Molecular based allergic diagnosis

Molecular-based allergy
diagnostics
Suparat Sirivimonpan, MD.
18/10/2013

A WAO - ARIA - GA2LEN consensus
document on molecular-based allergy
diagnostics

World Allergy Organization Journal 2013, 6:17

Topic outline
Introduction
 Definitions and concepts
 Clinical application








Increase accuracy and resolve cross-reactivity
Assess the risk and type of reaction
Specific immunotherapy

Micro-array technology

Diagnosis
•

Clinical : History, Physical examination

•

Allergic test
•

•

Skin prick test
Blood test

crude extracts

•

Specific IgE

•

Component resolve diagnosis (CRD)

Currently available tests detect only sensitization,
not clinical allergy, cannot predict prognosis and severity

Nature
Allergenic
source

contain a mixture of allergenic and
non-allergenic molecules

Introduction
Molecular allergy (MA) diagnostics is increasingly entering
routine care
•

More than 130 allergenic molecules are commercially
available for in vitro sIgE testing
•

Clinicians and immunologists specifically trained in
allergology must keep abreast of the new and rapidly
evolving evidence available for MA diagnostics
•


Introduction
WAO - ARIA - GA2LEN consensus document :
practical guide for the indication, determination, and
interpretation of MA diagnostics
aimed for clinicians specifically trained in allergology
•


Definitions and concepts
•

Allergen extract
•
•

A crude, unfractionated mixture of allergenic and nonallergenic proteins, polysaccharides, and lipids
extraction from an allergen source

•Allergenic
•
•

molecule (allergen component)

A molecule (i.e., protein or glycoprotein) bind sIgE antibodies
 “allergen”
Allergens can be isolated from
•

natural allergen sources (native, purified allergen)

•

recombinant DNA technology (recombinant allergen)


Allergen component
Almost anything containing proteins can be an allergen
source
- each allergen source contains many different allergenic
protein (allergen component)
- each allergen component commonly has several different
epitopes
- epitope is the actual three-dimensional binding site for an
antibody

Clinical and Experimental Allergy;2010 ,40: 896-904

Stability of allergens
•Acid
•

pH :

susceptible to acid pH (peptic digestion)  not able to cross
gastric barrier
(except possibly in patients treated with antacid drugs)

•Temperature
•

(cooking or boiling) :

Temperature susceptibility  does not maintain its
allergenicity after cooking/heating procedures


Allergen component

Protein stability
- allergen that stable to heat and digestion are more
likely to cause severe reaction
- allergen that heat and digestion labile are more
likely to be tolerated or only cause mild/local symptom


•

Primary allergen :
•

original sensitizing molecule (i.e., driving trigger)

•Secondary
•

sensitization :

cross-reactivity


Cross-reactivity
•

phenomenon of an IgE antibody recognizing, binding,
and inducing an immune response to similar allergenic
molecules (homologues) present in different species
(reacts to both Bet v 1 in birch pollen and Cor a 1 in hazelnut  PR10)

•

≥ 50-70% sequence homology between primary
structures of proteins


Cross-reactivity
•

IgE cross-reactivity often occurs between the following:
a) Allergenic molecules in closely related species

b) Well preserved molecules with similar function present in
widely different species that belong to the same protein family
(tropomyosin : Der p 10 in house dust mite ,Pen m 1 in black tiger shrimp)


Co-sensitization
Genuine sensitization to more than one allergen
source (e.g., timothy grass and birch)
• sensitization is not due to cross-reactivity
•


Co-sensitization
Genuine sensitization to more than one allergen
source (e.g., timothy grass and birch)
• where the sensitization is not due to cross-reactivity
•


sIgE sensitization
presence of allergen-specific IgE (sIgE) antibodies in the blood
• presence or absence of clinical symptoms
•

a) Mono-sensitization:
- Sensitization to one allergen source (D. pteronyssinus) or to a
closely related taxonomical family or group of allergen sources
(i.e., mites)

b) Poly- (or multi-) sensitization:
- Sensitization to three or more allergen sources (e.g., mite,
birch, and grass pollen).


sIgE detection based on allergen extracts
Singleplex or multiplex platforms
• such as CAP, radioallergosorbent test (RAST)
• interchangeably
•

•

sIgE detection based on allergenic molecules
Singleplex or multiplex platforms
• identify cross-reacting molecules
•


Systematic allergen nomenclature
•World

Health Organization and International Union of
Immunological Species (WHO/IUIS)
• Using their Latin family name (genus and species)
• Arachis hypogaea : Ara h (peanut)
• A number : distinguish various allergens from the same species
(e.g., Ara h 1, Ara h 2, etc.)
• The numbers are assigned to the allergens in the order of their
identification
• Allergenic molecules are classified into protein families, according
to their structure and biological function
•www.allergen.org

