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Презентация на тему In vitro Diagnosis of Drug Allergy: Current Status and Perspectives

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Dr. Fleisher has no conflicts of interest related to this presentation
In vitro Diagnosis of Drug Allergy: Current Status and Perspectives Thomas A. Dr. Fleisher has no conflicts of interest related to this presentation Drugs as ImmunogensBiologics: foreign macromolecules (e.g. antibodies, recombinant proteins) act directly as Use of in vitro Testing for Drug AllergyTesting in the setting of Immediate Reaction to DrugGell and Coombs type 1 reaction that occurs rapidly Tryptase TestingMature tryptase reflects mast cell degranulation and is elevated in a Allergen Specific IgE TestingIn vitro “equivalent” of immediate skin testingDoes not subject Basophil Activation TestTest evaluates basophils present in either whole blood or separated Basophil Activation TestSteiner, M. et al. J Vis Exp 2011 Gating “lymphocytes” Basophil Activation TestAdvantagesDoes not subject patient to any risksFunctional test that resembles BAT in Radiocontrast Media ReactionsEvaluation of 26 patients with history of immediate Delayed Immunologic Reaction to DrugsMost commonly linked to cellular response (Gell and Focus of in vitro Testing Confirm that the clinical findings are the Varied concentrations of pure drug, incubate at 37ºC with 5% CO2Peripheral blood Lymphocyte Transformation Test (LTT)Must use controls to establish lack of drug induced Evaluation of LTT in Different Types of Delayed Hypersensitivity Drug Reactions27 patients LTT Used to Identify the Drug  that Induced DRESSTwo patients receiving LTT SummaryLTT appears to be a suitable complement to other testing in Alternatives to LTT (3H Thymidine)Evaluation of upregulation of a T cell activation Varied concentrations of pure drug, incubate at 37ºC with 5% CO2PBMCI- Activation CD69 Upregulation in Response to DrugEvaluation of a phenytoin-allergic patient following 48 Summary of LTT AlternativesCD69 upregulation appears to perform similar to LTT with Immunopathogenesis of SJS/TEN	Bullous skin processes (SJS/TEN) associated with drugs appear to be “Real Time” Test to Diagnose SJS/TENThe serum level of granulysin is ~100X In the FutureMultiplex cytokine evaluation following in vitro culture (e.g. IFN-γ, IL-2, Summary in vitro Testing in Drug Allergy Immediate drug reactionsSpecific IgE testing: ConclusionsThe clinical story remains the most important starting point evaluating possible drug ReferencesFujita Y, et al. Rapid immunchromatographic test for serum granulysin is useful
Слайды презентации

Слайд 2 Dr. Fleisher has no conflicts of interest related

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

to this presentation


Слайд 3 Drugs as Immunogens
Biologics: foreign macromolecules (e.g. antibodies, recombinant

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

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)

Слайд 4 Use of in vitro Testing for Drug Allergy
Testing

Use of in vitro Testing for Drug AllergyTesting in the setting

in the setting of an immediate drug reaction
Testing in

the setting of a delayed drug reaction
Testing on the horizon


Слайд 5 Immediate Reaction to Drug
Gell and Coombs type 1

Immediate Reaction to DrugGell and Coombs type 1 reaction that occurs

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)


Слайд 6 Tryptase Testing
Mature tryptase reflects mast cell degranulation and

Tryptase TestingMature tryptase reflects mast cell degranulation and is elevated in

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

Слайд 7 Allergen Specific IgE Testing
In vitro “equivalent” of immediate

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

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

Слайд 8 Basophil Activation Test
Test evaluates basophils present in either

Basophil Activation TestTest evaluates basophils present in either whole blood or

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

Слайд 9 Basophil Activation Test
Steiner, M. et al. J Vis

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

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


Слайд 10 Basophil Activation Test
Advantages
Does not subject patient to any

Basophil Activation TestAdvantagesDoes not subject patient to any risksFunctional test that

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

Слайд 11 BAT in Radiocontrast Media Reactions
Evaluation of 26 patients

BAT in Radiocontrast Media ReactionsEvaluation of 26 patients with history of

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


Слайд 12 Delayed Immunologic Reaction to Drugs
Most commonly linked to

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

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)

Слайд 13 Focus of in vitro Testing
Confirm that the

Focus of in vitro Testing Confirm that the clinical findings are

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)

Слайд 14





Varied concentrations of pure drug, incubate at 37ºC

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

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

PBMC


Cells



Lymphocyte Transformation Test (LTT)

Add 3H thymidine


T cell

T cell

6 days


Слайд 15 Lymphocyte Transformation Test (LTT)
Must use controls to establish

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

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


Слайд 16 Evaluation of LTT in Different Types of Delayed

Evaluation of LTT in Different Types of Delayed Hypersensitivity Drug Reactions27

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


Слайд 17 LTT Used to Identify the Drug that Induced

LTT Used to Identify the Drug that Induced DRESSTwo patients receiving

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


Слайд 18 LTT Summary
LTT appears to be a suitable complement

LTT SummaryLTT appears to be a suitable complement to other testing

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

Слайд 19 Alternatives to LTT (3H Thymidine)
Evaluation of upregulation of

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

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

Слайд 20





Varied concentrations of pure drug, incubate at 37ºC

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

with 5% CO2






PBMC
I- Activation in vitro
II- Quantify Response


PBMC
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


Слайд 21 CD69 Upregulation in Response to Drug

Evaluation of a

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

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



Слайд 22 Summary of LTT Alternatives
CD69 upregulation appears to perform

Summary of LTT AlternativesCD69 upregulation appears to perform similar to LTT

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)

Слайд 23 Immunopathogenesis of SJS/TEN
Bullous skin processes (SJS/TEN) associated with

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

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


Слайд 24 “Real Time” Test to Diagnose SJS/TEN
The serum level

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

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


Слайд 25 In the Future
Multiplex cytokine evaluation following in vitro

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

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)

Слайд 26 Summary in vitro Testing in Drug Allergy
Immediate

Summary in vitro Testing in Drug Allergy Immediate drug reactionsSpecific IgE

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)


Слайд 27 Conclusions
The clinical story remains the most important starting

ConclusionsThe clinical story remains the most important starting point evaluating possible

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

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