1 Clinical Pharmacology & Therapeutics 2011 Vol: 90(3):455-460. DOI: 10.1038/clpt.2011.155

In Vitro Testing for Hypersensitivity-Mediated Adverse Drug Reactions: Challenges and Future Directions

Clinical Pharmacology & Therapeutics, the most cited journal publishing primary investigation in pharmacology and pharmacy, is the authoritative, cross-disciplinary journal in experimental and clinical medicine devoted to publishing advances in the nature, action, efficacy and evaluation of therapeutics.

Mentions
Figures
Figure 1: Pathogenesis and in vitro tests used for the diagnosis and prediction of immune-mediated drug hypersensitivity reactions (DHRs) (see text for details). A, activation; APC, antigen presenting cells; BAT, basophil activation test; D, detoxication; HSP, heat shock protein; iPTA, in vitro platelet toxicity assay; LTA, lymphocyte toxicity assay; LTT, lymphocyte transformation test; NO, nitric oxide; ROS, reactive oxygen species; TCR, T cell receptor.
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References
  1. Davies, E.C., Green, C.F., Taylor, S., Williamson, P.R., Mottram, D.R. & Pirmohamed, M. Adverse drug reactions in hospital in-patients: a prospective analysis of 3695 patient-episodes. PLoS ONE 4, e4439 , (2009) .
    • . . . Adverse drug reactions (ADRs) account for 6.5% of all hospital admissions and occur in 10–20% of hospitalized patients.1,2 Most ADRs (85–90%) are predictable, dose-dependent, and related to the pharmacology of the drug (augmented or type A reactions), but 10–15% are unpredictable, unrelated to the known pharmacology of the drug, and do not have a clear dose dependency (bizarre or type B reactions).3 DHRs and drug hypersensitivity syndrome (DHS) together constitute a major subtype of the type B reaction . . .
  2. Pirmohamed, M. et al. Adverse drug reactions as cause of admission to hospital: prospective analysis of 18 820 patients. BMJ 329, 15-19 , (2004) .
    • . . . Adverse drug reactions (ADRs) account for 6.5% of all hospital admissions and occur in 10–20% of hospitalized patients.1,2 Most ADRs (85–90%) are predictable, dose-dependent, and related to the pharmacology of the drug (augmented or type A reactions), but 10–15% are unpredictable, unrelated to the known pharmacology of the drug, and do not have a clear dose dependency (bizarre or type B reactions).3 DHRs and drug hypersensitivity syndrome (DHS) together constitute a major subtype of the type B reaction . . .
  3. Rawlins, M., Thompson, J. Mechanisms of adverse drug reactions. In: Textbook of Adverse Drug Reactions. (ed. Davies, D.) 18-45 (Oxford University Press, Oxford, UK, 1991) , .
    • . . . Adverse drug reactions (ADRs) account for 6.5% of all hospital admissions and occur in 10–20% of hospitalized patients.1,2 Most ADRs (85–90%) are predictable, dose-dependent, and related to the pharmacology of the drug (augmented or type A reactions), but 10–15% are unpredictable, unrelated to the known pharmacology of the drug, and do not have a clear dose dependency (bizarre or type B reactions).3 DHRs and drug hypersensitivity syndrome (DHS) together constitute a major subtype of the type B reaction . . .
  4. Pichler, W. Drug hypersensitivity reactions: Classification and relationship to T-cell activation. In: Drug Hypersensitivity. (ed. Pichler, W.) 168-189 (Karger, Basel, Switzerland, 2007) , .
  5. Coombs, R. & Gell, P. Classification of allergic reactions responsible for clinical hypersensitivity and disease. In: Clinical Aspects of Immunology. (eds. Gell, P., Coombs, R. & Lachmann, P.) 761-781 (Blackwell, London, 1975) , .
