Medizinische Universität Graz Austria/Österreich - Forschungsportal - Medical University of Graz
Gewählte Publikation:
SHR Neuro Krebs Kardio Lipid Stoffw Microb
Ausch, C; Dandachi, N; Buxhofer-Ausch, V; Balic, M; Huber, K; Bauernhofer, T; Ogris, E; Hinterberger, W; Braun, S; Schiessel, R.
Immunomagnetic CD45 depletion does not improve cytokeratin 20 RT-PCR in colorectal cancer.
Clin Chem Lab Med. 2007; 45(3):351-356
Doi: 10.1515/CCLM.2007.059
Web of Science
PubMed
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FullText_MUG
- Führende Autor*innen der Med Uni Graz
- Dandachi Nadia
- Co-Autor*innen der Med Uni Graz
- Balic Marija
- Bauernhofer Thomas
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- 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 genePBGD 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 b 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.
- Find related publications in this database (using NLM MeSH Indexing)
- Antigens, CD - blood Antigens, CD - isolation & purification
- Antigens, CD45 - blood Antigens, CD45 - isolation & purification
- Bone Marrow Cells - pathology
- Centrifugation, Density Gradient -
- Colorectal Neoplasms - genetics Colorectal Neoplasms - immunology
- Humans -
- Immunomagnetic Separation - methods
- Keratin-20 - blood Keratin-20 - genetics
- Reverse Transcriptase Polymerase Chain Reaction -
- Find related publications in this database (Keywords)
- CD45 depletion
- colorectal cancer
- cytokeratin 20 reverse transcriptase-PCR
- Ficoll density
- gradient separation