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

¹ Hospital Großhansdorf, Center for Pneumology and Thoracic Surgery, Großhansdorf, Germany. ² Firestone Institute for Respiratory Health, St Joseph's Healthcare-McMaster University, Hamilton, Ontario, Canada

CORRESPONDENCE: O. Holz, Research Laboratory, Hospital Großhansdorf, Centre for Pneumology and Thoracic Surgery, Wöhrendamm 80, D-22927, Großhansdorf. Fax: 49 4102692295. E-mail: olaf.holz@t-online.de

Keywords: Fixation, flow cytometry, freezing, lymphocyte subsets, sputum induction

Received: November 10, 2003
Accepted April 14, 2004

This study was supported by Landesversicherungsanstalt (LVA) - Freie und Hansestadt Hamburg and Labor Dr. Kramer und Kollegen, Geesthacht, Germany.

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 –20°C 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 T-suppressor 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.