influence of centrifugation conditions for plasma processing on … · 2019. 11. 21. · figure 2:...

Influence of Centrifugation Conditions for Plasma Processing on ccfDNA Yield1Daniel Groelz, 1Tomasz Krenz, 1Eric Provencher and 1Thorsten Voss1PreAnalytiX GmbH, Hombrechtikon, Switzerland

IntroductionCirculating, cell-free DNA (ccfDNA) in blood has become widely accepted for biomarker analysis in academic and clinical research and beyond. Considering the usual low sample input and required high sensitivity for target detection, optimal outcomes require validation and control of the entire workflow. The PAXgene Blood ccfDNA System (RUO) integrates preanalytical steps from sample collection to ccfDNA extraction for subsequent analysis by quantitative PCR (qPCR), digital PCR (ddPCR) and next-generation sequencing (NGS).

Here we present the influence of centrifugation conditions for plasma processing on ccfDNA yield and compatibility with primary tube handling on the QIAsymphony® SP instrument using the QIAsymphony PAXgene® Blood ccfDNA Kit* and corresponding protocols.

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

Stability claims for the PAXgene Blood ccfDNA Tube are 37°C for up to 1 day and 2–30°C for up to 7 days.

Workflow Options for the PAXgene Blood ccfDNA System

10 ml blooddrawn intoPAXgene

Blood ccfDNATube

Transport ofblood to

laboratory

Centrifugationto isolateplasma

10 ml blooddrawn intoPAXgene

Blood ccfDNATube

Transport ofblood to

laboratory


Transferplasma tonew tube


Transferplasma tonew tube

60 min

20 min

Optional 2nd Centrifugation

Option A

Option BDirect PrimaryTube Processing

Manual orautomated

ccfDNAisolation

AutomatedccfDNAisolation

QIAsymphonySP

Figure 1. Option A is the workflow described in the QIAsymphony PAXgene Blood ccfDNA Kit Handbook using the optional second centrifugation step. Option B is the new primary tube handling workflow using the PAXgene Blood ccfDNA Tube to be processed directly on the QIAsymphony SP instrument. Time savings based on processing a 24 sample batch.

Results

Study 4 – Compatibility with NGS workflow

• Different brake settings during centrifugation have minimal effect on hemolysis, plasma and ccfDNA yield from blood collected and stored in PAXgene Blood ccfDNA Tubes

• The highest brake force led to an angled plasma/cell border

• Efficient stabilization of blood cells mediated by the PAXgene Blood ccfDNA stabilization technology led to minimal changes in analyzed parameters over storage time

• After blood storage for 7 days and double centrifugation at 1,900 × g, ccfDNA yield was comparable to ccfDNA yield observed directly after blood draw.

• All GeneReader NGS acceptance criteria were met for every sample, including region of interest with coverage of bases >500× (>90%), region of interest with coverage of bases >200× (>95%) and reads above average quality 25 (>80%).

• Across all samples, 17 ± 5 different variants in 7 ± 1 different genes were identified in healthy donors using the AIT panel.

Figure 2: Angled plasma/cell border. Plasma separation after 15 minutes centrifugation at 1,900 × g with brake setting 19 (full brake). Red arrow shows affected plasma/cell border

Figure 13: Stability of ccfDNA shown as relative copy number change of 18S rDNA 66 bp fragments in the PAXgene Blood ccfDNA Tube. Copy numbers from 60 healthy blood donors were determined directly after blood collection (day 0) and after blood storage for 7 days at 25°C (day 7). The mean value (mean = 1.045) is shown as a blue dashed line. Ideal ratio would be 1.0 (no change versus day 0).

Figure 14: NGS compatibility of the liquid biopsy workflow. Blood from 60 healthy donors was stored in PAXgene Blood ccfDNA Tubes for 7 days at 25°C prior to ccfDNA extraction. NGS was performed on the GeneReader system using the GeneRead QIAact Actionable Insights Tumor (AIT) panel. Sequencing data was analyzed with the QIAGEN Clinical Insight Analyze (QCI-A) tool for quality control (QC) parameters (A, B) and gene variant detection (C). System QC thresholds for 3 of the most important NGS QC parameters (coverage 500×, coverage 200×, reads above average quality 25) define “passing” or “out-of-specification” samples. Variant analysis was carried out for every individual sample and all gene variants of the panel. The AIT panel contains 773 variant positions in 12 genes.

Figure 3: Hemolysis. Mean plasma absorption at 414 nm (absorption maximum of hemoglobin) by blood storage and plasma processing protocols.

