FAQ (veelgestelde vragen)
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FAQ (veelgestelde vragen) - LeucoSep®/OncoQuick®

1. Is it possible to enrich tumor cells from minced tumor tissue by means of OncoQuick®?

No! Tumor cells possess an equal buoyant density as the surrounding organ tissue cells. At best the blood cells remaining in the tissue (erythrocytes and lymphocytes) might be depleted.

2. What is the explanation for the term "Minimal Residual Disease" (MRD)

MRD describes a certain stage of a tumoral disease characterized by disseminated tumor cells remaining in the patient's body after surgical resection or chemo-/radiotherapy of a primary tumor. Very often these remaining cells are resistant against the therapy and potentially can cause metastases.

3. What is the purpose of the porous barrier inserted in the OncoQuick®-tube?

  • the porous barrier has no impact on the separation process
  • allows more convenient filling of the tube (sample can be poured-in carefully)
  • prevents recontamination of the enriched cell fraction
  • does not act as a filter or sieve

    • 4. How is it possible to avoid the loss of OncoQuick®-enriched tumor cells during a following cytospin step?

  • use a careful cytospin protocol
  • adapt speed of cytofuge to sensitive cells (600 to 800 rpm with Shandon-Cytofuge) to avoid disruption of cells
  • use concentrated cell suspensions (1 000 000 cells/mL; 100 - 200 µl cell suspension/slide)
  • lengthen centrifugation time (6-10 min)

    • 5. How is it possible to avoid the loss of RNA derived from tumor cells that have been enriched by means of OncoQuick®?

  • well known problem: current RNA-isolation kits often loose RNA working with small cell numbers
  • RNA isolation has been successfully performed using Roche HiPure RNA isolation kit (Cat. # 1828665) or Stratagene StrataPrep total RNA Microprep kit

    • 6. What about a tube that is optimized for small blood sample volumes (0.5 to 5 mL)?

    Due to the limited number of disseminated tumor cells in the blood of tumor patients (experience: in 25 mL of blood of breast cancer patients maximal up to 100 tumor cells have been enriched) it is really questionnable to work with small sample volumes, thereby limiting dramatically the probability to find the rare tumor cells.

    7. What can I do if I am not able to get rid of air bubbles below the porous barrier by centrifugation?

  • air bubbles should not be too big (approx. size of a pea)
  • they will usually be pressed out if you carefully fill the tube with the sample specimen
  • if this does not happen: remove the tube

    • 8. How can I get rid of platelets that contaminate the enriched cell fraction?

  • most important for users who want to pick tumor cells – platelets often stick to these cells
  • avoid harvesting the whole plasma layer by pouring-off the whole upper phase
  • selectively harvest interphase with a serological pipette
  • excessive contamination with platelets can be eliminated by performing a second centrifugation in a 4 to 20% sucrose gradient, tumor cells will be pelleted

    • 9. Is there any special recommendation for the use of OncoQuick® with bone marrow or low-volume blood samples?

    We recommend the removal of the porous barrier:

  • open the tube under a sterile hood
  • press laterally on the barrier with a sterile serological pipette
  • the barrier flips and can be removed easily with a sterile pair of tweezers
  • dilute the bone marrow with an equal volume of buffer
  • carefully overlay the sample on top of the separation medium and proceed as indicated in the manual

    • 10. Which are the advantages of OncoQuick® vs. conventional density gradient centrifugation (e.g. Ficoll, current

  • Simplified filling due to porous barrier
  • Enhanced thinning of mononuclear blood cells
  • Faster screening of the enriched cell fraction
  • Cost saving due to limited need for diagnostics (e.g. antibodies)
  • Reduction of false positive artifacts

    • 11. Which are the advantages of OncoQuick® compared to immunomagnetic beads (MACS, Dynabeads)?

  • Significantly faster enrichment of tumor cells (45 min vs. 90-120 min)
  • No need for additional lab equipment (magnet rack, separation columns)
  • No need for expensive magnetic beads
  • Limited number of tumorspecific antibodies
  • No blocking of diagnostically important epitopes by magnetic antibodies used for tumor cell enrichment

    • 12. What is the advantage of screening patient blood for tumor cells and not bone marrow?

  • Venipuncture is much more tolerable for the patient compared to bone marrow aspiration
  • Venipuncture can be done by a nurse, bone marrow aspiration needs a whole medical team, sterile conditions, local anaestesia etc.
  • Cost saving

    • 13. Is it possible to use Leucosep® to perform the enrichment of tumor cells from peripheral blood?

  • Leucosep® was developed for the enrichment of mononuclear blood cells (separation medium=Ficoll)
  • Leucosep® is suitable for the separation of white blood cells from red blood cells
  • OncoQuick® is suitable for the separation of circulating tumor cells from blood cells

    • 14. Can I use OncoQuick® to perform the enrichment of leukemic cells from whole blood or bone marrow

    This does not work since the buoyant density of leukemic and hematopoietic cells is similar.

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