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Prevalence of blood contamination in adult saliva andquantitative measurement of salivary hemoglobin level by using
an anti-human hemoglobin monoclonal antibody
Hidenori Yamada, DDS, PhD (1), Ayako Okada, DDS, PhD (1), Takatoshi Murata, DDS,PhD (1), Kimiyuki Uetani, DDS (2), Mami Kotoh, DDS, PhD (2), Naoyuki Nishitsuji,DDS (2), Yoshihide Yabuki, DDS (2), Yoichi Fukuzawa, DDS, PhD (2), Hirokazu
Yoshino, DDS (2), Koshi Kubo, DDS (2), Mitsuhiro Abe, DDS, PhD (2), Takatoshi One,DDS, PhD (2), Hiroaki Nagai, DDS, PhD (2), Masataka Yajima, DDS (2), YoshiakiNomura, DDS, PhD (1), and Nobuhiro Hanada, DDS, PhD (1)(1) Department of Translational Research, Tsurumi University School of Dental Medicine, Yokohama,and (2) Minato-ku Shiba Dental Association, Tokyo, Japan
Purpose: Blood contamination in saliva is more common in individuals with periodontal destruction. This study
aimed to determine the prevalence of salivary hemoglobin and periodontal pathogens in Japanese adults.
Materials and Methods: The study participants were 249 subjects older than 20 years who participated in anoral health promotion festival organized by the Tokyo Minato-ku Shiba Dental Association. We collected 249
saliva specimens from the 249 subjects in the middle of Tokyo, Japan. For bacterial detection, 24 specimens
were selected. Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans were detected in 24
specimens by using polymerase chain reaction.
Results: The salivary human hemoglobin (hHb) levels in 94 subjects (37.8%) were lower than the detectablelevel (<1 µg/mL). The remaining 155 subjects (62.2%) were suspected of having periodontal disease. If weadopt a cutoff hHb level of 2.0 µg/mL, 101 subjects (40.6%) would be positive for periodontal disease. Of the 24
saliva specimens, 21 (87.5%) and 3 (12.5%) tested positive for P. gingivalis and A. actinomycetemcomitans,respectively.
Conclusion: Our data indicate that screening tests for blood contamination in adult saliva were useful for oral
and general health.
(Asian Pac J Dent 2016; 16: 9-13.)
Key Words: anti-human hemoglobin monoclonal antibody, hemoglobin, periodontal disease, saliva

Introduction
Martí et al. [1] studied a population of 184 adult patients composed of 101 totally edentulous patients with nooral mucosa lesions and 83 dentulous patients with clinically healthy gingival tissues. Of the dentulous patients,
67% tested positive for hemoglobin contamination in saliva. On the other hand, none of the patients in the totally
edentulous group exhibited hemoglobin in saliva. These data suggest that periodontal conditions are an essential
factor for the presence of hemoglobin contamination in saliva. In healthy humans, the epithelial barrier provides
the first line of defense against the invasion of pathogenic and commensal bacteria. Periodontal pathogens,smoking, and biting force induce inflammatory responses that lead to the destruction of the oral epithelial barrier.
As a result of the inflammatory responses, saliva can often be contaminated with hemoglobin. Hemoglobin
contamination in saliva is more common in individuals with periodontal destruction. Hemoglobin is a protein
composed of four protein chains containing an iron atom. The iron atom is specifically removed from thehemoglobin molecule and used by the periodontal pathogens for their growth.
A new method of immunological detection of hemoglobin was reported in 1991 [2]. The detectable
hemoglobin concentration with this method was 0.5 µg/mL. Nagata and Tanaka [3] produced monoclonal
antibodies against human hemoglobin (hHb). They analyzed 785 fecal specimens by using colloidal gold
agglutination and compared the results with those obtained using latex agglutination. Hemoglobin was detected9Yamada et al.

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in 75 specimens by using colloidal gold agglutination, in 76 specimens by using latex agglutination, and in 70
specimens by using both methods. The overall agreement between the two methods was 98% [3].
In this study, we applied the method for immunological detection of hemoglobin in fecal specimens to saliva
specimens, and report the prevalence of salivary hemoglobin and periodontal pathogens in Japanese adults.

