Metal Allergy and the Lymphocyte Transformation Test

Lymphocyte tranformation tests in dentistry

Lymphocyte tranformation tests in dentistry

Is the Lymphocyte Transformation Test (LTT) suitable for the determination of an intolerance, in particular, an intolerance to metals?

First of all – NO! The development of the lymphocyte transformation test (LTT) was based on the observation in the year 1960 that a substance in plants (phytohaemagglutinin) causes the activation of leucocytes. Leucocytes are important cell components of our immune system.

Since then, this test has been used by numerous working groups in labs for the evaluation of cell-mediated immune responses. The principle of the test rests on the fact that lymphocytes, which were sensitized with a certain antigen and subsequently, mutated into so-called memory cells, are stimulated to form in blasts when they come into contact with that antigen once more. Blasts are cells that, in turn, can produce new lymphocytes.

Like the numerous biological processes in a transforming cell, there are various chemical and physical methods to measure the transformation in blasts. For example, one can measure metabolic products, proteins, cell changes, etc. These measurements allow conclusions as to how far the transformation of the memory cell into a blast has progressed. Since there are a great number of different measuring methods by now, the “International Union of Immunological Societies” (IUIS) recommended a LTT that measures the incorporation of tritium-labeled thymidine. This method has since been used for the determination of the lymphocyte function.

Another area of application of the LTT is the verification of a specific sensitization of patients against foreign antigens (infectious agents, allergens) or autoantigens (autoimmune diseases). The test has proven itself for about 40 years, particularly with regard to the diagnosis of diseases due to allergies against medications. In environmental medicine, too, the hope was that the test would provide hints regarding the cause of unresolved diseases, where allergic reactions against “environmental substances” (metals, food products, toxins, etc.) were suspected.

Blasts can produce new lymphocytes

Blasts can produce new lymphocytes

Due to methodical problems, there are an almost immeasurable number of test modifications now, which in turn result in differing interpretations, also with regard to the significance and validity of the method. In the following, we will discuss the relevance of the LTT concerning various issues, as well as its indications within the framework of environmental medicine.

Execution of the LTT

In order to verify a specific sensitization (in the following, referred to as a “specific LTT”), the substance to be tested or its chemically pure metabolites are added to the lymphocyte assays in various dilutions (e.g. 0.1, 1.0, 10, 100, 1000 μg/ml) and, as an internal check, only a medium is added instead of the antigen (“spontaneous proliferation”). These dilution series are necessary in order to find the optimal relationship between the lymphocyte amount and the antigen concentration in the culture showing the highest cell partition rate.

The test is conducted in parallel cultures with lymphocytes of a healthy control person, who has never come into contact with the respective substance. The cells are incubated for six days at 37 °C and labeled with radioactive 3H-thymidine (integrated in their DNA in proportion to the proliferation of the lymphocytes) 18 hours prior to the expiration of the culture. The measured integration rate of the thymidine (counts per minute = cpm) allows for the calculation of the stimulation index (SI): SI = integrated radioactivity in cultures with antigen/integrated radioactivity in cultures without antigen. Pathologic SI-values above 3 may be interpreted with certainty as positive; values between 2 to 3 have a high probability to show a positive LTT.

In order to avoid the use of radioactive material (tritium-labeled thymidine), there are newer approaches to measure the integration of bromodeoxyuridine by means of ELISA, or, to determine the proliferation by means of FACS (fluorescence activated cell sorting). However, there is very little documented experience with these methods and therefore, they have not gained widespread acceptance yet.

Invariably, the specific LTT is only used to provide evidence for the responsiveness and existing sensitization of lymphocytes, but that does not automatically mean an allergy! In other words, this test may not be used automatically – which also applies to other tests for the verification of a sensitization – as an indicator for an allergy (effector response).

Such “positive” findings, which only verify an exposure, are primarily observed in substances that (almost) anybody comes in contact with (e.g. food products, pollen, metals, foreign proteins, etc.). Whether an effector response is induced, i.e. whether symptoms of an allergy appear, depends on other factors.

Furthermore, there are many cases in which “false negative” results are obtained with the LTT, i.e. there is a clear allergic reaction to a certain substance, but it cannot be proven with the LTT!

Significance of the specific LTT for proving a food or pollen allergy

In the lab, a sensitization, e.g. to protein or hazelnuts, can often be shown in test persons who do not experience any symptoms after an exposure to antigens. Likewise, when lymphocytes are incubated with pollen or mites antigens, a positive finding in the LTT cannot always be equated with a clinical symptomatology.

Significance of the specific LTT for proving an allergic reaction to metals

One issue that is also heatedly debated in scientific literature is the specificity of the LTT for proving a reactivity against metals or a resulting illness. It is undisputed that an exposure to metals can trigger autoimmune reactions. However, there is no clear evidence to what extent the use of the LTT makes sense to prove the clinical manifestation of such a reaction, since (as was already explained above) it only shows the sensitization and thus, the exposure – but not the effector response.

Metal allergy is a controversial topic in dentistry

Metal allergy is a controversial topic in dentistry

Therefore, it was possible to clearly demonstrate for nickel (as for food products as well) that an increased cell division of the lymphocytes, vis-à-vis nickel in the LTT, cannot be equated with the appearance of clinical symptoms. There is a high probability that this also applies to many other metals such as, for example, gold, palladium, or amalgam.

In principle, it must be said that there is still a considerable need for research regarding the application of the LTT for the verification of a metal-induced illnesses, before it can be generally recommended as a diagnostic test in this area. All of the tests presently conducted are dubious and will only cost you money and energy!

Significance of the specific LTT for proving an allergic reaction to animal or plant antigens

As a matter of principle, an exposure to foreign proteins (chondroprotective agents, thymus preparations, mistletoe or other plant extracts) leads to immunological reactions in the sense of a sensitization and thus, to a positive LTT result as well, without any “side effect reaction,” i.e. any effector response having to appear. Therefore, for example, a positive reaction to bee or wasp venom in the LTT can only be observed in persons who had already been stung by these insects in the past, but it is not necessarily an indication that such individuals will also have an allergic reaction to these substances.

Significance of the specific LTT for proving a medicinally induced allergy

Today, the main indication area of the “specific” LTT as a diagnostic test lies in the area of medicinal allergies – and only there! The LTT is primarily indicated in cases concerning medications that must be continually prescribed due to certain illnesses (anti-rheumatic drugs, antibiotics, anti-arrhythmic drugs, antiepileptic drugs). In such cases, one should simultaneously test for similar substance groups in order to determine whether the patient has already been sensitized to related (cross-reactive) compounds. Furthermore, the LTT is suited for use in patients that have undergone multiple therapies or in patients, who are receiving combination drugs in order to identify the specific antigen that has lead to the triggering of a side effect reaction.

Therefore, the significance of the LTT for environmental medicine is still not clear. Whether and to what extent the test will be able to make a contribution in this area – to improve the identification of allergic reactions to environmental substances – would still have to be determined through extensive studies. Such studies would have to be conducted for each eligible substance, both in exposed test persons with clinical symptoms, as well as in exposed test persons without clinical symptoms; in test persons with similar symptoms without exposure, as well as in healthy test persons (without exposure, without symptoms). This also applies to all enhancements or modifications of the test (cytokine detections, lymphocyte subpopulations, etc.). Therefore, the need for research regarding these issues must be explicitly emphasized once more.

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