Rare Non-Cryptic NUP98 Rearrangements Associated With Myeloid Neoplasms and Their Poor Prognostic Impact
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Ann Lab Med 2019; 39(1): 91-95
Published online September 13, 2018 https://doi.org/10.3343/alm.2019.39.1.91
Copyright © Korean Society for Laboratory Medicine.
Mi-Ae Jang, M.D.1,*, Eun-Ae Han, M.D.1,*, Jin Kyung Lee, M.D.2, Kwang Hwan Cho, Ph.D.3, Hee Bong Shin , M.D. Ph.D.1*, and You Kyoung Lee, M.D. Ph.D.1*
1Department of Laboratory Medicine and Genetics, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Korea.
2Department of Laboratory Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Seoul, Korea.
3Department of Radiation Oncology, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, Bucheon, Korea.
Correspondence to: Corresponding author: You Kyoung Lee, M.D. Department of Laboratory Medicine and Genetics, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, 170 Jomaru-ro, Wonmi-gu, Bucheon 14584, Korea. Tel: +82-32-621-5941, Fax: +82-32-621-5944, cecilia@schmc.ac.kr
Co-corresponding author: Hee Bong Shin, M.D. Department of Laboratory Medicine and Genetics, Soonchunhyang University Bucheon Hospital, Soonchunhyang University College of Medicine, 170 Jomaru-ro, Wonmi-gu, Bucheon 14584, Korea. Tel: +82-32-621-5942, Fax: +82-32-621-5944, shinhb@schmc.ac.kr
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Cytogenetic dosimetry is useful for evaluating the absorbed dose of ionizing radiation based on analysis of radiation-induced chromosomal aberrations. We created two types of
Keywords: Chromosome aberration, Cytogenetic dosimetry, X-ray, Dicentric chromosome, Translocation, Correlation
Cytogenetic dosimetry is an important technology for estimating the ionizing radiation dose absorbed by an individual and is based on chromosomal damage following chronic or acute exposure [1,2,3]. In Korea, cytogenetic dosimetry has been used by a few national centers, such as the Korea Institute of Radiological & Medical Sciences (KIRAMS) and the Radiation Health Institute (RHI), to provide a dose assessment for individuals employed in the nuclear power industry and to prepare for potential exposure via radiation emergency [4,5]. However, very few clinical laboratories have been equipped to provide biodosimetry services.
Of the several available cytogenetic dosimetry techniques, a dicentric chromosome (DC) assay and FISH translocation (TR) assay have recently been approved as new health technologies by the committee for New Health Technology Assessment (nHTA) pursuant to the Medical Services Act of August 2016 [6]. The main objective of cytogenetic dosimetry is to assess the irradiated dose reflecting the damage caused by ionizing radiation and to recommend appropriate treatment for exposed patients [7,8]. However, there are some challenges in disseminating these new practices in most clinical laboratories in Korea: (1) lack of adequate radiation generators, (2) unavailability of reference standards reflecting
We constructed
After obtaining informed consent, heparinized peripheral blood samples were collected from two males (36 and 22 years) and two females (28 and 24 years) in a tertiary-care hospital in Bucheon, Korea. Since chromosomal aberrations are known to be affected by factors such as age, sex, and smoking status [2], we chose study participants considering these factors. None had a history of smoking, and they had never been subjected to radiotherapy or chemotherapy. The samples were aliquoted into 11 separate tubes (one control and 10 for acute single exposure to doses of 0.05, 0.1, 0.25, 0.5, 0.75, 1, 2, 3, 4, and 5 Gy). X-rays were generated using a 6MV LINAC (Siemens, Concord, CA, USA) at a dose rate of 0.5 Gy/min. The samples were placed at the center of a 20×20 cm radiation field at a source-sample distance of 100 cm. Following irradiation, lymphocyte culture, harvesting, staining, and scoring processes were performed according to the guidelines of the International Atomic Energy Agency (IAEA) [2]. Briefly, 1 mL blood from each dose-point was cultured for 48 hrs in a culture tube containing 9 mL of medium (RPMI-1640). Next, 80 µL of colcemid (10 µg/mL) were added to 10 mL of each culture (final concentration, 0.08 µg/mL) during the final 24 hours of the culture period. Following hypotonic treatment with 0.075 M KCl, the cells were fixed in a 3:1 methanol-acetic acid solution. The cells were prepared on a slide, stained with Giemsa, and the number of DCs was scored per 1,000 metaphases. Once more than 100 DCs were identified in samples above the 2 Gy dose-point, analysis was stopped, and the proportion per scored cells was calculated.
