exceptional healthcare, personally delivered prenatal arrays: the bgl experience 20 th november...

Exceptional healthcare, personally delivered www.nbt.nhs.uk/genetics

Prenatal arrays:

The BGL experience

20th November 2014

Rob Moore

Genetic Technologist

Exceptional healthcare, personally delivered

Contents

• EACH at BGL EACH samples DNA extractions EACH aCGH• Prenatal array development Extractions Processing throughput Processing time

http://www.nbt.nhs.uk/genetics


EACH

Evaluation of Array Comparative Genomic Hybridisation in

Prenatal Diagnosis of Foetal Anomalies.

“Evaluate the efficacy of prenatal array CGH and address the hypothesis that array CGH detects more de novo pathogenic chromosomal imbalances than standard karyotyping”

•AF, CVS & FB samples from pregnancies with positive ab-scans and/or NT > 3.5mm.•In total 186 EACH samples 79 AF, 105 CVS, 2 FB•127 proceeding to aCGH 68 AF, 55 CVS, 2 FB



EACH sample work flow



EACH Extractions

EACH CVS: 2.5 – 4mg of sample

Lab developed manual protocol •Labour intensive and time consuming.•Overnight incubation step + a further half day of lab work.•Produces high quality un-sheared DNA.

iGENatal: 2 – 3ml AF + 10ml supernatant

iGEN Biotech phenol based extraction kit•Labour intensive, uses hazardous chemicals.•Produces un-sheared DNA, varied levels of phenol contamination.•Quantified by Qubit due to phenol contamination.

Manual Cultured Cell : nunc tube culture

Same protocol as EACH CVS, reduced incubation (30mins)



EACH arrays

Quantity of DNA determined type of array processing.

Standard array processing•500ng total DNA loaded onto array (Agilent 8x60k oligo array).•Identical to current postnatal array processing.

Reduced array processing•150ng total DNA loaded onto array (Agilent 8x60k oligo array).•Requires a custom reference in order to be processed.

Parental follow up arrays•Identical to current PvP array processing.



Extractions•Current EACH extraction protocols are too slow/too labour-intensive.•Extractions need to be streamlined and automated.

Pooling QFPCR and aCGH samples for QFPCR EZ1 extraction•~ 5ml AF or ~3mg CVS to be run on EZ1 tissue protocol.

Prenatal array development




Processing throughput•Current aCGH ~50 samples per week.•Processing runs of 24 samples twice a week.

Future sample numbers•5+ Prenatal samples a week (highly variable)•20 - 30 solid tissue samples a week•?20 NGS confirmation array samples a week

HT processing•48 sample processing•Increased automation/robot time Dilutions, PCR set up, sample/ref combination




Sample processing time•Current 3 day aCGH processing

•Potential 2 day aCGH processing


Day 1

Sample labelling

Day 2

Array hybridisation

Day 3

Array washing

Day 1

Sample & Array labelling hybridisation

Day 2

Array washing

Exceptional healthcare, personally delivered http://www.nbt.nhs.uk/genetics

Acknowledgements

A big thank you to all BGL staff (past and present) for all your help and for making the EACH project bearable…..roll on prenatal arrays

exceptional healthcare, personally delivered prenatal arrays: the bgl experience 20 th november...

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