4 Components - Resolved Diagnosis (CRD) Component-Resolved Diagnosis uses defined recombinant allergens to dissect the individual patient’s IgE reactivity profile with the aim of identifying the disease-eliciting molecules.Valenta R et al, Clin Exp Allergy 1999Molecules, genuine markers tell you the truth !Mari A., VIP Customers day, Brussels, 17th November 2009.The presence of IgE to cross-reactive allergen components can be determined and used to predict clinically relevant sensitization to allergen sources which contain immunologically related allergens.
5 Biochemical property of the allergens Allergens, data basesPeanut allergensBiochemical property of the allergens
6 « specific » IgE for extracts From a laboratory point of view…What does one value of sIgE means for allergen extracts ?How to manage this sIgE rate ?Concerning the care of the patient ?Concerning the risks ?Impact on overall patient management and health outcomes (severity assessment, risk, therapy choices, improve patient’s quality of live).Is there an interest of Component-resolved diagnosis in the laboratory routine practice?How can we realize CRD in the laboratory?Our experience with peanut sensitization…+ sIgE for f13, anamnesis, componentsOur question is :”Should we recommend the strict avoidance of peanut ?”
7 Clinical Cases (I) 1. Julie, F : f13 (peanut) = 27.4 kUA/L. SPT + : peanut, walnut, hazelnut, sesame seed, weeds, birch, pine nut.SPT - : soybean, egg white et walnut.Asthma, gastrointestinal intolerance and erythema after ingestion of nuts.
8 Clinical cases (II) 2. Laurent, H : Maud, F : f13 (peanut) = 1.22 kUA/L.sIgE + : birch, apple, pear, peach, apricot, hazelnut, walnut and recently to celery and to fennel.Atopy, polysensitised, oral allergy syndrome (OAS) and a recent evolution to breathing difficulties.Maud, F :f13 (peanut) = 1.23 kUA/LsIgE + : pollens of weeds, cow milk, carrot, orange, tomato, wheat, potato, garlic, onion, celery, banana, peach and peanut.Sensitization to a wide variety of foods from plants.Gastro-intestinal disorders.
9 Clinical cases (III) Eddy, M : f13 (peanut) = 0,44 kUA/L SPT+ : birch, apple, peach, banana, garlic, peanutSwollen lipsSensitization to birch…What can we conclude?
10 Clinical cases (IV) 5. Nadia, F : In those 5 clinical cases… f13 (peanut) = 1,73 kUA/Lf17 (hazelnut) = 4,23 kUA/LsIgE + : apple, peach, hazelnut, peanutSPT +++ : peanut, hazelnut, walnut.Atopy, polyallergy, rhinoconjonctivitis, OAS, urticaria et angiœdemaIn those 5 clinical cases…sIgE rates for peanut extracts are they proportional to the gravity of the allergy ?What are the risks for those patients?Eviction of diet or not ?How to manage the patient?What will be the future of the patient ??
11 Predict cross reactions Storage proteinProtein found in seeds serving as source material during the growth of a new plantOften stable and heat-resistant proteins causing reactions also to cooked foodsrSes i 3rBer e 1Cor a 9Ara h 1 Ara h 2 Ara h 3nGly mβ-conglycininnGly m glycinin
12 Predict cross reactions PR-10 protein, Bet v 1 homologueA heat labile protein, cooked foods are often tolerated.Breiteneder H, Biotechnol Adv, 2005Often associated with local symptoms such as oral allergy syndrome (OAS).Often associated with allergic reactions to fruits, vegetables and spices in northern Europe.Cor a Cor a 1.04Gly m 4Api g 1Ara h 8Dau c 1Bet v 1Pru p 1Mal d 1Pyr c 1Pru ar 1Pru av 1
13 Oral Allergy Syndrome (OAS) Oral Allergy Syndrome : allergic reactions that occur rapidly, within minutes of eating a trigger food.An itching or burning sensation in the lips, mouth, and/or pharynx.Associated to a sensitization in one or more proteins like PR-10 (Bet v 1-like) (birch).Hazelnut, 46%Apple, 39%Peach, 24%Cherry, 22%Nut, 21%Pear, 20%Almond, 19%Plume, 17%Brazil nut, 16%Potato, 15%Carrot, 13%Peanut, 10%Strawberry, 10%Orange, 9%Apricot, 7%PR-10allergic reactions that occur rapidly, within minutes of eating a trigger food. The most common reaction is an itching or burning sensation in the lips, mouth, and/or pharynx.Allergies to a certain pollen are associated with OAS reactions to certain foods. For Alder pollen: almonds, apples, celery, cherries, hazel nuts, peaches, pears, parsleyBirch pollen: