FISH unveils a unified method for multi-marker biodose assessment

Abstract

Accurate dose assessment following radiation disasters or accidents is crucial for informed medical interventions. Cytogenetic biomarkers, such as dicentrics (dic), translocations, and chromosomal fragments, are essential for radiation biodosimetry in various exposure scenarios. However, quantifying these markers via separate staining and detection methods presents challenges in terms of efficiency and consistency. This study aimed to quantify multiple cytogenetic markers, including dic, balanced and unbalanced translocations and acentric fragments, from the same metaphases via fluorescence in situ hybridization (FISH). By enabling multimarker dose estimation from a single sample, this approach minimizes interexperimental variation and improves overall accuracy. Independent calibration curves were generated for each marker, enabling precise dose estimation with smaller class intervals, in accordance with the IAEA and ISO guidelines. The method was validated by estimating doses for five blinded samples via both standard cytogenetic methods and protein biomarkers (γH2AX and 53BP1). The multimarker approach yielded the closest estimates with 2-7% variation from true doses, providing the most accurate results among all cytogenetic techniques. This unified FISH-based approach enhances the precision of dose estimation for both recent and past radiation exposures, offering a more reliable tool for diverse biodosimetry applications.

Keywords: Cytogenetic markers; Dose-repose-calibration curve; Fluorescence in situ hybridization (FISH); Human radiation-exposure-dosimetry; Retrospective biodosimetry.

Fig. 1

Dose response curves generated for ⁶⁰Co-γ-ray-induced (A) BT, (B) UT, (C) dic-F and (D) acentric fragments, scored in the same metaphases processed with FISH, for chromosome pairs 1 and 2.

Fig. 2

Representative metaphase spreads hybridized for two-color FISH. (A) Stable aberrations include (A1) control metaphase (no aberration), (A2) one BT between the green and black chromosomes, and (A3) one BT between the red and black chromosomes. (B) Unstable aberrations consisting of (B1) one dic-F involving green and red chromosomes with a bicolour acentric fragment, (B2) one dic-F involving green and black chromosomes with a bicolour acentric fragment, and (B3) one UT between red and black chromosomes.

Fig. 3

Representative metaphase spreads hybridized for three-color FISH using whole chromosome paint probes tagged with green (FITC), red (SpO), or a combination of both (FITC and SpO) fluorophores for chromosome pairs 1, 2, and 4. (A) Control metaphase with no aberration. (B) One BT between the yellow and blue chromosomes. (C) One BT between red and blue chromosomes. (D) One BT, between the green and blue chromosomes. (E) One UT between the green and blue chromosomes. (F) One dicentric between the red and blue chromosomes, accompanied by a bicolour acentric fragment. Metaphases with UT (E) and dicentric (F) data were excluded from the scoring.

Fig. 4

Representative metaphase spreads hybridized with mFISH (24-color FISH), where all 24 chromosome types were labeled with distinct colors. Metaphases exhibit (A) control with no aberration, (B) one BT between chromosomes 1 and 2, (C) two BTs between chromosomes 3 and 8 and between chromosomes 5 and 16, and (D) one BT between chromosomes 1 and 3, accompanied by an acentric fragment from chromosome 7.

Fig. 5

Dose estimation for five blinded samples (BD1-BD5) via various cytogenetic markers. Doses were measured by quantifying yields from BT, UT, dic-F, Fr, and Avg-FISH in metaphases processed with two-color FISH. Additional estimates were obtained via independent methods: dic-G, MN analysis of binucleated Giemsa-stained cells, and immunofluorescence detection of γH2AX and 53BP1 foci in G0 lymphocytes.

Fig. 6

(A) Relative error (%) in dose estimates from BT, UT, dic-F, acentric fragment, and Avg-FISH using the unified two-color FISH method compared with independent methods: dic-G, MN (Giemsa-stained), γH2AX foci (immunofluorescence), and 53BP1 foci (immunofluorescence). (B) Relative error (%) for blinded samples (BD1 to BD4). Higher doses (BD1–1 Gy and BD3–2 Gy) resulted in more accurate estimates, whereas lower doses (BD2–0.25 Gy and BD4–0.5 Gy) resulted in greater deviations. The variation in the estimated doses was ranked as follows: BD3

Fig. 7

(A & B) Representative G0 lymphocytes from samples BD5, BD2, BD4, BD1, and BD3, illustrating the respective numbers of 53BP1 (red) and γH2AX (green) foci observed after 1 h of incubation postirradiation under optimal conditions. (C) Representative metaphases: (C1) control with no dic, (C2) one dic with an acentric fragment, and (C3) two dic with two acentric fragments. (D) Representative binucleated lymphocytes: (D1) control with no MNs, (D2) one MN, (D3) two MNs, (D4) mononucleated lymphocytes, (D5) trinucleated lymphocytes, and (D6) tetranucleated lymphocytes. Mononucleated, trinucleated, and tetranucleated lymphocytes were excluded from the dose estimations.

Fig. 8

Graphical summary of comparative evaluation of Giemsa and FISH-based methods for detection of various chromosomal aberrations in biodosimetric applications.