Jump to ContentJump to Main Navigation
Show Summary Details
More options …

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.

12 Issues per year

IMPACT FACTOR 2016: 3.432

CiteScore 2016: 2.21

SCImago Journal Rank (SJR) 2016: 1.000
Source Normalized Impact per Paper (SNIP) 2016: 1.112

See all formats and pricing
More options …
Volume 43, Issue 6 (Jun 2005)


Standardized quantification of circulating peripheral tumor cells from lung and breast cancer

Katharina Pachmann
  • Klinik für Innere Medizin II, Friedrich Schiller Universität Jena, Jena, Germany
/ Joachim H. Clement
  • Klinik für Innere Medizin II, Friedrich Schiller Universität Jena, Jena, Germany
/ Claus-Peter Schneider
  • Zentralklinik Bad Berka GmbH, Bad Berka, Germany
/ Babette Willen
  • Tranfusionsmedizinisches Zentrum Bayreuth, Bayreuth, Germany
/ Oumar Camara
  • Frauenklinik der Friedrich Schiller Universität Jena, Jena, Germany
/ Ulrich Pachmann
  • Tranfusionsmedizinisches Zentrum Bayreuth, Bayreuth, Germany
/ Klaus Höffken
  • Klinik für Innere Medizin II, Friedrich Schiller Universität Jena, Jena, Germany
Published Online: 2014-06-11 | DOI: https://doi.org/10.1515/CCLM.2005.107


Detection and quantitation of circulating tumor cells from solid epithelial tumors could become a valuable tool for therapy monitoring if the procedure can be standardized. In the present work we assessed the influence of preanalytical handling, storage and white blood cell isolation on analysis of a population of spiked tumor cell-line cells and intrinsically present epithelial cells in the peripheral blood of breast and lung cancer patients and the sensitivity of their detection. Sucrose density separation did not enrich epithelial cells, and even depleted them, leading to a gross underestimation of their numbers (3/13 positive, between 2.9 and 50cells/mL) in comparison to red blood cell lysis (13/13 positive, between 77,200 and 800cells/mL). Short-term storage of whole blood samples for up to 7days had little influence on the number of epithelial cells recovered. The effectiveness of magnetic bead enrichment was dependent on the number of relevant cells and the volume used for enrichment. Red blood cell lysis and fluorochrome-labeled antibody staining in a no-wash procedure with subsequent laser scanning cytometry allowed the detection of circulating epithelial cells in 92% of breast and lung cancer patients. Two examples of how this method can be applied for the longitudinal analysis in individual patients are shown, with an increase in numbers preceding relapse and a decrease paralleling tumor reduction. The proposed simple and easy method allows close monitoring, which may help in real-time analysis of the response of solid tumors, especially their systemic component, to therapy and hopefully will contribute to more individually tailored therapy.

Keywords: circulating tumor cells; standardization; therapy monitoring.


  • 1

    Gross HJ, Verwer B, Houck D, Hoffman RA, Recktenwald D. Model study detecting breast cancer cells in peripheral blood mononuclear cells at frequencies as low as 10 –7. Proc Natl Acad Sci USA 1995; 92: 537–41. CrossrefGoogle Scholar

  • 2

    Riethmueller G, Schlimok G, Kufer P, Gruber R, Holz E, Pantel K. Minimal residual disease in solid cancer: the diagnostic and therapeutic opportunity for monoclonal antibodies. Ann Oncol 1996; 7(Suppl 1): 17. Google Scholar

  • 3

    Ghossein RA, Rossai J. Polymerase chain reaction in the detection of micrometastases and circulating tumour cells. Cancer 1996; 78: 10–6. CrossrefGoogle Scholar

  • 4

    Ross AA. Minimal residual disease in solid tumour malignancies: a review. J Hematother 1998; 7: 9–18. CrossrefGoogle Scholar

  • 5

    Pantel K, von Knebel Doeberitz M, Izbicki JR, Riethmüller G. Disseminierte Tumorzellen: Diagnostik, prognostische Relevanz, Phänotypisierung und therapeutische Strategien. Chirurg 1997; 68: 1241–50. CrossrefGoogle Scholar

  • 6

    Bessa X, Elizalde JI, Boix L, Pinol V, Lacy AM, Salo J, et al. Lack of prognostic influence of circulating tumor cells in peripheral blood of patients with colorectal cancer. Gastroenterology 2001; 120: 1084–92. CrossrefGoogle Scholar

  • 7

    Moldenhauer G, Momburg F, Moller P, Schwartz R, Hammerling GJ. Epithelium-specific surface glycoprotein of M r 34,000 is a widely distributed human carcinoma marker. Br J Cancer 1987; 56: 714–21. Google Scholar

  • 8

    Pachmann K, Heiß P, Demel U, Tilz G. Detection and quantification of small numbers of circulating tumor cells in peripheral blood using Laser Scanning Cytometry (LSC ®). Clin Chem Lab Med 2001; 39: 811–7. CrossrefGoogle Scholar

