ID01

ID02

ID03

ID04

ID05

ID07

ID08

ID09

ID11

ID13

ID15

ID16

ID19

ID21

ID23

ID24 Trypan blue

Trypan blue

Trypan blue

Guava ViaCount

Trypan blue

CASY Model TTInovatis

Trypan blue

Trypan blue

Trypan blue

Trypan blue

Trypan blue

Trypan blue

Trypan blue

Guava PCA-96System

Trypan blue

Guava PCA-96System

Countingmethod

37

37

37

37

37

37

37

RT

4

37

RT

RT

37

37

RT

37

mediumtemp (°C)

X-vivo 15

X-vivo 15

RPMI 1640

AIM-V

CTL wash medium +benzonase

X-vivo 15

RPMI 1640

RPMI 1640

IMDM

IMDM + 2.5% HS + DNAse

Iscove + 10% AB serum

RPMI 1640

X-vivo 15

RPMI 1640 + AB serum

PBS

CTL wash medium +benzonase

Thawing medium

2

20

18

2

3

3

18

6

19

14

2

2

21

20

3

17

Restingtime (h)

serum

serum

serum

serum

serum-free

serum

serum

serum

serum

serum

serum

serum

serum-free

serum

serum

serum-free

Preferredprotocol: serum /

serum-free

Supplementary Table 1A: cell handling conditions. A questionnaire was completed by eachparticipating centre to provide details of individual protocols and reagents used in the IFN-ELISPOT assay: Thawing medium and temperature, counting method, resting conditions andpreferred condition. The IDs from the three labs normally using a serum-free protocols areunderlined.

ID01

ID02

ID03

ID04

ID05

ID07

ID08

ID09

ID11

ID13

ID15

ID16

ID19

ID21

ID23

ID24

NO SERUM PLATES READERTEST WITH SERUM ANTIBODIES

serum medium plate type ReaderVendormedium

10% human AB serum PAN X-vivo 15 BD ELISPOT plate 551849 CTL Immunospot S3Becton DickinsonX-vivo 15

10% human AB serum AIM-V Millipore MAIP N4550 AID ERL02MabtechIMDM

10% human AB serum PAA AIM-V Millipore MSIP S4W10 AID ERL02Becton DickinsonIMDM

5% human AB serum sigma X-vivo 15 Millipore MSIP N4W CTL Immunospot S4MabtechX-vivo 15

10% human AB serum X-vivo 15 Millipore MSIP S4 CTL Immunospot S2MabtechRPMI 1640

10% human AB serum PAN X-vivo 15 Millipore MAHA S4510 BioSys 3000PharmingenIscove

10% human AB serum CCpro X-vivo 15 Millipore MSIP N4B50 CTL Immunospot S5MabtechIMDM

10%human AB serum PAA X-vivo 15 Millipore MSHA S4510 BioSys 5000MabtechIMDM

10% human AB serum LONZA X-vivo 15 Millipore MAIP S4510 AID ELR02MabtechRPMI 1640

10% human AB serum X-vivo 15 Millipore MSIP S45 CTL Immunospot S2MabtechRPMI 1640

5% human poolserum X-vivo 15 Millipore MSIP S45 Zeiss ELISPOT reader 4.1Mabtechpre-coated plates

RPMI 1640

10% human AB serum BioWhit. CTL-test medium Millipore MSIP S45 AID ELR03Mabtechpre-coated plates

RPMI 1640

10% FBS Gibco Invitrogen AIM-V Millipore MSIP S4W AID ELR04Mabtechpre-coated plates

RPMI 1640

10% human AB serum X-vivo 15 Millipore MSIP S4510 A EL VIS Eli Scan V2.0MabtechRPMI 1640

10% human AB serum X-Vivo 15 Millipore MSIP S45 CTL Immunospot S4Mabtechpre-coated plates

X-vivo 15

5% human AB serum CCpro X-vivo 15 BD ELISPOT plate 512447 Zeiss ELISPOT reader 4.4Becton DickinsonX-vivo 15

