A new experimental protocol as an alternative to the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic assay to assess the haematotoxic potential of new drugs.
In this work, a first attempt to set-up a new in vitro experimental protocol with culture in liquid medium and flow cytometry analysis of bone marrow progenitors is described. This protocol is proposed as an alternative to the colony-forming unit-granulocyte/macrophage (CFU-GM) clonogenic in vitro assay currently used to assess the toxic potential of new drugs in the bone marrow. This new experimental approach should enable to speed up the procedure of the in vitro haematotoxic potential assessment, to reduce inter-experimental variability and to enhance result accuracy. Preliminary results obtained demonstrated that the progenitor cell count by flow cytometry replacing the light microscopy granulocyte/macrophage colony count represents a tremendous improvement in terms of accuracy and standardisation. Moreover, differential counts of cell sub-populations can be performed by using specific monoclonal antibodies. Furthermore, this method demonstrated to be time-saving, since 4 day cell incubation period is required instead of 7-14 day incubation in the CFU-GM clonogenic assay. On the basis of results obtained so far, the new experimental protocol proposed looks a promising alternative to the CFU-GM clonogenic assay currently used.
Dal Negro
Gianni
G
GlaxoSmithKline S.p.A., Medicines Research Centre, Via A. Fleming, 4, 37135 Verona, Italy. gdn9073@gsk.com
Vandin
Luca
L
Bonato
Monica
M
Repeto
Paolo
P
Sciuscio
Davide
D
D000818:Animals; D019070:Cell Lineage; D003114:Colony-Forming Units Assay / Q000379:methods*; D003470:Culture Media; D004317:Doxorubicin / Q000633:toxicity; D005434:Flow Cytometry; D006098:Granulocytes / Q000187:drug effects* / Q000502:physiology; D006412:Hematopoietic Stem Cells / Q000187:drug effects*; D008264:Macrophages / Q000187:drug effects* / Q000502:physiology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D015215:Zidovudine / Q000633:toxicity
D003470:Culture Media; D015215:Zidovudine; D004317:Doxorubicin