Journal of Hematology - Supplements - Haematologica

43
washing the cell suspension and the positive cells
rosetted with the Dynal paramagnetic microspheres,
coated with sheep anti-murine antibody.
The CD34 + cells were magnetically separated
from the cell suspension using the ISOLEX 300
selection separator. Negative cells were removed
by washing and target cells were separated from
immunomagnetic beads by cleaving the protein
linkage by chimopapain (Chymocell-T). The
colony-forming units (CFU) of granulocytes and
macrophages (GM) present in BM and PBSC
were measured by plating cells (10 4 cells/plate)
in methylcellulose medium containing GM-CSF,
and interleukin-3. Colonies were evaluated and
counted after 14 days, using an inverted microscope.
The number of CD34 + cells infused was
measured both in purified BM and in leukapheresis
products using a fluoresceinated MoAb
(Beckton-Dickinson), as previously described.
Data were gated on forward scatter lymphocytes
and monocytes; ten thousand cells were evaluated
for each sample. The number of T-lymphocytes
after E-rosetting for T-cell depletion was
measured using MoAb anti-CD3. 11,12
In the remaining transplants, we performed the
CD34 + cell selection using the Clinimacs system
(Miltenyi) because of the potentially better purity
and recovery of cells that are virtually not
affected by isolation procedures. The positive
cells are specifically labeled with supermagnetic
iron-dextran particles which are covalently conjugated
to mouse anti-human CD34 antibody.
After magnetic labeling, the Clinimacs system
automatically passes cells through a separation
column which is held in a strong permanent
magnet. The magnetically labeled cells are
retained within the column and separated from
the unlabeled cells, which flow through. The
retained cells are evaluated from the Clinimacs
system by removing the column from the magnetic
field, washing the cells and collecting them.
Results
Mobilization, collection and T-depletion of
PBSCs
In the total apheresis collection, the mean
number of CD34 + cells was 13.42×10 6 /kg. The
mean number of PB CD34 + cells collected after
positive selection using the Isolex 300 System
was 12.8×10 6 /kg recipent’s weight (range: 2-40)
with a mean purity of 97.3%. After T-cell depletion
using the E-rosetting method, the mean
number of the infused CD3 + cells was 0.385
×10 5 /kg (range, 0-0.98). Patient DCL received
PBSC without any T-cell depletion, while patient
CA received PBSC depleted with Campath-1M.
When the Clinimacs system was used the mean
number of PB CD34 + cells collected was 14.05
×10 6 /kg recipent’s weight (range: 8.1-20). No T-
cell depletion was performed and the mean
number of the infused CD3 + cells was 0.53
×10 5 /kg (range, 0.06-1). The total apheresis
mean number of CFU-GM infused was 8.1
×10 5 /kg of weight.
Collection and T-cell depletion of BM
When the Isolex 300 System was used, a mean
of 11.4×10 6 bone marrow cells CD34 + /kg of
recipient’s weight was infused. All grafts were
treated with Campath1M for T-depletion and
the number of CD3+ cells was undetectable.
When the Clinimacs system was applied for
CD34 + cell selection, the mean number of
infused bone marrow CD34 + cells was 9.85
×10 6 /kg of recipient’s weight with a mean number
of infused CD3 + cells of 3.66×10 5 /kg.
In the total bone marrow aspirates, the mean
number of infused CFU-GM was 3.59×10 5 /kg
of recipient's weight.
Engraftment and chimerism
The 9 patients achieved peripheral blood neutrophil
counts greater than 0.5×10 9 /neutrophils/L
at a mean of 14.6 days (range, 6 to 22
days). RFLP analysis of peripheral granulocytes
and lymphocytes on day 14 confirmed engraftment
of donor-CD3 derived cells in all cases.
Granulocytes were autologous in 2 cases, split
chimerism was observed in 1 case and complete
chimerism in the remaining 6 cases.
Immune reconstitution
Phenotypic and functional analyses of posttransplant
lymphocyte subsets were performed.
In 8 patients who survived more than 120 days
after BMT, the mean number of CD2 + cells was
284/mm 3 , that of CD3 + cells 426/mm 3 , CD4 +
299/mm 3 , CD8 + 125/mm 3 , CD19 + 94/mm 3 ,
and CD16 + 132/mm 3 .
Graft-vs.host disease
Six patients developed GVHD. One patient
showed acute skin grade I GVHD, 2 patients
developed grade II GVHD (1 skin and 1 skin plus
gut) and 3 patients developed grade III acute
skin GVHD; two of three patients with grade III
GVHD were successfully treated with methylprednisolone.
Only one patient developed acute
grade IV skin GVHD after the first transplant and
he was treated with steroids and ATG after the
2 nd and 3 rd transplants.
Toxicity and clinical outcome
The conditioning regimen was well tolerated
by all patients. Eight of the nine patients are alive
and well 1-37 months after BMT (mean follow
up 7.5 months). One patient died of disseminated
CMV infection early after BMT.
haematologica vol. 85(supplement to n. 11):November 2000