The Quest for Clinical Relevance

Transcription

1 Allergy Testing in Laboratory The Quest for Clinical Relevance

2 1989 A Good Year Current Concepts Lecture Allergy

3 1989 a good year

4 WHY ME? Current Concepts Lecturers 1989 Andrew Wootton David Gillis Clinical and Laboratory aspects of allergy

5 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

6 What Testing For Allergy 1989 TtlS Total Serum IgE IE Specific IgE to allergen (RAST) (Skin Prick testing )

7 What diseases are caused by Allergy? IgE mediated allergy Atopy Anaphylaxis Pseudo allergy

8 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

9 Diseases caused by allergy Atopy Is it increasing? Increased asthma Increased rhinitis Increased eczema

10 Common Allergens Couch House Dust Mite Rye Grass Bee

11 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.

12 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

13 What is the role of Allergy Testing? Is allergy causing clinical i l problem? What allergen is causing it?

14 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

15 What Laboratory Allergy tests? Serum total IgE Tests for specific IgE against allergen

16 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

17 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

18 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

19 Increasing Sensitivity of Specific IgE Testing

20 RAST testing Originally Assay Steps Allergen solid phase Specific antibodies from serum sample Separation of bound from free Radiolabelled anti human IgE

21 LabelledAnti IgE Serum Allergen Measuring Specific IgE against Allergen The Original RAST Testing

22 But insensitive as not enough allergen on the disc to suck up all the antibody!

23 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

24 Solid phase: ImmunoCAP Reference: L. Sevéus & A. Sandell, 1992

25 Quantitative Standardisation against Total Serum IgE Parallelism UniCAP IgE (ku /l) A d specific Measure , Dilution factor Calibrator (ku/l) d1, e1, e5, f1, f2, f14, g3, m2, t3, w1, w6, 25666

26 Pharmacia CAP System Units of IgE WHO

27 Reproducibility Concentration (k A U/l) 25 d1, January Lot number

28 Quantitative Results reported in internationally standardized units. Parallel serum dilution curves for calibrator and allergens

29 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

30 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

31 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

32 Probabilityof clinicalreaction to allergen correlates with Probability of clinical reaction to allergen correlates with concentration of specific IgE to allergen

33 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

34 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

35 Molecular Allergology Allergy as you ve never seen it The Third Improvement Component Resolved Diagnosis Micro Array testing

36 Two Types of IgE antibodies Germinal Centre and not Germinal Centre Low affinity Cross reactive High affinity More Specific

37 An allergen source

38 contains of which thousands only a few of are molecules allergenic Allergenic molecule = component

39 From allergen source to components

40 Allergen extract and components for an improved IgE antibody profile Molecular Allergology

41 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

42 Specific & Cross reactive reactive components Specific Specific Cross reactive Specific Cross reactive

43 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

44 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

45 Cross reactive reactive components (e.g. PR 10) Soy, birch and peanut contain components from the PR 10 protein family with high similarity

46 III. Protein stability Stable Labile Labile Labile Stable

47 Risk assessment

48 Protein stability Labile protein Local reaction Stable protein Systemic reaction

49 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

50 Molecular Allergology helps you to Assess the clinical risk for reaction Explain symptoms due to cross reactivity Identify the right patients for Specific Immunotherapy

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52 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.

53 Peanut Allergen Components

54 Does High Concentration of IgE against Specific Component Predict a Positive Challenge than Specific IgE Against Whole Allergen?

55 Ara H2 testing Versus Skin testing Versus Specific IgE to Whole Peanut Number needed to challenge after testing Dang et al

56 ARAH2 testing ti has better ROC curve analysis that Specific IgE to whole Peanut Dang et al

57 Different Study Different cutoff Different cut offs for different ages Different pretest tprobability bilit Different co morbitities Reproducibility oftesting Klemans et al

58 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 ku A /l < 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

59 Are certain patterns of antibodies against Subcomponents more clinically relevant than other Patterns?

60 Microarray Technology Knopp et al 2012

61 True Latex Allergy Versus False Positives Knopp et al 2012

62 Double Positive Results in Venom testing both Bee and Wasp Posiitive

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64 Most Patients with Unterpretable Results were Most Patients with Unterpretable Results were positive for subcomponents Vesv5 or Vesv1

65 Specific IgE More Clinically Relevant?? GETTING THERE BUT NOT YET!!!