For the TR assay, samples were processed as described above, and metaphases were prepared on a slide using the same protocol described for the DC assay. Two slides were prepared for each sample, and the TR assay was carried out with a mixture of commercially available fluorophore-labeled probes (chromosome 1, red; chromosome 2, green; chromosome 4, yellow; Metasystems, Altlussheim, Germany). The number of TRs was scored per 1,000 metaphases. The detailed scoring rules for exchange-type aberration were as follows: (1) only stable cells were counted, (2) TR between three chromosomes was scored as two TR equivalents, (3) inversion or insertion was scored as one TR equivalent, and (4) deletions were not included in the analyses.
Metaphases were captured using the Metafer Image Analysis System (Metasystems). Two investigators independently interpreted the DC and TR results. Discordant interpretations were reanalyzed in several ways, including comparison of images and a final review by a third experienced investigator in the cytogenetic laboratory.
Statistical analysis of the data used to generate the dose-response curves was conducted using R version 2.10.1 (R Foundation for Statistical Computing, Vienna, Austria) and the analysis scripts as mentioned in the IAEA guidelines [2]. To determine whether the DC or TR frequency followed a Poisson distribution as expected for X-ray irradiation, the dispersion index (α2/y) and the normalized unit of this index (u) were obtained for each dose. Pearson's correlation was calculated between the delivered dose and chromosomal aberration frequency.
Following
Unlike physical measurements routinely conducted for individuals with occupational exposure, most people who are exposed during radiation emergencies do not carry personal dosimeters [7]. Therefore, biological dosimetry using chromosome damage is important not only to determine whether individuals have been over-exposed to radiation but also to consider individual differences in susceptibility [2].
We present two types of dose-response curves compatible with two methods: a DC assay for assessing the acute phase within two months of exposure [7] and chromosome painting TR analysis using three probes (one each for chromosome pairs 1, 2, and 4), which can estimate a radiation exposure dose received several years earlier [12]. A previous Japanese study demonstrated considerable variation between individuals in the frequency of chromosomal aberrations formed, especially at low doses of ≤1 Gy [13]. We included a total of seven points below 1 Gy in our study (0, 0.05, 0.1, 0.25, 0.5, 0.75, and 1); however, no significant inter-individual differences were observed in DC and TR frequency at these doses (See
Laboratories should have their own dose-response curves to avoid inter-laboratory variations [2,8] as several factors are known to impact the resulting dose-response curves, including the intrinsic environmental conditions in each laboratory, choice of reagents, technical procedures and equipment, scoring criteria, and the subjective nature of microscopic identification of chromosome aberrations [9,14].
In order to approximate
No potential conflicts of interest relevant to this article were reported.
This work was supported by the Soonchunhyang University Research Fund.
Dose-response calibration curves and metaphase images of chromosomal aberrations induced in human lymphocytes by X-ray exposure. (A and B) Dose response curves with the 95% confidence interval delimited by dotted lines. The black dots (●) denotes data points with standard errors of the mean. (A) Dicentric yields, Y=(0.0011±0.0004)+(0.0119±0.0032)D+(0.0617±0.0019)D2, where Y is the dicentric yield, and D is the absorbed dose in Gy. (B) Translocation of chromosomes 1, 2, and 4, Y=(0.0015±0.0004)+(0.0048±0.0024)D+(0.0237±0.0014)D2, where Y is the translocation yield and D is the absorbed dose in Gy. (C) Dicentric chromosomes and their respective fragments are marked with an arrows and arrowheads, respectively. (D) Metaphases with painted chromosomes—1 (red), 2 (green), and 4 (yellow)—and an apparent two-way translocation involving chromosomes 2 and 4 (arrows).
