Equis ISSN 2398-2977

Lymphocyte stimulation test (LST)

Synonym(s): Lymphocyte blastogenesis, lymphocyte proliferation

Contributor(s): Alastair Foote, Vetstream Ltd

Overview

  • The lymphocyte stimulation test (LST) measures the ability of lymphocytes to respond in vitroto an activation stimulus. 
  • A suboptimal response may indicate primary immunodeficiency or secondary suppression of the immune response.

Sampling

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Tests

Methodologies

  • The LST may be performed in a number of ways.
  • Some tests are performed on whole blood samples, whereas others involve the extraction and use of the mononuclear cell fraction (monocytes, T and B lymphocytes) of the blood sample.
  • This is achieved by differential centrifugation over a density gradient medium.
  • The test is performed in a microplate system using small volumes, eg typically 100-200 ul.
  • One form of LST uses a substance called a mitogen to non-specifically activate the lymphoid cells in the sample.
  • Mitogens are generally derived from plants, and bind to carbohydrate molecules on the surface of lymphocytes to trigger their activation (blastogenesis) and subsequent division (proliferation).
  • Three mitogens are in common use for this purpose:
    • Concanavalin A (ConA) and phytohemagglutinin (PHA) are considered T cell mitogens.
    • Pokeweed mitogen (PWM) is considered to be a B cell mitogen.
    • Other mitogens include lipopolysaccharide (LPS) and staphylococcal enterotoxin (SEB).
  • The lymphocytes are suspended and maintained in tissue culture medium.
  • The culture medium is usually supplemented with serum (usually fetal calf serum or serum obtained from normal animals of the same species as the lymphocytes being assayed).
  • The lymphocyte cell suspension is then aliquoted into multiwell plates and culture media and mitogenic substance are added to relevant culture wells.
  • The conditions for the culture, eg number of lymphocytes/well, type and concentration of mitogen/well, are all predetermined.
  • Cultures without mitogen are included for control purposes.
  • The total volume for each well should remain constant, with only the concentration of lymphocytes and or mitogen changing from well to well.
  • All wells on a plate should be set up in triplicate for statistical analysis.
  • Duplicate cultures using lymphocytes from normal animals should be established for control purposes.
  • Culture periods vary but are usually between 2-5 days in a tissue culture incubator at 37°C and 5% CO2.
  • Within the last few hours (typically 16-18 h but can be as little as 5 h) of the culture period, a set quantity of radiolabeled (tritiated) thymidine is added to each well in the plate, except for a triplicate of wells that act as controls.
  • The radiolabeled thymidine is incorporated into newly synthesized DNA as the cellular division continues in the wells.
  • The culture is then stopped and the contents of each well are mechanically aspirated through a filter mat.
  • Cells are collected onto the filter mat whilst the culture fluid, including unincorporated thymidine, drains through the filter and is collected as waste.
  • The filter mat is sealed into a plastic bag, containing a scintillation cocktail and each mat is passed through a machine that measures the emitted beta radiation.
  • The amount of beta radiation emitted is proportional to the amount of radiolabeled thymidine incorporated into the lymphocytes in culture and thus is proportional to their rate of division.
  • The result is generally recorded as a stimulation index (SI) which takes into account the level of background cell division occurring in control wells not exposed to mitogen.
  • A stimulation index of greater than 3 is generally considered a significant result.
  • Non-radioactive means of assessing lymphocyte division have also been developed, eg it is possible to measure cytokine production, eg IL-2, and release into the culture fluid as an index of lymphocyte proliferation.
  • The second form of LST is one that tests the ability of the lymphocytes to respond to a specific antigen to which the animal has been previously exposed.
  • Vaccine antigens are often used in this way as most animals have been vaccinated.
  • This test also measures the function of antigen presenting cells (APC) in the sample.

The stimulator cells are usually irradiated to prevent them dividing, so that any measured beta emission is indicative of lymphocyte (aka responder cells) proliferation and not a mixture of the two.

  • The culture methodology is similar to that described above; however, a specific antigen is added to the culture wells in place of mitogen.
  • The antigen is first taken up and processed by the APC that then present the antigen on their surface as a peptide fragment derived from the antigen that is associated with a class II molecule of the Major Histocompatibility Complex (MHC).
  • This in turn is recognized by the antigen specific T cell receptor of those lymphocytes in the well that are pre-programmed to respond to that antigen.
  • In similar fashion, these lymphocytes undergo blastogenesis and start to proliferate, and this is measured by incorporation of radiolabeled thymidine as above.

Availability

  • The test will not be widely available on a commercial basis, and will be restricted to research institutions.

There are no laboratories in the UK offering LST on a commercial basis.

Validity

Sensitivity

  • The mitogen-driven LST is a fairly crude index of the ability of lymphocytes to respond in a non-specific fashion to stimulation, however any impairment in this ability relative to controls should be considered as having clinical significance.
  • The assay does not discriminate between a primary functional defect in the cells, or a secondary suppression of their activity (anergy) by other non-specific factors.
  • Depression in mitogen responsiveness may be recognized in a wide range of disease states, including infectious, inflammatory and neoplastic diseases.
  • In some diseases it has been recognized that soluble serum factors are responsible for this suppression, as cultures of lymphocytes from affected animals show normal mitogenic responsiveness when incubated with serum from normal animals (as opposed to autologous serum from the same animal).

Specificity

  • Mitogen-driven LST is completely non-specific.
  • It tests for the ability of all T lymphocytes or all B lymphocytes (regardless of antigen specificity) to respond.
  • The antigen-driven LST, by contrast, is a selective measure of the ability of that subpopulation of lymphocytes pre-programmed to recognize the antigen to respond.

Result Data

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Further Reading

Publications

Refereed papers

  • Recent references fromPubMed.
  • Davis E G, Zhang Y, Tuttle J, Hankins K & Wilkerson M (2008)Investigation of antigen specific lymphocyte responses in healthy horses vaccinated with an inactivated West Nile virus vaccine.  Vet Immunol Immunopathol126(3-4), 293-301PubMed.
  • Witonsky S G et al(2008)Horses experimentally infected with Sarcocystis neurona develop altered immune responses in vitro. J Parasitol94(5), 1047-1054PubMed.
  • Bell S C, Savidge C, Taylor P, Knottenbelt D C & Carter S D (2001)An immunodeficiency in Fell ponies: a preliminary study into cellular responses. Equine Vet J33(7), 687-692PubMed.
  • Kristensen F et al(1982)The lymphocyte stimulation test in veterinary immunology. Vet Immunol Immunolpathol3(1-2), 203-277PubMed.


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