    • . . . However, a general classification of “allergic reactions” has been adopted to describe DHRs (Table 1).5 The Gell and Coombs classification for immune-mediated reactions does not provide a mechanistically comprehensive classification system for DHRs, but it is a clinically relevant system that classifies DHRs into immediate, accelerated, and delayed reactions as related to the temporal relationship between introduction of the causative agent and appearance of the symptoms (Table 1).6 . . .
  6. Levine, B.B. & Ovary, Z. Studies on the mechanism of the formation of the penicillin antigen. III. The N-(D-alpha-benzylpenicilloyl) group as an antigenic determinant responsible for hypersensitivity to penicillin G. J. Exp. Med. 114, 875-904 , (1961) .
  7. Elzagallaai, A.A., Rieder, M.J. & Koren, G. The in vitro platelet toxicity assay (iPTA): a novel approach for assessment of drug hypersensitivity syndrome. J. Clin. Pharmacol. 51, 428-435 , (2011) .
    • . . . The second category includes the lymphocyte toxicity assay (LTA) and the recently described in vitro platelet toxicity assay (iPTA).7 This article focuses on the current status of the in vitro diagnosis of immune-mediated DHRs; we describe these tests and their strengths and weaknesses as well as possible future directions for this quickly evolving field. . . .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
    • . . . We have found that platelets from hypersensitive patients respond to in vitro chemical insult in a fashion similar to that of PBMCs; however, the degree of cell death is greater and easier to detect.7 We speculate that platelets have lower capacity for detoxication of reactive metabolites. . . .
  8. Elzagallaai, A.A., Knowles, S.R., Rieder, M.J., Bend, J.R., Shear, N.H. & Koren, G. Patch testing for the diagnosis of anticonvulsant hypersensitivity syndrome: a systematic review. Drug Saf. 32, 391-408 , (2009) .
    • . . . Factors that contribute to the difficulty of diagnosing DHS include variable clinical pictures, overlap with symptoms of other clinical conditions (e.g., infection or malignancy), and the typical delayed temporal relationship between administration of the causative drug and the appearance of symptoms.8 An active debate on whether this constellation of symptoms represents different degrees of severity of the same disease or distinct pathological entities is ongoing.9,10 Systemic rechallenge is not ethically acceptable because of the possibility of severe adverse effects to the patient . . .
  9. Elzagallaai, A.A., Garcia-Bournissen, F., Finkelstein, Y., Bend, J.R., Rieder, M.J. & Koren, G. Severe bullous hypersensitivity reactions after exposure to carbamazepine in a Han-Chinese child with a positive HLA-B*1502 and negative in vitro toxicity assays: evidence for different pathophysiological mechanisms. J. Popul. Ther. Clin. Pharmacol. 18, e1-e9 , (2011) .
    • . . . Factors that contribute to the difficulty of diagnosing DHS include variable clinical pictures, overlap with symptoms of other clinical conditions (e.g., infection or malignancy), and the typical delayed temporal relationship between administration of the causative drug and the appearance of symptoms.8 An active debate on whether this constellation of symptoms represents different degrees of severity of the same disease or distinct pathological entities is ongoing.9,10 Systemic rechallenge is not ethically acceptable because of the possibility of severe adverse effects to the patient . . .
    • . . . Although some exceptions exist, the metabolism of a drug into its active metabolites usually represents the first step in the cascade of events leading to development of DHR.19 The “reactive metabolite hypothesis” posits that DHR develops as a result of imbalance between metabolic activation (or toxication) and detoxication of drugs in the biological system, leading to the accumulation of one or more toxic reactive metabolites.20,21 DHS is always associated with either drugs that are known to be electrophilic or those that are readily bioactivated to electrophilic metabolites capable of covalently modifying endogenous macromolecules, including proteins and DNA.22 It is important to understand that “reactive metabolites” may not be the principal direct activators of the immune system; parent, nonreactive drugs can activate isolated T-cells in vitro even in the absence of bioactivation.23 However, chemically reactive electrophilic metabolites seem to be the major, if perhaps not the only, products capable of supporting two important pathways in the immune system activation process: generation of haptenated endogenous proteins (that act as antigens, signal 1) and generation of danger signals from stressed and dying cells (signal 2, Figure 1).9 Signal 2 can also be generated by factors such as trauma, bacterial and viral infections, coadministered drugs, and environmental pollutants . . .