Clinical application
increased accuracy in allergy diagnosis and prognosis
• plays an important role in three key aspects of allergy
diagnosis:
•

(1) Increase accuracy and resolve cross-reactivity
(2) Assess the risk and type of reaction
(3) Specific immunotherapy
(patients and triggering allergens)


Allergic individuals may produce IgE antibodies to allergens that
are either unique to a single species or common to many
•

Proteins within the same family share common epitopes
 same IgE antibody can bind to similar structures present in
allergens from different allergen sources
•

These cross-reactive allergens give valuable information on
potential sensitization and clinical reactions to several different
sources
•


•


S ta b le


L a b ile


While, MA studies have not fully elucidated the
underlying mechanism governing cross-reactivity and
symptom presentation
 analysis of the epitopes of key allergens will
provide insight into this issue
•

Currently, a large number of purified or recombinant
allergens are commercially available
•


Food


A e ro a lle rg e n s


O th e r alle rg e n s


Clinical uses of MA :
• identify the offending allergenic molecule
• distinguish specific molecules from markers of crossreactivity


In the field of pollen-related food allergy : increase accuracy of
diagnosis
•

* peanut allergic patients,
- Ara h 2  genuine marker,systemic reactions
-Ara h 8  cross-reactivity among food allergens ,Fagales tree pollen
 mild, oral reactions
•measuring IgE responses to certain food allergens may reduce the
need for food challenges
•


•

In patients sensitized to different pollen species
• detecting new relevant sensitizations
• by ruling out clinically irrelevant sensitizations caused by nonsymptomatic cross-reactive allergens
•

For example, MA diagnostics can help to distinguish baker’s
asthma from pollen or wheat allergy


In conventional SPTs, some allergens can be poorly represented
in extracts because of the biological variability of allergen source
•

* Dog allergic patients
•

•

•

Can f 5  prostate-derived allergen produced by male dogs
 38% of dog allergic patients
Allergen extracts used in skin tests : dog hair
 skin tests routinely fail to identify patients’ sensitivity to
Can f 5 (low concentration in dog hair)
IgE response to Can f 5 using MA diagnostics may enhance
accuracy of dog allergy diagnosis

All allergy diagnostics, including MA, should be evaluated within
framework of a patient’s clinical history
•

•

IgE sensitization  not necessarily imply clinical responsiveness

This is of particular importance, since allergic patients respond in
individualized manner to exposure to allergens from various sources
•i.e., every individual produces their own unique IgE antibody profile
at molecular level
•


Ability to improve risk assessment, particularly for food allergies
• Different foods contain unique allergenic molecules that are stable
or labile to heat & digestion
• stability of a molecule & patient’s clinical history : risk of
systemic versus local reactions
• Labile  local reactions (typically oral symptoms) and cooked
food is often tolerated
• Stable allergens  systemic reactions
•

MA diagnostics may decrease the need for provocation testing and
improve recommendations for allergen avoidance
•


Information may only be applicable to the specific population which
has been studied
• Disease expression differ according to local exposures patterns
characteristic of the geographical region
• ex. Peanut (USA, Spain, Sweden)
•


Food allergens
Allergens resistant to heat and digestion often trigger more severe
allergic reactions

Storage protein, LTP

Food allergens
In addition, the amount of a molecule present in a food
source is also a parameter to take into consideration
•


Food allergens

Peanut
Ara h1,2,3 (storage proteins) genuine peanut reaction
• Ara h 8 (PR-10,Bet v 1-homologue)  milder or local symptoms
•

•

LTP (Ara h 9)  southern Europe, prevalent sensitizing allergen
 systemic and more severe reactions
•

•

Further studies in other geographical regions

Profilin or CCD sensitization  no or local oral symptoms
 heated peanuts may be
tolerated


Food allergens

Soy
•

Gly m 5 and/or Gly m 6  severe reactions

•

Gly m 4 (PR-10)  OAS
•

•

In birch pollen-allergic individuals, the combination of Gly m 4
sensitivity and intake of large amounts of mildly processed
soy, such as soy drinks, can induce a severe reaction

Profilin or CCD  no, or local oral symptoms
 heated soy may be tolerated


Food allergens

Hazelnut
•

Cor a 1 (PR-10)  local reactions like OAS

•

Cor a 8 (LTP) , storage proteins (Cor a 9,Cor a 14)
 severe symptoms

•

Profilin (Cor a 2) or CCD alone
 no or local oral symptoms
 heated hazelnuts may be tolerated.


