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Lymphocyte stimulation test (LST)

ISSN 2398-2950

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Synonym(s): Lymphocyte blastogenesis, lymphocyte proliferation

Overview

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

Uses

Alone

  • The LST may be used to investigate suspected primary or secondary immunodeficiency.
  • Antigen-specific LST may be used to assess a specific immune response, eg the response to vaccine antigen.
  • The LST assesses the ability of T and/or B lymphocytes to respond in vitroto an activation stimulus.
  • An antigen-specific LST will also measure the function of antigen presenting cells (APC, eg macrophages or dendritic cells).

In combination

  • The LST can be included as part of a panel of diagnostic procedures to investigate immunodeficiency other tests, eg phagocytosis assay, flow cytometry, northern/southern blotting?, may be used to assess the humoral (antibody and complement) or phagocytic arms of the immune system.

Sampling

Source of test material

  • The LST is most commonly performed to assess the function of blood lymphocytes. Therefore, a sample of peripheral blood is required.
  • The laboratory should be contacted prior to sample submission.
  • The test may be adapted for application to suspensions of lymphocytes obtained from lymphoid tissue, eg lymph node, bone marrow or spleen, but this is generally a research procedure.

Quantity of test material

  • The ideal volume of blood is 10 ml and this should be collected into preservative free Heparin, unless the laboratory advises otherwise.

Sample collection technique

  • Control samples will be required, eg at least 2 samples from clinically normal, age- and sex-matched animals should be submitted.

Quality control

Timing of test

  • The LST should be performed as soon as possible after blood sample collection and certainly within 24 hours.

Sample storage

  • Lymphocytes can be isolated from the blood sample and snap frozen in cyroprotectant medium for long-term storage in liquid nitrogen. This is generally only available in research institutions.

Sample transport

  • The blood sample may be sent through the post using standard precautions for shipment of animal blood.

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 phytohaemagglutinin (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 hours but can be as little as 5 hours) 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 for 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, which can cloud the issue).
  • 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 canine disease states including infectious, inflammatory and neoplastic diseases.
  • In some diseases, eg chronic demodicosis, 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 dogs (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

Normal (reference) values

  • The test result is generally given as a stimulation index (SI) as described above.
  • An animal with a SI of greater than 3 would be regarded as having normal functional capacity of lymphocytes.
  • The tests from the control animals established in parallel must be significantly positive to validate the test.
  • Lymphocyte responses to mitogens are generally considered to reduce with aging in the dog, and may be influenced by breed and diet.

Errors and artifacts

  • There has been little experience with antigen-specific LST in the dog and cat.
  • Studies in which vaccine antigens have been used to attempt to elicit a recall (memory) response in such cultures, have given variable results in outbred pet animals (as opposed to those housed in a standard laboratory environment and given controlled exposure to antigen).

Further Reading

Publications

Refereed papers

  • Recent references from VetMedResource and PubMed.
  • Wernette C M et al (2002) CpG oligodeoxynucleotides stimulate canine and feline immune cell proliferationVet Immunol Immunopathol 84, 223-36 PubMed.
  • Lobetti R G (2001) Pneumocystis carinii infection in miniature dachshundsCompend Contin Educ Pract Vet 23, 320-324.
  • Kim H W, Chew B P, Wong T S et al (2000) Modulation of humoral and cell-mediated immune responses by dietary lutein in catsVet Immunol Immunopathol 73,331-341 PubMed.
  • De Luna R, Vuotto M L, Ielpo M T L et al (1999) Early suppression of lymphoproliferative response in dogs with natural infection by Leishmania infantumVet Immunol Immunopathol 70, 95-103 PubMed.
  • Kearns R J, Hayek M G, Turek J J et al (1999) Effect of age, breed and dietary omega-6:omega-3 fatty acid ratio on immune function, eicosanoid production and lipid peroxidation in young and aged dogsVet Immunol Immunopathol 69, 165-183 PubMed.
  • Corato A, Shen C-R, Mazza G, Barker R N & Day M J (1997 )Proliferative responses of peripheral blood mononuclear cells from normal dogs and dogs with autoimmune haemolytic anaemia to red blood cell antigensVet Immunol Immunopathol 59, 191-204 PubMed.
  • Greeley E H, Kealy R D, Ballam J M, Lawler D F & Segre M (1996) The influence of age on the canine immune systemVet Immunol Immunopathol 55, 1-10 PubMed.
  • Kristensen F et al (1982) The lymphocyte stimulation test in veterinary immunologyVet Immunol Immunolpathol 3, 203-277 PubMed.

Other sources of information

  • Day M J (1999) Clinical Immunology of the Dog and Cat. Manson Publishing, UK.