Magnetic bead RT-PCR: establishment of a new method for detecting circulating melanoma cells.
This study was performed to detect circulating melanoma cells in peripheral blood using a novel method based on magnetic-activated cell separation (MACS) followed by a nested reverse transcriptase-polymerase chain reaction (RT-PCR) for tyrosinase and MART-1 mRNA. Samples to be tested were enriched for tumour cells either by isolating melanoma cells using two anti-melanoma antibodies (MART-1 and HMB-45) or by CD45 depletion of the non-melanoma cell fraction. The tumour cell-enriched fractions were subjected to mRNA isolation using oligo-deoxythymidylate (oligo-dT) magnetic beads followed by a nested RT-PCR. Sensitivity was assessed by spiking experiments and compared with a commonly used total RNA isolation system previously established in our department. Positive isolation of melanoma cells showed insufficient sensitivity, whereas negative isolation by depletion of leukocytes showed a detection limit of at least one melanoma cell per millilitre of whole blood. In further experiments, the depletion assay was applied to 25 peripheral blood samples of melanoma patients. The preliminary data obtained from the new method indicate a comparable detection rate to the established total RNA extraction method. However, not all the results were concordant. Therefore, future experiments need to be performed with a statistically greater number of patients.
Georgieva
J
J
Department of Dermatology, University Medical Centre Benjamin Franklin, The Free University of Berlin, Fabeckstrasse 60-62, 14195 Berlin, Germany.
Milling
A
A
Orfanos
C E
CE
Geilen
C C
CC
D000328:Adult; D000368:Aged; D000951:Antigens, Neoplasm; D015704:CD4 Antigens / Q000276:immunology; D005260:Female; D006801:Humans; D018189:Immunomagnetic Separation; D058965:MART-1 Antigen; D008297:Male; D008545:Melanoma / Q000097:blood* / Q000473:pathology; D058950:Melanoma-Specific Antigens; D008863:Microspheres; D008875:Middle Aged; D009363:Neoplasm Proteins / Q000276:immunology; D009367:Neoplasm Staging; D009360:Neoplastic Cells, Circulating; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger / Q000097:blood; D012334:RNA, Neoplasm / Q000097:blood; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
D000951:Antigens, Neoplasm; D015704:CD4 Antigens; D058965:MART-1 Antigen; C548667:MLANA protein, human; D058950:Melanoma-Specific Antigens; D009363:Neoplasm Proteins; D009838:Oligodeoxyribonucleotides; D012333:RNA, Messenger; D012334:RNA, Neoplasm; C027903:oligo (dT)