validation and flow cytometric quality control for cd3/cd19 · • non target cell bag (ntcb) ......
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Validation and flow cytometric
quality control for CD3/CD19
depleted grafts
Ulrike Köhl
Laboratory of Stem Cell Transplantation and Immunotherapy
Paediatric Haematology and Onkology, Frankfurt, Germany
• Graft manipulation is essential to avoid severe GvHD, especially
in case of haploidentical stem cell transplantation (haploSCT)
• Quantification of the various cell subpopulations is of major
importance in order to enable the composition of an optimized graft
• Advanced flow cytometric analyses compared to currently used
methods to evaluate residual immune cells in engineered grafts
Köhl 21.05.2011
IntroductionQuality control of stem cell grafts
Qualification and validation of residual T cells in
CD3/CD19 depleted grafts
Risk Factors:
• Increased GvHD
(Graft-versus-Host Disease)
• Immunosuppressive therapy lead
to impaired immune function
Benefit:
• Immunocompetent cells such as
monocytes, dendritic cells, NK cells
are remaining in the transplant and
enhance the GvT/L (Graft-versus-
Tumour / -Leukaemia effect)
GvHD Grade IV
kindly provided by J. Gratama
Lymphocytes (i.e. NK)
Tumour cellLehrnbecher T, Koehl U et al. Lancet Oncol 2008
HaploSCT with CD3/CD19 depleted grafts
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AimsHaploSCT with CD3/CD19 depleted grafts
Phase II study (EUDRACT CT 2006-000393-76)Primary clinical aim (7x106/kg BW CD3/CD19 depl. CD34+ cells)
Technical aims:
• Influence of different variables on
yield and T cell removal:
CD3/CD19 depl. vs. CD34 sel.
(GMP conditions)
• Quantification of residual T cells
using advanced flow cytometry
CD34 selection CD3/CD19 depletion
Immunomagnetic purification
NTCB = Non target cell bag; CCB = Cell collection bag
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CD3/19 depleted graft
CD34 selected graft
Stem cell graft: CCB (cell collection bag)
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Leukapheresis
Quality control
CD3/CD19 depletion
• Leukapheresis product
• CD3/CD19 depleted graft (Cell collection bag (CCB))
• Non target cell bag (NTCB)
•Flow cytometric quality control (FC500 )
- dual platform analyses (tetraChrome 45/4/8/3 )
- single platform analyses (Fluorepheres beads)
•
CD45 CTRL IgG1 7AAD IgG1 CD45 CD34 CD19 7AAD CD20
CD45 IgG1 CD14 7AAD CD56 CD45 CD3 CD14 7AAD CD56
Take care! Masked CD3 antigens on T cells (CD3 microbeadsconjugated with OKT3) use 8G12 clone (SK7)
based on Koehl U et al. Int J Hematol 2008
FITC / PE / ECD / PC5 / PC7
Quality control before and after CD3/CD19 depletion Methods
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prior to depl DTS LS LS
1.0×1004
1.0×1005
1.0×1006
1.0×1007
1.0×1008
1.0×1009
1.0×1010
1.0×1011C
D3
+ a
bsolu
te c
ell
num
bers
T cell depletion during graft purification
- 5.12 log- 4.74 log- 3.58 log
CD3/CD19 depletion
n=133
CD34 selection
n=33
DTS = Depletion Set LS = Large Scale enrichment Set
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Validation, Verification and Quality control
Specify and implement
Ensure that
specifications are met
Document the results
• Purpose
• System/ material
description
• Study plan/ method
• Responsibilities
(individuals)
• Procedure/SOP
(step by step…)
Köhl 21.05.2011
Distinct phases of validation
New
Method
Design
Method and
Instrument
Installation
Method
Preparation
Method
Start-Up
Method is
Operational
Method
System is
Defined
Installation
Qualification
IQ
Operational
Qualification
OQ
Performance
