Allergy Testing in Laboratory The Quest for Clinical Relevance 1989 20130 3
1989 A Good Year Current Concepts Lecture Allergy
1989 a good year
WHY ME? Current Concepts Lecturers 1989 Andrew Wootton David Gillis Clinical and Laboratory aspects of allergy
What is Allergy? IgE mediated disease IgE to allergen attaches to mast cells When allergen cross links IgE, histamine and other mediators are released Causing allergic rhinitis, allergic asthma, g g, g, eczema and anaphylaxis
What Testing For Allergy 1989 TtlS Total Serum IgE IE Specific IgE to allergen (RAST) (Skin Prick testing )
What diseases are caused by Allergy? IgE mediated allergy Atopy Anaphylaxis Pseudo allergy
Allergic Disease Atopy Clinical IgE mediated disease allergic rhinitis, allergic asthma and atopic eczema Evidence of Sensitisation Skin testing or specific IgE testing in serum Family history of atopic disease
Diseases caused by allergy Atopy Is it increasing? Increased asthma Increased rhinitis Increased eczema
Common Allergens Couch House Dust Mite Rye Grass Bee
Allergic Disease Anaphylaxis Severe life threatening reaction to allergy May occur in non atopic individuals Food in atopic children Food, medication and insect venom in adults in non atopic patients.
What Causes Anaphylaxis? More common eg peanut allergy increased dtwice in 20 years Cohort of patients with allergy childhood to adulthood Differentallergens cashew nut, brazil nut Cephasporins versus penicillin Seafood Chlorhexidine versus latex
What is the role of Allergy Testing? Is allergy causing clinical i l problem? What allergen is causing it?
What is the role of allergy testing History Skin Prick Testing? Positive skin test to allergen Serum testing for specific IgE to allergen if in doubt
What Laboratory Allergy tests? Serum total IgE Tests for specific IgE against allergen
Indications for Serum testing 1989 Skin testing unavailable Interfering medications antihistamines Widespread Eczema Young children Dermatographism Substances causing anaphylaxis Increasing use as skin testing not available
Why was serum testing second fiddle??? Not as sensitive as skin testing Poor standardisation Poor relationship between positive test and clinical i l allergy Many people p positive test and no allergy
The Problem with IgE Testing 1989 Not as sensitive as skin testing Patient has specific IgE to allergen but does not react to allergen if exposed Patients has specific IE IgE to allergen but the allergen is not causing the symptoms
Increasing Sensitivity of Specific IgE Testing
RAST testing Originally Assay Steps Allergen solid phase Specific antibodies from serum sample Separation of bound from free Radiolabelled anti human IgE
LabelledAnti IgE Serum Allergen Measuring Specific IgE against Allergen The Original RAST Testing
But insensitive as not enough allergen on the disc to suck up all the antibody!
RAST testing The First improvement Increase the Solid Phase Allergen Each allergenic protein has to be in molar excess binding g is affinity independent Increased allergen in the solid phase to suck up all specific IgE in serum Presence of allergen components Use of serum pool for Quality controls
Solid phase: ImmunoCAP Reference: L. Sevéus & A. Sandell, 1992
Quantitative Standardisation against Total Serum IgE Parallelism UniCAP IgE (ku /l) A d specific Measure 100 10 1 01 0,1 1 10 100 1000 Dilution factor Calibrator (ku/l) d1, 25774 e1, 27731 e5, 26843 f1, 24751 f2, 24052 f14, 24905 g3, 15273 m2, 17847 t3, 29450 w1, 12814 w6, 25666
Pharmacia CAP System Units of IgE WHO
Reproducibility Concentration (k A U/l) 25 d1, January 1998 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Lot number
Quantitative Results reported in internationally standardized units. Parallel serum dilution curves for calibrator and allergens
The Problem with IgE Testing 1989 Not as sensitive as skin testing Patient has specific IgE to allergen but does not react to allergen if exposed Is increased specific IgE more clinicallyrelevant? Patients has specific IgE to allergen but the allergen is not causing the symptoms
Does high concentration of specific IgE antibody mean clinical relevance? Improvement Number 2 Compare level of specific IgE antibody to result of controlled challenge IgE antibody concentrations to peanut above which h there is a 95% chance of positive clinical challenge
RAST Clinical Correlation Specific IgE levels correlate closely with results of inhalation challenge in cat allergy Mite specific IgE antibody levels correlate significantlywiththemiteallergen the allergen contents of reservoir dust in the homes of mite sensitive people HOWEVER not perfect and differences in cutoffs between labs
