Clinical and Vaccine Immunology, April 2008, p. 585-589, Vol. 15, No. 4
1071-412X/08/$08.00+0 doi:10.1128/CVI.00161-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Optimal Blood Mononuclear Cell Isolation Procedures for Gamma Interferon Enzyme-Linked Immunospot Testing of Healthy Swedish and Tanzanian Subjects
C. Nilsson,1*
S. Aboud,2
K. Karlén,1
B. Hejdeman,3
W. Urassa,2 and
G. Biberfeld1
Department of Immunology and Vaccinology, Swedish Institute for Infectious Disease Control and Karolinska Institute, 171 82 Solna, Sweden,1 Department of Microbiology and Immunology, Muhimbili University of Health and Allied Sciences, Dar es Salaam, Tanzania,2 Venhälsan, Karolinska Hospital and Karolinska Institute, Stockholm, Sweden3
Received 13 April 2007/ Returned for modification 17 August 2007/ Accepted 8 February 2008
Determination of antigen-specific T-cell responses is an important part of vaccine assessment. High levels of recovery, viability, and functionality of peripheral blood mononuclear cells (PBMCs) are essential for reliable assessment of cell-mediated immune responses. Here, we sought to find the cell preparation technique best suited for two clinical vaccine trial sites: Stockholm, Sweden, and Dar es Salaam, Tanzania. Standard Ficoll-Paque gradient centrifugation, BD Vacutainer cell preparation tube (CPT), and Greiner Bio-One LeucoSep tube techniques were tested. Cell yield and viability were recorded. Gamma interferon (IFN-
) enzyme-linked immunospot (ELISPOT) testing was used to assess cell functionality. No differences in mean recovery or mean viability of fresh PBMCs were observed between Ficoll-Paque gradient centrifugation and CPT techniques as used in Stockholm. In Dar es Salaam, recovery of PBMCs isolated by use of the Ficoll-Paque gradient technique was higher than that seen with CPT (1.58 ± 0.6 versus 1.34 ± 0.4 million cells/ml of blood [P = 0.0469]), and the viability of PBMCs processed by Ficoll-Paque gradient was higher than that seen with CPT-purified cells (95.8% ± 2.3% versus 92.6% ± 4.8% [P = 0.0081]). Furthermore, LeucoSep cell separation gave higher levels of yield (1.10 ± 0.3 versus 0.92 ± 0.3 million cells/ml of blood [P = 0.0022]) and viability (95.7% ± 2.0% versus 93.4% ± 3.2% [P = 0.0012]) than Ficoll-Paque cell separation. The cells purified by the different techniques at the two sites performed equally well in IFN- ELISPOT assays. Both techniques generated cell preparations with excellent yield, viability, and functionality in Stockholm. In Dar es Salaam, CPT did not perform as well as Ficoll-Paque separation. In a subsequent comparison, LeucoSep performed better than Ficoll-Paque separation. Our findings emphasize the need for on-site assessment of PBMC purification techniques for optimal evaluation of cell-mediated immune responses.
* Corresponding author. Mailing address: Dept. of Immunology and Vaccinology, Swedish Institute for Infectious Disease Control and Karolinska Institute, 171 82 Solna, Sweden. Phone: 46 8 4572612. Fax: 46 8 337460. E-mail: charlotta.nilsson{at}smi.ki.se
Published ahead of print on 20 February 2008.
Clinical and Vaccine Immunology, April 2008, p. 585-589, Vol. 15, No. 4
1071-412X/08/$08.00+0 doi:10.1128/CVI.00161-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»