Gingival crevicular fluid sampling techniques

1. COMPARISON OF GINGIVAL
CREVICULAR FLUID SAMPLING
METHODS IN PATIENTS WITH SEVERE
CHRONIC PERIODONTITIS
Arndt Guentsh, Martin
Kramesberger, Aneta Sroka,
Wolfgang Pfister, Jan Potempa, and
Sigrun Eick
J Periodontol, July 2011
Presented By:
Pallavi Prashar

2. Introduction
• In periodontal disease, gingival crevicular fluid
(GCF) is an inflammatory exudate.
• GCF contains substances from the host and
supra- and subgingival bacteria.
• Host constituent include
▫ molecule from blood and periodontal tissues
▫ inflammatory and immune cells that infiltrate into
periodontal tissue are found in GCF together with
markers of inflammation, including
 Enzymes
 Cytokines
 Interleukins (Ils)
• Products of tissue breakdown.

3. • The analysis of GCF and subgingival microflora has
become more important in diagnosis and therapy of
periodontal diseases.
• The presence of large number of periodontopathic
bacteria in GCF, such as Actinobacillus
actinomycetemcomitan (AAC) and member of red
complex organism including P.gingivalis, T.forsythia,
T.denticola indicates clinically important microbial
infection.
• Arginine-specific gingipains (Rgps) are an important
virulence factors of P.gingivalis that plays major role in
maintenance of inflammatory conditions in
periodontitis. They are able to impair neutrophil
function and degrade the extracellular matrix and
bioactive peptides such as complement factor-C5,
prekallikerin, and kininogens. Furthermore Rgps can
inactivate IL-6 and inhibitor of neutrophil proteases.

4. • In periodontitis there are increased levels of
expression and synthesis of IL-1, TNF-alpha,
and IL-6 and 8 in periodontal tissue.
• An effective host response to bacteria is
primarily mediated by neutrophil and
characterized by influx of neutrophil into
gingival crevices.
• Elastase levels are among the highest of any
proteinase activity determined in GCF during
periodontal inflamation. The assessment of the
granulocyte elastase activity in GCF can serve as
marker of intracrevicular granulocyte activity.

5. Different techniques have been employed for sampling
the content of periodontal pocket.
• Subgingival bacteria can be sampled with curettes or
paper points, whereas cytokines and host enzymes
were usually collected with filter paper strips.
• There are considerable variations in the application of
the paper-strip method of collection which can be
intracrevicular (strip is inserted unless resistance is
felt) or extracrevicular (strip kept at enterance of
crevice).
• Washing technique is an possible alternative when
other sampling method fails.

6. Aim of the study
• To identify a method that could be used for
different purposes (e.g., microbiota and
immunologic variables)
• Moreover, different sampling techniques were
tested to effectively detect a parameters of
special interest, in this case Rgps.
• Because of missing data in literature , an
additional aim of this study was to investigate
the level of Rgps and its correlation to
P.gingivalis in GCF.

7. Materials and Methods
• Subject recruitment
▫ Thirty six subjects aged 38 to 56 years with chronic periodontitis
were recruited.
▫ The definition of chronic periodontitis was based on the
classification system of the International Workshop for
Classification System of Periodontal Diseases and Conditions from
1999.
▫ Patients with generalized chronic periodontitis were included if
they were >35 years of age and demonstrated attachment loss of
>5 mm at >30% of sites.
▫ After the hygiene phase plaque was <35%.
• To ensure similar periodontal conditions for comparison of
sampling methods, each molar per quadrant had a site with
probing deapth between 5 and 7 mm.
• Subjects with significant disease (e.g., diabetes melluitus,
cancer, or coronary heart disease), antibiotic therapy within the
past 6 months, and females who were pregnant and lactating
were excluded.
• Only non-smokers with no history of smoking were included.

