Some aspects of stem cell
collection and transplantation
- a single centre experience
Dept. of Haematology and Oncology
Charles University Plzen, Czech Republic
Daniel Lysak
Saint- Petersburg, 8 Jun 2004
TRANSPLANT CENTERS IN CZECH REP.
Czech Republic
Praha (6x)

Plzeň

Hradec Králové



Olomouc

Brno
10.2 million inhabitants
10 transplant centers
TRANSPLANT RATES - EUROPE
Total SCT per 10 million
0 or no report
1-50
51-200
201-400
> 400
Transplant rates in European
countries 2001. A.Gratwohl,
Leukemia, 2003
TRANSPLANT RATES - CZECH REP.
599
600
562
535
500
patients
417
390
400
356
300
76
4333
330
293
143
136
100
304
90
46
365
300
216
199
200
336
280
273
392
56
57
76
86
81
100
113
144
143
0
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
years
total transplantations
autologous patients
allogeneic patients
TRANSPLANT RATES - OUR CENTRE
60
50
52
49
47
47
44
48
45
patients
40
39
30
31
29
22
20
19
17
4
0 0
4
2 1
1
9
1
19
16
15
11
10
0
29
3
10
109
9
2223
7
4
1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003
year
auto
alo related
1992- 4/2004: 445 autologous
transplantations
289 allogeneic
alo unrelated
INDICATIONS FOR SCT
our centre
diagnosis
alo (%)
Europe*
auto (%) alo (%)
auto (%)
CML
36
0
21
1
AML
24
8
25
8
ALL
10
0
18
2
NHL/M.H.
15
49
10
42
MM
3
43
5
27
MDS
6
0
7
0.4
AA
6
0
5
0
• indications for SCT are similar in comparison with other European countries
• alo transplants:
more CML x ALL
• auto transplants: more MM x solid tumors
*A. Gratwohl, Blood, 2002
STEM CELL SOURCE
allogeneic transplantations
autologous transplantations
40
60
35
50
30
40
patients
patients
25
20
15
30
20
10
10
5
0
0
1992 1993
1994 1995 1996
1997 1998 1999 2000
year
2001 2002 2003
BM
PBSC
1993
1994
1995
1996
1997
1998
year
1999
2000
2001
2002
BM
2003
PBSC
PBSC COLLECTION
180
160
140
aphereses
120
100
80
60
40
20
0
1993
1994
1995
1996
1997
1998
1999
2000
2001
2002
2003
PBSC total
15
108
148
164
176
160
146
101
122
155
149
auto
15
106
146
162
167
146
136
85
91
85
80
alo related
0
2
2
2
9
14
10
7
15
38
36
alo unrelated
0
0
0
0
0
0
0
6
10
27
24
DLI
0
0
0
0
0
0
0
3
6
5
9
1993- 2003: 1444 procedures – 1219 autologous
– 225 allogeneic
AUTOLOGOUS COLLECTION MOBILIZATION REGIMEN
cyclophosphamide 2.5 – 5.0 g/m2
79 % pts
HAM
5 % pts
HD-ara-c
10 % pts
other
6 % pts
+ G-CSF 10 ug/kg/day (splitted in two doses)
Monitoring before apheresis:
•
•
•
•
CBC measured daily from day +7 (CY regimen)
when WBC recovered to > 1 x 109/l – CD34+ in PB enumerated
apheresis started if CD34+ count reached > 10 (20) cells/ul
target dose: 4.0 x 106 (MM pts – double transplant)
3.0 x 106 (other pts) CD34+ cells/kg bw per SCT
AUTOLOGOUS COLLECTION - PATIENTS
all pts
good mobilizers
CD34+ > 20 /ul
poor mobilizers
CD34+ < 20 /ul
53
54 (20 – 72)
50 (20 – 64)
MM
43 %
51 %
10 %
NHL/M.H./CLL
49 %
44 %
67 %
AML
8%
5%
23 %
disease stage at dg. (stage 0-3/4)
26 % / 74 %
25 % / 75 %
ns
time from diagnosis (months)
5.3 (1 – 126)
8.4 (0.4 – 75)
ns
no. of prior chemotherapy cycles
4.0 (2 – 14)
4.5 (2 – 12)
ns
42 %
37 %
61 %
p=0.0168
58 %
63 %
39 %
PLT count (x 109/l)
224 (57 - 774)
174 (53 – 418)
p=0.0005
Hb level (g/l)
128 (79 – 174)
125 (92 – 161)
ns
median age
diagnosis:
disease status: CR
PR/SD
ns
groups comparable in majority parameters, only different variables:
• premobilization PLT count
• premobilization disease status
• higher representation of AML and lymfoma pts in poor mobilizers group
( stem cell toxicity of previous therapy: ara-c, FLU, ...)