Probabilityof clinicalreaction to allergen correlates with Probability of clinical reaction to allergen correlates with concentration of specific IgE to allergen
But many people below cut offs and still react to foods or aeroallergens Patients above cut offs (>95%) don t need to be challenged eg Peanut IgE>15 KUA/l they react anyway Patients negative for specific IgE don t need to be challenge Many patients in between have to be challenged to work out if they react
The Problem with IgE Testing 1989 Not as sensitive as skin testing Patient has specific IgE to allergen but does not react to allergen if exposed Are certain subcomponents of allergens more clinically relevant? Patients has specific IgE to allergen but the allergen is not causing thesymptoms
Molecular Allergology Allergy as you ve never seen it The Third Improvement Component Resolved Diagnosis Micro Array testing
Two Types of IgE antibodies Germinal Centre and not Germinal Centre Low affinity Cross reactive High affinity More Specific
An allergen source
contains of which thousands only a few of are molecules allergenic Allergenic molecule = component
From allergen source to components
Allergen extract and components for an improved IgE antibody profile Molecular Allergology
Four important aspects of components: I. Specific II. Cross reactive III. Different stabilities IV. Different amounts Components are proteins, belonging to different protein families based on homology
Specific & Cross reactive reactive components Specific Specific Cross reactive Specific Cross reactive
I. IgE abs to Specific components indicate genuine sensitization Peanut Ara h 2 Honey bee Api m 1 Egg Gal d 1 Timothy Phl p 1 Birch Bet v 1 Gal d 1 = Gallus domesticus, allergen component # 1 Ara h 2 = Arachis hypogaea, allergen component # 2
II. IgE abs to Cross reactive reactive components IgE antibodies from one source may react to similar proteins in another = cross reactivity reactivity (or cross sensitization) sensitization) Similar components may be present also in distantly related species
Cross reactive reactive components (e.g. PR 10) Soy, birch and peanut contain components from the PR 10 protein family with high similarity
III. Protein stability Stable Labile Labile Labile Stable
Risk assessment
Protein stability Labile protein Local reaction Stable protein Systemic reaction
Clinical consequences Sensitization to specific components: Guides avoidance recommendations Indicates good Specific Immunotherapy (SIT) outcome Sensitization to cross-reactive components only: Suggests further investigation to find primary sensitizer
Molecular Allergology helps you to Assess the clinical risk for reaction Explain symptoms due to cross reactivity Identify the right patients for Specific Immunotherapy
Component Resolved Diagnosis BUT. A technique looking for a use More sensitive in picking up patients negative by other techniques More specific positive more likely to translate into a clinical senario than other techniques.
Peanut Allergen Components
Does High Concentration of IgE against Specific Component Predict a Positive Challenge than Specific IgE Against Whole Allergen?
Ara H2 testing Versus Skin testing Versus Specific IgE to Whole Peanut Number needed to challenge after testing Dang et al
ARAH2 testing ti has better ROC curve analysis that Specific IgE to whole Peanut Dang et al
Different Study Different cutoff Different cut offs for different ages Different pretest tprobability bilit Different co morbitities Reproducibility oftesting Klemans et al
Using peanut components in clinical practice Patient Caroline, 16 years Emma, 16 years Anamnesis Local reaction to peanuts Pollen and peanut allergies Local reaction to peanuts Pollen and peanut allergies Component testing Ara h 1 Ara h 2 Ara h 3 Ara h 8 Ara h 9 ku A /l < 0.1 < 0.1 < 0.1 33 < 0.1 ku A /l 2.1 12.3 3.3 10.3 < 0.1 Diagnosis Pollen associated peanut allergy Genuine peanut allergy Advice Suitable for re introduction Emergency medication unnecessary Strict peanut avoidance Emergency medication necessary Improved diagnoses and altered advice
Are certain patterns of antibodies against Subcomponents more clinically relevant than other Patterns?
Microarray Technology Knopp et al 2012
True Latex Allergy Versus False Positives Knopp et al 2012
Double Positive Results in Venom testing both Bee and Wasp Posiitive
Most Patients with Unterpretable Results were Most Patients with Unterpretable Results were positive for subcomponents Vesv5 or Vesv1
Specific IgE More Clinically Relevant?? GETTING THERE BUT NOT YET!!!