C AR O LY N A. K E E V E R - TAY L O R , P H D

M E D I C AL C O L L E G E O F W I S C O N S I N B L O O D AN D

M AR R O W T R AN S P L AN TAT I O N P R O G R AM

Challenges for Product Evaluation by

Flow Cytometry for Cellular Therapy

Product Processing Laboratories

I S C T R E G I O N A L W O R K S H O P

M E M P H I S , T N

2 0 1 6

Facility/Program Information

• Support for combined pediatric and adult

• FACT & CAP/CLIA accredited for processing and testing

• Processing and transplant activities for 2015 include:

• Transplants Total 299 • Autologous 173

• Allogeneic 126

• DLI 18

• Total Procedures 1300 • Marrow harvests 26

• Cryopreservation 389

• Thawing (Dex/Alb Wash) 332

• Fresh Infusions 127

• Cell Separations (CliniMACS) 12

• Additional Activities

• Peripheral Blood CD34 414

• Product Flow 544

• Inoculate Sterility Cultures

• Materials Management

• Quality Program

• Personnel

• Director 1

• Medical Director 1

• Assistant Director 1

• Laboratory Manager 1

• QA/QC Specialist 1

• Cellular Therapy Specialists 5

• Research Technologist 1

Objectives of Presentation

• Describe types of product flow analysis required by/for

laboratory

• Identify specific challenges unique to analysis of HPC

products

• Identify challenges of thawed product testing and very

rare event analysis

Types of Product Analysis

• Products

• HPC, Apheresis

• HPC, Marrow

• HPC, Cord

• MNC, Apheresis

• Whole Blood

• Stage of assessment

• Fresh

• Thawed

• Cultured

• Reason for assessment

• Product release

• Need for additional collections

• Common manipulations

• Subset enriched

• MNC

• CD34+ cells

• NK cells or other subsets

• Subset reduced

• CD3+ cells, B cells or other subsets

• Procedure or process validation

• Stability studies or expiration time

Product Testing and Analysis Challenges

• Panels

• Antibodies

• Fluorochromes

• Combinations per tube

• Custom trial specific

• Methods

• Staining

• Use of kits

• Wash steps

• Lyse steps

• Analysis methods

• Automated

• Manual • Single platform

• Dual platform

• Availability of flow services

• Within the laboratory

• External to the laboratory

• Clinical flow lab

• Research flow lab

• On or off site

• Hours of service

• Technical issues

• Dead cells

• Contaminating cells

• High background staining

• Rare events

Most Common Assessments

• Peripheral Blood CD34 • Prior to first collection, or prior to each collection

• Viability high, hematology analyzer cells counts accurate

• Lysis required

• Washes not required

• Dual or single platform methods give similar results

• Fresh Product CD34- Same as above except for: • Every collection

• Lysis not required when CD45 in every tube

• Both assays require rapid turnaround time due to clinical decisions regarding

initial or subsequent collections. PB CD34 may be rare event analysis.

Example PB-CD34 Low vs High

Events:300,000 Events:24 CD34/µL=4

Events:100 CD34/µL=65 Events:300,000

Example CD34 Product

Events:256 CD34/µL=999 Events:100,000

Events:77 CD34/µL=191 Events:100,000

Validation Studies

• Fresh Products

• Viability

• Range of detection

• Effect of storage

• Frozen Products

• Staining Methods

• Acquisition Methods

• Stability and expiration

Example Method Validation-Viability by 7-AAD

• Determine conditions to kill but not lyse 100% of cells

• Determine when to add and how much to add

0 15 30 45 600

25

50

75

100

Minutes at 56oC

%V

iabi

lity

Tryp

an B

lue

100

101

102

103

104

0

200

400

600

800

1000

PBMC.001…alive

FL3-H: 7AAD

SS

C-H

: Sid

e S

catte

r

99.4

0

500

1000

1500

2000

2500

3000

3500

0 0.25 0.5 0.75 1

MC

F 7-

AA

D+

7-AAD Concentration µg/mL

7-AAD Mean Channel Fluorescence

7-AAD sensitivity

• Cross titer dead into living cells

% Dead % Living

98.0% 2.0%

80.0% 20.0%

60.0% 40.0%

35.0% 65.0%

20.0% 80.0%

12.0% 88.0%

7.0% 93.0%

4.0% 96.0%

2.0% 98.0%

1.5% 98.5%

1.0% 99.0%

0.5% 99.5%

0.1% 99.9%

Dilution Series

Whole Curve

3.2 17.9 64.8

0.9965

Example Validation- CD34 sensitivity

R² = 0.9987

0.00%

0.50%

1.00%

0.00% 0.20% 0.40% 0.60% 0.80% 1.00%

CD34 Linearity & Fit to expected

R² = 0.981

0.00%

0.10%

0.20%

0.30%

0.00% 0.10% 0.20% 0.30%

CD34 Linearity & Fit to expected

Dilution of mobilized

HPC(A) into non-

mobilized PBMC

Effect of overnight product storage Validation

• Evaluated CD34, CD3, and MNC content at receipt and after overnight refrigerated

storage

• Selected products with a range of MNC content (>70%, <70%). Duplicate tubes to

measure inter and intra assay variation

• Results, inter assay cv <5% before and after storage. Good correlation of results over

wide range of MNC content.