Materials and Methods
Participant recruitment
The Tokyo Minato-ku Shiba Dental Association holds annual festivals as oral health promotion efforts toimprove oral health through saliva screening. The participants of this study were 249 subjects older than 20years who participated in an oral health promotion festival in response to recruitment materials (public bulletins,posters, flags, and solicitations).
Saliva specimens
We collected 249 saliva specimens from subjects of various ages, both sexes, and occupation in the middle ofTokyo, Japan, in May 2013. A standardized bolus of a gum base containing neither fragrant nor taste ingredientswas given to the subjects. Stimulated whole saliva was collected by chewing on the gum base for 5 minutes.
Measuring salivary hemoglobin
For the examination of salivary hemoglobin levels, the collected saliva specimens were maintained at 4˚C. These
specimens were immediately transported to the examination laboratory. Salivary hHb levels were measured byusing the NS-Plus automated analyzer and saliva immunochemical testing system (Ness Coat Saliva Hemo Plus,
Alfresa Pharma Co., Osaka, Japan). Ness Coat Saliva Hemo Plus was used as a diagnostic device for quantitative
detection of the hHb in human saliva by using the immunochemical method. Colloidal gold labeled withmonoclonal antibody against hHb was agglutinated with hemoglobin and changed its color from red to gray. The
color that diminished with the presence of an antigen was unchangeable from 30 min to 4 h. The detectable
hemoglobin concentration with this method was 1.0 µg/mL.
Polymerase chain reaction
The polymerase chain reaction kits for detection of periodontal pathogens in saliva were purchased from a
medical examination company (Genesis Healthcare, Co., Tokyo, Japan). Porphyromonas gingivalis andAggregatibacter actinomycetemcomitans were detected by using PCR.
Ethical considerations
The study was approved by the ethics committee of Tsurumi University School of Dental Medicine (approvalNo. 430) and conducted in accordance with the Declaration of Helsinki.

Results
Finally, 249 saliva specimens were examined. The distribution of the salivary hHb levels is shown in Fig. 1. One
specimen with outlier values that remarkably differed from other data had an hHb level of 876.0 µg/mL. This
sample was excluded for the following analysis.
The salivary hHb levels in 94 subjects (37.8%) were <1.0 µg/mL. If we adopt a cutoff hHb level of 1.0 µg/mL,
155 subjects (62.2%) would be suspected as having periodontal disease. Meanwhile, if we adopt a cutoff hHblevel of 2.0 µg/mL, 101 subjects (40.6%) would test positive. Bacterial pathogens are important targets for
detection and identification in medicine. Saliva specimens that tested positive for P. gingivalis and A.

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actinomycetemcomitans were assessed qualitatively for comparison with different salivary hHb levels.

Fig. 1
Distribution of the salivary humanhemoglobin levels

Table 1

Cross tabulation of the presence of periodontal bacteria according to salivary human hemoglobin level

Salivary human hemoglobin level

Porphyromonas gingivalis

Aggregatibacter actinomycetemcomitans(µg/mL)+−+−0.1-0.2 µg/mL

111111>80 µg/mL

10221

Of the 24 specimens, 21 (87.5%) and 3 (12.5%) tested positive for P. gingivalis and A.
actinomycetemcomitans, respectively (Table 1). The overall detection rate of P. gingivalis was high (21/24specimens), whereas that of A. actinomycetemcomitans was low (3/24 specimens). Owing to the extremely wide
distribution of the values, we could not find any significant difference between the two groups of salivary
hemoglobin levels in terms of the prevalence of periodontal bacteria.

