High resolution confocal laser scanning microscopy analysis of DNA excision repair capability in small volume marrow samples exposed to DNA directed treatment moieties.
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.


OBJECTIVE
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.


CONCLUSIONS
Multiple well SCGE followed by frozen section, high resolution CLSM allows for rapid analysis of high numbers of multiple drug exposure permutations clinically required.
Lannon
C L
CL
Department of Pathology, Dalhousie University, IWK Grace Health Centre, Halifax, Nova Scotia, Canada.
Ball
L M
LM
Pyesmany
A F
AF
Yhap
M
M
Langley
R
R
van Velzen
D
D
D001854:Bone Marrow Cells / Q000166:cytology* / Q000473:pathology*; D020552:Comet Assay / Q000295:instrumentation / Q000379:methods*; D004260:DNA Repair; D006801:Humans; D018613:Microscopy, Confocal / Q000379:methods*; D012680:Sensitivity and Specificity