Analysis of in vitro lymphocyte proliferation as a screening tool for cellular immunodeficiency

Clinical Research Program, Children's Hospital Boston, Boston, MA, USA
Clinical Immunology (Impact Factor: 3.99). 04/2009; DOI: 10.1016/j.clim.2008.11.003

ABSTRACT Measuring lymphocyte response to mitogens and antigens is a mainstay of screening for cellular immunodeficiency. Few reports analyze performance as a screening tool in diverse patient cohorts. We studied proliferation assays performed at Children's Hospital Boston from 1996 to 2003 using mitogens phytohemagglutinin (PHA), concanavalin A (CONA) and pokeweed mitogen, and antigens tetanus (TT) and diphtheria (DT) toxoids, and compared a subset of patients with T cell dysfunction with adult controls using receiver operating characteristic analysis. Results were correlated with clinical data. CONA was superior to PHA in identifying patients with immunodeficiency. TT was second best. Interpretation based on raw CPM, a stimulation index, or reference to simultaneous controls all performed equally. Combining data from multiple mitogens and/or antigens did not enhance performance. Proliferation testing is a useful component of screening for cellular immunodeficiency, but is not a sensitive predictor of cellular immune compromise or risk of opportunistic infection.

Download full-text


Available from: Henry A. Feldman, Jun 13, 2014
1 Follower
  • Source
    • "Reference values from whole blood obtained from more than 100 healthy donors stimulated with a standardized mitogen and antigen panel are shown in Table 1. Since [ 3 H]-thymidine incorporation is considered the " golden standard " for diagnosis of severe immunodeficiency [12] [13], PBMC and whole blood from five healthy donors were stimulated with PHA, PWM and Con A, according to their respective protocols. Fig. 1B shows the correlation between the number of T-lymphocyte blasts and cpm obtained from the mitogen stimulation assay. "
    [Show abstract] [Hide abstract]
    ABSTRACT: The golden standard for functional evaluation of immunodeficiencies is the incorporation of [(3)H]-thymidine in a proliferation assay stimulated with mitogens. Recently developed whole blood proliferation assays have the advantage of parallel lymphocyte lineage analysis and in addition provide a non-radioactive alternative. Here we evaluate the Flow-cytometric Assay for Specific Cell-mediated Immune-response in Activated whole blood (FASCIA) in a comparison with [(3)H]-thymidine incorporation in four patients with severe combined immunodeficiency. The threshold for the minimum number of lymphocytes required for reliable responses in FASCIA is determined together with reference values from 100 healthy donors when stimulated with mitogens as well as antigen specific stimuli. Finally, responses against PWM and SEA+SEB stimuli are conducted with clinically relevant immunomodulatory compounds. We conclude that FASCIA is a rapid, stable and sensitive functional whole blood assay that requires small amounts of whole blood that can be used for reliable assessment of lymphocyte reactivity in patients.
    Clinical Immunology 06/2014; DOI:10.1016/j.clim.2014.05.010 · 3.99 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ultrastructural and mechanical properties of single resting, activated and apoptosis lymphocyte have been investigated by atomic force microscopy (AFM). Using topographic imaging, we showed that the surface of the resting lymphocyte is smooth, while lymphocyte activation and apoptosis are often accompanied by changes in cell morphology. The apoptosis lymphocyte is rougher than those of the two other morphotypes, and coated with many big particles. Using spatially resolved force-distance curves, we found that the valve of the activated lymphocyte is about two to three times stiffer (Young's modulus of approximately 20 kPa) than those of the two other morphotypes (5-11 kPa). These results can improve our understanding of the mechanical properties of cells during growth and differentiation.
    Journal of Biomechanics 06/2009; 42(10):1513-9. DOI:10.1016/j.jbiomech.2009.03.051 · 2.50 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: T lymphocyte proliferations can be measured by [(3)H]thymidine incorporation. However, many labs avoid this technique because of the need to use radioactive substrates. In addition, [(3)H]thymidine incorporation method does not permit simultaneous characterization of the proliferating cells. We developed the 5-ethynyl-2'-deoxyuridine (EdU) and Cu(I)-catalyzed cycloaddition "click" reaction assay to measure T-cell responses by flow cytometry. Spleen cells from normal, immune-deficient purine nucleoside phosphorylase (PNP) defective (PNP-/-) mice or PNP-/- mice with partial immune reconstitution were stimulated with anti-CD3 antibodies. The correlation (r) between [(3)H]thymidine and EdU incorporations into stimulated T cells was measured and the stimulation index (SI), the ratio between stimulated and non-stimulated cells, was calculated. Flow cytometry was used to characterize the proliferating cells. EdU and [(3)H]thymidine incorporation into normal spleen cells were strongly correlated (r=0.89). Following stimulation, EdU incorporation into spleen cells from normal and immune-reconstituted PNP-/- mice was significantly increased compared to PNP-/- immune-deficient mice. Immune-deficient PNP-/- mice had increased [(3)H]thymidine and EdU incorporation into non-stimulated spleen cells, indicative of spontaneous proliferation. Analysis of EdU incorporation showed that the increased proliferation was due primarily to cells expressing CD3, CD4 and IgM. EdU-Click technology accurately measures proliferation of murine T lymphocyte and can be used as an alternative to [(3)H]thymidine assays. The EdU-Click technology also allows identification of proliferating cells.
    Journal of immunological methods 08/2009; 350(1-2):29-35. DOI:10.1016/j.jim.2009.07.008 · 2.01 Impact Factor
Show more