Diagnostic Method for Hypersensitivity in

Allergy Reaction

Lusiana Lim
Product Manager
EUROIMMUN South East Asia Pte Ltd

6th July 2019

Workshop: Hypersensitivity Reaction in Allergy Disease, Indonesia

Infectious Antigen Molecular genetic

Autoimmunity Allergology diagnostics Automation
Serology detection
Outline

1. About EUROIMMUN

2. Overview of Allergy Situation in Indonesia

3. Profiles for Allergy Diagnostics Testing

4. Case Studies

5. Summary
EUROIMMUN

 Established in 1987,Lübeck Germany

 Laboratory partners for IVD for autoantibody

diagnostics, infectious serology, allergy, https://www.britannica.com/place/Lubeck

antigen detection and molecular diagnostics

 Distribution in 150 countries

 Focuses on Quality, R&D and Guidelines

More Than 30 Years of Innovation and Beyond

2019 RA Analyser 10
Random Access Instrument for Autoimmune Serological Diagnostics
2018 EUROArray

2017 First ZIKA Serological CE-IVD Test Kit

2016 First MERS-COV Serological CE-IVD Kit

2014 EUROPattern
Automated Immunofluorescence Slide Processor & Interpretation
2009 EUROPattern Software
Evaluation software for ANA (Anti-nuclear Antibodies)
Accreditation of ifQ
2008 Institute for Quality Assurance Lubeck

2005 Institute for Experimental Immunology

Scientific activities of researching new antigens and developing innovative methods
2003 First SARS Serological Diagnostic CE-IVD Kit

1996 EUROPLUS Technology

combination of tissue sections and antigen spots

BIOCHIPs
1983 for tissue sections, antigen dots, blot strips and nucleotide arrays
TITERPLANE Technique
1979 Immunofluorescence substrates are placed onto the droplets from above)
EUROIMMUN Academy

Annual educational sharing of latest research results and

developments in latest research results of serological
biomarkers

Singapore Germany
EUROIMMUN Quality Assessment

Participation from 40
over countries
EUROIMMUN System Solutions

Reagents
+
Automation
+
Software
All from one source +
Support

IIFT ELISA Immunoblot Array RealTime-PCR

Autoimmune & Infectious Diagnostics

Autoimmune Infectious
Infectious Diagnostics
Allergy Diagnostics
 Allergy Prevelance

Allergy in Germany
45
Allergy prevalence(%)

40
35
30
25
20
15
10
5
0
1980 1990 2000 2010

• Worldwide, sensitization rates to one or more allergens are currently 40-50 %

• About 60 % of allergic patients are polysensitized
• Allergy: Third most common chronic disease in children under 18 years

Source: GA²LEN (Global Allergy and Asthma European Network,http://www.aaaai.org/about-aaaai/newsroom/allergy-statistics

 Allergy worldwide

Allergies now affect up to 30% to 40% of the population worldwide

with the children and young adults bear the greatest burden of
these diseases as the escalation of the prevalence of the diseases is
mostly seen in these populations (Gatot Soegiarto et. al. 2019. Asia Pac Allergy)

https://www.cbc.ca/natureofthings/features/map-of-allergies-around-the-world
 Allergy Epidemiology in Indonesia

The prevalence of allergic diseases in school children of metropolitan in

Indonesia shows a similar pattern to that of developed countries
Gatot Soegiarto et. al. 2019. Asia Pac Allergy

“Indonesia has the lowest prevalence

of asthma, allergic rhinitis, and
eczema symptoms as showed by the
results of The International Study of
Asthma and Allergies in Childhood
(ISAAC) in 2007.

Surabaya is the second biggest city

in Indonesia, but unfortunately was
not involved in the ISAAC study”

Gatot Soegiarto et. al. 2019. . The prevalence of allergic diseases in school children of metropolitan in Indonesia shows a similar
pattern to that of developed countries Asia Pac Allergy. 2019 Apr;9(2):e17. https://doi.org/10.5415/apallergy.2019.9.e17
Epidemiology of Global Allergic Diseases in
Children

Disease Name Time of Onset Incidence rate

Allergic asthma 6-7 years old Up to 25%
Allergic rhinitis and conjunctivitis 6-7 years old Up to 20%
Skin allergic disease 6-7 years old Up to 18%
Chronic diarrhea Infant 3-5%
Food allergy Child 3-5%

Pediatric Allergy and Immunology 24 (2013) 195–209

Allergy

Chronic condition involving an abnormal reaction to an ordinarily

harmless substance called an allergen.

