IMPORTANCE OF IN VITRO LABORATORY METHODS IN ALLERGOLOGY

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IMPORTANCE OF IN VITRO LABORATORY METHODS IN
ALLERGOLOGY
Dr. Sándor Sipka
http//rimm.dote.hu
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Johansson et al.
Hypersensitivity hyperreactive reaction of the
organisen elicited by a trigger of enviroment
Allergic hypersensitivity (immunologic mechanism
defined or strongly suspected)
Nonallergic hypersensitivity (immunologic
mechanism excluded)
IgE-mediated
Not IgE-mediated
T celle.g.,contact dermatitis, celiac
Nonatopic
Atopic
Insect sting
Eosinophile.g., gastroenteropathy
Helminths
IgG-mediatede.g.allergic alveolitis
Drugs
Other
Other
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Allergy a pathologic state in certain group of
(atopic) patients responding with inflammatory
reactions on certain types of antigens
(allergens) being otherwise neutral for a greater
part of the population.   Backround a diathesis
of polygenic type (inherited pathologic state)
with - increased production of allergen
specific IgE - raised production of total IgE
stimulated by IL-4 - overproduction of
inflammatory cells - increased sensitivity of
tissues to certain types of mediator
substances  
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Types of allergic reactions   Type I.
reaction IgE mediated allergic reaction Type II.
reaction cytotoxic reaction induced by IgG and
IgM Type III. reaction tissue damages caused by
immunocomplexes Type IV. reaction delayed type
hypersensitivity induced by Th1 lymphocytes
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Type I. allergic reaction 
The reaction is mediated by allegen specific
IgE   The reaction is of immediate type ( the
symtoms of inflammation appear within 4 hours
after the allergen challange)   The symptomes are
elicited by mediator substances released from
mast cells, basophils, eosinophils, macrophages
or platelets.   Mediators of mast
cells/basophils eosinophils macrophages
platelets
histamine, triptase ECP
proteases serotonine
PGD2, LTC4 MBP
PGD2, PGE2 histamine
PAF ROS
TxA2, LTB4 TxA2
IL-1, IL-4, IL-5 LTC4, PAF LTC4,
PAF ROS TNFa, IFNg
IL-5 IL-1, TNF

ROS
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Types of allergens Drugs (penicilline,
etc) Inhalative allergens pollens (ragweed,
mugwort, etc) animal epithelium (cat,
dog,etc) mites fungi (mucor, aspergillus,
etc) textile/cotton Insects
bee, wasp Nutritive allergens milk, egg,
soybeen, etc. Atopy pathologic hypersensitivity
to allergic reactions. It is a diathesis. Anaphyla
xy a lifethretening state when enormously high
amouts of the inflammatory mediators get into the
circulation, skin, lung and gastrintestinal
truct a.) IgE mediated b.) not IgE mediated
forms (mediated by complement and other factors)
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Laboratory diagnosis Serum total IgE
(nephelometry, turbidimetry) Allergen specific
IgE (RAST, ELISA, FIA, dot-blot) Activity
markers increased levels of eosinophil cationic
protein (ECP) and tryptase

