Standardization of cytokine flow cytometry assays.
BACKGROUND
Cytokine flow cytometry (CFC) or intracellular cytokine staining (ICS) can quantitate antigen-specific T cell responses in settings such as experimental vaccination. Standardization of ICS among laboratories performing vaccine studies would provide a common platform by which to compare the immunogenicity of different vaccine candidates across multiple international organizations conducting clinical trials. As such, a study was carried out among several laboratories involved in HIV clinical trials, to define the inter-lab precision of ICS using various sample types, and using a common protocol for each experiment (see additional files online).


RESULTS
Three sample types (activated, fixed, and frozen whole blood; fresh whole blood; and cryopreserved PBMC) were shipped to various sites, where ICS assays using cytomegalovirus (CMV) pp65 peptide mix or control antigens were performed in parallel in 96-well plates. For one experiment, antigens and antibody cocktails were lyophilised into 96-well plates to simplify and standardize the assay setup. Results ((CD4+)cytokine+ cells and (CD8+)cytokine+ cells) were determined by each site. Raw data were also sent to a central site for batch analysis with a dynamic gating template. Mean inter-laboratory coefficient of variation (C.V.) ranged from 17-44% depending upon the sample type and analysis method. Cryopreserved peripheral blood mononuclear cells (PBMC) yielded lower inter-lab C.V.'s than whole blood. Centralized analysis (using a dynamic gating template) reduced the inter-lab C.V. by 5-20%, depending upon the experiment. The inter-lab C.V. was lowest (18-24%) for samples with a mean of > 0.5% IFNgamma + T cells, and highest (57-82%) for samples with a mean of < 0.1% IFNgamma + cells.


CONCLUSIONS
ICS assays can be performed by multiple laboratories using a common protocol with good inter-laboratory precision, which improves as the frequency of responding cells increases. Cryopreserved PBMC may yield slightly more consistent results than shipped whole blood. Analysis, particularly gating, is a significant source of variability, and can be reduced by centralized analysis and/or use of a standardized dynamic gating template. Use of pre-aliquoted lyophilized reagents for stimulation and staining can provide further standardization to these assays.
Maecker
Holden T
HT
BD Biosciences, San Jose, USA. holden_maecker@bd.com
Rinfret
Aline
A
D'Souza
Patricia
P
Darden
Janice
J
Roig
Eva
E
Landry
Claire
C
Hayes
Peter
P
Birungi
Josephine
J
Anzala
Omu
O
Garcia
Miguel
M
Harari
Alexandre
A
Frank
Ian
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Baydo
Ruth
R
Baker
Megan
M
Holbrook
Jennifer
J
Ottinger
Janet
J
Lamoreaux
Laurie
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Epling
C Lorrie
CL
Sinclair
Elizabeth
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Suni
Maria A
MA
Punt
Kara
K
Calarota
Sandra
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El-Bahi
Sophia
S
Alter
Gailet
G
Maila
Hazel
H
Kuta
Ellen
E
Cox
Josephine
J
Gray
Clive
C
Altfeld
Marcus
M
Nougarede
Nolwenn
N
Boyer
Jean
J
Tussey
Lynda
L
Tobery
Timothy
T
Bredt
Barry
B
Roederer
Mario
M
Koup
Richard
R
Maino
Vernon C
VC
Weinhold
Kent
K
Pantaleo
Giuseppe
G
Gilmour
Jill
J
Horton
Helen
H
Sekaly
Rafick P
RP
D001793:Blood Preservation; D015925:Cryopreservation; D016207:Cytokines / Q000097:blood*; D003586:Cytomegalovirus Infections / Q000097:blood / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry / Q000379:methods / Q000592:standards*; D005612:Freeze Drying; D006801:Humans; D007202:Indicators and Reagents; D007753:Laboratories; D008214:Lymphocytes / Q000737:chemistry; D010750:Phosphoproteins / Q000097:blood; D015203:Reproducibility of Results; D013048:Specimen Handling; D013601:T-Lymphocytes / Q000737:chemistry*; D014763:Viral Matrix Proteins / Q000097:blood
D016207:Cytokines; D007202:Indicators and Reagents; D010750:Phosphoproteins; D014763:Viral Matrix Proteins; C065418:cytomegalovirus matrix protein 65kDa