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. Johansson et al. Hypersensitivity: hyperreactive reaction of the organisen elicited by a trigger of enviroment. Allergic hypersensitivity (immunologic mechanism defined or strongly suspected). - PowerPoint PPT PresentationTRANSCRIPT
IMPORTANCE OF IN VITRO
LABORATORY METHODS IN
ALLERGOLOGY
Dr. Sándor Sipka
http://rimm.dote.hu
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 cell:e.g.,contact dermatitis, celiac
Eosinophil:e.g., gastroenteropathy
IgG-mediated:e.g.allergic alveolitis
Other
Nonatopic Atopic
Insect sting
Helminths
Drugs
Other
Johansson et al.
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 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 TNF, IFN IL-5 IL-1, TNF ROS
Types of allergens:Drugs (penicilline, etc)Inhalative allergens: pollens (ragweed, mugwort, etc)
animal epithelium (cat, dog,etc)mitesfungi (mucor, aspergillus, etc)textile/cotton
Insects bee, waspNutritive 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 mediatedb.) 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 activitydemonstration 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 practicePosition:Number of your allergic patients/year: 5/a.<1000 5/b. 1000-5000 5/c> 5000
Seasonal inhalant allergens SIA Perennial inhalant allergens PIA 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
Seasonal inhalant allergens SIA Perennial inhalant allergens PIA 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
Questionnaire The most frequent allergens in your clinical pratctice
The main methods used for diagnosis of allergyType 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 releaseMeasurement of leukotriene releaseMeasurement of CD63 expression
Lymphocyte activation tests:
T cell proliferation assaycytokine releasenitroblue tetrazolium (NBT) testQuantitative PCR for IL-4
Chip technique with recombinant allergens
Indications for using of allergen specific IgE measurements
Patients with any types of skin diseasesLack of allergens used for skin testsDiversity of the result of skin test and the anamnesisAt the beginning and the end of specific immunotherapyWhen 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 361 100%
Positivity 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 1461 100.0%
Positivity 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 66 100%
Positivity 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 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
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
V. Occurence of allergens in 1-6 years old children with inhalative allergy in Debrecen II.
3rd Department of Internal Medicine
Regional Immunology Laboratory