Assessment of Spleen Tyrosine Kinase Expression in Basophils using Flow Cytometry

Abstract

RATIONALE: In basophils, spleen tyrosine kinase (syk) expression correlates with maximal histamine release. This suggests that syk may play a role in determining the severity of atopic disease. Our goal was to develop a high throughput method of assessing basophil syk expression, sensitive enough to differentiate between syk levels in basophils from healthy and asthmatic donors.METHODS: Basophils from peripheral blood mononuclear cell (PBMC) samples were stained for intracellular syk using flourescein isothiocyanate (FITC) conjugated syk antibodies. The stained cells were then analyzed using flow cytometry. Basophils were identified as a lineage negative, CD123 positive staining population. PBMC were obtained from healthy non-atopic volunteers, asthmatic patients, and patients with Eosinophilic Gastroenteritis (EG).RESULTS: Basophils stained for syk had between 1.7 and 2.1 times greater fluorescence than isotype control samples. Staining of basophils from healthy controls, asthmatics, and EG patients did not reveal significant variations in syk expression between these groups. Syk staining of dendritic cells and B-cells was brighter than that of basophils.CONCLUSIONS: Using flow cytometry, we found no significant difference in basophil syk expression between healthy controls, asthmatics, and EG patients. However, Basophil syk fluorescence was only marginally above the noise level established by the isotype control. This suggests that flow cytometry may not be a sensitive enough method to accurately determine syk expression in basophils. RATIONALE: In basophils, spleen tyrosine kinase (syk) expression correlates with maximal histamine release. This suggests that syk may play a role in determining the severity of atopic disease. Our goal was to develop a high throughput method of assessing basophil syk expression, sensitive enough to differentiate between syk levels in basophils from healthy and asthmatic donors. METHODS: Basophils from peripheral blood mononuclear cell (PBMC) samples were stained for intracellular syk using flourescein isothiocyanate (FITC) conjugated syk antibodies. The stained cells were then analyzed using flow cytometry. Basophils were identified as a lineage negative, CD123 positive staining population. PBMC were obtained from healthy non-atopic volunteers, asthmatic patients, and patients with Eosinophilic Gastroenteritis (EG). RESULTS: Basophils stained for syk had between 1.7 and 2.1 times greater fluorescence than isotype control samples. Staining of basophils from healthy controls, asthmatics, and EG patients did not reveal significant variations in syk expression between these groups. Syk staining of dendritic cells and B-cells was brighter than that of basophils. CONCLUSIONS: Using flow cytometry, we found no significant difference in basophil syk expression between healthy controls, asthmatics, and EG patients. However, Basophil syk fluorescence was only marginally above the noise level established by the isotype control. This suggests that flow cytometry may not be a sensitive enough method to accurately determine syk expression in basophils.