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Allergy School on Allergic Reactions to Drugs From Phenotype to Genotype Evaluation of Drug Hypersensitivity by flow cytometry (technical session) Enrique Gomez 19 - 21 September 2013 Carlos Haya - Hospital. (IBIMA) Malaga. Adverse Drug Reactions (ADR) A response to a drug that is noxious and unintended and occurs at doses normally used in humans for the prophylaxis, diagnosis or therapy of disease, or modification of physiological function. (World Health Organization, 1972). A percentage of all ADR are produced by an altered immunologic response to the drug and are named Allergic drug reactions with an immunological basis or ADRIB (Gruchalla R., J Allergy Clin Immunol. Allergy 2003). HOSPITAL ADMISSIONS ADR 6% HOSPITALIZED PATIENTS ALLERGIC REACTION 10-15% Bigby M, et al. JAMA. 1986 ALLERGIC REACTIONS CLASSIFICATION Type 1 Type 3 Type 2 Type 4 Gell and Coombs 1963 CLASSIFICATION OF ALLERGIC REACTIONS TIME INTERVAL IMMEDIATE: <1h IgE mediated Th2 (IL4, IL5, IL10, IL13) NON-IMMEDIATE: > 1 h – days T-cell mediated Th1 (IFNγ, TNFα, IL12) Levine BB. N Engl J Med 1966 ADVERSE REACTIONS TO DRUGS T-cell mediated IgE mediated Clinical diseases & pathological mechanisms Urticaria/angioedema Rhinitis / bronquial asthma Anaphylactic Shock Mast cell and basophil degranulation Multiform Erithema Exanthema Urticaria Fix drug eruption DRESS Cytotoxic CD4 or CD8 (perforin or granzyme B) and/or eosinophils Acute generalised exanthematic pustulosis Toxic epidermal necrolysis T cells involvement and recruitment and activation of neutrophils T cell involvement with massive keratinocyte apoptosis. In vivo Diagnostic tools in hypersensitivity to drugs Clinical history Skin tests Drug provocation test In vitro Immunoassays Basophil activation test Lymphocyte transformation test Monitoring of the response What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream. Flow Cytometer Single cell focusing. Lasers excitation produce a single wavelength that is read by detectors. Mixture of wavelengths, needs subsequent optical filtering. Flow Cytometer Flow cytometers use separate fluorescence (FL-) channels to detect light emitted. The number of detectors will vary according to the machine and its manufacturer. Fluorochrome Alexa405 BC Krome orange BD Horizon 450 BD Horizon V500 Pacific blue Pacific orange SYTOX Blue/DNA complex 7-AAD ALEXA FLUOR 488 CFSE ECD eYFP FITC PE PE-Alexa610 PE-Cy5 PE-Cy5.5 PE-Cy7 PE-Texas Red PerCP PerCP-Cy5.5 Propidium Iodide (PI) R-PE APC Alexa647 APC-Alexa700 APC-Alexa750 APC-Cy5.5 APC-Cy7 Laser (nm) 405 405 405 405 405 405 405 488 488 488 488 488 488 488 488 488 488 488 488 488 488 488 488 633 633 633 633 633 633 Excitation (nm) 402 398 404 415 410 400 444 548 495 494 496 513 485 496 496 496 496 496 496 490 490 496 650 650 650 650 650 650 Emisssion max (nm) 421 528 488 500 455 551 480 647 519 521 620 527 518 575 628 670 690 760 615 675 690 617 575 660 668 723 775 690 785 Flow Cytometer Flow Cytometer application Cell counting Cell sorting Biomarker detection Protein engineering Nano-structures detection Evaluation of hypersensitivity to drugs by flow cytometry IgE mediated Basophil Activation Test (BAT) T-cell mediated Lymphocyte Transformation Test (LTT) Basophil Activation Test (BAT) Leukocyte (Polimorphonuclear granulocyte) Constitute 0.1 -1% of peripheral blood cells Contain preformed mediators like Prostaglandin, Leukotrienes, Tryptase, Histamine. Release of the granular contain after contact with allergens. BAT in drug allergy Sensitivity 40 – 60 % depending on the drug Specificity 85 – 100% Test considerations Use heparin /EDTA blood Acid Citrate Dextrose (ACD) Test should be done as soon as possible after blood drawing à 3h is recommended Use only freshly reconstituted drugs Allergen Considerations No matrix/bound drugs, only liquid or solid phase pH has to be between 7.0 – 7.5 Light exposure over time Drug concentrations have to be evaluated when using