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