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

Köhl 21.05.2011

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

Köhl 21.05.2011

CD3/19 depleted graft

CD34 selected graft

Stem cell graft: CCB (cell collection bag)

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

Problem solving:

Dump Channel

(CD14)

Problem:

Discrimination of monocytes

Discrimination of the monocyte population

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

Quantification of residual T cells

Cell Collection bag

example

Köhl 21.05.2011

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

Köhl 21.05.2011

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

Köhl 21.05.2011

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