Evaluation of Drug Hypersensitivity by flow cytometry (technical session)

Transcription

1 Allergy School on Allergic Reactions to Drugs From Phenotype to Genotype Evaluation of Drug Hypersensitivity by flow cytometry (technical session) Enrique Gomez September 2013 Carlos Haya - Hospital. (IBIMA) Malaga.

2 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

3 ALLERGIC REACTIONS CLASSIFICATION Type 1 Type 2 Type 3 Type 4 Gell and Coombs 1963

4 CLASSIFICATION OF ALLERGIC REACTIONS TIME INTERVAL IMMEDIATE: < 1 h 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

5 ADVERSE REACTIONS TO DRUGS Clinical diseases & pathological mechanisms T-cell mediated IgE mediated Urticaria/angioedema Rhinitis / bronquial asthma Anaphylactic Shock Multiform Erithema Exanthema Urticaria Fix drug eruption DRESS Acute generalised exanthematic pustulosis Toxic epidermal necrolysis Mast cell and basophil degranulation Cytotoxic CD4 or CD8 (perforin or granzyme B) and/or eosinophils T cells involvement and recruitment and activation of neutrophils T cell involvement with massive keratinocyte apoptosis.

6 Diagnostic tools in hypersensitivity to drugs In vivo Clinical history Skin tests Drug provocation test Immunoassays In vitro Basophil activation test Lymphocyte transformation test Monitoring of the response

7 What Is Flow Cytometry? Flow ~ cells in motion Cyto ~ cell Metry ~ measure Measuring properties of cells while in a fluid stream.

8 Flow Cytometer Single cell focusing. Lasers excitation produce a single wavelength that is read by detectors. Mixture of wavelengths, needs subsequent optical filtering.

9 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 Laser (nm) Excitation (nm) Emisssion max (nm) Alexa BC Krome orange BD Horizon BD Horizon V Pacific blue Pacific orange SYTOX Blue/DNA complex AAD ALEXA FLUOR CFSE ECD eyfp FITC PE PE-Alexa PE-Cy PE-Cy PE-Cy PE-Texas Red PerCP PerCP-Cy Propidium Iodide (PI) R-PE APC Alexa APC-Alexa APC-Alexa APC-Cy APC-Cy

10 Flow Cytometer

11 Flow Cytometer application Cell counting Cell sorting Biomarker detection Protein engineering Nano-structures detection

12 Evaluation of hypersensitivity to drugs by flow cytometry IgE mediated Basophil Activation Test (BAT) T-cell mediated Lymphocyte Transformation Test (LTT)

13 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.

14 BAT in drug allergy Sensitivity % depending on the drug Specificity %

15 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

16 Allergen Considerations No matrix/bound drugs, only liquid or solid phase ph has to be between 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

17 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

18 Mechanism of IgE mediated reactions 1 st allergen contact (sensitisation) DC B7 CD28 T-cells T IL-4 B Pre-activated basophil IgE FcεRI

19 Mechanism of IgE mediated reactions: 2 nd allergen contact Release of mediators CD63 Activated basophil CD63

20 BAT protocol for flow cytometry mab-anti IgE / CD203c(FITC) mab-anti CD63 (PE) pre-stimulation Drug stimulation Lysis of Red cells Labelled basophils

21 Flow cytometry Eosinóphils Polimorphonuclears Monocytes Lymphs - Basophils

22 Flow cytometry Negative Control 7.79 % Positive Control Positive Control 63.2 % Dypirone Patient

23 BAT Strategies Activation marker: CD63+, CD203c+ Selection marker: CCR3+ aige CD123+/HLADR - CRTH2 + CD3 -

24 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)

25 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).

