Effect of freezing of sputum samples on flow cytometric analysis of lymphocyte subsets

Abstract

Sputum samples should be processed shortly after induction to prevent cell degradation. For intermediate storage, freezing of homogenised samples or immediate fixation have been shown to be suitable for cytospins. The aim of this study was to investigate whether freezing or immediate fixation of sputum affect the analysis of lymphocyte subsets by flow cytometry.Selected plugs from 24 sputum samples were homogenised. One aliquot was processed immediately and analysed by flow cytometry. A second aliquot was homogenised, frozen at -20uC after addition of dimethylsulfoxide and stored for a median time of 6 days. In six samples a third aliquot was fixed in formalin after induction and stored for up to 72 h before further processing.Compared to immediate processing, percentages of total lymphocytes and Tsuppressor cells were elevated after being frozen, with a minor decrease in the T4/T8 ratio. Proportions of total lymphocytes, T-helper and T-suppressor cells correlated between native and frozen samples, intra-class correlation coefficients being 0.74, 0.85 and 0.70, respectively. The formalin-fixed aliquots could not be analysed with the antibodies used.In conclusion, freezing seems to be a suitable technique to store sputum samples for flow cytometry of CD3, CD4 and CD8 lymphocyte subsets. Its effects were minor compared to the variation between subjects. Sputum cells can be analysed by flow cytometry to obtain data on lymphocyte subsets [1, 2] and intracellular cytokine production [2]. For differential cell counts on cytospins it is recommended that sputum be processed soon after expectoration to prevent degradation of cells [3]. Freezing or fixation of sputum has been suggested to elongate time until processing, allowing, for example, transport to a specialised laboratory [4,5]. This aim of this study was to assess the effect of intermediate storage on flow cytometric analysis of lymphocyte subsets using the freezing and the fixation methods [4,5]. Materials and methods SubjectsA total of 24 sputum samples from 18 subjects were analysed. Six subjects were healthy (two males/four females; mean¡SD age 28¡6 yrs; forced expiratory volume in one second (FEV1) 106¡15% predicted), nine had asthma of different severity (five males/four females; age 40 ¡ 11 yrs; FEV1 73¡14% pred) and three had allergic rhinitis without asthma (3 males; age 32¡7 yrs; FEV1 110¡17% pred). All subjects gave their informed consent and measurements were approved by the local Ethics Committee. Sputum inductions were performed by inhalation of 3-5% or 0.9% saline. Selected plugs were separated into two aliquots immediately after homogenisation with Sputolysin1 (20 min at room temperature, 6.5 mM dithiothreitol; Calbiochem, Darmstadt, Germany) [6,7]. One aliquot was mixed 1:1 with PBS containing 1% BSA and 30% dimethylsulfoxide (DMSO; Sigma, Munich, Germany), and stored frozen at -20uC (median storage time 6 days (range 1-16)). The second aliquot (native sputum) was processed as usual [6].In six samples a third aliquot was taken before ho...

Flow cytometry of sputum: assessing inflammation and immune response elements in the bronchial airways

“…Some studies have used specific cell lineage markers (e.g. : CD3, CD4, CD14, HLA-DR or other markers) to identify and gate specific cells of interest (Barry et al 2002; Barry and Janossy2004; Frankenberger et al 2004; Janossy et al 2008; Holden et al 2008; McCarthy et al 2007) and a few more recent studies (Leckie et al 2003; Dominguez et al 2004; Jaksztat et al 2004; Lay et al 2007; Alexis et al 2009; Antoniou et al 2005; Dua et al 2010) employed more intricate approaches; however, none have provided detailed descriptions of a comprehensive and rigorous approach using FCM for differentiating sputum leukocytes from debris.…”

Identifying leukocyte populations in fresh and cryopreserved sputum using flow cytometry

“…In addition, although freezing has been reported as a suitable storage strategy prior to assessment of sputum lymphocyte subsets (8) and DCC (14,15), the effects of freezing on FCM assessment of other leukocyte populations in IS has not been reported. Furthermore, little is known about the viability of individual cell populations in IS, as light microscopic assessment only allows overall cell viability to be determined.…”

“…However, there is little detailed information available regarding FCM gating for leukocyte populations in IS, although strategies for bronchoalveolar lavage (BAL) have been published (3,4). 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).…”

Tendencias temporales de las concentraciones de citocinas Th1 y Th2 en esputo inducido de pacientes asmáticos durante infecciones víricas agudas de las vías respiratorias superiores

“…La congelació n de una muestra de esputo inducido inmediatamente despué s de su obtenció n para su procesamiento posterior a conveniencia de los té cnicos de laboratorio evita los problemas causados por el contenido enzimá tico de este fluido corporal, que digeriría los componentes celulares y del fluido si la muestra permaneciera sin congelar. Diferentes equipos de investigació n han verificado esta té cnica en los ú ltimos añ os [9][10][11][12] .…”