Download Evaluation of Drug Hypersensitivity by flow cytometry

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