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J Lab Meb 2006;30(2):101-106 © 2006 by Walter de Gruyter - Berlin - New York. DOI 10.1515/JLM.2006.014

Significance of the patch test and the lymphocyte transformation test in the diagnostic of type IV-sensitization.

Statement of the German professional association for enviromental medicine


Frank Bartram1, Hans-Peter Donate2, Kurt E. Müller3, Claus-Hermann Bückendorf4,
Peter Ohnsorge5, Wolfgang Huber6, Volker von Baehr7

1 Praxis für Allgemeinmedizin/Umweltmedizin, Augustinergasse 8, 91781 Weissenburg
2 Praxis für Allgemeinmedizin/Umweltmedizin, Saarbrücker Strasse 9, 66679 Losheim am See
3 Praxis für Dermatologie/ Umweltmedizin, Scherrwiesenweg 16, 88316 Isny
4 Praxis für Allgemeinmedizin/Umweltmedizin, Wolfsbrook 2, 24113 Kiel
5 Praxis für Hals-Nasen-Ohrenheilkunde/Umweltmedizin, Juliuspromenade 54, 97070 Würzburg
6 Praxis für Innere Medizin/Umweltmedizin, Maaßstraße 28, 69123 Heidelberg-Wieblingen
7 Institut für Medizinische Diagnostik, Abteilung Immunologie, Nicolaistrasse 22, 12247 Berlin

Summary

The epicutaneous test is the most commonly used method for the detection of type IV sensitisation to allergens and haptens.
This test is in the hands of experienced investigators mostly very helpful for the evaluation of the role of allergens in contact allergies. However, standardised in-vitro methods are also required especially for the identification of potentially sensitising toxic or carcinogenic substances.
Recent developments in cell culture technology have led to the establishment of modern cellular techniques carried out in specialised laboratories particularly in environmentally-challenged patients as a powerful alternative for the assessment of type IV sensitisation. At the same time as considering the potential of such in-vitro assays one should bear in mind their limitations too.
The lymphocyte transformation test (LTT) represents nowadays an important alternative for the detection of a specific type IV sensitisation. Advantages and disadvantages of both procedures regarding their diagnostic specificity and sensitivity and the arising conclusions for application in routine diagnostics will be discussed. Keywords epicutaneous test, lymphocyte transformation test, type IV sensitisation, allergen, hapten

Epicutaneous test (ECT)

When diagnosing allergic contact eczema (type IV allergy), the epicutaneous test (patch test) is most frequently used. The test principle is based on the fact that allergen-specific T lymphocytes migrate to the patch of skin to which the test allergen has been applied where they induce a macroscopically apparent skin infiltrate after 24 to 72 hours. When assessing positive test reactions, allergic and irritative skin responses must be differentiated. Many contact allergens are also skin irritants, particularly for patients with sensitive skin. When evaluating an epicutaneous test, to avoid false positives and false negatives the examiner must consider the varying penetrative ability and immunological responsivity of different skin types as well as the different sensitivity and reproducibility of the individual test allergens. For these reasons, epicutaneous testing should remain the preserve of examiners who are well trained in allergology and who are familiar with the limits of this established routine test.

The lymphocyte transformation test (LTT)

The LTT is currently the only comprehensively validated in vitro method for detecting specific cellular sensitisations. It is based on the principle of antigen (allergen) induced cell division of specific T lymphocytes and analysis of the induced DNA synthesis. A positive reaction in the LTT verifies the presence of allergen-specific T memory cells in the patient’s blood. The optimised LTT versions in use since 2002 have achieved increased sensitivity and specificity thanks to the addition of recombinant interferon-alpha to the lymphocyte culture (1, 2, 3).
Until a few years ago, the LTT had reduced sensitivity but was still equivalent to the skin prick test. The specificity was limited due to borderline reactions that were not uncommon. The diversity in the methods used and the lack of standardisation at the time explain the very different evaluations of the LTT prior to 2000. Along with studies that attested to the high sensitivity and specificity of the LTT as well as its clinical relevance even then (4, 5, 6), there are also publications reporting on false positive results (7, 8).
Over the last 5 years, the importance of the LTT as well as the body of data on the sensitivity and specificity has changed significantly. Further developments in cell culture techniques, the quality of the antigens used and not least improved measurement methods have all contributed to this. The liquid scintillation devices previously used for tritium counting have been replaced by highly sensitive, automated solid-phase b counters. As a result of these methodological developments, the LTT was accredited in its current standardised form as a test procedure by expert reviewers from the Deutsche Akkreditierungsstelle Chemie GmbH (German accreditation body for chemistry) in spring 2003 according to DIN EN 17025 and since January 2005 according to DIN EN 15189.