Observed

Expecte

d

Rare Event Analysis

• CD3 Reduction followed by CD56 enrichment in the setting of haploidentical transplantation

• T cells defined as CD3+CD56-

• Limit 1 x 104 T cells/kg

• Target CD56+ cells of 5.0 x 106/kg

• Approach to improve sensitivity • Acquire minimum of 150,000 debris free events

• Use a combination of CD14 and CD33 to remove monocytes and immature myeloid cells from analysis gate

• Use detection antibodies for CD3 and CD56 that recognize different epitopes than antibodies used for depletion or enrichment

NK Enrich Example Analysis

Starting HPC, MNC

FS vs SS CD45 vs SS 7-AAD vs SS CD14/CD33 vs SS CD3 vs CD56

NK Cells, Apheresis

Validation of Methods to Assess Recovery in Thawed Products

• Reasons for performing flow cytometry on thawed products • Assessment of cellular content post thaw when uncertain, e.g. CD34+ cells for cord blood, CD3+

cells for DLI products

• Validation of new or modified cryopreservation procedures

• Establishment of post thaw expiration

• Stability assessment of cryopreserved products

• Challenges performing flow cytometry assessment of thawed products include: • Staining methods may introduce artifacts

• Technical issues such as dead cells or debris that can clog cytometer

• TNC from hematology analyzer may not be accurate when doing dual platform cytometry

• Handling of sample while still having it be representative of the product

• Approaches • Single platform versus dual platform methods to avoid hematology analyzer problems

• Avoid using lysing reagents that may further damage cells

• Use no wash method (required if beads already in tube)

• Careful handling of product or product sample to reduce cell loss due to clumping

Wash/Lyse versus No Wash/No Lyse- Cord Blood

TNC Frozen: 1.01 x 109 CD34 Frozen: 1.44 x 106

TNC Recovered: 4.3 x 108 CD34 Recovered: 1.67 x 106 %CD34 Recovered: 116%

TNC Recovered: 3.5 x 108 CD34 Recovered: 0.89 x 106 %CD34 Recovered: 61.9%

7-AAD vs SS CD45 vs SS CD34 vs SS CD45Dim vs SS LFS vs LSS

No Wash/No Lyse Single Platform

51.1

%

98.7

%

0.30

%

0.26

%

0.26

%

62.1

%

96.2

%

0.39

%

0.35

%

0.35

%

Wash/Lyse Dual Platform

Cord Blood CD34 Recovery- Dual vs Single Platform

Cord Dual Cord Single0

100

200

300

P value < 0.0001

Mean ± SEM of column A

Mean ± SEM of column B

124.1 ± 8.904 N=36

59.88 ± 3.724 N=35

%C

D34

Rec

over

y

Lyse & Wash No Lyse

No Wash

No Lyse/No Wash

Dual Single0

1.0×1007

2.0×1007

3.0×1007

4.0×1007

5.0×1007

P value 0.0549

Platform

Cel

ls p

er m

L

Improved (realistic) results with single platform method does not appear to be completely

due to more accurate cell counts since counts are only marginally higher. Might be due to

removing lysing and/or wash steps

Stability Post Dex/Albumin Wash Validation

0 60 120 180 240 3000

20

40

60

80

100

%MNC%TNC%CD34

N=5

Post Thaw Time (minutes)

%R

ecov

ery

Via

ble

Cel

ls

Full Study on HPC(A) products Immediate vs 90 min HES vs Dex Study

Flow assessment after a period of equilibrium seems to improve before stabilizing.

Also seen in HES validation study @ 90 min for CD34 recovery and viability, but not

for TNC, MNC, and overall viability.

Single Platform Gating, Dextran 8.3%/4.3% HSA Arm Immediate Post Wash

CD34 FSC CD45

7-AAD CD45 CD34

CD45 FSC

CD

14

Non-Viable Gate

Other Suggestions for Flow Analysis of Thawed Cells

• Include more events typically considered as debris

• Found that dead CD34 under-estimate since scatter with debris

• Our own findings do not support this as we get good agreement between total CD34 frozen and the sum of living and dead CD34 as gated in previous example

• Include anti-glycophorin Ab in Cord Blood assessments for removal of NRBC’s and unlysed RBC from analysis

• Did not find significant differences from using only CD45 to remove RBC from gates in MCW experience

• For non-washed product stability, dilute samples in protein containing medium and maintain all reagents and tubes in the cold

• Could help. Since MCW washes all thawed products we validated only immediate post thaw samples encompassing the 15 minutes set for post thaw expiration

• Found that after the first staining period of 15 minutes, largely failed to get useful data when directly thawed products stored and resampled at later intervals without product or product sample dilution