**2.6 Statistical analysis**

*Gingival Disease - A Professional Approach for Treatment and Prevention*

Study participants are recruited by direct invitation and pamphlets from pediatric allergy-immunology clinic at Dr. Soetomo General Hospital (Indonesia). Data collectors reviewed the clinic appointment schedules to identify subjects with HDM allergy and met with subjects and their parents or families after their physician visit for a screening to assess their eligibility. Patients were enrolled after considering various inclusion and exclusion criteria such as mentioned in (**Table 1**). By total sampling, 32 subjects were obtained from this hospital. They were prescreened from January 31 to February 28, 2017 at pediatric allergyimmunology clinic at Dr. Soetomo General Hospital (Indonesia). Based on the inclusion and exclusion criteria mentioned in (**Table 1**), 22 subjects were eligible to participate in the study. Informed consent is given as a voluntary agreement to participate in this research, out of which, 20 parents agreed and 2 parents

Data were collected by taking venous puncture for measuring total serum IgE and IgG4 from subjects' blood samples and taking saliva for measuring lipopolysaccharide of *Porphyromonas gingivalis* concentrations [23]. Total serum IgE and IgG4 were assessed by direct-sandwich ELISA (R&D System Europe Ltd., Abingdon, UK) according to the manufacturer's protocol. Briefly, total serum IgE was detected by diluting plasma (1:200), transferring it to pre-coated plates, and adding the supplied conjugate. However, total serum IgG4 were detected using monoclonal antibody against IgG4, followed by additions of blocking solution, diluted plasma sample (1:100,000) or standards, and conjugate, with washing between the steps. Total serum IgE and IgG4 ELISAs were developed with the supplied TMB substrate and stop solutions. Total serum IgE and IgG4 concentrations were determined using assay-specific 7-point calibration curves generated with the

> Any sign of allergic diseases (asthma, hay fever, food allergy, eczema) within 1 month before observation

> Taking any antihistamines or steroid within 1 month

Experienced dental scaling and root planning within

The presence of low-grade fever due to infections

Recent blood disorders or congenital abnormalities

Any medical conditions that may be harmful to be involved in this study, in which phlebotomy is

before observation

contra-indicated

6 months before observation

rather than gingivitis/periodontitis

**Inclusion Criteria Exclusion Criteria**

Children aged 6–16 years at any gender and

Have been diagnosed with HDM allergies by

Periodontally healthy children without any sign

Understand and able to cooperate to the research

Positive culture of *Porphyromonas gingivalis* from salivary samples in the absence of gingival

Parents or legal representatives have signed the written consent in accordance with our

ethnic

diseases

protocol

institutional policies

*Eligibility criteria for study participants.*

positive skin-prick test

of chronic gingival diseases

**2.4 Participants**

declined to participate in the study.

**2.5 Data collection**

**34**

**Table 1.**

The obtained data were tabulated and analyzed using Statistical Package of Social Science (SPSS version 17, IBM, New York, USA). First, univariable linear regression procedures were conducted to examine associations between circulatory Ig-E and all determinants. In advance, dummy variables were created for all categorical determinants. Second, a multiple linear regression analysis with a stepwise exclusion method was conducted with all continuous and dummy variables. Determinants that seemed relevant for prediction of activation were kept in the model. Statistical significance was set at *p* < 0.05 [23].
