The ELISPOT proficiency panel was conducted with a group of 31 centers. Twenty-six participating laboratories were located in 9 European countries (Denmark, France, Germany, Italy, The Netherlands, Spain, Sweden, Switzerland and the United Kingdom). Fourteen of these laboratories were prior participants of CIP proficiency panels, and twelve laboratories participated for the first time. In addition, five laboratories (4× US and 1× Germany) were recruited from the CRI-CIC proficiency panel program that collaborated in this study. Each laboratory received an individual laboratory ID number and was assigned to one of three subgroups of similar size (10, 12 and 10 laboratories, respectively). One participating laboratory analyzed PBMCs from all three subgroups and generated three completed independent data sets. One participant observed an enormous background spot production in all tested donor–antigen combinations, which made the evaluation of the results impossible. This data set was therefore excluded from the final analysis. Consequently, we obtained 32 evaluable data sets from the 31 participating laboratories. The following “Materials and methods” section was prepared compliant to the MIATA guidelines for structured reporting of T-cell experiments .
PBMCs were isolated from buffy coats obtained from thirteen healthy HLA-A*0201 donors after informed consent at the Transfusion Center, University Medical Center Mainz, Germany. Within 24 h after collection, PBMCs were separated by Ficoll gradient centrifugation and cryopreserved in three different freezing media at 15 million (Mio) cells per vial (A = 90 % heat-inactivated human AB serum (pooled from blood donations from local donors) + 10 % DMSO, B = CryoMaxx II (PAA, Pasching, Austria), C = 10 % human serum albumin (CSL Behring, Marburg, Germany) + 10 % DMSO + 80 % RPMI (Gibco Invitrogen, Darmstadt, Germany) using an automated controlled-rate freezing device (Sy-Lab 14S-B, Neupurkersdorf, Austria). The three media were selected based on results from the survey asking for preferences in participating laboratories.
PBMCs were transferred to the vapor phase of liquid nitrogen and stored until shipment on dry ice to European laboratories (2–20 h transfer time) or shipment in liquid nitrogen shippers for the four US laboratories (32–56 h transfer time). Shipped PMBCs were stored at −80 °C after receipt and thawed after duration of 2–12 weeks at the day of the experiment.
All donor PBMCs were thawed and pretested at least 2 times in IFN-gamma ELISPOT for reactivity against the HLA-A*0201-restricted model epitopes hCMVpp65495–503 (NLVPMVATV), FLU M158–66 (GILGFVFTL) and EBV BMLF1280–288 (GLCTLVAML). Six donors were selected based on a cell viability of >90 % as determined with a Guava counter in at least 2 independent thawed samples for all three selected freezing medium conditions. Distributed samples in each of the three subgroups were confirmed to have reactivity in four different donor–antigen combinations.
Each participating laboratory received PBMCs from two of the six preselected donors, each frozen in the three different freezing media (A, B and C) and three peptides (CMV, FLU, EBV) for antigenic stimulation. Participants had to thaw all cells using their preferred thawing procedure and determine the number of recovered PBMCs as well as the viability (%) after thawing and resting (a resting phase was recommended but not mandatory for laboratories that have SOPs that do not utilize a resting phase). Eighteen centers performed manual counting using a microscope and Trypan blue exclusion, 11 centers used Guava Counters, and three centers used other methods (CD45/7AAD, Nexcelom Cellometer, Vi-Cell XR). Results obtained for cell viability and recovery in the three tested medium conditions were compared using an unpaired, two-sided t test (p = 0.05).
For experiments performed at the central laboratory (Mainz), PBMCs from six healthy HLA-A*0201 buffy coats donors (donors 1–6) were collected after informed consent was obtained. The buffy coats were obtained from the Transfusion Center, University of Mainz, Germany. Within 24 h after collection, PBMCs of each donor were separated by Ficoll gradient centrifugation and cryopreserved in 7 different freezing media at 16 × 106 cells per vial: the first three freezing media (A, B and C) correspond to the freezing media tested in the proficiency panel (see above). In addition, the following four freezing media were utilized: (D) CryoKit ABC (CTL, Bonn, Germany), (E) 90 % inactivated FCS + 10 % DMSO, (F) 12.5 % Albumin bovine Fraction V (Serva, Heidelberg, Germany) + 77.5 % RPMI + 10 % DMSO, (G) 12,5 % BSA + 77.5 % RPMI + 5 % DMSO + 5 % hydroxyethyl starch (Fresenius Kabi, Bad Homburg, Germany). PBMCs were frozen using an automated controlled-gradient freezing device (Sy-Lab 14S-B, Neupurkersdorf, Austria) and then transferred into the vapor phase of liquid nitrogen.
