It has been debated
whether gingival crevicular fluid represents a transu-
date of gingival interstitial fluid (particularly in
healthy gingival tissues), but once gingival inflamma-
tion is detected clinically, gingival crevicular fluid is
considered a true inflammatory exudate (as reviewed
by Griffiths (13)).
The composition of gingival crevicular fluid is gen-
erally regarded as being reflective of the inflammatory
state of the gingival and periodontal tissues and
has therefore been of great interest to periodontal
researchers who wish to improve understanding of
periodontal pathogenesis and develop novel diagnos-
tic strategies for periodontal diseases. Gingival crevic-
ular fluid was serendipitously discovered in the late
1950s during investigations of the subgingival micro-
biota in the gingival pockets in a dog (4, 21). Studies
of gingival crevicular fluid began in earnest in the
early 1960s (9) and expanded rapidly through the
1970s, with the publication of the landmark mono-
PERIODONTOLOGY 2000
graph on gingival crevicular fluid by Cimasoni in 1974
(7). As technology improved (particularly advances in
analytical techniques for small volumes of biological
fluids), research groups throughout the world focused
on analysis of gingival crevicular fluid as a means by
which to study inflammation in the periodontal tis-
sues. Collection of gingival crevicular fluid was
regarded as a noninvasive and relatively simple pro-
cedure – a statement which may be debated by those
who have substantial experience in collecting, analyz-
ing and interpreting gingival crevicular fluid data.
The advantage of analysis of gingival crevicular
fluid is that it provides site-specific information about
the inflammatory status at any particular periodontal
site (i.e. analysis at the level of the periodontal site
without having to take a biopsy of the gingival tissue).
This may also be regarded as a disadvantage, how-
ever, as unless gingival crevicular fluid is collected
from a large number of periodontal sites (which
would be prohibitively time-consuming and costly in
clinical practice), limited information at the full-
mouth level will be obtained. Collection of gingival
crevicular fluid has been undertaken in the past using
a variety of methods, including gingival washing tech-
niques, capillary tubes or micropipettes, and the use
of absorbent filter paper strips (13, 16). The most fre-
quently used technique in the periodontal literature
is the filter paper method, and this will form the basis
for discussion in this paper. Whilst early researchers
used this method to simply quantify the amount of
gingival crevicular fluid collected from a periodontal
site, for example, using the ninhydrin stain or fluores-
cein labeling methods (25), these techniques pre-
vented subsequent biochemical analysis of the
composition of the collected gingival crevicular fluid
sample. An alternative method for quantifying gingi-
val crevicular fluid volume on filter paper strips was
required, and this niche was filled by the Periotron
65
Wassall & Preshaw
series of instruments (Oraflow Inc., Smithtown, NY,
USA), initially with the Periotron 600, then with the
Periotron 6000 and presently with the Periotron 8000.
Typically, after volume determination (if per-
formed), gingival crevicular fluid samples on filter
paper strips are placed in individual cryotubes (with
or without buffer), snap frozen in liquid nitrogen and
then stored in a !80°C freezer until required for anal-
ysis. As any researcher who has worked with gingival
crevicular fluid will know, there are a large number of
variables in the pathway of analysis (from sampling to
volume measurement, to storage and subsequent
biochemical analysis) and each step must be consid-
ered. It is important to note that evidence to support
any specific ‘recommended’ protocol is generally
lacking, and it is precisely this lack of certainty that
will be discussed and debated in this paper. However,
for all studies involving gingival crevicular fluid, it is
important that comprehensive validation studies are
performed before gingival crevicular fluid sampling,
as part of the research project, to ensure that mean-
ingful results can be generated. Furthermore, a core
principle of scientific reporting is that the methods
sections of research papers should provide enough
detail for an interested scientist working in the same
field to be able to replicate any given study in their
own research center. Regarding studies in the peri-
odontal literature reporting gingival crevicular fluid
data, it is usually the case that insufficient informa-
tion is presented for colleagues to replicate the study
(see below), which can result in confusion. It is there-
fore important that the standard of reporting is
improved in studies in which gingival crevicular fluid
is collected.
Review of reporting methods for
gingival crevicular fluid collection
and analysis
A review of papers describing gingival crevicular fluid
collection and analysis was undertaken to provide
information regarding the current standard of report-
ing in the periodontal literature. Clearly, analysis of
gingival crevicular fluid is important for understand-
ing the underlying mechanisms of periodontitis and
for providing opportunities to develop tools to predict
disease activity. The use of inconsistent methods will
impact on the interpretation of results, prevent
accurate comparison of data between studies and
potentially limit the conclusions that can be made
from a larger body of evidence. Within this context,
66
we considered it useful to assess the quality of the
reporting of methods used to describe gingival crevic-
ular fluid collection, recovery, quantification and
analysis. This was not intended to be a comprehen-
sive or systematic review, although a structured
approach was followed in the search strategy. The
main objective was to review recent literature to iden-
tify trends in the reporting of gingival crevicular fluid
collection and analysis methodology. Given the huge
number of studies that have analyzed gingival crevic-
ular fluid, we restricted our search to studies of
interleukin-1beta in gingival crevicular fluid as an
exemplar.
We utilized the following characteristics to identify
studies using the PICO (Participants, Intervention,
Comparisons, Outcomes) approach. Participants:
male or female subjects undergoing gingival crevicu-
lar fluid interleukin-1beta analysis. Intervention:
undergoing clinical nonsurgical periodontal manage-
ment. Comparisons: before treatment vs. after treat-
ment. Outcomes: studies that reported changes in
gingival crevicular fluid interleukin-1beta levels.
Other parameters in the search included: study
design (comparison studies); length of follow up
(any); language (English); publication status (fully
published); and years considered (2010–2014).
Searches were performed in key electronic databases,
including MEDLINE, Embase, Web of Knowledge and
Trip, and were supplemented by hand searching ref-
erence lists of retrieved articles. The following search
terms and keywords/phrases were used: Periodonti-
tis/therapy, Periodontal disease, inflammation,
Cytokines, Interleukin-1, comparison studies. Ten
articles were identified that reported on gingival
crevicular fluid interleleukin-1beta collection pre-
and post-treatment in this time period. Data were
abstracted, and the key findings are reported in
Table 1.
It is immediately apparent that there is consider-
able variation in the methods used for collection, pro-
cessing and analysis of gingival crevicular fluid
samples by different research groups. It could be
argued that this is to be expected as different groups
use different analytical systems and may have needed
to optimize their protocols for analysis of gingival
crevicular fluid. However, this makes it very difficult
for other researchers to utilize the same methods
and, particularly when reporting of precise methodol-
ogy is vague or incomplete, standardization of meth-
ods of analysis becomes almost impossible. Some of
the analytical parameters that require consideration
include:
 Analysis of gingival crevicular fluid

