Post on 08-Dec-2018
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
Composition Instructions for washing (manually as well
as with automated Sepax100).
“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 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
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
Current methods for estimating potency ... and their pitfalls
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
Generous: 732 events Strict: 617 events (CB unit aliquot, post-thawing)
B)
A) Clone 8G12:
860 events
... more sensitive ?
Clone 581:
755 events
... more specific ?
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)
0 100 200 300 400 5000
2
4
6
8
10
CB4
CB5
CB6
CB7
CB8
CB9
CB10
CB1
CB2
CB3
ideal
seeded CD34+cells/per ml
CF
U p
er
seed
ed
CD
34
+
CFU/CD34+ ratio(thawed)
0 100 200 300 400 500 6000
2
4
6
8
10
CB4
CB6
CB7
CB8
CB9
CB10
CB1
CB2
ideal
seeded CD34+cells/per ml
CF
U p
er
seed
ed
CD
34
+
Though, correlation
CFC/HSC varies with
seeding-density!
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
Factor affecting potency: Cell loss
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.
Recovery total nucleated cells (CellDyn)
Whole
blo
od
Whole
blo
od + H
ES
Post
red
uctio
n
Pre
free
ze +
DM
SO
Thawed
Post
was
h
0
20
40
60
80
100
120
**
Reco
very
[%
]
Recovery CD34+-cells (CellDyn + FCM)
Whole
blo
od
Whole
blo
od + H
ES
Post
red
uctio
n
Pre
free
ze +
DM
SO
Thawed
Post
was
h
0
20
40
60
80
100
120
* *
Reco
very
[%
]
This loss also affects the population
of CD34-positive cells on a
comparable level.
T.F.Radke – Estimating potency
Using Annexin V, it clearly shows that viability of HSC is not as high as
estimated with 7-AAD alone.
Viability CD34 (7-AAD)
Whole
blo
od
Whole
blo
od + H
ES
Post
red
uctio
n
Pre
free
ze +
DM
SO
Bag
thaw
ed u
ndilute
d
Bag
thaw
ed p
ost w
ash
0
20
40
60
80
100
Via
bilit
y [
%]
Viability CD34 (AnnV + 7-AAD)
Whole
Blo
od
Whole
blo
od + H
ES
Post
red
uctio
n
Pre
free
ze +
DM
SO
Bag
thaw
ed u
ndilute
d
Bag
thaw
ed p
ost w
ash
0
20
40
60
80
100
Via
bilit
y [
%]
Factor affecting potency: Loss in viability
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
Problem: How to predict potency ahead of thawing
Cell count recovery segments vs. aliquots vs. bag
Segm
ents
Aliq
uots
Thawed
bag
Was
hed b
ag
0
20
40
60
80
100
Cell
co
un
t re
co
very
[%
]
Live CD34 (AnnV/7-AAD) in bag (thawed/washed), aliquot and segments
Segm
ents
Aliq
uots
Thawed
bag
Was
hed b
ag
0
20
40
60
80
100
Via
bili
ty [
%]
Can segments/aliquots reproducibly give information on quality ?