1. The SAFE test
Rachel Dunn

2. The SAFE lab
S t Georges
A ntenatal
F etal
E valuation
NHS lab dedicated to the NIPT of T13, T18 and T21
Scientist Lead
John Short Technical and R&D Manager
Technical Lead
Rachel Dunn - Band 6 Genetic technologist
3 x Band 4 Genetic Technologists
1 x Band 3 Admin Staff

3. SAFE Start
New laboratory - Open since November 2015 for sample analysis.
Brainchild of Professor Basky Thilaganathan, Consultant obstetrician at St Georges.
Wanted a dedicated NIPT lab at St Georges, co-ordinated with the SWTRGS in particular John Short – Technical R&D Manager
Tender (OJEU) -Provision of complete service
53 Questions, 6 Sections
Technical requirements
Instrumentation
Quality Assurance
Data Analysis and Storage
Service and Support
General
Awarded to Premaitha
Full laboratory refurbishment and kitting out

4. NIPT Cell free fetal DNA (cffDNA)comes from the placenta.
First detectable from 4-5 weeks gestation.
By 10 weeks gestation cffDNA reaches required levels for the SAFE test.
Cleared from maternal circulation within the first hour after birth.

5. Current Screening
Involves biochemical testing of the mothers hormone levels combined with an ultrasound
Usually 85 % accurate.
If high risk (1/150)– offered an invasive procedure amnio/CVS
These both carry a risk of miscarriage
SAFE test
Involves taking 1 x 10 ml tube of blood from the mother
99% accurate as you are analysing the baby's DNA.
Only need to have the invasive procedure if SAFE test comes back as high risk
Non Invasive therefore removes the miscarriage risk and stress

6. Premaitha Workflow Manager
The IONA/SAFE Workflow
Maternal Plasma
DNA Extraction
Library Preparation
Sequencing
Analysis
10 mL blood sample is collected from the expectant mother.
Fully automated DNA extraction is carried out on the QIAsymphony®.
Library preparation is completed using the IONA® Library Preparation Kit on the NGS Sciclone®. Sample quantitation is done with LabChip® GX Touch.
Completed library is prepared for downstream sequencing using the ION Chef™ and then analyzed on the ION Proton™ systems.
Automated data analysis with the IONA® Software applies stringent QC criteria and supplies an individual sample report for each patient.
Premaitha Workflow Manager
3 Days
© Premaitha Health

7. Premaitha Workflow Manager
Sample Receipt
Barcode Generation
Sample Tracking
Paperless
Audit
Certified Medical Device

8. Fully traceable

9. Workflow
8 patients are sequenced on each chip on the Proton using whole genome shotgun sequencing
Therefore each step of the workflow is ran in batches of 8.
Using current protocols and equipment we can only run 16 samples through the lab per day. 2 x pools and chips of 8

10. Day 1: Plasma Separation
Streck tube - contains a preservative that stabilises nucleated blood cells, this prevents the release of genomic DNA, allowing isolation of high-quality cell-free DNA for up to 14 days.

11. Extraction of Cell-Free DNA from Plasma
Day 1 : QIAsymphony:
Extraction of Cell-Free DNA from Plasma
Plasma (+ Carrier RNA solution), reagents, consumables, waste containers and elution plates loaded into respective slots
Plasma
Eluted DNA (65μl)
A mixture of maternal and fetal DNA is extracted from maternal plasma sample

12. Day 2 Part One : The Sciclone: Automated NGS Library Construction
Samples are, amplified, quantified and pooled
Automated process – 6 hours total
The eluate plate is placed on the Sciclone along with reagent plates, bead plate, tips and plastic ware for quantification and PCR.
Reagent plate contains –
End repair buffer and enzymes, ligase and polymerase required to produce the blunt ends.
Adapters to uniquely identify each patient that will end up in the pool of 8.
PCR primers and mixes to amplify the patient samples.
Bead plate contains–
Enough beads for each patient and the 2 patient pools.
Reagent plate layout