8. Clinical Assessment
• Probing depths were measured with UNC 15 probe at six sites
per tooth.
SAMPLE COLLECTION
• Patients were randomized per lot into one of the three
groups.
▫ In Group 1 paper strips were compared to paper points.
▫ In Group 2 paper strips were compared to washing techniques.
▫ In Group 3 paper points were compared with washing
techniques.
• GCF was sampled in each patient using two sampling
techniques on only molars with probing depth from 5 to
7mm.
• One method was performed at upper and lower molars on
right side and the other method was performed at
corresponding molar on left side of oral cavity.
• After 1 week, the collection of samples were repeated using
the opposite sites.
• Samples were collected in the morning 2 to 3 hours after
breakfast.

9. • The sites to be sampled were isolated with cotton rolls and
gently air dried.
• Paper strips and paper points were gently placed for 30 seconds
into pocket until minimum resistance were felt.
• Samples were eluted at 4ºC overnight into 500 µL phosphate-
buffered saline (PBS).
• After being centrifuged for 4 minutes, the paper points/strips were
removed; both paper points/strips and supernatants were kept frozen
at -20̊C until assayed.
• Crevicular washes were obtained with following method
▫ A gel-loading capillary tip was carefully inserted into the crevices at a
level~1 mm below the gingival margin.
▫ In each case five sequential washes with 10µL of 0.9% NaCl were
performed using a micropipette.
▫ The washes were transferred into a microcentrifuged tube and centrifuged
for 4 minutes, after which supernatant were immediately frozen and kept
at-20̊C until analyzed.
• All samples containing blood were discarded and sampling was
repeated 2 days later.

10. MICROFLORA
• DNA was extracted using DNA-extraction system
according to recommendations of manufacturer from
paper points/strips after elution and 5µLof the washes.
• A real-time PCR was carried out using a real-time rotary
analyzer.
• PCR amplification was carried out in a reaction volume of 20
µL consisting of 2µL template DNA and 18 µL reaction mixture
composed of 2 µL PCR buffer , 2.75mM magnesium chloride,
0.2mM nucleotides.
• Negative and positive controls were included in each batch of
specimens.
• The positive control consisted of 2µL genomic DNA in
concentrations in range of 10^2 to 10^7 bacteria of the reference
strains, and negative control was 2µL of sterile water; each
control added 18µL reaction mixture.
• The cycling conditions comprised an initial denaturation step at
95 ̊C for 5 minutes followed by 45 cycles at 95 ̊C for 15 seconds
, at 65 ̊C for 20 seconds using a touhdown for five cycles and at
72 ̊C for 20 seconds.

11. NEUTROPHIL ELASTASE
• Neutrophil granulocyte elastase (NE) activity was
measured with a microplate assay using by using
chromogenic substrate N-methylsuccinyl-L-alanine-L-
proline-L-valine-para-nitroaniline.
• The assay was performed in a total volume of 150µL with a
0.75mM final substrate concentration in 50mM Tris-HCl, pH
7.5.
• The rate of p-nitroanilide released was recorded at 405 nm by
using microplate reader.
• One unit was calculated as the amount of enzyme that
hydrolyzes 1nmol substrate in 1 minute.
IL-6 and IL-8
• Concentration of IL-6 and IL-8 were determined by
commercially available enzyme-linked immunosorbent assay
(ELISA) kit as described in the manufacturer’s instruction.
• The detection level of kit was ~2pg/mL.

12. ANTIBODY USED IN ELISA ASSAY
• Antibodies (immunoglobulin Y [IgY]) anti-HRgpA
were raised in chickens as described by Pike et al.
• The IgY specific for the Rgp catalytic domain were
purified by affinity chromatography using
immobilized RgpB.
• Anti-RgpB mouse monoclonal antibody was produced
on site in a monoclonal facility.
• Because the caspase like domain of RgpB is essentially
identical with the catalytic domain of RgpA, the
obtained mAb reacted with equal affinity with both
Rgp.