  10. Peyrière, H. et al.; Network of the French Pharmacovigilance Centers. Variability in the clinical pattern of cutaneous side-effects of drugs with systemic symptoms: does a DRESS syndrome really exist? Br. J. Dermatol. 155, 422-428 , (2006) .
    • . . . Factors that contribute to the difficulty of diagnosing DHS include variable clinical pictures, overlap with symptoms of other clinical conditions (e.g., infection or malignancy), and the typical delayed temporal relationship between administration of the causative drug and the appearance of symptoms.8 An active debate on whether this constellation of symptoms represents different degrees of severity of the same disease or distinct pathological entities is ongoing.9,10 Systemic rechallenge is not ethically acceptable because of the possibility of severe adverse effects to the patient . . .
  11. Edwards, R.G., Spackman, D.A. & Dewdney, J.M. Development and use of three new radioallergosorbent tests in the diagnosis of penicillin allergy. Int. Arch. Allergy Appl. Immunol. 68, 352-357 , (1982) .
    • . . . Radioallergosorbent testing, cellular fluorescent assay–IgE, and enzyme-linked immunosorbent assay are commonly used technologies that are known to have high positive predictive value and low negative predictive value.11 Consequently, positive results strongly indicate immune mediation of the reaction, but negative results do not exclude this possibility . . .
  12. Fontaine, C. et al. Relevance of the determination of serum-specific IgE antibodies in the diagnosis of immediate beta-lactam allergy. Allergy 62, 47-52 , (2007) .
    • . . . As an example, one commercially available test (CAP-FEIA, Phadia) was found to have sensitivity between 0 and 25% and specificity ranging from 83.3 to 100% in diagnosing immediate reactions to β-lactam antibiotics, and the variation was reported to be dependent on the clinical manifestations.12 Measurement of drug-specific IgE antibodies is widely used for the diagnosis of immediate reactions to β-lactam antibiotics, muscle relaxants, and some NSAIDs. . . .
  13. Abuaf, N. et al. Validation of a flow cytometric assay detecting in vitro basophil activation for the diagnosis of muscle relaxant allergy. J. Allergy Clin. Immunol. 104, 411-418 , (1999) .
    • . . . This latter test has been useful as an allergenic diagnostic approach and has been validated clinically for type I reactions to muscle relaxants,13 β-lactam antibiotics,14 pyrazolones, and certain NSAIDs.15,16 The disadvantages of the basophil activation test include its relatively low sensitivity and its availability for only a limited number of drugs (Table 2).17 . . .
  14. Torres, M.J. et al. The diagnostic interpretation of basophil activation test in immediate allergic reactions to betalactams. Clin. Exp. Allergy 34, 1768-1775 , (2004) .
    • . . . This latter test has been useful as an allergenic diagnostic approach and has been validated clinically for type I reactions to muscle relaxants,13 β-lactam antibiotics,14 pyrazolones, and certain NSAIDs.15,16 The disadvantages of the basophil activation test include its relatively low sensitivity and its availability for only a limited number of drugs (Table 2).17 . . .
  15. Sanz, M.L., Gamboa, P. & de Weck, A.L. A new combined test with flowcytometric basophil activation and determination of sulfidoleukotrienes is useful for in vitro diagnosis of hypersensitivity to aspirin and other nonsteroidal anti-inflammatory drugs. Int. Arch. Allergy Immunol. 136, 58-72 , (2005) .
    • . . . This latter test has been useful as an allergenic diagnostic approach and has been validated clinically for type I reactions to muscle relaxants,13 β-lactam antibiotics,14 pyrazolones, and certain NSAIDs.15,16 The disadvantages of the basophil activation test include its relatively low sensitivity and its availability for only a limited number of drugs (Table 2).17 . . .