Qualification
PQ
Ongoing
Analyzer
Controls
IQ/OQ Phase PQ Phase
Method and Instrument Validation
Vendor Responsibility Validation Approval
Implementation
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min. 3x (10x) measurement
proove for outlier values
Samples (ranging from no to high amount of T cells)
min. 3x (10x) measurement
proove for outlier values
Homogenisation,
take two samples
each
New method
single platform
45/3/14/7AAD/56
Currently used method
dual platform
tetraChrome 45/4/8/3 + 14
Comparison single versus dual platform approach
• Measurement on the same day (Intra-assay precision)
• Measurement on different days (Inter-assay precision)
• Measurement in different laboratories by various individualsKöhl 21.05.2011
Documentation
Parameter Result
Viabilility %
Leukocytes /µl
CD3+ cells /µl
CD3+ cells %
CD3-CD56+ NK cells /µl
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SSF
S
Gate: Lymph
T helper cells
SS
Gate: ungated Gate: Lymph + Check FSC Lymph
CD45 FITC
Gate: Lymph + Check FSC Lymph Gate: Lymph + Check FSC LymphCD3 PC5
CD
4 P
E
CD3 PC5
CD
8 E
CD
CD3 PC5
Gate: CD45+
CD
14 P
C7
SS
Dual platform TetraChrome 45/4/8/3 /14
CD3 T cells (%): Flowcytometry; absolute WBC counting electronically
CD3 PC5
cytotoxic T cells
Flowcytometric quantification of T cells„single platform“ in support to the „ISHAGE“ protocol
„Single platform ISHAGE“ protocol: Absolute cell number and CD34
measurement, both are done simultaneously using flowcytometry
Keeney et al. Cytometry 23:61-70 (1998) Barnett et al. Br J Haematol 106:1059-62 (1999)
Brando et al. Cytometry 42:327-46 (2000) Gratama et al. Cytotherapy 5:55-65 (2003)
7AAD 45-FITC 34-PE CD45 FITC
FS 45-FITC FS Time
CD45 IgG1 CD14 7AAD CD56 CD45 CD3 CD14 7AAD CD56
FITC / PE / ECD / PC5 / PC7
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Viability CD45+ Viability and CD45+ Beads
ungated Viability and CD45+Viability and CD45+ and
CD3+
Viability and CD45+ and
CD3+ and Cluster CD3
Viability and CD45+ and
WBC Viability and CD45+Viability and CD45+ and
CD56+
Viability and CD45+ and
CD56+ and Cluster CD56
Not Beads NK-ZellenViability and CD45+ and
WBC
Viability and CD45+ and CD56+
and Cluster CD56 and CD3-CD56+
CD45-FITC CD3-PE CD14-ECD 7AAD CD56-PC7
Quantification of absolute cell numbers Results
Basis plots in all protocols
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CD45+ region
Border line of the right region has to be separated from the fluorespheres (CAL-beads)
Left region should include CD45dim
Lymphocyte region
Gate: ViabilityGate: Viability and CD45+
Debris
WBC region
(Leukocyte population))
Region enhanced (right and upper)
Check FSC region
Boder line cells and debris
No relevance for monocytes
All count regions are
linked with the Check
FSC Region! Beads = Stem Count Fluorospheres
Precise CAL factor has to be given
Value of the CAL region ≥95%
Include only single beads run in
CAL region
doublets
Gate: BeadsGate: Beads
Stop: 7 min; 250 000 – 700 000 CD45+ events
CD45 CD3 CD14 7AAD CD56
Viability CD45+ Viability and CD45+ Beads
ungated Viability and CD45+Viability and CD45+
and CD3+
Viability and CD45+ and
CD3+ and Cluster CD3Viability and CD45+ and
WBC Viability and CD45+Viability and CD45+
and CD56+Viability and CD45+ and
CD56+ and Cluster CD56
based on Koehl et al. Int J Hematol 2008
Quantification of absolute cell numbers
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Problem solving:
Dump Channel
(CD14)
Problem:
Discrimination of monocytes
Discrimination of the monocyte population
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CCB
