Research paper Lymphocyte proliferation specific for recall, CMV and HIV antigens in miniaturized and automated format Giuseppina Li Pira a, ⁎, Nadia Starc a , Antonella Conforti a , Alice Bertaina a , Sergio Rutella a, b , Franco Locatelli a, c , Fabrizio Manca a a Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children Hospital, Rome, Italy b Catholic University Medical School, Rome, Italy c University of Pavia, Italy article info abstract Article history: Received 8 July 2012 Received in revised form 27 July 2012 Accepted 31 July 2012 Available online 7 August 2012 Lymphoproliferation assay (LPA) is used to test specific T-cell responses. LPA is performed in 96-well plates with 2–5×10 5 PBMC/well. In order to test numerous antigens, as in the case of epitope mapping or screening of antigenic panels from relevant pathogens, PBMC numbers may not be sufficient. We developed a miniaturized and automated procedure to perform LPA in 384- and 1536-well plates with one fourth to one twentieth of PBMC numbers used for standard assays. Here, we demonstrate that the procedure is reliable and robust using recall antigens and protein and peptide antigens from CMV and HIV. By using HIV specific T-cell lines, we also demonstrate that sensitivity ranges overlap with those of standard LPA and that as few as 3 specific cells/well provide a positive signal. This procedure is consistent with our policy to miniaturize assays for specific T-cell immunity, as we have already established for cytokine secretion assays. © 2012 Elsevier B.V. All rights reserved. Keywords: Lymphoproliferation Miniaturization T-cell immunity T-cell lines CMV HIV 1. Introduction Lymphoproliferation assay (LPA) has been used for decades as one of the most reliable assays for the measurement of cellular immune response (James, 2002; Kern et al., 2005; Thiel et al., 2004). High sensitivity and high stimulation indexes are major advantages, in addition to the fact that the actual proliferative capacity (i.e. potential for clonal expansion) of antigen- stimulated T-cells is directly measured. Alternative methods, other than tritiated thymidine incorporation, have been pro- posed in order to avoid the use of a radioactive reagent (Givan et al., 1999; Lyons, 2000; Lyons and Doherty, 2004; Wallace et al., 2008). The number of peripheral blood mononuclear cells (PBMC) needed to run a conventional LPA in 96-well plates can be a limiting factor as frequently experienced in the clinical immunology laboratory. This may be due to the large number of antigens that need to be tested, as in the case of epitope mapping using peptide libraries, or when T-cells are challenged with antigen panels derived from different pathogens to test cellular immune competence. The same difficulty is encountered when the number of available PBMC is limited as with children who can provide small blood volumes, or with lymphopenic, immune compromised subjects, whose PBMC yield is low. For these reasons, we developed a procedure for LPA in small formats such as 384- and 1536-well plates, that require one fourth to one twentieth of PBMC in comparison to conventional 96-well plates. The feasibility of this scale reduction and its comparison with the conventional method are illustrated here. 2. Materials and methods 2.1. Media and reagents Cell cultures were performed in RPMI-1640 (BioWhittaker, Verviers, Belgium) supplemented with 10 mM L-glutamin, Journal of Immunological Methods 384 (2012) 135–142 ⁎ Corresponding author at: Department of Pediatric Hematology and Oncology, IRCCS Bambino Gesù Children Hospital, Piazza S. Onofrio 4, 00165 Rome, Italy. Tel.: +39 06 68593782. E-mail address: lipira@email.it (G. Li Pira). 0022-1759/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.jim.2012.07.022 Contents lists available at SciVerse ScienceDirect Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim