This study investigated whether detecting hemoglobin (Hb) in gingival crevicular fluid (GCF) could improve the diagnostic accuracy of periodontal disease when used along with probing depth (PD) and bleeding on probing (BOP). The study found Hb was detected in over 60% of GCF samples from BOP-negative sulci considered periodontally stable, and over 85% of BOP-negative sulci requiring periodontal management. This suggests examining Hb from microbleeding along with PD and BOP may provide a more accurate periodontal diagnosis by detecting early tissue damage not seen with BOP alone. The addition of Hb measurement to clinical exams could improve validity and serve as a diagnostic strategy before periodont

1. CORRELATION BETWEEN GCF
HEMOGLOBIN CONTENT AND
PERIODONTAL CLINICAL
PARAMETERS
Hiroshi Ito DDS PhD, Yukihiro Numabe DDS PhD, Shuichi Hashimoto DDS PhD,
Satoshi Sekino DDS PhD, Etsuko Murakashi DDS PhD, Hitomi Ishguro DDS PhD,
Daisuke Sasaki DDS PhD, Takashi Yaegashi DDS PhD, Hideki DDS PhD, Masaru
Mezawa DDS PhD, Yorimasa Ogata DDS PhD, Hisashi Watanabe DDS PhD,
Satsuki Hagiwara DDS PhD, Yuichi Izumi DDS PhD, Yuka Hiroshima DDS PhD,
Jun-Ichi Kido DDS PhD, Toshihiko Nagata DDS PhD, Kazushi Kunimatsu DDS PhD.
Journal of Periodontology
November 2016, Vol. 87, No. 11, Pages 1314-
1319
https://doi.org/10.1902/jop.2016.160092

2. INTRODUCTION
Probing depth(PD) and bleeding on probing(BOP) are essential clinical
parameters used for periodontal diagnosis. This study investigated
whether detection of hemoglobin(Hb) in gingival crevicular fluid(GCF)
along with PD and BOP would improve diagnostic accuracy.

3. Periodontitis is inflammation of the gingiva with attachment loss, and
is clinically diagnosed with bleeding on probing(BOP). Examination of
BOP is an important clinical parameter which points to the success or
failure of periodontal therapy, reveals the presence of inflammation
in the bottom of pocket and aids in planning and change of
treatment.

4. AIM/PURPOSE OF STUDY
The purpose of the present study was to determine whether
detection of Hb in GCF along with PD and BOP would more
accurately detect periodontal disease activity. The associations
between Hb and BOP evaluations of less than and more than cutoff
value were analyzed statiscally and accuracy of both evaluations
was compared to the clinical diagnosis.

5. MATERIALS AND METHODS
A cross-sectional study design was employed to analyze Hb in GCF and its association with clinical
parameter, especially BOP.
1)SAMPLE:
A total of 184 non-smokers (mean age: 63.0 +/- 11.3 years, 73 males: 63.2 +/- 14.4 years,
Females: 62.8 +/- 8.8 years) who were receiving regular periodontal maintenance therapy were examined.
All participants were healthy and were not receiving and medication at the time of study.
2)EXCLUSION CRITERIA:
1)Pateints with systemic diseases such as Diabetes, Immunological disorders, Hepatitis, Cardiac disease,
2)Pregnant and lactating women and women taking oral contraceptice drugs,
3)Patients who had received antimicrobial therapy for past 3 months, and
4)Individuals whoe self reported the use of tobbaco products.

6. MATERIALS AND METHODS
3)MEASUREMENTS OF CLINICAL PARAMETERS:
A)Following clinical parameters were recorded for 401 sites of anterior teeth without restorations in maxilla/
mandible of 184 patients: PI, GCF amount, GI, PD, CAL and BOP.
B)After PI measurement, GCF was collected from 401 pockets using paper strips, and was expressed as
microliters. Samples of GCF contaminated with whole blood were excluded.
C)After GCF sampling, GI was recorded in 401 sites, and PD and CAL were measured using a pocket probe.
D)Finally BOP was recorded.

