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Basophil Activation Test
Marta Ferrer, MD, PhD Clinica Universidad de Navarra
Medical School
Hausmann OV et al Immunol Allergy Clin North Am 2009;29:555-‐‑66
CCR3+/CD3- CD123+/HLA-DR- IgE+/CD203+
CD63 CD203 p38 MAPK CD300
Detect basophil activation
Identification markers Activation markers
Method
Basal
Alternatives: Buffy coat leukocytes Leukocyte enriched plasma Whole Blood
Isolation Buffer+IL3 prewarming
Stimulation Anti-IgE CD123 HLA-DR CCR3
Buffer+IL3 prewarming
Basal Anti-IgE
Anti-IgE CD123 HLA-DR CCR3
Buffer+IL3 prewarming
Basal Anti-IgE
Stimulation Allergen, drug
Allergen drug
15-‐‑40 min 37°C
centrifugation
supernatant pellet
BAT
Anti-IgE CD123 HLA-DR CCR3
Buffer+IL3 prewarming
Basal Anti-IgE
Allergen, drug
Allergen drug
Basal Anti-IgE Allergens drug
Alternative: Anti-‐‑CD63-‐‑PE Anti-‐‑CD123-‐‑PerCP Anti-‐‑DR-‐‑FITC Anti CD203c,aCD45 CCR3,CRTH2...
anti-CD63 PE anti-IgE, CD123
Basophil labeling
R1
Region 1: Lymphocytes
R2
Region 2: Cells with anti-IgE receptor
Software CellQuest: 50000 cells
Flow cytometer analysis
Negative control
1.6%
75.% Drug
84%
Positive control anti-‐‑IgE
Steps and differences 1. Blood sample preparation 50-100 µl (whole heparinized blood vs. leukocyte
isolation) 2. Preincubation with IL-3 vs. prewarming of blood 3. Samples and reagents to 37C, allergen is added, and basophils are
incubated for 15-30 minutess at 37C in the water bath. 4. Range concentration 5. Staining with monoclonal antibodies coupled to fluorochromes and lysis of
contaminating erythrocytes. 6. Select basophils (anti-CCR3/anti-CD3, anti-CD123/anti-HLA-DR). 7. Activation markers CD63, CD203c, or P-p38MAPK
8. Minimal number of basophils 200 per test tube but it is preferred that 600 to 1000 of results.
9. Include positive control (anti-IgE or anti-FcεRI) 10. Thresholds for positivity vs. ROC
CD63 CD203c
Basophil activation markers CD63 CD203c LAMP family tetraspanin
Ectoenzyme
Expressed only upon activation Depends upon P38-‐‑MAPK phosphorilation
Expressed under resting conditions and up-‐‑regulated if activation
Tmax: 25-‐‑30 min
Tmax: 10-‐‑20 min
Basophils, monocites, platelets
Specific for basophils and mast cells
IL-‐‑3 -‐‑ IL3 + Ebo DG et al. Allergy, 2006
MacGlashan D Jr. Clin Exp Allergy. 2010
CD63 vs. CD203c
0.8%
65.9%
71.2%
31.6%
74.4%
94.4%
Negative control
Negative control
Anti IgE
Anti IgE
Parietaria
Allergen
CD
203c
PE
CD
63 P
E CD63
CD203c
De Weck Al, Sanz ML. Clin Exp Allergy 2003
CD63 vs. CD203c
Hausmann OV et al Immunol Allergy Clin North Am 2009
p38-‐‑MAPK
Allow conjugates
Steiner M. J Vis Exp. 2011
Conjugates
Steiner M. J Vis Exp. 2011
European BAT Interest group (Eurobat)
• Optimal drug concentration • Positivity thresholds • Minimal basophil number • Drug solubility • Blood storage sample and preparation • Whole blood vs. Isolated leukocytes • Preincubation conditions:
o IL3 or prewarming o Time from 10 to 30 minutes
Betalactams
Sanz ML et al. Clin Exp Allergy 2002
CAP: 38% and 87% BAT: 50% and 94% CAP+BAT: 65.5% 34.5% with positive skin test were negative for both techniques
Pat. Cntrl. Test Sensitivity Specificity Sensitivity CAP+BAT
Ref.
58 30 CAP, ST 53 93.3 65.5% Sanz, 2002
70 40 CAP, RAST, ST, DBPT
49 91 65.7% Torres, 2004
27 14 ST, DBPT CD63: 33 CD203c: 67
CD63: 79 CD203c: 100
Abuaf 2008
Group n Positive BAT
ST positive 53 27 ST negative CAP/RASTpositive
10 6
ST and CAP/RAST negative 7 1
Cephalosporin 77%
Torres MJ et al. Cl Exp Allergy 2004
Torres MJ et al. Cl Exp Allergy 2004
Neuromuscular blocking agents
Pat. Ctrls. Tests Sensibility Specificity Ref
NMBA 41 23 ST 64 93 Abuaf 1999
NBMA 50 17 ST 54 100 Monneret, 2002
NMBA 21 10 ST, HRT CD63: 79 CD203c: 36
100 Sudheer, 2005
NMBA 47 40 ST, DBPT 36-‐‑86 93.3 Kvedariene, 2006
Rocuronium
14 8 ST 91.7 100 Ebo, 2006
NMBA 26 23 ST, SIgE 68 100 Sainte-‐‑Laudy, 2008
Ebo DG et al Allergy. 2006
BAT sensitivity and time interval
Kvedariene V et al. Allergy. 2006
NSAIDs • Low sensitivity (15 % to 55%) • Positive in healthy controls • Useful hypersensitivity to an isolated NSAID:
pyrazolones Drug Patients Controls Test
s Sensibility Specificity Ref
Metamizol 26 30 ST 42.3 100 Gamboa, 2002
Pyrazolones 51 56 ST 54.9 86 Gomez, 2009
Discussion of clinical cases
BAT: ü Complementary technique for immediate drug reactions ü Sensitivity: 54% muscle relaxants , 50% beta-‐‑lactams ü Specificity 80-‐‑90%, confirmed by several studies ü Specially high for cephalosporine ü Combined with skin tests and sIgE increases sensitivity ü Allows cross reactivity studies among muscle relaxants ü Useful when no alternative in vitro diagnosis test is available ü Early diagnosis is crucial ü Needs to standardize the protocol ü There is a need of large multicenter studies to validate the
technique and provide a definitive assessment of its sensitivity.
