Allergic Disease

Prof. Rusudan Karseladze

Iv. Javakhishvili Tbilisi State University
Institute of Pediatrics
Bronchial asthma: age-specific
practice
(review)

Prof. Rusudan Karseladze

Prof. Lia Zhorzoliani
Tbilisi State University
Institute of Pediatrics
Batumi, 2016
Allergic diseases
18 cen. 2140 y b.c. –
pharaoh of Egypt
died with
anaphylactic
1906 - „allergy“ –
(„allos ergeia“) -
Pirquet,1906
Allergic Diseases:
A Global Public
Health Concern
 Why is allergy a major problem
worldwide?
 There has been a steady increase the prevalence of
allergic diseases globally.

 It is estimated that 30-40% of the world population is

now affected by one or more allergic conditions.

 A high proportion of the increase in allergic disease is

in young people. In the near future, the burden of
allergic diseases is expected to greatly increase as
these patients become adults.
 Asthma Burden

• According to WHO, the world's 300 million people suffer from asthma.

• lethality is from 0.2 to 18%.

• The prevalence of asthma is increasing in all countries, ranging from 1 to

18%. Significantly reduces the
quality of life
• Asthma is the most common.
•       in childhood and is 5 to 10% of the child population.

• In 50-80% of children with asthma symptoms first appear under the age
of 5 years.

GINA 2014
 The economic burden
of allergy
A few global facts and figures for two common allergic diseases, asthma and rhinitis :
Country Year costs Population Disease Direct costs* Indirect costs** Total costs
calculated (2010) estimated

Australia 2007 23 million All allergies A$1.1 billion A$8.3 billion A$9.4 billion

Finland 2005 5.3 million All allergies €468 million €51.7 million €519.7 million

South 2005 50 million Asthma US $1.78 billion

Korea Allergic Rhinitis US $266 million

Israel 7.5 million Asthma US $250 million

Mexico 2007 103 million Asthma US $35 million

USA 2007 310.2 million Asthma US $14.7 billion US $5 billion US $19.7 billion
2005 Allergic Rhinitis US $11.2 billion Up to US $9.7 Up to $20.9
billion billion

* Direct costs: Expenditure on medications and health care provision

** Indirect costs: Cost to society from loss of work, social support, loss of taxation income, home
modifications, lower productivity at work, etc
Source: WAO White Book on Allergy (Member Society Reports)
ACAAI AAAAI WAO

EAACI
ATS CIA

ALA ERS

NHLBI EFAAA

NIAID Organizations UCB Institute of Allergy UCB

ARIA
Interashma

GA2LEN
WHO

GOLD GARD
GINA
 International documents Global Initiatives

 GINA - Global Initiative for Asthma

 ARIA _WHO’s Global Initiative for
Allergic Rhinitis and its impact on
Asthma
 GOLD – Global Initiative for Obstructive
Lung Diseasses
 Global Initiative for Chronic Obstructive
Diseasses (COPD)
 GARD –

• DOCUMENTS: PRACTALL- EAACI / AAAAI CONSENSUS,

WAO –World Allergy Organization, UCB, GLORIA – Global
Resources of Allergy
 Defining the Problem:
The WAO White Book on Allergy

The WAO White Book on Allergy outlines

data indicating that allergy poses a major
global public health issue;

it provides high level recommendations to

governments, health authorities and other
health care providers.
 Bronchial Asthma: international
documents

Allergy 67 (2012) 976-997 © 2012 John Wiley & Sons A/S

AAH

GINA 2014/2015/2016
 Allergic Disease
• Dramatic increase in 60%
allergic disease over 50%
the past three 50%
decades, why is
this? 40% 30% - 35%
• Genetics 30%
• Environmental 22%
factors - pollution 20%
14%
17%
• Changes in Lifestyle 8%
• Occupational 10%
3,5% 3% 5%
0%
  0%
1926 1958 1968 1981 1985 1987 1989 1995 2000 2050
 Allergy is a systemic disorder

Nose
Pharynx
Allergic rhinitis
Asthma
Lungs
Oesophagus

Stomach
Food allergy
Skin
Eczema
Urticaria
Allergic dermatitis

“Global diseases” – due to the large spectrum of symptoms

affecting the whole body
 The Main Immunological
Mechanism

Micro
b
Unkno
protein
wn

Protect Th 1 Th 2 Allergy

Immunne reaction of Adults : Th1/Th2 balance

The Main Immunological Mechanism
of newborns

Protec
t
Th 1 Th 2 allergy

Th2-assimetry
 PPathogenesis
Early and late reactions
early: (madiated—histamin, maybe leucotriens)
late: (mediated—leucotriens and cytocins )
– eozinofiluri infiltracia, anTeba

Secretion in right upper

lobe in an asthmatic patient.
 Allergy-systemic process

Lungs
Bronchial asthma
Cutaneus LOR-organs
Atopic Dermatitis Allergic Rhinitis,
Allergic evstachitis
Urticaria

