T-cell depletion of allogeneic bone marrow grafts: enrichment of mononuclear cells using the COBE Spectra cell processor, followed by immunoselection of CD34(+) cells.
BACKGROUND
Enrichment in mononuclear cells (MNC) is a prerequisite for CD34(+) cell selection from BM allografts. However, centrifugation over a density gradient (Ficoll), does not comply with good manufacturing practice (GMP), because Ficoll is not a clinical grade reagent. We evaluated the COBE Spectra cell separator as an alternative and evaluated the recovery of MNC and CD34(+) cells. CD34(+) cell selection was then performed using either the Nexell Isolex 300i or the CEPRATE SC (CellPro) device.


METHODS
20 allogeneic BM grafts were processed, with an initial enrichment of MNC using the COBE Spectra processor, followed by immunoselection of CD34(+) cells (13 Isolex 300i and seven CEPRATE SC). We evaluated total nucleated cells (TNC), MNC and CD34(+) cells recoveries when using COBE Spectra and compared CD34(+) cells recovery and T-cell depletion when using Isolex 300i or CEPRATE SC.


RESULTS
Median recoveries of the product using the COBE Spectra were 31.2% (18.1-68.9%) for TNC, 76.9% (29.5-148.1%) for MNC and 79.6% (60.9-191%) for CD34(+) cells. Median recoveries of CD34(+) cells were 39.4% (15.1-75.8%) and 36.7% (26.2-64.3%) with the Isolex 300i and CEPRATE SC devices, respectively. CD34(+) purity was 85% (64.4-94.5%) and 84.4% (66.8-86.8%). Colony-forming unit granulocyte/macrophage (CFU-GM) recoveries were 10.9% (0.2-152%) and 36% (0.0-67.3%). Median log depletion for T-cell numbers, B-cell numbers and natural killer (NK) cell numbers were 2.8 log (2.1-3.8), 2.0log (0.95-3.1) and 3.1log (2.40-4.8), respectively.


CONCLUSIONS
Processing with COBE Spectra produces a cell population that is suitable for CD34(+) cell selection. The Isolex 300i and CEPRATE SC devices demonstrate similar performances. The two-step procedure allows allo-BMT in patients with high risk of GvHD.
Olivero
S
S
Centre de Thérapie Cellulaire, Institut Paoli-Calmettes, Centre Régional de Lutte Contre le Cancer Provence-Alpes-Côte d'Azur, 232 Boulevard de Ste Marguerite, BP 156. 13273 Marseille Cedex 9, France.
Novakovitch
G
G
Ladaique
P
P
Sainty
D
D
Viret
F
F
Faucher
C
C
Stoppa
A M
AM
Blaise
D
D
Chabannon
C
C
D018952:Antigens, CD34 / Q000096:biosynthesis; D016026:Bone Marrow Transplantation / Q000379:methods*; D002452:Cell Count; D002469:Cell Separation / Q000379:methods; D005434:Flow Cytometry / Q000379:methods; D006086:Graft vs Host Disease / Q000276:immunology; D006801:Humans; D018189:Immunomagnetic Separation / Q000379:methods; D007963:Leukocytes, Mononuclear / Q000166:cytology*; D008212:Lymphocyte Depletion / Q000379:methods*; D013053:Spectrophotometry / Q000379:methods; D013601:T-Lymphocytes / Q000166:cytology* / Q000378:metabolism; D014184:Transplantation, Homologous / Q000379:methods
D018952:Antigens, CD34