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Innovative Two-Step Negative Selection of Granulocyte
Colony-Stimulating Factor-Mobilized Circulating Progenitor Cells:
Adequacy for Autologous and Allogeneic Transplantation
Alessandro Rambaldi,
Gianmaria Borleri,
Gianpietro Dotti,
Piermario Bellavita,
Ricardo Amaru,
Andrea Biondi, and
Tiziano Barbui
From the Divisione di Ematologia e Centro Trasfusionale, Ospedali
Riuniti Bergamo, Bergamo, Italy; and Clinica Pediatrica
Università di Milano, Monza, Italy.
A major obstacle in purifying either autologous or allogeneic
hematopoietic stem cells from granulocyte colony-stimulating factor
(G-CSF) mobilized circulating progenitor cells (CPC) is represented by
the huge cellularity present in each apheretic product. To obtain a
significant debulking of unwanted cells from the leukapheresis, we
developed a modified protocol of immune rosetting whereby human
ABO-Rh- compatible red blood cells (RBCs) are treated with chromium
chloride and then coated with murine monoclonal antibodies (MoAbs)
against leukocyte antigens. When experiments were performed with
leukaphereses obtained from normal donors or from T-cell acute
lymphoblastic leukemia (T-ALL) patients, RBCs were coated with murine
MoAbs against human mature myeloid cells (CD11b) and T cells (CD6);
whereas, in the case of patients with B-precursor ALL, B-cell
non-Hodgkin's lymphoma (B-NHL), or multiple myeloma (MM), RBCs were
coated with anti-CD11b only. After incubation with CPC, rosetting cells
(myeloid precursor cells, granulocytes, monocytes, and T cells) were
removed by Ficoll-Hypaque density gradient centrifugation with a blood
cell processor apparatus, COBE (Lakewood, CO) 2991. After this step, a
significant reduction of the initial cellularity was consistently
obtained (range, 72% to 97%), whereas the median absolute recovery of
the CD34+ cells was above 85% (range, 64 to 100), with a
10-fold relative enrichment ranging from 3% to 41%. In a second step,
CPC can be further purged of contaminating T or B cells by incubation
with lymphoid-specific magnetic microbeads (anti-CD2 and -CD7 to remove T cells; anti-CD19 to remove B cells) and elution through a type-D depletion column (composed of ferromagnetic fiber) inserted within a
SuperMACS separator device (Miltenyi Biotech, Bergisch-Gladbach, Germany). By this approach, a highly effective (three to
four logs) T-cell depletion was achieved in all experiments performed with normal donors or T-ALL patients (median loss of CD3+
cells: 99.8% [range 99.2 to 100]) and an equally efficient B-cell depletion was obtained from B-precursor ALL, B-NHL, or MM patients. At
the end of the procedure the T- or B-cell depleted fraction retained a
high proportion of the initial hematopoietic CD34+ stem
cells, with a median recovery above 70% (range 48% to 100%) and an
unmodified clonogenic potential. In five patients (two follicular NHL
and three ALL) the purified fraction of stem cells was found disease
free at the molecular level as assessed by polymerase chain reaction
(PCR) analysis of the t(14;18) chromosome translocation or
clono-specific DNA sequences of IgH or T-cell receptor  and  chain genes. Purified autologous and allogeneic CPCs were transplanted in three and six patients, respectively, who showed a prompt and sustained hematologic engraftment. In conclusion, this method represents a simple and reproducible two-step procedure to obtain a
highly efficient purging of T or B cells from G-CSF expanded and
mobilized CPCs. This approach might lead to the eradication of the
neoplastic clone in the autologous stem cell inoculum as well as for
T-cell depletion during allogeneic transplantation.
Blood, Vol. 91 No. 6 (March 15), 1998:
pp. 2189-2196
© 1998 by The American Society of Hematology.
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