Emphysema is characterised by a loss of alveolar structure, as reflected in elastic recoil and gas exchange. As fibroblasts play a key role in the maintenance of structure, the current authors hypothesised that their proliferation might be constitutively impaired in lung emphysema.Using explant cultures, lung fibroblasts were obtained from resected lungs of 10 patients with emphysema (median forced expiratory volume in one second (FEV1) 40% predicted) and 10 control patients (FEV1, 95% pred). The doubling time (DT) was measured over 4 days under standard conditions (10% foetal calf serum) prior and after cryopreservation. Additionally, in seven samples per group the total population doubling level (PDL) was determined.In emphysema, mean¡SEM DT was 33.6¡2.8 h compared with 24.8¡1.4 h in controls. The differences in DT were preserved after cryopreservation. Groups also differed in the initial slope of the PDL plot during long-term culture (up to 35 days). However, the median (range) maximum PDL did not differ significantly between groups (13.8 (7.4-22.6) versus 20.2 (11.2-25.5)).The current authors, therefore, suggest that the reduced proliferation rate in vitro of lung fibroblasts from patients with emphysema reflects a persistent, intrinsic failure of cellular replacement and maintenance in this disease, possibly in relation to pre-term aging.

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...