Supplementary Table 1B: Results from questionnaires (ELISPOT conditions). A questionnaire was completed byeach participating centre to provide details of individual protocols and reagents used in the IFN- ELISPOT assay: Testmedia (with serum and serum-free), type of plates, antibody vendor and ELISPOT reader. The IDs from the three labsnormally using a serum-free protocols are underlined.

n.d.n.d.10161068912BGP + BGP + BGP16PP + BG

P + BG910BG8363BGBG7966BGBGBGP + BG

n.d.32P + BGn.d.55n.d.11P + BGP + BG1514n.d.7

n.d.36BG7n.d.153n.d.31P + BG43140n.d.10

4841P + BGBG18419034BGP + BGP + BG38BGBGP + BG

9832103109810P1241944

n.d.n.d.P + BG2BG64P + BG8P + BGPP + BG12P + BG6

2629BGP123742415P + BG21726BG8

n.d.5361214812837389BG4236n.d.18

23BG3P + BG11111521BGP + BGP + BG15BG7P + BG

3641BGBG98116P23P + BG132025BG11

P + BGP + BGP + BGP + BGBGP + BGP + BGP + BGP + BGBGP + BGP + BGP+ BGBG

1733P3P + BGP + BG162022512486

44BG2776BGBG8BGBG9BG4

P + BG12PPn.d.41n.d.PP + BGP3Pn.d.BG

2433541161242128P + BGP + BG242067

noserumserum

noserumserum

noserumserum

noserumserum

noserumserum

noserumserum

noserumserum

ID01ID02ID03ID04ID05ID07ID08ID09ID11ID13ID15ID16ID19ID21ID23ID24

D3 / CMV D4 / Flu D5 / FluD2 / CMVD1 / CMV D2 / Flu D3 / Flu

Supplementary Table 2A: Overview of specific spot counts. The reported frequencies of antigen-specific T-cells.expressed as spots per 100,000 PBMC, are shown for all 16 centres that submitted complete data sets for the sevendonor-antigen combinations where antigen-specific T cells were present in detectable numbers (D1/CMV, D2/Flu,D2/CMV, D3/Flu, D3/CMV, D4/Flu, D5/Flu). For each donor-antigen combination the results are shown for test withserum and without serum. Where insufficient cells were recovered to perform the test in triplicate the results wereeliminated from further analysis (not done, n.d.). A two-sided T-test for unpaired samples was performed on alltriplicates to eliminate variability through inconsistent seeding; triplicates with p-value of >0.05 are indicated in thetable as (P). Antigen-specific spots were determined where the number of spots provided by PBMC plus antigen were>3-fold of the medium only background; triplicates that were <3-fold background are indicated as (BG).

D3 / CMV D4 / Flu D5 / FluD2 / CMVD1 / CMV D2 / Flu D3 / Flu

10 4 11 11 5 4 9 8 14 10 8 7 12 8

16 12 16 16 16 16 16 13 16 13 16 16 14 1163 33 69 69 31 25 56 62 88 77 50 44 86 738 6 22 25 15 24 20 21 100 108 6 5 25 23

4 2 11 14 26 36 10 10 42 45 5 3 17 14

50.53 23.93 49.12 54.42 175.78 148.66 51.58 46.71 41.70 42.13 89.83 67.76 61.27

detected by

from

%mean

SD

CV 61.33

Supplementary Table 2B: Overview of specific spot counts.The table gives a summary of the group results (all 16 participants shown in suppl. Table 2A) for the seven reactivedonor-antigen combinations (D1/CMV, D2/Flu, D2/CMV, D3/Flu, D3/CMV, D4/Flu, D5/Flu). For each donor-antigencombination the group results are shown for test with serum and without serum. The table shows the number ofcentres that were successfully able to detect a response (first row), the number of tests performed (second row), thepercentage of centres that were able to successfully detect a response (third row), the mean frequency of antigen-specific T cells (fourth row), the standard deviation of reported frequency of antigen-specific T cells (fifth row) andthe coefficient of variation as calculated from all positive responses (sixth row).

noserumserum

noserumserum

noserumserum

noserumserum

noserumserum

noserumserum

noserumserum