Allergens can include aeroallergens such as dust mite, mold, and

tree weed and grass pollen, as well as food allergens such as milk,
egg, soy, wheat, nut or fish proteins.
Allergy symptoms

Hay fever Conjunctivitis Eczema Urticaria

Asthma Anaphylaxis Food allergies : vomiting, diarrhea,

respiratory symptoms or
anaphylaxis immediately after
ingestion of the culprit allergen

https://www.gesundheitsstadt-berlin.de/fast-jeder-dritte-deutsche-hat-eine-allergie-11257/
http://www.augenkrankheiten-ratgeber.de/bindehautentzundung-konjunktivitis/
https://www.dr-gumpert.de/html/ekzem_im_gesicht.html
http://urtikaria-netzwerk-bb.de/ueber-urtikaria/einfuehrung.html
http://www.focus.de/gesundheit/asthmasprays-erste-hilfe-wenn-die-luft-weg-bleibt_id_5771310.html http://diets-doctor.com/de/pages/851921
Types of Hypersensitivity & Characteristics

Type I Type Type Type IV

II III

Allergy Cytotoxic reaction Immune complex Cellular Immunity

IgE-mediated IgG/IgM-mediated IgG/IgM immune T-Cell mediated

complex
Within few minutes Within few hrs Within few hrs 24 hrs-several days

Allergic rhinitis HDN、Transfusion Streptococcal Contact Allergy

、hayfever Reaction nephritis、SLE
 Type I Hypersensitivity Mechanism

First
allergen APC
1. Sensitisation
contact

T-cell
 1st allergen contact

B-cell
 Memory development
Second
allergen
contact  No symptoms induced
IgE

2. Allergy
Mast cell
Basophile

Mediators
(Histamine etc.)  Sensitisation
 Clinical symptoms

Blood vessels Muscles Mucous gland Nerve fiber

Types of Allergen

Ingestion Inhaled
allergen allergen

Allergen

Contact Injectable
allergen allergen
Why Test for Allergen?

▪ Specific Diagnosis of Allergic Diseases

▪ Allergy testing is a prerequisite for the diagnosis of allergic diseases

▪ Specific Treatment for Allergic Diseases

▪ Avoid allergen measures

▪ Specific immunotherapy (desensitization treatment)

▪ Patient education

▪ Early assessment of high-risk infants who may develop into allergic

diseases in the future
 Allergy Diagnosis - Guidelines

Anamnesis Skin Prick

In-Vitro Provocation
Diagnosis test

http://www.drsavant.in/latest-update/allergy-patch-testi/24
 Allergy Diagnosis - Guidelines

Study incidence of systemic reactions to skin prick tests

Rate of systemic reactions after SPT was 77 per

100,000 patients, UK allergy clinic 2007-2013
Skin Prick

Tazocin: showing a rapidly

extending wheal with
widespread erythema

Treated with intramuscular

Within minutes she developed
epinephrine, intravenous
rhinoconjunctivitis, wheeze, dyspnea,
antihistamines, intravenous
and tachycardia
steroids, and nebulizers
Sellaturay P et. al. The incidence and features of systemic reactions to skin prick tests. Ann Allergy
Asthma Immunol. 2015 Sep;115(3):229-33. doi: 10.1016/j.anai.2015.07.005
 Allergy Diagnosis - Guidelines

Anamnesis Skin Prick

In-Vitro Provocation
Diagnosis test

http://www.drsavant.in/latest-update/allergy-patch-testi/24
Singleplex versus Multiplex Testing

Singleplex Multiplex

• Single allergen tubes • Ready-to-use profiles:

• Challenge: Indication- and country-specific
Optimal combination of allergen for • Comprehensive results
testing within a single incubation
• Evaluation:
Further testings required?
Singleplex versus Multiplex Testing
Multiplex
• Ready-to-use profiles: Indication-
and country-specific
• Comprehensive results within a
single incubation
Advantages:
• Simultaneous determination of
specific IgE to a multitude of
different allergens
• Extensive, individual
sensitization profiles from a
single analysis
• Identification of cross-
reactivities
• Precise identification of specific
immunotherapy, particularly in
cases of polysensitization (DPA-Dx
profiles)
EUROLINE – Focused and Comprehensive
EUROLINE – Multiparameter Test

= Immunoblot-based test for a semi-quantitative measurement of specific serum IgE

Indicator CCD marker

Membrane chips with allergens (optimal condition for each allergen)

Features of the EUROLINE:

• Efficiency:  up to 54 allergens on one profile within one incubation

 cost and time efficient
• Optimisation:  small sample-volume required, ideal for pediatrics

• Processing:  easy-to-handle, ready-to-use profiles

 manual processing or individual automation solutions

Efficient Automation

Semi-automated Fully automated

EUROLine Master EUROBlot ONE

Semi-automated Fully automated

Obtain > 2300 single results in less than 4.5 hours

Test evaluation: EUROLineScan Software
Test evaluation: EUROLineScan Software
EUROLINE – Multiparameter Test