first time Use several concentrations Attention!! Be sure to use the truly metabolite culprit of the reactions Advantages Functional in vitro test which best reflects the in vivo mechanisms Only valuable in vitro diagnostic tool for drugs Useful in case of low IgE serum levels Very flexible handling of drugs In general, standardized protocols Fast analysis acquisition at flow cytometer Mechanism of IgE mediated reactions 1st allergen contact (sensitisation) DC B7 CD28 T-cells T IL-4 B IgE Pre-activated basophil FcεRI Mechanism of IgE mediated reactions: 2nd allergen contact Release of mediators CD63 Activated basophil CD63 BAT protocol for flow cytometry mAb-Anti IgE / CD203c(FITC) mAb-Anti CD63 (PE) pre-stimulation Drug stimulation Labelled basophils Lysis of Red cells Flow cytometry Eosinóphils Polimorphonuclears Monocytes Lymphs - Basophils Flow cytometry Negative Control Positive Control 7.79 % Positive Control 63.2 % Dypirone Patient BAT Strategies • Activation marker: CD63+, CD203c+ • Selection marker: CCR3+ aIgE CD123+/HLADRCRTH2+CD3- BAT in IgE mediated reactions Drug allergic reactions Natural Allergens Inhalants: grasses, weeds, trees, mites, … Environmental: latex, … Foods: peanut, milk, egg, wheat, … Hymenoptera venoms … Monitoring & evaluate: - diseases - treatment (e.g. Immunotherapy) Concluding REMARKS BAT is an in vitro test useful in immediate responses (less than 1h after drug intake - Th2). BAT determines the basophil activation, after stimulation with the suspicious drug, responsible of the reaction. BAT permits us, to identify cross-reactivities and to test multiple conditions (allergens/Drugs) at the same time. There are different strategies according with the antibodies applied, to identify the basophils activation. BAT have to be carried out as soon as possible, because lost of sensitivity is associated with longer period after reactions (drugs). Mechanisms of T-cell mediated reactions in drug allergy Haptenization APC proteins Drug-protein Adduct < 1000D APC HLA-DR APC B7.1/B7.2 HLA-DR TCR CD4 T-cell CD28 IMMUNE RESPONSE Landsteiner K and Jacobs J. J Exp Allergy 1935; 61 Immune recognition APC APC APC HLA-DR B7.1 B7.2 B7.1 HLA-DR B7.2 Signal 1 TCR CD28 Signal 2 Co-stimulatory signals CD4 T-cells Activation and proliferation of specific T-cell clones Non Co-stimulatory signals HLA-DR TCR CD4 T-cells CD28 Tolerization, anergy or deletion of specific T cell clones Evaluation of T-cell responses Immunohistochemistry Real-time PCR Cell culture Flow-cytometry LTT Lymphocyte Transformation Test (LTT) It is a measurement of specific T-lymphocytes proliferation in culture conditions It detects not only proliferation but also key players of the reaction Proliferation can be measured by different means and through different markers SI=[% Stimulated Cells% / % Non-stimulated Cells ] Proper SI value depend of several variables (precursors, affinity of drug and TCR, Type of Reaction Th1 > Th2); SI > 2 positive Attention!! High SI ≠ higher severity Protocol & considerations (LTT) LTT has been demonstrated to be suitable in different pathologies and with different drugs: LTT IN DRUG HYPERSENSITIVITY SENSITIVITY SPECIFICITY 60-70% 85% • Sensitivity depends on the drug responsible • High values in LTT are associated with high precursor frequency of drug specific T cells. • Drug-specific cells persist for as long as 20 years in peripheral blood even after strict avoidance of the drug Beeler A et al. J Allergy Clin Immunol 2006;117:455-62. Pichler WJ et al. Allergy 2004;59:809-820. Protocol & considerations (LTT) Samples • Blood collection: tubes with Anticoagulant (EDTA, Heparin, others) • Isolation of PBMC by density gradient (Fycoll, others) • Avoid macrophages (PGE2, recommended <25%) • Freshly processed or stored as PBMC in liquid-N2. Medium & drugs • Medium Supplemented (L-Glutamine, essential aa, Antibiotics, autologous sera or others commercial serum 10% (complement-free 30’ at 560C. Filtered 0.22 um) • Drugs