1. BÜHLMANN CAST® Assays Technical Presentation
03/06/2016

2. Type 1 Allergy Mechanism
APC
Sentinel Cells
Allergens
‘Insults’
sIgE antibodies
T Lymphocytes
B Lymphocytes/Plsma Cells
Specific Immune Response
Activated &
Degranulated Mast Cells
Basophils
Effector Cells
Mediator Release:
Histamin, Leukotriens
Lab Training 16 - MR

3. BÜHLMANN CAST®: Functional IVD Cell Based Assays in Allergy Diagnosis
The CAST® assay is the diagnostic support of the allergologist to predict the patient allergic reaction to a substance in a safe blood test.
With CAST® assay we have standardized an in vitro functional assay to evaluate and quantify the potential of an allergen to activate the patient specific basophils, the circulating effector immune cells in allergy.
Activated &
Degranulated Basophils
CAST® Allergens
Basophil Activation Test (BAT)
Flow CAST®
Mediator Release:
Leukotrienes
Immunoassays
CAST® ELISA
Lab Training 16 - MR

4. Flow CAST® Principle: CCR3 / CD63
a) BÜHLMANN Allergen a)
Selection Marker anti-CCR3-PE:
Detects all basophils whithin the blood samples
Activation Marker anti-CD63-FITC:
Detects only basophils which have been activated by the allergen b)
Lab Training 16 - MR

5. Flow CAST® Principle: CCR3 / CD63
Stimulation Control anti-FcεRI mAb:
Immunological activation of the basophils: Mimics the cross-linking of two bound IgE with the allergen directly via FcεRI receptor
Stimulation Control fMLP (tripeptide):
Non-immunological activation of the basophils: The mechanism is not known.
Detects the viability of the basophils. e)
Lab Training 16 - MR

6. Flow CAST® Samples per PatientPB
Patient Background (PB)
Stimulation Buffer
Blood
Staining Reagent
PC1
PC2
Patient Controls (PCs)
Stimulation control
(PC1: anti-FcεRI mAb; PC2: fMLP)
Blood
Staining Reagent Ax
Allergen
Allergen x
Blood
Staining Reagent
Lab Training 16 - MR

7. Flow CAST® Analysis Basophils Eosinophils Neutrophils Lymphocytes
FSC (rel. volume)
Lymphocytes
Monocytes
SSC (rel. granularity)
Lab Training 16 - MR

8. Flow CAST® Analysis Basophils Eosinophils Neutrophils Lymphocytes
FL2+; SSClow
FL2+; SSChigh
Basophils
Eosinophils
CCR3-PE (FL2)
Neutrophils
Lymphocytes
Monocytes
SSC (rel. granularity)
Lab Training 16 - MR

9. Flow CAST® Analysis Activated & degranulated basophils FL1+FL2-
CD63
CD203c
Activated & degranulated basophils
CD63-FITC (FL1)
FL1-
FL2-
FL1-FL2+
Not activated
basophils
CCR3-PE (FL2)
Lab Training 16 - MR

10. Flow CAST® Analysis Patient Background (PB) ~ 2% Patient Control (PC)
FSC (rel. volume)
CCR3-PE (FL2)
CCR3-PE (FL2)
CD63-FITC (FL1)
Patient
Control
(PC)
(a-FceRI)
SSC (rel. granularity)
SSC (rel. granularity)
CCR3-PE (FL2)
Lab Training 16 - MR

11. Flow CAST® Analysis Patient Background (PB) ~ 2% Patient Control (PC)
FSC (rel. volume)
CCR3-PE (FL2)
CCR3-PE (FL2)
CD63-FITC (FL1)
Patient
Control
(PC)
(a-FceRI)
>10%
sample is evaluable
SSC (rel. granularity)
SSC (rel. granularity)
CCR3-PE (FL2)
Lab Training 16 - MR