= Immunoblot-based test for a semi-quantitative measurement of specific serum IgE

Indicator CCD marker

Membrane chips with allergens (optimal conditionfor each allergen)

Features of the EUROLINE:

• Efficiency:  up to 54 allergens on one profile within one incubation

 cost and time efficient
Targeted and comprehensive analysis of patient’s situation
• Optimisation:  small sample-volume required, ideal for pediatrics

• Processing:  easy-to-handle, ready-to-use profiles

 manual processing or individual automation solutions

What is the allergy situation in
Indonesia?
 The prevalence of allergic diseases in school children of metropolitan
in Indonesia shows a similar pattern to that of developed countries
Gatot Soegiarto et. al. 2019. Asia Pac Allergy

Prevalence of allergic sensitization in Surabaya school Common allergens in sensitized Surabaya school children
children

There was a trend of increasing allergic sensitization
among school children and undergrad students
(against 45 allergen extract tested)
House dust mites were the most common allergens in
secondary school children and undergraduate student.
Cockroach and mold spore were quite dominant in all of
the groups, and being the most common allergen in
primary school children

Gatot Soegiarto et. al. 2019. . The prevalence of allergic diseases in school children of metropolitan in Indonesia shows a similar
pattern to that of developed countries Asia Pac Allergy. 2019 Apr;9(2):e17. https://doi.org/10.5415/apallergy.2019.9.e17
The prevalence of allergic diseases in school children of metropolitan
in Indonesia shows a similar pattern to that of developed countries
Gatot Soegiarto et. al. 2019. Asia Pac Allergy

Inhalation allergen testing is important

in Indonesia because of the high
sensitization in the population and the
relatively higher risk of causing life-
threatening conditions
 Allergy Testing in Indonesia

Serum specific IgE responses to inhalant allergens sensitization

Iris Rengganis
Suriani Alimuddin
Agus J. Susanto

Patients (19–60 YO) with a history of respiratory allergy in

Jakarta, Indonesia between September and December 2016

Specific IgE Testing at Allergy and Immunology Clinic

Cipto Mangunkusumo Hospital, Jakarta

Evaluate specific IgE production of common inhalant allergens in

patients with asthma and/or allergic rhinitis in Jakarta, Indonesia

Rengganis, et al. 2017. Specific IgE to inhalant allergens. Medical Journal of Indonesia
 Allergy Testing in Indonesia

Serum specific IgE responses to inhalant allergens sensitization

House Dust Mite

Dermatophagoides pteronyssinus
Blomia tropical
Dermatophagoides farinae

Accounts for 73%

IgE Sensitization in
this study

Rengganis, et al. 2017. Specific IgE to inhalant allergens. Medical Journal of Indonesia
 Allergy Testing in Indonesia

Serum specific IgE responses to inhalant allergens sensitization

Co-sensitization among
inhalant allergens is also
common, and 80% occurs
with dust mites.

Importance of
multiplex testing

Rengganis, et al. 2017. Specific IgE to inhalant allergens. Medical Journal of Indonesia
EUROLINE Profiles

Different countries, different needs!

…more than 90 indication and country-specific profiles

 Atopy Indonesia 1 Atopy
Order #: DP 3707-1601 E

Allergen (n = 32) Allergen (n = 32) Allergen (n = 32)

Bermuda grass Honey bee venom Codfish
Timothy grass Cockroach (German) Tuna
Grass mix 5: Sweet vernal grass, Candida albicans Salmon
Bermuda grass,Timothy grass, Mould mix 1: Penicillium notatum, Crab
Cultivated rye Cladosporium herbarum, Aspergillus Shrimp (Pacific)
Acacia fumigatus, Alternaria alternata Lobster
White pine (Australian) Wheat flour Duck meat
Oil palm Gluten Beef, cooked
Dermatophagoides pteronyssinus Egg white Pork, cooked
Dermatophagoides farinae Cow‘s milk Chicken
Dermatophagoides microceras nBos d 4 Alpha-lactalbumin (Milk) Lamb meat
Tyrophagus putrescentiae nBos d 5 Beta-lactoglobulin (Milk) Cheddar cheese
Glycyphagus domesticus nBos d 8 Casein (Milk) Tomato
Blomia tropicalis Chocolate Garlic
Horse Peanut Strawberry
Dog Soybean Kiwi
Cat Hazelnut Shellfish mix 1: Spiny
nBos d 6 BSA (Milk) Feather mix 1: Almond lobster, Oyster, Clam
Chicken feathers, Duck feathers, Goose Baker‘s yeast Coffee
feathers, Glutamate CCD marker Indicator
Kapok
Inhalation Profile Inhalation

