image quality [18]. In contract, three-dimensional techniques like Computed Tomography (CT) or Cone Beam Computed Tomog- raphy (CBCT) data provide an accurate 3D assessment of the airway in all three planes; coronal, sagittal and axial [17]. However, the radiation dose for standard CT scan of the maxillofacial region is high and repeated scanning is a concern. Medium field-of-view CBCT radiation ranged from 69 to 560 microsieverts (mSv); whereas, a similar field-of-view medical CT produced 860 mSv [19]. CBCT has been shown to be effective in localization of impacted teeth and in assessing the outcome of alveolar bone grafting and the eruption path of the canine in grafted alveolar clefts [20,21,22]. Previous studies have shown that 3D imaging using CBCT is a simple and effective method to accurately analyze the airway [23,24]. Advantages of CBCT include X-ray beam limitation, image accuracy, rapid scan time, dose reduction, display modes unique to maxillofacial imaging, and reduced image artifact [25]. Recently, there have been multiple studies using CBCT to assess the airway in relation to facial morphology in individuals with sleep apnea [26,27]. To date, we have not found any published CBCT studies of the airway of individuals with CLP, despite the frequently occurring airway problems in these individuals [4,5,6,7]. We hypothesized that individuals with CLP have smaller pharyngeal cross-sectional areas and volumes compared to non-cleft controls. Methods This was retrospective study of subjects recruited from the Craniofacial Center and Orthodontic clinic. Written informed consent was obtained for all participants of the study which was approved by CHR. We obtained ethics approval for our study from the ethics committee at UCSF, (CHR # h44601-34031-02). Previous published data on volume in non cleft individuals report an average volume of 20.9 mm3 with a S.D. of 3.6 mm3. We made the assumption that the airway would be 10% smaller in CL/P with the same S.D. as the control. This calculation resulted in a sample size of 29, in order to detect a difference. The experimental group included 19 individuals (12 male and 7 female with CLP; 16 with unilateral and 3 with bilateral complete clefts). The exclusion criteria included clefting associated with diagnosed syndromes and no prior adenoidectomy and/or tonsillectomy. All individuals with CLP had maxillary expansion and alveolar bone grafting. The control group included 19 individuals who were gender, age, and Sella-Nasion (SN) matched with the experimental group. The inclusion criteria included Angle Class I malocclusion with beginning CBCT records for starting orthodontic treatment and no prior orthodontic treatment, no habitual mouth breathing recorded and no prior adenoidectomy and/or tonsillectomy. CBCT scans with 0.4 mm slices were obtained at one time point on the Hitachi MercuRay CBCT machine (Hitachi Medical Corporation, Japan). Scans were standardized with an 868-inch field of view and contained 512 axial sections in 0.4 mm thick increments. DICOM files were imported into CB Works 3.0 (Seoul, Korea), software capable of volume rendering. First, a CBCT technician de-identified the files, removing name, sex and date of birth from the DICOM. Next, files were reoriented to a standardized view. In the sagittal view, the vertebral column was realigned perpendicular to the horizontal axis. If the vertebral column was curved, the pharynx was reoriented perpendicular to the horizontal axis. It was preferred to use the vertebral column as a landmark because it is a hard tissue. 3D Airway in Clefts The airway was segmented and volume rendered in multiplanar reconstruction (MPR) mode. The threshold tool was used to select the airway space. The histogram was set to include data within the boundaries of 1025 to 2524 HU. These values were saved as a preset in CB Works. Next, the Volume of Interest (VOI) tool was used to grossly remove data outside of the airway. The airway data were further refined and superior and inferior borders were defined using the Regional Edit tool. The superior border was defined as a line connecting Sella and Posterior Nasal Spine. The inferior border was defined as a line connecting the most anterior region of the airway formed by the thyroid cartilage to the transverse arytenoid muscle at the level where the esophagus splits from the airway (Fig. 1). The SSD function was used to create a 3D rendering of the airway space. The volume (cm3) and smallest cross sectional area was determined from the total airway segmentation. In order to define the location of the smallest cross sectional area, the airway was further segmented into three sections: nasopharynx, oropharynx and hypopharynx. The superior border of the nasopharynx was defined as the posterior choana and inferiorly as the horizontal line along the hard and soft palates. The oropharynx was defined superiorly by the soft palate and inferiorly by the vallecula. The hypopharynx was defined superiorly by the hyoid bone and vallecula and inferiorly by the junction of the larynx and esophagus (Fig. 1). The linear measurement tool in the CB Works 3.0 software was used to determine SN in order to properly match the experimental and control groups. Results The Pearson Correlation Coefficient and Lin Concordance were used to test the reliability of the methods used in choosing landmarks that further lead to measurements of airway volumes and cross sectional areas. Seven randomly selected cases were re- studied and airway volumes and cross sectional areas re-measured with a Pearson Correlation Coefficient of .99 and a Lin Concordance of .99 for both measurements. The mean anterior cranial base length (SN) was 55 and 56 mm, respectively, for the cleft and non-cleft individuals, indicating no significant size different between the groups. Figure 1. Landmarks of pharyngeal airway as shown in sagittal view. 1) Center of Sella to Posterior Nasal Spine 2) Hard Palate 3) Vallecula 4) Junction of larynx and esophagus. doi:10.1371/journal.pone.0043405.g001 PLOS ONE | www.plosone.org 2 August 2012 | Volume 7 | Issue 8 | e43405