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

  • Christoph Ausch , Nadia Dandachi , Veronika Buxhofer-Ausch , Marija Balic , Klaus Huber , Thomas Bauernhofer , Emil Ogris , Wolfgang Hinterberger , Stephan Braun and Rudolf Schiessel
Published/Copyright: March 22, 2007
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Clinical Chemistry and Laboratory Medicine (CCLM)
From the journal Volume 45 Issue 3

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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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

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Published Online: 2007-03-22
Published in Print: 2007-03-01

©2007 by Walter de Gruyter Berlin New York

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https://doi.org/10.1515/CCLM.2007.059
Keywords for this article
CD45 depletion; colorectal cancer; cytokeratin 20 reverse transcriptase-PCR; Ficoll density gradient separation

Articles in the same Issue

  1. From human genetic variations to prediction of risks and responses to drugs and the environment
  2. Nutrigenomics – 2006 update
  3. How to comprehensively analyse proteins and how this influences nutritional research
  4. Genotypes, obesity and type 2 diabetes – can genetic information motivate weight loss? A review
  5. The Gene-Diet Attica Investigation on childhood obesity (GENDAI): overview of the study design
  6. Polymorphisms in the APOA1/C3/A4/A5 gene cluster and cholesterol responsiveness to dietary change
  7. Nutri-epigenomics: lifelong remodelling of our epigenomes by nutritional and metabolic factors and beyond
  8. Emerging role of cathepsin S in obesity and its associated diseases
  9. Association analysis of hepatitis virus B infection with haplotypes of the TBX21 gene promoter region in the Chinese population
  10. Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality
  11. Real-time multiplex PCR assay for genotyping of three apolipoprotein E alleles and two choline acetyltransferase alleles with three hybridization probes
  12. Immunomagnetic CD45 depletion does not improve cytokeratin 20 RT-PCR in colorectal cancer
  13. Analysis of the components of hypertransaminasemia after liver resection
  14. Fine characterization of mitral valve glycosaminoglycans and their modification with degenerative disease
  15. Oxidative stress evaluated using an automated method for hydroperoxide estimation in patients with coronary artery disease
  16. Secretory phospholipase A2 activity and release kinetics of vascular tissue remodelling biomarkers after coronary artery bypass grafting with and without cardiopulmonary bypass
  17. Clustered components of the metabolic syndrome and platelet counts in Japanese females
  18. International Standard for serum vitamin B12 and serum folate: international collaborative study to evaluate a batch of lyophilised serum for B12 and folate content
  19. Multicentre physiological reference intervals for serum concentrations of immunoglobulins A, G and M, complement C3c and C4 measured with Tina-Quant® reagents systems
  20. In vivo and in vitro allergy diagnostics: it's time to reappraise the costs
  21. Experience with post-market surveillance of in-vitro diagnostic medical devices for lay use in Germany
  22. Evaluation of the high-sensitivity, full-range Olympus CRP OSR6199 application on the Olympus AU640®
  23. How to improve the teaching of urine microscopy
  24. In vitro determination of allergen-specific serum IgE. Comparative analysis of three methods
  25. Efficacy of a new blocker against anti-ruthenium antibody interference in the Elecsys free triiodothyronine assay
  26. Clinical indications for plasma protein assays: transthyretin (prealbumin) in inflammation and malnutrition: International Federation of Clinical Chemistry and Laboratory Medicine (IFCC): IFCC Scientific Division Committee on Plasma Proteins (C-PP)
  27. Meeting Report: From human genetic variations to prediction of risks and responses to drugs and the environment
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