Immunomagnetic detection of micrometastatic cells in bone marrow in uveal melanoma patients

Purpose: Our objective was to introduce immunomagnetic separation (IMS) in ocular research by evaluating the possibility of detecting tumour cells in bone marrow (BM) and peripheral blood (PB) samples and validating the captured cells as melanocytic cells. Methods: Mononuclear cell (MNC) fractions isolated from BM and PB in uveal melanoma patients were examined for tumour cells using our IMS method. Sheep-anti-mouse IgG antibody-coated super paramagnetic particles were conjugated to an anti-melanoma antibody. Microscopy of the magnetic fraction isolated from MNCs was performed to identify and count the number of bead-rosetted cells. The finding of at least two rosettes with coated beads in a 20-mu l fraction of a sample was registered as a positive test. The melanocytic nature of the tumour cells was ascertained with a double labelling procedure using fluorescent microparticles. Results: Using IMS in a study of 328 patients, tumour cells were at initial diagnosis found in BM and PB in 29.9% and 1.6% of cases, respectively. In positive samples, a median of 56 tumour cells (range 2-500) were identified. The captured cells were documented to be of melanocytic origin by the simultaneous binding of fluorescent beads coated with another melanoma-associated antibody. Conclusions: The IMS method was sensitive and efficient in the detection of occult melanoma tumour cells in BM. The validity of the immunomagnetic technique was strengthened by verifying the melanocytic characteristics of the isolated cells. The IMS procedure identifies intact, vital tumour cells, permitting further molecular characterization, an advantage which makes this method attractive for extended use. The clinical relevance of the findings will be further investigated in follow-up studies with repeated sampling and characterization of the isolated tumour cells.