Analysis of melanoma cells in peripheral blood by reverse transcription-polymerase chain reaction for tyrosinase and MART-1 after mononuclear cell collection with cell preparation tubes: a comparison with the whole blood guanidinium isothiocyanate RNA isolation method

Melanoma cell detection In peripheral blood by tyrosinase reverse transcrip tion-polymerase chain reaction (RT-PCR) is usually performed on RNA isolate d from whole blood using a guanidinium isothiocyanate (GITC)/phenol extract ion method or from Ficoll Hypaque isolated mononuclear cells. The first met hod 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 loadi ng on Ficoll Hypaque. We examined whether CPTs can be used to determine mel anoma cell dissemination in peripheral blood. We first investigated whether melanoma cells were retained in the mononuclear cell layer. All six morpho logically different melanoma cell lines studied in the spiking experiments were retained in the upper layer. In further experiments, we were able to d etect 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 wa s extracted from paired blood samples from 24 analysable stage III and stag e 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 quali ty 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 CPT/RNeasy compared with whole blood/GI TC (six versus three), whereas tyrosinase mRNA was found less often in CPT/ RNeasy RNA (two versus eight). Taken together these results suggest that th e CPT isolation method is suitable for the isolation of mononuclear cells, including melanoma cells. (C) 2000 Lippincott Williams & Wilkins.