Table 1. Summary of key data relating to gingival crevicular fluid collection and analysis from published studies in the
periodontal literature

Author, year Gingival crevicular fluid collection Gingival crevicular fluid Analytical technique and reporting
(ref. no.) and volume measurement elution of data

Kinney et al., Samples were obtained from the Gingival crevicular fluid was Commercial array (RayBiotech
2014 (19) mesiobuccal aspect of each tooth eluted by washing Quantibody Human Cytokine
Gingival crevicular fluid was Periopapers from eight Array). Results are expressed as
collected, using a 30-s sampling selected clinical sites with pg/ml but it is not clear if these
protocol, on Periopaper strips 11 ll of proteinase inhibitor were total mediator content per
placed into the sulcus/pocket until (1% aprotinin and 0.5% 30-s sample (i.e. the concentration
light resistance was felt. Volume phenylmethanesulfonyl calculated from the assay itself) or
was quantified using Periotron fluoride) for a total elution concentrations in the gingival
6000 and Periopapers were stored volume of 55 ll per site. crevicular fluid sample (i.e.
dry at !80°C Then, each sample was corrected for the original gingival
centrifuged at 2,000 rpm and crevicular fluid volume according
4°C for 5 min. The eight to the Periotron data). No data
samples were then pooled to were presented on detection limits
obtain a total elution volume of the assay
of 440 ll per patient (8 9 55 ll)
Biyikoglu et al., Samples were obtained from two Gingival crevicular fluid was Commercial ELISA kit (BD
2013 (3) single-rooted teeth with probing eluted by addition of 200 ll Biosciences). Results were
depth >5 mm of phosphate-buffered saline expressed separately as either total
Gingival crevicular fluid was and shaking on an ELISA interleukin-1beta content (pg/two
collected, using a 30-s sampling plate shaker for 45 min (no sites) or concentrations (pg/ll of
protocol, on Periopaper strips information on temperature) gingival crevicular fluid) following
placed into the orifice of the Gingival crevicular fluid conversion based on gingival
gingival pocket samples were used neat for crevicular fluid volume from the
Volume was quantified using the the assay (interpreted to Periotron calibration curve. Data
Periotron 8000 and pooled indicate that the eluate was presented regarding minimum
Periopapers were stored dry at used for assay without detection limit (0.8 pg/ml). No
!40°C further dilution) information was given regarding
how values below the level of
detection were handled
Fiorini et al., Samples were obtained from four Gingival crevicular fluid was Commercial ELISA kit (BD
2013 (12) sites per subject (the four deepest eluted by addition of 200 ll Biosciences Human Inflammatory
pockets) of phosphate-buffered saline Cytokine kit). Results expressed as
Gingival crevicular fluid was and 2 ll of pg/ml but it is not clear if these
collected, using a 30-s sampling phenylmethanesulfonyl were total mediator content per
protocol, on Periopaper strips fluoride (serine protease 30-s sample (i.e. the concentration
placed into the periodontal pocket. inhibitor) and incubated for calculated from the assay itself) or
Volume was quantified using the 30 min. Then, Periopapers concentrations in the gingival
Periotron 8000 and Periopapers were transferred to a second crevicular fluid sample (i.e.
were stored dry, frozen (no tube, containing the same corrected for the original gingival
information on storage solutions, for a further crevicular fluid volume according
temperature was given) 30 min (no information was to the Periotron data). No data
given on temperature). The were presented on the detection
contents of the two tubes limits of the assay
were then combined and
50 ll was used for analysis
Eltas & Orbak, Samples were collected from Gingival crevicular fluid was Commercial ELISA kit (but name/
2012 (10) mesiobuccal sites of four teeth, in eluted at the time of manufacturer not specified). No
test and control quadrants, with collection by placement into units presented with the data, so
probing depths 4–6 mm microcentrifuge tubes not clear what the data represent.
Gingival crevicular fluid was containing 200 ll of No data presented on detection
collected, using a 30-s sampling phosphate-buffered saline, limits of assay.
protocol, on Periopaper strips vortexing for 30 s and
inserted into periodontal pockets centrifugation at 3,000 g for
until resistance felt. Volume was 5 min (temperature not
quantified using the Periotron 8000 stated). Supernatants were
and gingival crevicular fluid was then stored at !80°C
eluted before freezing