26 Mechanisms of T-cell mediated reactions in drug allergy

27 Haptenization APC proteins < 1000D Drug-protein Adduct HLA-DR APC APC HLA-DR B7.1/B7.2 TCR T-cell CD4 CD28 IMMUNE RESPONSE Landsteiner K and Jacobs J. J Exp Allergy 1935; 61

28 Immune recognition APC APC APC B7.1 HLA-DR B7.2 HLA-DR Signal 1 TCR CD4 T-cells B7.1 B7.2 CD28 Signal 2 Co-stimulatory signals HLA-DR TCR CD4 T-cells Non Co-stimulatory signals CD28 Activation and proliferation of specific T-cell clones Tolerization, anergy or deletion of specific T cell clones

29 Evaluation of T-cell responses Immunohistochemistry Real-time PCR Cell culture Flow-cytometry LTT

30 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

31 Protocol & considerations (LTT) LTT has been demonstrated to be suitable in different pathologies and with different drugs:

32 LTT IN DRUG HYPERSENSITIVITY SENSITIVITY 60-70% SPECIFICITY 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: Pichler WJ et al. Allergy 2004;59:

33 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-n 2. Medium & drugs Medium Supplemented (L-Glutamine, essential aa, Antibiotics, autologous sera or others commercial serum 10% (complement-free 30 at 56 0 C. Filtered 0.22 um) Drugs have to be pure substances diluted in medium, DMSO or others. Prepare Freshly STOCK solutions and make dilutions.

34 Protocol & considerations (LTT) Culture cells conditions: 96 Plates U-shaped well bottom 2x x10 5 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

35 Lymphocyte Transformation Test (LTT) Peripheral blood mononuclear cells (PBMC) PBMC PBMC+anti-CD3 PBMC+Drug Proliferation [Thimidine incorporation /CFSE]

36 LTT Flow cytometry determination H 3 -Thymidine incorporation: Based in DNA duplication. Cells incorporate H 3 -Thym in sequences of new chains of DNA. Cells are incubated hours more Scintillation fluid and β-radiation detector

37 LTT Flow cytometry determination Carboxyfluorescein succinimidyl ester (CFSE): It is a fluorescent cell alive staining dye, stored in the cytoplasm of cells. Nº of generations Day 2 Day 3 Day 4 No proliferation CFSE intensity Proliferation CFSE intensity

38 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

39 Kim,M et al. AAAI resp. 2012

40 Monocytes + B cells vs. DC as APC in the LTT (PBMC) Classic LTT T lymphocytes Monocytes + IL4 and GM-CSF 6 days Mo-imDC

41 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

42 LTT in NIR to Heparins (Dendritic Cells) 20 Patient 1 20 Patient 2 S.I S.I Dp Bp Np Tz Ep Sd Patient Dp Bp Np Tz Ep Sd Patient S.I. 10 S.I Dp Bp Np Tz Ep Sd Patient Dp Bp Np Tz Ep Sd Patient 6 mo/bcell imdc S.I. 10 S.I Dp Bp Np Tz Ep Sd 0 Dp Bp Np Tz Ep Sd 20 Patient 7 20 Control S.I Mean S.I Dp Bp Np Tz Ep Sd 0 Dp Bp Np Tz Ep Sd Lopez S et al. Br J Dermatol. 2009

43 LTT with CFSE NIR to AMOXICILLIN CD3 CD4 CD8 LY+AX-ADH.001 LY+AX-ADH.001 LY+AX-ADH.001 without DCs 5.02% M1 M2 4.33% M1 M2 1.24% M1 M CFSE FITC CFSE FITC CFSE FITC LY+DC+AX-ADH.001 LY+DC+AX-ADH.001 LY+DC+AX-ADH.001 with DCs M % 57.13% M1 M1 M2 6.19% M1 M CFSE FITC CFSE FITC CFSE FITC

44 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..

45 100 Monitoring acute response Analysis in different compartments % Cells % Cells % Cells CD4 CD8 CD45RO Days % Cells Days % Cells CD Days CLA Peripheral Blood Blister fluid Days Days Mayorga C et al. Curr Opin Allergy Clin Immunol 2006

46 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.

47 Thanks to all of you