almonds, apples, apricots, avocados, carrots, celery, cherries, chicory, coriander, fennel, fig, kiwifruit, nectarines, parsley, parsnips, peaches, pears, peppers, plums, potatoes, prunes, soy, wheat; Potential: hazel nuts, and walnutsGrass pollen: fig, melons, tomatoes, orangesMugwort pollen : carrots, celery, coriander, fennel, parsley, peppers, sunflowerRagweed pollen : banana, cantaloupe, cucumber, honey dew, watermelon, zucchini; Potential: Dandelions or chamomile teaPossible cross-reactions (to any of the above): berries (strawberries, blueberries, raspberries, etc), citrus (oranges, lemons, etc), grapes, mango, figs, peanut, pineapple, pomegranates, watermelonrBet v 1rPru p 1rGly m 4rAra h 8rApi g 1.01rAln g 1rCor a 1nAct d 8rMal d 1rDau c 1
14 Predict cross reactions LTP (non-specific Lipid Transfer Protein, nsLTP)A protein stable to heat and digestion causing reactions also to cooked foodsOften associated with systemic and more severe reactions in addition to OASLTPs often cause food allergy to fruit in the absence of pollen allergy.Often associated with allergic reactions to fruit and vegetables in southern Europe.LTPZea m 14nPru p 3rCor a 8rPar j 2nArt v 3Ara h 9Mal d 3Zea m 4Jug r 3Cor a 8Pru p 3Pru av 3Mal d 3Pru ar 3Ara h 9
15 Predict cross reactions ProfilinSeldom associated with clinical symptoms but may cause demonstrable or even severe reactions in a small minority of patientsProfilins are present in a broad range of pollens and foods including trees, grasses and weeds.There is a wide range of homology between profilins from different fruits and vegetables.Ara h 5PFLrBet v 2rPhl p 12rMer a 1rPhl p 12rBet v 2nOle e 2rMer a 1rHev b 8rMal d 4rPru p 4nAct d 9nAna c 1nAra h 5profilin in cross-reactivity between Latex profilin and other plant profilins.rPhl p 12rBet v 2nOle e 2rMer a 1rMal d 4rPru p 4nAct d 9nAna c 1Fra a 4nAra h 5Cor a 2rApi g 4rDau c 2rHev b 8rMal d 4Pru p 4rOle e 2rCor a 2rPyr c 4
17 In-vitro cross-reactions CCD (MUXF3) :Marker of sensitization to Carbohydrate determinants (cross-reacting carbohydrate determinants). Glyco-epitopes.Present in most plants,sIgE directed towards glycans appear to show the widest pattern of cross-reactivity among allergenic extracts and are often responsible for observed in-vitro cross-reactions.« in-vivo » effects ?Seldom associated to clinical symptoms …But, publications ?To compare to the clinical informations and to SPT…Test if…Sensitization to fruits and vegetables, seeds.Sensitization to latex in a pollinic patient without occupational risk exposure.Sensitization to Hymenoptera venom with/without sensitization to pollens.
18 Arachis Hypogea Allergenicity : Ara h 9 : nsLTPStable to heat and digestion.Severe reactions >> systemic reactions !Ara h 8 : ProfilinCross-reactions between many fruits and vegetables.CCD (MUXF3)Cross-reactive Carbohydrate DeterminantsSeldom associated to symptoms.Present in most plants.Ara h 1 : Storage protein (vicillin)Major allergenAra h 2 : Storage protein (conglutin)+ frequent, sensitizing potential ++, stability ++ to digestion.Ara h 3 : Storage protein (glycinin)Minor allergen, rare sensitizations.Ara h 8 : PR-10Major allergen of peanut in birch pollen allergic patients.Sensitive to heat and digestion.Allergenicity :Ara h 2 is 100 times stronger than Ara h 1Koppelman SJ et al, Clin Exp Allergy 2004,34:583Ara h 2 possesses high proteolytical stabilityLehmann K et al, Biochem J, 2006:After roasting peanuts, allergenicity of Ara h 2 is multiplicated by 3Maleki SJ et al, J Allergy Clin Immunol, 2003
19 Laboratory practice : How to diagnose peanut allergy ? Peanut (f13) + Ara h 2 (f423)Peanut (f13) : NegAra h 2 : NegPeanut (f13) : PosAra h 2 : NegPeanut (f13) : PosAra h 2 : PosLow risks of severe reactionsRisk of severe reaction?Very high risk of severe reactionsHow to explore? RecommendationsHow to explore ?RecommendationsTest : RiskAra h 1 (f422)Ara h 3 (f424)Ara h 9 (f427)Ara h 8 (f352)CCD (Ro214)Birch (north Europe) : test Ara h 8 (f352)
20 Clinical Cases (1) 1. Julie, F : f13 (peanut) = 27.4 kUA/L. SPT + : peanut, walnut, hazelnut, sesame seed, birch, weeds, pine nut.SPT - : soybean, egg white et walnut.Asthma, gastrointestinal intolerance and erythema after ingestion of nuts.What are the risks ? Birch?
