To date no study has investigated the actual technical success rate of IA injections into the joint space. Knowledge of technical success is relevant to determine potential efficacy confounders due to inappropriate injection. The aim of this study was to determine the success rate of intra-articular injection of an investigational treatment of knee osteoarthritis pain into the knee joint with and without ultrasound guidance, using radiographic documentation of intra-articular air injection as the reference. Between December 2020 and March 2022, 216 patients underwent intra-articular injection of an investigational treatment, a cryopreserved Amniotic Suspension Allograft (ASA, Organogenesis, Canton, MA) into the knee joint, either under ultrasound guidance or with palpation-based injection without imaging guidance. Injection of the study treatment was performed in a phase 3 prospective double-blind, multicenter, placebo-controlled, parallel group, randomized control trial (RCT) in subjects with moderate to severe symptomatic knee osteoarthritis. Injection was performed by physicians or physician-assistants in Orthopedics, Internal Medicine, Rheumatology, or Radiology. Inclusion criteria were the presence of radiographic knee OA (Kellgren and Lawrence grade 2 or above) and knee pain. About half of the recruited patients underwent ultrasound guided injection and the other half had palpation-guided injection without imaging guidance. 1 mL of 1% lidocaine was used to anesthetize the skin and periarticular tissues with a 27g needle prior to using a 21g 1.5-inch needle or 22g 3-inch needle for joint aspiration and injection of either ASA (2mL ASA + 2 mL saline) or 4 mL placebo (USP normal saline). After the injection of study treatment, and with the needle in the same exact location, 10 mL of air were injected, and a lateral X-ray of the knee was performed within 10 minutes to document the presence of intra-articular air. Radiographs were read by a musculoskeletal radiologist blinded to injection technique. Patients’ age, gender, body mass index (BMI), and radiographic knee OA grade were recorded. Collected data (success rate of intra-articular injection) were stratified according to guidance method (with or without ultrasound), approach of injection (medial, lateral suprapatellar, anterior), specialty of injector (Orthopedics, Rheumatology, Internal Medicine, or Radiology), experience of injector (<10 years, 10 years or more but <20 years, 20 years or more), sex, BMI, and Kellgren and Lawrence grade. One-sided Cochran-Armitage test for trend was used to evaluate if there was decreasing success rate with increasing BMI, and to evaluate if there was increasing success rate with increasing injector experience. Included were 216 patients whose mean age was 59.8 (SD ± 10.2) years, and mean BMI 30.6 (4.8). 134 patients were male (62.0%). 94 patients had US-guided injection and 117 patients had palpation-guided injection. Details of demographic characteristics are summarized in Table 1. Of these, 159 injections were intra-articular and 42 extra-articular. 3 injections were indeterminate since air was not visible on the X-ray and excluded from analysis. Although US-guided injection had higher success rate (80/94, 85.1%) than palpation-based injection (89/117, 76.1%), there was no statistically significant difference (p=0.10). There was no difference of success rate with respect of injection approach (medial or lateral, p=0.75). No statistically significant difference in success rate was seen regarding injector specialty, patient sex, BMI, or radiographic OA grade (Table 1). Table 2 also shows more experienced injectors had better success rate than less experienced injectors albeit not statistically significant (p=0.11). There was no trend for decreasing success rate of intra-articular injection with increasing BMI (3 categories), with p-value=0.43. Neither ultrasound-guided nor palpation-guided injection provides 100% success rate for intra-articular injection of the knee as demonstrated by intra-articular air visualization on radiography. The more experienced operators have a higher success rate, with a threshold of 10 years of experience, while specialty or training seems to be less relevant. While ultrasound-guided injection showed somewhat higher success rates compared to palpation-guided injection, documentation of intraarticular injection using air seems paramount to avoid subsequent extra-articular injection of investigational or disease modifying osteoarthritis drug (DMAOD) compound and thus, maximize efficacy. No funding was received for the analysis of this Study. AG has received consultancies from Pfizer, Novartis, Regeneron, AstraZeneca, Merck Serono, and TissueGene and is shareholder of Boston Imaging Core Lab (BICL), LLC. FWR is shareholder of BICL, LLC. and has received consultancies, speaking fees, and/or honoraria from Calibr –California Institute of Biomedical Research and Grünenthal, GmbH. MDC is shareholder of BICL, LLC. CORRESPONDENCE ADDRESS: [email protected]