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Optimizing Utilization of Laboratory Investigations in Neonatal Intensive Care Unit. OBJECTIVE: To optimize utilization of laboratory tests by measuring baseline rates and appropriateness of investigations, assessing the barriers to rational use, and developing and implementing an educational package for resident doctors. DESIGN: Quality improvement study. SETTING: Neonatal intensive care unit (NICU) from August, 2015 to December, 2016. PARTICIPANTS: All neonates admitted in NICU and resident doctors working in NICU. INTERVENTION: Addressing barriers, educational package, posters and group discussions. MAIN OUTCOME MEASURES: Laboratory test rates for hematology, biochemistry and blood gas. Proportion of tests judged to be inappropriate. RESULTS: At the baseline, median (IQR) laboratory test rate patient/day was 0.6 (0.2-1.5) and one-fifth of tests were classified as inappropriate. Mechanical ventilation and sepsis were independent predictors of laboratory test rates but could explain only 35% of the disparities, indicating variations in clinical practice. Following a short period of intervention, hematology investigations showed a trend towards reduction, though overall test rates did not change significantly. CONCLUSION: Addressing barriers, creating awareness and educational interventions were able to bring down hematology laboratory test rates in a short period. A longer period of sustained intervention is required to demonstrate significant effects on test ordering behavior.