Results and harmonization guidelines from two large-scale international Elispot proficiency panels conducted by the Cancer Vaccine Consortium (CVC/SVI).
The Cancer Vaccine Consortium of the Sabin Vaccine Institute (CVC/SVI) is conducting an ongoing large-scale immune monitoring harmonization program through its members and affiliated associations. This effort was brought to life as an external validation program by conducting an international Elispot proficiency panel with 36 laboratories in 2005, and was followed by a second panel with 29 participating laboratories in 2006 allowing for application of learnings from the first panel. Critical protocol choices, as well as standardization and validation practices among laboratories were assessed through detailed surveys. Although panel participants had to follow general guidelines in order to allow comparison of results, each laboratory was able to use its own protocols, materials and reagents. The second panel recorded an overall significantly improved performance, as measured by the ability to detect all predefined responses correctly. Protocol choices and laboratory practices, which can have a dramatic effect on the overall assay outcome, were identified and lead to the following recommendations: (A) Establish a laboratory SOP for Elispot testing procedures including (A1) a counting method for apoptotic cells for determining adequate cell dilution for plating, and (A2) overnight rest of cells prior to plating and incubation, (B) Use only pre-tested serum optimized for low background: high signal ratio, (C) Establish a laboratory SOP for plate reading including (C1) human auditing during the reading process and (C2) adequate adjustments for technical artifacts, and (D) Only allow trained personnel, which is certified per laboratory SOPs to conduct assays. Recommendations described under (A) were found to make a statistically significant difference in assay performance, while the remaining recommendations are based on practical experiences confirmed by the panel results, which could not be statistically tested. These results provide initial harmonization guidelines to optimize Elispot assay performance to the immunotherapy community. Further optimization is in process with ongoing panels.
Janetzki
Sylvia
S
ZellNet Consulting, Inc, Fort Lee, NJ, USA. sylvia@zellnet.com
Panageas
Katherine S
KS
Ben-Porat
Leah
L
Boyer
Jean
J
Britten
Cedrik M
CM
Clay
Timothy M
TM
Kalos
Michael
M
Maecker
Holden T
HT
Romero
Pedro
P
Yuan
Jianda
J
Kast
W Martin
WM
Hoos
Axel
A
D019496:Cancer Vaccines / Q000276:immunology*; D002470:Cell Survival / Q000276:immunology; D019411:Clinical Laboratory Techniques / Q000592:standards* / Q000706:statistics & numerical data; D004797:Enzyme-Linked Immunosorbent Assay / Q000592:standards*; D006306:Health Surveys; D006801:Humans; D007391:International Cooperation; D007753:Laboratories / Q000592:standards*; D007958:Leukocyte Count; D007963:Leukocytes, Mononuclear / Q000276:immunology; D010455:Peptides / Q000276:immunology; D011786:Quality Control; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D012952:Societies
D019496:Cancer Vaccines; D010455:Peptides