  16. De Weck, A.L. et al. Nonsteroidal anti-inflammatory drug hypersensitivity syndrome. A multicenter study. I. Clinical findings and in vitro diagnosis. J. Investig. Allergol. Clin. Immunol. 19, 355-369 , (2009) .
    • . . . This latter test has been useful as an allergenic diagnostic approach and has been validated clinically for type I reactions to muscle relaxants,13 β-lactam antibiotics,14 pyrazolones, and certain NSAIDs.15,16 The disadvantages of the basophil activation test include its relatively low sensitivity and its availability for only a limited number of drugs (Table 2).17 . . .
  17. Mayorga, C. et al.; Immunology Committee of the Spanish Society of Allergology and Clinical Immunology of the SEAIC. In vitro diagnosis of immediate allergic reactions to drugs: an update. J. Investig. Allergol. Clin. Immunol. 20, 103-109 , (2010) .
  18. Bircher, A.J. Lymphocyte transformation test in the diagnosis of immediate type hypersensitivity reactions to penicillins. Curr. Probl. Dermatol. 22, 31-37 , (1995) .
    • . . . Although this test is used mainly for assessment of delayed T cell–mediated reactions (see below), positive LTT results also occur with type I (IgE-mediated) reactions.18 Although this may appear contradictory, it must be recalled that the production of drug-specific antibodies requires activation of T cells. . . .
  19. Naisbitt, D.J., Williams, D.P., Pirmohamed, M., Kitteringham, N.R. & Park, B.K. Reactive metabolites and their role in drug reactions. Curr. Opin. Allergy Clin. Immunol. 1, 317-325 , (2001) .
    • . . . Although some exceptions exist, the metabolism of a drug into its active metabolites usually represents the first step in the cascade of events leading to development of DHR.19 The “reactive metabolite hypothesis” posits that DHR develops as a result of imbalance between metabolic activation (or toxication) and detoxication of drugs in the biological system, leading to the accumulation of one or more toxic reactive metabolites.20,21 DHS is always associated with either drugs that are known to be electrophilic or those that are readily bioactivated to electrophilic metabolites capable of covalently modifying endogenous macromolecules, including proteins and DNA.22 It is important to understand that “reactive metabolites” may not be the principal direct activators of the immune system; parent, nonreactive drugs can activate isolated T-cells in vitro even in the absence of bioactivation.23 However, chemically reactive electrophilic metabolites seem to be the major, if perhaps not the only, products capable of supporting two important pathways in the immune system activation process: generation of haptenated endogenous proteins (that act as antigens, signal 1) and generation of danger signals from stressed and dying cells (signal 2, Figure 1).9 Signal 2 can also be generated by factors such as trauma, bacterial and viral infections, coadministered drugs, and environmental pollutants . . .
  20. Knowles, S.R., Uetrecht, J. & Shear, N.H. Idiosyncratic drug reactions: the reactive metabolite syndromes. Lancet 356, 1587-1591 , (2000) .
    • . . . Although some exceptions exist, the metabolism of a drug into its active metabolites usually represents the first step in the cascade of events leading to development of DHR.19 The “reactive metabolite hypothesis” posits that DHR develops as a result of imbalance between metabolic activation (or toxication) and detoxication of drugs in the biological system, leading to the accumulation of one or more toxic reactive metabolites.20,21 DHS is always associated with either drugs that are known to be electrophilic or those that are readily bioactivated to electrophilic metabolites capable of covalently modifying endogenous macromolecules, including proteins and DNA.22 It is important to understand that “reactive metabolites” may not be the principal direct activators of the immune system; parent, nonreactive drugs can activate isolated T-cells in vitro even in the absence of bioactivation.23 However, chemically reactive electrophilic metabolites seem to be the major, if perhaps not the only, products capable of supporting two important pathways in the immune system activation process: generation of haptenated endogenous proteins (that act as antigens, signal 1) and generation of danger signals from stressed and dying cells (signal 2, Figure 1).9 Signal 2 can also be generated by factors such as trauma, bacterial and viral infections, coadministered drugs, and environmental pollutants . . .