‘Non-specific mitogenic effects’ can be excluded in the LTT

In the older technical literature, there are articles (no studies) that postulate that non-specific mitogenic effects occur with the LTT when testing for metals. This can be reliably excluded by the validated, standardised concentrations of the allergens used. These are also not ‘mitogenic effects’ because the cells that proliferate in the LTT are exclusively CD4 T helper cells. Positive LTT reactions without clinical contact allergies are based on balanced sensitisations with an immunological tolerance is maintained by CD25+ regulatory T cells (9). IL-10-secreting CD4 cells are also involved (10). The postulated ‘non-specific activations’ in the LTT must also not be confused with the toxic irritative inflammatory reactions that occur in the skin prick tests because non-specific effects in the LTT can be excluded by using standardised test concentrations and parallel antigen inhibition tests (11).

Sensitivity and reproducibility of the epicutaneous test

Despite great advances in the standardisation of the test allergens, the ‘weak points’ of epicutaneous testing are the subjective test evaluation and the varying nature of the skin of the test persons. The opinion still frequently voiced these days, that epicutaneous testing is in principle superior to the LTT in its predictive ability, must be critically examined.
Several clinical trials show that the sensitivity of epicutaneous testing is too low for a ‘gold standard’. Negative epicutaneous tests with existing clinically verified sensitisation have been repeatedly described (12, 13, 14). Rustemeyer showed for nickel that of 74 patients with clinically verified nickel sensitisation, only 40 had a positive reaction of the skin, which corresponds to a sensitivity of only 54% (10). Bourke showed that epicutaneous testing with various contact allergens, each carried out in duplicate on both sides of the spine on the patient’s back, had an intra-assay reproducibility of about 92% (15). Multicentre studies yielded figures of 84% (16) or only 56% (17). In the study conducted by Gollhausen at the Dermatology Clinic at the Ludwig Maximilians University Munich, patients were examined twice at intervals of one week. In a review published by Iris Ale in 2004, the ratio of non-reproducible reactions from nine studies examined varied from 4.2% to 43.8% (18). It is highly probable that the ‘deviations’ determined in these controlled trials are even higher for routine clinical diagnostics.

Use and scientific reputation of the lymphocyte transformation test (LTT)

The LTT has proven to be equivalent to epicutaneous testing in diagnosing immunologically based medicinal product reactions and superior in its sensitivity (19, 20, 11). This resulted in the positive evaluation by the Robert Koch Institute for this issue (21). However, in the same expert statement the evidence for allergic reactions to environmental pollutants(including metals) was assessed critically ‘because there is no adequate test material available’. That a high sensitivity is ascribed to the identical test for haptens such as derivatives of medicinal products but not for others such as metals is incomprehensible. Precisely for metals such as chrome, nickel and cobalt, more recent studies show that the LTT has a higher sensitivity compared to epicutaneous testing (22, 23).
In the expert opinion from the RKI mentioned, it is also postulated that positive LTT results ‘only indicate exposure’ that does not always have to be linked to clinical symptoms. A sensitisation verified in the LTT (as well as in the skin prick test!) does not actually have to be associated with clinical symptoms but it does not cast doubt on the sensitisation that is present. It is known that not every sensitisation leads to an allergy. That positive LTT results ‘only indicate exposure’ is also not conclusive because in this case the rate of positive reactions to dental metals, nickel or even cadmium (present in cigarette smoke) is undoubtedly far higher than would have to be demonstrated. The prevalence of positive responses in the LTT is far below the high number of people with relevant exposure (4, 22).
To detect a type IV sensitisation to beryllium, the LTT is undisputedly the tool of choice (24, 21). There is no conclusive explanation of why this is the case for beryllium but not other metals. Metals are particularly suitable for LTT testing because, unlike medications, they are not metabolised. At the Institute for Clinical Immunology of the University Hospital Essen, the correlation of the various test methods LTT, epicutaneous testing and cytokine analyses to each other and to the clinical results was recently examined (25). There was a good correlation of the results of the LTT, the epicutaneous test and the cytokine analyses. Compared to the clinical picture, both the LTT and the epicutaneous test correlated (epicutaneous test, r=0.73, p<0.0001; LTT r=0.74, p<0.0001).
For intolerances to perfume (26), iodine-containing contrast agent (27) and methacrylates (28), it has been demonstrated that the LTT is useful for the often problematic differentiation between allergic and irritative reactions.
All recent studies show that the validity of the LTT results determined is more dependent on the quality of the test implementation than on the methodology itself. This is no different to other laboratory methods, however. The undifferentiated scepticism found in older publications regarding the LTT is no longer justified in light of the methodological developments and recent studies.

Sensitisation does not have to be associated in every case with local symptoms at the contact site.

