Michaela Lucas

Clinical Immunologist/Immunopathologist

Pathwest, QE2 Medical Centre, Princess Margaret Hospital

School of Medicine and Pharmacology, School of Pathology and Laboratory Medicine University

of Western Australia; IIID, Murdoch University

Allergy in Australia-The Present

1.13

In Australia:• Children < 1 yr: 10%• Children < 5 yrs: 4-8%• Adults: up to 2%

Anaphylaxis

• Difficult/noisy breathing

• Swelling of tongue

• Swelling/tightness in throat

• Difficulty talking and/or hoarse voice

• Wheeze or persistent cough

• Persistent dizziness or collapse

• Pale and floppy (young children)

Mild or moderate signs and symptoms may not always precede anaphylaxis

Anaphylaxis is the most severe form of allergic reaction (immediate IgE mediated

reaction)

Symptoms and signs of anaphylaxis:

Time trends Anaphylaxis in Australian Hospitals

Cause of anaphylaxis 1998-2012 - admission by age groupMullins R et al., JACI 2015

Diagnosis - identification of allergic triggers

• Suspected allergy should always be confirmed

• Clinical history

• Tests to identify IgE sensitisation to an allergen– Skin Prick Testing (SPT)– Serum allergen specific IgE (a blood test formerly known

as RAST; IgE/IgG4)

• Medically supervised food allergen challenge

Positive allergy tests do not always result in clinical allergy (sensitisation versus reaction)

Skin prick testing

• Molecular and component-resolved diagnostics tests for serum screening are

increasingly available

• AraH2 (peanut component) sensitivity of 100% and specificity of 70-80%

• Omega-5-gliadin (wheat) diagnostic relevance for exercise induced anaphylaxis

• rGLYm4 for allergy to soy in birch allergic individuals

• may be helpful for fruit, vegetable, nut, soy and seafood allergies

• Basophil activation assays (BAT) are promising, but currently mainly a

research tool

Promising new diagnostics

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Investigation of IgE mediated reactions

Basophil activation assays

Immediate allergic reactions

– Result of IgE production by antigen-specific B cells after sensitisation

– Following re-exposure, the Ag (haptenated) crosslinks IgE bound to mast

cells and basophils, leading to the release of preformed mediators

– Natural history: IgE antibody responses wane over time, however

sensitisation may persist for years

Introduction-Basophil biology

Basophil activation Mediator release

Granules

(e.g. histamine) Lipids (e.g.

Leukotriene C4)

Cytokines (e.g.IL4)

Stimulus

Basophil activationNewly or increased cell-surface marker expression

CD11b

CD13

CD63

CD69

ST2 (IL33R)

CD203c

CD164

CD107a/c

Stimulus

Novel marker expression

Piecemeal and anaphylactic degranulation

Piecemeal Degranulation

Histamine release

No CD63 upregulation

CD203c? upregulation

Anaphylactic degranulation

Histamine release

CD63 upregulation

Basophil

CD63

CD63

Principle of Basophil activation testing

• Flow based assay

• Measures activation of basophils upon allergen stimulation

• Uses two activation markers CD63 and CD203c

• CD63 is also expressed on platelets, eosinophils and monocytes; assays

therefore use additional basophil markers (CD123, CCR3, CRTH2, IgE and

CD203c)

• CD203c exclusively expressed on basophils, including resting at low levels

Commercially available assays

• Uses whole blood (100 microliter) or separated PBMCs

• Uses the expression of CCR3 to identify basophils and expression

of CD63 to identify activation

• “enhanced assay” adds third marker CD203c

• Some oddities:

– Adds anti-CD63 antibody at the same time as cells are

stimulated

Basophil activation assay

• CD63 and CD203c expression differs in response to IL3 priming

• In commercial kits, IL3 is often used to increase sensitivity but it

blunts CD203c response

• Needs to be processed within 4 hours (then basophil reactivity starts

to decline)

• Anti-FceRI antibodies and fMLP (Formyl-Met-Leu-Phe) are used as

positive controls, stimulation buffer alone is the negative control

Principle of Basophil activation testing

Histaflow

• Measures Histamine release

• an enzyme-affinity-gold method based on the high affinity of diamine

oxidase (DAO) for its substrate histamine

• Effectively couples DAO to a fluorochrome which then binds histamine

• Histamine release can be measured by flow-cytometry in combination with

phenotypic analysis

D.Ebo et al.,Journal of Immunological Methods 375 (2012)

Basophil testing - food allergy

Common food triggers

Whilst 90% of food allergic reactions are caused by the foods below, any food may cause an allergic reaction

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Basophil activation distinguishes peanut allergic from tolerant patients

• BAT correctly diagnosed 89% of patients

– 96.7% if non-responders to positive control were excluded

• 2-step strategy (either SPT or Ara h2 sIgE then BAT if required) correctly diagnosed 95% of

patients (Santos AF et al JACI 2014;134:645-52)

