Year : 2010 | Volume
: 2 | Issue : 3 | Page : 197--201
Lymphocyte chromosomal aberration assay in radiation biodosimetry
Paban K Agrawala, JS Adhikari, NK Chaudhury
Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Delhi - 110 054, India
N K Chaudhury
Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Delhi - 110 054
Exposure to ionizing radiations, whether medical, occupational or accidental, leads to deleterious biological consequences like mortality or carcinogenesis. It is considered that no dose of ionizing radiation exposure is safe. However, once the accurate absorbed dose is estimated, one can be given appropriate medical care and the severe consequences can be minimized. Though several accurate physical dose estimation modalities exist, it is essential to estimate the absorbed dose in biological system taking into account the individual variation in radiation response, so as to plan suitable medical care. Over the last several decades, lots of efforts have been taken to design a rapid and easy biological dosimeter requiring minimum invasive procedures. The metaphase chromosomal aberration assay in human lymphocytes, though is labor intensive and requires skilled individuals, still remains the gold standard for radiation biodosimetry. The current review aims at discussing the human lymphocyte metaphase chromosomal aberration assay and recent developments involving the application of molecular cytogenetic approaches and other technological advancements to make the assay more authentic and simple to use even in the events of mass radiation casualties.
|How to cite this article:|
Agrawala PK, Adhikari J S, Chaudhury N K. Lymphocyte chromosomal aberration assay in radiation biodosimetry.J Pharm Bioall Sci 2010;2:197-201
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Agrawala PK, Adhikari J S, Chaudhury N K. Lymphocyte chromosomal aberration assay in radiation biodosimetry. J Pharm Bioall Sci [serial online] 2010 [cited 2022 Aug 13 ];2:197-201
Available from: https://www.jpbsonline.org/text.asp?2010/2/3/197/68501
The discovery of X-ray by Wilhelm Conrad Roentgen in 1895 led to many similar discoveries related to ionizing radiation. The impact of discovery of Roentgen was phenomenal. In 1896, Ernest Rutherford, a physicist from New Zealand, was working under J.J. Thompson at the Cavendish Laboratory in 1898-1899 and he discovered three other types of radiation, alpha, beta and gamma. Becquerel, Curie and many other pioneered the discovery of radiation-related phenomena. Roentgen was thus honored with the first Nobel Prize for physics in 1901.
The application of X-ray in medical diagnostic was immediately started by physicians for bone imaging. The harmful effects of X-ray, to the surprise of users, were noticed within 4 months of the discovery. The effect of X-ray on skin was observed and the first case of radiation-induced skin cancer was diagnosed in 1902. Around the same time, in 1898, Marie Curie discovered the naturally occurring radioactive material "radium" and Pierre Curie demonstrated radiation-induced burn by voluntarily exposing his arm to radium for long hours in 1900. The use of X-rays in medicine (to develop into the field of radiation therapy) was pioneered by Ajor John Hall-Edwards in Birmingham, England. In 1908, he had to have his left arm amputated owing to the spread of X-ray dermatitis. In summer 1895, while working with cathode discharge tubes, E. Grubbe developed a curious dermatitis of the hands which neither he nor his physician understood and he died of cancer in 1960. Becquerel, in 1901, found a reddening of the skin under his waistcoat, where he had pocketed a radiation source. Danlos of the Hospital Saint-Louis investigated whether the effects of radium on the skin could be utilized for the treatment of certain skin diseases. Thus, many of the pioneers in radiation research became martyrs to their work. A memorial to these martyrs was erected in Hamburg in 1936, listing 178 names.
Majority of the health hazards of radiation exposure in the initial period were due to the ignorance and underestimation of the potential of ionizing radiation to cause damage to living systems. Further studies in animal models and, more prominently the aftermath of Nagasaki bombing on August 6, 1945, and after 3 days the Hiroshima bombing witnessed by the world, certainly forced the scientific communities to consider ionizing radiation as a potential hazard to mankind and led to the formation of radiation protection guidelines. During the early 1900s, Marie Curie emphasized the need of dose estimation and over the years several physical and chemical dosimetry methods have been developed. With expansion of use of ionizing radiation in hospitals for diagnostics and therapy and in industries for sterilization of various types of consumable products and more so in energy sector as nuclear power plants, the concerns of unwanted exposure to human is increasing. In addition, the military use of nuclear materials is concern for everyone. The unwanted accident at the Chernobyl Nuclear power plant in 1986 was the largest in all dimensions after World War II. The accident at the Chernobyl nuclear reactor on April 26, 1986, remains the most hazardous radiation accident to date. An enormous amount of literature has emerged analyzing the causes of the accident, the sequence of events on that fateful night, the amount of radioactive material released into the environment, the health and environmental consequences of the accident, and the implications of the accident for nuclear reactor safety and the future of atomic energy. This accident also clearly demonstrated how an accident can affect the neighboring countries, and thus becomes a general concern for the entire world community. A close survey of all radiation-related accidents since the discovery of X-ray reveals an ever increasing frequency of radiation incidents. A number of new categories of such incidents have been observed. These are related to use of possible improvised nuclear devices, radiological devices in public places by terrorists or they can otherwise cause nuclear emergency. The worldwide use of ionizing radiation for beneficial purposes has also led to hundreds of instances in which one or more persons were accidentally overexposed. More than 380 instances are reported in literature.
