Modifications of chromosome number or structure are known to occur in all organisms. In humans, such changes may occur spontaneously or be associated with exposure to chemical, biological, and physical agents. Ionizing radiation is a particularly powerful agent because of its unique ability to cause direct or indirect damage to chromosomal DNA. The damage to DNA increases with the absorbed dose. Biological dosimetry is an essential tool for radiation dose assessment when exposures are unexpected and physicians are unable to rely on instruments of physical dosimetry (eg, film badges, other kinds of cumulative radiation dosimeters). Lymphocytes can serve as biosensors of radiation exposure because of their sensitivity, accessibility, and whole-body distribution, as well as their ability to record chromosomal DNA damage in a reproducible, dose-dependent manner.
Because of constant exposure to ambient terrestrial and cosmic radiations, most humans have a very low spontaneous frequency (approximately 0.5 to 1 per 10
3 cells) of dicentric chromosomes in their peripheral blood lymphocytes whether or not they have been directly exposed to medical or industrial sources.
15,16 The numbers of these dicentric chromosomes are independent of both age and sex and are relatively easy to identify. The DCA was first introduced in the 1960s and has now been validated by its use in a number of radiation accidents that occurred in Goiânia, Brazil
17; Chernobyl, Ukrainev
18; and Tokai-mura, Japan.
19 The DCA remains the standard assay for estimating radiation dose.
20 After whole-body exposure to low-LET radiation, doses down to about 100 to 200 mGy (10-20 rem) are detectable with the DCA.
A DCA involves the following steps: (1) lymphocyte culture, (2) cell fixation, (3) slide preparation and staining, and (4) scanning individual cells at the metaphase stage for the presence of chromosomes with 2 or more centromeres. To perform the DCA, peripheral blood is drawn and put into tissue culture for 48 hours. During this time, lymphocytes are artificially stimulated to divide using the chemical phytohemagglutinin and then the division process is stopped in metaphase with the chemical colcemid. Cells with chromosomes that have undergone double-strand breaks and asymmetrical DNA exchanges are unable to divide at metaphase. The dicentric chromosomes are counted and then compared with dose-response curves that have been predetermined on the basis of measures of human blood that has been irradiated to known doses with a variety of radiation sources. At the Radiation Emergency Assistance Center/Training Site (REAC/TS), we use human blood that has been irradiated with cobalt-60 (Co-60 or 60Co) γ-rays, 250 keV x-rays, and californium-252 (Cf-252 or 252Cf) neutrons at various doses to develop the dose-response curves against which a patient's numbers of dicentric chromosomes are compared.
Figure 7 reproduces a photomicrograph of a metaphase spread used for the DCA.
Although the DCA is the standard of radiation biodosimetry,
21 it is labor intensive, time consuming, and costly. Other cytogenetic biodosimetry techniques are available, including premature chromosome condensation, fluorescence in situ hybridization, and the micronucleus assay. Each technique has its own advantages and disadvantages and not all are available to all cytogenetics laboratories. Unfortunately, there are only 2 federally funded cytogenetic biodosimetry laboratories in the United States: REAC/TS in Oak Ridge, Tennessee, and the Armed Forces Radiobiology Research Institute in Bethesda, Maryland. There are more than 100 laboratories in the United States that are accustomed to performing cytogenetics techniques; however, they are in the business of analyzing chromosomes for the purposes of family genetic counseling and are not in the business of performing radiation biodosimetry. Efforts are underway at REAC/TS to collaborate with these genetics laboratories using online distance scoring of chromosomes to increase cytogenetic throughput nationally.
22 These efforts are in preparation for radiologic/nuclear incidents such as those that were discussed in part 2 of this series.
23
The number of abnormal (eg, dicentric) chromosomes increases in a dose-responsive manner up to about 5 Gy, at which point a person is considered to have acute whole-body irradiation. Above that dose, circulating lymphocytes have usually been so depleted that by the time the DCA is performed, a person does not have enough lymphocytes that will grow in culture. Therefore, at doses ⩾5 Gy, other cytogenetic techniques must be used.
Table 2 shows the effects of γ-ray dose on dicentric chromosome frequency.