Particularly in dental clinics, it is often assumed that a clinically relevant sensitisation absolutely must be associated with oral symptoms. This is not the case because the hapten presentation of the oral mucous membrane and the epidermis differ due to particular immunological features (29, 13). The reason lies in the different lipid composition of the mucous membranes and epidermis (30), more rapid removal of allergens due to the greater blood flow of the reticular layer (31) and the primary immune response influenced by about 400 bacterial species in the oral cavity (32). The Langerhans cells primarily responsible for the initial local inflammatory reaction have functional differences between the epidermis and the mucous membrane. Langerhans cells in the mucous membrane express greater amounts of Fc epsilon RI receptor (33, 34) and are able to induce an allogeneic T cell stimulation in vitro (35). Comparative provocation tests showed that the allergen concentrations required to trigger a mucous membrane response are 5 to 12 times higher than those required for the skin (36).
To develop an allergic mucous membrane response to a foreign substance, it is also critical that the primary contact, that is, the formative immunological recognition process, takes place via the mucous membrane, the skin or the intestine. Both in the mouse model and in humans, the reactivity of the oral mucous membrane can be suppressed by previous intestinal allergen exposure (37, 38).
The differences cited explain why positive epicutaneous test reactions are not always associated with oral manifestations but that at the same time contact allergic mucous membrane responses are not always apparent in every case in epicutaneous testing (16).

LTT and epicutaneous testing can only demonstrate sensitisation but not an allergy.

Evidence of an immunological sensitisation, such as that produced by the LTT and the skin prick test, is not obligatorily associated with clinical local or system symptoms. An allergy diagnosis can only be made in light of the clinical results and the medical history. Allergy tests support the diagnosis because sensitisation is an essential prerequisite for an allergy. In our opinion, epicutaneous testing and an LTT carried out in a cell culture laboratory specialising in the procedure are two methods that complement one another well in difficult cases.
Our experience over the previous four years shows that an LTT carried out in a standardised manner is important particularly for the following issues:

  1. Negative epicutaneous test result with urgent clinical suspicion of a contact allergy
  2. Dubious positive results in the epicutaneous test (toxic reactions?)
  3. Preventive testing before using dental restoration material or queries from professional organisations
  4. Testing of potentially sensitising or carcinogenic substances

For preventive queries, the LTT should be used.

In principle, for preventive queries regarding existing type IV sensitisations, such as those carried out primarily in dentistry, epicutaneous testing should be used with restraint becausethe application of the test substance to the skin presents a potential risk of sensitisation (39, 40). Agrup showed in one study in which epicutaneous tests were carried out twice for the standard allergens that with repeated testing there was a significant number of new sensitisations after 6 months. The prevalence of iatrogenic sensitisation is 5% for cobalt, 4.6% for p-phenylene diamine, 2.3% for chrome and 9.9% for p-aminoazobenzene, for example (39). Additional case descriptions are available for benzoyl peroxide, butyl hydroquinone, composite mix (41), 4-tert-butylcatechol (42), various plant extracts (43), budesonide (44), formaldehyde (45), nickel (10) and acrylates (46). For penicillin, therapeutic cutaneous application is still considered malpractice due to the risk of sensitisation (41). In animal trials, sensitisation to chrome, mercury and nickel can be induced, for example (47). In light of this, the common practice of routine epicutaneous testing of the complete standard series of allergens must be critically evaluated.
The procedure of confirming a positive LTT result using an epicutaneous test should therefore only be done in exceptional cases, e.g. with borderline results, because amplification of the clinical symptoms has been demonstrated for various contact allergens as a result of exposure to the test contact allergen (48, 49, 50). Anyway, the limited sensitivity and lack of reproducibility (12, 13) restrict the indications for such ‘follow-up tests’.

From the standpoint of curative environmental medicine, to evaluate positive test results, the following take-away messages are recommended:

  1. For preventive examinations with potentially sensitising substances, all tests with carcinogenic materials and the testing of patients with predisposing immunological disorders, the LTT as a non-stressful procedure should be preferred to epicutaneous testing.
  2. A reliably positive result in the epicutaneous test ensures sensitisation. Dubious positive results and results suspected of being due to toxic responses in epicutaneous testing should be confirmed by the LTT even for curative cases.

The quality standards must be high for cellular laboratory procedures!

The use of the LTT in clinical diagnostics places stringent requirements on the treating doctor in terms of the indication and on the laboratory carrying out the test to ensure consistently high quality for the test implementation. The LTT is reserved for specific issues due to the high costs compared to epicutaneous testing. Considering the literature described here, it would be wrong, however, to not implement this modern diagnostic in vitro method.
However, what must be stipulated is that this demanding cell culture method is performed exclusively by accredited medical institutes which have sufficient experience using the method. The mandatory ring and comparative tests with test laboratories prescribed for laboratories accredited according to DIN ISO 15189 are a critical prerequisite for effective quality management and reliable consideration of the test results in clinical practice.

Important note:

Crucial parts of the RKI statement mentioned in the text were corrected in 2008.

The statement from the German Professional Association of Environmental Medicine was nevertheless printed here in the original form from 2006 so as not to violate copyright.

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