AUTOLOGOUS COLLECTIONS - RESULTS I.
all pts
good
mobilizers
poor
mobilizers
1st collection day, median
10 (8 – 28)
10
12
total CD34+ cells x 106/kg
6.8 (1.3 – 35.8)
7.7
3.5*
no. of aphereses per patient
2 (1 – 6)
1 (1 – 3)
2 ( 1 – 6)
CD34+ cells x 106/kg per LP
3.1 (0.5 – 35.8)
4.3
1.3*
7%
2%
29 %
45 (10 – 387)
64
16*
failure of collection (< target)
peak CD34+ count in PB (cells/ul)
*p<0.0001
AUTOLOGOUS COLLECTIONS - RESULTS II.
Variables predictive for successful mobilization and collection:
a) premobilization variables
PLT count
Hb level (in myeloma patients)
previous chemotherapy stem cell toxicity
x
age, sex, disease stage at dg., no of prior CHT: 0 correlation
b) preapheresis variables
CD34+ cells count in PB correlated with CD34+ yield (r2 = 0.8)
x
WBC, PLT count: 0 correlation
yield prediction (linear. regression STEP – WISE):
CD34+ (x106/kg) = 0.99 + 0.002* weight (kg) + 0.064* CD34+ (/ul)
Number of CD34+ cells in PB versus number of CD34+
cells obtained in a single apheresis on the same day
40
6
C34+ cells x 10 /kg
35
30
2
R = 0,8022
25
20
15
10
5
0
0
100
200
CD34+ cells/ul in PB
300
400
ALLOGENEIC COLLECTION MOBILIZATION REGIMEN
Mobilization regimen:
G-CSF 10 ug/kg/day (single dose) for 5- 6 days
Monitoring before apheresis:
• CBC and CD34+ measured daily from day +4
• apheresis started on day +5 (or on day +4 if WBC
exceeds 50 x 109/l)
• target dose: 5.0 x 106 CD 34+ cells/kg bw of the
recipient
• maximum of 2 aphereses performed
• maximum WBC count: 70 x 109/l (or STOP G-CSF)
ALLOGENEIC COLLECTIONS - DONORS
all donors
donors >= 50 years
number of donors
105
32
sex (male/female)
64/41
10/22
41 (16 – 75)
55 (50 – 75)
64/41
31/1
weight (kg), median
76 (55 – 130)
86 (59 – 105)
peripheral/central venous access
67 % / 33 %
54 % / 46 %
3.2 (2.5 – 4.4)
3.0 (2.5 – 3.8)
median age
HLA match. sibling/unrelated
blood volumes processed
ALLOGENEIC COLLECTIONS - RESULTS I.
all donors
donors >= 50
years
CD34+ cells x 106/kg donor b.w per LP
2.2 (0.3 – 16.7)
1.9 (0.4 – 10.2)
CD34+ cells x 106/kg recipient b.w.
5.0 (0.7 – 17.5)
4.4 (0.7 – 10.6)
1 LP to reach target
28 %
19 %
failure of collection (< target)
45 %
56 %
64 (11 – 204)
56 (11 - 204)
141 (77 – 257)
136 (77 – 257)
CD34+ count in PB (cells/ul) on day+5
PLT count at day +6 (x109/l)
CD3+ cells x 108/kg recipient b.w.
