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Flexible bronchoscopy (FB), developed in the 1960s, is widely used in the clinical practice of pediatrics and has demonstrated fundamental value in clinical diagnoses and treatment. However, as an invasive procedure, the use of FB is limited due to concerns regarding the tolerance of the procedure and the possible complications in neonatal units. Thus, the present study aimed to investigate the clinical safety and efficacy of flexible bronchoscopy (FB) in a neonatal intensive care unit (NICU). Neonates (n=54) who received FB in the NICU of Shanghai Children's Hospital between January 2012 and December 2016 were enrolled as the experimental group and another 54 neonates who required nebulization and tracheal secretion suction treatments were the control group. Indicators including blood gas, complete blood count, C-reactive protein (CRP), X-ray, patient breathing rate, temperature and blood pressure were monitored prior to and following the procedure. No significant differences in sex, gestational age, birth weight or postnatal age were observed between the experimental group and the control group (P>0.05). Among the 54 FB patients, several cases with side effect were identified, including 18 (33.3%) with respiratory tract stenosis, nine (16.7%) with malacia and stenosis and six (11.1%) with esophagotracheal fistula. Among the 54 members of the control group, 44 neonates (81.4%) were discharged with improved condition, five (9.3%) succumbed and five patients (9.3%) abandoned the treatment and left the hospital. Bronchoalveolar lavage demonstrated consistent results with respiratory secretion culture or tracheal tube culture. In comparison between the experimental and the control groups, no significant difference in pH, partial pressure of carbon dioxide (PCO2), partial pressure of oxygen (PO2) and HCO3- was observed, while there were no statistical differences in the values of pH, PCO2 and HCO3- (P>0.05). However, PO2 was significantly increased, and CRP was significantly reduced, following FB procedure compared with prior to FB (P<0.05). No pneumothorax, shock, other severe complications, fever or diffused pneumonia were observed during or after FB. The data from the present study demonstrated that FB is a safe and effective strategy for the diagnosis and differentiation of neonatal respiratory diseases in NICU. [ABSTRACT FROM AUTHOR]