21 Very high risk of severe reactions How to explore? Recommendations Clinical cases (1)Peanut (f13) = 27,4 kUA/LAra h 2 (f423) = 14,2 kUA/LPeanut (f13) : PosAra h 2 : PosVery high risk of severe reactionsSensitization to Ara h 8 due to birch allergy (PR-10).Sensitization to Ara h 2 : Storage protein (conglutin), Stable ++ to digestion, Diet eviction of peanut because risks of severe reactions +++.Sensitization to 3 major allergens of peanut.How to explore? RecommendationsCross-reaction : storage proteins ??Tests : RisksAra h 1 = 3,46 kUA/LAra h 3 = 0,6 UA/LAra h 9 <0,1 kUA/LAra h 8 = 2,27 kUA/LCCD < 0,1 kUA/LMajor allergens of peanutStorage proteinspeanut PR-10 protein !
22 Clinical cases (2) 2. Laurent, H : f13 (peanut) = 1.22 kUA/L. sIgE + : birch, apple, pear, peach, apricot, hazelnut, walnut and recently to celery and to fennel.Atopy, polysensitised, oral allergy syndrome(OAS) and a recent evolution to breathing difficulties.What are the risks?What about the Birch?Recent evolution of the allergy?
23 Risk of severe reactions? How to explore? Recommendations Clinical cases (2)Peanut (f13) = 1,22 kUA/LAra h 2 (f423) < 0,10 kUA/LSensitization to peanut after birch allergy (PR-10 proteins).Sensitization to other PR-10 :- Apple (Mal d 1), pear (Pyr c1), peach (Pru p 1), apricot (Pru ar 1), hazelnut (Cor a 1) and recently to celery (Api g 1)In this case, evolution of the OAS with respiratory distress explained by recent sensitization to Api g 1.Peanut (f13) : PosAra h 2 : NegRisk of severe reactions?How to explore? RecommendationsTests : RiskAra h 1 < 0,1 kUA/LAra h 3 < 0,1 kUA/LAra h 9 < 0,1 kUA/LAra h 8 = 8,4 kUA/LCCD < 0,1 kUA/LPR-10, Bet v 1-homologue
24 Clinical cases (3) Maud, F : f13 (peanut) = 1.23 kUA/L sIgE + : pollens of weeds, cow milk, carrot, orange, tomato, wheat, potato, garlic, onion, celery, banana, peach and peanut.Sensitisation to a wide variety of foods from plants.Gastro-intestinal disorders.What are the risks ?Multiple sensitizations to fruits and vegetables.