67
Wassall & Preshaw
Table 1. (Continued)
Author, year Gingival crevicular fluid collection Gingival crevicular fluid
(ref. no.) and volume measurement
Fentoglu et al., Samples were collected from Gingival crevicular fluid was
2012 (11) mesiobuccal and distopalatal sites eluted at time of collection
at three teeth in each quadrant
Gingival crevicular fluid was
collected, using a 30-s sampling
protocol, on Periopaper strips
placed into the gingival orifice.
Volume was quantified using the
Periotron 8000, and gingival
crevicular fluid was eluted before
freezing
Konopka et al., Samples were collected from the
2012 (20) mesiobuccal site, but the number
of teeth/samples, or whether
pooled, is not clear. Gingival
crevicular fluid was collected, using
a 30-s sampling protocol, on
Periopaper strips placed into the
gingival crevice until mild
resistance was felt. Volume was not
measured. Periopapers were stored
dry at !80°C
Oliveira et al., Samples were collected from the
2012 (26) mesiobuccal aspect of every tooth
in two randomly selected
quadrants Gingival crevicular fluid 30 min, then centrifugation
was collected, using a 30-s
sampling protocol, on Periopaper
strips placed 1–2 mm
subgingivally. Volume was
quantified using the Periotron
8000, and Periopapers were stored
dry at !80°C
Toker et al., Samples were collected from upper
2012 (31) anterior teeth only, but it is not
clear as to the number of teeth/
samples or whether samples were
pooled
Gingival crevicular fluid was
collected, using the 30-s sampling
protocol, on Periopaper strips
placed into the pocket until mild
resistance was felt
Volume was not measured
Periopapers were stored dry at
!80°C
68
elution
by placement into
microcentrifuge tubes
containing phosphate-
buffered saline (volume not
stated), followed by
vortexing and centrifugation
at 1,500 g for 10 min
(temperature not stated).
Supernatants were then
stored at !80°C
Gingival crevicular fluid was
eluted by addition of 500 ll
of phosphate-buffered saline
and gentle shaking for 1 h at
room temperature
Gingival crevicular fluid was
eluted by addition of 60 ll of
assay buffer, vortexing for
at 11,200 g for 10 min (no
information on temperature
was provided)
Gingival crevicular fluid was
eluted by addition of 500 ll
of phosphate-buffered
saline, gentle shaking for
1 min, followed by
centrifugation at 2,000 g for
5 min (no information on
temperature was provided)
Analytical technique and reporting
of data
Commerical ELISA kit (Orgenium).
Results were expressed as pg/ml
but it is not clear if these were total
mediator content per 30 s sample
(i.e. the concentration calculated
from the assay itself) or
concentrations in the gingival
crevicular fluid sample (i.e.
corrected for the original gingival
crevicular fluid volume according
to the Periotron data). Data
presented regarding lower
detection limit (<7 pg/ml). No
information given regarding how
values below the level of detection
were handled
Commercial ELISA kit (R&D Systems
Quantikine). Results were
expressed as pg/sample but it is
not clear if this was per gingival
crevicular fluid sample or per
patient (i.e. pooled samples). Data
are presented on assay sensitivity
(1 pg/ml). Sites with interleukin-
1beta levels below this threshold
were recorded as 0
Commercial multiplex bead
immunoassay (Luminex, Millipore)
Results were expressed as pg/ml
but it is not clear if these were total
mediator content per 30-s sample
(i.e. the concentration calculated
from the assay itself) or
concentrations in the gingival
crevicular fluid sample (i.e.
corrected for the original gingival
crevicular fluid volume according
to the Periotron data). Samples
below the lowest standard of the
assay (0.13 pg/ml) were recorded
as 0. Samples above the highest
standard of the assay (2,000 pg/ml)
were recorded as 2,000 pg/ml
Commercial ELISA kit (DIAsource).
Results were expressed as pg/site
for the total amount of interleukin-
1beta. Data were presented
regarding assay sensitivity
(0.35 pg/ml). Sites with
interleukin-1beta levels below this
threshold were recorded as 0
 Analysis of gingival crevicular fluid

Table 1. (Continued)

Author, year Gingival crevicular fluid collection Gingival crevicular fluid Analytical technique and reporting
(ref. no.) and volume measurement elution of data

Rosalem et al., Samples were collected from five Gingival crevicular fluid was Commercial multiplex bead
2011 (28) inflamed shallow sites (pooled in eluted at the time of immunoassay (Luminex;
500 ll of phosphate-buffered collection by standing for Millipore). Results were expressed
saline) and five inflamed deep sites 40 min at room temperature as pg/site for the total amount of
(pooled in 500 ll of phosphate- in phosphate-buffered saline interleukin-1beta, but it is unclear
buffered saline) followed by centrifugation at if this relates to a value for the
Gingival crevicular fluid was 3,000 g for 5 min pooled five sites or per individual
collected, using the 30-s sampling (temperature not stated). site. There was no calculation of
protocol, on Periopaper strips Supernatants were stored at mediator concentration using
placed into the pocket until mild !20°C gingival crevicular fluid volume. No
resistance was felt data were presented on the
Volume was quantified using the detection limits of the assay, other
Periotron 8000, and gingival than indicating minimum
crevicular fluid was eluted before (0.13 pg/ml) and maximum
freezing (2,000 pg/ml) standards
Thunell et al., Samples were collected from four Gingival crevicular fluid was Commercial multiplex bead
2010 (30) diseased and two healthy sites eluted at the time of immunoassay (Millipore). Results
Gingival crevicular fluid was collection by placement into were expressed as pg/30 s sample.
collected, using the 30-s sampling 300 ll of 0.01 M sodium No data were presented on the
protocol, from Periopaper strips phosphate buffer containing detection limits of the assay
placed into the gingival crevice 140 mM NaCl and protease
until mild resistance was felt. inhibitor, followed by
Volume was quantified using the vortexing for 10 s, shaking
Periotron 8000 and gingival for 20 min, then
crevicular fluid was eluted before centrifugation at 5,800 g for
freezing 5 min (temperature not
stated). Supernatants were
stored at !80°C

" the timing of gingival crevicular fluid sample col-
lection in relation to other clinical parameters
(best done before probing depths are recorded to
avoid blood contamination, but this may necessi-
tate additional appointments so that probing
depths can be recorded in advance to permit
selection of appropriate sites for gingival crevicu-
lar fluid sampling).
" whether or not gingival crevicular fluid volume
is recorded (several studies recorded gingival
crevicular fluid volume but did not appear to
utilize this information in any analysis, which
begs the question as to whether there was any
point in measuring gingival crevicular fluid
volume, other than for providing information
about gingival crevicular fluid volume for its
own sake).
" whether gingival crevicular fluid samples are
stored and analyzed individually or pooled.
" whether gingival crevicular fluid samples are
stored dry (i.e. Periopapers placed in microtubes
with no buffer), or whether they are placed in
buffer and then stored.
" the temperature of storage (samples are recom-
mended to be stored at !80°C).
" the timing of elution of gingival crevicular fluid
(e.g. immediately after collection and before freez-
ing, or after freezing/thawing and immediately
before analysis).
" the protocol for eluting gingival crevicular fluid
from Periopaper strips (shaking, vortexing, cen-
trifuging, duration and temperature).
" use of protease inhibitors in the elution protocol.
" identifying limits of detection (lower and upper)
for the analytical technique, and deciding how to
handle mediator values that are outside these
limits.
" reporting data as total mediator content per 30-s
sample, or converting the concentration output
from the analysis back to a concentration in gingi-
val crevicular fluid based on the original gingival
crevicular fluid volume.
It is important to note that each research group will
need to consider these issues as they plan their pro-
jects, and pilot studies will need to be performed to
optimize the methods to ensure the best repeatability

