ALLERGY -basic mechanisms and tests

1. Allergy : Basic
mechanisms and
tests
BY DR.T VENUKUMAR

2. Allergy
 Allergy is defined as hypersensitivity reaction initiated by
specific immunologic mechanisms.
 It describes objectively reproducible symptoms or signs
initiated by an exposure to a defined stimulus at a dose
tolerated by normal person.
 Hypersensitivity is an altered immune response Which
produces physiogical/histopathological damage in the hoSt.
 Atopy is a tendency to become sensitized and produce
allergen specific sIgE in response to ordinary exposure to
allergens

4. Aetiology of sensitization
 Type 1 IgE mediated immediate hypersensitivity occurs only in a sensitized
individual .
 Immune response to innocuous antigens leading to Sensitization are
multifactorial like host factors
 – genetic predisposition (chr 11q2-13 encodes beta sub unit of high
affinity IgE receptor)and (chr 5 encodes TH2 promoting cytokines such as
IL4, balance between TH1 /TH2, p40)
 environmental factors – exposure to inf diseases since childhood,
 Upto 40% of population in industrialized countries – tendency to atopy.
 An atopic person usually ha a high total IgE level, strong familial tendency
and increased susceptibility to allergic diseases like AR and atopic
dermatitis

5. Hygiene hypothesis
 Prevalence of allergic diseases is higher in children Who grew up in
hygienic environment than in less hygienic regions where infectious
diseases are more common.
 Th1(pyoinflamatory)>Th2 (defence and allergic).
 Counter regulation hypothesis –regulatory cytokines Il 10 and TGF –B are
upregulated in presence of infection
 Atopy Is less common despite th2 inflammation.

7. Effectors of allergic diseases
• The essential effector cells of immediate hypersensitivity are cells
expressing high affinity IgE receptors (FcεRI) such as mast cells,
basophils and eosinophils.
• Other effectors include antigen presenting cells (e.g. dendritic cells)
and Th2 cells that promote allergen-specific IgE synthesis by B cells.

8. Mast cells
• Mast cells are haemopoietic stem cell derivatives and mature locally.
They reside near the surface where our body is exposed to pathogens
and allergens.
• Based on the distribution either mucosal mast cells or tissue mast cells.
• They are often found in close proximity to blood vessels.
• FcεRI is capable of being tightly bound to sIgE even in the presence of
a very low serum concentration.
• On exposure to their specific allergens, crosslinking of the receptor
and signalling follow to produce clinical phenomena

9.  • FcεRII is a low affinity IgE receptor (CD23).
 • It is present in many cells including B cells, activated T cells,
monocytes, eosinophils, platelet and follicular dendritic cells.
 • Mast cells granules contain preformed mediators that are responsible
for the immediate symptoms of allergic reactions.
 • Mast cells also produce other mediators upon FcεRI signalling.
 • Mediators that are rapidly synthesized are involved in the immediate
symptoms of allergic reactions.
 • Other mediators may lead to late phase responses and chronic
allergic inflammation

11. Basophils
 • Basophils develop from bone marrow granulocytemonocyte
progenitor.
 • There is increasing evidence that basophils involve significantly in
allergic diseases.
 • They respond to many stimuli including cytokines, antibodies,
proteases and antigens.
 • Cross-linking of IgE , FcεRI receptors on basophils
produce mediators .
 • Basophils have been identified in nasal washes of patients with
allergic rhinitis.

12. Eosinophils
 • Eosinophils are granular leucocytes that
mainly reside in submucosal connective
tissue in respiratory, gastrointestinal and
urogenital tracts.
 • They play an essential role in defence against
helminths.
 • Eosinophils also express FcεRI receptors and
are capable of releasing mediators

15. T-Lymphocytes
 • T cells are central to the pathogenesis of allergic diseases since they
are the only cells capable of recognizing antigens presented by antigen
presenting cells.
 • Maturation of a naive T cell takes place on activation by signals
including specific peptide-MHC via T cell receptor and costimulatory signal
via CD28.
 • T helper cells (CD3+CD4+) differentiated into many types depending
upon the type of immune response taking place and cytokine
environment.
 • Th1 cells differentiate in the presence of interferon gamma (IFN-γ)
and IL-12, and produce predominantly IFN-γ and interleukin 2 (IL-2).

16.  • Th2 cells differentiate in the presence of IL-4 and produce IL-
4, IL-5
and IL-13 cytokines preferentially.
 • Recruitment of Th2 cells, their activation in tissues and
generation of Th2 cytokines are main features of both allergic
rhinitis and asthma.
 • Low dose antigen exposure via mucosal surface with inhaled
allergen is typical of Th2 response in allergic rhinitis,
 high dose allergen delivered by injection favour Th1
response.

