Comparison of two density gradient centrifugation systems for the enrichment of disseminated tumor cells in blood.
BACKGROUND
The detection of disseminated tumor cells in peripheral blood is limited by the presence of very few tumor cells within a large number of blood cells. Therefore, tumor cell detection calls for enrichment systems with effective depletion of blood cells and high tumor cell recovery.


METHODS
We compared the new density gradient centrifugation method OncoQuick with the standard method of Ficoll. The enriched cell fractions were quantified. Tumor cell spiking experiments examined the recovery of tumor cells as detected by immunocytochemistry and cytokeratin-20 reverse transcriptase-polymerase chain reaction (RT-PCR). Clinical application of OncoQuick was evaluated in 37 peripheral blood samples of patients with gastrointestinal carcinomas.


RESULTS
The depletion of mononuclear cells (MNCs) in the enriched cell fraction after OncoQuick centrifugation was 632-fold, with an average cell number of 9.5 x 10(4), compared with Ficoll, with a depletion factor of 3.8 and a mean number of 1.6 x 10(7) MNCs. The mean tumor cell recovery rates were 87% for OncoQuick and 84% for Ficoll. The increased depletion of MNCs with OncoQuick centrifugation further simplified immunocytochemical evaluation by reducing the number of cytospins and increasing the tumor cell density. Due to the reduced number of co-enriched MNCs by OncoQuick, the blood volume, which could be analyzed in one RT-PCR reaction, was increased up to 30 ml. Examination of peripheral blood samples from 37 patients with gastrointestinal tumors showed a cytokeratin-20 detection rate of 30% and a significant correlation with the presence of distant metastases (P < 0.02).


CONCLUSIONS
OncoQuick significantly reduced the co-enriched number of MNCs, with a high tumor cell recovery rate. Processing blood from tumor patients with OncoQuick increased the chance of detecting circulating tumor cells.
Rosenberg
R
R
Chirurgische Klinik und Poliklinik, Klinikum rechts der Isar, Technische Universität München, Munich, Germany.
Gertler
R
R
Friederichs
J
J
Fuehrer
K
K
Dahm
M
M
Phelps
R
R
Thorban
S
S
Nekarda
H
H
Siewert
J R
JR
D002452:Cell Count; D002469:Cell Separation; D002499:Centrifugation, Density Gradient / Q000295:instrumentation / Q000379:methods*; D005362:Ficoll; D006801:Humans; D007150:Immunohistochemistry; D007381:Intermediate Filament Proteins / Q000235:genetics / Q000378:metabolism; D053554:Keratin-20; D018365:Neoplasm, Residual / Q000175:diagnosis; D009360:Neoplastic Cells, Circulating / Q000473:pathology*; D012333:RNA, Messenger / Q000378:metabolism; D012334:RNA, Neoplasm / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D007381:Intermediate Filament Proteins; C507499:KRT20 protein, human; D053554:Keratin-20; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D005362:Ficoll