can you produce the risk of bias according to the Agency for Healthcare Research and Quality for the study “Gingival Crevicular Fluid Levels of Human Beta-defensin 2 and 3 in Healthy and Diseased Sites of Individuals with and without Periodontitis” while i provide you with the following data from the study :
1.Study design and sampling strategy
“This cross-sectional study comprised participants from a previous study (Costa et al. 2018), totalling 40 individuals selected from the Periodontology Clinic at the Federal University of Minas Gerais. Sample size was calculated using statistical software (GPower 3.1 Statistical Power Analysis for Windows, Düsseldorf, Germany) consider- ing mean values from previous studies (Dommisch et al. 2005, Brancatisano et al. 2011, Ertugrul et al. 2013), an effect size of 0.30, a significance level of 0.05 and a study power of 0.80. Based on these parameters, a sample size of ~20 individuals per group was determined to be necessary. Sampling strategy was as previously described (Costa et al. 2018). Briefly, during the period of data collection (August 2012 to December 2014), approximately 360 individuals sought dental care in the university, were determined to be eligible and invited to participate in the study. According to their availability and accessibility during the dental care routine, individuals who met the inclusion criteria were added to the study groups until the required sample size was reached. Individuals were excluded if they were pregnant or lactating women, smokers and former smokers or if they have any systemic diseases. Exclusion was also applied to individuals using drugs that could influence periodontal health, having received periodontal treatment within the last 6 months or having used antibiotics and anti- inflammatory drugs within the last 3 months.”,
2. Periodontal clinical examination
“All participants underwent a full-mouth periodontal examination and the parameters of probing depth (PD), clinical attachment level (CAL), and bleeding on probing (BOP) were recorded at 4 sites (buccal, distal, lingual, mesial) for all teeth present except any third molars. Intraoral radiographs were taken to detect bone loss (Costa et al. 2018). One single trained examiner performed all examinations with a manual periodontal probe (PC- PUNC 15 Hu-Friedy®, Chicago, USA). Evaluations of PD and CAL parameters were performed and repeated within a 1-week interval for 10 individuals randomly selected during the recruitment of participants, in order to determine intra-examiner agreement. Analyses retrieved kappa values for PD and CAL greater than 90% and intra- class correlation coefficient greater than 89%. Those individuals were not included in the study. Participants were divided into 2 groups (n = 20): periodontally healthy individuals (H) and individuals with periodontitis §. Periodontally healthy individuals (H) should have a minimum of 20 natural teeth, PD ≤3 mm and no more than 20% of sites with BOP. It is important to stress that CGF samples were collected in these individuals from sites with no BOP. Individuals with periodontitis should have a minimum of 15 natural teeth and at least 4 teeth with 1 or more sites with PD ≥4 mm and CAL ≥3 mm in the same site. The diagnosis of periodontitis was based on clinical and radiographic parameters as previously established (American Academy of Periodontology Task Force Report on the Update to the 1999 Classification of Periodontal Diseases and Conditions, 2015). Diagnosis and definition of periodontitis were updated according to the new classification scheme for periodontal diseases and conditions (Tonetti et al. 2018) and participants’ periodontal condition was confirmed.”,
3. Gingival crevicular fluid sampling
“GCF sampling was carried out 1 week after the periodontal examination among all participants. GCF fluid samples were obtained from healthy sites (Hh) of each periodontally healthy individual and from healthy sites, sites with gingival inflammation but no attachment loss (Pg) and sites with gingival inflammation and attachment loss (Pp) of each individual with periodontitis. For each sample category, 4 representative sites were selected in each participant and analyzed together (pool samples) to provide a mean value per category/individual (Hh [n=20],Ph[n=20],Pg[n=20],Pp[n=20]).A total of 320 sites were sampled (80 in H individuals and 240 in P individuals).
Healthy sites (Hh and Ph) for GCF collection showed PD and CAL ≤3 mm without BOP and without alveolar bone loss. In P individuals, sites with gingival inflammation but no attachment loss (Pg) for GCF collection showed PD and CAL ≤3 mm and BOP; sites with gingival inflammation and attachment loss (Pp) showed PD ≥4 mm, CAL ≥3 mm and BOP. The 4 Pp sites were selected according to the deepest PD at different non-adjacent teeth, preferably 1 site in each quadrant. Clinical parameters of sampling Pp sites were: mean PD 6.01±2.03 mm and mean CAL 6.40±2.31 mm). The 4 Hh and Ph sites (healthy sites), as well as the 4 Pg sites (sites with gingival inflammation but no attachment loss), were also collected at different non- adjacent teeth, preferably 1 site in each quadrant (Costa et al. 2018).
Before GCF sampling, all selected sites were isolated with cotton rolls to avoid contamination with saliva, supragingival plaque was removed and the tooth was gently air-dried. Paper strips (Periopaper, PerioCol Collection Strip, Oraflow, Plainview, NY, USA) were inserted into the gingival sulcus/pocket until mild resistance was felt and left in position for 30 seconds. Care was taken to avoid mechanical injury. Paper strips contaminated with blood were discarded (Griffiths 2003, Wassall and Preshaw 2016). The total GCF volume collected by each strip was measured with a calibrated device (Periotron 6000, IDE Interstate, Amityville, NY, USA). Representative samples of the same site category (Hh, Ph, Pg, Pp) in the individual were placed into a dry Eppendorf tube (4 paper strips per tube) and frozen at −80°C until laboratory analysis.” ?