17. Dendritic cells
 • Dendritic cells are professional antigen presenting cells (APC), which are
abundant in the epithelium and submucosa of both upper and lower
respiratory mucosa in patients with allergic diseases.
 classified as myeloid derived DC1 cells (derived from human blood monocytes) or
DC2, plasmacytoid cells (derived from lymphoid cells).
 • DC1 cells produce a high level of IL-12 and favour Th1 development.
 DC2 cells are low IL-12 producers and support preferential Th2 differentiation.
 • However, in dendritic cells subpopulations, differential function may depend on
their location, their degree of maturation and local cytokine environment.

18.  • Immature cells express high levels of immunoglobulin receptors and are
highly endocytic , efficient antigen capture.
 • In contrast, mature cells express high levels of MHC class II and have
upregulated CD86 expression and produce abundant cytokines, features
consistent with their role in allergen presentation and immune modulation.
 • Rapid maturation (within 2 hours) was observed in dendritic cells
 within the bronchial wall and not peripheral lung.

19.  • Rapid dendritic cells recruitment to the bronchial mucosa in
atopic asthmatic patients has also been observed within the time
frame of late asthmatic responses following local segmental
allergen challenge.
 • Plasmacytoid dendritic cells (CD123) IL-3R α-chain+CD45RA+ cells
 increase dramatically in number following local allergen challenge
 repeatedly for 7 days.
 • This is of particular interest since plasmacytoid dendritic cells
matured in vitro can induce preferential Th2 development

20. Immunoglobulin E (IgE)
 • IgE concentration is the lowest immunoglobulin isotype in human
serum,(approximately 100–400ng/mL)
 • The majority of IgE is tissue-bound with a half-life of approximately 2 days.
 • FcεRI is a high affinity receptor for IgE, which is mainly present on
mast cells, basophils and eosinophils.
 • But low-level expressions are detected in dendritic cells, monocytes,
and macrophages that are in different molecular structures.
 • The IgE also bind to a low affinity receptor FcεRII (CD23) on B cells,
monocytes and macrophages.

21.  • IgE is developed from isotype switching of IgM.
 • B cells secrete IgM following antigen recognition .
 • Isotype switching is influenced by specific Th2 cells and the cytokine environment.
IgE production is favoured by IL-4 and Il-13 derived from Th2 cells.
 • It is also perpetuated by IL-4 and IL-13 from mediator release as a result of
degranulation in allergic reaction.
 • IgE production generally occurs in the bone marrow and the draining regional
lymph nodes.
 • But local IgE production has been reported in local forms of allergic conditions
(e.g. allergic rhinitis).
 • Local IgE synthesis has been considered as the cause in some situations – allergic
symptoms without sensitization on skin prick test and increased sIgE;
 some atopic individuals develop rhinitis whereas other develop asthma or eczema,
and others may have sensitization without any clinical manifestation.

22. Histamine
 • Histamine is generated from histidine decarboxylase and stored in granules
of mast cells and basophils.
 • Elevated histamine levels correlate well and indicate higher sensitivity than
tryptase in anaphylaxis.
 • Tryptase level is elevated in 60% whereas histamine and PAF are elevated in 70%
and 100% respectively in anaphylaxis.
 • However, histamine is highly soluble and rapidly diffuses away after the release.
 • The majority of histamine is metabolized within minutes of release by many
enzymes such as histamine N-methyl transferase,monoamineoxidase
and diamine oxidase

23.  • The short plasma half-life and sample handling issue are major limiting
factors for histamine measurement in routine laboratory use.
 • Urinary metabolites (N-methyl histamine) can be detected several hours
after anaphylaxis.
 • But there are potential confounding factors for this measurement such as
the effect of histamine producing bacteria, histamine containing foods and
so on.
 • In scombroidosis, the urinary histamine level can be increased despite a
normal serum tryptase level.