  21. Shapiro, L.E. & Shear, N.H. Mechanisms of drug reactions: the metabolic track. Semin. Cutan. Med. Surg. 15, 217-227 , (1996) .
    • . . . Although some exceptions exist, the metabolism of a drug into its active metabolites usually represents the first step in the cascade of events leading to development of DHR.19 The “reactive metabolite hypothesis” posits that DHR develops as a result of imbalance between metabolic activation (or toxication) and detoxication of drugs in the biological system, leading to the accumulation of one or more toxic reactive metabolites.20,21 DHS is always associated with either drugs that are known to be electrophilic or those that are readily bioactivated to electrophilic metabolites capable of covalently modifying endogenous macromolecules, including proteins and DNA.22 It is important to understand that “reactive metabolites” may not be the principal direct activators of the immune system; parent, nonreactive drugs can activate isolated T-cells in vitro even in the absence of bioactivation.23 However, chemically reactive electrophilic metabolites seem to be the major, if perhaps not the only, products capable of supporting two important pathways in the immune system activation process: generation of haptenated endogenous proteins (that act as antigens, signal 1) and generation of danger signals from stressed and dying cells (signal 2, Figure 1).9 Signal 2 can also be generated by factors such as trauma, bacterial and viral infections, coadministered drugs, and environmental pollutants . . .
  22. Uetrecht, J. Idiosyncratic drug reactions: current understanding. Annu. Rev. Pharmacol. Toxicol. 47, 513-539 , (2007) .
    • . . . Although some exceptions exist, the metabolism of a drug into its active metabolites usually represents the first step in the cascade of events leading to development of DHR.19 The “reactive metabolite hypothesis” posits that DHR develops as a result of imbalance between metabolic activation (or toxication) and detoxication of drugs in the biological system, leading to the accumulation of one or more toxic reactive metabolites.20,21 DHS is always associated with either drugs that are known to be electrophilic or those that are readily bioactivated to electrophilic metabolites capable of covalently modifying endogenous macromolecules, including proteins and DNA.22 It is important to understand that “reactive metabolites” may not be the principal direct activators of the immune system; parent, nonreactive drugs can activate isolated T-cells in vitro even in the absence of bioactivation.23 However, chemically reactive electrophilic metabolites seem to be the major, if perhaps not the only, products capable of supporting two important pathways in the immune system activation process: generation of haptenated endogenous proteins (that act as antigens, signal 1) and generation of danger signals from stressed and dying cells (signal 2, Figure 1).9 Signal 2 can also be generated by factors such as trauma, bacterial and viral infections, coadministered drugs, and environmental pollutants . . .
  23. Pichler, W.J., Adam, J., Daubner, B., Gentinetta, T., Keller, M. & Yerly, D. Drug hypersensitivity reactions: pathomechanism and clinical symptoms. Med. Clin. North Am. 94, 645-64, xv , (2010) .
    • . . . Although some exceptions exist, the metabolism of a drug into its active metabolites usually represents the first step in the cascade of events leading to development of DHR.19 The “reactive metabolite hypothesis” posits that DHR develops as a result of imbalance between metabolic activation (or toxication) and detoxication of drugs in the biological system, leading to the accumulation of one or more toxic reactive metabolites.20,21 DHS is always associated with either drugs that are known to be electrophilic or those that are readily bioactivated to electrophilic metabolites capable of covalently modifying endogenous macromolecules, including proteins and DNA.22 It is important to understand that “reactive metabolites” may not be the principal direct activators of the immune system; parent, nonreactive drugs can activate isolated T-cells in vitro even in the absence of bioactivation.23 However, chemically reactive electrophilic metabolites seem to be the major, if perhaps not the only, products capable of supporting two important pathways in the immune system activation process: generation of haptenated endogenous proteins (that act as antigens, signal 1) and generation of danger signals from stressed and dying cells (signal 2, Figure 1).9 Signal 2 can also be generated by factors such as trauma, bacterial and viral infections, coadministered drugs, and environmental pollutants . . .