13. LEVEL OF Rgp
• The level of the P.gingivalis proteases Rgp in the GCF was
determined by ELISA technique.
• 100µL from the paper GCF eluate and 10µL from each washing
GCF of diluted 10 fold to 100µL by addition of phosphate-
buffered saline was used.
• The wells of the microtiter plates were coated with chicken
antibody anti-Rgp in the final concentration of 1 µg/mL in
carbonate buffer(pH9.6) for 12 hrs at 4 ̊C.
• A negative control and GCF samples are added to wells and a
plate incubated for 12 hrs at 4 ̊C.
• After additional washing, secondary mAb anti-Rgp catalytic
domain was added to each well.
• The reaction was stopped by addition of 100µL of 1% H2SO4,
and absorbance was read at 450 nm.

14. IN VITRO STUDY
• In addition, in vitro experiment was performed to determine
the recovery levels of known concentrations of P.gingivalis,
AAC, IL-6 and IL-8, neutrophil elastase, and RgpB from paper
points.
• Bacteria was used in a range of 10^4 to 10^7 per microliter
• IL-6 and-8 were diluted to final concentrations of 62.5, 125, and
250pg/µL
• RgpB and human neutrophil elastase were tested at conc. 0.3 and 1.2
ng/µL and between 0.025 and 0.1µg/µL, respectively.
• The suspension or dilution media always contained 10% human
serum.
• 1µL of the final solution or suspension of tested bacteria or protein
was placed on point and paper strip.
• Further processing of the sample was made as described before for
clinical samples.
• An independent analysis was made at least in triplicate.

15. STASTICAL ANALYSIS
• Clinical data were expressed as mean standard
deviations.
• Laboratory variables were presented as median
including quartiles.
• Groups were compared using the paired Wilcxon test.
• The corelation among tested variables was made
using the Spearman test.
• Statistical software was used for all statistical
analyses.

16. RESULTS
• Subjects of all groups showed similar clinical signs; no difference
among groups was found to be statistically significant.
COMPARISON OF PAPER BASED METHODS
• The cytokines, IL-6 and IL-8 were detected more often when paper
strip were used compared to when paper points were used.
• The cytokines, IL-6 was measured in conc. <250pg/sites by mean
of paper strips and 154pg/site by mean of paper points (no
significance; P=0.311)
• IL-8 was analyzed insignificantly higher levels (P=0.001) by using
paper strips (<350pg/site) compared to paper points (246pg/site).
• The level of cytokines measured by both methods correlated
positively(IL-6:R=0.565, P<0.001; IL-8:R=0.337, P=0.0019).
• The neutrophil elastase activity was measurable in 85% of paper
strip sample and in 98%of paper point samples.
• Levels were slightly high when using paper points(not significant;
P=0.130)

17. • Measured bacterial load did not differ
significantly between the two sampling
method(.gingivalis: P=0.375; AAC:P=0.627)
• Two-third of the samples were positive for P.gingivalis
in contrast, AAC was detectable in 25% of paper point
samples and in 33%of the paper strip sample.
• Rgps were found in only two paper point eluates(4%)
and 11 paper strips eluates (22.9%)
• All Rgps positive samples were also positive for
P.gingivalis, which meant that Rgps was detected in
6%of positive samples by using paper points and in
35% by using paper strips.

18. Levels of IL-6 - and -8, activity of NE, bacterial loads of P. gingivalis and A.
actinomycetemcomitans, and levels of arginine-specific cysteine proteases (Rgps
derived from Pg) in GCF determined by using paper points and paper strips for
sampling materials. lg = logarithmic steps.

19. WASHING AS A SAMPLING METHOD
• Cytokines were detected in higher levels using the
washing method compared to the paper based methods.
These difference were more profound for IL-8(significant
difference compared to paper strips [P=0.012] and paper
points[P<0.001])
• The neutrophil elastase activity was significantly
lower in samples collected using the washing method
compared to paper strips (P=0.001)
• Bacterial organisms were found in low numbers, but
nevertheless, there was a good correlation to presence of
specific bacteria collected with other sampling
methods(p.gingivalis;P=0.004 compared to paper strips
and P-=0.001 compared to paper points; AAC:P=0.001
compared to paper strips ans P=0.002 compared to paper
point)

