Estimating potency of cord blood transplants: Current ... · T.F.Radke – Estimating potency The cord blood bank`s challenge Primary task: Banking cord blood transplants with the

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


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.


Data provided

Final report : (Exemplary single volume-reduced CB unit for pediatric patient)

HLA-data Volume & additives Cell counts Disease markers etc.


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.)


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).”


Current methods


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


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!)


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




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 ?



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!


Potential improvements


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.


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!


Factors affecting 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.


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


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.


Segments and aliquots


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 ?


Acknowledgements

Gesine Kögler

Riccardo Saccardi

Sergio Querol

Richard Duggleby

Svenja Peters

David Barbosa

Documents

Estimating potency of cord blood transplants: Current ... · T.F.Radke – Estimating potency The cord blood bank`s challenge Primary task: Banking cord blood transplants with the