In vitro Diagnosis of Drug Allergy: Current Status and Perspectives

Июль 23, 2022

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In vitro Diagnosis of Drug Allergy: Current Status and Perspectives

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2. Dr. Fleisher has no conflicts of interest related to this presentation
3. Drugs as Immunogens Biologics: foreign macromolecules (e.g. antibodies, recombinant proteins) act directly as immunogen Drugs (non-biologics)
4. Use of in vitro Testing for Drug Allergy Testing in the setting of an immediate drug
5. Immediate Reaction to Drug Gell and Coombs type 1 reaction that occurs rapidly upon exposure to
6. Tryptase Testing Mature tryptase reflects mast cell degranulation and is elevated in a systemic allergic reaction
7. Allergen Specific IgE Testing In vitro “equivalent” of immediate skin testing Does not subject patient to
8. Basophil Activation Test Test evaluates basophils present in either whole blood or separated mononuclear cells Validated
9. Basophil Activation Test Steiner, M. et al. J Vis Exp 2011 Gating “lymphocytes” Gating basophils Negative
10. Basophil Activation Test Advantages Does not subject patient to any risks Functional test that resembles the
11. BAT in Radiocontrast Media Reactions Evaluation of 26 patients with history of immediate radiocontrast media (RCM)
12. Delayed Immunologic Reaction to Drugs Most commonly linked to cellular response (Gell and Coombs Type IV
13. Focus of in vitro Testing Confirm that the clinical findings are the result of an immunologic
14. Varied concentrations of pure drug, incubate at 37ºC with 5% CO2 Peripheral blood mono-nuclear cells (PBMC)
15. Lymphocyte Transformation Test (LTT) Must use controls to establish lack of drug induced toxicity and to
16. Evaluation of LTT in Different Types of Delayed Hypersensitivity Drug Reactions 27 patients in three groups:
17. LTT Used to Identify the Drug that Induced DRESS Two patients receiving multiple drugs including anticonvulsants
18. LTT Summary LTT appears to be a suitable complement to other testing in delayed drug reactions
19. Alternatives to LTT (3H Thymidine) Evaluation of upregulation of a T cell activation antigen in response
20. Varied concentrations of pure drug, incubate at 37ºC with 5% CO2 PBMC I- Activation in vitro
21. CD69 Upregulation in Response to Drug Evaluation of a phenytoin-allergic patient following 48 hrs of stimulation
22. Summary of LTT Alternatives CD69 upregulation appears to perform similar to LTT with the advantage of
23. Immunopathogenesis of SJS/TEN Bullous skin processes (SJS/TEN) associated with drugs appear to be linked to cytotoxic
24. “Real Time” Test to Diagnose SJS/TEN The serum level of granulysin is ~100X greater than sFasL
25. In the Future Multiplex cytokine evaluation following in vitro culture (e.g. IFN-γ, IL-2, IL-4, IL-5, IL-8,
26. Summary in vitro Testing in Drug Allergy Immediate drug reactions Specific IgE testing: safe test but
27. Conclusions The clinical story remains the most important starting point evaluating possible drug allergy In vitro
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Dr. Fleisher has no conflicts of interest related to this presentation

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Drugs as Immunogens
Biologics: foreign macromolecules (e.g. antibodies, recombinant proteins) act directly

as immunogen
Drugs (non-biologics)
Hapten – drug (e.g. β-lactam antibiotics, quinidine) combines with a host macromolecule
Pro-hapten – processed drug (e.g. sulfonamides, phenytoin) combines with a host macromolecule
Drugs can act directly to stimulate an immune receptor (pharmacologic interaction with immune receptors = p-i concept)

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Use of in vitro Testing for Drug Allergy
Testing in the setting

of an immediate drug reaction
Testing in the setting of a delayed drug reaction
Testing on the horizon

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Immediate Reaction to Drug
Gell and Coombs type 1 reaction that occurs

rapidly upon exposure to a specific drug
Standard approach to evaluate is immediate skin testing (penicillin major and minor determinants are validated, other drugs ?)
In vitro methods of evaluation include:
Tryptase to establish mast cell degranulation
Allergen (drug) specific IgE testing
Basophil activation test (BAT)