Inhalation India Inhalation Venezuela 1

Order #: DP 3120-1601 E Order #: DP 3135-1601-1 E
Allergen (n=20)
Timothy grass Allergen (n = 32) Allergen (n = 32)
Cultivated rye Bermuda grass Aspergillus fumigatus
Cultivated corn Rye grass Candida albicans
Eucalyptus Alternaria alternata
Johnson grass
False ragweed
Mugwort Dermatophagoides pteronyssinus
Carnation flower
sunflower Dandelion Dermatophagoides farinae
House dust mite mix 1: Dermatophagoides Plantain (English) Blomia tropicalis
pteronyssinus , Dermatophagoides farinae Honey bee venom
Rough pigweed
Dermatophagoides farinae White-faced hornet
Cat
Cockroach (German) Common wasp venom
Dog
Cat Paper wasp venom
Dog Budgerigar feathers Fire ant
Pigeon feathers Chicken feathers Mosquito
Chicken feathers Cockroach (American)
Duck feathers
Aspergillus fumigatus Ascaris
Trichophyton mentagrophytes Goose feathers
Echinococcus
Cotton Latex
Penicillium notatum Anisakis
Straw dust CCD marker Indicator
Jute Cladosporium herbarum
Sheep wool
CCD Marker Indicator
Case Studies
Case Study 1

Antoni 15 Allergens Positive False positive?

CCD Blocker

0 Allergens Positive
Case Study 1 – CCD Blocker

Identification of patients with anti-CCD IgE antibodies

CCD marker band on every EUROLINE profile

A positive CCD marker indicates the presence of anti-CCD IgE antibodies

Elimination of anti-CCD IgE antibodies to avoid false positive results?

Anti-CCD Absorbent (ZD 3001)

What is CCD?

• Secretory pathway: Glycan structures are added to the protein

• Glycan pattern in invertebrates and plants is different from human
= Cross-reactive Carbohydrate Determinants (CCDs)

Specific IgE antibodies to peptide epitopes

= allergic patients

Anti-CCD IgE
• usually clinically not relevant
• highly cross-reactive
(„mimicry“ of polysensitisations )
• approximately in 20 % of allergic patients
 Anti-CCD Absorbent

• Stability: - storage of reconstituted reagent up to 6 months on -20 °C

• Processing: - simple one-step preincubation

• Evaluation: - automated interpretation by EUROLineScan-Software

• Internal check:- CCD marker on each profile allows tracking neutralisation efficiency

The patient must avoid far fewer allergens than originally diagnosed
Case Study 2

Peanut allergy

Lisa Tom

Lisa is sensitized to several storage Tom is sensitized to Bet v1 from birch

proteins (Ara h1, h2, h3) (Ara h8 homologue) which triggers
cross reaction
Case Study 2 Cross Reactive Components

Birch Peanut

Bet v1
Bet v2 Ara h2
Ara h8

Cross-reactive components:

Structural similarity between a wide range of allergen sources

DPA-Dx Pediatrics

 Risk assessment of severe allergic reactions

 Only small amount of serum (100 – 400 uL) required – ideal for pediatrics
 From sample to report < 3 hours
 Standardised evaluation according to EAST (Enzyme Allergosorbent Test)
class system, using the EUROLineScan software
DPA-Dx Pediatrics
Lisa
Strict avoidance of allergen source.
To always carry an emergency set
Tom
Milk symptoms of allergy upon contact
with peanut due to cross reactivity.
Diagnosed with pollen-associated food
allergy which can be alleviated by SIT
(Specific Immunotherapy)
DPA-Dx Pediatrics

Milk Allergen Components Egg Allergen Components

heat labile
• Lactoferrin (Bos d)
• α-Lactalbumin (Bos d 4)
• β-Lactoglobulin (Bos d 5)
• Serum Albumin (Bos d 6)
heat labile
• Ovalbumin (Gal d 2)
• Conalbumin (Gal d 3)
• Lysozyme (Gal d 4)

Babies allergic to
heat labile milk
component can
heat stable
consume after the
Babies allergic • Ovomucoid (Gal d 1)
milk is heated
heat stable to certain egg
• Casein (Bos d 8)
component can
avoid vaccines
that contains it
EUROIMMUN DPA-Dx Profiles

DPA-Dx Insect Venoms

DPA-Dx Pediatrics

DPA-Dx Peanut DPA-Dx Food DPA-Dx Pollen

Summary

 Simultaneous determination of specific IgE to a multitude of different

allergens
 Sample efficiency
 Cost and time efficiency

 Semi and fully automated incubation, fully automated results evaluation

 Advantages of indication for possible CCD interaction
 Molecular analysis (DPA-Dx) for efficient treatment & patient
management
 Results guaranteed by participation in Institute for Quality Assessment, IfQ