69
Wassall & Preshaw
and recoverability of mediator analysis in gingival
crevicular fluid. It is also important that these various
parameters are described in the manuscript that will
eventually be published to enable other researchers
to replicate the methods and improve consistency of
reporting.
Collection of gingival crevicular
fluid
Research publications typically describe the process
of collection of gingival crevicular fluid well. As noted
above, the most common method of collection is by
using filter paper strips. Originally, these were pieces
of laboratory filter paper of various types cut to size
by researchers. It has been reported that recovery of
proteins in gingival crevicular fluid samples varies
considerably according to the type of filter paper on
which the gingival crevicular fluid sample is collected
(18), but this problem is likely to be less of an issue
now as most researchers appear to use Periopapers
(Oraflow Inc.). These are presterilized filter paper
strips of standard size that can absorb fluid volumes
of up to approximately 1.2 ll (larger Periocol paper
strips are also available for collecting larger volumes
of up to 2.0 ll). As Periopapers are so widely used, we
will limit further discussion in this paper to gingival
crevicular fluid collection with Periopapers only.
Typically, Periopapers are placed just into the
entrance of the sulcus/pocket until mild resistance is
felt (i.e. utilizing the intracrevicular method of place-
ment) (13). Attempts to position Periopapers to the
known depth of a particular periodontal pocket are
typically unsuccessful because the paper has a ten-
dency to crumple up under the pressure of insertion
as it becomes increasingly saturated with fluid. Fur-
thermore, contamination with blood is also very com-
mon if trying to position a Periopaper to the full
depth of a pocket. Therefore, the Periopapers are typ-
ically inserted no more than 1–2 mm into the sulcus/
pocket to permit absorption of gingival crevicular
fluid from the site, with gentle placement until mild
resistance is felt.
The length of time that the Periopaper is held in
place is an important consideration. Some authors
have advocated holding the Periopaper in place until
it is ‘visibly wet’, but, more commonly, the Periopaper
is held in place for a fixed period of time (typically
30 s). As evidenced in Table 1, most researchers cur-
rently adopt a standardized method in which the
Periopaper is held in place for 30 s before removal.
This runs a potential risk of the Periopaper becoming
70
saturated at very inflamed sites with abundant gingi-
val crevicular fluid flow (although, in fact, a much
more likely explanation for saturation of Periopapers
is inadequate isolation and drying of the site to
remove saliva before sampling). On the other hand, if
the Periopaper is held in place ‘until visibly wet’, it
can take a very long time to see any evidence of gingi-
val crevicular fluid wetting the paper, particularly at
noninflamed periodontal sites. Therefore, on balance,
the most appropriate method is to adopt a standard-
ized sampling protocol, with the Periopaper being
placed 1–2 mm into the sulcus/pocket (after careful
drying and isolation) and held in place for 30 s.
Many researchers report that samples visibly con-
taminated with blood are rejected, but beyond this,
little detail is provided. For example, it would be use-
ful to know exactly how many samples are affected.
Furthermore, it is not usually possible, at the same
patient visit, to obtain a non-contaminated sample
from the same site if a previous sample is heavily con-
taminated with blood. Most researchers would agree
that frank bleeding, resulting in a red-stained
Periopaper, should lead to that Periopaper being dis-
carded, as clearly blood has been collected rather
than gingival crevicular fluid. However, there are
occasions (particularly at inflamed sites) in which a
very small amount of light-pink/reddish-brown
discoloration might be noted just at the very tip or
corner of the Periopaper, beyond which there is
visible wetting, indicating that gingival crevicular
fluid has been collected; in this scenario it may be
perfectly acceptable to retain this Periopaper for
analysis. Plaque deposits can also influence recorded
gingival crevicular fluid volumes (8, 15, 29), and
therefore large, visible supragingival plaque deposits
should be carefully removed with a curette before
sampling (13).
Quantification of gingival
crevicular fluid volume
The volume of gingival crevicular fluid absorbed by
Periopaper strips can be quantified using a Periotron
device. The current Periotron model is the Periotron
8000, although there are probably many Periotron
6000 models still in use in research centers through-
out the world. In essence, the Periotron is an elec-
tronic instrument that measures the effect of wetness
of filter paper strips on the capacitance between the
‘jaws’ of the device, between which the filter paper is
placed after the sample has been collected. In order
to calculate volume from the Periotron read-out, the
 Analysis of gingival crevicular fluid

device must be calibrated with known volumes of
fluid pipetted onto Periopaper strips to generate a
standard curve. Typically, serum has been used for
this purpose, being considered to have a similar vis-
cosity to gingival crevicular fluid, although the
manufacturer states that distilled water, saliva or
serum may all be used. Various types of regression
models have been proposed to solve the equation for
the calibration curve that is generated (2, 5, 6, 14, 17,
27), and the 8000 series is supplied with software for
calculating gingival crevicular fluid volumes using a
4th order polynomial regression. Some practical steps
for Periotron calibration are shown in Box 1, and a
typical Periotron calibration curve is shown in Fig. 1.
It is important to remember that errors associated
with the calibration process (such as evaporation
from the Periopaper or when dispensing very small
volumes onto the Periopapers) become magnified in
percentage terms at the lower end of the calibration
curve, and most researchers consider that the Peri-
otron is most accurate within the range of approxi-
mately 0.1–1.2 ll (13).
It is important that, once the gingival crevicular
fluid sample has been collected, the Periopaper is
transferred as quickly as possible to the Periotron
to minimize evaporation, where it is then posi-