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Tryptase Testing
Mature tryptase reflects mast cell degranulation and is elevated in

a systemic allergic reaction
Current laboratory test most widely available measure total tryptase (not mature tryptase)
Released within 30-60 minutes following activation and half life is ~2 hours allows longer “testing window”
Levels above normal range (vary among labs: 10-11.4 ng/mL) are consistent with anaphylaxis (or increased mast cell numbers) but the sensitivity is not high
More sensitive test for anaphylaxis: mature tryptase level or a total tryptase rise over baseline of > 2 ng/mL

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Allergen Specific IgE Testing
In vitro “equivalent” of immediate skin testing
Does not

subject patient to risk and does not have a potential of inducing sensitization
Limited range of drugs available impacts utility: β-lactams (penicilloyl G & V, ampicilloyl, amoxocilloyl), ACTH, cefator, ceftriazone, chlorhexidene, ethylene oxide, gelatin, insulin, neuromuscular blocking agents, tetanus toxoid)
Tests generally have high specificity with lower sensitivity - negative test does not rule out allergy

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Basophil Activation Test
Test evaluates basophils present in either whole blood or

separated mononuclear cells
Validated for aeroallergens, hymenoptera venoms, foods, latex, some drugs (generally based on a generated drug-protein complex)
Commercial assay (not FDA approved in USA): uses expression of CCR3 to identify basophils and expression of CD63 to identify activation after incubating cells the with drug complex
“Enhanced assay” adds a third marker, CD203c

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Basophil Activation Test
Steiner, M. et al. J Vis Exp 2011
Gating

“lymphocytes” Gating basophils Negative control

Drug-HSA Negative control Positive control

Positive control: 52.5% CD63+, SI - 5501/386 = 14.2

Positive drug BAT: 20.6% CD63+; SI - 1893/386 = 4.9

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Basophil Activation Test
Advantages
Does not subject patient to any risks
Functional test that

resembles the in vivo pathway
Relatively good sensitivity with high specificity
Positive BAT depends on type of allergen
Aeroallergens/foods >15% CD63+ basophils
Venoms >10% CD63+ basophils
Drugs (β-lactams, analgesics) >5% CD63+ basophils
Disadvantages
Must have viable, non-activated cells (24 hr “window”)
More limited availability since it requires a flow cytometer and generation of drug-protein (hapten-carrier) complex
Negative test does not rule out drug allergy

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BAT in Radiocontrast Media Reactions
Evaluation of 26 patients with history of

immediate radiocontrast media (RCM) reactions: BAT using five different RCM products (tested months later)
BAT results: 15/26 patients had a positive BAT
1:100 RCM: patients = 13.1% CD63+/SI=8.1 (p=0.01)
controls = 2.7% CD63+/SI=1.5
1:10 RCM: patients = 19.2% CD63+/SI=9.0 (p=0.001)
controls = 3.7% CD63+/SI=2.3
Receiver Operator Curve (ROC) area under the curve was 0.79 = test with moderate accuracy

Pinnobphun P, et al. Ann Allergy Asthma Immunol 2011, 106:387

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Delayed Immunologic Reaction to Drugs
Most commonly linked to cellular response (Gell

and Coombs Type IV reaction involving T cells)
These reactions have been subdivided into
Type IVa: mediated by Th1 response
Type IVb: mediated by Th2 response
Type IVc: mediated by cytotoxic cell response
Type IVd: mediated by neutrophilic inflammation
Additional data now suggests that some reactions involve conventional TcR activation (e.g. where there is an HLA link) and others involve direct drug-immune receptor interaction (p-i concept)

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Focus of in vitro Testing
Confirm that the clinical findings are

the result of an immunologic response (rather than a pharmacologic or idiosyncratic response)
Identify the causative drug in settings where multiple drugs have been administered
Current testing methods
Lymphocyte transformation test (LTT)
CD69 upregulation flow cytometry test
Cytokine production
Evaluation of cytotoxicity (or its products)

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Varied concentrations of pure drug, incubate at 37ºC with 5% CO2
Peripheral

blood mono-nuclear cells (PBMC)

