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Single-Cell Flow Cytometry Profiling of BAL in Children
Shivanthan Shanthikumar1,2,3, Matthew Burton4, Richard Saffery5,3
1Respiratory and Sleep Medicine, Royal Children's Hospital, Melbourne, Victoria, Australia.
Insights
This study presents a novel flow cytometry method to analyze immune cells in children's bronchoalveolar lavage (BAL) fluid. This technique aids in understanding childhood pulmonary diseases by characterizing various cell types and assessing cryopreservation effects.
Area of Science:
- Pediatric Pulmonology
- Immunology
- Cell Biology
Background:
- Childhood pulmonary diseases lead to significant morbidity, mortality, and long-term impairment.
- Understanding the pathophysiology of these diseases is crucial for effective clinical management.
- Limited knowledge exists on cell populations in pediatric bronchoalveolar lavage (BAL) and the impact of sample handling.
Purpose of the Study:
- To develop and validate a flow cytometry protocol for the purification and characterization of diverse immune cell populations in pediatric BAL.
- To assess the impact of cryopreservation on cell phenotype and frequency in pediatric BAL samples.
- To establish a comprehensive method for investigating the cellular basis of childhood pulmonary diseases.
Main Methods:
- Development of a multi-parameter flow cytometry panel for simultaneous quantification of alveolar macrophages, T cells (CD4, CD8), B cells, NK cells, dendritic cells, granulocytes, and monocytes (CD16+/CD16-).
- Application of the protocol to bronchoalveolar lavage (BAL) samples from children.
- Evaluation of the effects of cryopreservation on the identified cell populations.
Main Results:
- Successful simultaneous quantification of multiple immune cell types in pediatric BAL using flow cytometry.
- Characterization of alveolar macrophages, T cells, B cells, NK cells, dendritic cells, granulocytes, and monocytes.
- Data on the stability and changes in cell phenotype and frequency following cryopreservation.
Conclusions:
- The described flow cytometry method provides a robust tool for detailed cellular analysis in pediatric BAL.
- This approach addresses a critical knowledge gap regarding cell characterization and cryopreservation effects in pediatric lung research.
- The protocol facilitates deeper investigation into the pathophysiology of childhood pulmonary diseases, potentially improving diagnosis and treatment.
Abstract:
Childhood pulmonary diseases not only cause childhood morbidity and mortality but also can cause long-term pulmonary impairment. The clinical management of many childhood pulmonary diseases is hampered by a limited understanding of the underlying pathophysiological mechanisms. Flow cytometry, which can be used to phenotype individual cell populations or isolate cells for downstream analysis, represents a crucial technology that can help to elucidate the pathophysiology of these conditions. Here, we describe a flow cytometry-based method for purification and characterization of cell populations in BAL from children. This includes assessment of the effect of cryopreservation on cell phenotype and frequency, a knowledge gap recently identified by an American Thoracic Society report on flow cytometry in lung samples. To our knowledge, this is the first study to simultaneously quantify alveolar macrophages, T cells (CD4 and CD8), B cells, natural killer cells, dendritic cells, granulocytes, and monocytes (CD16+/CD16-) in the BAL of children. The protocols described can be used to advance investigation of the pathophysiology of childhood pulmonary diseases.