Peanut allergy severity associated with basophil reactivity

Santos AF JACI 2015;135:179-86

Basophil activation is specific for egg and peanut allergic patients

Impact of study population – egg white sIgE >0.35kUL: sensitivity 93.5%, specificity 92.5%(Ocmant et al. Clin Exp Allergy 2009, Vol. 39, 8)

Egg allergic subjects

(Ovalbumin)

Peanut allergic subjects

BAT in food allergy

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•

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• Distinguish degree of cow’s milk reactivity- Ford LS et al JACI 2013 131:180-6

• Diagnose subtypes of wheat dependent exercise induced anaphylaxis- Chinuki et al JACI 2012

129:1404-6

Summary-BAT in food allergy

• Performs well for selected food allergens in experienced hands

• Low up front cost, existing expertise

• Limited clinical utility if ≅ sIgE

• Fits a niche

– Broadly sensitised patients

– Population cohorts in place of OFC

– Serial monitoring in immunotherapy

• Note: has also been evaluated for venom allergy

Basophil testing - drug allergy

Classification of drug HR

Drug hypersensitivity reactions (DHRs) are heterogeneous!

Clinically, DHRs can be classified as:

1. Immediate DHRs (BAT is helpful here)

– urticaria, angioedema, rhinitis, conjunctivitis, bronchospasm,

gastrointestinal symptoms (nausea, vomiting, diarrhea, abdominal pain),

anaphylaxis, anaphylactic shock;

– they typically occur within 1–6 h after the last drug administration.

Classification of drug HR

2. Non-immediate DHRs (BAT will be negative)

– varying clinical picture (often involve the skin)

– Spectrum of disease with varying pathogenesis (Type II to IV cell

reactions)

– they may occur at any time as from 1 h after from the initial drug

administration (this often depends on exposure history)

Considerations: Drugs as Immunogens

• Biologics: foreign macromolecules (e.g. antibodies, recombinant

proteins) that act directly as immunogen

• Drugs (non-Biologics)

– Hapten/protein/drug complex (e.g. beta-lactam antibiotics,

quinidine)

– Pro-hapten-processed drug combines with a host

macromolecule (e.g. sulfonamides, phenytoin)

• Pharmacological interaction with an immune receptor (p-i TCR or p-i

HLA; e.g. Abacavir)

Drug preparation

• Requires cytotoxicity assessment and dose dependent testing to

determine optimal concentrations

• Several commercial reagents are available, but expensive

Allergy to NSAIDs and Aspirin

• Results for BAT testing inconclusive

• May be only useful for severe reactions

• Poor sensitivity even when CD203c is included into the assay

• Reactivity is dose-dependent and likely influenced by the fact that

Prostaglandin E2, a natural inhibitor of basophils, is inhibited by

COX-1 inhibitors

• HSA conjugated drugs have been used and shown to work in some

case reports but not others, e.g. severe diclofenac HSR

Allergy to Radiocontrast Media

• Heterogenous group (IgE and non-IgE mediated)

• Four studies

• Sensitivity 46.2% to 61.5%; specificity 88.4% to 100%

• Correlation with skin testing less strong (complementary

role in testing?)

Allergy to Fluoroquinolone Antibiotics

• Ciprofloxacin, moxifloxacin, norfloxacin etc.

• Skin tests are limited due to their skin-irritation properties (88% false

+ rate in intradermal testing)

• Seven studies

• Sensitivity ranges between 0-100% depending on study

• Role of CD203c versus CD63

• Specificity in all studies high (over 88%), therefore excellent

negative predictive value for drug provocation testing

Allergy to Beta-lactam antibiotics

• 9 studies

• Sensitivities ranged from 28.6 to 55% (closer to 50% in larger

studies)

• Specificity over 90%

• Can be positive even when the skin testing is negative

• May be adapted to a wider range of beta lactam antibiotics (e.g.

cephalosporins)

Allergy to Neuromuscular blocking agents

• Seven clinical trials

• Sensitivity ranges from 36.1 to 91.7%

• Heterogenous studies (inclusion criteria, drugs tested)

• In those with clearly proven NMBA anaphylaxis, sensitivity was

36.1% which increased to 85.7% when tested within 3 years

• High correlation to skin testing, but more sensitive and specific

Allergy to Anti-neoplastic agents and others

• Multiple case reports

• Biologic agents

• Anti-neoplastic agents

• Other drugs: corticosteroids, anti-histamines, gelofusine,

heparin, chlorhexidine, pholcodine etc

Conclusions BAT – drug allergy

• Advantages

– In-vitro testing, safe

– Functional test that resembles the in-vivo pathway

– Relatively good sensitivity with high specificity

– Allows testing of drugs for which no skin testing is available

• Disadvantages

– Performance depends on type of allergen, drugs may need to be

haptenated

Questions?