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Blood Basophil Stability in BAT TestMichele Romano, PhDProduct ManagerEuroBAT Meeting 201818/10/2018A COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Synergy is the Key for Spreading BAT in Clinical PracticeAllergySymptoms/HypersensitivityreactionIs it serious?how long it can last?Is there a cure?Is the cure effective?StronglyimpairedQOLNo allergydiagnostic workflowClinical historySkin test/sIgEOFCbasophilsmarkersBAT ty ofthe BATallergensScientific validity ofthe analyteA COMMITMENT TO DIAGNOSTICS – SINCE 1976DiagnosticDevelopmentClinical researchBlood Basophil Stability in BAT Test - MR
Basophil Activation Test: a Diagnostic Perspective“Improve the quality, safety and reliability of medicaldevices”IVDR2022 is the horizon of new InVitro Diagnostic MedicalDevices Regulationsnew risk classification system for in vitro diagnosticmedical devices in line with international guidancereinforcement of the rules on clinical evidence,including an EU-wide coordinated procedure forauthorization of multi-centre clinical sectors/medical-devices/regulatory-framework se-public-health0 enA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Basophil Activation Test: a Diagnostic PerspectiveFlow CAST is standardization of BAT assayTechnical performancePre-analyticsClinical performanceBlood Stability: Kind of blood Time from blood taking Temperature of storage Is blood logistic feasible?some confusion and misleading infoA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Basophil Stability in Whole BloodEDTAHeparinACDStability over 4 days of storageDifferent temperature of storage 2-8 C, 20 C, 28 CBAT was analyzed by Flow CAST reagentsBlood takingD0D1BAT testing:A COMMITMENT TO DIAGNOSTICS – SINCE 1976D2RecoveryActivationD3D4Neg Ctrl (buffer)Anti FceRI Ab, 5 dosesAllergen, G6, 5 dosesBlood Basophil Stability in BAT Test - MR
Flow CAST Procedure and Reagents1. Stimulation & StainingStimulation Bufferwith Heparin, IL-3 and Ca2 with Heparin and Ca2 Stimulation Controlanti-FcεRIfMLPStaining 647Anti-CD63/CD203c-PE-DY6472. LysisLysing Reagent3. AnalysisWash BufferA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Flow CAST Protocol1. Stimulation & StainingPipetting scheme:50 ml Stimulus (neg Control, anti FceRI, Allergen)100 ml Stimulation Buffer50 ml EDTA/Heparin/ACD Blood20 ml Staining Reagent15 minutes incubation at 37 C2. Lysis2 ml of Lysing Solution5-10 minutes incubation at RT5 minutes centrifugation at 500gReconstitution of cell pellet with Wash Buffer (volume according to the instrument)3. Flow Cytometry AnalysisA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
PatientBackground(PB) 2%CCR3-PE (FL2)FSC (rel. volume)Flow CAST AnalysisSSC (rel. granularity)PatientControl(PC)(a-FceRI)CD63-FITC (FL1)SSC (rel. granularity)CCR3-PE (FL2)CCR3-PE (FL2)A COMMITMENT TO DIAGNOSTICS – SINCE 1976Lab Training 16 - MR
EDTA blood has the Best Recovery of BasophilsA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Basophil Activation is Retained Over DaysEDTAHeparinACDA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Basophil Stability is Influenced by Storage TemperatureEDTA2-8 C20 C28 CA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Basophil Stability is Influenced by Storage TemperatureHeparin2-8 C20 C28 CA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Basophil Stability is Influenced by Storage TemperatureACD2-8 C20 C28 CA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
BAT Test is Feasible Over Days After Blood TakingBasophils are stable in terms of recovery and activation over days24/48 hours of storage allow reliable BAT test resultsDifferent kinds of blood result in differences in basophil recovery and basophil activationEDTA shows the best recovery with the Flow CAST test systemThe temperature of storage is another sources of can influence variability in basophilactivationA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Blood Stability – Anti IgECD63CD203cWith IL3W/o IL3A COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Blood Stability – AllergenCD63CD203cWith IL3W/o IL3A COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Variability in Basophil Sensitivity1008060402000,010,101,0010,00 100,00 1000,00anti-FceRI (ng/ml)A COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Flow CAST BAT Testing Allow Blood LogisticStatistically significant recovery of basophil at least at 24/48hrsCD63 is the preferred activation marker for blood storage, CD203c lose activationwindow in 24 hoursEDTA shows the best recovery with the Flow CAST test system2-8 C is the appropriate temperature of storage for blood24/48 hours of blood storage is feasible for allergen specific basophil reactivitymeasurement24/48 hours of blood storage has an impact on variability of allergen specific basophilsensitivity. This variability, together with other source of variability must be taken inaccount in follow up study based on basophil sensitivity.A COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
CONTACTA COMMITMENT TO DIAGNOSTICS – SINCE 1976Blood Basophil Stability in BAT Test - MR
Waiver of LiabilityThis document and content are copyright of BÜHLMANNLaboratories AG [2018]. All rights reserved. Allinformation contained is believed to be accurate.Neither BÜHLMANN nor its employees, agents, ordirectors however are liable for misprints or errorscontained within.A COMMITMENT TO DIAGNOSTICS – SINCE 197620
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