13. Day 2 Cont: Quantification
Samples are removed from the Sciclone for the PCR step and post PCR and size selection quantification on the GX touch
Sample libraries below the cut off of 0.65 post PCR are automatically put back for re-extraction and will not be included in the pools.
The GX touch communicates directly with the Sciclone and feeds back the quantification values.
The Sciclone is then programmed to automatically normalise the samples and produce diluted pools ready for sequencing preparation.
Sample passed Quantification
Sample failed Quantification

14. Ion Chef Eppendorf tube
Day 2 Part Two : Loading the ION Chef with normalised pooled patient libraries.
The Chef puts samples from chef tubes (Prepared by the Sciclone) onto chips, ready for the Ion Proton sequencer
Ion Chef Eppendorf tube
Ion Torrent Chip
Ion Chef can prepare 2 chips (16 samples) at a time.
1 chip contains 1 pool from 8 libraries
Ion Chef –run overnight
( hours)

15. ION Chef
Utilises a technique called Emulsion PCR (emPCR) where DNA fragments are clonally amplified onto beads ensuring there is enough signal to be picked up by the Proton Sequencer.
Millions of bead with millions of different fragments
The beads are then added to the sequencing chip ready for analysis by the Proton

16. Day 3: DNA sequencing on the ION Proton
The Proton is prepared and the chips are then loaded for sequencing.
One chip at a time: 2.5 hours to run each chip and 4 hours to download data

17. ION Proton
Uses semi conductor chip similar to that of a digital camera.
Chip covered in millions of wells.
In each well sits a bead with prepared DNA on it.
dNTPs are washed over the chip.
If base matches releases H+ ion and changes the pH in the well
This PH change is detected and a base is called.
Sequence of DNA is produced

18. The ‘BEAST’
Once the Proton has sequenced the DNA the ‘Beast’ then analyses the data.
Maps the sequence reads to the various area of the genome
Called the ‘Beast’ as it is a very large powerful and noisy computer!
Takes 4 hours per chip to process the data.

19. The SAFE lab Scientist……… aka the IONA PC
Once the Beast has analysed the data it is then the turn of the IONA PC, this sits in the SAFE office
It is also a very powerful and noisy PC – it is a medical device therefore no solitaire, internet explorer etc.
The SAFE lab Scientist – it looks at the data produced by the Beast and uses algorithms to determine the ratios of the chromosomes T13, T18 and T21.
This information is then combined with the maternal age to produce High Risk/Low Risk reports
It is a screen therefore if high risk an invasive procedure recommended
Fully CE IVD marked and has been thoroughly tested

20. Data Analysis – IONA PC
IONA® Software is a custom, dedicated bioinformatics package, employing highly efficient multi-core analysis algorithms.
Compliant with medical device development standards
Automatically processes sequenced DNA fragments into a self-contained report
Fast, user-friendly data entry and operation
Customisable results reports and exportable data
Localised data analysis
No bioinformatics personnel required

21. Example Report

22. MyNIPT Portal

23. SAFE lab stats Reports produced– as of 22.04.2016 - 548 T21 - 12
Sex Determination reports produced since the start of January for private patients.
130
70 Females, 60 Males
3 x samples unable to produce a report
2 x patients repeated through the process and successful the 2nd time so only 1 re bleed so far

24. TATs
Target TAT is < 5 days

25. Challenges New staff, new test, new workflow, new clinics……..all new!
3 x robots, 2 x analysers, 3 x PC programmes, Humans…
What could go wrong?!!
Regular speaking terms with engineers from Life technologies, Qiagen, Premaitha and Perkin Elmer.
Writing SOPs….general paperwork
Validation and verification of lab, process and equipment
Determining checks required for IQC within the lab
Meeting a 5 day TAT

26. The Future
Increase in sample numbers and an expanding service as more providers offer the test routinely.
Room for expansion and doubling up of equipment, co-ordinating the high throughput nature of test. Coping with yet more machines!
Using the biochemical test results as part of the analysis to provide a more accurate probability assessment.
Incorporating new technologies within the process
Introduction of v3 Proton chip < hands on time as no chip prep required
increase in capacity on the Sciclone from 16 to 32 samples.
UKAS Accreditation ISO – THE BIG ONE

27. Thank you for listening
Any Questions……….