TYPE 1 HYPERSENSITIVITY REACTION INVESTIGATIONS
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TYPE 1 HYPERSENSITIVITY REACTION INVESTIGATIONS
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Type I Hypersensitivity Reaction - Investigations
Mechanism (Brief Overview)
Type I (immediate) hypersensitivity results from antigen binding to IgE on the surface of mast cells and basophils via high-affinity FcεRI receptors. Cross-linking of IgE by allergen triggers degranulation, releasing preformed mediators (histamine, tryptase, heparin) and de novo mediators (prostaglandin D2, leukotrienes).
Classification of Investigations
Investigations are divided into In Vivo and In Vitro tests.
A. IN VIVO TESTS
1. Skin Prick Test (SPT) / Puncture Test
The most widely used, rapid, and sensitive test for IgE-mediated allergy. A small break in the epidermis (forearm or upper back) is made with a lancet, allowing penetration of the allergen. The allergen binds to mast cell-bound IgE; if sufficient crosslinking occurs, degranulation produces the wheal-and-flare reaction.
- Reading: Measured at 15-30 minutes. A wheal ≥3 mm in diameter (compared to histamine positive control) is positive.
- Controls: Histamine (positive) and saline (negative) controls are mandatory.
- Late-phase reaction (LPR): Deep tissue swelling beginning 1-2 hours after testing, resolving in 24-48 hours; IgE-dependent but not used for diagnosis.
Contraindications/Limitations:
- Patients who had anaphylaxis within the last 30 days (skin becomes unreactive for 2-4 weeks)
- Dermographism, urticaria, cutaneous mastocytosis, atopic dermatitis
- Medications that blunt results: antihistamines, tricyclic antidepressants, omalizumab, topical steroids in test area, beta-blockers (increase anaphylaxis risk with testing)
2. Intradermal Test (Intracutaneous Test)
A small amount of allergen is injected under the skin surface. More sensitive than SPT but less specific. Used when SPT is negative but clinical suspicion remains high.
3. Patch Test
Used for allergic contact dermatitis (delayed/Type IV). Allergen patches placed on the back for ~48 hours, read at 72-96 hours. This is NOT a Type I test but is listed here for comparison.
4. Nasal/Bronchial Provocation Challenge
- Nasal provocation testing: Used to diagnose Local Allergic Rhinitis (LAR) - a condition where SPT and serum sIgE are negative but local IgE production occurs in nasal mucosa. Definitive diagnosis by positive nasal challenge or detection of antigen-specific IgE in nasal secretions.
- Bronchial challenge: Used for suspected occupational asthma. Direct exposure to inhaled allergen/chemical.
5. Food Challenge (Oral Provocation)
The gold standard for IgE-mediated food allergy diagnosis. Performed under expert supervision. A positive skin test or RAST to a food the patient rarely ingests should be confirmed by oral food challenge.
B. IN VITRO TESTS
1. Total Serum IgE
Measures the overall quantity of IgE in blood. Useful as a screening tool.
- Normal: varies with age; elevated levels ≥100-150 IU/mL suggest atopy.
- Elevated in: atopic disorders (asthma, hay fever, eczema), parasitic infections, allergic bronchopulmonary aspergillosis (ABPA), IgE myeloma, Hyper-IgE syndrome, Wiskott-Aldrich syndrome.
- Limitations: Significant overlap between allergic and non-allergic individuals. A normal total IgE does NOT exclude allergy - patients may have high allergen-specific IgE with normal total IgE.
- Methods used: ELISA, near-infrared particle immunoassay (NIPIA), fluorescence immunoassay (FEIA), chemiluminescence immunoassay (CIA).
2. Allergen-Specific IgE (sIgE) - "RAST" / ImmunoCAP
The most important in vitro investigation for Type I hypersensitivity.
Classic RAST (Radioallergosorbent Test) - now largely replaced but principle remains:
- Antigen-bound paper disc is placed in a test tube with patient serum
- Allergen-specific IgE in the serum binds to antigen on the disc
- Excess non-specific IgE is washed away
- Radiolabeled anti-IgE antibody is added and binds to the patient's IgE
- Excess anti-IgE washed away; bound radiolabel measured by gamma counter
Modern assays use enzyme-linked immunoassay (ELISA) or FEIA rather than radiolabels. Commercial systems include:
- ImmunoCAP (Thermo Fisher/Phadia) - most widely used
- Immulite (Siemens)
- HYTEC-288 (Hycor/Agilent)
All three are automated, use the same WHO IgE reference standard (75/502), and offer similar sensitivity.
Advantages over skin testing:
- No risk of anaphylaxis
- Not affected by skin conditions (dermographism, eczema)
- No need to stop medications before testing
- Can test multiple allergens simultaneously
Disadvantage: More expensive; longer turnaround.
Key point: A positive sIgE only indicates sensitization, not necessarily clinically significant allergy.
3. Serum Tryptase
The best biological marker of anaphylaxis (acute Type I reaction).
