Blood Basophil Stability in BAT Test

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


Synergy is the Key for Spreading BAT in Clinical Practice


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


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


“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


Basophil Activation Test: a Diagnostic Perspective

Flow CAST® is standardization of BAT assay

Technical performance

Pre-analytics

Clinical performance


Blood Stability:

• Kind of blood

• Time from blood taking

• Temperature of storage

• Is blood logistic feasible?

some confusion and misleading info


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 taking

BAT testing:

Recovery

Activation

D0 D1 D2 D3 D4

Neg Ctrl (buffer)

Anti FceRI Ab, 5 doses

Allergen, G6, 5 doses


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



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



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


EDTA blood has the Best Recovery of Basophils



Basophil Activation is Retained Over Days


EDTA Heparin

ACD


Basophil Stability is Influenced by Storage Temperature

EDTA


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


Heparin

A COMMITMENT TO DIAGNOSTICS – SINCE 1976 Blood Basophil Stability in BAT Test - MR

2-8°C 20°C

28°C


ACD


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 Stability – Anti IgE


With IL3

W/o IL3

CD63 CD203c


Blood Stability – Allergen


With IL3

W/o IL3

CD63 CD203c


Variability in Basophil Sensitivity


0

20

40

60

80

100

0,01 0,10 1,00 10,00 100,00 1000,00

anti-FceRI (ng/ml)


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.



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A COMMITMENT TO DIAGNOSTICS – SINCE 1976 20

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information contained is believed to be accurate.

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Blood Basophil Stability in BAT Test