NIST-FDA Cell Counting Workshop: Sharing practices in cell counting measurements 

April 10, 2017-NIST West Square, Gaithersburg MD 20899

Case study 4

Memorial Sloan Kettering: Brigitte Senechal

CTCEF Cell Therapy and Cell Engineering Facility 

Case study 4 / Brigitte Senechal
CTCEF / Isabelle Riviere

Cell Therapy and Cell Engineering Facility (2015)

Production of CAR T cells from apheresis product 

GTF Gene Transfer and Somatic Cell Engineering Facility

Research lab

Production

(manufacturing /QC testing)

R&D Patient Monitoring

Case study 4 / Brigitte Senechal

Microscope cell counting (Trypan blue)

Cellometer Nexcelom (AOPI)

Ac-T diff Beckman Coulter

Cellometer Nexcelom (AOPI)

Microscope cell counting (Trypan blue) Microscope cell counting (Trypan blue)

Cellometer Nexcelom (AOPI)
Case study 4 / Brigitte Senechal

Lab space/equipment

Different lab

Multiple pieces of the same equipment 

Different methods (optical/diffraction, impedence, fluorescence-AOPI, TB...)

Operators

15 operators

Cell sample properties :

•Fresh v frozen

•Cell lines v patient isolated

•Manipulated v un-manipulated

•Homogeneity / over time / over source

•(species)

Multiple factors that may affect cell counting measurements

...

Verification

SOP 

training
Case study 4 / Brigitte Senechal

Production

start

Production

end

CAR T cell dose

REQUESTED

Bag with CAR T-cell 

dose requested

(cryopreserved)

Infusion

CAR T cell dose

INFUSED

Release

Thawing

Request for production Request for infusion

Viability

Fresh

(CofA>80%)

Dose assigned

(#CAR per kg)

Patient weight

(kg)

Patient weight

(kg)

Pass >70%

Viability (vial)

Cryopreserved

(CofA/release criteria)

CAR cell dose preparation and release: process

current method: trypan blue

Production

(manufacturing /QC testing)
Microscope cell counting (Trypan blue) / our reference

Cellometer Nexcelom (AOPI)?

CAR cell dose preparation and release

Case study 4 / Brigitte Senechal

Counting Instructions (SOP-6010):

Trypan blue

1:1 cell:tb

Read within 3 minutes

2 chambers

Average %

AOPI (Nexcelom)

1:1

1 reading

Dilution if >10e7

Release Instructions (SOP-5008):

Cryopreserved Viability-trypan blue

>70% pass (CofA)

<70% / 2nd operator

<70% / 2nd vial thawed

<70% / 2nd operator

IRB approval

QA report to PI

PI requests deviation

•Subjective

•Great inter-operator variability

•Fully manual

•Time consuming

Microscope cell counting (Trypan blue)

-> not subjective

-> minimal inter run variability

-> automated

-> rapid

Health & safety: TB v PI
Case study 4 / Brigitte Senechal

End of production

Request for production / 2016 Request for infusion / 2016

Viability

Fresh

(CofA>80%)

Viability (vial)

Cryopreserved

(CofA/release criteria>70%)

Microscope cell counting (Trypan blue) v Nexcelom (AOPI):study design

•Data for 39 CAR products / fresh

•Data for 23 CAR products / cryopreserved

•Data paired for 23 products

TB: duplicate (as per SOP)

N: triplicate
Case study 4 / Brigitte Senechal

Data for 39 fresh CAR T cells products

Comparison of viable cell counts with two methods

Absolute viable cell counts are comparable for 39 products assessed in parallel

(the small difference of 2.9% is limit significant p=0.044)

TB N

median 7,250,000 7,386,667

average 12,007,179 12,481,111

min 1,070,000 1,296,667

max 73,750,000 67,200,000

n 39

ttest 0.044

median of difference 2.9%

1

10

100

1 10 100

Nexcelom

Trypan blue exclusion

Viable cell count 10e6 perml 

FRESH

Corr.0.993
Case study 4 / Brigitte Senechal

Data for 39 fresh CAR T cells products

(continued)

Out of the 39 products tested, 17 were tested without respecting dilution instructions for the Nexcelom

Same comparison was repeated for 22 products tested with respect to dilution instructions

Again absolute viable cell counts are comparable for 22 products

The small difference of 2 % is not significant (p=0.256)

TB N

median 5,772,500 5,516,667

average 11,544,318 11,836,515

max 1,070,000 1,296,667

min 73,750,000 67,200,000

n 22

ttest 0.256

median of difference 2.0%

1

10

100

1 10 100

Nexcelom

Trypan blue exclusion

Viable cell count 10e6 per mL 

FRESH- 22 products

Corr.0.992

Comparison of viable cell counts with two methods

viable cell counts condition the formulation, therefore

the two counting methods will allow the preparation of 

the same cell dose
Case study 4 / Brigitte Senechal

Data for 39 fresh CAR T cells products

Corr.0.748

http://www.nexcelom.com/Applications/cell-viability-3-

comparing-trypan-blue-and-aopi-staining-methods.php

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100

Nexcelom

Trypan blue exclusion

Percent viability FRESH

Comparison of % viability with two methods

Percent viable cells are not comparable due to Nexcelom

detecting more dead cells in fresh CAR T cells products
Case study 4 / Brigitte Senechal

Data for 39 fresh CAR T cells products

(continued)