IFN-gamma ELISPOT assay
Participants were asked to quantify antigen-specific T-cell responses against the three peptides (stock solution at 1 μg/μl in 10 % DMSO) that were shipped together with the PBMCs on dry ice to the European laboratories and in liquid nitrogen to the 5 US laboratories. Peptides had to be used at a final concentration of 1 μg/ml. The positive control could be chosen by the participants. In order to facilitate the analysis of data generated, participants received a plate layout that included six replicates of the MOCK control (cells plus medium and no peptide), three peptide antigens added as triplicates and 1 well of positive control for each of the six donor-freezing medium conditions. The laboratories were free to use their own protocol and reagents according to their laboratory SOPs. They had to complete a questionnaire to provide basic information on the ELISPOT operating procedure, such as plates, antibodies, incubation time and staining procedure. For experiments performed at the central laboratory, Multiscreen HA-plates MAHA S45 (Millipore, Darmstadt, Germany) were coated with 50 μl per well of antihuman IFN-γ (7.5 μg/ml, clone Mab 1-D1K, Mabtech) on day 1. The plate was stored overnight at RT. On Day 2, the coating antibody was discarded. The plate was washed 3 times with PBS (Gibco Invitrogen, Darmstadt, Germany) and blocked with X-Vivo (Lonza, Basel, Switzerland) containing 2 % HSA for 1–4 h at 37 °C, 5 % CO2. The PBMCs were thawed and the number of recovered PBMCs as well as the viability (%) after thawing and 2-h resting determined. The cells were rested at a concentration of 1 Mio/ml in OpTmizer™ CTS™ T-Cell Expansion SFM (Invitrogen, Darmstadt, Germany) for 2 h at 37 °C, 5 % CO2 in 50 ml tubes. The median cell recovery after thawing was 13.3 × 106 with a median viability of 95 %. After resting, the median cell loss was 25.1 %. Cell counts and viability was obtained using a Guava counter EasyCyte 5HT and the ViaCount kit. After resting, the PBMCs were washed and resuspended at 2 × 106 cells/ml in OpTmizer. 150 μl PBMCs per well were added to a final cell number of 300,000 cells per well. 50 μl per well of the peptides hCMV pp65495–503 (NLVPMVATV) and FLU M158–66 (GILGFVFTL) were added as triplicates. SEB was added to one well as positive control to a final concentration of 1 μg/ml. In six wells, cells plus medium was added, without peptide (medium control). The plates were incubated at 37 °C, 5 % CO2 overnight. On Day 3, the plate was washed and 60 μl per well of the detection antibody Biotin antihuman IFN-γ (1 μg/ml, clone Mab 7-B6-1, Mabtech) was added. After 2-h incubation at 37 °C, 5 % CO2, the plate was washed and 100 μl per well of the enzyme avidin-alkaline phosphatase (1:100, Sigma) was added. After 1-h incubation at RT and washing the plate, 100 μl per well of the BCIP/NBT (Sigma) was added according to the manufacture’s instructions. After 3–5 min, the staining reaction was stopped by washing the wells under running water. No internal assay controls were used except for six medium control wells to determine the background spot production.
Participants analyzed the plates using their preferred protocol, hardware and software. The results obtained by the ELISPOT reader were controlled by human auditing in 28 of 31 laboratories. Representative ELISPOT filter plates from the proficiency panel phase and the series of experiments performed in Mainz are shown in supplementary figures 1 and 2.
For experiments performed in the central laboratory, the filter plates were analyzed with the CTL ELISPOT reader using the ImmunoSpot 5.0.3 software and a locally established SOP for plate reading. The results obtained by the reader were verified by human auditing. A representative data set is shown in supplementary figure 2.
Analysis of data
The ELISPOT analysis was performed based on the spot numbers reported by the participants.
For experiments performed at the central laboratory, median background reactivity was 2 spots per 100,000 cells, with a range of 0–33 spots. Antigen-specific spots were determined by subtracting the mean spot number in the six medium control wells from the mean spot number in the experimental triplicates. The response determination in this panel was made using a previously published approach for response determination (p value of <0.05 . A Web-based interface for facilitated response determination can be found at http://www.scharp.org/zoe/runDFR/. Raw data of all experiments can be provided upon request.
Participating laboratories operated under different principles, varying from exploratory research to good clinical laboratory practice (GCLP) and good laboratory practice (GLP). Some laboratories used established laboratory protocols, and other laboratories worked with standard operating protocols (SOPs). Most participants reported to be experienced in the ELISPOT technology. Only two participants had no experience.
The central laboratory is working under exploratory research conditions. Work steps for PBMC preparation (cell isolation, freezing, thawing) were performed using laboratory SOPs. The cell staining protocol and filter plate analysis were performed per SOP. The ELISPOT assay protocol was qualified prior to use. To this end, the standardized assay was used in series of experiments with more than 20 donors to define the expected background spot production and intra- and inter-assay variation in the hands of defined operators. All experimental steps from handling of starting material through testing and acquisition of data were conducted by the same experienced operator.