Figure 5: Yield and stability of ccfDNA were measured with assays amplifying the same 66 bp 18S rDNA fragment. A) qPCR using the QIAGEN Rotor-Gene Q instrument, ideal ratio would be 1.0 (no change versus day 0). B) ddPCR using the BioRad QX 200 DR Droplet Digital PCR System.

Figure 4: Plasma yield per tube by blood storage and plasma processing protocols.

MethodsBlood from healthy consented donors was drawn into PAXgene Blood ccfDNA Tubes (PreAnalytiX®)* or Cell-Free DNA BCT® (Streck®) and was processed into plasma directly after phlebotomy or stored for 6–7 days at room temperature (15–25°C) to simulate a typical processing delay in a routine setting. Different plasma processing protocols were tested in 4 studies (Table 1).

The level of hemolysis in the separated plasma was analyzed by photometric measurement of the absorption at 414 nm, which is the absorption maximum for hemoglobin. ccfDNA was isolated from PAXgene plasma on the QIAsymphony SP (QIAGEN®) automated platform using the corresponding QIAsymphony PAXgene Blood ccfDNA Kit* and protocol and from Streck plasma using the QIAsymphony DSP Circulating DNA Kit (QIAGEN) and protocol. Yield and stability of ccfDNA generated in Studies 1–3 were measured by qPCR (QIAGEN Rotor-Gene® Q) and ddPCR (BioRad QX 200 DR Droplet Digital PCR System) with assays amplifying a 66 bp fragment of the 18S rDNA sequence. Compatibility with NGS was evaluated by sequencing ccfDNA samples from Study 4 on the QIAGEN GeneReader™ NGS System, including the GeneRead™ QIAact Actionable Insights Tumor (AIT) Panel* for PCR target enrichment and library preparation on the QIAcube® instrument, QC with capillary electrophoresis, sequencing on the GeneReader instrument* and data management with the QIAGEN Clinical Insight (QCI™) Analyze* tool.

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

Study 1 Brake settings

Blood collection & storage25 donors

10 PAXgene Blood ccfDNA Tubes5 tubes: direct processing (t0), 5 tubes: 6 days at 25°C

Plasma processing (Sigma 4K15C centrifuge)

Two centrifugation steps

15´at 1,900 × g, 10´ at 1,900 × g Maximum brake (19)

15´ at 1,900 × g, 10´ at 1,900 × g Medium brake (9)

One centrifugation step

15´ at 1,900 × g Maximum brake (19)

15´ at 1,900 × g Medium brake (9)

15´ at 1,900 × g No brake (0)

ccfDNA extraction 4.8 ml plasma QIAsymphony PAXcircDNA_STA_4800

Study 2 Centrifugation forces

& times

Blood collection & storage 25 donors

8 PAXgene Blood ccfDNA Tubes, 2 Streck Cell-Free DNA glass tubes4 PAXgene tubes: direct processing (t0), 4 PAXgene & 2 Streck tubes: 7 days at 25°C

PAXgene plasma processing (Sigma 4K15C centrifuge)

Two centrifugation steps15´ at 1,900 × g, 10´ at 1,900 × g

Medium brake (9)

One centrifugation step




Streck cfDNA spin 1 & spin 2 protocols* (Sigma 4K15C centrifuge)

Two centrifugation steps

Spin 1: 20´ at 300 × g, 10´ at 5,000 × g Medium brake (9)

Spin 2: 10´ at 1,600 × g, 10´ at 16,000 × g Medium brake (9)

ccfDNA extractionPAXgene plasma: 2.4 ml QIAsymphony PAXcircDNA_STA_2400

Streck tubes: 2.0 ml QIAsymphony circDNA_2000

Study 3 Primary tube work-

flow

Blood collection & storage

17 donors

10 PAXgene Blood ccfDNA Tubes

5 tubes: direct processing (t0), 5 tubes: 7 days at 25°C

Plasma processing (Sigma 4K15C centrifuge)

One centrifugation Medium brake (9)

15´ at 1,900 × g 5´ at 1,600 × g15´ at 1,600 × g 5´ at 3,000 × g15´ at 3,000 × g

ccfDNA extraction 4.0 ml / 2.4 mlQIAsymphony primary tube PAXcircDNA_STA_4000

or 2400

Study 4 NGS workflow

Blood collection & storage60 donors

10 PAXgene Blood ccfDNA Tubes1× direct processing, 1× 7 days at 25°C

Plasma processing (Sigma 4K15C centrifuge) Two centrifugation steps15´ at 1,900 × g, 10´ at 1,900 × g