24. Serum Tryptase
 • Tryptase is produced by mast cells and basophils.
 • In humans, tryptase exists in two isotypes (alpha and beta), which
are in two forms – mature and immature.
 • Beta tryptase is the predominant isotype in mast cells.
 • Protryptase is the immature form and is spontaneously secreted by
unstimulated mast cells.
 • Mature tryptase is stored in the intracellular granules and released
on degranulation.
 • Therefore it is normally undetectable. The normal reference range of
total tryptase is 1–13ng/mL

25.  • The total tryptase levels peak 30–60 minutes after the onset
of
 symptoms due to degranulation, and reduce afterwards with a
half-life of about 2 hours.
 • Tryptase levels can be measured in both serum and plasma.
 • Normal tryptase levels do not exclude anaphylaxis.
 • Other causes of increase tryptase levels include
mastocytosis, acute myelocytic leukaemia, myelodysplastic
syndrome and the myeloid varaints of hypereosinophilic
syndrome

26.  • Elevated total tryptase has also been reported in some conditions such as
onchocerciasis treatment and administration of recombinant stem cell factor.
 • There are two mechanisms leading to mediators release in mast cells and basophils.
 • The FcεRI signalling as a result of the sIgE-antigen complex is the underlying
mechanism in the IgE mediated hypersensitivity.
 • However, mediator release can also occur by other factors that are capable of IgE
independent direct stimulation.
 • These include viral proteins (e.g. HIV gp120), bacterail proteins (e.g. LPS),
drugs (e.g. opiate), chemical and physical stimuli, complement activation and
neuropeptides, cytokines.

27. High affinity receptor (FcεRI) signalling
in allergic reaction
• Allergic reactions are overlapping and synergistic physiological effects resulting from mediators
release due to the activation of mast cells and basophils through a mechanism generally
understood to involve FcεRI signalling.
• This signalling has been extensively investigated using passive cutaneous and passive systemic
anaphylaxis models.
• Unlike other immunoglobulin isotypes, the majority of IgE is tightly bound to FcεRI.
• There is no signalling until these receptors are cross-linked by their specific antigens.
• The high level of IgE has potential to enhance IgE dependent effector function.

28. • FcεRI is a tetrameric receptor consisting of three subunits: a IgE
binding unit (α chain); a signal transducing unit (β chain); and two
signal regulator units (γ chain).
• Allergen–sIgE binding is followed by early signalling events including
phosphorylation of cytosolic domains of the receptor known as
immunoreceptor tyrosine-based activation motif (ITAM).
• This phophorylation is mediated by Src family proteins.
• There are two types of Src family protein in these events, namely
signal-initiating kinase (Lyn) and signal-propagating kinase (Syk).

29. • This is followed by the recruitment of many signalling proteins to
form multi-molecular signalling complexes.
• The downstream effect of this signalling results in an activation of
protein kinase C and liberation of intracellular calcium.
• These lead to degranulation within minutes.
• In addition eicosanoid generation and activation of transcription
factors for the production of cytokines and other mediators of allergic
reaction follow within hours

30. Roles of sensory nerves
• Activation of sensory nerves is an important mechanism in rhinitis in
many ways.
• These include generation of acute rhinitis symptoms, nasal hyper-
reactivity to non-specific triggers, release of neuropeptides such as
substance ‘p’ and neurokinin from inflamed sensory nerves.
• Neurogenic inflammation is self-perpetuating and represents an
important component of the allergen-IgE interaction.
• Nerve growth factor can be detected in nasal fluids in chronic allergic
rhinitis

31. • It is increased after allergen challenge.
• Therefore immediate response is due to a complex interaction
between mediators release and target organs such as the sensory
nerve, the vasculature and mucus secreting glands.
• Hyper-responsiveness of target organs associated with late response
and day-to-day allergic disease is likely to relate to a combination of
both inflammation and increased sensory nerve activation.
• The contribution of these components varies in different individuals.

32. Clinical features of IgEmediated
hypersensitivity
 Mainly due to effect of the mediator released and may involve many
systems.
 These include cutaneous, respiratory, cardiac, gastrointestinal systems.
 Onset of immediate Allergy depends on route of exposure to an allergen,
usually occurs with in minutes but incase of iv drug it occurs in seconds.
 ECG changes : T inversion, SV arrhythmias and BBB are reported in
anaphylactic shock

33.  Late phase reactions had been mainly described in respiratory and
cutaneous allergy.
 Post mortem findings of fatal anaphylaxis- odematous obstruction of
airways, laryngeal edema,eosinophilic infiltration around bronchi, and
swelling of liver,spleen,intestine.