Blood film eosinophylia
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FIRST ALLERGEN STIMULUS
SECOND ALLERGEN STIMULUS
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Type II. allergic reaction
Mechanism cytolytic and cytotoxic reactions
induced by IgG and IgM, causing tissue
damages - complement mediated cytolysis
(classic pathway) - stimulation of PMN, Eo cells
and monocytes/macrophages by activated C3 - IgG
bindig to effector cells killer cells, PMN, Eo
cells and monocytes/macrophages   Allergens
drugs chinine, furosemide, gold salt,
indomethacine, sulphonamides, salicylate,
chloramphenicole   Laboratory diagnosis measurem
ent of complement activity demonstration of the
activation of PMN, Eo, monocytes/macrophages
ADCC  
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Type III. allergic reaction
Mechanism tissue damages caused by
immunocomplexes
sedimentation of IC in circulation
sedimentation of IC in tissues   Allergens
drugs, antibiotics, benzotiazine, hidantoine,
bacteria streptococcus, etc
viruses hepatitis B,C,
etc.   Laboratory diagnosis
Measurement of IC level in serum
Measurement of complement factor activity
in serum Histology
microscopic IC verification    
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Type IV. allergic reaction
Mechanism delayed type hypersensitivity
induced by the cytokines of Th1 cells. The
symptoms appear within 12-24 hours after the
allergen challange.   Forms a.) Contact
sensitivity   Hapten-carreir complexes processed
by Langerhans cells to Th1 lymphocytes cytokine
release   antigens nickel, gutta percha, oils,
Hg salts, stains, drugs, cosmetics
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b.) Tuberculin-type reaction   Mononuclear
(monocyte-lymphocyte) cell infiltration at the
site of antigen penetration Antigen bacterial
proteins, chemicals (circorium, berillium) The
same mechanism is involved in the rejection of
transplants   Laboratory diagnosis  
Histology (mononuclear cell infiltration)
Lymphoblast transformation induced by the
antigen Measurement of cytokine
production
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Questionnaire The most frequent allergens in your
clinical pratctice
Country
City Place of work 3/a.Universitiy
Department 3/b. Hospital 3/c. Private
practice Position Number of your allergic
patients/year 5/a.lt1000 5/b. 1000-5000 5/cgt 5000
Seasonal inhalant allergens SIA Seasonal inhalant allergens SIA Perennial inhalant allergens PIA Perennial inhalant allergens PIA Food allergens FA Food allergens FA
name of allergen Percent of frequency name of allergen Percent of frequency name of allergen Percent of frequency
Common ragweed Ambrosia elatior House dust mite D.farinae Egg FA1
Meadow grass Poa pratensis House dust mite D.pteronyssinus Milk (casein) FA2
Mugwort Artemisia vulgaris Cat epithelium and dander Haselnut FA3
Cultivated rye Secale cereale Dog epithelium and dander Soya bean FA4
Plantain Plantago lanceolata Chicken feather Wheat FA5
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Questionnaire The most frequent allergens in your
clinical pratctice
Seasonal inhalant allergens SIA Seasonal inhalant allergens SIA Perennial inhalant allergens PIA Perennial inhalant allergens PIA Food allergens FA Food allergens FA
name of allergen Percent of frequency name of allergen Percent of frequency name of allergen Percent of frequency
Dandelion Taraxacum vulgare Duck feather Peanut
Rye-grass Lolium perenne Goose feather Orange
Cocksfoot Dactylis glomerata Parrot feather Tomato
Birch Betula Cockroach Codfish
Hazel Corylus Aspergillus Almond
Willow Salix alternaria pork
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The main methods used for diagnosis of allergy
Type I. reaction in vivo cutaneous Prick test  
in vitro serum total
IgE
allergen
specific IgE

allergen specific IgG
produced mainly in infants
against milk, egg, soybeen,
etc mast cell/basophil
activation products

histamine, tryptase, leukotrine
eosinophil
activation products ECP     Type III. reaction
in vitro allergen-IgG/IgM complex measurement

(ELISA, Ouchterlony)   Type IV. reaction in
vivo epicutaneous skin test (contact allergens)
in vitro
lymphocyte proliferation assay (for drugs)

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Methods used for the determination of allergen
specific IgE
Types of methods   Allergens used
purified natural extracts
molecules produced by recombinant
technique
mix of allergens   Anti-IgE immunoassays
radioimmunoassay (RIA)
enzyme linked immunosorbent assay
(ELISA)
fluorescence immunoassay (FIA)
chemiluminescence
immunoassay (CHLIA) Values of measurements 1.
kU/l 2. Spec. IgE positivity class
0-6    Criteria of use
sensitivity
specificity
positive predictive value
negative predictive value 
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SPECIFIC IgE POSITIVITY CLASSES
kU/l Allergen-spec. IgE class Valuation
lt0.36 0 Not detectable
0.36-0.71 1 low
0.72-3.59 2 moderate
3.6-17.99 3 high
18-49.99 4 very high
50-99.9 5 very high
gt100 6 very high
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Other forms of in vitro tests for allergy
Basophil activation tests Measurement of
histamine release Measurement of leukotriene
release Measurement of CD63 expression   Lymphocy
te activation tests  T cell proliferation
assay cytokine release nitroblue tetrazolium
(NBT) test Quantitative PCR for IL-4 Chip
technique with recombinant allergens
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Indications for using of allergen specific IgE
measurements
Patients with any types of skin diseases Lack of
allergens used for skin tests Diversity of the
result of skin test and the anamnesis At the
beginning and the end of specific
immunotherapy When the skin test may provoke
anaphylaxis (e.g penicilline) At infants  At
patients when the physical or mental conditions
exclude the skin test taking
antihistamines, benzodiazepines or
corticosteroid, etc.    The value of
a negative specific IgE test The clinician
can exclude the allergic pathomechanism (testing
for food allergy)
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The problems with allergen specific IgE
determinations
CROSS REACTIVITY BETWEEN ALLERGENS e.g.
ragweed water melone, banana, cucumber
peanut chestnut, soya bean, pea The lack of
chemically well defined allergens   The in vitro
tests are more expensive than the skin
tests   The possibility for misusing during the
ordering of these tests. Lack of widely accepted
and used international standards for the
tests.   The international external quality
controls did not come into general use.
(NEQUAST, Pharmacia, QualiCont)
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The clinical value of allergenspecific IgG
measurements
This antibody is not in a direct relation to
allergy.   The production of anti-milk, -egg,
-soybeen,- tomato IgG is possible by an increased
intestinal permeability, by an inflammation,
mainly in infants. Therefore, it reflects a
state of an increased enteral permeability for
food antigens.   The association of specific IgG
with IgE, however, may predict the prolonged
persistance of a hypersensitivity to milk, for
example.  
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The fundamental in vitro laboratory tests of
allergy
Serum total IgE Serum allergen specific IgE
Verifies the allergen ( children)
Early definition of allergen
(chicken egg, bovine milk)
Can be used for series measurements ( in a
population) Following up the
specific immunotherapy   Markers of activity
serum ECP and tryptase levels  
Free radical production
(chemiluminescence) of  
peripheral phagocytes  
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I. Occurence of allergens in children with food
allergy less than 1 year old in Debrecen
Number of patients 361 100
Positivity 18 5