Medium brake (9)ccfDNA extraction 4.8 ml plasma QIAsymphony PAXcircDNA_STA_4800

Table 1: Experimental setup of studies 1–4*Protocols according to IFU for Streck Cell-Free DNA BCT

Conclusions• Centrifugation parameters are important for optimal plasma quantity

and quality and are dependent on the tube type used for blood collection

• Main findings from the different centrifugation conditions tested: – High brake settings can lead to angled plasma/cell border – Low centrifugation forces and times (under 1,600 × g for less than 15 min)

can lead to reduced plasma yield and insufficient separation of plasma from cellular fraction, which can lead to gDNA contamination of ccfDNA by transferred white blood cells

– No tube breakage was observed for PAXgene Blood ccfDNA Tubes – Centrifugation in range of 1,600 × g to 3,000 × g for 15 min has no impact on

hemolysis and results in no significant differences for total yield and in situ stability (yield change over storage time)

• Recommended protocol: – 15 minutes at 1,600–3,000 × g with medium brake setting – Optional: Second centrifugation, 10 minutes at 1,600–3,000 × g with medium

brake setting

• Data from the first 3 studies imply that the recommended centrifugation parameters compared to the procedure described in the product literature will not impact: – Plasma yield and quality – Compatibility with automated ccfDNA isolation procedures including primary

tube handling workflow – Downstream performance with qPCR and digital droplet PCR

• Eluates from the PAXgene Blood ccfDNA System are compatible with the GeneReader NGS workflow

DisclaimerFor Research Use Only. Not for use in diagnostic procedures.For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN and PreAnalytiX kit handbook or user manual.Handbooks and user manuals are available at www.qiagen.com and www.preanalytix.com or can be requested from QIAGEN Technical Services or your local distributor.Trademarks are the property of their respective owners.© 2018 PreAnalytiX GmbH. PreAnalytiX, the PreAnalytiX Logo and all other trademarks are property of PreAnalytiX GmbH, Hombrechtikon, CH.

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HemolysisCentrifugation Settings Have No Influence on Hemolysis

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Mean Plasma Yield Centrifugation Settings Have No Influence on Plasma Yield


Study 2 – Influence of different centrifugation conditions during plasma processingStudy 1 – Influence of brake use during plasma processing• Hemolysis, plasma and ccfDNA yield from blood collected and stored in PAXgene Blood ccfDNA Tubes is independent of tested centrifugation forces, times and

repetitions

• Streck tubes show more hemolysis and reduced plasma yields; DNA yields after storage increased and showed higher variance compared to yields obtained from replicate samples collected in PAXgene Blood ccfDNA Tubes


Figure 8: Yield and stability of ccfDNA were measured with assays amplifying the same 66 bp 18S rDNA fragment. A) qPCR using the QIAGEN Rotor-Gene Q instrument, ideal ratio would be 1.0 (no change versus day 0). B) ddPCR using the BioRad QX 200 DR Droplet Digital PCR System.

Figure 7: Plasma yield per tube by blood storage and plasma processing protocols.

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Mean Plasma Yield Lower Plasma Yield in Streck Tubes


Study 3 – Compatibility of different centrifugation conditions with primary tube handling workflow• Hemolysis in PAXgene Blood ccfDNA Tubes is independent of tested centrifugation forces,

times and repetitions

• Centrifugation for 5 minutes at 1,600 × g resulted in reduced plasma yields, turbid plasma fractions and diffuse plasma/hematocrit differentiation

Figure 9: Turbid plasma fractions and diffuse plasma/hematocrit border. Plasma separation after 5 minutes centrifugation at 1,600 × g.


Figure 12: Yield and stability of ccfDNA were measured with assays amplifying the same 66 bp 18S rDNA fragment. A) qPCR using the QIAGEN Rotor-Gene Q instrument, ideal ratio would be 1.0 (no change versus day 0). B) ddPCR using the BioRad QX 200 DR Droplet Digital PCR System. *Due to the low plasma volume and impacted plasma quality, no PCR analysis was performed for the 5 min at 1,600 × g condition.

Figure 11: Plasma yield per tube by blood storage and plasma processing protocols. Red boxes mark reduced plasma yields

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Mean Plasma Yield Low Centrifugation Forces and Times Reduce Plasma Yield


influence of centrifugation conditions for plasma processing on … · 2019. 11. 21. · figure 2:...

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