Supplementary Figures To

Supplementary Figures to

Variable resistance to freezing and thawing of CD34 positive stem cells and lymphocyte subpopulations in leukapheresis products

Supplementary Figure 1A. Analysis report of CD34+ cell and CD45+ cell viability measurement. Flow cytometry analysis was performed on a single platform using CD45

FITC/CD34 PE antibodies, 7-AAD viability dye, and Trucount beads (BD Biosciences, San Jose,

CA) according to the modified ISHAGE protocol. A report of a representative post thaw product sample is shown. Data.006 Enter These Values Data (Total .006 CD45) Trucount = 48450.00 No gate

R2 Dilution Factor = R1 R5

0 1 2 3 10.00 Sample 0 1 2 3 10 10 10 10 10 10 10 10 4 4 10 10 CD34 PE Volume = CD45 FITC Data.006 Data.006 R2*G1 50.00

R3 File: Data.006 Sample ID: 15/134-4 Patient ID: XXXXXXXXXX Trucount: 48450 Dilution Factor: 10 Sample Volume: 50 Acquisition Date: 04- Dec-15 Total Events: 159356 R1

0 1 2 3 10 10 10 10 0 200 400 600 800 4 1000 10 FSC-Height Beads 23682 14.86 CD45 FITC Total CD34 1667 1.05 Data.006 Viable Lymphs 25070 15.73 Data.006 Total CD45 134952 84.69 No gate Debris 0 0.00

R6 Inspect All Dotplots Results Total CD45 = 55218.52 /uL

0 200 400 600 800 0 1 2 3 10 10 10 10 1000 CD45 Viability = 53.02 % 4 FSC-Height 10 CD45 FITC Data.006 Viable CD34 = 342.89 /uL Data.006 Viable cells R1*R2*R3 Viable CD45 = 29274.59 /uL

R8 Total CD34 = 682.09 /uL

0 1 2 3 CD34 Viability = 50.27 % 10 10 10 10 0 1 2 3 4 10 10 10 10 10 4 7-AAD 10 Viable CD34 of Viable CD45 = 1,17 % 7-AAD

Total CD34 of Total CD45 = 1.24 %

Supplementary Figure 1B. Analysis report of lymphocyte subpopulation measurement. Flow cytometry analysis was performed in a dual platform assay using TriTest reagents (CD3/CD4/C45,

CD3/CD8/CD45, CD3/CD19/CD45, CD3/CD16+C56/CD45; BD Biosciences) and BD MultiSET software for data analysis.

Reports of a representative post thaw product sample are shown. MultiSET™ Physician Report Director: Prof. Dr. J. Oldenburg Software: MultiSET V1.1.2 Operator: LE Cytometer: FACScan (#81343)

Sample 6 Date Acquired: Die, 17. Nov Name: 15/134-4 2015 13:47 Uhr Sample XXXXXXXX Date Analyzed: ID: Case XX Number: 3 Color Die, 17. Nov 2015 Panel TBNK Reference Range Name: WBC: Type: BD User 3 Inputs: 6.1 x10 /µL Percent Lymphs: 25.6 Abs Cnt Resu % (cells/µL) Reference lt / Range Nam R 55% e a t 84% i o 690

13% 41%

190 1140

6% 25%

90 660

5% 27%

90 590

Lymphosum: 99.9 CD3 % Lymph 3 Difference: CD3 1001 - 1050 Abs Cnt Range: 1.41 T Helper/Suppresso r Ratio: Comments: Laboratory Director: MultiSET™ Lab Report Prof. Dr. J. Oldenburg Director: LE Software: MultiSET V1.1.2 Operator: Cytometer: FACScan (#81343)

S D Die, 17. Nov 2015 13:47 a at Uhr m e p A l c e q ui N re a d: m e : 6 Sam D Die, 17. Nov 2015 ple a ID: t e

A n a l y z e d : Case Ref. Range Type: BD Number: XXXXXXX XXXX Panel 3 User 6 Lymphs Name: Inputs: . (%): 25.6 C 1 o WBC l (x1000): o r T B N K

CD3/ D Data 15/134-4 06.01 CD4/ a File: CD45 t a

S e t [ 1

] Reagent Lot ID: 46970 Attr Def File: 3/4/45 Tri v2.0 Events Acquired: 15000

C a s e

N u m b e r :

P D 1 a a 5 n t / e a 1 3 l F 4 il - N e 4 a : 0 m 6 e . : 0 2

S a m p l e

I D : CD3/CD19/CD45 Data Set [ 1 ] Data File: 15/134-4 06.03 Re Attr age Def nt File: Lot 3/19/ ID: 45 619 Tri 81 v2.0 Eve nts Acq uire d: 165 00

C D 15/134-4 D a 06.04 3 t a / C F D il e 1 : 6 + 5 6 / C D 4 5 Re Attr age Def nt File: Lot 3/16+ ID: 56/45 257 Tri 17 v2.1 Eve nts Acq uire d: 165 00 9 3 9 1001 - 1050 . 9 1.4

Multi-tube QC /Suppressor L Ratio: y m p h o s u m : C D 3 % L y m p h Di ff er e n c e: C D 3 A b s C nt R a n g e: T H el p er after thawing (gray).

The right peak shows

the CD3+ events.

Supplementary Figure

2. Overlay analysis of

CD3+ events in cryopreserved apheresis products.

CD3+ events of apheresis products from two healthy volunteers were compared in samples taken before freezing (black) and Supplementary Figure

3. NK cell post thaw recovery and liquid storage time. Post thaw

CD16+CD56+ cell recovery was determined in 90 cryopreserved apheresis products. Liquid storage time shows the time between end of apheresis and end of cryopreservation.