Estimating potency of cord blood transplants: Current standards and

T.F.Radke – Estimating potency
Cord Blood Infusions:
Current Gold Standards in Preparing and Testing of Cord Blood Products for
Transplantation
Estimating potency of cord blood
transplants: Current standards and
challenges
Teja Falk Radke
José Carreras Cord Blood Bank
University Medical Center Düsseldorf, Germany
T.F.Radke – Estimating potency
The cord blood bank`s challenge
Primary task:
Banking cord blood transplants with the best quality
achievable according to current standards.
but also:
1.) Providing quality data on these units!
2.) Providing clear instructions for infusion!
 Transplant centers/physicians should be able to estimate
potency of the CBU and must be aware of potential risks.
 Data/Instructions provided must be reliable, reproducible,
and unmistakable.
T.F.Radke – Estimating potency
Data provided
Final report : (Exemplary single volume-reduced CB unit for pediatric patient)
HLA-data
Volume & additives
Cell counts
Disease markers etc.
T.F.Radke – Estimating potency
Data provided
Repository labeling : (Exemplary for double-bag volume-reduced CB unit)
Data on content, including active
components (TNC, erythrocytes,
platelets etc.) and reagents (e.g.
DMSO, Hespan etc.)
T.F.Radke – Estimating potency
Instructions provided
“Instruction Insert and Product Information”
10 pages with comprehensive instructions
Identification, Indication, Information on use, Precautions, Side effects,
Pharmacological and toxicological properties
Instructions for washing (manually as well
as with automated Sepax100).
Composition
“Formal Request for
Procurement”
“4. The transplant center must agree that
the CB unit will be thawed and
processed prior to the transplantation
using the thawing procedure from
Düsseldorf, which is identical to the
Rubinstein protocol (it is possible to receive
a ´dummy CB unit` for practice use or
training).”
T.F.Radke – Estimating potency
Current methods
T.F.Radke – Estimating potency
Current methods for estimating potency
Cell count
Total cell count of nucleated cells (TNC) with and without erythroblasts using
haematology analyzers
Content CD34+-cells :
Amount of haematopoietic stem and progenitor cells (CD34+) by flow cytometry
according to ISHAGE protocol. Additionally, viability is assessed by 7-AAD.
Colony-forming cells:
Ability to form colonies in an in vitro colony-forming unit (CFU)-assay
T.F.Radke – Estimating potency
Current methods for estimating potency
... and their pitfalls
Cell count
WBC : White blood cell count,
either based on WOC or WIC/NOC
WOC : White blood cells Optical Count  all cells
 Can be falsely increased by resistant red blood cells
(ResRBC) or decreased by fragile WBC (e.g. after
thawing)
WIC/NOC : White blood cells Impedance Count /
Nucleated cells Optical Count  all nucleated cells
 Can be falsely increased by nucleated red blood cells
(Cord blood contains high amounts of NRBC!)
T.F.Radke – Estimating potency
Current methods for estimating potency
... and their pitfalls
BUT: Together with WBC and CFU dose, NRBC content is a significant
predictor of time to myeloid engraftment.
TNC value might be more important than a perfect WBC value!
WIC values are better
reflecting CB potency
T.F.Radke – Estimating potency
Current methods for estimating potency
... and their pitfalls
Content CD34+-cells:
Results can be influenced by A) antibody used and B) gating strategy
A)
Clone 8G12:
860 events
... more sensitive ?
Clone 581:
755 events
... more specific ?
B)
Generous: 732 events
Strict: 617 events
(CB unit aliquot, post-thawing)
T.F.Radke – Estimating potency
Current methods for estimating potency
... and their pitfalls
Colony-forming cells:
Ability to form colonies in an in vitro colony-forming unit (CFC)-assay
CFU/CD34+ ratio
(after volume reduction)
CFU/CD34+ ratio
(thawed)
10
CB1
CB2
CB3
CB4
CB5
8
6
CB6
CB7
CB8
CB9
CB10
4
ideal
2
0
CFU per seeded CD34+
Though, correlation
CFC/HSC varies with
seeding-density!