7. MATERIALS AND METHODS
4)HB INSPECTION IN GCF:
The amount of Hb in GCF was measured with an Hb-detection kit using immunochromatography.
When 1ng/pocket or more Hb was present, the pocket site was denoted as positive for detection of Hb.
The means amount of Hb in GCF samples from all 401 sites was 48.6 +/- 85.4 ng/pocket.
5)STATISTICAL ANALYSIS:
A)The statistical anaylsis was performed by 2 authors (HI, YN). The correlation between clinical
parameters and Hb detection was anaylzed using Spearman's correlation coefficient. The Mann-
Whitney U test was used for between group comparisions.
B)The Receiver operating characterstis (ROC) curve, Youden index, and cut off values of the clinical
parameters were created based on PD and GCF.
C)A clinically healthy site was defined a having a PD less than or equal to 4mm and GCF less than 0.2
microliter, a periodontitis progression risk site was defined as having PD greater or equal to 5mm and
GCF more than 1.0 microliter.
D)Statisical analysis was considerd at the probability level, p<0.05 .

8. RESULTS
1)CORRELATION BETWEEN THE CLINICAL PARAMETERS AND HB CONTENT IN GCF:
All Hb inspections in GCF were carried out prior to the examinations of clinical parameters( GI,PD,CAL,BOP,PI)
Significant correlation were observed between every clinical parameter and Hb content in GCF. However, the
correlation coefficient between BOP(+) and HB content was 0.172, which was the lowest coefficient in
comparision to other parameters.
PI 0.5 ± 0.6
GCF (μl) 1.54 ± 2.04
PD (mm) 3.7 ± 1.7
GI 0.8 ± 0.7
CAL (mm) 4.6 ± 2.3
BOP 0.2 ± 0.4
CC 0.244
SP **0.000
CC 0.431
SP **0.000
CC 0.350
SP **0.000
CC 0.299
SP **0.000
CC 0.313
SP **0.000
CC 0.172
SP **0.001
1)CLINICAL PARAMETERS: Mean +/- SD
2)Hb values: 48.6 +/- 85.4 ng/pocket.
3)CC (Coefficient of correlation)
4)SP (significant probability)
5) ** P<0.01
6)n= 401 sites

9. 2)COMPARISION BETWEEN THE GROUPS THAT WERE CLASSIFIED AS PERIODONTAL DISEASE BASED ON PD
AND GCF:
On examination of sites, the clinical parameters(PI,GI,CAL) in periodontitis progression risk sites
(123 sites; PD ≥ 5mm and more than 1.0 µl of GCF)were siginifcantly higher than those of clinically
healthy sites (45 sites; PD ≤ 4mm and less than 0.2 µl of GCF)
The clinically healthy sites: PD ≤4mm & GCF < 0.2µl; n=45 sites
The periodontitis progression risk site: PD ≥ 5mm & GCF ≥ 1.0 µl; n= 123 sites
** p value < 0.01 (≤4mm & <0.2 µl vs ≥5mm & ≥ 1.0 µl)
PI
GI
CAL
PD.GCF MEAN SD
≤4mm & <0.2 µl 0.2 0.4
≥5mm & ≥ 1.0 µl 0.7 0.6
≤4mm & <0.2 µl 0.4 0.7
≥5mm & ≥ 1.0 µl 1.3 0.5
≤4mm & <0.2 µl 2.8 1.5
≥5mm & ≥ 1.0 µl 6.6 1.6
P-value Siginificant difference
0.000 **
0.000 **
0.000 **

10. 3)SETTING OF CUT OFF VALUES FOR PD AND GCF:
The cutoff values for PI,GI, and CAL calculated by clinically healthy sites and periodontitis progression risk
sites were 0.500, 0.500, 4.500 mm. And likelihood ratios were 3.375, 2.937, 7.871. These values were
used to create the ROC curves and Youden indexes. A total of 107 sites with values less than cutoff values
were classified in periodontically stable group, and 123 sites had values more than cutoof values and were
classified as periodontal management required group.
PI GI CAL
Cut off values 0.500 0.500 4.500
Area under the
curve
0.745 0.829 0.953
sensitivity 0.675 0.978 0.976
specificity 0.800 0.667 0.876
+ predictive value 0.902 0.889 0.952
- predictive value 0.474 0.909 0.929
Proper diagnosis
rate
0.708 0.893 0.946
Likelihood ratio 3.375 2.937 7.871

