Development of an automatable micro-PCC biodosimetry assay for rapid individualized risk assessment in large-scale radiological emergencies

Abstract

In radiation accidents and large-scale radiological emergencies, a fast and reliable triage of individuals according to their degree of exposure is important for accident management and identification of those who need medical assistance. In this work, the applicability of cell-fusion-mediated premature chromosome condensation (PCC) in G0-lymphocytes is examined for the development of a rapid, minimally invasive and automatable micro-PCC assay, which requires blood volumes of only 100μl and can be performed in 96-well plates, towards risk assessments and categorization of individuals based on dose estimates. Chromosomal aberrations are visualized for dose-estimation analysis within two hours, without the need of blood culturing for two days, as required by conventional cytogenetics. The various steps of the standard-PCC procedure were adapted and, for the first time, lymphocytes in blood volumes of 100μl were successfully fused with CHO-mitotics in 96-well plates of 2ml/well. The plates are advantageous for high-throughput analysis since the various steps required are applied to all 96-wells simultaneously. Interestingly, the use of only 1.5ml hypotonic and Carnoy’s fixative per well offers high quality PCC-images, and the morphology of lymphocyte PCCs is identical to that obtained using the conventional PCC-assay, which requires much larger blood volumes and 15ml tubes. For dose assessments, appropriate calibration curves were constructed and for PCC analysis specialized software (MetaSystems) was used. The micro-PCC assay can be combined with fluorescence in situ hybridization (FISH), using simultaneously centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes. This allows dose assessments on the basis of accurate scoring of dicentric and centric ring chromosomes in G0-lymphocyte PCCs, which is particularly helpful when further evaluation into treatment-level categories of exposed individuals is needed. The micro-PCC assay has significant advantages for early triage biodosimetry when compared to other cytogenetic biodosimetry assays. It is rapid, cost-effective, and could pave the way to its subsequent automation.

Use of human lymphocyte G0 PCCs to detect intra- and inter-chromosomal aberrations for early radiation biodosimetry and retrospective assessment of radiation-induced effects

“…Interestingly, the frequency of rings remained essentially the same in ex vivo irradiated lymphocytes irrespective of the post-recovery times used for PCC preparation (8 hrs and 24 hrs). Recently, Pantelias and Terzoudi [46] developed an automatable micro-PCC assay for rapid individualized dose estimation during large-scale radiological emergencies. This technique will enhance the practical applicability of G0 PCC as an effective triage tool.…”

High Resolution and Automatable Cytogenetic Biodosimetry Using In Situ Telomere and Centromere Hybridization for the Accurate Detection of DNA Damage: An Overview

“…The automation of PCC–CHO following uniform staining [ 66 ] and subsequent TC staining [ 12 ] has now been initiated; however, the implementation of this technique and its current use requires further technical development.…”

“…In the fusion approach, Chinese hamster ovary (CHO) cells in the M-phase of their cell cycle are fused with the target interphase cells using a fusogen-like polyethylene glycol or inactivated Sendai virus. Prior to fusion, mitotic CHO cells have been synchronized and stocks are frozen and stored until used [ 28 , 66 ].…”

Interphase Cytogenetic Analysis of G0 Lymphocytes Exposed to α-Particles, C-Ions, and Protons Reveals their Enhanced Effectiveness for Localized Chromosome Shattering—A Critical Risk for Chromothripsis

“…For this purpose, we use a clearly detectable cytogenetic endpoint of exposure in order to obtain reliable RBE values of different radiation qualities, as well as to investigate the mechanisms underlying the induction of chromosome shattering and the formation of chromothripsis-like chromosomal alterations. Towards this goal, the fusion PCC assay is employed to visualize and analyze chromosome fragmentation directly in G0 human lymphocytes, without the requirement of exposed cells entering into mitosis [ 37 , 38 ]. Specifically, lymphocytes isolated from whole blood were exposed to various doses (up to 6 Gy) of α-particles (4.70 MeV, 92 keV/μm), accelerated C-ions (56.5 MeV, 295 keV/μm), and protons (2.2 MeV, 28.5 keV/μm).…”