IgE
Cardiovascular Eyars
system Allergic
conjunctocotis
Gastro-
intestinal tract

Bosquet J, Allergy 2002, Aug 57;8:661-662

 Genetics (1)
• Family history of allergic disease is a
strong risk factor for developing asthma
• Danger of developing asthma particularly if
one or both parents are atopic
• Children with atopic dermatitis at risk of
asthma -– “the allergic march”
 Genetics (2)
• No single "allergy or asthma chromosome".
Several markers demonstrated in small
selected populations - much further work is
required
• The genetics of allergy and asthma are
polygenic - influence many factors such as
IgE secretion, cytokines and inflammatory
cell profiles
 Environment (1)
• Children & adults 90% spent time indoors
• Allergens in dust (dust mite faeces) or pets (particularly
cats) - increased risk of allergic sensitization in
proportion to exposure.
• Most children and adolescents with asthma sensitized
to indoor allergens - avoidance often leads to
improvement in airway disease.
• Modern housing generally poorly ventilated with fitted
carpets and central heating - house dust mite infestation
 Environment (2)
• Children exposed to tobacco smoke more likely to
develop wheezing and impaired lung function
• Outdoor allergens –seasonal variation and weather
• Account for 10-20% of allergic disease in Europe -
mainly hay fever. 
• Increased pollution not responsible for increase in
allergic disease - pollutants worsen respiratory
symptoms in asthmatics and reduce lung function
 Changes in Lifestyle (1)
• Hygiene hypothesis - Past 30 years - changes in pattern
of childhood infection, many no longer experienced
• Exposure to certain infections may protect against the
development of allergies.
• Respiratory viruses may be a risk factor for the
development of asthma
• Vaccination programmes not thought to have direct
effect on the development of allergic disease
 Changes in Lifestyle (2)
• Intake of fresh fruit and vegetables has declined
leading to lower anti-oxidant levels.
• Certain fatty acids are able to shift the immune
system towards allergic susceptibility
• Food preservatives may effect gut flora leading
to allergic sensitization rather than development
of tolerance
 Changes in Lifestyle (3)
• The immune system is severely
compromised by poor nutrition
• Paradoxically the vast improvement in
nutrition in the last fifty years might have
led to the immune systems of some
individuals "over reacting" to benign
substances i.e. allergens
 Every child has the potential risk of developing
allergies

Low risk Moderate risk High risk

An allergy One of the Both parents
is not parents has an have allergy
allergy risk symptoms

Allergy in Family

Children with allergy

diseases
%
 Allergic Disease
• Seen in 30-35% of the population
• Perennial & seasonal allergic rhinitis
• Allergic (extrinsic asthma)
• Atopic and contact dermatitis
• Urticaria
• Food intolerance
 Allergy
• Elevated IgE levels seen in allergy and
parasitic infection
• Binds to mast cells and basophils
• Often specific for harmless
environmental factors - allergens
Identified Genes of Atopy
 Allergens
• Environmental substances

• Usually benign

• Sub-group of individuals exhibit a

hypersensitivity reaction (type 1)
Allergens
Mite faeces (digestive
enzymes)
Pollen
Animal dander (cats)
Insect stings
Food
 IgE
Allergen

Mast Cell

Histamine
Crosslinking
release
 Allergy

Inflammation
Beneficial Harmful

Removal of insult 1.Persistence or

2.constant exposure

RESOLUTION HYPERSENSITIVITY
 IgE
• Very low serum concentration – 0.00005 mg/ml)
• Sensitises mast cells and basophils by binding via Fc
portion to high affinity receptor – FcR1
• Serum half life of a few days
• Binding protects IgE from destruction by serum
proteases
• Sensitisation can last for many months
• Detected by skin prick test or radio absorbant test
(RAST)
Allergy – an inappropriate
immune response
 Allergy – an inappropriate
immune response
• Parasite larvae – proteases

• House dust mite – faeces (skin) – proteases

• Pollen – proteases

• Cat saliva - proteases

Mast cells and basophils
Mast Cell
Mast cells 
Release pre-formed mediators
(histamine) and lipids together
with several TH2 cytokines
 Anaphylaxis
• Very acute and severe reaction to allergen
• Peanuts, shellfish, penicillin, insect stings
• Allergen moves from gut to blood stream
• Massive histamine release from mast cells
and basophils
• Vasodilatation leads to dramatic drop in
blood pressure
• Often fatal if not treated with adrenaline
 Allergic rhinitis
• Seasonal (pollen, spores) or perennial
(house dust mite)
• Mucus production (Runny nose, nasal
stuffiness
• Itching & sneezing
• Treat with antihistamines or nasal steroids
 Urticaria
• Wheal and flare
• Itching
• Allergen-induced
• Idiopathic – pressure, cold etc.
• Food – shellfish, strawberries, peanuts
• Treat with antihistamines
 Atopic dermatitis
• Allergen –induced particularly milk protein
from the gut enters blood stream –deposited
in skin – mast cell degranulation
• Exfoliating eczema and itching
• Treat with antihistamines
• May progress to asthma
 Drug Allergy – Clinical types

• Cutaneus reactions
(80%)
• Anaphylaxis
(9 - 15%)
• Respiratory system
(6 - 9%)
• Drug Hyperthermia
(2 - 6%)
Skin prick test
 Allergic Inflammation
• Much more complex than histamine
release

• Involvement of a whole host of cells,

cytokines, chemokines and mediators
Granule proteins Cytokines
IL-3, IL-4, IL-5
MBP, ECP, EPO GM-CSF, IL-6
IL-12, TGF-

Epithelial damage/loss Attract/activate eosinophils

Muscarinic M2 Airway remodelling, IgE,
dysfunction/ AHR Th2 polarisation

LTC4, PAF Chemokines

Eotaxin, RANTES

Mucus hypersecretion
Airway narrowing Attract/activate eosinophils
Attract/activate pro-inflammatory cells
 Mast Cells
Mediators: histamine, prostaglandins,
PAF, LTC4 & LTD4

Mucosal oedema, vasodilation,

mucus secretion, bronchial
smooth muscle contraction
 Mast Cells
Cytokines (e.g. IL-4, IL-5,
TNF, IL-8): LTB4, PAF

