Assessing the Applicability of FISH-based Prematurely Condensed Dicentric Chromosome Assay in Triage Biodosimetry

Abstract

The dicentric chromosome assay (DCA) has been regarded as the gold standard of radiation biodosimetry. The assay, however, requires a 2-d peripheral blood lymphocyte culture before starting metaphase chromosome analyses to estimate biological doses. Other biological assays also have drawbacks with respect to the time needed to obtain dose estimates for rapid decision on the correct line of medical treatment. Therefore, alternative technologies that suit requirements for triage biodosimetry are needed. Radiation-induced DNA double strand breaks in G0 lymphocytes can be detected as interphase chromosome aberrations by the cell fusion-mediated premature chromosome condensation (PCC) method. The method, in combination with fluorescence in situ hybridization (FISH) techniques, has been proposed in early studies as a powerful tool for obtaining biological dose estimates without 2-d lymphocyte culture procedures. The present work assesses the applicability of FISH-based PCC techniques using pan-centromeric and telomeric peptide nucleic acid (PNA) probes in triage mode biodosimetry and demonstrates that an improved rapid procedure of the prematurely condensed dicentric chromosome (PCDC) assay has the potential for evaluating exposed radiation doses in as short as 6 h after the collection of peripheral blood specimens.

Construction of Calibration Curve for Premature Chromosome Condensation Assay for Dose Assessment

“…Earlier reports demonstrated that variability could influence the results of PCC assays and this should be taken into account while planning the experiments. It was shown that the initial yield of PCC fragments soon after irradiation was high, but drop quickly even at 3 h postirradiation and reached plateau at 6–8 h and stayed stable up to 48 h.[ 7 8 ] The rejoining kinetics of excess PCC fragments were highlighted in earlier publications[ 4 9 10 ] and suggested that PCC assay is sensitive to the delay time between exposure and analysis. Therefore, experimental investigation should be performed in more realistic condition to the accident situation taking into account the kinetics of PCC fragments.…”

“…In the recent years, PCC method has attracted much attention in laboratories because of its potential use in biological dosimetry. Several modifications of this method such as analysis of PCC-ring fragments,[ 8 9 ] analysis PCC-dicentric chromosomes using peptide nucleic acid centromere and telomere probes[ 10 11 ] were recently introduced into practice, and such improvements have increased the capability of this technique in estimating the dose more precisely. Therefore, the introduction of PCC fusion assay as a platform for other PCC-based techniques is the first step toward further strengthening of the biodosimetry laboratories worldwide.…”

Triage biodosimetry using centromeric/telomeric PNA probes and Giemsa staining to score dicentrics or excess fragments in non-stimulated lymphocyte prematurely condensed chromosomes

“…Recently, the development of centromeric/telomeric (C/T) peptide nucleic acid (PNA) probes and their simultaneous use in lymphocyte PCCs in combination with FISH, has offered a new dynamic to the PCC-method, enabling the analysis of dicentrics and centric ring chromosomes directly in non-stimulated lymphocytes, with a level of accuracy and ease not possible previously [20][21][22]. In the present work, we make use of the PCC-method in combination with PNA probes and the FISH technique in order to construct a dose response curve for up to 10 Gy.…”

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

“…Initially, Pantelias et al [34] used the centromeric heterochromatin banding technique (C-banding) for analyzing the frequency of IR induced dicentric chromosomes. Subsequently, the fluorescence in situ hybridization (FISH) technique was employed using DNA probes specific for human centromeres and telomeres for DC detection in PCCs obtained from non-stimulated lymphocytes [35–37]. Karachristou et al [35] demonstrated the utility of PCC-FISH technique for triage biodosimetry by constructing a dose response curve up to 10Gy of γ-rays.…”