CFU‐GM Assay for Evaluation of Drug Myelotoxic Activity

Abstract

To study hematotoxicity of compounds on the myeloid cell compartment, the authors describe a standard procedure developed as a workable good laboratory practices-compliant protocol to determine the in vitro myelotoxic effect of drugs and chemicals. Specific protocols are presented to prepare human and murine myeloid progenitors (CFU-GM) for testing in a validated CFU-GM assay. Details are given for performing a screening test when toxicity data are not available and for passing on to an accurate inhibitory concentration-determination phase. To quantify the potential hematotoxicity of xenobiotics from their direct adverse effects on CFU-GM, the unit describes how to manage the results by means of an algorithm able to predict the acute xenobiotic exposure levels that cause maximum tolerated decreases (MTD) in absolute neutrophil count (ANC). A protocol describes a miniaturized application of the procedure in 96-well plates for high-throughput screening of compounds or for testing compounds that are available in very small quantities.

Beyond AOPs: A Mechanistic Evaluation of NAMs in DART Testing

“…To assess an impact of chemical exposure on immune system related genes including cytokines, chemokines and interleukins, assays pertaining to its individual components can be considered. Lymphocytic and myeloid cells can be isolated from peripheral blood mononuclear cells (PBMCs) and can be tested to determine toxicity to each of these lineages specifically, upon chemical exposures ( Hassan et al, 2007 ; Pessina and Bonomi, 2007 ). Other targeted functional assays for specific mechanisms include human lymphocyte activation (HuLA) assay, an antigen recall assay, similar to the in vivo T-cell-dependent antibody response (TDAR) where multiple immune cell types are needed to produce responses; multiple cytokines (IL-2, IFN-γ, IL-1β, and IL-8) assay; and the BioMap panel of assays where test systems are constructed with one or more primary cell types from normal human donors stimulated with cytokines or growth factors recapitulate relevant signalling networks that naturally occur in human tissue or disease states and can be explored for specific biomarker readouts ( Collinge et al, 2010 ; Kimura et al, 2018 ; Singer et al, 2019 ; Collinge et al, 2020 ).…”

Stem cells in myelotoxicity

“…This very successful study was able to predict MTD for 20 of 23 compounds, thus providing an assay that may be of significant benefit to those entering a Phase I clinical study where there is limited information on human MTD (Pessina et al, 2003). Standard operative procedures are available (Pessina and Bonomi, 2007). In order to assess if the prediction model defined by Pessina et al (2003) is relevant using rat hematopoietic stem cell, a validation study, using 10 of 20 drugs tested in CFU-GM validation is performing in 2008.…”