Melanoma cell detection in peripheral blood by
tyrosinase reverse transcription-polymerase chain reaction (RT-PCR) is usually performed on
RNA isolated from whole blood using a
guanidinium isothiocyanate (
GITC)/
phenol extraction method or from
Ficoll Hypaque isolated mononuclear cells. The first method contains environmentally harmful
reagents, and the second is laborious in the preanalytical steps. Cell preparation tubes (CPTs) are ready-to-use
Ficoll Hypaque-based tubes that avoid the time-consuming and critical loading on
Ficoll Hypaque. We examined whether CPTs can be used to determine
melanoma cell dissemination in peripheral blood. We first investigated whether
melanoma cells were retained in the mononuclear cell layer. All six morphologically different
melanoma cell lines studied in the spiking experiments were retained in the upper layer. In further experiments, we were able to detect low dilutions of added SK-MEL-28 cells more consistently after nested RT-PCR for
tyrosinase or MART-1 in the
RNA isolated from mononuclear cells from CPTs than from
RNA isolated with the
GITC method. In addition,
RNA was extracted from paired blood samples from 24 analysable stage III and stage IV
melanoma patients and analysed for the presence of
tyrosinase and MART-1
RNA using both the
CPT/RNeasy and the whole blood/
GITC method. The quality of the
CPT/RNeasy
RNA was better than the
RNA isolated from whole blood with
GITC/
phenol. However, the RT-PCR results were less unequivocal: MART-1
mRNA was more often detected with CPTIRNeasy compared with whole blood/
GITC (six versus three), whereas
tyrosinase mRNA was found less often in
CPT/RNeasy
RNA (two versus eight). Taken together these results suggest that the
CPT isolation method is suitable for the isolation of mononuclear cells, including
melanoma cells.