- Tryptase is a mast cell-specific serine protease stored in granules; released during degranulation.
- Peaks at 60-90 minutes after onset of anaphylaxis.
- Half-life: ~2 hours - must be measured close to onset of symptoms and again ~2 weeks after recovery (baseline).
- Elevated baseline tryptase indicates increased risk for severe anaphylaxis (especially insect-sting triggered) and should prompt evaluation for an occult mast cell disorder (e.g., systemic mastocytosis).
- Chronically elevated tryptase is seen in systemic mastocytosis and hereditary alpha-tryptasemia.
4. Plasma/Serum Histamine
- Peaks very early (~10 minutes) after onset of anaphylaxis.
- Returns to normal within 1 hour (much shorter window than tryptase).
- Urinary histamine (or urinary histamine metabolites) may remain elevated for up to 24 hours - a more practical sample.
- Less commonly used in routine clinical practice compared to tryptase.
5. Basophil Activation Test (BAT)
An emerging in vitro assay becoming a reliable second-line test.
- Patient blood is incubated with the suspected allergen + IL-3 (to enhance expression)
- Activated basophils up-regulate surface markers CD63 and CD203c
- Detected and quantified by flow cytometry
- CD63 is the best clinically validated marker; CD203c is also reliable.
- Sensitivity 77-98%, specificity 75-100% - superior to sIgE tests in distinguishing clinically allergic from tolerant-but-sensitized individuals.
- Useful when SPT and sIgE are discordant; essential for confirming drug allergy (sometimes the only available test).
- Also used to monitor response to omalizumab in chronic idiopathic urticaria.
6. Basophil Histamine Release Test
- Measures release of histamine from peripheral blood basophils incubated with allergen.
- Requires samples to be tested within 24 hours (living cells).
- Not standardized; considered an investigative tool only.
- Useful in chronic urticaria (detects functional IgG antibodies to FcεRI alpha in ~30-40% of patients).
7. Component-Resolved Diagnosis (CRD)
- Detects sIgE at the individual allergen protein/component level using purified or recombinant allergen components (e.g., Ara h2 for peanut allergy).
- Can distinguish cross-reactivity from true co-sensitization.
- Helps predict risk of systemic reactions and guides immunotherapy selection.
- Expensive; not first-line. Reserved for polysensitized patients, unclear symptoms, or non-responders to treatment.
- Example: Can f 5 (a prostatic protein from male dogs) - up to 38% of dog-allergic patients are mono-sensitized to Can f 5, meaning they tolerate female dogs.
8. Eosinophil Count and Eosinophil Cationic Protein (ECP)
- Blood eosinophilia is supportive (not diagnostic) of atopic disease.
- ECP (eosinophil cationic protein) reflects eosinophil activation; elevated in both immediate and late-phase IgE-mediated reactions.
- ECP is a protein of 133 amino acids (18.5 kDa) originating from activated eosinophils.
Summary Table
| Investigation | Type | What it detects | Timing/Notes |
|---|---|---|---|
| Skin Prick Test (SPT) | In vivo | Allergen-specific IgE on mast cells | Read at 15-30 min; wheal ≥3 mm = positive |
| Intradermal test | In vivo | sIgE; more sensitive than SPT | Second-line when SPT negative |
| Nasal/Bronchial provocation | In vivo | Local IgE response | For LAR and occupational allergy |
| Oral food challenge | In vivo | Food allergy confirmation | Gold standard for food allergy |
| Total serum IgE | In vitro | Overall IgE burden | Normal does NOT exclude allergy |
| Specific IgE (ImmunoCAP/RAST) | In vitro | Allergen-specific sensitization | Indicates sensitization, not clinical allergy |
| Serum tryptase | In vitro | Mast cell degranulation / anaphylaxis | Peaks 60-90 min; t½ = 2 hours |
| Urinary histamine | In vitro | Histamine release | Elevated up to 24 hours |
| Basophil Activation Test (BAT) | In vitro | CD63/CD203c on stimulated basophils | Best sensitivity/specificity; flow cytometry |
| Basophil histamine release | In vitro | Basophil degranulation | Investigative only; not standardized |
| Component-resolved diagnosis | In vitro | Individual allergen proteins | Expensive; third-line |
| Blood eosinophil count/ECP | In vitro | Eosinophil activation | Supportive evidence only |
Sources:
- Tietz Textbook of Laboratory Medicine, 7th Edition - Allergy Tests section
- Goldman-Cecil Medicine, International Edition - Laboratory Evaluation
- K.J. Lee's Essential Otolaryngology - Diagnosis of Allergic Disease
- Cummings Otolaryngology Head and Neck Surgery - Basophil Activation Test / Component Resolved Diagnosis
- Quick Compendium of Clinical Pathology, 5th Edition - Type I Hypersensitivity
- Harrison's Principles of Internal Medicine, 22E (2025) - Diagnosis of Anaphylaxis
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