1.4.1 Assays to diagnose drug hypersensitivity reactions and
assess the phenotype of antigen-specific immune cells.
There are various methods by which it is possible to diagnose whether a person is hypersensitive to a particular substance. Patch testing is one of these, and requires an incubation period of 24-48 hours of the allergen in question on a small area of the skin of an individual, using a non-toxic concentration determined by prior non-allergic volunteer testing. A positive reaction is one in which the exposed skin develops a reaction which presents as erythema. This test does however depend on quite a few factors such as T-cell presence in the skin, ability of the drug to penetrate the skin, and the ability of T-cells to present the drug, among others. Although a negative patch test does not rule out hypersensitivity, a positive patch test is seen as a very reliable indication that an individual is hypersensitivity to a particular substance (Pichler and Tilch, 2004).
As well as the patch test, there are two other skin-based tests; prick and intradermal. The prick test is carried out by placing a needle through a solution containing the allergen before pricking the skin. This test is commonly used as it is the safest of the skin tests, but it is associated with low sensitivity. Usually an intradermal test is a result of a failure to determine hypersensitivity following a prick test. The intradermal test has increased sensitivity but is deemed less safe with the potential to induce a false positive reaction (Brockow et al., 2002). If the administered concentration is low or the allergen is unable
47 to efficiently enter the skin, these skin tests can be associated with false-negative results leaving a potentially hypersensitive patient at risk of being exposed to a compound to which they are allergic.
In the event of unsuccessful skin testing, drug provocation tests (DPT) may be performed where an individual is simply given the drug. These tests are always carried out under medical care due to the implications of a reaction being positive. This occurs whether the test is done with the culprit drug or a structurally related moiety. There are obvious dangers and ethical implications for use of the DPT, which may require hospitalisation of an individual for the duration of the test. However, due to its ability not only to produce allergic symptoms but also to produce other manifestations associated with a reaction, the DPT is known as a gold standard test to diagnose hypersensitivity (Aberer et al., 2003).
Re-administration of a drug to a hypersensitive patient is not always harmful and can be used to treat the patient through a process known as desensitisation. Drug desensitisation allows for the use of a compound associated with hypersensitivity by inducing temporary tolerance, which can be particularly beneficial when a drug is essential for the successful treatment of a patient. In practice this requires that the drug is given to a patient over a brief time-frame in small, but increasing doses, until the patient has been exposed to, and tolerated, a therapeutic dose. For drugs which are associated with the onset of hypersensitivity within 24 hours, a rapid dosing regimen to achieve optimal dose within 4-12 hours is often used. It is this continuous exposure of the individual to the drug that maintains the state of tolerance (Cernadas et al., 2010). While the mechanisms of desensitisation are not well defined, protocols for this procedure are well characterised for the use of drugs associated with immediate reactions (Castells et al., 2008). In contrast, the role of desensitisation to drugs
48 associated with delayed-type reactions is less established, but what data has been published suggests that this method is both safe and effective for these drugs too (Whitaker et al., 2011b).
All of the above tests including skin tests and drug desensitisation have one shared major disadvantage, the potential to induce a harmful reaction in humans. To avoid this, in vitro tests are currently being developed and/or optimised which may allow us to better understand the phenotype and function of the immune cells implicated in hypersensitivity reactions. For patients who suffer from type IV T-cell mediated reactions it is possible to isolate T-cells from a peripheral blood sample, or to collect T-cells from the blister fluid or skin samples. This allows a direct T-cell phenotype to be established ex vivo utilising cells obtained from the site of reaction (Brockow et al., 2002; Yawalkar et al., 2000).
1.4.2 In vitro diagnosis of hypersensitivity.
1.4.2.1 Lymphocyte transformation test.
A commonly used in vitro T-cell test is the lymphocyte transformation test (LTT). It has a reported specificity of 85% and a sensitivity of 74%, a 12% increase in comparison to skin tests (Nyfeler and Pichler, 1997; Romano et al., 2004). It involves a blood sample from a patient from which PBMCs are cultured with the culprit drug for five days before measurement of cell proliferation. This test relies on the presence of sensitised memory T-cells within the PBMC population to respond to the drug (Luque et al., 2001). The disadvantages of the LTT include the requirement for radioactive isotopes for the measurement of proliferation, relatively lengthy test duration, and that it only provides a positive or negative reaction result without mechanistic insight into the aetiology of a
49 reaction. However, as this assay is based on the the presence and proliferation of T-cells and PBMCs, the LTT can be used to detect both immediate and delayed-type reactions. In delayed-type reactions the LTT is much more sensitive than skin tests, however the sensitivity for immediate reactions is just 64.5% compared to 77.4% for skin tests (Luque et al., 2001).