CFU per seeded CD34+
10
8
CB1
CB2
6
CB4
CB6
CB7
CB8
CB9
CB10
4
ideal
2
0
0
100
200
300
400
seeded CD34+ cells/per ml
500
0
100
200
300
400
seeded CD34+ cells/per ml
500
600
T.F.Radke – Estimating potency
Potential
improvements
T.F.Radke – Estimating potency
Single-platform enumeration of cells
Bead-based systems for flow cytometry:
Commercially available inert beads are added to sample (defined
number/defined volume)
 Number of beads recorded allows calculation of tested volume.
T.F.Radke – Estimating potency
Assessement of apoptotic cells
Annexin V (AnnV) : Stains phosphatidylserine (PS). PS is present on
the cytosolic side of the cell membrane, but gets exposed on the
outside in case of apoptosis.
7-AAD only vs. 7-AAD + AnnV
True viability might be much lower!
T.F.Radke – Estimating potency
Factors affecting
potency
T.F.Radke – Estimating potency
Factor affecting potency: Cell loss
Recovery total nucleated cells (CellDyn)
120
100
Recovery [%]
A certain loss of total cells is connected to
processing, resulting in a recovery of app.
85% after volume reduction and app. 75%
after thawing and washing.
*
*
80
60
40
20
Th
aw
ed
Po
st
w
as
h
W
ho
le
W
ho
le
bl
oo
bl
d
oo
d
+
Po
H
ES
st
re
Pr
du
e
ct
fr
ee
io
n
ze
+
D
M
SO
0
Recovery CD34+-cells (CellDyn + FCM)
120
Recovery [%]
100
*
*
This loss also affects the population
of CD34-positive cells on a
comparable level.
80
60
40
20
h
w
as
ed
Po
st
Th
aw
W
ho
W
le
ho
bl
le
oo
bl
d
oo
d
+
Po
H
ES
st
r
Pr
ed
e
uc
fr
tio
ee
n
ze
+
D
M
SO
0
T.F.Radke – Estimating potency
Factor affecting potency: Loss in viability
Using Annexin V, it clearly shows that viability of HSC is not as high as
estimated with 7-AAD alone.
Viability CD34 (AnnV + 7-AAD)
Viability CD34 (7-AAD)
80
80
60
40
Viability [%]
100
60
40
0
0
ho
le
W
ho
le
ho
le
ho
le
W
W
B
lo
bl
od
oo
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+
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H
ES
st
re
Pr
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ct
fr
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io
ez
B
n
ag
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+
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D
aw
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ed
ag
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th
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aw
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te
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20
bl
oo
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+
Po
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B
ed
ag
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nd
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ed
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d
po
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w
as
h
20
W
Viability [%]
100
T.F.Radke – Estimating potency
Factor affecting potency: Temperature/Time
There are no definite requirements in
regard to ambient temperature prior to
processing. Though, storage at 4°C
resulted in less non-viable leukocytes as
compared to room temperature (20-24°C).
Analyzing apoptosis in CD34-positive
cells over time prior to processing, it
could be confirmed that storage at 4°C or
room temperature showed no significant
difference. Storage exceeding 48h,
however, resulted in increased apoptosis.
T.F.Radke – Estimating potency
Segments and
aliquots
T.F.Radke – Estimating potency
Problem: How to predict potency ahead of thawing
Can segments/aliquots reproducibly give information on quality ?
Live CD34 (AnnV/7-AAD) in bag (thawed/washed), aliquot and segments
100
80
80
as
he
d
ed
Se
gm
ba
g
W
W
as
he
d
ba
g
ed
Th
aw
A
en
ts
0
ba
g
0
liq
uo
ts
20
en
ts
20
ba
g
40
Th
aw
40
60
liq
uo
ts
60
A
Viability [%]
100
Se
gm
Cell count recovery [%]
Cell count recovery segments vs. aliquots vs. bag
T.F.Radke – Estimating potency
Acknowledgements
Gesine Kögler
Riccardo Saccardi
Sergio Querol
Richard Duggleby
Svenja Peters
David Barbosa

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