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Background: Assessment of resistance to drug moieties in tissue culture is complicated by limited sample, clonal selection and alteration of cycling fraction and cycle duration in clonally mixed lesions. DNA damage assessment by single cell gel electrophoresis (SCGE) of excised DNA is limited by non-linear analysis in fluorescent light microscopy. Confocal Laser Scanning Microscopy (CLSM) with high N.A. magnification allows for quantitation of total excised DNA fragment size distribution but is still limited by the large volume required for labour intensive SCGE, precluding multi-exposure clinical testing., Aims: To optimise sample requirement for SCGE and CLS., Methods: Standard slide mounted bed gels were punched with multiple coded 6 mm wells and filled with suspensions of cells subjected to drug/concentration variations. After SCGE, 30 microns frozen sections were prepared of each well and mounted in ethidium bromide solution on multi-well hydrophilic slides to allow for short working distance of high resolution CLSM in a Zeiss Axiovert L410 SM. Testing for feasibility, reproducibility and consistency used both cultured standard leukaemic cell lines, normal human control marrow and clinical samples., Results and Conclusion: Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.

Background: High resolution Confocal Laser Scanning Microscopy (CLSM) may be applied to testing of drug resistance in vitro in clinical setting. Rapid analysis of DNA damage by precise quantitation of excised DNA in bone marrow samples exposed to potential treatment moieties directly after isolation but the relative sensitivity of the integrated method is as yet untested., Aims: To test the clinical applicability of SCGE/high resolution CLSM for differences in drug resistance in marrow cells., Methods: Cells from normal bone marrow samples were exposed for identical periods and at 4 concentrations to either 1 hour of standard Daunorubicin (.5, 1, 1.5, 2 micrograms/ml) or 8 hours DaunoXome (courtesy of NeXstar Inc, USA) (.05, .1, .15, .2 microgram/ml). After 2 and 6 hours recovery, cells were harvested for SCGE, randomization, analysis of tail length, total excised DNA and fragment size distribution using high resolution CLSM., Results: Tail length and fragment size distribution was not, but total excised DNA was significantly increased after 0.1 microgram/ml Liposomal Daunorubicin (DaunoXome) compared to 1.0 microgram/ml Daunorubicin., Conclusion: SCGE/high resolution CLSM effectively demonstrated differences in Daunorubicin resistance of human marrow cells to alternative formulations. The method has potential for use in clinical testing of neoplastic cell drug resistance.