  24. Horton, J.K., Rosenior, J.C., Bend, J.R. & Anderson, M.W. Quantitation of benzo(a)pyrene metabolite: DNA adducts in selected hepatic and pulmonary cell types isolated from [3H]benzo(a)pyrene-treated rabbits. Cancer Res. 45, 3477-3481 , (1985) .
    • . . . The clinical manifestations of DHS are probably primarily mediated by the immune system, although in some cases a direct toxic effect of the reactive species generated from the drug during metabolism may be manifested clinically.24 As an example, the immune response may be responsible for the skin reaction, whereas enhanced formation of cytotoxic metabolites may result in liver or kidney dysfunction . . .
  25. Shear, N.H. & Spielberg, S.P. Anticonvulsant hypersensitivity syndrome. In vitro assessment of risk. J. Clin. Invest. 82, 1826-1832 , (1988) .
    • . . . It has been established for several decades that cells from patients with DHS (peripheral blood mononuclear cells, PBMCs) are more susceptible to in vitro toxicity from the reactive metabolite(s) of the suspected drug than are cells from healthy individuals (controls) who have tolerated the drug.25 These observations prompted the development of the LTA.26 . . .
  26. Spielberg, S.P., Gordon, G.B., Blake, D.A., Goldstein, D.A. & Herlong, H.F. Predisposition to phenytoin hepatotoxicity assessed in vitro. N. Engl. J. Med. 305, 722-727 , (1981) .
  27. Elzagallaai, A.A., Knowles, S.R., Rieder, M.J., Bend, J.R., Shear, N.H. & Koren, G. In vitro testing for the diagnosis of anticonvulsant hypersensitivity syndrome: a systematic review. Mol. Diagn. Ther. 13, 313-330 , (2009) .
  28. Neuman, M.G., Malkiewicz, I.M. & Shear, N.H. A novel lymphocyte toxicity assay to assess drug hypersensitivity syndromes. Clin. Biochem. 33, 517-524 , (2000) .
    • . . . A preselected increase in the percentage of cell death of incubated patient cells (vs. controls) is considered an indication of patient susceptibility.28 The predictive value of this test remains difficult to define because of the lack of a gold-standard test for comparison and because of the technical complexity of the test. . . .
  29. Elzagallaai, A.A. et al. Predictive value of the lymphocyte toxicity assay in the diagnosis of drug hypersensitivity syndrome. Mol. Diagn. Ther. 14, 317-322 , (2010) .
  30. White, J. Platelet structure. In: Platelets (ed. Michelson, A.) 45-74 (Academic Press, Burlington, MA; 2007) , .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
  31. McNicol, A. Platelet preparation and estimation of functional responses. In: Platelets (eds. Watson, S., Authi, K.) 1-26 (Oxford University Press, Oxford, UK, 1996) , .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
  32. Capron, A., Joseph, M., Ameisen, J.C., Capron, M., Pancré, V. & Auriault, C. Platelets as effectors in immune and hypersensitivity reactions. Int. Arch. Allergy Appl. Immunol. 82, 307-312 , (1987) .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
  33. Pitchford, S.C. Defining a role for platelets in allergic inflammation. Biochem. Soc. Trans. 35, 1104-1108 , (2007) .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
  34. Pitchford, S.C. et al. Platelets are essential for leukocyte recruitment in allergic inflammation. J. Allergy Clin. Immunol. 112, 109-118 , (2003) .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
  35. Tamagawa-Mineoka, R., Katoh, N. & Kishimoto, S. Platelets play important roles in the late phase of the immediate hypersensitivity reaction. J. Allergy Clin. Immunol. 123, 581-7, 587.e1 , (2009) .
