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Depletion of neutrophils from induced sputum using a novel bead and sieve-separation system

: Holz, Olaf; Schaumann, Frank; Janben, Ole; Lavae-Mokhtari, Bianca; Witte, Lena; Krug, Norbert; Heinrich, J. M.; Hohlfeld, Jens M.

American Journal of Respiratory and Critical Care Medicine 185 (2012), Abstract A2781
ISSN: 1073-449X
ISSN: 0003-0805
ISSN: 1535-4970
American Thoracic Society (ATS International Conference) <2012, San Francisco/Calif.>
Fraunhofer ITEM ()

RATIONAL: To obtain macrophages/monocytes from induced sputum of well characterized patient groups for preclinical in-vitro tests of novel drug compounds, low stress cell isolation methods are required.
In this study we tested if the novel Pluribead® (Pluriselect) methodology can also be used for the depletion of neutrophilic granulocytes from induced sputum.
METHODS: We analysed sputum with high neutrophil counts of 11 healthy subjects that was collected 6 hour following a LPS inhalation challenge. After homogenisation with DTT, the sputum was mixed with CD3 and CD15 coupled catcher-beads for 30 min under soft rotation. Cells were then placed on a 30 µm sieve, which retained the bead coupled cell populations.
RESULTS: The median (IQR) percentage of neutrophils before separation was 69 (60; 74) %. After depletion with Pluribeads, neutrophils were reduced to 8 (1; 16) %. The proportion of lymphocytes were not reduced. Using flow cytometry we observed that CD3 positive cells could not be detected in homogenized sputum, which is compatible with the observed lack in binding of these catcher beads.
CONCLUSION: The Pluriselect method is suited for the depletion of neutrophils from induced sputum and enables a quick and effective way to enrich the macrophage/monocyte fraction for further analysis. Lymphocyte depletion by CD4/8 beads and the activation state of macrophages after the procedure will need to be tested.