Attract and activate neutrophils &

eosinophils
 Connective tissue Mucosal
Mast Cell Mast Cell

Gut & lung

Ubiquitous T cell dependent
Long lived >40 days Short lived <40 days
3x104 IgE receptors 25x105 IgE receptors
High histamine content Lower histamine content
Heparin & high tryptase Chondroitin sulphate
Lower tryptase
 Histamine
• Skin – wheal, erythema, pruritis
• Eye - conjunctivitis, erythema, pruritis
• Nose – nasal discharge, sneeze, pruritis
• Lung – bronchospasm of smooth muscle
 Histamine
• Therapeutic intervention in allergy
often focused on blocking the effects
of histamine
• Histamine also functions as a
neurotransmitter in CNS
• Very important in maintaining a
state of arousal or awareness
 First Generation Antihistamines

• The first H1 antagonist synthesised by

Bovet & Staub at the Institut Pasteur
• Too weak or toxic
• Phenbezamine first effective antihistamine
• Mepyramine maleate, diphenhydramine &
tripelennamine developed in 1940’s
• Still in use today
 First Generation Antihistamines
• Easily cross the blood–brain barrier.
• Sedative and anticholinergic effects (sedating antihistamines).
• Short half-lives.
• Limited use in the treatment of allergic symptoms.
• Still widely used, mainly as over-the-counter products, often in combination with other drugs.
 Second Generation Antihistamines

• Highly effective treatments for allergic disease

• Do not cross blood-brain barrier
• Lack significant CNS & anticholinergic effects
• Long half life
• Among the most frequently prescribed and
safest drugs - expensive
 Other treatments
• Nasal steroids – must be given before
season – relieve nasal blockade
• Antihistamines combined with anti-
leukotriene drugs
• Avoidance -mattress covers, specialised
Hoovers, wood floors,
 Global Resources in Allergy (GLORIA™)

Global Resources In Allergy (GLORIA™) is the

flagship program of the World Allergy
Organization (WAO). Its curriculum educates
medical professionals worldwide through
regional and national presentations. GLORIA
modules are created from established
guidelines and recommendations to address
different aspects of allergy-related patient care.
 World Allergy Organization (WAO)

The World Allergy Organization is an

international coalition of 74 regional
and national allergy and clinical
immunology societies.
 WAO’s Mission

WAO’s mission is to be a global

resource and advocate in the field of
allergy, advancing excellence in clinical
care, education, research and training
through a world-wide alliance of allergy
and clinical immunology societies
 Nomenclature of allergy

Hypersensitivity

Allergic hypersensitivity Non-allergic hypersensitivity

(immunological mechanism (immunological mechanism
defined or strongly suspected) excluded)

IgE-mediated Not IgE-mediated

Johansson SGO et al. Allergy 2001 and JACI 2004

 Atopy

Atopy is a personal and/or familial tendency, usually

expressed anytime in life from childhood and
adolescence, into maturity, to become sensitized and
produce IgE antibodies in response to ordinary
exposures to allergens, usually proteins.
As a consequence, atopic persons can develop IgE-
mediated allergic diseases including asthma,
rhinoconjunctivitis, or eczema.
WAO Nomenclature Review Committee
Johansson et al. J Allergy Clin Immunol 2004;113:832-6
Allergic Disease Progression with Age

Saarinen UM et al. Lancet 1995

Pathophysiology of an Allergic
Reaction
The Essential Components of Allergy
Diagnosis

Clinical History and Physical Examination

Symptoms versus Exposure

Diagnostic Confirmatory Test

Skin Test (Puncture, Intradermal)
Allergen-specific IgE antibody serology

Provocation Test
Oral, Nasal, Bronchial Challenge
 Key Concepts in Allergy Diagnosis
• A proper allergy history involves determining the symptom complex, any
relationship to allergen exposure and a careful physical examination,
looking for the specific signs of allergy.
• Once allergic disease is suspected, a confirmatory test (skin test or IgE
antibody serology) is performed to verify sensitization by the presence of
allergen specific IgE antibody.1-3
• Where it can be performed and interpreted, skin prick testing (SPT) remains
the primary confirmatory test because it is fast, safe, sensitive, minimally
invasive and results correlate with nasal and bronchial challenges.
• Quantitative IgE antibody serology is an accepted alternative.
• SPT and/or IgE serology are essential adjuncts to history and physical exam
when making the diagnosis of allergy.
• Provocation
1. Oppenheimer tests are sometimes
Ann Allergy 2006;S1:6-12,needed toClin
2. Bousquet confirm sensitization.
Allergy 17:529-36, 1987

3. Cockroft Am Rev Respir Dis 135:264-7., 1987

 Allergy History
• Demographics (age)
• Symptoms: frequency and severity
• Pattern: intermittent, persistent or seasonal
• Response to environmental factors:
– Temperature changes, odors, humidity, alcohol
• Occupation and hobbies
• Identification of allergens/irritants in the home,
office or environment
• Treatment, past and present: efficacy, compliance,
side effects
 Allergy Symptoms
Clinical History Drives the Diagnosis
• Hypersensitivity to an injected, ingested, or inhaled
antigen in response to a first exposure.
– Skin: itch, rash, swelling, redness
– Eyes: itchy, tears, watery, redness, crusting
– Nose: runny, itchy, congestion, sneezing
– Lung: wheezing, cough, tightness, shortness of breath
– Stomach-Intestines: nausea, vomiting, bloating, diarrhea
– Heart-Blood Vessels: anaphylaxis, syncope, faintness,
death
Allergy Physical Examination:
The Everted Eyelid
Allergy Physical Examination: The Swollen
Nasal Mucosa
 What is an Allergen?
An antigen causing an allergic disease is called an “allergen”.
Most allergens initiating an IgE-antibody mediated allergic
reaction are glycoproteins with a molecular weight of 5 to 100
kD, most around 20 kD.
Many pollen allergens are surface enzymes.