Gate: viable CD45+
NTCB
MFI=12,3
Problem solving: Use of a positive control
CD3 expression
PBSC
MFI=47,2
PBSC
NTCB
Gate: viable CD45+
NTCB
Problem:
NTCB = non target cell bag
CCB = cell collection bag
CD3 AB for flowcytometry:
8G12 clone (SK7)
Steric effects of CD3 antibody binding
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Problem: Subjective gating of the „X-Coordinate lower left”
4,06/µl 8,92/µl
Problem solving: Use of an internal CCB control
4,06/µl
Gate: NK-ZellenGate: viable CD45+
Determination of the borderline CD3neg to CD3pos
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Gate: NK-Zellen
3rd) Internal control
CCB
Gate: viable CD45+
2nd) Positive control
NTCB
Gate: viable CD45+
1st) CD14 Dump Channel
CCB
1. Discrimination of the monocyte population
2. Steric effects of CD3 antibody binding
3. Determination of the borderline CD3neg to CD3pos
Strategies and gates for residual T cell detection
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Quantification of residual T cells
Cell Collection bag
example
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Stop: 7 min or 100 CD3+ events; 250 000 – 700 000 CD45+ events
0 40 80 120 160 2000
40
80
120
160
200
dual platform [CD34 +/µl]
sin
gle
pla
tfo
rm [
CD
34
+/µ
l]
50 100 150 200
-1000
-500
0
500
1000
AverageD
iffe
ren
ce
PB and PBSC freshly: good correlation
p > 0.97
Bochennek K et al BMT 2005
Dual versus single platform: CD34 enumeration
Bland Altman comparison
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Dual versus single platform: CD3 enumeration
Bland Altman comparison
2500 5000 7500
-1750
-1250
-750
-250
250
750
1250
1750
Average CD3+cells/µl
Dif
fere
nc
e B
-A (
ce
lls
/µl)
2500 5000 7500
-1750
-1250
-750
-250
250
750
1250
1750
Average CD3+ cells/µl
Dif
fere
nce
B-A
(cells/µ
l)
45/3/14/7-AAD/56 versus 45/4/8/3/14
PB and PBSC fresh PB, PBSC thawed orpurified grafts fresh
Koehl et al. Int J Hematol 2008
PB and PBSC fresh: good correlation
PB, PBSC thawed or exceedingly engineered grafts: no correlation
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0.001 0.01 0.1 1 10 1000.01
0.1
1
10
100
1000
10000
CD3 [%]
Via
ble
CD
3+ c
ells
/µl
Precision for CD3+ T cell enumeration (single platform)
Range for T cell enumeration (n = 10 per measuring point)
Detection limit: 0.2 0.1 CD3+ cells/µlKöhl 21.05.2011
Data 1
CCB0
10
20
30
40
CD
3+
T c
ell
s/µ
l
Data 1
PBSC0
10000
20000
30000
40000
50000
60000
70000
CD
3+
T c
ell
s/µ
l
Data 1
CCB0
20
40
60
80
100
Via
bil
ity
[%
]
63 304/µl68.9/µl
0.7%
x:sd:vc:
19.3/µl2.3/µl11.8%
96,4%1.2%1.3%
Intra-assay precision for CD3+ T cell enumeration(single platform)
n = 7 n = 7 n = 7
Köhl 21.05.2011
Qualification and validation of residual T cells in
CD3/CD19 depleted grafts requires enhanced flow
cytometric technology for T cell detection
Essential of “step by step” procedure of material
description, study plan/SOPs and
result documentation
Distinct phases of validation (IQ, OQ, PQ),
validation approval and implementation
ConclusionFlow cytometric quality control of
CD3/CD19 depleted grafts
Köhl 21.05.2011
T. Klingebiel
P. Bader, D. Schwabe, J Soerensen, T. Lehrnbecher, M. Becker,
S. Kuci, H. Kreyenberg, S. Zimmermann, K. Bochennek; H.P. Grüttner
Paediatric Haematology and Oncology
Frankfurt, Germany
Transfusion Medicine
Red Cross Center
Frankfurt and
Dresden, Germany
E. Seifried, T. Tonn
C. Seidl, H. Bönig, H. Bialleck,
P. Becker, R. HenschlerKöhl Lab: R. Esser, S. Klöss, S. Hünecke, M. Bremm, C. Brehm, E. Auth,
A. Quaiser, S. Wehner, S. Betz, O. Zimmermann, S. Erben, R. Lehne