Box 1. Practical steps for Periotron calibration

" Before use, turn on the Periotron device, according to the manufacturer’s instructions
" Zero the Periotron by placing a dry Periopaper between the jaws and adjusting the dial to the left or right
until the display reads zero
" Use a microliter syringe to dispense known quantities of fluid (e.g. 0.1–1.4 ll) onto individual Periopapers.
A suitable syringe is the Hamilton microliter syringe, which can be mounted in a retort stand at eye level
for easier viewing of the meniscus
" Typically, serum has been used for calibration purposes, being considered to have a similar viscosity to
gingival crevicular fluid. However, the Periotron manufacturer states that it makes little difference
whether the test fluid is distilled water, serum or saliva. Do your own experiments to identify what works
best according to availability
" Once the test fluid has been dispensed onto the Periopaper, transfer the Periopaper rapidly to the Peri-
otron and place between the jaws of the sensor
" Place it between the jaws of the sensor in a standardized position (e.g. the 3 o’clock position, with the
black line on the Periopaper positioned at the perimeter of the lower jaw of the device)
" Close the jaws of the Periotron onto the Periopaper and wait for the Periotron score to be displayed
" Repeat this step at least twice (i.e. in duplicate) for each volume that is used for calibration, although some
researchers prefer to repeat each volume in triplicate
" Between each sample, clean the jaws of the Periotron with an alcohol swab and allow to dry
" The manufacturer recommends using volumes of 0.50, 0.75, 1.00 and 1.25 ll in triplicate for calibration
purposes, and for each volume recording the mean Periotron score. This can then be utilized by the Peri-
otron Professional software (Oraflow Inc.) to compute a standard curve. Subsequently, when a sample of
unknown volume is collected from a patient, the volume of fluid (ll) is calculated by interpolation from
the standard curve
" If performing your own calibration, you may prefer to calibrate using a greater number of volumes (e.g.
0.1–1.4 ll, at 0.1-ll increments). Then, plot the results to permit generation of a standard curve
" The manufacturer states that calibration only needs to be performed once and then if you wish to check
that calibration has not changed over time, you can measure the volumes of two or more samples of
known volume. Individual researchers may choose to calibrate the Periotron more regularly (e.g.
before/during/after a study, or at 3-monthly or even 1-monthly intervals). There is no specific guidance
that can be given, other than to say that it is important to ‘get to know’ your Periotron. If the device is
new, then it would be important to perform calibration several times (e.g. three to four times at weekly
intervals) to assess if there is any variation between calibration curves. Similarly, if it has not been used for
some time, then performing a number of calibration exercises will be important to ensure that the device
is functioning effectively

71
Wassall & Preshaw

160 Many researchers snap freeze gingival crevicular

140 fluid samples chairside, but if this is not possible,
then the sample should be stored on ice and trans-
120
ferred to the laboratory as soon as possible, where it
Periotron units

100 may be snap frozen and then stored either in liquid

80
60
40
20
0 once the sample is removed from storage and is
0 0.5 1 1.5
Serum volume (µl)
Fig. 1. Calibration curve for Periotron. Scatter plot for
Periotron units generated for 16 different volumes of serum
(ranging from 0.1 to 1.6 ll), each measured in triplicate.
tioned between the jaws of the Periotron in a stan-
dardized way (e.g. at the 3 o’clock position with
the black line of the Periopaper coincident with
the perimeter of the jaw of the device). Once the
digital readout has been displayed and recorded,
the sample should then be transferred into an
appropriate cryovial ready for freezing and storage.
The jaws of the Periotron should then be cleaned
by closing them onto an alcohol swab and ensur-
ing they are completely dry, before the next sample
is measured.
nitrogen or in a !80°C freezer, or simply placed
directly into the freezer. Recoverability studies should
be performed beforehand to ensure that whatever
method is used, it is possible to obtain reproducible
and measurable levels of the mediator(s) of interest
2
analyzed.
A further consideration is whether the Periopa-
pers should be stored ‘dry’ in the microtube, or
whether the receiving microtube should contain
any fluid into which the Periopaper is placed.
Typically, it is convenient to place the Periopapers
into the assay buffer for the analytical procedure
that will be employed later, for example, a known
volume of autoclaved and filtered phosphate-buf-
fered saline. It is important to stress that recover-
ability experiments (in which known quantities of
the analyte of interest are ‘spiked’ onto the Peri-
opapers which then proceed through the storage
and analysis procedure) are performed before
commencing the clinical study, to ensure that the
methods to be used are effective for the mediators
to be analyzed.

Box 2. Practical tips for gingival crevicular fluid collection

" Before use, turn on the Periotron device, according to the manufacturer’s instructions
" Zero the Periotron by placing a dry Periopaper between the jaws and adjusting the dial to the left or right
until the display reads zero
" Select the sites to be sampled in advance, so that probing is not required immediately before sample col-
lection; to minimize contamination with blood, gingival crevicular fluid samples should be collected
before periodontal probing
" Isolate the site to be sampled with cotton rolls and a saliva ejector, and dry with a gentle stream of air.
Carefully remove any visible deposits of supragingival plaque with a curette before sampling
" Place a Periopaper carefully into the sulcus/pocket until mild resistance is felt (1–2 mm into the pocket)
and hold in place for 30 s
" Transfer the Periopaper quickly to the jaws of the Periotron to minimize evaporation errors, and position
it in a standardized position (e.g. the 3 o’clock position with the black line on the Periopaper positioned at
the perimeter of the lower jaw of the device)
" Record the Periotron units displayed by the device, then open the jaws and place the Periopaper into a
microtube for elution and/or freezing
" Between samples, clean the jaws of the Periotron with an alcohol swab and allow to dry
" If it is not planned to measure GCF volume, then place the Periopaper directly into a microtube for elution
and/or freezing

72
 Analysis of gingival crevicular fluid

Typically, Periopapers will be placed individually Table 2. Recoverability of interleukin-6 following elu-
into separate microtubes (i.e. one sample per tube) tion from spiked Periopapers
so that information relating to the specific periodon- Method Spiked Mean recovered %
tal site is generated from each Periopaper. However, interleukin-6 interleukin-6 Recoverability
sometimes researchers pool Periopapers that are (pg/ml) (pg/ml)
then stored, eluted and analyzed together. The Method A 233.33 146.59 62.83
advantage of this is that costs are reduced when the
116.67 26.89 23.05
samples are analyzed; however, the major disadvan-
tage is that site-specific information is lost by pool- 58.33 8.07 13.83
ing and processing samples collectively, which runs Method B 233.33 123.97 53.13
against the generally stated benefit of gingival crevic- 116.67 26.09 22.36
ular fluid analysis as a site-specific procedure. In
58.33 6.05 10.37
general, it is best that gingival crevicular fluid sam-
ples should be stored and processed individually, to Method C 175.00* 92.88 53.07

provide site-specific information. Some practical tips 87.50* 34.63 39.57

regarding gingival crevicular fluid sampling and 43.75* 14.17 32.39
volume determination using the Periotron are Method D 175.00* 78.76 45.01
presented in Box 2.
87.50* 17.28 19.75
43.75* 8.11 18.53
Analytical techniques for gingival *The spiked interleukin-6 concentration is lower for methods C and D, com-
pared with methods A and B, because these samples were diluted by the addi-
crevicular fluid mediators tion of 50 ll of 1% bovine serum albumin as part of the elution protocol.