20. P.gingivalis AND ARGININE-SPECIFIC CYSTEINE
PROTEASE (Rgps)
• When comparing different methods of sampling, R-
gingipains were detectable in 49% of GCF washes, 13%of
paper-point samples, and 26% of paper-strips samples.
• To overcome limitations of individual methods, at least
partially, the higher values of each of the 144 sites
obtained by two methods were used for further analysis.
• Thus 70 sites (49%) tested positive for Rgp, and 109 sites
(76%) tested positive for P.gingivalis, which yielded a 64%
overlap of sites infected with P.gingivalis in which Rgps
were found.
• The analysis of all sites revealed that the concentration of
the Rgp gingipain correlated positively with bacterial load
of P.gingivalis (R=0.429; P<0.001)

21. Levels of arginine-specific cysteine protease (Rgps derived from P.gingivalis) in
GCF in relation to the load of P. gingivalis

22. IN VITRO EXPERIMENTS
• The mean recovery rate of cytokines was higher from
paper strips than from paper points.
• The difference was more significant for IL-8 compared to
IL-6.
• Concentration dependent defects were clearly visible.
• From paper strips, the lower concentration were
completely released; while in contrast, nearly 100% of Il-8
was eluted of 62.5 pg applied on paper strips, and at 250
pg applied on paper points, only 152 pg (60%) of cytokines
were recovered.
• Bacterial loads still attached to papers after elution were
found to be between 80.75% and 87.25%
• Bacterial load are normally counted as log states, and thus,
deviations appear to be smaller.
• Recovery rate of RgpB from paper was low, independent of
concentration of applied enzyme.

23. Recovered levels of cytokines applied in vitro onto paper points and paper strips.

24. DISCUSSION
• The quantity and quality of GCF are highly affected by method
• The wide range of volumetric distribution, the site-specific nature,
and the impact of a distinct sampling site on the volume were
described as important features of GCF collection and analysis.
• A standardization of the extent of probing depth, degree of gingival
inflammation, and distinct sampling may improve the reliability of
GCF methology.
• For this reason, only patients with comparable probing depths, good
oral hygiene (plaque index <0.35%),18 and low gingival
inflammation (samplecollection after the hygiene phase) to avoid
contamination of GCF samples with blood were chosen.
• A standardized time for collection of GCF by means of paper-based
methods was applied because the clinical situation was better
represented by the analysis of GCF based on the time of sampling
than on volume.
• This procedure allowed an instant freezing of samples to prevent
proteolysis.
• Nevertheless, the lack of sample standardization according to the
protein content or collected volume was a limitation of the study.

25. • Two paper-based sampling methods were compared using a regular
nitrocellulose paper point and a filter paper strip.
• These methods were quick and easy to use, were applied to individual sites,
and were not traumatic when correctly used.
• In addition, GCF was collected by an intracrevicular washing technique.
This technique uses the installation and continuous reaspiration of definite
solutions (e.g., Hanks’ balanced salt solution31 or PBS32) at the gingival
crevice.
• The method is highly sensitive, but requires participation of a trained,
experienced investigator to collect samples.
• The GCF collection with filter paper strips is probably the most preferred
sampling method.
• Several studies used this method to analyze the level of different cytokines
and other biomarkers in GCF. Significantly, amounts of IL-6 and -8 in GCF
reported in these studies were comparable with our data.
• Compared to the use of paper points, the use of filter paper strips resulted in
higher IL-8 levels. This finding was supported by the in vitro analysis; the
recovery rate of IL-8 was much lower from endodontic paper points
compared to paper strips.
• An earlier study also reported an incomplete recovery of proteins from
paper points supposedly because of binding of GCF proteins to the paper.
• The difference in elution of IL-6 and IL-8 was most likely due to difference
in the structure, charge distribution, and hydrophobicity of these cytokines
molecules.

