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

Abstract

Pretreatment with dithiothreitol (DTT) is necessary to dissolve mucus in samples of induced sputum prior to analysis. However, DTT may affect cell surface markers which are essential for lymphocyte subtyping. Therefore, the aim of this study was to evaluate the effect of DTT on an appropriate panel of surface markers. Peripheral blood leukocytes were used because these cells, in contrast to sputum cells, could be obtained without DTT treatment.Peripheral blood from healthy donors was incubated with either DTT according to standard sputum procedures or phosphate-buffered saline (PBS), washed and incubated with fluorochrome-labelled antibodies. After lysis of erythrocytes, analysis was performed using a calibrated flow cytometer. Leukocyte populations were identified by their light scattering properties. For analysis, fluorescence intensity was compared between DTT-and PBS-treated samples.After treatment with DTT, fluorescence intensity was significantly increased in CD16-positive granulocytes; it was reduced in CD2-positive lymphocytes, CD45-positive lymphocytes and CD14-positive monocytes (p#0.001). These changes occurred in all samples. The fluorescence intensity of CD3-, CD4-, CD8-, CD19-, CD56-and histocompatibility leukocyte antigen DR-positive lymphocytes was not altered by DTT. However, there were statistically significant (p<0.001), although small, changes in the percentages of leukocytes.The present data demonstrate that, although dithiothreitol as used in sputum analysis affects some surface markers of peripheral blood leukocytes, comparability between samples concerning lymphocyte surface markers is preserved. Therefore, it is suggested that treatment of sputum samples with dithiothreitol does not invalidate the immunocytochemical analysis of lymphocytes. Eur Respir J 2000; 16: 324±329. The method of induced sputum is widely used as a noninvasive procedure for obtaining biological samples from the airways [1,2] in order to determine their cellular and biochemical composition in relation to airway diseases [3,4]. As sputum cells are embedded in airway secretions, the material has to be liquified in order to prepare singlecell suspensions for flow cytometry. This can be achieved by incubation with the potent reducing agent dithiothreitol (DTT, 2,3-dihydroxybutane-1,4-dithiol) [5]. Therefore, DTT is widely used in sputum processing [6±9]. However, owing to its reducing properties, DTT could affect the three-dimensional structure of proteins, which is maintained by disulphide bonds, thereby leading to changes in the availability and integrity of epitopes which could hamper immunological detection procedures [10].Indeed, the data which are available indicate effects of DTT or dithioerythritol (DTE) on surface markers assessed by flow cytometry on eosinophils or neutrophils even if cell viability remains unchanged [11±13]. Data regarding markers that are essential for lymphocyte subtyping have been published in abstract form [14]. Recent data have shown improved cell and sputum supernatant inflamma...

Flow cytometry analysis of leukocytes in induced sputum from asthmatic patients

“…Sputum cells were then stained with different panels of antibodies (Table 1) and expression of these markers was measured on gated CD45+ cells. Although, treatment with DTT has been demonstrated to cause statistically significant changes in certain surface antigens, all studied markers (CD45, CD66b, CD125 and CD14) remained above the detection limit (Loppow et al 2000).…”

“…Sputum material has to be liquefied in order to prepare single cell suspensions and this can be achieved by incubation with the potent reducing agent dithiothreitol (DTT, 2,3-dihydroxybutane-1,4-dithiol) (Tockman et al 1995). However, DTT has been shown to alter the detection of cellular markers (Loppow et al 2000). It is therefore not surprising that the distinction between eosinophils, neutrophils and macrophages in the sputum from asthmatic patients remains technically challenging.…”

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

“…To date, most reports using FCM to analyze IS have focussed on one particular cell subset, such as macrophages (5,6), neutrophils (7), or lymphocytes (8)(9)(10)(11). This may be due in part to the specific difficulties associated with the FCM analysis of IS, including contamination with nonviable cells, or the use of DTT (dithiothreitol) for mucus dispersal, which affects FCM detection of certain cell markers (12).…”

Update on sputum methodology

“…As sputum has to be homogenized before flow cytometry, a possible effect of the DTT treatment on surface markers has to be considered. After treatment of blood leukocytes with DTT, both increased and decreased levels of surface markers could be detected, indicating that DTT is able to destroy epitopes, but can also result in a better access of antibodies to their respective antigen [52]. In most studies published so far on sputum lymphocyte subsets, checksum controls were not included.…”