Sensitive methods for the precise quantification of

mediators in gingival crevicular fluid, such as cytoki-
nes, are necessary because cytokine levels may be low
and sample volumes are small. Various techniques
have been used for detecting and quantifying cytoki-
nes in biological samples, including bioassay,
radioimmunoassay and ELISA, as well as multiplex
assays for the simultaneous quantification of
multiple cytokines. Most research groups tend to use
ELISA-based technologies for assaying mediators in
gingival crevicular fluid, and therefore the ELISA
technique will be the focus of this review.
ELISAs are immunoassays based on the capture
of test antigen (or standards of known quantity) by
antibody coated onto the wells of microtiter plates.
After a washing step to remove any free antigen, a
second antibody is added and this binds to the
antigen already present on the plate. The plate is
then washed to remove unbound antibody and a
ligand is added, which binds to the bound anti-
body and itself is covalently coupled to an enzyme
such as peroxidase. Free ligand is washed away,
then bound ligand is visualized by the addition of
a chromogen, a colorless substrate that is acted on
by the enzyme portion of the ligand to produce a
colored end-product. The color intensity in the
reaction wells is determined by optical density
scanning of the plate, and the quantity of the test
antigen is determined by comparison with a stan-
dard curve. ELISAs are widely used in periodontal
research, and are available for a large number of
analytes that are of relevance in periodontal dis-
ease pathogenesis. Given that gingival crevicular
fluid volumes obtained from patients are small and
that low cytokine levels within samples usually pre-
clude serial dilutions, then typically only a single
ELISA can be used for assessing a single gingival
crevicular fluid sample.
Therefore, high-throughput multiplex immunoas-
says have been developed that allow for simultane-
ous quantification of multiple cytokines in a
sample. Using ELISA technology, two basic assay
formats have been developed for simultaneous
quantification of multiple cytokines: planar array
assays; and microbead assays. In planar assays, dif-
ferent capture antibodies are spotted at defined
positions on a two-dimensional array, such as a
precoated microtiter plate. In the microbead assays,
the capture antibodies are conjugated to different
populations of microbeads, which can be distin-
guished by fluorescence intensity in a flow cytome-
ter. Both formats use a standard curve of known
concentrations of cytokines to quantify unknown
cytokine levels. The high cost of these multiplex
assays could, however, limit their use in large
clinical studies. Whichever assay system is used, the
gingival crevicular fluid must first be eluted from
the Periopaper strips so that the ‘eluate’ can be
analyzed for mediator content.

73
Wassall & Preshaw

Box 3. How to convert ELISA output to the concentration in the original gingival crevicular fluid sample

Suppose we have just completed an ELISA analysis of a particular mediator eluted from a gingival crevicular fluid
sample collected on a Periopaper. The samples were eluted in 200 ll of ELISA buffer. The information that we have
available at this stage is: (i) the ELISA concentration (in pg/ml); and (ii) the gingival crevicular fluid volume (in ll).
The method below explains how to convert this ELISA output to the concentration of the mediator in the original
sample of gingival crevicular fluid.
(Please note that the method described below will change if the ELISA output is not in pg/ml.)

1 Periopapers were eluted in 200 ll of buffer, and this 200 ll goes into the ELISA, with the end result being
an ELISA concentration in pg/ml.
If all we want to do is report the ‘mediator content per 30-s gingival crevicular fluid sample’, we can just report the
ELISA concentration of X pg/ml, and explain, in the methods, that the sample was eluted in 200 ll of buffer and we
are reporting the ELISA data in pg/ml per 30-s sample.

2 However, in this case, we wish to express the mediator concentration as a concentration in terms of the
original gingival crevicular fluid volume that was measured using the Periotron. Gingival crevicular fluid
volumes are measured in ll.
We assume that all the mediator in a sample comes from the original gingival crevicular fluid sample. We also
assume that the gingival crevicular fluid volume (usually <1 ll) is negligible compared with the elution volume
(200 ll), which seems a reasonable assumption.
If the output from the ELISA revealed that the concentration was X pg/ml of a particular mediator, it means that in
1 ml there would be X pg of the mediator.
However, we eluted in 200 ll; therefore, in that 200 ll, there must be X/5 or, to put it another way, X 9 0.2 pg of
the mediator.
Also, all of that mediator came from a certain volume of gingival crevicular fluid (measured in ll).
Therefore, the concentration of the mediator in the original gingival crevicular fluid sample =

Total amount of mediator in 200 ll

Gingival crevicular fluid volume (in llÞ

To calculate the numerator, we take the ELISA concentration of the mediator and multiply by 0.2 (equivalent to
dividing by 5, because 200 ll is one-fifth of 1 ml).
We know the denominator because that is the gingival crevicular fluid volume from the Periotron.

