The in vitro efficacy of drug candidates relative to hematopoietic stem cell proliferation and differentiation is currently assayed through use of the clonogenic "colony assay." The extremely low throughput of this assay precludes its use in library screening and much drug discovery work. A rapid-throughput assay of progenitor cell differentiation based on the quantification of hematopoietic lineage-specific markers has been developed. The CELISA assay employs a single incubation with a lanthanide-conjugated primary antibody and subsequent time-resolved fluorescence spectroscopy. The rapid-throughput nature of this assay is enhanced by the use of cell culture-compatible filter plates to reduce the number of manipulations as compared to currently available cell-based assays. The culture and assay are done in 96-well plates, and the quantitation process requires approximately 1 hour. The myeloid, erythroid, and megakaryocytic lineages can be objectively quantified; data from the assay correlate extremely well with data generated through use of the traditional colony assay. This assay makes possible both rapid-throughput drug discovery and toxicity screening in the area of hematopoiesis.
References
1.
Anderson DJ, Gage FH, Weissman IL: Can stem cells cross lineage boundaries?Nat Med2001;7:393-395.
2.
Henry D: Haematological toxicities associated with dose-intensive chemotherapy: the role for and use of recombinant growth factors. Ann Oncol1997;8:S7-S10.
3.
Kies MS, Grinblatt D, Runge-Morris M, et al: Phase 11 study of intravenous melphalan (NSC-8806) in the treatment of patients with advanced squamous carcinoma of the head and neck. Invest New Drugs1994;12:45-47.
4.
Ringborg U, Lewensohn R: Factors responsible for bone marrow toxicity after treatment of myeloma patients with different alkylating agents. Acta Med Scand1978;203:276-278.
5.
Scheding S, Media JE, Nakeff A: Acute toxic effects of 3′-azido-3′-deoxythymidine (AZT) on normal and regenerating hematopoieses. Exp Hematol.1994;22:60-65.
6.
Silverstein MN, Petitt RM, Solberg LA, et al: Anagrelide: a new drug for treating thrombocytosis. N Engl J Med1988;318:1292-1294.
7.
Eaves CJ: Assays of hematopoietic progenitor cells. In Beutler E.Lichtman MA, Collier BS, et al (eds): Williams' Hematology (5th ed). New York: McGraw-Hill, 1995:22-26.
8.
Phillips JE, Greenwalt DE, Pitt AM: Fluorescent cell-based assays for high-throughput analysis [abstr 334141. AAPS Pharmsci2000;Suppl:271.
9.
Hemmila I: Fluoroimmunoassays and immunofluorometric assays. Clin Chen1985;31:359-370.
10.
Warren MK, Rose WL, Beall LD, et al: CD34+ cell expansion and expression of lineage markers during liquid culture of human progenitor cells. Stem Cells1995;13:167-174.
11.
Brasier AR, Ron D: Luciferase reporter gene assay in mammalian cells. Methods Enzymol1992;216:386-397.
12.
Chasis JA, Mohandas N: Red blood cell glycophorins. Blood1992;80:1869-1879.
13.
Freyssinier JM, Lecoq-Lafon C, Amsellem S, et al: Purification, amplification and characterization of a population of human erythroid progenitors. Br J Haematol1999;106:912-922.
14.
de Serres M, Ellis B, Dillberger JE, et al: Immunogenicity of thrombopoietin mimetic peptide GW395058 in BALB/c mice and New Zealand white rabbits: evaluation of the potential for thrombopoietin neutralizing antibody production in man. Stem Cells1999;17:203-209.
15.
Zermati Y, Fichelson S, Valensi F, et al: Transforming growth factor inhibits erythropoiesis by blocking proliferation and accelerating differentiation of erythroid progenitors. Exp Hematol2000;28:885-894.
