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The effect of dithiothreitol on VLA-4 detection in peripheral blood and induced sputum cells
J Domínguez-Ortega1, F León, A Alonso-Llamazares
1Department of Allergy. Hospital Clínico San Carlos. Madrid. Spain. fajavdom@hotmail.com
Background:
Induced sputum has been shown to be a reliable technique for investigating airway inflammation non-invasively. Flow cytometry could provide useful information in this area. However, the viscosity of the sample entails the use of a mucolytic agent. Dithiothreitol (DDT) is the most frequently used agent although it could affect detection of different inflammatory markers.
Methods:
To measure the effect of DDT on the detection of certain adhesion molecules in eosinophils and lymphocytes, sputum was induced from seven non-smoking asthmatic and non-asthmatic subjects treated with 0.1 M DDT. The samples were analyzed by flow cytometry. Whole blood samples from the same subjects were also processed with DTT and analyzed by flow cytometry.
Results:
Very late activated antigen-4 (VLA-4) levels on eosinophils in intracellular and surface staining were much lower than expected. VLA-4 on lymphocytes was also altered but less so than on eosinophils. VLA-4 levels were also decreased on blood cells after DTT treatment. No abnormalities were found in the detection of CD29 on eosinophils and the beta7-chain in lymphocytes.
Conclusions:
Flow cytometry could be used as a complementary method to induced sputum in the investigation of airway inflammation. However, DTT could interfere with the detection of some inflammatory markers, as is the case with VLA-4.