3 The final formula is therefore as shown below – this calculates the mediator concentration in the original
gingival crevicular fluid sample (expressed as pg/ll):
ELISA $ 0:2
½pg=ll&
GCF

ELISA is the concentration from the assay; and GCF is the gingival crevicular fluid volume from the Periotron
in ll.

described in sufficient detail to permit replication of

Elution of gingival crevicular fluid
from periopapers
Whereas clinical gingival crevicular fluid sampling
protocols (e.g. details of placement of Periopaper into
the gingival crevice for 30 s) are generally well
reported in the literature, methods used to recover
(elute) biomarkers from Periopapers are rarely
the experiment. This is important, as the method used
to elute mediators from the filter paper strips into
supernatant before analysis can have a significant
effect on the mediator levels subsequently measured.
To study the impact of elution methods on media-
tor levels, we performed a study to compare the
effects of four different elution methods on inter-
leukin-6 measurements in spiked samples. The

74
 Analysis of gingival crevicular fluid

Box 4. Worked example of conversion of ELISA output to concentration in the original gingival crevicular fluid sample

Let us assume that we have a Periopaper that contains 0.6 ll of gingival crevicular fluid, as measured by a calibrated
Periotron. Also, that the concentration of mediator ‘Q’ in that sample calculated from the ELISA was 2,045 pg/ml.
This means that in 1 ml, there would be 2,045 pg of the mediator Q.
However, we did not have 1 ml; rather, we had 200 ll of eluting buffer (plus a negligible volume of gingival crevicu-
lar fluid, 0.6 ll).
Therefore, there must have been 2,045/5 = 409 pg of the mediator Q in the 200 ll of sample + buffer.
We assume that all of this 409 pg came from the gingival crevicular fluid sample, which had a volume of 0.6 ll.
Therefore, the concentration of the mediator Q in the original gingival crevicular fluid sample must have been: 409/
0.6 = 681.67 pg/ll of gingival crevicular fluid.
If we run this through the equation that was generated in Box 3

ELISA $ 0:2
½pg=ll&
GCF

(ELISA, concentration from assay in pg/ml; GCF, gingival crevicular fluid volume from the Periotron, in ll),
we get the same result:

2; 045 $ 0:2
¼ 681:67 pg=ll of gingival crevicular fluid
0:6

Thus, we could report the mediator data for this sample in any of three different scenarios:
1 If the sample was collected using a 30-s sampling protocol, we could report a mediator content of 409 pg
per 30-s sample.
2 Alternatively, and again if the sample was collected using a 30-s sampling protocol, we could report a medi-
ator concentration based on the ELISA output of 2,045 pg/ml per 30-s sample.
3 Finally, we could calculate the concentration according to the original gingival crevicular fluid volume, in
which case we would report a mediator concentration of 681.67 pg/ll of gingival crevicular fluid.
Clearly, very different numerical values are produced in each scenario, and while this may not be a problem within
the context of a single research study (provided that the results are reported thoroughly), it certainly makes it extre-
mely difficult to compare the results between studies that used different methods. A further source of problems if
reporting according to scenario 3 is that when gingival crevicular fluid volumes are very low (e.g. in the case of
healthy gingival tissues, or following successful periodontal therapy), then calculated gingival crevicular fluid con-
centrations of mediators can become artificially inflated (particularly toward the lower limits of detection of the
Periotron), resulting in difficulty when interpreting results.

results of this experiment are shown in Table 2. In
brief, 1 ll of known concentrations of interleukin-6
were spiked onto Periopapers, which were then
placed into 150 ll of phosphate-buffered saline. Four
different elution techniques (based on those given in
published papers: A, B, C or D – see below) were then
performed. Following the elution protocol, the
Periopaper was removed and the concentration of
interleukin-6 was measured immediately in the elu-
ate/supernatant using an interleukin-6 ELISA (Duo-
Set Human interleukin-6; R&D Systems, Abingdon,
UK). The four elution techniques were as follows:
" Method A: centrifuge for 60 min at 300 r.p.m.
(8 g) and then for 2 min at 12,000 r.p.m.
(13,201 g), at a temperature of 4°C.
" Method B: centrifuge for 2 min at 12,000 r.p.m.
(13,201 g), at a temperature of 4°C.
" Method C: as method A, but with addition of 50 ll
of 1% bovine serum albumin before commencing
the elution protocol to reduce nonspecific
binding.
" Method D: as method B, but with addition of 50 ll
of 1% bovine serum albumin before commencing
the elution protocol to reduce nonspecific
binding.
(Note: g is dependent upon both the r.p.m. and the
radius of the sample in the centrifuge rotor; reporting
r.p.m. alone does not provide sufficient information
for other researchers to be able to replicate the meth-
ods – it is important to report both g and r.p.m.)

75
Wassall & Preshaw

Table 2 shows the mean interleukin-6 concentra-
tions recovered and the per cent recoverability for the
four methods, based on two independent experi-
ments for each condition, each assayed in triplicate.
In broad terms, and taking into consideration the full
range of interlukin-6 concentrations in the spiked
samples, method C resulted in the best recoverability
of interleukin-6 from the Periopapers. In other words,
both the addition of 50 ll of 1% bovine serum albu-
min (to reduce nonspecific binding) and the centrifu-
gation of samples for 60 min at low speed before a
short, high-speed centrifugation, improved the recov-
erability of interleukin-6 from Periopapers, particu-
larly at lower concentrations.
It is important to note that this elution protocol will
not be optimal for all gingival crevicular fluid media-
tors, and research groups should perform and publish
similar experiments to optimize the recoverability of
gingival crevicular fluid analytes from Periopapers
within the context of their own laboratory techniques
and the assay that is to be used for quantifying medi-
ators in gingival crevicular fluid. Given that the level
of cytokines within gingival crevicular fluid samples
often approaches the lower detection level of assay
systems and also that the small volume obtained from
gingival crevicular fluid samples limits the number of
cytokines that can be analyzed by ELISA, the use of
an elution method that provides optimal recoverabil-
ity of mediators of interest is highly important.
Researchers should therefore perform spiking experi-
ments to optimize their elution protocol, which will
typically involve various combinations of shaking/
vortexing/low- and high-speed centrifugation at a
controlled temperature. Further consideration should
be given to the addition of substances such as bovine
serum albumin (to reduce nonspecific binding) and
proteinase inhibitors (e.g. aprotinin) to prevent/re-
duce degradation of mediators of interest in gingival
crevicular fluid samples during processing, storage
and analysis. There is no option but to perform pilot
studies to optimize the protocol for gingival crevicular
fluid analysis according to the mediator(s) being stud-
ied and the analytical techniques that are available.
All such information should be reported in the final

Box 5. Key aspects to report in studies of gingival crevicular fluid mediator analysis

