Rational application of flow cytometry in allergy

Didier EBOImmunology – Allergology – RheumatologyUniversity of Antwerp - Belgium

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

HLA-Dr / CD123/ CD63

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

Drug allergy

• 15% ADR• > no IgE available• Skin tests

- Culprit, cross-reactive compounds, alternatives- Serial dilutions (SPT, IDT)

• Provocation tests

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%

Rocuronium

• NMBA• 2 NH4+ epitopes• Severe (life-threatening) anaphylaxis• Cross-reactivity• Diagnosis > skin tests

- BAT- sIgE

Rocuronium

_+ (other nmba)18NMBA controls*

* Other cause (e.g. NRL, dyes, chlorhexidine, …)

++24Allergics

_+21Controls*

Skin testHistoryNGroup

Adapted from: Ebo DG et al, Allergy 2006

21 24 18

rocu - rocu + nmba

ROC 4%

17/21 (3 non-resp)

81% 71%

Adapted from: Ebo DG et al, Allergy 2006

21 24 18

rocu - rocu + nmba

ROC 4%

Ebo DG et al, Anaesth 2007

*

* neg. skin test rocuronium, pos skin test vecuronium

Individual cases

• Chlorhexidine

• Rifamycin

• Pentastarch, succinylated gelatin

• Heparin and LMWH

• Patent blue

• Hyaluronidase

• CsA (cremophore: polyethoxylated castor oil)

• Recombinant Hep B vaccine

• Valcyclovir

• Prednisolone

Ebo DG et al, Allergy (in press)

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

Ebo DG et al, Allergy 2005

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

Non-responder: refractory status

• 4-6 weeks

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.

Gating the wrong cell

See also Abuaf N, CEA 2008

Expression of activation marker

• CD63: bi-modal• CD203c, pp38 MAPK: homogeneous

Ebo DG et al, (submitted)

Drug allergy:

• Diagnostic• Cross-reactivity• Alternatives

• Combination of markers

Complementary to other tests

Absence of other tests

Equivocal, false neg. results

Less provocation tests

Thanks