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: 3.67). 04/2009; 131(1):41-49. DOI: 10.1016/j.clim.2008.11.003


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.

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Available from: Henry A. Feldman, Jun 13, 2014
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    • "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. "
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    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.
    Full-text · Article · Jun 2014 · Clinical Immunology
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    • "Cell-mediated immunity is important in the development of zoster and its manifestations.1 T-lymphocytes of helper/inducer phenotype (CD4+ or Leu3a+) proliferate in response to VZV,33–36 although proliferation testing is not a sensitive predictor of cellular immune compromise or risk of opportunistic infection.37 Helper/inducer T-lymphocytes are prominent in skin biopsy specimens from active herpes zoster lesions.34 "
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    • "Besides identifying quantitative anomalies in various immune cell populations by flow cytometry, functional assessment of these cell populations is equally important and can be achieved, for the most part, by the same methodology, though other methods can also be used. For example, measurement of lymphocyte proliferation to mitogens, such as Phytohemagglutinin (PHA), Pokeweed mitogen (PWM) and Concanavalin A (Con A), and antigens, such as Candida albicans (CA) and Tetanus toxoid (TT) to ascertain T cell immune competence in PIDs [110] has long been performed by DNA incorporation of radiolabeled thymidine (3H-T) after stimulation of peripheral blood mononuclear cells (PBMCs) with the appropriate agent. Elimination of techniques involving radioactivity is always beneficial to the clinical laboratory, and flow cytometry-based methods, primarily using the intracellular fluorescent dye, CFSE (carboxyfluorescein diacetate succinimidyl ester), are now available for measuring cellular proliferation [111-113]. "
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