There are two kinds of effects due to radiation exposure: deterministic and stochastic. Deterministic effects occur only at high radiation doses. There is general scientific consensus that no matter how small, radiation exposure always increases the risk of cancer.  Clinical signs and symptoms due to radiation exposure occur after a few hours to days depending upon the radiation dose absorbed. Medical management requires quick and accurate dose assessment from all suspected individuals and this is a problem for radiation biodosimetry lab. The accepted generic multiparameter and early-response approach includes measuring radioactivity and monitoring the exposed individual, observing and recording blood counts with white blood cell differentials, sampling blood for the chromosome aberration cytogenetic bioassay using the "gold standard".
As far as the procedures for usage of such ionizing radiation sources, X-ray (for medical diagnostics), gamma (treatment and therapy of cancers) rays are concerned, medical technologists are today much safer as compared to say, 50 years ago. With strict guidelines available and adherence, the radiation workers are much safe. At the same time, various nuclear technologies have provided sensitive radiation detector and monitoring devices for safety and personal dosimetry. However, such physical dosimeters cannot be provided to accidental scenario for the entire population. It only provides data on radiation exposure of an individual for a radiation worker involved in routine duties. Moreover, these physical dosimeters cannot account for the individual variations in radiation response. Besides, one needs to have the physical dosimeter on his/her body in an appropriate position at the time of exposure to radiation. Again, the physical dosimeters can be misused under circumstances where a radiation worker can place the dosimeter in the radiation environment without being present physically so as to claim compensation or escape from work. All these factors have necessitated the search of a biological radiation dosimeter that can estimate the actual absorbed radiation dose. Like the physical dosimeters, the biological dosimeters need to qualify certain criteria before they can be considered for estimation of the absorbed dose. First of all, the technique should be minimally invasive, if at all the end point under investigation requires it to be so. The end point to be considered should be purely radiogenic so as to avoid any false positive/erroneous data arising due to some factors other than radiation. The end point should show linearity/positive correlation with the absorbed dose at least over a broad dose range. The end point analysis should be easy enough requiring minimal training of the personnel conducting the analysis; the assay protocol should not require much sophistication and should be rapid. If the end point can be examined at a much later period following accidental radiation exposure, i.e., if the kind of radiation signature in the biological system being considered for use as a dosimeter persists over a protracted period, it can be useful for retrospective dose estimation as well. Researchers worldwide have put forward several assay protocols till date, which qualify few or several of these criteria for consideration as a biological radiation dosimeter. A radiological catastrophe could result in proportions of victims ranging from none to as many as 40% of the exposed population.  Medical management will require a capability to stratify victims rapidly into different dose categories in the dose range 0-5 Gy, to discriminate for acute radiation syndrome treatment vs. long-term surveillance within a clinically relevant time span.  Therefore, in the event of a mass casualty incident, the need to process large samples for diagnostic dose assessment is of paramount importance.  During the time course, a series of observations have been made and a number assay protocols have been attempted to provide support for medical management of radiation exposed subjects. A detailed discussion of all those assay protocols is beyond the scope of the current review. Here, we will be discussing the history of development of the dicentric assay in human lymphocyte metaphase preparations, which forms the gold standard of radiation biodosimetry and the recent advancements accomplished so as to make the assay more rapid, simple and reliable, over the last several decades.
From classical cytogenetics to molecular cytogenetics
Ionizing radiation deposits energy in biomolecules (DNA, proteins and lipids). The exact nature and quantity depend very much on the radiation quality and dose. Distribution of ionization and damages depend on the linear energy transfer of radiation. Amongst a variety of radiation-induced damages to DNA, the double strand breaks cause chromosomal aberrations visible at metaphase.  Various types of chromosomal aberrations occur and dicentric is the main aberration used in biodosimetry and will be discussed in the following sections.