Despite the low correlation between % viability determined by the 2 methods,

The same three products will not pass the cut-off of 80%, thus 100% agreement

TB N

median 92.19 80.67

average 90.87 79.75

max 73.34 34.03

min 97.71 93.90

ttest 2.09E-10

median of difference - 11.0%

fresh frozen

release criteria >80% >70%

TB 92% 100%

N 92% 92%

concordance 100% 92%

TB NEXCELOM

73.34 57.13

79.50 52.53

79.51 34.03

30

40

50

60

70

80

90

100

30 40 50 60 70 80 90 100

Nexcelom

Trypan blue exclusion

Percent viability FRESH

Comparison of % viability with two methods

 Three products did not pass the 80% 

criteria with both methods
Case study 4 / Brigitte Senechal

% viabilities are highly correlated between the two methods after thawing 

Products

The same 8 products will not pass the 70% release criteria

Only one product was below 70% with the Nexcelom but passed the release 

criteria for the trypan blue, bringing the concordance to 96%

release criteria >70%

TB 65%

N 56%

concordance 96%

Thawed product Fresh product

TB N TB N

45.00 38.20 79.50 52.53

58.37 44.75 73.34 57.13

59.84 54.50 88.68 77.33

61.89 60.60 91.71 80.67

67.51 69.90 86.58 86.77

67.57 62.20 90.90 70.70

68.50 61.20 91.05 76.67

69.30 65.20 90.97 73.40

70.61 73.80 94.28 82.00

70.62 71.70 93.15 81.97

72.89 78.30 92.64 93.90

73.48 78.20 93.89 89.73

74.22 70.30 88.14 79.03

76.31 74.60 92.00 90.83

80.32 83.90 94.18 93.17

80.59 73.90 89.05 80.23

80.93 66.50 93.04 70.13

80.97 79.40 95.34 91.00

81.56 80.00 93.67 89.63

86.24 73.70 95.81 80.50

87.69 78.60 91.27 88.03

88.36 88.80 97.06 90.23

89.35 88.40 95.84 87.17

Data for 23 cryopreserved CAR T cells products

50

60

70

80

90

100

50 60 70 80 90 100

Nexcelom

Trypan blue exclusion

% viability CRYOPRESERVED

TB N

median 73.48 73.70

average 73.57 70.29

min 45.00 38.20

max 89.35 88.80

ttest 0.006

median of difference - 1.7%

Corr.0.893

Comparison of % viability with two methods after thawing 23 cryopreserved products
Case study 4 / Brigitte Senechal

Data for 23 cryopreserved CAR T cells products

% viability with two methods correlate well on thawed products but not on fresh products

Corr.0.893 Corr.0.761

50

60

70

80

90

100

50 60 70 80 90 100

Nexcelom

Trypan blue exclusion

% viability FRESH

50

60

70

80

90

100

50 60 70 80 90 100

Nexcelom

Trypan blue exclusion

% viability CRYOPRESERVED

TB N

median 73.48 73.70

average 73.57 70.29

min 45.00 38.20

max 89.35 88.80

ttest 0.006

median of difference - 1.7%

TB N

median 92.00 81.97

average 90.96 80.99

min 73.34 52.53

max 97.06 93.90

ttest 1.18E-06

median of difference - 10%
Case study 4 / Brigitte Senechal

Fresh-cryopreserved data for 23 CAR T cells products

Comparison of viability before and after cryopreservation with the same assay

50

60

70

80

90

100

50 60 70 80 90 100

Nexcelom / thawed

Nexcelom / fresh

Effect of storage

Corr.0.894

Fresh thawed

52.53 38.20

57.13 44.75

70.13 66.50

70.70 62.20

73.40 65.20

76.67 61.20

77.33 54.50

79.03 70.30

80.23 73.90

80.50 73.70

80.67 60.60

81.97 71.70

82.00 73.80

86.77 69.90

87.17 88.40

88.03 78.60

89.63 80.00

89.73 78.20

90.23 88.80

90.83 74.60

91.00 79.40

93.17 83.90

93.90 78.30

ttest 3.55E-09

median of difference -10%

NEXCELOM

Upon thawing there is on average

a 10% decrease of the viability when 

measured with Nexcelom
Case study 4 / Brigitte Senechal

Fresh-cryopreserved data for 23 CAR T cells products

50

60

70

80

90

100

50 60 70 80 90 100

Nexcelom / thawed

Nexcelom / fresh

Effect of storage

Corr.0.894

ttest 3.55E-09

median of difference -10%

NEXCELOM

40

50

60

70

80

90

100

40 50 60 70 80 90 100

Trypan blue / thawed

Trypan blue / fresh

TRYPAN BLUE

Storage +?

Corr.0.730

Hypothesis:

PI more sensitive than TB to detect dead cells ?

PI is less specific than TB?

1 F/T cycle increases correlation between the 2 assays: 

the discrepant signal wiped out?

ttest 2.13E-10

median of difference -17%

the viability measured on thawed products by trypan 

blue is not well correlated to the viability before 

thawing

decrease of viability after thawing seems higher with 

the Trypan blue (-17%) than with the Nexcelom (-10%)

! the viability of fresh products determined by Trypan 

blue is higher than with Nexcelom (~+10%) [slide 10]
Case study 4 / Brigitte Senechal

Preliminary Conclusions

Trypan blue to Nexcelom / formulation and release of cryopreserved CAR T cells

Viable cell count is not affected -> formulation of the same CAR T cell dose

% viability fresh significantly different with nexcelom detecting more dead cells

(-11%, p<0.001)

But 100% agreement for a provisional release criteria of 80% on fresh products

% viability cryopreserved slightly different between the two methods

(-1.7%, p=0.006)

With excellent agreement of 96% for a release criteria of 70% (only 1 discrepant)

..to confirm with more products

...to gain confidence and understanding of discrepant data observed on fresh 

products: AOPI detected more dead cells than TB in T cells expanded in vitro