Distribution and frequencies of DCs in human peripheral blood lymphocytes following X-ray irradiation
Dose (Gy) | Cells scored | DC cell distribution | Total DC* | DC/cell | α2/y† | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
0 | 1 | 2 | 3 | 4 | 5 | ||||||
0 | 4,000 | 3,994 | 6 | 0 | 0 | 0 | 0 | 6 | 0.0015 | 1.00 | −0.06 |
0.05 | 4,000 | 3,995 | 5 | 0 | 0 | 0 | 0 | 5 | 0.0013 | 1.00 | −0.05 |
0.1 | 4,000 | 3,990 | 10 | 0 | 0 | 0 | 0 | 10 | 0.0025 | 1.00 | −0.11 |
0.25 | 4,000 | 3,966 | 34 | 0 | 0 | 0 | 0 | 34 | 0.0085 | 0.99 | −0.37 |
0.5 | 4,000 | 3,921 | 77 | 2 | 0 | 0 | 0 | 81 | 0.0203 | 1.03 | 1.32 |
0.75 | 4,000 | 3,845 | 153 | 2 | 0 | 0 | 0 | 157 | 0.0393 | 0.99 | −0.61 |
1.0 | 4,000 | 3,669 | 323 | 8 | 0 | 0 | 0 | 339 | 0.0848 | 0.96 | −1.67 |
2.0 | 1,800 | 1,350 | 397 | 50 | 3 | 0 | 0 | 506 | 0.2811 | 0.95 | −1.42 |
3.0 | 1,000 | 536 | 344 | 104 | 15 | 1 | 0 | 601 | 0.6010 | 0.92 | −1.89 |
4.0 | 600 | 215 | 228 | 104 | 49 | 3 | 1 | 600 | 1.0000 | 0.93 | −1.19 |
5.0 | 400 | 73 | 140 | 113 | 47 | 19 | 8 | 623 | 1.5575 | 0.88 | −1.66 |
Distribution and frequencies of chromosome TRs in human peripheral blood lymphocytes following X-ray irradiation
Dose (Gy) | Cells scored | TR cell distribution | Total TRs* | TR/cell | α2/y† | ||||||
---|---|---|---|---|---|---|---|---|---|---|---|
0 | 1 | 2 | 3 | 4 | 5 | ||||||
0 | 4,000 | 3,995 | 5 | 0 | 0 | 0 | 0 | 5 | 0.0013 | 1.00 | −0.05 |
0.05 | 4,000 | 3,991 | 9 | 0 | 0 | 0 | 0 | 9 | 0.0023 | 1.02 | 0.45 |
0.1 | 4,000 | 3,990 | 10 | 0 | 0 | 0 | 0 | 10 | 0.0025 | 1.00 | −0.11 |
0.25 | 4,000 | 3,985 | 15 | 0 | 0 | 0 | 0 | 15 | 0.0038 | 1.00 | −0.16 |
0.5 | 4,000 | 3,964 | 36 | 0 | 0 | 0 | 0 | 36 | 0.0090 | 0.99 | −0.40 |
0.75 | 4,000 | 3,934 | 66 | 0 | 0 | 0 | 0 | 66 | 0.0165 | 0.98 | −0.73 |
1.0 | 4,000 | 3,865 | 133 | 2 | 0 | 0 | 0 | 137 | 0.0343 | 1.00 | −0.22 |
2.0 | 1,975 | 1,773 | 193 | 9 | 0 | 0 | 0 | 211 | 0.1068 | 0.98 | −0.66 |
3.0 | 1,135 | 906 | 208 | 20 | 1 | 0 | 0 | 251 | 0.2211 | 0.96 | −0.88 |
4.0 | 416 | 270 | 126 | 19 | 1 | 0 | 0 | 167 | 0.4014 | 0.86 | −1.96 |
5.0 | 199 | 99 | 78 | 21 | 1 | 0 | 0 | 123 | 0.6181 | 0.78 | −2.24 |