Sutton coined the term "cytogenetics" to the study of chromosomes, after Boveri (1902)  and Sutton (1902)  independently postulated the "chromosome theory of inheritance". The term "karyotype" for the chromosome complement of eukaryotic cells was coined by Levitsky (1924).  However, the exact diploid chromosome number of man was confirmed to be 46, only in 1956 by Tjio and Levan, applying the "pre-fixation hypotonic shock" technique developed by Hsu and Pomerat in 1955.  The finding that colchicine can arrest cells at metaphase  significantly added to the study of mitotic chromosomes. Nowell (1962)  demonstrated that phytohemagglutinin can stimulate peripheral blood lymphocytes to divide in culture and this study expedited the study of human chromosomes. Until 1971, it was difficult to study the chromosomal rearrangements like deletions, duplications, insertions, etc. The observation made by Caspersson et al. in 1968  using quinacrine, and by Drets and Shaw (1971)  using Giemsa, that human chromosomes produce definitive banding pattern, made the study of chromosomal rearrangements feasible. Pardue and Gall (1970)  were able to localize mouse satellite DNA onto metaphase preparations by hybridizing radiolabeled DNA and successively several workers used cloned DNA for hybridization. The use of hazardous radiolabeling technique was sooner replaced by the use of biotin conjugation of nucleotides.  Further developments in methods for enzymatic incorporation of biotin in nucleic acid,  its immunofluorescence detection and use of Fluorescein Isothiocyanate (FITC) labeled avidin  led to the emergence of fluorescence in situ hybridization (FISH) technique.
Chromosomes can be best visualized under microscope at metaphase stage of cell division and metaphase preparations in larger numbers, though were available, it often remained an arduous task to obtain a good karyogram. The overall staining techniques applied in those days, at best allowed classification of human metaphase chromosomes into seven groups, based on sizes and centromere positions. Caspersson, hoping for a nonrandom distribution of base pairs along the length of the chromosomes, suggested inducing specific differential staining patterns of parts of metaphase chromosomes. Ed Modest, an organic chemist at the Children's Cancer Foundation, synthesized quinacrine mustard (QM) which was tried on chromosome preparations in Stockholm. ,, Metaphase chromosomes of Vicia, Scilla, and man showed differential fluorescence along each chromosome. Soon thereafter, several other banding techniques were developed, of which the Giemsa-induced G banding and the R (or reverse) banding are most widely used. ,,,,,,
Radiation induced chromosomal aberration in human lymphocytes
The pioneering work of Muller in Drosophila led to the first report on radiation (X-ray) induced chromosomal aberrations in the year 1926.  It was only during 1970s that radiation researchers revealed many different types of lesions induced in DNA by ionizing radiation. Though many damages can be repaired and lead to normal functioning, strand breaks, viz., double strand breaks are not repairable and lead to chromosomal aberrations which can be observed at metaphases during cell cycle progression in radiation exposed cells. Radiation can cause different types of chromosomal aberrations which are further classified as stable and unstable. Dicentric chromosomal aberrations form when exchange takes place between the centromeric pieces of two broken chromosomes. This is accompanied by acentric pieces. At high dose, multicentric chromosomes can be observed. In addition, centric rings are also formed, but are much rarer. In 1962, it was suggested by Bender and Gooch  that dicentric chromosomes in peripheral lymphocytes could well be used for the detection and dose assessment of human radiation exposures and these authors have used this method for the first time in the sense of biological dosimetry on the occasion of the so-called Recuplex criticality accident at Hanford, USA. A further significant development of the method was the introduction of the fluorescence plus Giemsa (FPG) staining.
The dicentric aberrations arise when exchange takes place between the centromeric pieces of two broken chromosomes. That is during repair of DNA strand breaks, misrepair of two chromosomes and abnormal chromosome replication can lead to dicentric chromosomes (a chromosome with two centromeres). Although radiation induces many types of chromosomal changes in addition to dicentric chromosomes, dicentrics are considered the most sensitive and most specific for assessing radiation dose. The lymphocyte-dicentric assay of Bender and Gooch remains the "gold standard" for early response and definitive dose assessment.
