Detection of defective granulocyte function with flow cytometry in newborn infants

Yu-Ching Wu¹, Ya-Fang Huang², Chyi-Her Lin¹, Chi-Chang Shieh^1,3
1Department of Pediatrics, ²Institute of Basic Medicine and ³Institute of Microbiology and Immunology, National Cheng-Kung University Medical College, Tainan, Taiwan

Received: June 21, 2004 Revised: July 8, 2004 Accepted: September 15, 2004

Corresponding author: Dr. Chi-Chang Shieh, M.D., Ph.D., Department of Pediatrics, National Cheng-Kung University, 138 Sheng-Li Rd, Tainan 704, Taiwan. E-mail:

Reactive oxygen species (ROS) production by phagocytic leukocytes is a critical factor in immunity against microorganisms. Human neonates are susceptible to overwhelming infections, for which abnormal granulocyte function may play an important role. In this study, we aimed to identify a convenient and quantitative method to measure ROS production by human granulocytes after cellular activation. We first compared the results of a flow cytometric assay with 2',7'-dichlorodihydrofluorescein diacetate (H₂DCF) probe or dihydrorhodamine-123 (DHR123) and an enhanced chemiluminescence test using granulocytes from a patient with chronic granulomatous disease and normal granulocytes stimulated with different concentrations of phorbol myristate-13-acetate. Peripheral blood granulocyte respiratory burst ability from 37 newborn babies with different gestational ages was then quantified using flow cytometric assay with H₂DCF probe. We found that flow cytometric assay of ROS production is sensitive and correlates with chemiluminescence measurements. The results showed that activated granulocytes from neonates with higher birth body weights and more advanced gestational ages tend to have higher levels of ROS production in respiratory burst (p=0.0042 and 0.063, respectively). This study demonstrates that flow cytometry is suitable for detecting the functional defects in granulocyte ROS production in human neonates of different gestational age.

Key words: Chemiluminescent measurements, flow cytometry, newborn infant, premature infant, respiratory burst

J Microbiol Immunol Infect 2005;38:17-24.