Flow cytometric analysis of the effect of dithiothreitol on leukocyte surface markers

Pretreatment with dithiothreitol (DTT) is necessary to dissolve mucus in sa mples of induced sputum prior to analysis. However, DTT may affect cell sur face markers which are essential for lymphocyte subtyping. Therefore, the a im of this study was: to evaluate the effect of DTT on an appropriate panel of surface markers. Peripheral blood leukocytes were used because these ce lls, in contrast to sputum cells, could be obtained without DTT treatment. Peripheral blood from healthy donors was incubated with either DTT accordin g to standard sputum procedures or phosphate-buffered saline (PBS), washed and incubated with fluorochrome-labelled antibodies. After lysis of erythro cytes, analysis was performed using a calibrated flow cytometer. Leukocyte populations were identified by their Light scattering properties. For analy sis, fluorescence intensity was compared between DTT- and PBS-treated sampl es. After treatment with DTT, fluorescence intensity was significantly increase d in CD16-positive granulocytes; it was reduced in CD2-positive lymphocytes , CD45-positive lymphocytes and CD14-positive monocytes (p less than or equ al to 0.001). These changes occurred in all samples. The fluorescence inten sity of CD3-, CD4-, CD8-, CD19-, CD56- and histocompatibility leukocyte ant igen DR-positive lymphocytes was not altered by DTT. However, there were st atistically significant (p < 0.001), although small, changes in the percent ages of leukocytes. The present data demonstrate that, although dithiothreitol as used in sputu m analysis affects some surface markers of peripheral blood leukocytes, com parability between samples concerning lymphocyte surface markers is preserv ed. Therefore, it is suggested that treatment of sputum samples with dithio threitol does not invalidate the immunocytochemical analysis of lymphocytes .