2.66 (0.71 – 5.54) 2.58 (1.16 – 4.44)
side efects of G-CSF treatment
23 %
37 %
complication of apheresis
20 %
22 %
differencies in all parameters not significant
ALLOGENEIC COLLECTIONS - RESULTS II.
older donors compared to younger (> 50 vs < 30 years):
• WBC and CD 34+ kinetics similar peaking at day +5
• CD 34+ count at day +5 significantly lower (56 vs 80
cells/ul, p=0.048)
• median number of CD34+ (x106/kg bw of the recipient)
significantly lower (4.4 vs 6.3, p = 0.0404)
• 2 LP more frequently necessary
• median number of CD 3+ (x108/kg bw of the recipient)
comparable (2.58 vs 2.54, ns)
APHERESIS PROCEDURE
COBE Spectra (from 1995), Fenwal CS3000
LVL:
3 – 4 x TBV
ACD-A rate: 1.1 ml/l TBV/min
ACD-A:PB ratio: 1:14 – 15
inlet flow: 60 – 90 ml/min depending on ACD:blood
ratio and venous access
patients: intpatient basis
donors:
int/outpatient basis (venous access)
Ca prophylaxis: CaCl2 (2 amp) intermitent i.v. bolus
Minimal precollection blood counts:
PLT (x 109/l)
hematocrit (%)
patients:
> 20
30
donors:
> 100 (v.6), > 150 (v.4)
NA
Apheresis unit
2 collection rooms
airconditioning/filtration
MNC vs. AutoPBSC I.
procedures
inlet volume (l), median
MNC (v 4.7)
autoPBSC (v 6)
30 %
70 %
17 (13 – 22)
17 (13 – 23)
3.3 (0.98 – 32.4) 3.4 (0.4 – 35.8)
CD34+ cells x 106/kg bw/LP
ns
ns
57 (35 – 90)
75 (45 – 91)
209
240
time per one TBV (min), median
66 (51 – 126)
80 (64 – 108)
p= 0.0003
PLT contamination (x109/l), med
1434
677
p< 0.0001
patient related CD34+ recov. (%)
101 (21 – 268)
94 (15 – 254)
ns
10 %
9%
ns
postLP PLT (% of preLP count)
procedure time (min), median
complication rate
p< 0.0001
component CD34+ count x volume x 100
PR recovery = --------------------------------------------------------patient CD34+ count x TBV
MNC vs. AutoPBSC II.
MNC:
AutoPBCS:
PLT depletion:
higher
lower
apheresis time:
shorter
longer (+ ~ 15 %)
other parameters (CE, AE ..) not significantly different
Advantages of AutoPBSC:
1. automated interface establishment and higher separation factor
- increased stability of IF position - more consistent results
2. more of the operator attention to the patient or donor
3. reduced mistakes of staff members during collection
AutoPBSC:
pts/donors with: low PLT count
lower CE related to interface instability
MNC:
pts/donors with: limited procedure toleration (time)
QUALITY ASSURANCE OF THE GRAFT
Before collection (pt/donor PB):
ABO blood group
infectious disease: HIV, HBV, HCV, syphilis
After collection (apheresis product):
volume (ml)
WBC count (x 109/l)
total NC (x 108/kg)
CD34+ cells (%, ISHAGE based protocol, double platform)
CD34+ yield (x 106/kg)
CD3+ content (x 108/kg) – allogeneic collections
cell viability (7-AAD exclusion on flow cytometry)
sterility (Eu Pharmacopoeia approved method)
SUMMARY
1. PBSC are increasingly used to support autologous or
allogeneic transplantation.
2. Stem cell collection with apheresis is effective, safe
and well tolerated method with low complication rate.
3. COBE Spectra is a gold standard for PBSC collection,
providing comparable results among centers.
4. MNC and AutoPBSC have similar performance, version
selection can be affected by precollection variables.