can you produce the risk of bias according to the Agency for Healthcare Research and Quality for the study Gingival crevicular fluid levels of Interleukin-22 (IL-22) and human β Defensin-2 (hBD-2) in periodontal health and disease : A correlative study" while i provide you with the following data from the study :                                                                                                                                                                                                  1. Materials and methods "The study population consists of patients from the Department of Periodontology, Dr.D.Y.Patil Dental College and Hospital, Pimpri, Pune. The study protocol was designed in accordance with Helsinki declara- tion of 1975, as revised in 2013 and was approved by the university ethics committee (DYPV/EC/122/17).All participants willing to parti- cipate in our study signed an informed consent, were in good general heath, had no history of periodontal therapy, nor had they taken anti- biotics/anti-inflammatory drugs within 6 months. Smokers, pregnant women, patients with underlying systemic disease were excluded from the study. Sample size estimation was based on data provided by Chen et al., 2014 for expected means of GCF hBD-2 levels as measured by ELISA in chronic periodontitis, gingivitis and healthy control subjects (chronic periodontitis = 266.91 pg/mL ± 311.81 pg/mL, patients with chronic gingivitis:487.80 ± 420.66 pg/mL and healthy controls was 99.20 ± 99.98 pg/mL).14 Using a sample size of 25 subjects per group the overall estimated effect size was determined as f = 0.52 (F-test ANOVA) at p = 0.05 and 80% power, which was in the large range (> 0.40) and thus deemed acceptable.15 Since there are no preliminary data for correlation of IL- 22 and hBD-2 in each these groups, we es- timated the power of this sample to detect a significant large correlation effect (>0.5) and at n = 25, the sample had 80% power at p = 0.05 to determine a Pearson’s correlation coefficient of 0.53.",                                                                                                                                                                                                         2. Clinical measuresn"Clinical measurements made were plaque index (PI), gingival index (GI), bleeding on probing (BOP), probing depth (PD) and clinical at- tachment level (CAL). The measurements were taken at 6 sites per tooth and entered manually in the data sheet. Probing pocket depth and at- tachment level measurements were made to the nearest millimeter using an UNC 15 Probe. A total of 79 subjects were recruited in the study and were grouped according to their periodontal status. Group 1 (Healthy controls) in- cluded 27 subjects with a plaque index (PI) of < 1, gingival index (GI) of < 1, probing depth (PD) of ≤ 3 mm, and no loss of attachment. Group 2 (Gingivitis) included 25 subjects with a PI of ≥1, GI of ≥1PD ≤ 3 mm, clinical evidence of inflammation based on presence of BOP ≥ 50% of sites and no clinical attachment loss. Group 3 (Chronic periodontitis) had 27 subjects with a PI of ≥1, GI of ≥1, Probing depth (PD) ≥ 5 mm and clinical attachment level(CAL) ≥ 3 mm and presence of BOP ≥ 50% of sites with radiographic evidence of bone loss.” , is it possible to held the risk of bias ?"                                                                                                                                                                                                                                                              and 3. GCF collection :
"GCF collection was done a day after the periodontal examination. In healthy individuals, sites with PD ≤ 3 mm and no signs of inflamma- tion, in gingivitis patients, sites with bleeding on probing and in chronic periodontitis subjects, the sites with PD ≥ 5 mm were chosen for ac- quiring GCF. The area was isolated with gauze to avoid saliva con- tamination, after drying the area with air, supragingival plaque was removed with curette (Hu Friedy, USA), a calibrated volumetric mi- crocapillary pipette (Sigma Aldrich) was placed at the entrance of the gingival sulcus, gently touching the gingival margin. From each test site, 2 μl of GCF was collected. The collected GCF was then transferred to a sterile Eppendorf tube and stored at −70oc until the assay. Micropipettes with contaminants like blood or saliva were discarded.18 The GCF level of IL-22, hBD-2 was assayed using commercially available ELISA kit (E Lab Biosciences) [Catalog no: E-EL-H0106 (IL- 22), Catalog no: E-EL-H0996 (hBD-2]. The procedure was performed according to the instructions given in the kit." ?