Evaluation of Cellular Activity

by Peter Delves, PhD

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    In the nitroblue tetrazolium test (NBT), the pale yellow color of NBT changes to dark blue in neutrophils stimulated with phorbol myristate acetate (PMA) which is used to induce a reactive oxygen burst in these cells, producing reactive oxygen species. So a burst of respiration that normally occurs following phagocytosis of microorganisms. In a normal donor, we can see this dark blue precipitate that occurs over the neutrophils indicating that they have indeed mounted a respiratory burst producing reative oxygen species following this stimulation, artificial stimulation with PMA. In a patient with chronic granulomatous disease, which is an immunodeficiency in which patients are unable to produce reactive oxygen species, there is no reduction of the NBT. And therefore these dark patches do not appear over the neutrophils. The production of reactive oxygen species can also be measured using dihydrorhodamine 123. The T-cell receptor excision circle or TREC assay is used to analyze the output of T-cells from the thymus. TRECs are circular DNA molecules that are produced during T-cell receptor gene recombination. They can be measured using the polymerase chain reaction, PCR. And this assay is used to quantify recent thymic emigrants as a measure of T-cell output from the thymus. So here we have an example of the T-cell receptor α-chain gene locus. And as T-cells develop within the thymus they will recombine their T-cell receptor genes. And in the case of the T-cell receptor α-chain, this recombination will place a Vα gene segment adjacent to a Jα gene segment. And the intervening bit of DNA forms a circle, the T-cell receptor excision circle that we can see here. And the TREC assay measures the generation of these circles to give an indication of T-cell output from the thymus. Lymphocyte responses can be measured via the proliferation assay. In...

    About the Lecture

    The lecture Evaluation of Cellular Activity by Peter Delves, PhD is from the course Immunodiagnostics. It contains the following chapters:

    • Nitroblue Tetrazolium Test (NBT)
    • T-Cell Receptor Excision Circle (TREC) Assay
    • Lymphocyte Proliferation Assay
    • ELISPOT Assay to Measure Cytokine Release
    • Measuring Cytotoxic T-Cell Activity

    Included Quiz Questions

    1. Phytohemagglutinin
    2. ³H-thymidine
    3. ⁵¹Cr
    4. NBT
    5. EBV
    1. Phorbol myristate acetate
    2. Phytohemagglutinin
    3. ³H-thymidine
    4. Dihydrorhodamine 123
    5. IL-4
    1. TREC Assay
    2. NBT
    3. Lymphocyte proliferation assay
    4. ELISPOT assay
    5. Flow cytometry
    1. ELISPOT assay
    2. TREC assay
    3. NBT
    4. Lymphocyte proliferation assay
    5. Cytotoxic T cell activity measurement assay

    Author of lecture Evaluation of Cellular Activity

     Peter Delves, PhD

    Peter Delves, PhD

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