11. 4)DETECTION RATE OF HB IN BOP(-) AND BOP(+) SITES OF PERIODONTICALLY STABLE GROUP OR IN THE
PERIODONTICALLY MANAGEMENT REQUIRED GROUP:
1) In the periodontically stable group consisting of 107 sites, 98.1% (105 sites) were BOP (-) and 1.9%
(2 sites) were BOP(+). Hb was detected in 64.8% (68 sites) from 105 BOP(-) sites and in 50.0%
(1 GCF site) from 2 BOP(+) sites.
2)In the periodontal management required group consisting of 122 sites, 58.2% (71 sites) were BOP(-)
and 41.8% (51 sites) were BOP(+). Hb was detected in 88.7% (63 sites) from 71 BOP(-) sites and 90.2%
(46 sites) from the 51 BOP(+) sites.
1)68 sites BOP(-) -->Hb was 14.8 +/- 41.6 ng/pocket ; 1 site of BOP(+) --> was 31.8ng/pocket
2)63 sites of BOP(-) --> Hb was 73.0 +/- 98.1 ng/pocket ; 46 sites of BOP(+) --> Hb was 110.1 +/- 122.0 ng/pocket.
PERIODONTALLY STABLE GROUPS PERIODONTAL MANAGEMENt REQUIRED
GROUP
Detection sites of Hb (107) Detection sites of Hb (122 sites)
total Hb(+) Hb(-) Total Hb(+) Hb(-)
BOP(-) 105 sites 68 37 71 sites 63 8
BOP(+) 2 sites 1 1 51 sites 46 5

13. DISCUSSION
1)The BOP(+) considered to be one clinical sign of relapse during periodontal maintanence therapy, therefore
recording BOP in daily clinical practice has been recognized as most effective diagnostic tool.
2)Lang et al. Reported that when all results of examinations were conducted 4 times per year were BOP(-)
with attachment loss not more than 2mm in 98% sites. However inspite of BOP(-) attachment loss may still
occur in small number of sites. Therefore this study aimed to improve diagnostic accuracy by implementing
Hb examination before recording BOP.
3)Hb measurment in GCF was added as an inspection item because:
a)In previous study diagnosis based on BOP was inadequate because both (BOP- and BOP+ were found.
b)BOP indicates presence of bleeding after probing based on visual judgement and minute bleeding can be
overlooked.
c)it is not possible to know the presence or absence of occult bleeding caused by periodontitis with BOP inspe
ction

14. DISCUSSION
4)Hb was detected in 90.2% of GCF samples in pockets judged as BOP(+), but was observed also in 88.7%
GCF of BOP(-) pockets. There results suggest that the periodontal disease activity cannot be predicted
only by BOP inspection.
5)In this study, these Hb(+) group in BOP(-) sites means that the Hb is detected in periodontal pockets of
BOP(-) by Hb inspection prior to the BOP inspection. The result of these examinations indicate that the
initial periodontal tissue damage that cannot be detected by the BOP test had already occurred.
6)The present result is in agreement with previous reports that significant activities of inflammatory
parameters such as neutrophil elastase andor AST activity are detected in many GCF samples judged as
BOP(-)

15. DISCUSSION
7)Page at el. Observed that clinical discrimination between healthy and periodontically diseased conditions
in the early stage is difficult. Therefore we believe that implementation of Hb examination may improve the
validity of periodontal disease diagnosis and serve as a diagnostic strategy before the onset of periodontitis

16. CONCLUSION
1)In this study Hb was obeserved in more than 60% GCF samples in BOP(-) sulci in periodontal
stable group and in more than 85% GCF samples in BOP(-) sulci in periodontal management
required group.
2)These results suggest that simple and easy examination of Hb derived from micro bleeding in the
gingival sulci along conventional parameters of PD and BOP may serve as an index for accurate
periodontal diagnosis

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