circulating cell-free dna pre-analytics: importance of ccfdna … · 2017-07-24 · sample to...

Sample to Insight

Circulating Cell-Free DNA Pre-analytics:

Importance of ccfDNA Stabilization and Extraction for Liquid Biopsy

Applications

Martin Schlumpberger, Ass.Director R&D, QIAGEN GmbH

IQNpath 2017 - Circulating Cell-Free DNA Stabilization and Extraction 1

Sample to Insight

2

• QIAGEN products shown here are intended for molecular biology

applications. These products are not intended for the diagnosis,

prevention or treatment of a disease.

• For up-to-date licensing information and product-specific

disclaimers, see the respective QIAGEN kit handbook or user

manual. QIAGEN kit handbooks and user manuals are available

at www.qiagen.com or can be requested from QIAGEN

Technical Services or your local distributor.

IQNpath 2017 - Circulating Cell-Free DNA Stabilization and Extraction

Legal disclaimer

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Call for Workflow Standardization in routine healthcare

Diagnostic errors cause about 10% of all patient deaths and

about 17% of adverse events

Institute of Medicine (IOM) Report Sept. 2015

The pre-analytical phase accounts for 46% to 68% of such

errors observed during the total testing process

Medical Laboratory Observer, May 2014

SPIDIA & SPIDIA4P: Standardization and Improvement of Pre-analytical Procedures for

in vitro Diagnostics EU FP7-HEALTH (GA. no. 222916) & EU HORIZON 2020 (GA. no. 733112)

QIAGEN coordinating both initiatives (16 & 19 Partners)

22 CEN Technical Specifications and ISO Standards planned or developed

(highly consensus-driven international and European processes)

E.g., Specifications for pre-examination processes for venous whole blood in

Molecular in vitro diagnostic examinations

- Part 3: Isolated circulating cell-free DNA from plasma CEN/TS 16835-3: 2015


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Usually low concentrations in plasma, serum, urine and other body fluids (1-50 ng

DNA/ ml plasma)

Highly fragmented (<500bp)

Longer fragments (≥ 500bp) – possible from necrotic processes

Background DNA can be caused through cell lysis

Avoid release of cellular nucleic acids (gDNA)

Enable highly efficient large-volume nucleic acid extraction

Avoid fragment size bias to improve sensitivity

Circulating cell-free DNA: Workflow Challenges

Blood

Collection &

Stabilization

Sample

Logistics &

Storage

Plasma

Separation

ccfDNA

Extraction

Real-time

PCR, NGS,

others

Data

Analysis

4IQNpath 2017 - Circulating Cell-Free DNA Stabilization and Extraction

Sample to Insight


Blood

Collection &

Stabilization

Sample

Logistics &

Storage

Plasma

Separation

ccfDNA

Extraction

Real-time

PCR, NGS,

others

Data

Analysis

• Effective stabilization at RT minimizes background gDNA and maximizes ccfDNA yield from plasma

o White blood cells – helps prevent release of gDNA

o Red blood cells – helps minimize hemolysis

• Non-crosslinking NA preservation – no DNA modification

Unique stabilization of extracellular levels of ccfDNA

• Helps minimize risk of tube breakage

• Enhanced safety for healthcare and lab personnel

• Helps minimize contamination between samples

• Provides consistent blood draw volume

BD Vacutainer® plastic tube with BD HemogardTM safety closure

• Seamless integration into manual or automated prep with QIAamp® and

QIAsymphony® circulating DNA extraction technology

Integrated pre-analytical workflow

* For Research Use Only. Not for use in diagnostic procedures.