  • 9

    Schlimok G, Funke I, Pantel K, Strobel F, Lindemann F, Witte J, et al. Micrometastatic tumor cells in bone marrow of patients with gastric cancer: methodological aspects of detection and prognostic significance. Eur J Cancer 1991; 27: 1461–5. CrossrefGoogle Scholar

  • 10

    Thorban S, Rosenberg R, Busch R, Roeder RJ. Epithelial cells in bone marrow of oesophageal cancer patients: a significant prognostic factor in multivariate analysis. Br J Cancer 2000; 83: 35–9. Google Scholar

  • 11

    Sabile A, Louha M, Bonte E, Poussin K, Vona G, Mejean A, et al. Efficiency of Ber-EP4 antibody for isolating circulating epithelial tumor cells before RT-PCR detection. Am J Clin Pathol 1999; 112: 171–8. CrossrefGoogle Scholar

  • 12

    Braun S, Pantel K, Müller P, Janni W, Hepp F, Kentenich C, et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II or III breast cancer. N Engl J Med 2000; 342: 526–33. Google Scholar

  • 13

    Liotta LA, Kleinermann J, Sidel GM. Quantitative relationships of intravascular tumor cells, tumor vessels, and pulmonary metastases following tumor implantation. Cancer Res 1974; 34: 997–1004. Google Scholar

  • 14

    Baran J, Pituch-Noworolska A, Krzezowiak A, Wieckiewicz J, Stachura J, Pryjma J, et al. Detection of cancer cells in the blood by FACS sorting of CD45 cells. Int J Mol Med 1998; 1: 573–8. Google Scholar

  • 15

    Pantel K, Felber E, Schlimok G. Detection and characterization of residual disease in breast cancer. J Hematother 1994; 3: 315–22. CrossrefGoogle Scholar

  • 16

    Diel IJ, Kaufmann M, Costa SD, Holle R, von Minckwitz G, Solomayer EF, et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88: 1652–8. CrossrefGoogle Scholar

  • 17

    Lilleng PK, Harveit F. Missed micrometastases – the extent of the problem. Acta Oncol 2000; 39: 313–7. CrossrefGoogle Scholar

  • 18

    Nakamura T, Yasamura T, Hayashi K, Educhi R, Ide H, Takasaki K, et al. Immunocytochemical detection of circulating esophageal carcinoma cells by immunomagnetic separation. Anticancer Res 2000; 20: 4739–44. Google Scholar

  • 19

    Weitz J, Kienle P, Magener A, Koch M, Schrodel A, Willeke F, et al. Detection of disseminated colorectal cancer cells in lymph nodes blood and bone marrow. Clin Cancer Res 1999; 5: 1830–6. Google Scholar

  • 20

    Lopez-Guerrero JA, Gilabert PB, Gonzalez EB, Sanz Alonso MA, Perez JP, Talens AS, et al. Use of reverse-transcriptase polymerase chain reaction (RT-PCR) for carcinoembryonic antigen, cytokeratin 19 and maspin in the detection of tumor cells in leukapheresis products from patients with breast cancer: comparison with immunocytochemistry. J Hematother 1999; 8: 53–61. CrossrefGoogle Scholar

  • 21

    Jung R, Petersen K, Krueger W, Wolf M, Wagener C, Zander A, et al. Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes. Br J Cancer 1999; 81: 870–3. CrossrefGoogle Scholar

  • 22

    Racila E, Euhus D, Weiss AJ, Rao C, McConnell J, Terstappen LW, et al. Detection and characterization of carcinoma cells in the blood. Proc Natl Acad Sci USA 1998; 95: 4589–9. CrossrefGoogle Scholar

  • 23

    Fodstad O, Faye R, Hoifodt HK, Skovlund E, Aamdal S. Immunobead based detection and characterization of circulating tumor cells in melanoma patients. Recent Results Cancer Res 2001; 158: 40–50. Google Scholar

  • 24

    Bilkenroth U, Taubert H, Riemann D, Rebmann U, Heynemann H, Meye A. Detection and enrichment of disseminated renal carcinoma cells from peripheral blood by immunomagnetic cell separation. Int J Cancer 2001; 92: 577–82. CrossrefGoogle Scholar

  • 25

    Hermanek P, Hutter RV, Sobin LH, Wittekind C. International Union Against Cancer. Classification of isolated tumor cells and micrometastasis. Cancer 1999; 86: 2668–73. CrossrefGoogle Scholar

  • 26

    des Guetz G, Lacortes JM, Camilleri-Broet S, Bouillot JL, de Mestier P. Micrometastases in colonic cancers: diagnostic methods and prognostic elements. J Chir 2002; 139: 141–8. Google Scholar

  • 27

    von Knebel-Doeberitz M, Koch M, Weitz J, Herfarth C. Diagnosis and significance of minimal residual disease in patients with colorectal carcinoma. Zentralbl Chir 2000; 125(Suppl 1): 15–9. Google Scholar