I- Activation in vitro

II- Quantify Response

Harvest cells and count radioactivity, results: cpm or stimulation index (SI = drug stimulated cpm/unstimulated cpm)

PBMC

Cells

Lymphocyte Transformation Test (LTT)

Add 3H thymidine

T cell

6 days

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Lymphocyte Transformation Test (LTT)
Must use controls to establish lack of drug

induced toxicity and to rule out non-specific activation
Must have viable cells and requires sterile tissue culture
LTT has been successfully applied to drug associated:
Maculopapular exanthem
Pustular exanthem
Stevens Johnson syndrome (SJS)/toxic epidermal necrolysis (TEN)
Drug rash with eosinophilia and systemic symptoms (DRESS)
Positive LTT has generally been defined as a stimulation index (SI = cpm with drug/cpm with medium) > 2
Sensitivity is 60-70% under optimal conditions with a higher specificity
Negative test does not rule out T cell mediated drug response

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Evaluation of LTT in Different Types of Delayed Hypersensitivity Drug Reactions
27

patients in three groups: 8 maculopapular eruptions (MP), 6 SJS + 2 TEN, 11 DRESS
Evaluated by LTT at 1 week, 2-4 weeks, 5-8 weeks, 1 year and > 1 year following onset
Patients with MP and SJS/TEN had positive LTT at 1 week post-onset, response declined over time
Patients with DRESS were negative at 1 week and were positive at 5-8 weeks

Kano Y, et al. Allergy 2007, 62:1439

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LTT Used to Identify the Drug that Induced DRESS
Two patients receiving

multiple drugs including anticonvulsants and antibiotics associated with the development of DRESS
Evaluation by LTT utilized all drugs that had been given, each at 7 concentrations (1-200 μg/ml)
Studied 3 months after the clinical presentation
Causative drug was identified as ceftriaxone in one pt and piperacillin-tazobactam in the other pt
LTT assay proved valuable in defining the drug associated with DRESS (avoid in the future)

Jurado-Palomo J, et al. J Investig Allergol Clin Immunol 2010, 20:433

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LTT Summary
LTT appears to be a suitable complement to other testing

in delayed drug reactions
Time line of positivity may differ between the different types of delayed drug reactions
Positive test helps identify the offending drug but a negative test does not rule out drug related hypersensitivity
The test remains a research tool, it is not standardized and it requires tissue culture with results available after six or more days

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Alternatives to LTT (3H Thymidine)
Evaluation of upregulation of a T cell

activation antigen in response to in vitro drug exposure
CD69 up-regulation, an early product of T cell activation, measured by flow cytometry at 48 hrs
Ex vivo cytokine production
Cytokine secretion into the supernatant following mononuclear cell culture with drug (e.g. γ-IFN)
Elispot assay measures individual T cell production of a cytokine following in vitro drug stimulation

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Varied concentrations of pure drug, incubate at 37ºC with 5% CO2
PBMC
I-

Activation in vitro

II- Quantify Response

PBMC

Evaluate T cells by flow cytometry

CD69 upregulation expressed as percent CD69 positive T cells

T cell CD69 Upregulation

T cell

CD69

48 hours

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CD69 Upregulation in Response to Drug
Evaluation of a phenytoin-allergic patient following

48 hrs of stimulation
medium - negative control

Lochmatter P, et al. Immunol Allergy Clin N Am 2009, 29:537

Tetanus toxoid - positive control

Phenytoin - positive test

Unrelated drug clonazapam –
negative test

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Summary of LTT Alternatives
CD69 upregulation appears to perform similar to LTT

with the advantage of being a 48 hour assay and not requiring radionuclides
Cytokine production assays correspond to LTT but the actual cytokine produced does not appear to correlate well with the clinical phenotype (i.e. IFN-γ is typically produced with all types of delayed drug reactions)

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Immunopathogenesis of SJS/TEN
Bullous skin processes (SJS/TEN) associated with drugs appear to

be linked to cytotoxic T cell activity

sFasL

Porebski G, et al. Clin Exp Allergy 2011, 41:461

Soluble Fas ligand (sFasL) and granulysin have been found in the serum of patients with SJS/TEN

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“Real Time” Test to Diagnose SJS/TEN
The serum level of granulysin is