Some food allergens are remarkably stable and are stable even
after cooking.
A genetically predisposed (atopic) person can become IgE-
sensitized after several years of inhaling <1 µg of grass pollen
allergen per season.
Spectrum of Allergen Sources
 Allergen Extracts
• An allergen extract used for diagnosis or treatment is prepared by
incubating the allergenic material in a physiological buffer (e.g.,
phosphate buffered saline) followed by lipid extraction.

• The allergen content was commonly expressed in crude terms such

as protein nitrogen units (PNU) or weight:volume, but it may now
be expressed as micrograms of specific allergen per ml.

• Several commercial extracts used in skin testing are “standardized”

regarding allergen protein concentration, composition and lack of
irritating contaminants.

• In some countries such as the USA, grass, ragweed, dust mite and
cat allergens are currently standardized
 Selection of Aeroallergens
• An evidence-based approach that minimizes irrelevant test
antigens can reduce patient discomfort and costs.
• An understanding of pollen aerobiology and knowledge of
allergenic cross-reactivity between regional pollinating plant
families is necessary in selecting appropriate aeroallergen test
panels.
• Extensive allergenic cross-reactivity exists between northern
pasture grasses, permitting the use of a single northern grass
pollen for testing in most regions outside of southern regions
of North America and Europe
Practice Parameters for Allergy Diagnostic Testing
Ann Allergy 1995; 75:543-625
 Allergy Diagnosis - Definitions
• Sensitivity: proportion of subjects with allergy who test positive

• Specificity: proportion of subjects without allergy who test negative

• PPV: probability that a subject has allergy if they test positive

• NPV: probability that a subject does not have allergy if they test negative

• Efficiency: % of allergy patients correctly classified as diseased and

not diseased
Skin Testing and IgE Antibody Serology

Powerful adjuncts for confirming allergy in:

• Rhinitis and sinusitis
• Asthma, cough, dyspnea
• Eczema
• Food allergy
• Insect sting allergy
• Drug allergy (some i.e. beta-lactams and local anesthetics)
• Occupational (some)
• Anaphylaxis
Confirmatory Skin Testing
 Use of Skin Prick Tests (SPT)
• Diagnosis of allergy
• Confirmatory evidence (positive, negative) of IgE
sensitization in support of the clinical history
• Identifies the allergen against which IgE is specifically
directed, which is essential for allergen avoidance
measures
• Educational value: visual reinforcement strengthens
compliance of verbal advice
 General Rules for Successful SPT
• It is imperative that the technician performing the skin tests as well as the
clinician ordering/interpreting these tests understands the characteristics of
the specific tests they are administering.
• This includes:
– type of skin testing
– device used
– placement of tests (location and adjacent testing)
– the particular extracts (source, concentration) being used
– the potential confounder of medications that may suppress skin test
response.
 Skin Prick Testing
• SPT is easy to perform and rarely causes generalized reactions.

• Patients may have positive SPT but no clinical disease. A

positive SPT indicates the presence of IgE antibodies against
that allergen but does not indicate clinical sensitivity. A
correlation between the history and SPT is essential

• The results can be unreliable if the patient takes certain drugs,

such as anti-histamines and tricyclic anti-depressants.
Skin Prick Testing Solutions
Skin Prick Testing
Not all Allergens are Available as a Skin
Test Extract: Fruit Prick-Prick Test
Prick-Prick Test Reactions
Skin Testing with Natural Foods in Subjects Suspected of
Having Food Allergy

• 22 patients with highly suspected food allergies but with negative

SPT to commercial extracts had positive prick-prick skin tests with
fresh natural foods:
- 7 fish and seafood
- 4 fruit and vegetable
- 9 peanut and tree nuts
- 1 milk
- 1 egg

Rosen. J Allergy Clin Immunol 1994;93:1068

 Puncture Skin Testing Devices
• There are several different devices
available for skin prick testing. QTS GTK

• These devices result in varying

degrees of trauma to the skin with
differing levels of skin test
reaction.
MT QNT
• Thus, the physician should be 2
familiar with the characteristics of
the device used in his/her practice,
as each require different criteria for
AS QT ST GP
what constitutes a positive reaction.
 Intracutaneous Skin Testing (ICT)
• ICT should be interpreted cautiously. Many positive reactions
(up to 70% according to some published reports) are not
clinically relevant.

• Because ICT uses larger volumes of injected allergen

preparations, there may be some irritant reactions not
mediated by an allergic mechanism. Many drugs may directly
stimulate mast cells to release mediators.

• The incidence of severe systemic effects, while rare, are more

likely to occur with ICT than with SPT
 Comparison of SPT and ICT
Advantages of SPT Advantages of ICT
• Safer More sensitive:
• More rapid (300 to >1000 fold)
• Less discomfort to patient More reproducible
• Technically less demanding More positives
• More specific
• More allergens in one session
• Allergen more stable (50% glycerin)
• Positive and negative tests more easily
distinguished
• Steeper dose response curve
• Positive tests correlate better with
clinical disease
 Recording Skin Test Responses
Results of both SPT and ICT skin tests should be reported in the most quantitative
terms possible.