  • 28

    Jiao X, Krasna MJ. Clinical significance of micrometastasis in lung and esophageal cancer: a new paradigm in thoracic oncology. Ann Thorac Surg 2002; 74: 278–84. CrossrefGoogle Scholar

  • 29

    Tsavellas G, Patel H, Allen-Mersh TG. Detection and significance of occult tumor cells in colorectal cancer. Br J Surg 2001; 88: 1307–20. CrossrefGoogle Scholar

  • 30

    Iorgulescu DG, Kiroff GK. Minimal residual marrow disease: detection and significance of isolated tumour cells in bone marrow. ANZ J Surg 2001; 71: 365–76. CrossrefGoogle Scholar

  • 31

    Martin VM, Siewert C, Scharl A, Harms T, Heinze R, Öhl S, et al. Immunomagnetic enrichment of disseminated epithelial tumor cells from peripheral blood by MACS. Exp Hematol 1998; 26: 252–64. Google Scholar

  • 32

    Krag DN, Ashikaga T, Moss TJ, Kusminsky RE, Feldman S, Carp NZ, et al. Breast cancer cells in the blood: a pilot study. Breast J 1999; 5: 354–58. CrossrefGoogle Scholar

  • 33

    Iinuma H, Okinaga K, Adachi M, Suda K, Sekine T, Sakagawa K, et al. Detection of tumor cells in blood using CD45 magnetic cell separation followed by nested mutant allele-specific amplification of p53 and k-ras gene in patients with colorectal cancer. Int J Cancer 2000; 89: 337–44. Google Scholar

  • 34

    Siewert C, Herber M, Hunzelmann N, Fodstad O, Miltenyi S, Assenmacher M, et al. Rapid enrichment and detection of melanoma cells from peripheral blood mononuclear cells by a new assay combining immunomagnetic cell sorting and immunocytochemical staining. Recent Results Cancer Res 2001; 158: 51–60. Google Scholar

  • 35

    Zigeuner RE, Riesenberg R, Pohla H, Hofstetter A, Oberneder R. Isolation of circulating cancer cells from whole blood by immunomagnetic cell enrichment and unenriched immunocytochemistry in vitro. J Urol 2003; 169: 701–5. Google Scholar

  • 36

    Tsavellas G, Huang A, McCullough T, Patel H, Araia R, Allen-Mersh TG. Flow cytometry correlates with RT-PCR for detection of spiked but not circulating colorectal cancer cells. Clin Exp Metastasis 2002; 19: 495–502. CrossrefGoogle Scholar

  • 37

    Bertau P, Dumas F, Gala J-L, Eschwege P, Laour B, Philippe M, et al. Molecular detection of circulating prostate cells in cancer II: comparison of prostate epithelial cell isolation procedures. Clin Chem 1998; 44: 1750–3. Google Scholar

  • 38

    Benez A, Geiselhart A, Handgretinger R, Schiebel U, Fierlbeck G. Detection of circulating melanoma cells by immunomagnetic cell sorting. J Clin Lab Anal 1999; 13: 229–33. CrossrefGoogle Scholar

  • 39

    Gertler R, Rosenberg R, Fuehrer K, Dahm M, Nekarda H, Siewert JR. Detection of circulating tumor cells in blood using an optimized density gradient centrifugation. Recent Results Cancer Res 2003; 162: 149–55. Google Scholar

  • 40

    Vlems FA, Ladanyi A, Gertler R, Rosenberg R, Diepstra JH, Roder C, et al. Reliability of quantitative reverse-transcriptase-PCR-based detection of tumour cells in the blood between different laboratories using a standardised protocol. Eur J Cancer 2003; 39: 388–96. CrossrefGoogle Scholar

  • 41

    Schamhart D, Swinnen J, Kurth KH, Westerhof A, Kusters R, Borchers H, et al. Numeric definition of the clinical performance of the nested reverse transcription-PCR for detection of hematogenous epithelial cells and correction for specific mRNA of non-target cell origin asevaluated for prostate cancer cells. Clin Chem 2003; 49: 1458–66. CrossrefGoogle Scholar

About the article

Corresponding author: Katharina Pachmann, Abteilung für Experimentelle Hämatologie und Onkologie der Klinik für Innere Medizin II der Friedrich Schiller Universität Jena, Erlanger Allee 101, 07747 Jena, Germany Phone: +49-3641-9325821, Fax: +49-3641-9325827,

Received: 2004-11-30

Accepted: 2005-04-20

Published Online: 2014-06-11

Published in Print: 2005-06-01

Citation Information: Clinical Chemistry and Laboratory Medicine (CCLM), ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: https://doi.org/10.1515/CCLM.2005.107.

Export Citation

© Walter de Gruyter Berlin New York. Copyright Clearance Center

Comments (0)

Please log in or register to comment.
Log in