blood basophil stability in bat test
TRANSCRIPT
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Michele Romano, PhD
Product Manager
Blood Basophil Stability in BAT Test
18/10/2018
EuroBAT Meeting 2018
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Synergy is the Key for Spreading BAT in Clinical Practice
Blood Basophil Stability in BAT Test - MR
Diagnostic Development
Clinical research
BAT Assay Development
Technical performance
basophils
markers
allergens
Clinical validity of the BAT
Scientific validity of the analyte
Allergy
No allergy
Symptoms/
Hypersensitivity
reaction
Is it serious?
how long it can last?
Is there a cure?
Is the cure effective?
diagnostic workflow
Strongly
impaired
QOL
Clinical history Skin test/sIgE OFC
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Basophil Activation Test: a Diagnostic Perspective
Sources:
https://ec.europa.eu/growth/sectors/medical-devices/regulatory-framework_en
http://ec.europa.eu/growth/content/new-eu-rules-medical-devices-enhance-patient-safety-and-modernise-public-health-
0_en
Blood Basophil Stability in BAT Test - MR
“Improve the quality, safety and reliability of medical devices”
new risk classification system for in vitro diagnostic medical devices in line with international guidance
reinforcement of the rules on clinical evidence, including an EU-wide coordinated procedure for authorization of multi-centre clinical investigations
IVDR
2022 is the horizon of new In
Vitro Diagnostic Medical
Devices Regulations
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Basophil Activation Test: a Diagnostic Perspective
Flow CAST® is standardization of BAT assay
Technical performance
Pre-analytics
Clinical performance
Blood Basophil Stability in BAT Test - MR
Blood Stability:
• Kind of blood
• Time from blood taking
• Temperature of storage
• Is blood logistic feasible?
some confusion and misleading info
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Basophil Stability in Whole Blood
EDTA
Heparin
ACD
Stability over 4 days of storage
Different temperature of storage 2-8°C, 20°C, 28°C
BAT was analyzed by Flow CAST® reagents
Blood Basophil Stability in BAT Test - MR
Blood taking
BAT testing:
Recovery
Activation
D0 D1 D2 D3 D4
Neg Ctrl (buffer)
Anti FceRI Ab, 5 doses
Allergen, G6, 5 doses
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Flow CAST® Procedure and Reagents
1. Stimulation & Staining
Stimulation Buffer with Heparin, IL-3 and Ca2+
with Heparin and Ca2+
Stimulation Control anti-FcεRI
fMLP
Staining Reagent anti-CCR3-PE
anti-CD63-FITC
Anti-CD203c-PE-DY647
Anti-CD63/CD203c-PE-DY647
2. Lysis
Lysing Reagent
3. Analysis
Wash Buffer
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Flow CAST® Protocol
1. Stimulation & Staining
Pipetting scheme:
50 ml Stimulus (neg Control, anti FceRI, Allergen)
100 ml Stimulation Buffer
50 ml EDTA/Heparin/ACD Blood
20 ml Staining Reagent
15 minutes incubation at 37°C
2. Lysis
2 ml of Lysing Solution
5-10 minutes incubation at RT
5 minutes centrifugation at 500g
Reconstitution of cell pellet with Wash Buffer (volume according to the instrument)
3. Flow Cytometry Analysis
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Patient
Background
(PB)
Patient
Control
(PC)
(a-FceRI)
CCR3-PE (FL2)
CD
63
-FIT
C (
FL
1) SSC (rel. granularity)
FS
C
(rel.
vo
lum
e)
CC
R3
-PE
(F
L2)
SSC (rel. granularity)
~ 2%
CCR3-PE (FL2)
Flow CAST® Analysis
Lab Training 16 - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
EDTA blood has the Best Recovery of Basophils
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Basophil Activation is Retained Over Days
Blood Basophil Stability in BAT Test - MR
EDTA Heparin
ACD
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Basophil Stability is Influenced by Storage Temperature
EDTA
Blood Basophil Stability in BAT Test - MR
2-8°C 20°C
28°C
A COMMITMENT TO DIAGNOSTICS – SINCE 1976 Blood Basophil Stability in BAT Test - MR
2-8°C 20°C
28°C
Basophil Stability is Influenced by Storage Temperature
Heparin
A COMMITMENT TO DIAGNOSTICS – SINCE 1976 Blood Basophil Stability in BAT Test - MR
2-8°C 20°C
28°C
Basophil Stability is Influenced by Storage Temperature
ACD
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
BAT Test is Feasible Over Days After Blood Taking
Basophils are stable in terms of recovery and activation over days
24/48 hours of storage allow reliable BAT test results
Different kinds of blood result in differences in basophil recovery and basophil activation
EDTA shows the best recovery with the Flow CAST® test system
The temperature of storage is another sources of can influence variability in basophil
activation
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Blood Stability – Anti IgE
Blood Basophil Stability in BAT Test - MR
With IL3
W/o IL3
CD63 CD203c
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Blood Stability – Allergen
Blood Basophil Stability in BAT Test - MR
With IL3
W/o IL3
CD63 CD203c
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Variability in Basophil Sensitivity
Blood Basophil Stability in BAT Test - MR
0
20
40
60
80
100
0,01 0,10 1,00 10,00 100,00 1000,00
anti-FceRI (ng/ml)
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
Flow CAST® BAT Testing Allow Blood Logistic
Statistically significant recovery of basophil at least at 24/48hrs
CD63 is the preferred activation marker for blood storage, CD203c lose activation
window in 24 hours
EDTA shows the best recovery with the Flow CAST® test system
2-8°C is the appropriate temperature of storage for blood
24/48 hours of blood storage is feasible for allergen specific basophil reactivity
measurement
24/48 hours of blood storage has an impact on variability of allergen specific basophil
sensitivity. This variability, together with other source of variability must be taken in
account in follow up study based on basophil sensitivity.
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976
CONTACT
Blood Basophil Stability in BAT Test - MR
A COMMITMENT TO DIAGNOSTICS – SINCE 1976 20
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