~100X greater than sFasL in SJS/TEN making it an attractive target
An immunochromagraphic test for serum granulysin (>10 ng/mL) predicted SJS/TEN 2-4 days prior to mucocutaneous reuptions
This assay could prove useful in predicting when a drug reaction will lead to SJS/TEN

Fujita Y, et al. J Am Acad Dermatol 2011, 65:65

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In the Future
Multiplex cytokine evaluation following in vitro culture (e.g. IFN-γ,

IL-2, IL-4, IL-5, IL-8, IL-13, IL-17, etc) may reveal specifics about the type of immune response
Nature of drug derived epitopes inducing an immune reaction often are not well understood
Mass spectrometry (MS) has evolved as a powerful tool to evaluate proteomics and metabolomics
MS used to characterize the functional antigens derived from piperacillin (in CF patient serum) with the identification of multiple drug derived haptenic structures bound to albumin (Whitaker P, et al. J Immunol 2011, 187:200)

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Summary in vitro Testing in Drug Allergy
Immediate drug reactions
Specific IgE

testing: safe test but there are limited numbers of suitable drug conjugates available for testing
BAT: promising functional test that requires viable cells and a drug conjugate preparation for activation
Delayed drug reactions
Lymphocyte transformation test (LTT)
Most common research method to determine responsible drug
Issues remaining include: standardization, requirement for viable cells, six day sterile tissue culture period and use of radionuclides
CD69 upregulation may be equivalent to LTT – under study
In vitro cytokine production to drug – under study
Product of cytotoxic cells (granulysin) promising to help dx SJS/TEN prior to mucocutaneous symptoms (further study)

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Conclusions
The clinical story remains the most important starting point evaluating possible

drug allergy
In vitro testing can be complementary to in vivo testing and is evolving for the evaluation of both immediate and delayed drug allergy
There is currently no single laboratory test that reliably establishes the drug responsible for an immunologically mediated drug reaction

In vitro Diagnosis of Drug Allergy: Current Status and Perspectives

Содержание

Dr. Fleisher has no conflicts of interest related to this presentation

Drugs as ImmunogensBiologics: foreign macromolecules (e.g. antibodies, recombinant proteins) act directly

Use of in vitro Testing for Drug AllergyTesting in the setting

Immediate Reaction to DrugGell and Coombs type 1 reaction that occurs

Tryptase TestingMature tryptase reflects mast cell degranulation and is elevated in

Allergen Specific IgE TestingIn vitro “equivalent” of immediate skin testingDoes not

Basophil Activation TestTest evaluates basophils present in either whole blood or

Basophil Activation TestSteiner, M. et al. J Vis Exp 2011 Gating

Basophil Activation TestAdvantagesDoes not subject patient to any risksFunctional test that

BAT in Radiocontrast Media ReactionsEvaluation of 26 patients with history of

Delayed Immunologic Reaction to DrugsMost commonly linked to cellular response (Gell

Focus of in vitro Testing Confirm that the clinical findings are

Varied concentrations of pure drug, incubate at 37ºC with 5% CO2Peripheral

Lymphocyte Transformation Test (LTT)Must use controls to establish lack of drug

Evaluation of LTT in Different Types of Delayed Hypersensitivity Drug Reactions27

LTT Used to Identify the Drug that Induced DRESSTwo patients receiving

LTT SummaryLTT appears to be a suitable complement to other testing

Alternatives to LTT (3H Thymidine)Evaluation of upregulation of a T cell

Varied concentrations of pure drug, incubate at 37ºC with 5% CO2PBMCI-

CD69 Upregulation in Response to DrugEvaluation of a phenytoin-allergic patient following

Summary of LTT AlternativesCD69 upregulation appears to perform similar to LTT

Immunopathogenesis of SJS/TEN Bullous skin processes (SJS/TEN) associated with drugs appear to

“Real Time” Test to Diagnose SJS/TENThe serum level of granulysin is

In the FutureMultiplex cytokine evaluation following in vitro culture (e.g. IFN-γ,

Summary in vitro Testing in Drug Allergy Immediate drug reactionsSpecific IgE

ConclusionsThe clinical story remains the most important starting point evaluating possible

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