36. Local allergic rhinitis
 LAR has been described as localized nasal allergic response.
 Allergic sensitizations are not Detectable by serum specific IgE or skin testing
 Usually diagnosed by positive nasal allergen provocation test (NAPT).
 Studies shows that LAR accounts for 15% rhinitis cases.
 Pathophysiology: local specific IgE production in nasal mucosa with a TH2
inflamatory pattern response.
 Nasal secretions of these patients have detectable basal aeroallergen sIgE (eg:
house dust mite,cat,dog,grass etc.)
 sIgE levels rapidly increases after NAPT In 22% of LAR pts with perennial
symptoms and 35% of those with seasonal symptoms.
 Measurement of sIgE in LAR has high specificity but low sensitivity (40%)

37. Rhinitis & Asthma Link
 Approximately 30% patients with rhinitis develops asthma and 80% of
patients with perennial asthma have rhinitis.
 Its is important to recognize and treat the astma in patients presenting
with Allergic rhinitis.

38. Basic mechanisms of treatment of
allergic diseases
 Symptomatic therapy targeting the mediators – anti histamines and
adrenaline
 General immunosuppressive drugs –corticosteroods
 Therapies to modify the Pathigenesis of allergy – pollen desensitization for
seasonal allergic rhinitis.
Antihistamines
 Acts through h1 receptors
 Effective in partial suppression of immediate allergic responses in skin and
respiratory tract in AR.
 Little or no effect in late phase

39. Corticosteroids
 Binds to intracellular receptors leading to an inflamatory effect due to
alteration in transcription factors.

40. TESTS IN ALLERGY
DIAGNOSIS

41.  Demonstration of sensitization to a suspected allergen is required for the
diagnosis(Allergen specific IgE )
 But not all sensitized individuals are clinically allergic.
 Therefore confirmation of allergic diagnosis requires either a convincing
relevant history or a reproducible allergic phenomenon by a Provocation
test (controlled challenge test) .
 It is a gold standard test in allergic diagnosis.

42. Types of tests
Allergy Challenge Tests
(Provocative Tests)
(in Vivo Tests)
 Skin Provocation Tests o Skin Prick Test
(SPT) o Intra-Dermal Test (IDT)
 Bronchial Provocation Tests
 Nasal Provocation Tests (NPT)
Serum lgE Assays
(Serological Tests)
(in Vitro Tests)
 Total lgE
 Allergen Specific lgE

43. Skin testing
 Is a bioassay.
 Specific allergens introduced into skin , which cross links with FceRI if the
allergen specific IgE are present in sufficient quantities on cutaneous mast
cells.
 Transient wheel and flare develops – represent immediate phase .
 1-2hrs after testing- deep tissue swelling,warmth, pruritis and erythema
Which resolves in 24-48hrs. – late phase response (not used in diagnosis)

46. 2methods
1. SPT - most appropriate initial test
PPV- <50%
NPV- >95%
1. IDT – greater risk of systemic reactions, should only be performed after a
negetuve SPT.
has higher sensitivity than SPT but lower specificity.

47. SPT
 Both +ve (histamine 0.1%) and —ve (saline) controls are essential for correct
interpretation.
 +ve control should optimally elicit a wheal diameter > 3 mm.
 The device used to prick the skin may have a single or multiple heads (the
former preferred).
 Usually several antigens are tested. To avoid cross contamination, the anti usea
ens for every should antigen be applied application.at least 2 cm apart and a
separate lancet
 A drop of allergen extract is placed on the skin and the needle or lancet IS
gentlv passed through it to penetrate the epidermis without causing any
bleeding. The lancet is held against the skin for 1 second, with equal pressure
applied for each application.

48. Interpretation of SPT
 Negative: No reaction, or reaction not different from —ve control.
 Interrnediate: erythema and/or induration whose larger diameter is < 3
mm (or < 3 mm above the —ve control)
 Positive: induration whose longer diameter is > 3 mm (or > 3 mm above
the —ve control)
 Considering the wheal only in interpreting +ve test results t reproducibility.
 It may be better to record the exact reaction size.

49. For research purposes will take the mean of longest diameter and
diameter perpendicular to it .
 Cautions:
full emergency equipment and medications should be kept available..
Risk of systemic reactions 33:1lakh.
 Endpoint dilution technique a varient of IDT to identify the concentration
of extract that produces a reaction of defined size-not routinely used as
the risk of systemic reactions 0.5%.

51. Inta dermal test (IDT)
Technique
 Using a hypodermic (insulin) syringe and
needle, the skin is held tense and the
needle is inserted just under the dermis,
almost parallel to the skin surface, just far
enough to cover the beveled portion.
 Little amount of allergen extract is
injected to raise a small bleb.