25 Risk of severe reactions? How to explore? Recommendations Clinical cases (3)Peanut (f13) = 1,23 kUA/LAra h 2 (f423) < 0,1 kUA/LSensitization to CCD :- Marker of sensitization to Carbohydrate Determinants.- Multiple cross-reactions.- Seldom associated to clinical symptoms.Test CCD if sensitization to fruits and vegetables, seeds.Peanut (f13) : PosAra h 2 : NégRisk of severe reactions?How to explore? RecommendationsTests : RisksAra h 1 < 0,1 kUA/LAra h 3 < 0,1 kUA/LAra h 9 < 0,1 kUA/LAra h 8 < 0,1 kUA/LCCD = 1,93 kUA/LCCD
26 Clinical cases (4) 4. Eddy, M : f13 (peanut) = 0,44 kUA/L Swollen lips SPT+ : birch, apple, peach, banana, garlic, peanutSwollen lipsWhat are the risks ?Sensitization to birch…
27 Risk of severe reactions? How to explore? Recommendations Clinical Cases (4)Peanut (f13) = 0,44 kUA/LAra h 2 (f423) < 0,1 kUA/L?Sensitization to profilins (Bet v 2)Seldom associated with clinical symptoms except OAS and rarely severe reactions in a minority of patients.Presence of profilins in many fruits and vegetables.- Birch (Bet v 2), apple (Mal d 4), peach (Pru p 4), banana (Mus a 1), garlic (All c 4), peanut (Ara h 5).Peanut (f13) : PosAra h 2 : NegRisk of severe reactions?How to explore? RecommendationsBet v 2 = 4,6 kUA/LTestésAra h 1 < 0,1 kUA/LAra h 3 < 0,1 kUA/LAra h 9 < 0,1 kUA/LAra h 8 < 0,1 kUA/LCCD < 0,1 kUA/L
28 Clinical cases (5) 5. Nadia, F : f13 (peanut) = 1,73 kUA/L f17 (Hazelnut) = 4,23 kUA/LsIgE + : apple, peach, hazelnut, peanutSPT +++ : peanut, hazelnut, walnut.Atopy, polyallergy, rhinoconjonctivitis, OAS, urticaria et angiœdemaWhat are the risks ? …angioedema..;
29 Clinical cases (5) Peanut (f13) = 1,23 kUA/L Ara h 2 (f423) <0,1 kUA/LHazelnut (f17) = 4,23 kUA/LCor a 8 (f425) = 3,87 kUA/LPeanut (f13) : PosAra h 2 : NegHazelnut (f17) : PosCor a 8 : PosnsLTPRisk of severe reactions?Very high risk of severe reactionsHow to explore? RecommendationsSensitization to nsLTP :Marker of cross-reactions.Associated to severe reactions.Occurs mainly in South of Europe.Sensitization to apple (Mal d 3), peach (Pru p 3), hazelnut (Cor a 8) and peanut (Ara h 9).Test : RisksAra h <0,1kUA/LAra h 3 <0,1kUA/LAra h 9 = 8,2 kUA/LAra h 8 <0,1kUA/LCCD <0,1kUA/LnsLTP
30 Thanks to all the recombinants, More complex…Julien, M :Complex clinical case !!Atopy,Atopic dermatitis,Perannual rhinitis,Giant eczema.Food allergy (egg white, peanut, soybean, crab and garlic)How to evaluate the symptoms ?SPT Pneumallergens:+ : D1 et D2, dog, weeds.Suspiscious : birch.SPT Trophallergens :+ White egg, peanut, soybean, garlic.IgEs for extractsPeanut,f139.71Soybean,f144.43Crab,f231.98Garlic,f474.11Egg yellow,f750.61Pityrosporum orbiculare,m703.93Dermatophagoïdes pteronyssinus,d1>10Egg white,f15.37Schrimp ,f242.64Gx3:g1+g5+g6+g12+g1393.6Birch,t358.0Thanks to all the recombinants,We would be able to explain everything !!
31 In theory, this is possible… Request form ImmunoCAP™ComponentsPatient: Nom : Mister Albert GikAdresse: Birch Street 1…Birth date: 01/01/1950Some other components less useful in Belgium could be available.Don’t hesitate to ask to your Lab in case of specific clinical history.June 2009In theory, this is possible…Practically, …Docteur: Nom: Cavalier EtienneAdresse : CHU of LiègeDate de demande: 19/11/2009Signature:
33 Immuno Solid-phase AllergenChip (ISAC) Allergen moleculeAllergen-triplet afterfluorescence-reactionFluorescence-labeledsecondary anti-humanIgEIgE-antibody(in the patient’s serum)The allergen biochip is a solid substrate built from a glass plate that is chemically altered to retain allergenic protein molecules in a biologically active form. Picolitre quantities of each allergen preparation are deposited on spatially separate locations on the biochip. The resulting addressable microarray allows for many biological affinity reactions to be monitored in parallel (=multiplexing). In a single analytical step, the comprehensive allergen sensitisation profile of a patient can be measured using only a minute amount of serum or plasma; this is followed by automatic readout and report generation. The present chip used for in vitro diagnostics incorporates 103 allergens from about 50 different allergen sources.4 reaction sites