PAXgene Blood ccfDNA Tube (RUO)* Features

5

Sample to Insight

-10

10

30

50

70

90

110

130

150

EDTA t0

Problem: Need for Stabilization of ccfDNA

ccfDNA was extracted from EDTA plasma of 1 subject directly after blood draw (t0). 1 μl eluate was

analyzed using the Agilent® High Sensitivity DNA Kit.

ccfDNA fragment size profile from healthy donor

[FU]

ccfDNA


Sample to Insight

-10

10

30

50

70

90

110

130

150

EDTA t0

EDTA t6

Apoptosis of white blood cells leads to dilution of naturally occuring ccfDNA

ccfDNA was extracted from EDTA plasma of 1 subject directly after blood draw (t0) and after 6 days at

room temperature (t6). 1 μl eluate was analyzed using the Agilent® High Sensitivity DNA Kit.

Problem: Need for Stabilization of ccfDNA

ccfDNA

High-MW DNA

[FU]


Sample to Insight

-10

10

30

50

70

90

110

130

150

EDTA t0

EDTA t6

PAXgeneccfDNA t0

PAXgeneccfDNA t6

Solution: Prevent Blood Cell Lysis and Release of Genomic DNA

PAXgene® Blood ccfDNA Tubes (RUO)* help prevent release of gDNA into plasma

ccfDNA was extracted from EDTA and PAXgene plasma of 1 subject directly after blood draw (t0) and after

6 days at room temperature (t6). 1 μl eluate was analyzed using the Agilent® High Sensitivity DNA Kit.

High-MW DNA

ccfDNA

[FU]



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Blood cell stabilization prevents release of fragmented gDNA


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Stabilization of whole blood and ccfDNA during transport and storage


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Validation for qPCR analysis of methylation specific biomarkers (1)

11

mSHOX2 lung cancer: methylation specific therapy monitoring marker

• Blood from 29 consented lung cancer patients under treatment

• Collected in EDTA and PAXgene Blood ccfDNA Tubes

• Plasma processing immediately or after 7 days storage at RT

• ccfDNA quantified by real-time PCR with ERV (endogenous retrovirus) sequence

21

23

25

27

29

31

33

35

0 7

CT

Days at RT before plasma separation

ERV - Manual ccfDNA Isolation (D1-10)

EDTA (79 bp) PAXgene (79 bp)

EDTA (297 bp) PAXgene (297 bp)

23

25

27

29

31

33

35

37

0 7

CT

Days at RT before plasma separation

ERV - Automated ccfDNA Isolation (D11-29)

EDTA (79 bp) PAXgene (79 bp)EDTA (297 bp) PAXgene (297 bp)

Change of target CT over storage time for ccfDNA from plasma generated from EDTA and PAXgene Blood ccfDNA

Tubes. Real-time PCR assays amplifying 2 fragments of the single copy ERV sequence were used to measure DNA content of

original eluates after manual (A) and automated (B) ccfDNA isolation. Data courtesy of Dr. Fleischhacker, UKH Halle/Saale

A) B)


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Circulating Cell-Free DNA — Preanalytical Workflows

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Integrated

Workflow

No protocol

modifications or

pretreatments.

Optimized results.

Complete preanalytical workflow solutions from &

Blood

Collection &

Stabilization

Sample

Logistics &

Storage

Plasma

SeparationccfDNA

Extraction

Real-time

PCR, NGS,

others

Data

Analysis

PAXgene Blood ccfDNA Tubes (RUO)* 1–10 ml

Plasma

Input

QIAamp MinElute

ccfDNA Mini & Midi

Kit (MBA)†

NEW

PAXgene Blood ccfDNA Tubes (RUO) 1–10 ml

Plasma

EZ1® ccfDNA Mini &

Midi Kit (MBA) †

NEW

PAXgene Blood ccfDNA Tubes (RUO) 2.4 or

4.8 ml

Plasma

QIAsymphony

PAXgene Blood

ccfDNA (RUO)*

Manua

l

Auto

mate

d

* For Research Use Only. Not for use in diagnostic procedures.† For molecular biology applications. Not intended for the diagnosis, prevention or treatment of a disease.‡ Intended for in vitro diagnostic use.