Occurance of allergens () Occurance of allergens () Occurance of allergens ()
egg white (f1) 44.80
cow milk (f2) 20.7
hazelnut (f17) 13.8
wheat flour (f4) 6.9
peanut (f13) 6.9
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II. Occurence of allergens in 1-6 years old
children with food allergy in Debrecen (Hungary)
Number of patients 1461 100.0
Positivity 252 17.2
Occurance of allergens () Occurance of allergens ()
egg white (f1) 34.2
cow milk (f2) 21.0
wheat flour (f4) 3.1
strawberry (f44) 2.9
peanut (f13) 2.5
kiwi (f84) 2.5
peach (f95) 2.3
soyabean (f14) 1.9
hazelnut (f17) 1.6
tomato (f25) 1.4
melon (f87) 0.8
walnut (f16) 0.6
coconut (36) 0.6
almond (f20) 0.6
orange (f33) 0.2
fish (cod) (f3) 0.2
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III. Occurence of allergens in children with
inhalative allergy less than 1 year old in
Debrecen
Number of patients 66 100
Positivity 3 4.60

Occurance of allergens () Occurance of allergens () Occurance of allergens ()
goose feathers (e70) 33.3
chicken feathers (e85) 16.7
cow dander (e4) 16.7
cat epithelium (e1) 16.7
horse dander (e3) 16.7

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IV. Occurence of allergens in 1-6 years old
children with inhalative allergy in Debrecen I.
Number of patients 874 100
Positivity 131 15.00
Occurance of allergens () Occurance of allergens () Occurance of allergens ()
dermatophagoides pteronyssinus (d1) 15.4
deramtophagoides fariane (d2) 15.1
house dust (h1) 14.5 mites-epithelia-insects
cow dander (e4) 8.8
chicken feathers (e85) 7.7
common ragweed (w1) 5.7
horse dander (e3) 4.0
alternaria alternata (m6) 3.7
goose feathers (e70) 2.8
turkey feathers (e89) 2.6
cat epithelium (e1) 2.3
johnson grass (g10) 2.0
golden rod (w12) 1.7
cockroach (i6) 1.4
aspergillus fumigatus (m3) 1.1
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V. Occurence of allergens in 1-6 years old
children with inhalative allergy in Debrecen II.
salwort (w11) 1.1
dog epithelium (e2) 1.1
common pigweed (w14) 0.9
box-elder (t1) 0.9
goosefoot, lamb's quarters (w10) 0.9
oak (t7) 0.9
sweet vernal grass (g1) 0.6
cladosproium herbarum (m2) 0.6
plantain, ribwort (w9) 0.6
common silver birch (t3) 0.6
meadow grass (g8) 0.6
cultivated rye (g12) 0.6
rye grass (g5) 0.3
mugwort (w6) 0.3
duck feathers (e86) 0.3
bahia grass (g17) 0.3
penicillium notatum (m1) 0.3
timothy (g6) 0.3
velvet grass (g13) 0.3
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3rd Department of Internal Medicine
Regional Immunology Laboratory