DRK-Blutspendedienst West

Precipitation steps (PEG), centrifugation and careful resuspension of the invisible pelleted viral particles precede efficient extraction of viral RNA and DNA from minipools (n 96, 9.6 ml). Loss of nucleic acid during this time consuming stepsis another critical point.

We looked for an automated RNA/DNA extraction method,that could start right from the minipool sample (n = 96, 9.6 ml). Release of labile blood components calls for short assay time .

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SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

Magnetic bead technologyMagnetic bead technology

in viral RNA/DNA extraction from plasma minipoolsin viral RNA/DNA extraction from plasma minipoolsL. Pichl and V. SchottstedtL. Pichl and V. Schottstedt

German Red Cross Blood Transfusion Centre West, Central Laboratory, HagenGerman Red Cross Blood Transfusion Centre West, Central Laboratory, Hagen

DRK-Blutspendedienst West

Magnetic Separation Module I

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

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Rod HeadRod Head

Electromagnet

Tube track

DRK-Blutspendedienst West

Features

Polyethanol coated magnetic beads (1 µm Ø, hydrophilic)

12 Rod Head magnetizable

12 samples, up to 10 ml vol.in 50 ml tubes

One specific disposable tip per sample per complete separation

Eluate volume: 100 µl

Processing time: 1h 8 min

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

Zentrallabor HagenZentrallabor Hagen

DRK-Blutspendedienst West

Materials

Chemagen Magnetic Separation Module I

Chemagic Viral 10k Kit Special (www.chemagen.com)

LightCycler II (Roche Diagnostics), ABI SDS 7700 RealArt™ ParvoB19 LC PCR Kit

RealArt™ HAV LC RT PCR KitRealArt™ HBV TM PCR Kit

(www.artus-biotech.com)

Nucleic acid

Nucleic acid extraction

extraction

Amplification and

Amplification and

detection

detection

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

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DRK-Blutspendedienst West

Workflow

0 1h 2hrs 3hrs 4 hrs

Set up

Extraction

PAV B19

HAV

HBV

PCR

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

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DRK-Blutspendedienst West

SensitivityMagnetic Separation Module I + LightCycler II /ABI SDS 7700

Parameter PCR Cycler SpikeReferenceMaterial

95% detectionlimit*

[IU/ml single don.]

v.c. [%]

cp/threshold

PAV B19 LightCycler II WHO B19-DNA

NIBSC 99/800875 0.61 – 2.23

HAV LightCycler II WHO HAV-RNA

NIBSC 00/560260 0.45 – 2.76

HBV ABI SDS7700

WHO HBV-DNA

NIBSC 97/7461.274 1.02 – 2.71

* = calculated by probit analysis

Minipools of n = 93, EDTA-Plasma, triple-spike of diluted Ref. Mat., 100 µl each

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

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DRK-Blutspendedienst West

Robustness

102 Pools have been analysed for B19, HBV and HAV so far. None of them tested positive.

Cross contamination study was performed with two3 x 4 matrices of alternating negative and B19 spiked pools. Referring to a dose of 10EE 8 IU/ml per single donation all of the none spiked pool were negativein B19 LC-PCR.

Tracing back reactive results from pools to single donation via ”chessboard“ testing was performedfor HAV, HBV and B19.

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

Zentrallabor HagenZentrallabor Hagen

DRK-Blutspendedienst West

Outlook

Combination of Chemagen´s magnetic bead tech-nology with real time PCR without pre-extraction manipulation of the minipool (n = 96) reveals acceptable sensitivity and robustness for PAV B19, HAV and HBV;

Implementation of a robotic sample processor with the magnetic separation module for sample identification, aliquoting reagents and sample lysis;

Increasing throughput by optimizing time management.

SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04

Zentrallabor HagenZentrallabor Hagen

DRK-Blutspendedienst West

Thanks to:

Thorsten HageböckAndrea MatulinaYvonne Schmidt

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SoGAT XVII, Paris, 27/05/04 SoGAT XVII, Paris, 27/05/04