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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

Editor-in-Chief: Plebani, Mario

Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.


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Immunomagnetic CD45 depletion does not improve cytokeratin 20 RT-PCR in colorectal cancer

Christoph Ausch1 / Nadia Dandachi2 / Veronika Buxhofer-Ausch3 / Marija Balic4 / Klaus Huber5 / Thomas Bauernhofer6 / Emil Ogris7 / Wolfgang Hinterberger8 / Stephan Braun9 / Rudolf Schiessel10

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Corresponding author: Christoph Ausch, MD, Ludwig Boltzmann Institute of Surgical Oncology, Department of Surgery, Donauspital, SMZ-Ost, Langobardenstraße 122, 1220 Vienna, Austria Phone: +43-1-288023302, Fax: + 43-1-288023380

Citation Information: Clinical Chemical Laboratory Medicine. Volume 45, Issue 3, Pages 351–356, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2007.059, March 2007

Publication History

Received:
September 13, 2006
Accepted:
December 4, 2006
Published Online:
2007-03-22

Abstract

Background: Cytokeratin 20 reverse transcriptase polymerase chain reaction (CK20 RT-PCR) of blood and bone marrow specimens has been suggested for assessment of hematogenously disseminated tumor cell (DTC) spread in colorectal cancer (CRC) patients. Considerable discrepancies among the studies reported indicate a need for better evaluation procedures. We investigated whether mononucleated cell (MNC) enrichment by Ficoll density gradient centrifugation followed by immunomagnetic depletion of CD45-positive cells (extended enrichment) allows better detection of DTC-associated CK20 mRNA compared to MNC enrichment by Ficoll density gradient centrifugation alone (Ficoll enrichment).

Methods: We analyzed 53 samples [38 peripheral blood (PB), 15 bone marrow (BM)] from 38 CRC patients. Extended enrichment was performed for 30 specimens (PB and BM, n=15 each), and Ficoll enrichment for 23 blood specimens. Total RNA was extracted, reverse-transcribed and analyzed by real-time RT-PCR using a LightCycler instrument.

Results: Despite extended enrichment, 10 PB and 8 BM samples could not be analyzed because of low cellular yield. The depletion efficiency of CD45 separation was 2 log. RT-PCR of the housekeeping gene PBGD resulted in high and varied crossing point values (mean 37.1+3.0) for five PB and seven BM specimens. Ficoll enrichment yielded 23 analyzable blood specimens for which the mean crossing point value was 26.7+0.5 in PBGD RT-PCR. CK20 RT-PCR of 23 blood samples (all from Dukes D patients) revealed CK20 transcripts in four cases (17%).

Conclusions: Extended enrichment was not superior to Ficoll enrichment; in fact, the sensitivity was lower. Improvement of the reported CK20 RT-PCR assay of Ficoll-enriched MNC populations is warranted.

Clin Chem Lab Med 2007;45:351–6.

Keywords: CD45 depletion; colorectal cancer; cytokeratin 20 reverse transcriptase-PCR; Ficoll density gradient separation

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