Sample to Insight

Optimized extraction: QIAsymphony PAXgene Blood ccfDNA Kit (RUO)*

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Dedicated isolation technology works to streamline and

maximize ccfDNA recovery

• Optimized binding chemistry for use with PAXgene ccfDNA Tube

reagent

• Optimized input volumes to accomodate higher volume plasma

• Optional custom protocols for primary tube handling

Two protocol lines

• Standard protocol for small fragment isolation (≤500 bp)

• Large fragment protocols enable co-isolation of large fragments

(>500 bp) with flexible elution volume (60, 100, 150 µL)


PAXgene Blood ccfDNA Tubes (RUO) 2.4 or

4.8 ml

Plasma

QIAsymphony

PAXgene Blood

ccfDNA (RUO)


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QIAsymphony PAXgene blood ccfDNA

14

10 ml blood drawn into

PAXgene ccfDNA tube

Centrifugation to

isolate plasma

Transfer plasma to new tube

(Optional: repeat

centrifugation & plasma

transfer to new tube)

Plasma loaded onto

QIAsymphony SP to begin

automated ccfDNA isolation

ccfDNA for

use in NGS,

PCR, or other

Procedure

Option A

Option B

(Primary Tube

Processing)


https://en.wikipedia.org/wiki/File:Blood-centrifugation-scheme.png

Sample to Insight

ccfDNA Extraction: QIAamp MinElute® and EZ1® ccfDNA Kits

* For Research Use Only. Not for use in diagnostic procedures.† For molecular biology applications. Not intended for the diagnosis, prevention or treatment of a disease.


Sample to Insight

Fast and convenient protocol – Manual solution

Sample processing time – 4 samples (4 ml plasma)

25

38

48

51

61

72

20

15

20

20

15

20

0 10 20 30 40 50 60 70 80 90 100

QIAamp MinElute ccfDNA

Competitor Z

Competitor T

Competitor A

Competitor N

Competitor T

Incubation/centrifugation time

Hands on time

QIAamp MinElute ccfDNA Kit accelerates your ccfDNA extraction.

Time (minutes)


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Scalable sample input volume

7,35

18,15

36,60

64,05

R² = 0.9887

0

10

20

30

40

50

60

70

80

1 ml 2 ml 4 ml 8 ml

Total ccfDNA (ng)

10 ml or higher plasma input is possible.

Total yield with increasing plasma amounts

Plasma input


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Efficient recovery of ccfDNA – Highest yields

ccfDNA yield in eluate

Highest DNA yield ensuring detection of low frequency biomarkers.

15,72

14,91

8,92

8,58

6,39

4,74

0 2 4 6 8 10 12 14 16 18

QIAamp MinElute ccfDNA 20 µl

QIAamp Circulating Nucleic Acid 30 µl

Competitor Z

Competitor T

Competitor N

Competitor A

DNA (ng)

66bp66 bp


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Highest concentrations of ccfDNA

DNA concentration in eluate

Highest DNA concentration ensuring best performance in NGS analysis.

0,786

0,50

0,178

0,172

0,128

0,095

0,000 0,100 0,200 0,300 0,400 0,500 0,600 0,700 0,800 0,900

QIAamp MinElute ccfDNA 20 µl

QIAamp Circulating Nucleic Acid 30 µl

Competitor Z

Competitor T

Competitor N

Competitor A

DNA concentration (ng/µl)

66 BP66 bp


Sample to Insight

From Sample to Insight

Circulating cell-free DNA NGS workflow

• QIAamp MinElute

ccfDNA Kits

• EZ1 ccfDNA Kits

• QIAsymphony

• QIAcube

• EZ1 Advanced XL

• Ingenuity Variant

Analysis

• QIAseq

Targeted DNA

Panels

• QIAseq Library

Preparation

Kits

• CLC Genomics

Workbench

• PAXgene Blood

ccfDNA Tubes

ccfDNA

extractionInterpretation

NGS library

preparation

Data

analysis

Blood

collection

and

stabilization


Sample to Insight

Thank You!


circulating cell-free dna pre-analytics: importance of ccfdna … · 2017-07-24 · sample to...

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