• Reports of minimal usefulness include:

– Positive or negative
– 0 to 4+ (unless accompanied by an indication of what these numbers represent).
• Useful to report both wheal and flare measurements in mm:
– A superior method is to measure the reaction in mm across the cross-diameter
– Area (cross-diameter in mm) of the wheal and erythema is the most accurate
way to present results.
– Measurements of:
• the product of the orthogonal diameters
• the sum of the orthogonal diameters
• the longest diameters

–
Correlate very well with area (r values greater than 0.9 ). Ownby JACI 1982:69:536-
8
Are Skin Tests Easy to Interpret?
 Reproducibility of Skin Test Scoring and
Interpretation by Board-Certified/Eligible Allergists

• Methods:
– Series of SPT were digitally photographed
• 22 tests with controls
– a questionnaire regarding interpretation was sent to 70 allergists to
assess
• positive, negative or intermediate
• positive or whether a ICT test was desired

McCann Ann Allergy Asthma Immun 2002;89:368-71

 Reproducibility of Skin Test Scoring and
Interpretation by Board-Certified/Eligible Allergists
• Results:
– 33 interpretable responses
• 24 relied on a grading scale (0-4+);
• 2 measured in mm,
• 7 provided only interpretation with no grading
– Greatest agreement with median/mode score 4+
– Least agreement with median/mode score 1-2+
– Range of requested ICT test was 0-11 tests

• Conclusion:
– Significant variability in scoring and interpreting skin tests
– Reinforces the need to report skin test reactions by measuring and
recording reaction size in mm
McCann Ann All Asthma Immun 2002;89:368-71
 Inter-Individual Variation in SPT
Test result Nurse 1 Nurse 2 Nurse 3 Nurse 4 CV

Negative control 0.1 mm 0.4 mm 0.2 mm 0.2 mm 55.9%

Histamine 11.7 mm 9.7 mm 12.9 mm 14.5 mm 16.6%
Grass 2.1 mm 2.5 mm 4.7 mm 5.2 mm 42.8%
Mugwort 7.7 mm 4.8 mm 7.4 mm 9.1 mm 24.7%
Dog 1.5 mm 1.1 mm 3.0 mm 2.5 mm 43.3%
House dust mite 1.7 mm 2.2 mm 1.6 mm 2.8 mm 26.5%

CV = inter-individual coefficient of variation, Target < 25%; Vohlonen I et al. Allergy 1989; 44: 525-531
www.AAAAI.ORG
Suppression of Skin Tests by Medication

• Most antihistamines and anti-depressants suppress skin tests for 3-

7 days. Astemizole suppresses for 1-3 months.
• H2 antagonists have no, or a very minor, effect.
• Bronchodilators do not affect skin tests.
• Short-term and low dose oral corticosteroids have no effect.
– Reports vary on long-term high-dose use.

Cook J Allergy Clin Immunol 1973;51:71-7

Rao KS J Allergy Clin Immunol 1988;82:752-7
Miller J J Allergy Clin Immunol 1989;84:895-99
Slott RIJ Allergy Clin Immunol 1974;554:229-34
 Skin Test Safety

Review of surveys of fatal reactions to skin testing between

1959-2001
• 9 deaths associated with skin testing
• 1 death associated with SPT
– History of unstable asthma with FEV-1 36% 1 week prior
– Tested to 90 foods

Lockey JACI 1987;79:660-77

Reid JACI 1993;92:6-15
Bernstein JACI 2004;113:1129-36
 In-Vivo Provocation Tests
• Provocation tests involve the challenge of the affected organ by serial
dilutions of an allergen extract or by the actual, suspected allergen source
material, e.g. food or drug.
• A provocation test is time-consuming. It can result in dangerous clinical
reactions and should only be performed by experienced persons with
access to lifesaving equipment.

Due to space limitations, details of nasal, lung and insect sting challenge tests
will not be discussed further in this presentation.
 Food Allergy Diagnosis:
Oral Food challenges
Challenge types

Open
* useful when the history is vague and when the reaction is
likely to be negative (-ve specific IgE antibodies and unconvincing
history)

Single blind
* useful to confirm negative reactions
* useful to confirm non-subjective reactions

Double Blind Placebo Controlled (DBPCFC)

* gold standard - mandatory for research studies
* usually definitive; excellent for subjective reactions
* alternate placebo and active randomly
 Confirmatory
Total and Allergen-Specific IgE
Antibody Serological Testing
 Serological Tests Performed in
Diagnostic Allergy Laboratories
• Allergen-specific IgE (over 400 allergen specificities)
– Pollen (weeds, grasses, trees), Epidermals, Dust Mites, Molds, Foods,
Venoms, Drugs, Occupational allergens (Ispagula, Natural Rubber
Latex)
• Total Serum IgE (Xolair: anti-IgE; ABPA)
• Phadiatop (Multi-allergen screen) IgE (define atopy)
• Fx5e (Multi-allergen screening test for foods)
• Mast Cell Tryptase (indicator of anaphylaxis)
• Eosinophil Cationic Protein (eosinophil activation marker)
• Precipitin-IgG antibody (Hypersensitivity Pneumonitis, anaphylactic
reactions to dextran)
 Total Serum IgE Levels in Allergy
Patients with allergic asthma may have increased total serum IgE
concentrations, but this is not an allergy-specific finding:
• 60% of “allergic” asthmatics have increased IgE
• 40% of “allergic” rhinitis patients have increased IgE

Measurement of total serum IgE may be of value in patients with:

• Gastrointestinal symptoms/eosinophilic esophagitis
• Suspected occupational allergy with unclear genesis
• Anaphylaxis
• Allergic Bronchopulmonary Aspergillosis (ABPA)
• Allergic Fungal Sinusitis