1.4.2.2 Enzyme-linked immunosorbent assay.
An alternative in vitro diagnostic test is the enzyme linked immunosorbent assay (ELISPOT). As with the LTT, this can be performed using PBMCs from patient blood, but can also be integrated into assays looking at the activation of specific T-cell populations. The basis for this assay is the culture of PBMCs or T-cells with antigen on a membrane coated with a capture antibody for a particular cytokine of interest. Activated cells may release this cytokine which subsequently binds to the pre-coated antibodies. The removal of cells and addition of a detection antibody conjugated to an enzyme means that on the final step, addition of an enzyme-activated colour substrate, coloured spots appear in each well as a measure of cytokine release. By utilising antibodies with different binding-specificities the assay can be utilised to measure the release of an array of cytotoxic mediators such as FasL, Granzyme B, and perforin, as well as pro- inflammatory Th1 (IFN-γ, TNFα, IL-12) and anti-inflammatory Th2 (IL-4, IL-5, IL-13) cytokines (Bowen et al., 2008; Olleros et al., 2008). Thus a major advantage of this assay is that it does provide some mechanistic insight to the reaction at hand.
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1.4.2.3 Flow cytometry.
Flow cytometry allows the detection of specific cell surface markers by staining the cells with antibody-conjugated fluorescent, each specific for a particular cell surface marker. This can be used to correctly ascertain cell phenotype, such as CD45Ra and CD45Ro for naïve and memory T-cells, respectively, or to look at T-cell activation, which is associated with increased expression of markers such as HLA-DR, CD40L, CD25, CD69, and CD71 (Beeler and Pichler, 2006). It has been shown that resting T-cells as well as those cells exposed to drug from non-sensitised individuals do not up regulate these activation markers and so flow cytometry can be a useful tool to look at the exact phenotype of drug-specific T-cells. Previous studies have also utilised flow cytometry to assess the ability of a compound to activate professional APCs, with both CD40 and MHC class II molecule expression found to be increased subsequent to exposure to a well-known sensitising agent (Coulter et al., 2007a).
5, 6-Carboxyfluorescein diacetate succinimidyl ester (CFSE) staining can be a useful accompaniment to flow cytometry. CFSE is able to measure cellular proliferation, and can directly measure the number of rounds of cell division a cell has undergone as the cell CFSE content halves during each division (Beeler and Pichler, 2006). CFSE fluorescence has minimal variability within a stained population making it particularly easy to gate for proliferating cell populations (Quah and Parish, 2010). This can be particularly useful in combination with antibodies for other specific markers as the user can identify differences in marker expression between dividing cell populations. While the depletion of CFSE-intensity with each cell division forms the basis of this assay, this represents a major limitation as this leaves a low precursor frequency and thus limits detection to only the strongest responding cells (Lerch et al., 2007).
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1.4.2.4 T-cell cloning.
For defining the characteristics of a specific response it is useful to look at the single cell level. T-cell cloning requires a two week incubation with drug before the placement of a single cell per well in approximately twenty 96-well plates. Expansion of these T-cell clones allows the phenotype and function of a single cell that is responsive to a specific antigen to be analysed. The presence of a single TCR is useful for determining the cross reactivity of related antigens and therefore helps to define the important characteristics of the antigen (Beeler and Pichler, 2006). Clones are able to be restimulated every two weeks and can be kept alive for long periods of time but they are, however, susceptible to infection and exhaustion. Proliferation, cytokine release, and cell phenotype data can then be compiled for each clone using the assays listed previously. Indeed, it was data from clones deriving from patients with different forms of cutaneous hypersensitivity that helped to establish the expanded Gell and Coombs classification.
The successful implementation of any of these in vitro assays requires the identification of the susceptibility factors that make certain individuals develop hypersensitivity reactions while others, often the vast majority, remain tolerant. Two such factors which have been described in detail in relation to these reactions in recent years are the role of the HLA complex and drug metabolism.