26. • In addition, the recovery rate of the RgpB was only in the range of
23% to 26% of the predetermined concentrations.
• This low recovery of RgpB explained the low number of positive
samples collected by one of the paper-based methods.
• Intracrevicular washing was the only method that detected relevant
amounts of Rgps.
• Taking into account the molecular mass of Rgps and the volume of
GCF in periodontitis patients, a concentration <1.5 mM was found.
• This finding was highly significant because it allowed for the
prediction of whether a specific gingipains substrate would be
degraded in vivo.
• For example, a 150-fold less concentration would be sufficient to
cleave IL-6 and was more than high enough to destroy the
complement, protease inhibitors,8 and bactericidal peptides and
impair neutrophil functions in periodontal pockets.
• This underlined the importance of Rgps in vivo.
• Nevertheless, a correlation between the level of Rgps and the load of
P.gingivalis was not found.
• The levels of synthesized and released Rgps differ among strains and
depend on environmental conditions (e.g., the contact to epithelial
cells).

27. • In contrast to the superiority of the washing method in the
determination of gingipains, paper-based methods detected higher
levels of the NE activity.
• Earlier studies indicated that NE concentrations or activities in GCF
could be used to identify differences among disease activities within
patients.
• Therefore, this enzyme activity is an excellent qualitative measure of
gingival inflammation.
• Elastase is one of the proteolytic enzymes present in the
polymorphonuclear leukocyte (PMN) primary granules that is
released upon activation of the PMN and capable of degrading
extracellular matrix proteins of the connective tissue.
• The higher granulocyte elastase activity was previously observed in
patients with periodontitis (both aggressive and chronic) compared
to healthy controls.
• A positive correlation between the IL-8 level and NE activity was
found in the GCF of periodontitis patients, which was explained by
the intensity of the host inflammatory response induced by the IL-8–
elicited activity to activate granulocytes.
• Periodontal therapy reduced the levels of IL-8, suggesting a
relationship between this cytokine and periodontal status.

28. • The qualitative and quantitative composition of GCF with respect to
subgingival microbiota and host mediators is well known to reflect the
severity of periodontal disease.
• Unfortunately, the clinical significance of the analysis is often unclear
because different sampling methods are usually used to measure the
content of cytokines and to determine microflora of discrete periodontitis
sites.
• In the present study, the same sample was used for both analyses.
• Surprisingly, as shown in the in vitro assays, >80% of the bacteria were
still attached on the paper points or strips after the overnight elution with
PBS and before extraction of DNA.
• The pathogenic microflora was detected in nearly all patient samples with
both paper-based sampling methods.
• Papers are easy to insert into the gingival sulcus; the low costs of
endodontic paper points indicate their usage for determination
• of microflora only.
• The outcomes of paper points for microbiologic diagnostics were recently
compared to the sampling of subgingival biofilm with
• curets.
• The authors concluded that paper points were suitable for microbiologic
diagnostics.

29. • In this work, we found that supernatants of GCF washes were not
well suited for the determination of microflora.
• In the present study design, we added a centrifugation step to remove
cells and detect only soluble cytokines and NE in GCF so that bacteria
should not sediment, but they would if associated with host cells
and/or tissue debris.
• Indeed, the analysis of supernatants and sediments from five
additional GCF washing samples revealed that the majority of
bacteria were in pellets, and only <10% of bacteria were present in
supernatants.
• This result suggested that, before centrifugation, 5 mL of the washing
sample should be retained for microbiologic diagnostics.
• Keeping in mind the limitations of the method used for detection of
bacterial loads, it was not surprising that more samples were positive
for Rgps (total: 47 samples) than for P. gingivalis (total: 33 samples).
• The washing method was most suitable for detection of the Rgps.
• In comparison to paper-based sampling methods, the washing
method allowed for the collection of the highest amounts of
gingipains (significant difference: P = 0.018 for washes compared to
paper points).

30. CONCLUSIONS
• The washing technique is an alternative
sampling method of GCF for special purposes
when sampling by paper-based methods fails.
• Paper points are suitable for the determination
of the microflora and are recommended for daily
microbiologic analysis in dental practice.
• Paper strips are the method of choice for most
biomarkers in immunologic studies; a combined
determination of periodontopathic bacteria
seems possible.