In human lymphocyte population, the cells are in the G0 phase of cell cycle at the time of blood sampling after exposure. The chromosomal aberration induced by ionizing radiation will subsequently need to be analyzed after processing. For conversion of observed frequency of aberration into radiation dose, a calibration curve is to be established by in vitro exposures of whole blood. There are a number of procedures that have been used to process the blood specimen from lymphocyte culture to metaphase spread and finally data analysis. This technique is capable of estimating whole body doses of low LET radiation down to about 10 cGy based on analysis of 1000 metaphases. There are a number of issues in applying dose response curve for estimation of absorbed dose, which are as follows: radiation quality, whole body vs. partial body exposure, individual variation, chronic exposure and time (after few weeks to few months).
Dicentric chromosomal assay, the "gold standard" in radiation accidents and current status
There are several requirements for radiation biodosimetry: being specific to ionizing radiation, having a low background level, direct relationship with dose and radiation quality, being minimally non invasive, having a good reproducibility, comparability of in vitro and in vivo results, possibility of inter and intralab validation. Dicentric assay conforms to all of these and thus is considered "gold standard" in radiation biodosimetry.  The official record of practical use of chromosomal aberrations dates back to 1982 in Germany at cytogenetic laboratory of the Federal Office for Radiation Protection. The utility of cytogenetic assays for assessing dose in radiation mass casualties and guiding treatment decisions has been demonstrated in Chernobyl,  Goiania, Brazil  and Tokaimura, Japan. , After the fateful September 11, 2001 event in USA, various organizations within their country established sophisticated radiation Biodosimetry Laboratory under the Program Home Land Security. Similar efforts are now taken by other developed countries, viz., UK, Germany and France (automated, high-throughput, cytogenetic biodosimetry laboratory to process a large number of samples for conducting the dicentric assay using peripheral blood from exposed individuals, according to internationally accepted laboratory protocols, i.e., within days following radiation exposures). The components of an automated cytogenetic biodosimetry laboratory include blood collection kits for sample shipment, a cell viability analyzer, a robotic liquid handler, an automated metaphase harvester, a metaphase spreader, high-throughput slide stainer and coverslipper, a high-throughput metaphase finder, multiple satellite chromosome-aberration analysis systems, and a computerized sample tracking system. Laboratory automation using commercially available, off-the-shelf technologies, customized technology integration, and implementation of a laboratory information management system (LIMS) for cytogenetic analysis will significantly increase throughput. ,
Necessary Requirements for a Radiation Biodosimetry Laboratory and Future Directions
Rigorous quality control and assurance should be integral in a dose assessment plan for stratifying a population into treatment categories. , International Organization for Standardization (ISO)'s new standard will define the process and identify rigorous quality standards for using cytogenetic methods to rapidly assess radiation dose. Large volumes of samples plus the required record keeping using laboratory notebooks invariably pose quality control and assurance challenges. In contrast, electronic sample tracking and data management using barcodes provide an unbroken chain-of-custody of information for every sample, thereby increasing efficiency, confidence, speed and throughput, while minimizing data-transcription errors. International organizations, International Atomic Energy Agency (IAEA) and World Health Organization (WHO)  have contributed enormously and are still continuously updating several aspects of radiation biodosimetry including dicentric chromosomal aberration. The aim of these organizations is to formulate and facilitate the methodologies for radiation dosimetry and to establish a global network. In support to these efforts, ISO established a radiation protection program. 
Dicentric chromosomal assay has matured through a number of discoveries and developments in instrumentation (microscope, liquid handling systems) and software. This has greatly reduced time and labor and increased consistency but this assay cannot handle mass scale radiation casualties. New strategies of dicentric chromosomal assays and scoring through networking need to be explored without affecting the standard. In addition, the inherent limitation is that dicentic aberration and other related aberrations are unstable in nature and likely to be lost at cell division. Thus, for retrospective estimation after longer delays in blood sampling, the FISH technique will be more useful for stable chromosomal aberration (translocation). Other cytogenetic assays, micronuclei, premature chromosomal condensation need to be examined in the same laboratory. Assay of molecular biomarkers, viz., radiation responsive proteins, is another novel approach that needs to be undertaken along with cytogenetic assays. Already promising results are available in literature.  Because of practical usefulness for radiation biodosimetry particularly after immediate exposure, technological advancements, the prospects of dicentric chromosomal assay using human lymphocyte is very purposeful for radiation countermeasures. In our institute, we have undertaken initiatives toward establishing radiation biodosimetry facility and the first step in this direction, the conventional dicentric chromosomal assay, is in progress.
The authors are grateful to DRDO, Govt of India for financial support for the on going Radiation Biodosimetry project.
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