Total serum IgE may be measured to determine the dosage of omalizumab

 Some Disorders
with Elevated Total Serum IgE Levels
• Helminth infestation, e.g. Ascaris, Schistosoma
• Infections with Staphylococcal strains containing enterotoxins, so called
“super-antigens”
• Virus infections, e.g. cytomegalovirus (CMV)
• ABPA and Allergic fungal sinusitis
• Graft Versus Host Disease (GVHD)
• Hyper-IgE Syndrome
 Serological Testing for Allergen-IgE
Antibody is Recommended when In-Vivo
Tests Cannot be Used
• When the patient is taking anti-histamines or other confounding
medications for skin tests
• When the patient has eczema or dermographism
• Immediately (up to 6 weeks) following an anaphylactic event
• If the patient is morbidly afraid of skin testing
 Allergen-Specific IgE
In-Vitro and In-Vivo Tests
In-vitro In-vivo
IgE Antibody SPT
Serology
High sensitivity Yes Yes
High specificity Yes Yes
High reproducibility Yes Yes
Quantitative results in kIU/L Yes No
WHO Standard calibrated Yes No
Quality assurance test program Yes No

Can be used independently Yes No

of pharmaceutical treatment

Can be used independently Yes No

of patient skin status
Time factor 1-7 days 15-30 minutes
Cost factor more expensive inexpensive
Usefulness in motivating patients obscure dramatic
 Evolution in Specific IgE Antibody
Assay Technology
1st generation: manual chemistries
• RAST® =Radio Allergo Sorbent test, Phadia* 1974
• Hycor Hy-Tec (paper disc based)
• FAST = Allergenics/Biowhittaker, fluorescent allergosorbent test
• MAST = Hitachi: thread pipette
• EAST = Sanofi Dignostics Pasteur
• Magic Lite = ALK/Corning/Bayer
• Matrix = Abbott

2nd generation (semi-automated chemistries)

• Alastat, Diagnostic Products Corp. (DPC, biotin-allergen)
• Pharmacia CAP System® , ImmmunoCAPTM 3D Solid Phase, Phadia* 1989

3rd generation (autoanalyzers)

• Immulite 2000: Diagnostic Products Corporation
• ImmunoCAP 250TM, 1000): Phadia* 2001

*Phadia is the new name of Pharmacia Diagnostics (2006)

 Evolution from Qualitative to
•
Quantitative IgE Antibody
1974-80: PRU/ml: Phadebas Relative Units
Results
• 1980s-1990s: AU/ml: AU = allergen unit
Normalized Counts, ASM (adjusted counts)
sIgE/ml, IU/ml, FSU/ml, VRU/ml
EAU/ml; Classes
• 1997: Clinical and Laboratory Standards Institute: LA20A: Evaluation
Methods and Analytical Performance of Immunological assays for human
IgE Antibody: (www. CLSI.org)
• 1989-present: kIU/L or kIUa/L (interpolation from total serum IgE
heterologous dose response curve traceable to WHO 75/502 IgE Serum
Standard) [1U = 2.44 ng of IgE]
Design of Serological Assays for Allergen
Specific IgE Antibody
Step 1:
Separate allergen-specific
IgE from other antibodies
present with a solid phase
allergosorbent
Step 2:
A buffer wash separates
bound IgE from unbound
antibodies
Step 3:
Bound IgE is detected with
a labeled anti-human IgE
reagent
JACI 2003;111:S687-701
 Solid Phase Allergosorbent
• As with skin testing, the allergen used in the solid phase is critical to the
validity of the test:
– Characterization of allergen
– Ensure that critical antigen epitopes are on the solid phase
– Antigen epitopes should be present in excess to maximize binding of
IgE antibody
– If not in excess, can have competitive binding from antibody of other
isotypes (IgG)
– Antigen excess binds IgE in an affinity-independent fashion
Illustration of a Widely Patient IgE

Used Assay Allergen coupled to

(ImmunoCAP® System) ImmunoCAP

for Allergen Specific IgE Conjugate;

Enzyme Anti-IgE

Quantification
Patient IgE ab bound to
ImmunoCAP allergen

Fluorogenic substrate

Conjugate bound to
patient IgE

Conjugate enzyme reacts with

substrate forming a
fluorescent product
The Clinical Validity of IgE Antibody
Serology Testing
Sensitivity 89%
Specificity 91%

UniCAP® Specific IgE

Physician’s conclusion Positive Negative Total
Positive 1121 144 1265
Negative 360 3545 3905
Total 1481 3689 5170

The clinical performance of UniCAP® Specific IgE was documented in clinical trials in six
countries: Italy, Spain, Germany, The Netherlands, Sweden and Great Britain,
on 894 patients with suspected allergy.
Clinical sensitivity and sensitivity were calculated as agreement between the test result and a
specialist diagnosis based on the established diagnostic routines of the clinic.
Paganelli R et al., Allergy 1998; 53: 763-8
 Proficiency and Quality Control
• Clinical and Laboratory Standards Institute Guideline:
Evaluation of Methods and Analytical Performance Characteristics of
Immunological Assays for Human Immunoglobulin E Antibodies of
Defined Allergy Specificities

• Recommends quality control for daily performance of testing with minimal

performance targets for IgE antibody assays:
– Intra-assay and Inter-assay coefficient of variation in IgE antibody assays
should not exceed 10% and 15%, respectively.
– Laboratories encouraged to participate in an inter-laboratory external
proficiency testing program: 3 cycles per year, 5 or 6 sera per cycle, measure
total IgE, multi-allergen screen, specific IgE to 5 allergen specificities in each
cycle
– Laboratories should be credentialed (licensed) by appropriate government
agencies
– Document available from CLSI (www.CLSI.org I/LA20 guideline)
Interpretation of Allergen-Specific IgE
Antibody Results
• Presence of allergen-specific IgE antibodies in serum indicates
sensitization. It does not equal clinical symptoms.
• Serum IgE antibody is an absolute prerequisite for the development of
IgE-mediated symptoms.
• With precise, quantitative assays, IgE antibody production can be
detected at an early stage, even before clinical symptoms have fully
developed.
Controversial: Unproven In-Vitro Allergy Tests
1. Tests with no diagnostic value for any disease under any circumstance
(not based on sound scientific principles):

