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Rational application of flow cytometry in allergy

Rational application of flow cytometry in allergy. Didier EBO Immunology – Allergology – Rheumatology University of Antwerp - Belgium. IgE-mediated allergy: diagnosis. History Specific IgE (CAP) ‏ Skin test Mediator release assays histamine leukotriene Challenge tests.

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Rational application of flow cytometry in allergy

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  1. Rational application of flow cytometry in allergy Didier EBOImmunology – Allergology – RheumatologyUniversity of Antwerp - Belgium

  2. IgE-mediated allergy: diagnosis • History • Specific IgE (CAP)‏ • Skin test • Mediator release assays • histamine • leukotriene • Challenge tests All have draw backs None absolute Additional tests

  3. HLA-Dr / CD123/ CD63

  4. FCM in Barcelona • 4 sessions: • 9/6 Practical course: Cellular in vitro tests (119). • Principles and clinical applications • 10/6 Basic Plenary Symposium 3: Allergy diagnosis and monitoring (211/212)‏ • 10/6 In vitro allergy diagnosis (117). • Drug allergy • 10/6 (19h) EuroBAT Working group meeting (CCIB 124/125) • Drug, venom and food allergy • Over 30 abstracts

  5. Drug allergy • 15% ADR • > no IgE available • Skin tests • Culprit, cross-reactive compounds, alternatives • Serial dilutions (SPT, IDT)‏ • Provocation tests

  6. Stimulus Ref test Sens Spec N Ref -lactam H + ST 50 93 88 Sanz M, CEA 2002 -lactam H + ST + IgE + PT 49 91 110 Torres M, CEA 2004 -lactam 67 100 100 79 41 Abuaf N, CEA 2008 Metamizol H + PT 42 100 55 Gamboa P, Allergy 2003 NSAID H + PT 15-55 74-100 90 Gamboa P, CEA 2004 NMBA H 64 93 50 Abuaf N, JACI 1999 NMBA H + ST 54 100 60 Monneret G, Clin Imm 2002 NMBA H 79 100 24 Sudheer P, Anaesth 2005 NMBA H + ST 36-86 93 92 Kvedariene V, Allergy 2006 Rocuronium H + ST 92* 100 22 Ebo D, Allergy 2006 NSAID: non steroidal anti-inflammatory drugs NMBA: neuromuscular blocking agent * Including non-responders: 76%

  7. Rocuronium • NMBA • 2 NH4+ epitopes • Severe (life-threatening) anaphylaxis • Cross-reactivity • Diagnosis > skin tests • BAT • sIgE

  8. Group N History Skin test Controls* 21 + _ Allergics 24 + + NMBA controls* 18 + (other nmba)‏ _ * Other cause (e.g. NRL, dyes, chlorhexidine, …)‏ Rocuronium

  9. 17/21 (3 non-resp)‏ 81% 71% ROC 4% 21 24 18 rocu - rocu + nmba Adapted from: Ebo DG et al, Allergy 2006

  10. ROC 4% 21 24 18 rocu - rocu + nmba Adapted from: Ebo DG et al, Allergy 2006

  11. Ebo DG et al, Anaesth 2007

  12. * * neg. skin test rocuronium, pos skin test vecuronium

  13. Individual cases • Chlorhexidine • Rifamycin • Pentastarch, succinylated gelatin • Heparin and LMWH • Patent blue • Hyaluronidase • CsA (cremophore: polyethoxylated castor oil)‏ • Recombinant Hep B vaccine • Valcyclovir • Prednisolone

  14. Ebo DG et al, Allergy (in press)‏

  15. Ebo DG et al, Allergy 2005;60:703-704 Ebo DG et al, Allergy 2005

  16. Non-responders • Adults: 5-10% (patients, control individuals)‏ • Children: ? • Irrespective applied marker • CD63, CD203c, pp38 MAPK (preliminary data)‏ • Should be mentioned in publications !!! • Individual: test is lost • Studies: false-negatives

  17. Non-responder: refractory status • 4-6 weeks

  18. Non-responders: drugs • In vitro (supra therapeutical): yes • Desloratadin, promethazin • Atorvastatin, cervistatin • Majlesi Y, J Leuk Biol 2003 • In vitro (therapeutical): no • Desloratadin and glucocorticosteroids • Krauzelbinder et al. EAACI 2008 abstract 54.

  19. Gating the wrong cell See also Abuaf N, CEA 2008

  20. Expression of activation marker • CD63: bi-modal • CD203c, pp38 MAPK: homogeneous

  21. Ebo DG et al, (submitted)‏

  22. Drug allergy: • Diagnostic • Cross-reactivity • Alternatives • Combination of markers Complementary to other tests Absence of other tests Equivocal, false neg. results Less provocation tests

  23. Thanks

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