Food allergens

Walnut
•

Storage protein (Jug r 1, Jug r 2) or LTP (Jug r 3)
 severe reactions

Walnut allergens have not been available on the market until
recently, as is reflected by the lack of recent clinical studies
•

•

Profilin or CCD alone  no, or local oral, symptoms
 heated walnut may be tolerated


Food allergens

Wheat
•

ω-5-gliadin (Tri a 19)
 immediate allergic reactions in children
 systemic exercise-induced reactions in adults

•

LTP (Tri a 14)

 some degree of cross-reactivity with other food LTPs
 more knowledge is needed about its prevalence and
clinical implication
•

Profilin or CCD
 no, or local oral, symptoms
 heated wheat may be tolerated

Food allergens

Rosaceae fruits
Apple, peach, Appricot, Plum
• PR-10 proteins (Mal d 1, Pru p 1) or profilins (Pru p 4)
•

 local, oral reactions (sensitive to heat & digestion)
•

LTP (Pru p 3)

 typical of the Mediterranean area
 wide range of clinical expressions (from asymptomatic to anaphylaxis)
 risk marker for severe reactions including cofactor (e.g.,
exercise, alcohol or drugs) dependent anaphylaxis


Food allergens

Egg
High levels of sIgE antibodies to ovomucoid (Gal d 1)
 risk factor for persistent egg allergy, including
reactions to cooked/heated egg
• Undetectable levels indicate tolerance to cooked egg
•


Food allergens

Milk
Casein (Bos d 8) and beta-lactoglobulin (Bos d 5) sIgE
antibodies
 persistent allergy to milk, including heated milk
• undetectable levels indicate tolerance to baked milk
products
•


Food allergens

Fish
•

Parvalbumins (e.g., Gad c1 and Cyp c 1)
• major allergens in fish
• typically stable to heat and digestion
• high degree of cross-reactivity
whereby patients sensitized to one parvalbumin may also
react to parvalbumins from other fish, including carp, cod,
herring, plaice, mackerel, tuna, salmon, perch, and eel


Food allergens

Shellfish
Tropomyosin
- high degree of cross-reactivity across a wide
variety of species, including mites
•

Shrimp and other shellfish also contain other clinically
relevant allergens
- sarcoplasmic calcium-binding protein
- arginine kinases
•


Food allergens

Meat allergy
Galactose-α-1,3-galactose (α-Gal)
sugar structure found on glycoproteins and glycolipids of nonprimate mammals and new world monkeys, but not on humans
• It is assumed that sensitization to α-Gal can be induced by tick
bites or certain parasite infections
•

α-Gal– sensitized patients  delayed immediate type reactions to
red meat (beef, pork, goat, deer)
• Anti-α-Gal-IgE  severe allergic symptoms and with delayed-type
anaphylaxis
•


Food allergens

Meat allergy
α-Gal : present on the chimeric antibody cetuximab
• Patients sensitized to α-Gal may react with anaphylactic reaction
after the administration of cetuximab
• Testing for α-Gal before administration of cetuximab
•

•

Bovine serum albumin (e.g. Bos d 6)
•
•

•

heat labile allergen
present both in milk and beef
may cause cross-reactivity between different mammalian meat


Inhalants

Pet dander
Fel d 1 : asthma in cat-allergic individuals
• Lipocalins (Mus m 1, Equ c 1, Fel d 4, Can f 1, 2), kallikrein (Can f
5), and secretoglobin (Fel d 1) : severe asthma in Swedish children
•

•

More knowledge is needed in the area of pet allergy
• many of the patients are poly-sensitized to several pets
• clinical history is often inconclusive
• cross-reactivities between e.g. cat, dog and horse is not fully
clarified at the MA level


Inhalants

Pollen
Research in pollen allergy has focused on distinguishing genuine
allergens from those that are cross-reactive
• Little is known regarding specific markers of severe reactions
•

•

Some sensitivities to specific allergens (ex.Ole e 9 ,LTP Ole e 7)
• may be markers of more severe symptoms in pollen allergy
• increasing the risk of systemic reactions during
immunotherapy


Inhalants

Pollen
•

Profilin sensitization
•
•
•

common among pollen allergic patients
associated with mild or no clinical symptoms
minority of patients: risk factor for more severe reactions in
olive pollen-allergic individuals and in patients allergic to
certain plant foods like melon or citrus


Inhalants

Mites
No specific sensitization profile has been described as a
risk factor for lower airway disease or disease severity
•

