Our journey to Roche FLOW SystemPreparations for the implementation of the FLOW system

Annet Damhuis, LabPLus, Auckland

Introduction• Molecular Virology in LabPlus

– Current workflow – how do we move forward– The FLOW system – what does it offer

• Respiratory panel– In-house vs TIB Modular kit – performance– Additions to TIB Modular kit – RNA PC

• Implementation of FLOW and TIB assays

Current workflow• Sample preparation• Extraction on MP96• PCR setup• Data analysis• Reporting

Current workflow• Sample preparation• Extraction on MP96• PCR setup• Data analysis• Reporting

– Labour intensive– Time consuming– High contamination risk– Error prone

Moving forward

• Reduce hands-on time• Reduce contamination risk• Eliminate pipetting errors• Eliminate transcription errors• Improve workflow

The FLOW solution

Pro• Modular setup• In-house and kit based

assays• Process more samples

easily• Overlap runs• Walk away technology• Paperless system

Con• Test specific registration

critical• Not designed for swabs• No melt curve analysis

(yet)• Dead volumes• 96-well plates only

The FLOW solution

The FLOW solution

The FLOW brain• Recognises test requests based on

barcode• Links all machines• Creates run/setup files• Transmits results into LIS

Primary Sample Handler (PSH)

• Pipettes sample from original tube into MP96 sample cartridge– Linked to LIS system– Barcode check samples– Aliquot process control/polyA

• Extracts up to 96 samples• Run time ~ 1 hr• Low maintenance• Elution in plates or tubes

MagnaPure 96

PCR Setup Unit (PSU)

• Multiple master mixes• Multiple plates (max. 5)• Samples from several extraction plates

LightCycler 480 II

• Run setup– PCR conditions

• Plate setup– Sample names

• Analysis setup– Colour compensation– Results

Future planning• Data is analysed in FLOW software• Results automatically transcribed into LIS• Time saving• Cost saving

– Reagents are more expensive– Scientist hands-on time less

• Implementation timeline– Proposed February 2018

• Move other assays to FLOW

Our journey to Roche FLOW SystemComparison of in-house Respiratory Panel with TibMolBiol ModularDx RPAN

Hanna Antoszewska, LabPLus, Auckland

Objectives• First part validation was performed by RDNZ• Validate challenging samples : NPA, Bronchial

Washings, BAL, Autopsies (Lung, heart tissues)• Incorporate CMV and Bordetella Pertussis to TIB-

ModularDx• Validate RV and EV virus cross-reactivity• Check the stability of the RNA Processing Control

(Freshly made /Frozen)• Compare QCMD panels in both assays

Respiratory Panel (in-house)A. INF A/Internal ControlB. INF BC. PIV1D. PIV2E. PIV3F. RSVG. ADVH. CMVI. RVJ. EVK. hMPV/Extraction Control L. Bordetella Pertussis/ICM. RnasePN. CoV, MERS, BoV,

Parechovirus

TIB ModularDX composition

• MMx-1 (FluA, FluB, RSV, RNA PC)• MMx-2 (PIV1, PIV2, PIV3, hMPV, RNA

PC)• MMx-3 (EV, RV, ADV, RNA PC)• MMx-4 (HKU1, OC43, 229E, NL63, RNA

PC)• MMx-0 (H1N1, H3, RNA PC)

CMV results in TIB assay• CMV was multiplexed

with ADV, RV, EV and RNA PC in MMx-3

• It showed a good sensitivity with different viral loads

• It had no effect on detection of other targets in single and multiple infections

BP results in TIB assay

• BP in duplex with RNA PC

RV/EV validation• TIB assay showed minimal cross-reactivity

between RV and EV• TIB assay detected RV and EV at higher Cp

value (>1 log) than in-house assay • TIB missed 6/19 moderate level positive RV• TIB missed 2/5 moderate level positive EV• In limited dilutions experiment RV and EV were

detected with 1 log difference between assays

Comparison of Internal Extraction controls in-house and TIB Master mixes

In-house assay

IEC detected in all patient samples and EB

Comparison of Internal Extraction controls in-house and TIB Master mixes

TIB MMx-3

RNA PC only detected in some patient samples and EB

Comparison of Internal Extraction controls in-house and TIB Master mixes

TIB MMx-1

RNA PC detected in most patient samples and EB

Comparison of Internal Extraction controls in-house and TIB Master mixes

MMx-5

RNA PC detected in all patient samples and EB

Freeze/thaw RNA PC once has no effect on performance

QCMD Panels in TIBModularDX• INFA/B 100% concordance• RSV 100% concordance • B. Pertussis 100% concordance• ADV 100% concordance• PIV missed PIV4 • hMPV missed 2 core samples• RV missed RV72 core & educational,

RV8 educational• CoV missed CoV 229E educational

Conclusions• TIB assay performed well on all challenging samples• CMV was added to MMx-3 and run as multiplex with RV,

EV and ADV with a good sensitivity• BP was run as duplex with RNA PC• Results of RNA PC showed different patterns in different

MMxs• RNA PC is stable after one freeze thaw cycle• There were differences in RV and EV detection.

– TIB assay detected them with higher Cp– TIB assay shows minimal cross reactivity between RV and EV– TIB assay missed some types of RV and EV

• QCMD panels gave satisfactory results

Acknowledgements

• Dr M C Croxson (HOD, Molecular Virology)• Dr F Rahnama (Section leader, Molecular

Virology)• A Gourley (Roche Diagnostics NZ) • Molecular Virology Team (LabPlus)