Rational application of flow cytometry in allergy Didier EBO Immunology – Allergology –...
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Rational application of flow cytometry in allergy
Didier EBOImmunology – Allergology – RheumatologyUniversity of Antwerp - Belgium
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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
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HLA-Dr / CD123/ CD63
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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
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Drug allergy
• 15% ADR• > no IgE available• Skin tests
- Culprit, cross-reactive compounds, alternatives- Serial dilutions (SPT, IDT)
• Provocation tests
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Kvedariene V, Allergy 2006929336-86H + STNMBA
Sudheer P, Anaesth 20052410079HNMBA
Ebo D, Allergy 20062210092*H + STRocuronium
Monneret G, Clin Imm 20026010054H + STNMBA
Abuaf N, JACI 1999509364HNMBA
Gamboa P, CEA 20049074-10015-55H + PTNSAID
Gamboa P, Allergy 20035510042H + PTMetamizol
Abuaf N, CEA 200841100
79
67
100
-lactam
Torres M, CEA 20041109149H + ST + IgE + PT-lactam
Sanz M, CEA 2002889350H + ST-lactam
RefNSpecSensRef testStimulus
NSAID: non steroidal anti-inflammatory drugs
NMBA: neuromuscular blocking agent
* Including non-responders: 76%
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Rocuronium
• NMBA• 2 NH4+ epitopes• Severe (life-threatening) anaphylaxis• Cross-reactivity• Diagnosis > skin tests
- BAT- sIgE
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Rocuronium
_+ (other nmba)18NMBA controls*
* Other cause (e.g. NRL, dyes, chlorhexidine, …)
++24Allergics
_+21Controls*
Skin testHistoryNGroup
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Adapted from: Ebo DG et al, Allergy 2006
21 24 18
rocu - rocu + nmba
ROC 4%
17/21 (3 non-resp)
81% 71%
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Adapted from: Ebo DG et al, Allergy 2006
21 24 18
rocu - rocu + nmba
ROC 4%
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Ebo DG et al, Anaesth 2007
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*
* neg. skin test rocuronium, pos skin test vecuronium
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Individual cases
• Chlorhexidine
• Rifamycin
• Pentastarch, succinylated gelatin
• Heparin and LMWH
• Patent blue
• Hyaluronidase
• CsA (cremophore: polyethoxylated castor oil)
• Recombinant Hep B vaccine
• Valcyclovir
• Prednisolone
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Ebo DG et al, Allergy (in press)
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Ebo DG et al, Allergy 2005;60:703-704
Ebo DG et al, Allergy 2005
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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
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Non-responder: refractory status
• 4-6 weeks
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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.
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Gating the wrong cell
See also Abuaf N, CEA 2008
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Expression of activation marker
• CD63: bi-modal• CD203c, pp38 MAPK: homogeneous
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Ebo DG et al, (submitted)
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Drug allergy:
• Diagnostic• Cross-reactivity• Alternatives
• Combination of markers
Complementary to other tests
Absence of other tests
Equivocal, false neg. results
Less provocation tests
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Thanks