* Cytotoxic test,
* Antigen leukocyte cellular antibody test (ALCAT)

2. Tests intrinsically capable of valid measurements; effective/appropriate

for diagnosing certain diseases but not for allergy:

* Serum IgG antibodies

* Total serum immunoglobulin (IgG/IgA/IgM)
* Cytokine/cytokine receptor assays
* Lymphocyte subset counts, lymphocyte function assays
* Chemical analysis of body fluids/tissues
* Food immune complex assays

Practice Parameters for Allergy Diagnostic Testing. Ann. Allergy 1995; 75: 616
Cytotoxic Test: Unproven Diagnostic Utility
Method:
Buffy coat from centrifuged whole blood is placed on
Siliconized microscope slide previously coated with dried extracts of up
to 150 to 200 foods.

Interpretation:
Unstained cells are viewed microscopically at varying
intervals up to 2 hours for changes in shape and appearance of
leukocytes. Swelling, vacuolation, crenation, lack of movement =
evidence of allergy to food.

Concerns:
Incubation time, pH, osmolarity not standardized.
Controlled studies show results are non-reproducible and do not
correlate with clinical evidence of food allergy.

Practice Parameters for Allergy Diagnostic Testing. Ann. Allergy 1995; 75: 616
 Performance Characteristics
and
Clinical Utility of Diagnostic
(Skin and Serology) Confirmatory Tests
 Prick Skin Tests Correlate with Nasal
Challenge
1215
Nasal Challenge (Pollen Grains)

Relationship between nasal challenges

405 with pollen grains and skin prick test
Endpoints in patients allergic to
135 Dactylis glomerata

Bousquet Clin Allergy 1987;17:529-38

45

15

0
0 1 2 3 4 5 6 7 8
Prick Test Endpoint (Log3 Allergen Dose)
Rs= 0.54
p< 0.005
 Even SPT May Result in “False Positives”
in Respiratory Allergy
• Skin prick tests are positive in many patients who have no respiratory
symptoms:
- 42% with a positive family history for asthma or rhinitis may have +SPT
and no disease.
- 29% of those with a negative family history for asthma or rhinitis may
have +SPT.
• SPT results need to be interpreted carefully. There MUST be a
correlation with the history
• Skin tests are a diagnostic tool, an adjunct to the history, and do not
make the diagnosis
Adinoff & Nelson. JACI 1990;86:766
 SPT vs ICT Skin Tests in
Diagnosis of Allergic Diseases
In patients who had history of spring allergy-like symptoms but a
negative SPT to timothy grass, there was no difference in nasal challenge
or correlations between symptoms and pollen counts during the grass
season in those with positive or negative ICT to grass.

Nelson. JACI 1996;97:1193-1201.

 Evidence Based Medicine
• Likelihood ratio:
– the ratio of post-test odds after a test result to the pre-test odds
indicates how much the odds change after a test
• Likelihood ratios (positive – negative)
– >5.0 or < 0.2 generate moderate to large shifts in disease probability
– 1.0 to 2.0 and 0.5 to 1.0 generate very small and often clinically
insignificant changes in disease probability

Jaeschke JAMA 1994;271:703-7

 SPT vs ICT
Comparison Using Evidenced Based Medicine

Test Allergen + Likelihood - Likelihood

SPT Cat 4.93 0.08
IDT Cat 0.89 1.24
SPT Grass 6.82 0.28
IDT Grass 1.05 0.98

Conclusion:
SPT relate closely to disease, while ICT do not (there are
presently no available data in children or in allergens other
than cat and grass). Gendo Ann Int Med 2004;140:278-89
 Allergy Skin Testing (SPT vs ICT)
Conclusions
Skin Prick Tests:

- Diagnostically sensitive, safest, easy to perform and may even detect

asymptomatic sensitivity
- Correlate well with clinical rhinitis & asthma
- Correlate well with challenge tests and IgE antibody serology
measurements

Intradermal Skin Tests:

- Do not differentiate clinically allergic from non-allergic subjects in

epidemiologic studies or in studies of cat and grass sensitivity in
adults.
 IgE Antibody Determination Allows
Evaluation of Disease Prognosis
Early sensitization can be predictive of future allergies:
• IgE antibodies to food early in life may be associated with a high
risk of developing IgE antibodies to inhalants later in life.
• IgE antibodies to inhalants prior to symptoms also predict evolving
allergic disease.
• Even low levels of IgE antibodies to an allergen are of importance,
since they can predict a later development of symptoms caused by
this allergen.
 IgE Antibodies to Food Early in Life
Predict Later IgE Antibodies to Inhalants
Development of positive Dermatophagoides
pteronyssinus IgE Values at 5 yrs of age
RAST IgEs at
six months of age n No. %
Specific IgE for egg white
Positive finding 54 46 85*
Negative finding 54 8 15
Specific IgE for cow's milk
Positive finding 31 29 94*
Negative finding 77 25 32
Specific IgE for soy
Positive finding 16 16 100*
Negative finding 92 38 41
* p<0.001, by chi-square test
Reference: Data from Sasai et al., J Pediatr 1996; 128: 834-
840
Asymptomatic Skin Sensitization to Birch May Predict
Development of Birch Pollen Allergy in Adults

