CMCRC-Pilot grant

Rapid and minimally invasive early triage biodosimetry using Giemsa staining to score radiation-induced excess lymphocyte prematurely condensed chromosomes or Centromeric/Telomeric PNA probes to score accurately dicentrics and centric rings only in the over exposed individuals
2016-2018 | Research Subaward CMCRC program as support from a pilot grant from the Opportunity Funds Management Core of the Centers for Medical Countermeasures against Radiation, National Institute of Health; grant number U19AI067773.

For optimal radiation protection in emergency medicine programs it is imperative to focus on early triage biodosimetry for the best possible treatment of individuals involved in radiation accidents. Therefore, improved research strategies adopted for mass-casualty management are critically needed. Given that dicentric chromosomes, fragments and centric rings will be present in the blood samples at the time they reach the lab, the main objective of the project is to explore the applicability of cell fusion mediated PCC methodology for the visualization and quantification of these aberrations in G0-lymphocytes, in order to devise a rapid and minimally invasive biodosimetry tool for automatable early triage. Towards this goal, fingerstick-derived heparinised blood samples in volumes of less than 100μl are tested in multiple 96-tube racks, so that the development of this assay will pave the way to be used in an automated fashion. In fact, the PCC methodology enables the analysis of radiation-induced chromosomal aberrations directly in non-stimulated G0-lymphocytes within two-hours and without the two-day culture delay needed in metaphase analysis. Specifically, the yield of Giemsa stained prematurely condensed chromosomes (PCCs) in excess of 46 will be used as a rapid, inexpensive and minimally invasive assay. Following early triage, dose estimates can be reconfirmed based on accurate analysis of dicentric and centric ring chromosomes in non-stimulated (G0) lymphocytes. For this purpose the PCC-assay is combined with fluorescence in situ hybridization (FISH) using simultaneously centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes and applied for the over exposed individuals only since FISH is inherently labor intensive and expensive. Furthermore, the required calibration curves for a wide range of doses and post-irradiation times are be constructed and dose estimates are compared to those obtained using conventional metaphase analysis with Giemsa or C/T PNA probes at metaphase. The analysis of PCCs can be facilitated if tools for imaging automation, such as the slide-scanning platform Metafer of MetaSystems, are used. Specifically, the implementation of semi-automated imaging, based on automated detection of PCCs on slides, and on automated acquisition of high-resolution images for scoring of excess PCCs is validated.

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