34 ImmunoCAP® ISAC : Version 103. Proteins, Cross-reactions :11 PR-10 :(Bet v 1-like proteins) :Well represented, interest ++4 nsLTP :Some more ?Which ones?5 Profilins :So many profilins exist…Which ones are most representative in each botanic family ?5 Storage proteins2 Parvalbumins :Important !Which ones are most representative?4 Tropomyosins :5 serum albumins :Honey bee and paper wasp venoms :Lack…
36 Validation of the method Selection of 19 sera of patients (6 males/13 females)+ sIgE (>0.10 kUI/L) for recombinant allergens (ImmunoCAP ®250 Phadia).positive and negative sIgE for different kind of allergenic sources (latex, peanut, birch, Timothy grass, hazelnut, peach, soybean, cat, dog…).In all, we compared the results of 157 sIgE measured in CAP (recombinant allergens) with the ISAC®.We tested 2 samples with a high TotIgE rate (> kU/L) on ISAC ®.(rHev b 1; n=3, rHev b 3; n=3, rHev b 8; n=5), cross-reactive carbohydrate determinants (nAna c 2; n=2), soja (rGly m 4; n=4), peanut (rAra h 1; n=7, rAra h 2; n=7, rAra h3; n=5, rAra h 8; n=12), hazelnut (rCor a 8; n=3), Brazil nut (rBer e 1; n=1), peach (rPru p 1; n=11, rPru p 3; n=3), birch (rBet v 1; n=17, rBet v 2; n=11, rBet v 4; n=8), timothy grass (rPhl p 1; n=12, rPhl p2; n=2, rPhl p4; n=5; rPhl p 5; n=7, rPhl p 6; n=3, rPhl p11; n=1, rPhl p 12; n=5), Aspergillus fumigatus (rAsp f 6; n=2), Alternaria (rAlt a 1; n=3), cat (rFel d 1; n=7) or dog (rCan f 1; n=4, rCan f 2; n=2, rCan f 3; n=2).
37 Validation of the method Results:17 results out of the 122 measurements of positive sIgE in CAP® were found negative with ISAC ® (concordance of 86%).2 results out of the 35 measurements of negative sIgE in CAP® were found positive with ISAC ® (concordance of 94%).Until a rate of kU/L of TotIgE, we didn’t observe any unspecific binding.
38 Validation of the method DiscussionConcordance of all positive results was 86%.Can we explain the discordances?Cut-off of the ISAC ® higher (>0,30 USI) than cut-off of CAP ® (>0,10 kUA/L)Many measurements on ISAC ® were between the range of 0,10 and 0,30 USI.Concordance of all negative results was 94%.2 discordances concerned sera for which ISAC® found a positive Ara h1 and Ara h 3 (2 major allergens of peanut).Clinical importance of those 2 allergens !Look back the clinical anamnesis :The first patient had positive sIgE in CAP for Ara h 1 and Ara h 2, his oral provocation test (TPO) to peanut was positive.- Anamnesis well correlated with true positive Ara h 3 with ISAC ®The 2nd patient had undergone one TPO for peanut which was suspiscious, sIgE Ara h 8 ++, LTP ++, CCD +, polysensitized patient and large food eviction (hazelnut, peanut, sea food…)- Anamnesis correlated well with a possible true positive Ara h 1 with ISAC ®
39 Albert Gik Bet v 1 homologues (PR-10): Bet v 1, Aln g 1, Cor a 1, Mal d 1, Pru p 1, Gly m 4, Ara h 8, Api g 1Species markers :Gal d 1, Bos d lactoferrin, Fel d 1, Fel d 4, Can f 1, Mus m 1, Alt a 1Profilins :Bet v 2, Ole e 2, Hev b 8, Mer a 1, Phl p 12Der p 1, Der f 1, Der f 2, Der p 2, Eur m 2Pollens : Cyn d 1, Phl p 1, Phl p 2, Phl p 4, Phl p 5, Phl p 6 et Ole e 1Thanks to CRD, we can explain :Many food allergies due to sensitization to profilins, PR-10 proteins (OAS), tropomyosin (sea food), cow milk…Many weeds, mites, pets… which explain perannual rhinitis exacerbated during pollination.Tropomyosin : Der p 10
40 Conclusion (I)The way for the discover of sensitization profiles is open…Allergen extracts are still useful :In first intention,Don’t permit to conclude every time (multiple positivity of the tests, lack of precision),sIgE for extract not really significant.CRD is in our hands.Sensitizations can cause diagnosis errors (and therapeutic errors too).Protein families have been described, similarities of structure (homology of amino-acids or structural homology)Allergy to one protein can cause cross-reactions to other proteins of the same family.Recombinant allergens permit to focus (one or more) allergen(s) of one source:Available in CAP (and/or in ISAC) or to be developed…