IMPORTANCE OF IN VITRO
LABORATORY METHODS IN
ALLERGOLOGY
Dr. Sándor Sipka
http://rimm.dote.hu
Johansson et al.
Hypersensitivity:
hyperreactive reaction of the organisen elicited by a trigger of enviroment
Allergic hypersensitivity
(immunologic mechanism defined
or strongly suspected)
IgE-mediated
Nonatopic
Insect sting
Helminths
Nonallergic hypersensitivity
(immunologic mechanism
excluded)
Not IgE-mediated
Atopic
T cell:e.g.,contact dermatitis, celiac
Eosinophil:e.g., gastroenteropathy
IgG-mediated:e.g.allergic alveolitis
Drugs
Other
Other
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
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
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
histamine, triptase
PGD2, LTC4
PAF
IL-1, IL-4, IL-5
TNFa, IFNg
ECP
MBP
ROS
LTC4, PAF
IL-5
platelets
proteases
serotonine
PGD2, PGE2 histamine
TxA2, LTB4 TxA2
LTC4, PAF
ROS
IL-1, TNF
ROS
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.
Anaphylaxy: 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)
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
FIRST ALLERGEN
STIMULUS
SECOND ALLERGEN
STIMULUS
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:
measurement of complement activity
demonstration of the activation of PMN, Eo,
monocytes/macrophages
ADCC
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
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
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
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.<1000 5/b. 1000-5000 5/c> 5000
Seasonal inhalant allergens SIA
Perennial inhalant allergens PIA
name of allergen
name of allergen
Percent of
frequency %
Percent of
frequency %
Food allergens FA
name of allergen
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
Percent of
frequency %
Questionnaire The most frequent allergens in your clinical pratctice
Seasonal inhalant allergens SIA
Perennial inhalant allergens PIA
name of allergen
name of allergen
Percent of
frequency %
Percent of
frequency %
Food allergens FA
name of allergen
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
Percent of
frequency %
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)
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
SPECIFIC IgE POSITIVITY CLASSES
kU/l
Allergen-spec. IgE class
Valuation
<0.36
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
>100
6
very high
Other forms of in vitro tests for allergy
Basophil activation tests:
Measurement of histamine release
Measurement of leukotriene release
Measurement of CD63 expression
Lymphocyte activation tests:
T cell proliferation assay
cytokine release
nitroblue tetrazolium (NBT) test
Quantitative PCR for IL-4
Chip technique with recombinant allergens
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)
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)
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.
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
I. Occurence of allergens in children with food
allergy less than 1 year old in Debrecen
Number of patients
Positivity
361
100%
18
5%
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
II. Occurence of allergens in 1-6 years old children with food allergy in
Debrecen (Hungary)
Number of patients
Positivity
1461
100.0%
252
17.2%
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
III. Occurence of allergens in children with inhalative
allergy less than 1 year old in Debrecen
Number of patients
Positivity
66
100%
3
4.60%
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
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 (%)
dermatophagoides pteronyssinus (d1)
15.4
deramtophagoides fariane (d2)
15.1
house dust (h1)
14.5
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
mites-epithelia-insects
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
3rd Department of
Internal Medicine
Regional Immunology
Laboratory