have to be pure substances diluted in medium, DMSO or others . • Prepare Freshly STOCK solutions and make dilutions. Protocol & considerations (LTT) Culture cells conditions: • 96 Plates U-shaped well bottom • 2x105 -2.5x105 recommended: Vf= 250 uL. • Negative and positive controls have to be used • TT, PHA, PPD, LPS…. • Control of media to evaluate contaminations (Bacteria, yeast, mycoplasma, virus, fungi…) Evidence= Change color of medium Lymphocyte Transformation Test (LTT) Peripheral blood mononuclear cells (PBMC) PBMC PBMC+anti-CD3 PBMC+Drug Proliferation [Thimidine incorporation /CFSE] LTT – Flow cytometry determination H3-Thymidine incorporation: Based in DNA duplication. Cells incorporate H3-Thym in sequences of new chains of DNA. Cells are incubated 14-16 hours more Scintillation fluid and β-radiation detector LTT – Flow cytometry determination Carboxyfluorescein succinimidyl ester (CFSE): Nº of generations Day 3 It is a fluorescent cell alive staining dye, stored in the cytoplasm of cells. Day 4 CFSE intensity No proliferation Day 2 Proliferation CFSE intensity LTT - Flow cytometry Strategy Different specific moAb, labelled with different fluorochromes are designed to identify, almost, all known markers for human cells. The strategy to follow will depend of the cell population of interest. Th1 cells Th2 cells B-cells From http://www.biolegend.com Kim,M et al. AAAI resp . 2012 Monocytes + B cells vs. DC as APC in the LTT (PBMC) Classic LTT T lymphocytes Monocytes + IL4 and GM-CSF 6 days Mo-imDC LTT in NIR to AX (Dendritic Cells) SI>3 Positive Monocytes+B cells versus DC as APC in the LTT Rodríguez-Pena R et al, J Allergy Clin Immunol 2006 LTT in NIR to Heparins (Dendritic Cells) Patient 1 20 S.I. S.I. 15 10 5 5 Dp Bp Np Tz Ep 0 Sd Patient 3 20 Bp Np Tz Ep Sd Ep Sd Patient 4 S.I. 15 10 5 0 Dp 20 15 S.I. 15 10 0 Patient 2 20 10 5 Dp Bp 20 Np Tz Patient 5 Ep 0 Sd Dp Bp 20 Np Tz Patient 6 mo/Bcell imDC S.I. S.I. 15 10 10 5 0 5 Dp Bp Np Tz Ep Dp Bp Mean S.I. 10 Np Tz Ep Sd Ep Sd Control 20 15 S.I. 0 Sd Patient 7 20 15 10 5 5 0 15 Dp Bp Np Tz Ep Sd 0 Dp Bp Np Tz Lopez S et al. Br J Dermatol. 2009 LTT with CFSE NIR to AMOXICILLIN CD3 CD4 LY+AX-ADH.001 LY+AX-ADH.001 5.02% without DCs 101 104 100 LY+DC+AX-ADH.001 with DCs 62.76% 101 M2 102 103 CFSE FITC 101 M1 102 103 CFSE FITC 104 100 M1 100 101 101 102 103 CFSE FITC 6.19% M2 M1 M2 102 103 CFSE FITC 104 LY+DC+AX-ADH.001 57.13% 104 M2 M2 LY+DC+AX-ADH.001 M1 100 1.24% M1 102 103 CFSE FITC LY+AX-ADH.001 4.33% M2 M1 100 CD8 4 10 100 101 102 103 CFSE FITC 104 Flow cytometry for monitoring acute response • Blood samples are obtained at different time-points during the acute and resolution phases. • Samples can be stored at -80C and be processed all at the same time to avoid artificial variations due to the process. • Specific subset markers are used to identify them and their kinetic along the reactions. • …. Monitoring acute response Analysis in different compartments 100 % Cells CD4 100 % Cells CD8 100 75 75 75 50 50 50 25 25 25 0 0 0 1 100 3 7 15 30 Days % Cells 1 3 7 15 30 Days 75 Peripheral Blood Blister fluid 50 0 0 Days 7 15 30 7 15 30 Days % Cells CLA 50 25 3 3 75 25 1 CD45RO 1 100 CD69 % Cells 1 3 7 Days 15 30 Mayorga C et al. Curr Opin Allergy Clin Immunol 2006 Concluding REMARKS LTT is an in vitro test useful in Non-Immediate responses (more than 1h after drug intake – Th1). LTT determines the T-cell activation-proliferation, after stimulation with the suspicious drug. LTT allows to identify cross-reactivity and to test multiple conditions (allergens/drugs) at the same time. Flow cytometry permits to identify different subpopulations involves in T-cells responses, through the use of specific moAb labelled with different fluorochromes. Flow cytometer permits the monitoring of subset of cells along the T-cell reaction. Tandem LTT/Flow Cytometry is an important tool not only in research, but also in diagnosis of Non-Immediate allergic reactions. Thanks to all of you