Higher sIgE/IgG4 ratio for Der p 2 has been associated with
asthma
•

•

Der p 10 (tropomyosin)
•

•

minor allergen in mite-allergic patients
risk for allergic reactions to shellfish or snail, which can be
severe


Inhalants

Molds
Aspergillus fumigatus
Asp f 2, 4, and 6 may suggest ABPA
• Asp f 1 and/or Asp f 3 may be more indicative of allergic asthma
• These associations must still be confirmed in other patient
populations
•


Inhalants

Cockroach
Per a 2 correlates with severity of airway allergy in cockroachallergic patients
• Per a 2 is currently not commercially available for in vitro testing
• Per a 2 homologue Bla g 2 is available
•

Cross-reactive tropomyosin (Bla g 7)
 risk for allergic reactions to shellfish or snail, which can
be severe
•


Other allergens

Latex
•

Sensitization to Hev b 8 (profilin)
•

seems to be clinically irrelevant

•

not related to clinical latex reactions

The other latex allergens are linked to clinical reactions
• No association between allergens and severity of reactions
• The cross-reactive allergen responsible for the so called latex-fruit
syndrome are not fully clarified
•

•

although data indicate that Hev b 5, 6 and 11 play a role


Other allergens

Hymenoptera venoms
Most hymenoptera venom allergens possess CCDs
 responsible for a portion of clinically irrelevant IgE
antibody cross-reactivity between bee and wasp venom
•

Detection of recombinant venom allergens can discriminate
between genuine venom sensitization and cross-reactivity due to
CCDs in patients with double-positive IgE results from traditional
venom tests that are based on allergen extract
•


Specific immunotherapy (SIT)
Correct diagnosis, selection of truly eligible patients, and
identification of primary sensitizing allergen(s) are important
for optimal and cost-effective patient management
•

In some patients, a detailed clinical history and traditional extractbased IgE testing (SPT and/or in vitro sIgE) is sufficient to identify
the relevant allergen(s)
•

Patient demonstrates polysensitization : traditional diagnostic
tests based on allergen extracts and their clinical history is not
sufficient to clarify the nature of the sensitization
• may occur in a relatively high proportion of patients
•


Mixing numerous allergens appears to achieve good clinical
efficacy
•

•

there may be an inability to identify the responsible allergen in the case
of adverse events

Certain proteins (e.g., profilins, polcalcins, LTPs, PR10,
tropomyosins) are highly conserved in a wide variety of species
•

The use of recombinant/purified allergens would allow for the
discrimination between genuine sensitizations and cross
reactivities
•


Grass, Pollen

In the example mentioned above a patient with sIgE antibodies against Phl p 1 and Phl p 5 but no sIgE to Bet v
1 is truly sensitized to grass
If sIgE antibodies to Phl p 12 (profilin) were also detected, profilin sensitization would probably be responsible
for the positive SPT result obtained with birch extract, which contains profilin as well.
SIT would be prescribed for grass only


HDM

• if a patient is sensitized to a traditional house dust mite extract

but their IgE antibodies against Der p 10 (tropomyosin) and not to Der p 1, 2/
Der f 1, 2
•

SIT for mites should not be given, because mite extracts mainly contain Der
p 1, 2/Der f 1, 2 and have variable or low amounts of Der p 10
•


Hymenoptera venom

Sensitization to the major allergens Api m 1 of honeybee and Ves v 5 and/or
Ves v 1 of yellow jackets may be helpful in discriminating between true double
bee and wasp sensitization and cross-reactivity due to CCDs
•


Most commercial allergen extracts used in SIT are well
standardized for major allergens
• contain only minimal or variable amounts of minor
allergens
•

Patients with sensitization to minor allergens alone will
likely not receive sufficient amounts of allergen to
achieve a successful outcome by SIT
•


Theoretically, identification of molecules  tailored SIT??
• In practice, this does not seem feasible
1. number of possible combination of sensitization
profiles is large when taking into consideration all allergenic
sources
2. recombinant vaccines do not perform better than
traditional allergen extracts, as observed in some studies
3. each single recombinant/purified allergen would need
to be individually tested and registered, which carries a
substantial financial burden for manufacturers
• reality of patient-tailored SIT is still a distant prospect
•


MA diagnostics has been available on singleplex
platforms
• ImmunoCAP, ImmuLite, and HyTech platforms
• single allergens together with the
corresponding allergen extract
•

•

Multiplex technology :
• measure sIgE antibodies against multiple
allergens in a single assay
• testing of a large number of allergens using a
small amount of serum


ImmunoCAP Immuno-Solid Phase Allergen Chip
(ISAC) (Phadia AB)

Fluorescence is measured with a laser scanner and results are evaluated using a Microarray Image
Analysis (MIA) software, which provides an automatic readout of the results.