52. Advantages of SPT
 More specific: used to test for
aeroallergens (allergic rhinitis,
asthma) and food allergy
 Lower risk of systemic reactions
 Easier to perform, less invasive
 SPT is usually done first. A +ve
test circumvents the need for IDT
Advantages of IDT
 More sensitive: used to test for
drug allergy, eg, antibiotics, eg,
penicillins. It is contraindicated for
food allergy (high false +ve rate I
risk of systemic reactions)
 More reproducible
 IDT is mainly used in 2 situations:
- Drug allergy. - -ve SPT for highly
suspected Ag.

53. Nasal provocation test
 Each specific organ or location may have its own pattern of acquiring lgE sensitized
mast cells.
 Specific lgE may be locally produced within the nasal mucosa without being detectable
in skin or blood tests. So, NPT may occasionally be +ve for a specific Ag while allergic
skin tests and serologic lgE assays related to that Ag are —ve. There is no general +ve
correlation between skin test reactivity and symptoms of allergic rhinitis.
 NPT is intended to reproduce pathologic reactions of allergic nasal mucosa to defined
aeroallergens and occupationally relevant substances under standardized conditions.
 NPT may be a safer alternative to bronchial provocation when evaluating the relevance
of specific allergens to the etiology of bronchial asthma.

54. Serum specific IgE measurement
 Is an in vitro test to demonstrate sensitization.
 Serum IgE can be measured by sensitive immunoassays such as radioimmuniassay or
fluorescent immunoassay or enzyme immunoassay.
 Serum is incubated with allergen of intrest bound to solid phase.
 IgE with specificity to the allergen of intrest will remine on the solid phase, which is then
detected by labelled anti-IgE antibody.
 The level of the signal is proportional to the sIgE .
 False positive : if total IgE >1000kU/l due to nonspecific binding.
1. immunoCAP- cellulose sponge matrix. ( most widely used)
2. Immulite system –avidin solid phase.
3. HYTEC-288 - cellulose wafer
 Level igE is not directly correlated with the severity of allergy.

55. RAST (Radio-allergosorbent test)
 radioallergosorbent test (RAST) was described in 1967 by Wide, Bennich.
and Johansson as an in vitro diagnostic test for allergen antibodies.
 Allergens are covalently bound to solid-phase polysaccharides activated
by reaction with cyanogen bromide.
 In the first step of the RAST, solid-phase allergen reacts with serum, and
antibodies to the allergens (including IgE antibodies) bind. In the second
step of the RAST, after washing to remove unbound antibodies, the solid-
phase allergen-antibody complex reacts with radioiodinated affinity
chromatography-purified antibody to IgE. This complex is washed again,
and the bound radioactivity is measured. the quantity of radioactivity
bound to the solid-phase allergen-IgE complex is proportional to the
quantity ofIgE antibodies in the first step.

57. RAST scale (0-6)

58. Indications of sIgE measurement
 High risk of anaphylaxis
 Using medications that interfere with SPT
 Skin lesions unsuitable for SPT
 Small children

59. Basophil activation test (BAT)
 BAT is a invitee study of peripheral basophil responses to the stimuli.
 2 methods
1. Measurement of mediator release (eg:histamine)
2. Detecting the increase in cell surface molecules or newly expressed cell
surface molecules (Eg: CD63, CD 69,CD203)
 Whole blood of the patient is incubated with the allergen od intrest.
 Basophils are stimulated due to the cross linking of the specific IgE-FcęRI on
the basophil.
 Degranulation of basophil leads to expression of molecules on its surface.
 CD63 expression closely correlates with histamine release in degranulation.
 These can be detected by flow cytometry method.

60. Take a structured patient history including
symptom severity
Confirm the identity of the allergen with
an objective test:
a. First attempt to do skin prick test or measure slgE
b. consider an intradermal test in drug and insect venom
allergy
Consider BAT a If the allergen is known to
produce false positive results in skin testing
b. If there is no allergen source to use for skin or slgE testing
c. If there is discordance between the patient history and slgE or skin
tests
d. If the symptoms in the patient history suggest that skin testing may
result in systemic response
e. Before considering a provocation to confirm the causative allergen

61.  Sensitivity- 85% (AR), 50% (LAR)
 Specificity- 93%
 PPV – 0.92 (AR), 0.89 (LAR).
 NPV – 0.87 (AR), 0.62 (LAR).
BAT has been used to assess allergic response for allergens that are not
commercially avilable for sIgE measurement. These includes aerollergens
,drugs,venom,and food allergens.
Limitations:
 Not well standersized
 Needs fresh sample and skilled technicians
 Highly trained immunologist.

62. Non validated tests
 Vega test
 Kinesiology
 Hair analysis
 Provaction-neutralization
 Iridiology
 Leucocytotoxic testing
 Auriculo cardiac reflex