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.
BD Biosciences, San Jose, USA. holden_maecker@bd.com
Holden T
HT
Maecker
Aline
A
Rinfret
Patricia
P
D'Souza
Janice
J
Darden
Eva
E
Roig
Claire
C
Landry
Peter
P
Hayes
Josephine
J
Birungi
Omu
O
Anzala
Miguel
M
Garcia
Alexandre
A
Harari
Ian
I
Frank
Ruth
R
Baydo
Megan
M
Baker
Jennifer
J
Holbrook
Janet
J
Ottinger
Laurie
L
Lamoreaux
C Lorrie
CL
Epling
Elizabeth
E
Sinclair
Maria A
MA
Suni
Kara
K
Punt
Sandra
S
Calarota
Sophia
S
El-Bahi
Gailet
G
Alter
Hazel
H
Maila
Ellen
E
Kuta
Josephine
J
Cox
Clive
C
Gray
Marcus
M
Altfeld
Nolwenn
N
Nougarede
Jean
J
Boyer
Lynda
L
Tussey
Timothy
T
Tobery
Barry
B
Bredt
Mario
M
Roederer
Richard
R
Koup
Vernon C
VC
Maino
Kent
K
Weinhold
Giuseppe
G
Pantaleo
Jill
J
Gilmour
Helen
H
Horton
Rafick P
RP
Sekaly
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