Can you produce the risk of bias according to the Agency for Healthcare Research and Quality for the study “Impact of smoking on protein levels of beta-defensins in periodontal disease” while i provide you with the following data from the study: 
1. Study design
“The study protocol was approved by the Ethics Committee on Human Research of the School of Dentistry at Araraquara - UNESP (protocol number 01394712.5.0000.5416). The purpose and content of the study were explained to eligible subjects, and written informed consent was obtained from all participants for inclusion in the study. A total of 70 participants were recruited from June 2016 to March 2018 at the Periodontology Clinic, School of Dentistry at Araraquara - UNESP. The diagnosis of periodontitis was performed according to the classification of the 2017 World Workshop on the Classification of Periodontal and Peri-implant Diseases and Conditions 12 . All patients should present a minimum of 15 natural teeth. Twenty patients were included in the periodontally healthy control group. All subjects were free of systemic and periodontal diseases. They should present probing depth (PD) ≤ 3 mm, no bleeding on probing (BOP), no clinical attachment loss, plaque index (PI) and gingival index (GI) ≤ 15%. Fifty patients with periodontitis stage III or IV were included in periodontitis groups. In addition to the diagnostic criteria 12 , they should present at least five sites in non-adjacent teeth with PD ≥ 4 mm, BOP, and clinical attachment level (CAL) ≥ 4 mm, except for third molars. From the 50 patients, 25 had never smoked and 25 were smokers who should smoke at least 10 cigarettes/day for a minimum of 5 years.
None of the patients had a history of systemic diseases or immunological disorders. Subjects who had received antibiotics and anti-inflammatory drugs within the last 3 months or received periodontal treatment within the last 6 months were not included in the study. Patients using hormone replacement therapy, pregnant and lactating women and orthodontic patients were also excluded.
The sample size was calculated using statistical software (GPower 3.1 Statistical Power Analysis for Windows, Düsseldorf, Germany). Considering mean values of hBD expression and standard deviation from previous studies 13 , 14 , 15 , an effect size of 0.3 was obtained. Based on this effect size, a significance level of 0.05 and a study power of 0.80, a sample size of 20 individuals per group were determined to be necessary.”, 
2.Clinical examination
“One calibrated examiner performed all clinical examinations. The intra-examiner reproducibility was assessed by measuring the PD in six teeth (16, 21, 24, 36, 41 and 44), according to the Ramfjord index, in a total of six patients (36 sites/patient) on two different occasions in a time interval of 7 days. Data were submitted to Kappa concordance analysis (k=0.91). The parameters PD, BOP and CAL were assessed at six sites per tooth (mesio-buccal, mid-buccal, disto-buccal, mesio-lingual, mid-lingual, disto-lingual), while PI and GI were registered as absence (negative site) or presence (positive site) of visible plaque and gingival bleeding at four sites per tooth (mesial, distal, buccal and lingual), excluding the third molars. Measurements were performed with a manual periodontal probe (UNC15; Hu-Friedy, Chicago, IL, USA).”, 3. Gingival crevicular fluid (GCF) sampling :
“GCF samples were collected from healthy (n=5) sites of each periodontally healthy patient and from healthy (n=5) and diseased (n=5) sites of each periodontitis patient. Sites at different non-adjacent teeth, preferably one site per quadrant, were selected for GCF collection in each subject, one day after the periodontal examination. Healthy sites had no gingival inflammation, PD ≤ 3 mm, no BOP or clinical attachment loss; and diseased sites had gingival inflammation, PD and CAL ≥ 4 mm with BOP. Diseased sites were selected according to the deepest probing depth in each quadrant. A single examiner performed GCF sampling. The sites to be sampled were isolated with cotton rolls, the supragingival plaque was removed with a sterile curette and the surfaces were gently air-dried. GCF was sampled by inserting absorbent paper strips (Periopaper®, Oraflow Inc., New York, USA) into the gingival sulcus or periodontal pocket for 30 seconds. Paper strips contaminated with blood and saliva were rejected. An electronic device Periotron 8000 determined the GCF volume absorbed into each strip (Periopaper, ProFlow, Inc., Amityville, NY, USA), which was calibrated based on a protocol described before 16 (calibration of the electronic device by polynomial regression). The GCF was obtained as a pooled sample of the same category (healthy or diseased site) in each subject. The papers strips were immediately placed into a dry sterile polypropylene tube (5 paper strips per tube) and kept at -80 °C until analysis. The readings from the Periotron 8000 were converted to an actual volume (μL) by reference to a standard curve.”,
4. Immunosorbent assay for hBD 1 and hBD 2 quantification:
“First, the absorbed GCF was eluted from the paper strips by adding 750 μL (150 μL per strip) of 1x Phosphate-Buffered Saline (PBS) to each tube. The tubes were left on ice in a horizontal shaker for 30 minutes and then centrifuged at 4 °C, 13.000 g for 10 minutes. The GCF levels of hBD 1 and 2 were measured using the sandwich ELISA technique according to the manufacturer’s guidelines (Peprotech, Rocky Hill, NJ, USA). The absorbance was measured by a spectrophotometer at 450 nm wavelength. The levels of AMPs in each sample were determined using the concentration values of standards included in the kit contents. The results were expressed in pg/mL.” and
5. Statistical analysis
“The statistical analysis was performed using the software GraphPad Prism 7 (San Diego, CA, USA). For clinical data, normal distribution was analyzed using the D’Agostino and Pearson test. For normally distributed data, it was used One-way ANOVA followed by Tukey for healthy sites and unpaired t test for diseased sites on inter-group analyses and paired t test for intra-group analyses. For non-normally distributed data, Kruskal-Wallis and Dunn were used for healthy sites and Mann-Whitney tests was used for diseased sites on inter-group analyses, while the Wilcoxon test was performed for intra-group comparison. A p-value of less than 0.05 was considered significant. The effect size was estimated for the primary outcome of the study (AMPs level in GCF). Since non-parametric tests were used for intra and inter-group comparisons, the eta squared and the correlation coefficient r were calculated.”