A whole-blood lymphoproliferation assay for measuring cellular immunity against herpes viruses

Uninfected erythrocytes inhibit Plasmodium falciparum–induced cellular immune responses in whole-blood assays

“…Direct comparisons of PBMC assays and WBAs have shown good correlations for both proliferation and cytokine production, and WBAs have proven an effective simple tool for large scale studies of immune responses to whole mycobacteria and soluble antigen preparations. [10][11][12][13][14][15]31 WBAs have been shown to be suitable for measuring responses to a variety of parasites, 17 bacteria, 9 viruses, 18 all suggesting that antigen uptake, processing, presentation, and subsequent T-cell activation can occur in whole-blood assays. It was thus a considerable surprise to find, in 2 separate field studies, that cellular responses to malaria antigens in WBAs were markedly Figure 7.…”

“…Using whole-blood assays (WBAs) instead of PBMCs is faster, cheaper, and requires smaller blood volumes. Over the last 30 years, the WBA has been successfully used to evaluate cellular responses to mitogens, 4-8 bacterial, 9 mycobacterial, [10][11][12][13][14][15] parasite, 16,17 and viral antigens, 18,19 but in 2 separate field studies we have found that cytokine responses to malaria antigens in WBAs were unexpectedly low. 20,21 We therefore conducted a detailed comparison of cellular immune responses to live (intact) and lysed malaria-infected erythrocytes in WBAs and in peripheral blood mononuclear cell (PBMC) cultures to determine the suitability of WBAs for the study of T-cell responses to malaria antigens.…”

CMV Antigen-Specific CD4<SUP>+</SUP> and CD8<SUP>+</SUP> T Cell IFNγ Expression and Proliferation Responses in Healthy CMV-Seropositive Individuals

“…We now extend these observations to include simultaneous analysis of CMV-specific CD4 + and CD8 + T cell proliferation in response to these same three antigen preparations using the CFSE proliferation flow cytometry (LPFC) assay, which has been shown to give equivalent results to the standard tri-tiated thymidine uptake assays but is considerably easier to perform and allows phenotypic characterization of proliferating cells (17)(18)(19)(20). Using the LPFC assay, our results exceeded the sensitivity previously reported by most groups who have used standard tritiated thymidine uptake assays for measuring CMV-specific T cell proliferation in healthy CMV seropositive individuals (21)(22)(23)(24). Combining the results from the CFC and LPFC assays for responses to three different CMV antigen preparations, we observed that all 22 immunocompetent CMVseropositive volunteers (i.e., individuals with protective immunity against CMV end-organ disease and re-infection with exogenous CMV strains [1,4]) had positive T cell IFNγ and proliferation responses in at least three of the four measurements performed (CD4 + T cell IFNγ expression, CD8 + T cell IFNγ expression, CD4 + T cell proliferation, and CD8 + T cell proliferation).…”

Modified Whole Blood Microtechnique for Measurement of Mitogens and Allogeneic Lymphocyte Stimulated Proliferation of Human Lymphocytes

“…The first whole blood microtechnique response was measured using 200 μl of heparinized blood in neonates by Puri et al [5]. Diluted whole blood of 100 μl in 1.5 ml RPMI-1640 tissue culture media was used to measure lymphocyte proliferative response to herpes viruses [6], PHA, and promastigotes of Leishmania major described by Frankenburg [7]. Bloemena et al [8] were the first to compare the proliferative response of lymphocytes to anti-human lymphocyte serum and anti-CD3 mAbs using 1:10 (v/v) diluted whole blood microtechnique and the conventional density gradient separated lymphocyte method, which showed comparable results.…”