    • . . . In order to overcome many of the limitations of the LTA test and to simplify the procedure to encourage wider clinical use, recent research in our lab has focused on development and validation of peripheral blood platelets (thrombocytes) as a surrogate cell model for in vitro toxicity testing.7 Thrombocytes are metabolically active nonnucleated cells 2.0–5.0 µm in diameter and 0.5 µm thick, with an abundance of 150–450 × 109 cells/l.30 Given their small size and low density, they can be readily collected from peripheral blood using differential centrifugation.31 In addition to their effect on blood homeostasis, their role in inflammation, allergy, and hypersensitivity reactions has recently been recognized.32,33,34,35 Thrombocytes have active mitochondria and a complete apoptotic system, suggesting that they can serve as a cellular model for studying drug toxicity . . .
  36. Elzagallaai, A., Rieder, M., Koren, G. The in vitro platelet toxicity assay (iPTA): validation of the novel diagnostic test for drug hypersensitivity syndrome. Annual meeting of the American Society for Clinical Pharmacology and Therapeutics; 2-5 March, 2011; Dallas, TX, 2011 , .
    • . . . To validate the novel iPTA we used two approaches: (i) inclusion of rigorously identified DHS cases known to be caused by treatment with sulphonamide drugs (sulfa-DHS); and (ii) the use of the LTA, which we showed to have a positive predictive value of 100% in cases of DHS caused by sulfa drugs in patients who had been clinically re-exposed to the drugs.29 Using a 20% increase in cell death as a cutoff value, there was 85% agreement (11 out of 13) between the LTA and the iPTA results in the 13 sulfa-DHS cases we tested.36 In the two cases for which the two tests did not agree, the LTA was negative and the iPTA was positive and, of importance, these two were clinically confirmed as sulfa-DHS cases . . .
  37. Naisbitt, D.J. et al. Hypersensitivity reactions to carbamazepine: characterization of the specificity, phenotype, and cytokine profile of drug-specific T cell clones. Mol. Pharmacol. 63, 732-741 , (2003) .
    • . . . This technique also involves isolation of PBMCs using differential gradient centrifugation, and it is prone to the same technical complexity as the LTA; this is a limitation that confines its use to well-equipped research laboratories and makes its use in the clinical setting difficult.37 For a detailed description of this test procedure and its history please refer to Elzagallaai et al.27 . . .
  38. Birchler, A. Approach to the patient with a drug hypersensitivity reactions-clinical perspectives. In: Drug Hypersensitivity (ed. Pichler, W.) 352-365 (Karger, Basel, Switzerland, 2007) , .
    • . . . The evaluation and management of immune-mediated DHRs require a great deal of clinical and laboratory experience and expertise.38,39 The advantages and disadvantages of the currently used in vitro tests for DHRs are summarized in Table 2 . . .
  39. Schnyder, B. Approach to the patient with drug allergy. Med. Clin. North Am. 94, 665-79, xv , (2010) .
    • . . . The evaluation and management of immune-mediated DHRs require a great deal of clinical and laboratory experience and expertise.38,39 The advantages and disadvantages of the currently used in vitro tests for DHRs are summarized in Table 2 . . .
  40. Pirmohamed, M. Pharmacogenetics of idiosyncratic adverse drug reactions. Handb. Exp. Pharmacol. 196, 477-491 , (2010) .
    • . . . Genetic analysis has linked a few specific ADRs with particular polymorphisms for certain drugs in specific ethnic groups (e.g., HLA B*-1502 has been linked with carbamazepine-induced severe bullous reactions in Han Chinese, and HLA B*-5701 has been linked with hypersensitivity to abacavir).40 However, these studies have also made it clear that much more work is required in both basic and clinical research to enable us to better predict, prevent, and manage this type of ADR . . .
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