" Clinical gingival crevicular fluid sampling protocol: which teeth/sites; isolation/drying; plaque removal;
sequencing relative to other clinical assessments; details of placement of Periopaper; and duration of sam-
pling time (e.g. 30 s)
" Details of volume determination (if performed): Periotron model number; calibration details; and method
of calculation of gingival crevicular fluid volume
" Freezing protocol (e.g. snap freezing chairside, storage on ice and storage conditions in laboratory)
" Periopaper storage conditions: single or pooled; and dry or in buffer
" Gingival crevicular fluid recovery/elution protocol: whether eluted at time of sampling (i.e. before freez-
ing) or before analysis (i.e. after thawing); details of shaking/vortexing/centrifugation (including r.p.m.
and g); and temperature (e.g. controlled temperature or room temperature)
" Analytical technique used: manufacturer/name of kit; samples assayed singly or in duplicate; limits of
detection of the assay (lower and upper); details of how values below/above these limits are handled (e.g.
assign a score of 0 to values below the lower detection limit, and assign a score equal to the upper detec-
tion limit for samples above the upper detection limit)
" Whether results are expressed as:
" total mediator content per 30-s sample (pg/sample)
" mediator concentration as derived from the assay (e.g. pg/ml)
" mediator concentration following correction for the original gingival crevicular fluid volume (e.g. pg/
ll)
" In the situation of results being corrected for the original gingival crevicular fluid volume, details of how
this calculation was performed
" Be prepared to discard samples if the Periotron value indicates such a low score as to be beyond the
lower end of the standard curve, as this may result in artificially high calculated gingival crevicular fluid
concentrations

76

publication (either in the text of the paper itself or as
an online supplementary file) to support the interpre-
tation of results and allow other researchers to repeat
the experiment.
Correcting for original gingival
crevicular fluid volume (or not)
Many studies report the quantification of gingival
crevicular fluid volume on Periopapers using the
Periotron. The recording of gingival crevicular fluid
volume is useful in its own right, as a general indi-
cator of inflammation, but potentially can be more
useful for calculating mediator concentrations in
the gingival crevicular fluid sample. In broad terms,
two distinct approaches have evolved with respect
to reporting gingival crevicular fluid mediator con-
tent and concentrations. The first option is to sam-
ple gingival crevicular fluid for a fixed time period
(e.g. 30 s) and then report either total mediator
content (e.g. pg per 30-s sample) or the concentra-
tion as calculated from the assay (e.g. pg/ml per
30-s sample). The second option is to convert the
calculated concentration from the assay back into a
concentration based on the original gingival crevic-
ular fluid volume and then report, for example, as
pg/ll of gingival crevicular fluid. The precise
method of data reporting in this regard is usually
not described well in the periodontal literature.
Furthermore, it has been suggested previously that
reporting total mediator content is more accurate
than reporting concentrations in gingival crevicular
fluid (23, 24). Most authors now tend to sample for
a fixed period (usually 30 s) and then report
according to the first option described above (i.e.
total mediator content in a 30-s sample). However,
it is important to know how to take the output
from the analytical method (e.g. from an ELISA)
and convert this into a concentration based on the
original gingival crevicular fluid volume. A method
for doing this is presented in Box 3 with a worked
example presented in Box 4.
When presenting gingival crevicular fluid levels as a
concentration according to the original gingival
crevicular fluid volume, it is important to be aware
that very low gingival crevicular fluid volumes can
have a dramatic effect on the concentrations of gingi-
val crevicular fluid mediator calculated. This would
have particular consequences in prospective treat-
ment studies that are evaluating the impact of
improved periodontal health, following periodontal
Analysis of gingival crevicular fluid
therapy, on gingival crevicular fluid cytokine levels. In
such scenarios, even when following a 30-s gingival
crevicular fluid sampling methodology, the volume of
gingival crevicular fluid on the Periopaper may be so
low as to not register a Periotron value, or the Peri-
otron score may be so low as to be beyond the lower
limit of the calibration curve, resulting in increased
error and potential for artificially elevated gingival
crevicular fluid concentrations. Therefore, it is impor-
tant to have a protocol in place for handling either very
large or very small gingival crevicular fluid volumes.
In broad terms, the opinion of these authors is that
reporting total mediator content per 30-s sample
(reported either as pg per 30-s sample, or using the
concentration as calculated from the assay, i.e.
reporting as pg/ml per 30-s sample) is preferred to
correcting for original gingival crevicular fluid vol-
ume, as it removes a potential source of error from
the analysis (i.e. error associated with gingival crevic-
ular fluid volume determination). However, the deci-
sion regarding how to report the data will vary
according to the analyte of interest, and, as suggested
above, pilot studies must be performed to determine
the optimal method of analysis. If the decision is
made to correct the data for original gingival crevicu-
lar fluid volume, then it is recommended to present
both the total mediator content per 30-s sample and
concentration in gingival crevicular fluid, together
with gingival crevicular fluid volume data, so that a
full picture is presented to the reader (13).
Conclusions
The collection and analysis of gingival crevicular fluid
has massively improved our understanding of peri-
odontal pathogenesis and healing outcomes following
treatment, and will continue to play a significant role
in periodontal research in the years to come, particu-
larly when site-specific information is required.
However, it is technically challenging, with many
parameters that must be considered in relation to
sampling, elution, storage, analysis and reporting of
data (Box 5). There is no ‘one size fits all’ protocol,
and individual research groups must develop their
own methods based on the mediators of interest and
the analytical techniques that they have available.
With regard to reporting gingival crevicular fluid
mediator data, it is acceptable to either measure vol-
ume (e.g. with a Periotron) and then calculate media-
tor concentration according to the original gingival
crevicular fluid volume, or to report total mediator
77
Wassall & Preshaw
content per 30-s sample (not requiring volume
measurement, although this may still be performed
for the additional data it generates). Calibration stud-
ies are essential to optimize the collection and pro-
cessing of gingival crevicular fluid samples to ensure
optimal, reproducible and accurate analysis. The pre-
cise methods used should be reported in detail, either
in the methods section of the published paper or in
an online supplementary file, so that other research-
ers may reproduce the methodology.
Acknowledgments
The authors would like to thank Dr Arndt Guentsch
(Marquette University, Milwaukee, WI, USA) and Dr
Katrin Jaedicke (Newcastle University, Newcastle,
UK) for critical reviewing of this paper before
submission.
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