• Methods:
Asymptomatic adults were followed through use of daily diary cards
during 3 consecutive birch pollen seasons
15 +SPT for birch
15 non-atopic controls
6 birch pollen–allergic patients
At the 3-year follow-up visit, conjunctival and nasal challenges,
intradermal late-phase reaction evaluation, and measurement of
birch specific IgE were performed.
JACI 2003;111:149-54.
Asymptomatic Skin Sensitization to Birch May Predict
Development of Birch Pollen Allergy in Adults
Results:
– Asymptomatic +SPT subjects had both birch specific IgE levels and
positive conjunctival provocation testing.
– Sixty percent (n = 9) of the asymptomatic sensitized subjects
developed clinical allergy in the three year period.
– The development of clinical allergic disease was associated with an
initial birch skin prick test wheal diameter of >4 mm.
– IgE antibodies ≥ 0.7kU/L (class 2) was 87.5% predictive of allergy
development

Conclusion: Positive skin prick test in an asymptomatic patient may

indicate potential for development of allergy in the future.
JACI 2003;111:149-54
 A Comparison of SPT, ICT and IgE
Serology in the Diagnosis of Cat Allergy

Methods:
• 120 patients with asthma, with or without a history of cat allergy were
challenged with a characterized cat challenge model after a clinical history,
SPT, ICT and IgE Serology (ImmunoCAP® System).
• Challenge was positive if upper respiratory symptom score was >0.5,
mean lower respiratory symptoms score was > 0.4 or maximum fall in
FEV1 was > 15%
RA Wood, et al. JACI 1999;103:773
Diagnosis of Clinically Significant Cat Sensitivity
Category Positive Cat Challenge

SPT + 38/41 (92.7%)

SPT - 10/39 (25.6%)
ICT + 6/26 (23.1%)
ICT - 4/13 (30.8%)
IgE anti-Cat Serology + 27/27 (100%)
IgE anti-Cat Serology - 11/44 (25.0%)

• In patients with a negative SPT, there was no correlation between

positive or negative ICT and challenge results
RA Wood, et al. JACI 1999;103:773
 Utility of In vivo and In vitro Diagnostic
Methods for Cat Allergy: Conclusions

• Both the SPT and IgE antibody serology (Immuno-CAP System) exhibited
an equivalent excellent efficiency (83.1%, 83.4%) in the diagnosis of cat
allergy.
• ICT results added little to the diagnostic evaluation, exhibiting a low
diagnostic efficiency (38.5%)

RA Wood, et al. JACI 1999;103:773

Quantification of IgE Antibodies in
Diagnosing Food Allergy

Objective: Compare results of CAP system FEIA to outcome

of SPTs and Double Blind Placebo Controlled
Food Challenge (DBPCFC)

Population: 196/320 well characterized pediatric patients;

Age: mean 5.2 years [range: 0.6-18 years]
Gender: 117 male; 79 female

Evaluation: IgE mediated reactions by history, SPTs,

DBPCFC and open challenges

Sampson and Ho 1997 100: 444-51

 Performance Characteristics of
ImmunoCAP® at Cut-off 0.35 kU/L
egg milk peanut soy wheat fish

No. pts pos/neg 145/51 95/101 136/60 34/162 23/173 52/144

sensitivity 98 100 97 94 96 94

specificity 45 30 38 25 20 65

Positive Predictive 84 57 78 21 14 49
value
Negative Predictive 88 100 85 95 97 97
value

Sampson, Ho.1997;100
 Tests for Diagnosis of Food Allergy
Skin tests vs Challenge Test

• PPV of positive SPT - <50% vs DBPCFC

• NPV of negative SPT - >95% vs DBPCFC

Performance Characteristics of SPT at 3mm
vs DBPCFC
egg milk peanut soy wheat fish

No. pts pos/neg 90/34

53/53 20/2199 29/78 19/659 11/79
sensitivity % 98
96 0 76 90 0
specificity % 53
51 29 47 51 57

Predictive value
66 55 35 35 77
Positive 85
93 75 84 94 80
Negative 90

Sampson, Ho, 1997;100

 Size of SPT with 100% Likelihood of
Positive Open Challenge
milk egg peanut

Children 0-2yrs ≥6mm ≥ 5mm ≥ 4mm

Children all ages ≥ 8mm ≥ 7mm ≥ 8mm

Sporik et al Clin Exp Allergy 2000;30

 Predictive Values for CAP RASTS vs
Challenges
 95 % predictive value
– Egg: 7 Ku/L (2 Ku/L*)
– Milk: 15 Ku/L (5 Ku/L*)
– Peanuts: 14 Ku/L
– Fish: 20 Ku/L

Negative Predictive value = 95%

Sampson H. Ho D. JACI 2001
 Diagnosis of Allergic Diseases
Summary
• The decision that the patient’s symptoms and clinical
signs represent an allergic disease is made by an
experienced clinician on the basis of the case history,
physical examination, and symptoms following
allergen exposure.

• Measurement of IgE antibodies by SPT or serological

assays confirm the presence of specific IgE
antibodies and are an essential adjunct in making a
definitive diagnosis of allergic disease.
 Conclusion
• Atopy – propensity to produce high levels of
IgE from B cells
• Allergens mimic parasites – processed and
presented by APC (e.g. dendritic cells)
• Orchestrated by Th2 cells – cytokine release
• Effector cells – mast cells, basophils
• Mediators – cytokines, histamine,
leukotrienes, PAF etc.