ISAC
Using a standard calibration curve, results are reported within a
range of 0.3 - 100 ISAC Standardized Units (ISU-E)
• giving a semi-quantitative indication of IgE antibody levels
•


ISAC
•

ImmunoCAP
• results : kU/L
• not interchangeable
• correlate well

ImmunoCAP technology measures IgE binding under conditions
of excess of immobilized allergen
• ISAC uses low amounts of immobilized allergen allowing for
competition with allergen-specific isotypes other than IgE
•


When comparing ISAC with other sIgE measuring assays, concordance of results vary
between allergens tested

ISAC
Results of the ISAC assay are reproducible at a level that is
generally accepted and agreed upon
•

•

Special attention : samples contain low levels of sIgE
(0.3–1 ISU-E),  higher degree of variability

at low sIgE levels ImmunoCAP is more sensitive than ISAC
Comparative data with the ImmuLite or HyTech platforms are not
available in the literature
•


ISAC
ISAC is generally not recommended for monitoring quantitative
IgE levels over time in clinical routine
•

•Because

there is a higher degree of between assay variability for ISAC
compared to ImmunoCAP

The interpretation of the results may be challenging
1. clinical relevance of the different allergens must be
considered
2. results must be evaluated in relation to traditional
diagnostic tests
3. results must be evaluated with regard to the patient’s
clinical history (most important)
•


ISAC results for some allergen sources such as cashew nut,
sesame, dog, mugwort, and ragweed can be negative, even when
the extract-based test is positive
 triggering allergen is not present on the chip
•


Patients most likely to benefit from MA diagnostics
Patients who are poly-sensitized, have an unclear symptom
and/or sensitization pattern, or who do not respond to their
treatment
• Patients with documented poly-sensitization to one or more
inhalants, but also suffering from food allergy (i.e., from less
severe manifestations such as OAS to more severe, including
anaphylaxis, asthma or eczema)
•

Mono-sensitized patients with a clear case history and symptom profile may
not benefit from MA diagnostics compared to traditional diagnostic tests


Molecular-based allergy diagnostics
• MA diagnostics may offer additional information for
- early diagnosis of allergies
- aid in the monitoring of the evolution of the
allergic disease
- useful for preventive indications to the patient


Unmet needs!
1) Large-scale, population-based multicenter studies : categories of
patients
2) Large studies that include well-characterized patients and
healthy, sensitized controls representative of different geographical
regions
3) Cost-utility studies :
 compare effectiveness of MA diagnostics with the
traditional in vitro sIgE or SPT


Unmet needs!
4) Identification and clinical evaluation of the most relevant
allergens have to be further investigated in many allergen
sources.(e.g., nuts, molds, tree and weed pollen)
5) Training efforts in both the clinical and research settings
6) Development of clinical decision support is needed to prevent
misinterpretation and improve knowledge as the amount of
information obtained from MA diagnostics may be complex,
especially as the evidence for MA is rapidly progressing.


• International

guidelines recommendation

1st Step : a thorough clinical case history

2nd Step : allergen extract-based IgE tests (in vitro specific IgE or
skin prick test)
 identification of the allergen source
 SPT and in vitro sIgE tests provide similar information
associated advantages and disadvantages of both types of tests are
dependent on the clinical case

3rd Step : Molecular-based allergy (MA) diagnostics
 used for select patients in whom first- and second-line
investigations were inconclusive


For the majority of patients, first- and second-line investigation is sufficient to define the nature of a

patient’s allergy


For experienced users MA may be included in second-line testing

Conclusions
MA diagnostics is increasingly entering routine care
and can improve management of allergic patients
•

Benefit
(1) Increase accuracy and resolve cross-reactivity
(2) Assess the risk and type of reaction
(3) Specific immunotherapy
- small serum or blood sample
- broad spectrum analysis of a patient’s IgE profile


Conclusions
Sensitivity compared to ImmunoCAP is still often lower
• ISAC can have high clinical relevance by detecting
sensitization patterns to important allergens and crossreacting groups
•


Conclusions

Disadvantage
Interpretation of such sensitization in clinically
unresponsive patients is difficult or even impossible
• Relatively expensive compared with traditional tests
 economic consideration or budget limitations
•

the search for more, clinically relevant molecules is needed and
ongoing
•


Conclusions
Although MA diagnostics is a complex area, it provides
novel and relevant information for the allergist and will
soon become a standard tool in the allergist’s
armamentarium
•

Educational programs training allergists on the use and
interpretation of MA are highly needed
•
•


Molecular based allergic diagnosis

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