Discussion
Traditional screening methods for periodontal diseases that involve the use of periodontal probing may be
uncomfortable or even painful for subjects. In this study, a new method for immunological detection of occult
blood in saliva was introduced that used colloidal gold labeled with anti-hHb monoclonal antibody.
If the barrier between the bloodstream and oral mucosa is compromised by chronic inflammation such thatblood or plasma leaks into the saliva, then higher levels of biomarkers in the bloodstream could contaminate
saliva specimens. Among the blood or plasma biomarkers we tested, salivary hemoglobin and lactate
dehydrogenase levels correlated with periodontal destruction conditions [4,5]. These biomarkers also showed anassociation when the community periodontal index (CPI) method, which involves the use of periodontal probing,
was used [4].
In this study, the salivary hHb levels in 94 subjects (37.8%) were lower than the detectable level (<1 µg/mL),which indicated that 37.8% of the subjects had no oral inflammation. This means that 62.2% of the subjects had
some inflammation that led to periodontal disease. If we adopt a cutoff hHb level of 2.0 µg/mL, 101 subjects

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(40.6%) would test be suspected of having periodontal disease.
By contrast, salivary bacterial measurement was difficult to use for the periodontal disease screening test. Thedetection rate of P. gingivalis was 87.5% in total. In addition, the detection rate of P. gingivalis in the subjects
with salivary hHb levels within 0.1-0.2 µg/mL was 91.6%. These results indicate that most of the adults had P.
gingivalis infection. P. gingivalis is a well-known bacterium that causes periodontal diseases. To promote oral
health and periodontal disease prevention, subjects should undergo eradication therapy for oral cavity P.
gingivalis infections. Salivary hHb levels of >1 µg/mL should be reduced to below detectable levels (<1 µg/mL)
by tooth cleaning for prevention and interrupting the progression of periodontal disease.
A new therapy for dental biofilm management was established by our research group [6-10]. This therapy
was named 3DS (Dental Drug Delivery System) and validated by in vivo studies and clinical evaluations. In the3DS method, antibacterial agents are applied by using individual trays at chair-side and as home-care program.
By using individual trays, the dental biofilm that leads to gingivitis is controlled without changing the corecomposition of the oral microflora. We recommend applying this method in dental clinics worldwide.
Salivary screening has many advantages over other methods. However, mass screening is time-consuming
and entails high costs in terms of employment of dentists. Furthermore, before beginning the screening for
gingivitis and/or periodontitis, evaluation by the examiner is required. Mass screening using a questionnairemethod is simple and inexpensive but not sensitive for periodontal diseases. The progress of periodontal diseases
is difficult to determine because they are typically clinically silent. Screening tests for fecal occult blood is
popular worldwide. Similarly, screening tests for blood contamination in adult saliva should become popular in
the future.
Piazza et al. determined blood contamination in the saliva of patients with acquired immunodeficiency
syndrome (AIDS)-related complex/AIDS [11]. Patients with AIDS-related complex/AIDS have a mean salivary
hHb level of 190 µg/mL, which corresponds to 1.3 µL of blood per milliliter of saliva. If 10 mL of saliva is
exchanged between partners during kissing, an average of 13 µL of blood is transferred [11]. Piazza et al. also
detected blood contamination in the saliva of patients with human immunodeficiency virus (HIV) infection [12].
The data show that the anti-HIV-positive group had higher hHb concentrations than the control group. These
data indicate that screening tests for blood contamination in adult saliva are necessary for prevention not only ofperiodontal diseases but also of HIV/AIDS.
The reagent kits for measuring salivary hHb levels (provided by Alfresa Pharma Co.) that were used in thisstudy were approved according to the Pharmaceutical Affairs Law of Japan as official in vitro diagnostic reagent
for the evaluation of the periodontal conditions. This approval is a great advantage of salivary hHb screening
over other biomarker tests for periodontal diseases, which have not been approved according to the law.

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Correspondence to:
Dr. Nobuhiro Hanada
Department of Translational Research, Tsurumi University School of Dental Medicine
2-1-3 Tsurumi, Tsurumi-ku, Yokohama 230-8501, Japan
Fax: +81-45-573-2473 E-mail: hanada-n@tsurumi-u.ac.jp
Accepted February 12, 2016.
Copyright ©2016 by the Asian Pacific Journal of Dentistry.
Online ISSN 2185-3487, Print ISSN 2185-3479

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