Optimal blood mononuclear cell isolation procedures for gamma interferon enzyme-linked immunospot testing of healthy Swedish and Tanzanian subjects.
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-gamma) 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-gamma 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.
Nilsson
C
C
Dept. of Immunology and Vaccinology, Swedish Institute for Infectious Disease Control and Karolinska Institute, 171 82 Solna, Sweden. charlotta.nilsson@smi.ki.se
Aboud
S
S
Karlén
K
K
Hejdeman
B
B
Urassa
W
W
Biberfeld
G
G
D002469:Cell Separation / Q000379:methods*; D002470:Cell Survival / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods*; D006801:Humans; D007111:Immunity, Cellular / Q000276:immunology; D007371:Interferon-gamma / Q000737:chemistry*; D007963:Leukocytes, Mononuclear / Q000166:cytology* / Q000276:immunology*; D013548:Sweden; D013636:Tanzania
D007371:Interferon-gamma