The SAFE test
Rachel Dunn
The SAFE lab
S
A
F
E
t Georges
ntenatal
etal
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
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
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.
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
The IONA/SAFE Workflow
Maternal Plasma
10 mL blood sample
is collected from the
expectant mother.
DNA
Extraction
Library
Preparation
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.
Sequencing
Completed library
is prepared for
downstream
sequencing using
the ION Chef™ and
then analyzed on
the ION Proton™
systems.
Premaitha Workflow Manager
3 Days
© 2016 Premaitha Health
Analysis
Automated data
analysis with the
IONA® Software
applies stringent QC
criteria and supplies
an individual
sample report for
each patient.
Premaitha Workflow Manager
•
•
•
•
•
•
Sample Receipt
Barcode Generation
Sample Tracking
Paperless
Audit
Certified Medical Device
Fully traceable
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
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.
Day 1 : QIAsymphony:
Extraction of Cell-Free DNA from Plasma
Plasma
Plasma (+ Carrier RNA solution), reagents,
consumables, waste containers and elution
plates loaded into respective slots
Eluted DNA
(65μl)
A mixture of maternal and fetal DNA is extracted from maternal plasma sample
Day 2 Part One : The Sciclone:
Automated NGS Library Construction
1
End Repair
Buffer (10x)
2
3
4
5
6
7
8
9
T4 Ligase
Bst Polymerase
Adaptors
Adaptors
Adaptors
Adaptors
50µL
30µL
44µL
End Repair
Ligase Buffer (10x)
Enzyme Mix
A
30µL
130µL
B
30µL
50µL
30µL
C
30µL
50µL
30µL
D
30µL
50µL
30µL
E
30µL
50µL
30µL
F
30µL
50µL
30µL
G
30µL
50µL
30µL
H
30µL
50µL
30µL
PMH 005: 24µL
PMH 001: 24µL
PMH 013: 24µL
PMH 009: 24µL
PMH 006: 24µL
PMH 002: 24µL
PMH 014: 24µL
PMH 010: 24µL
PMH 007: 24µL
PMH 003: 24µL
PMH 015: 24µL
PMH 011: 24µL
PMH 008: 24µL
PMH 004: 24µL
PMH 016: 24µL
PMH 012: 24µL
Reagent plate layout
10
11
12
PCR Primers PCR MasterMix PCR MasterMix
50µL
130µL
130µL
50µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
130µL
•
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.
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 passed Quantification
• Sample libraries below the cut off of 0.65
post PCR are automatically put back for reextraction and will not be included in the
pools.
• The GX touch communicates directly with
the Sciclone and feeds back the
quantification values.
Sample failed Quantification
• The Sciclone is then programmed to
automatically normalise the samples and
produce diluted pools ready for sequencing
preparation.
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
(13-19 hours)
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
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
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
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.
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
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
Example Report
MyNIPT Portal
SAFE lab stats
• Reports produced– as of 22.04.2016 - 548
• T21 - 12
• T18 - 2
• T13 – 3
• 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
TATs
Target TAT is < 5 days
Average TAT (weekly)
7.00
6.00
5.00
4.00
3.00
2.00
1.00
0.00
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
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
o Introduction of v3 Proton chip < hands on time as no chip prep required
o increase in capacity on the Sciclone from 16 to 32 samples.
• UKAS Accreditation ISO 15189 – THE BIG ONE
Thank you for listening
• Any Questions……….