Estimating Radiation Exposure using Chromosomal Aberrations in Human Lymphocytes Team Members: Julie Asiello, Laura Banks, Leah Carmichael, Christopher Hudson, Amy Raught Our mentors: Adayabalam S. Balajee, PhD and Maria Escalona, MS, MLS, (ASCP)cm

Ionizing Radiation Sources of radiation DNA is susceptible to double strand breaks (DSB) if hit by radiation Ionizing radiation can strip electrons from atoms as they pass through them forming ions. Some sources of ionizing radiation can be x-rays, household items like granite countertops, and solar radiation. When DNA is ionized, the sugar-phosphate backbone can “break” and cause a double stranded break leaving gap in the chromosome.

Importance of Biodosimetry Increased exposure to ionizing radiation Threat of nuclear terrorism More accurate measure of absorption than physical dosimetry In todays world there are more and more people being exposed to Ionizing Radiation. Whether it is nuclear workers or the threat of Nuclear Terrorism, people have the possibility to be exposed. This research increases the ability to treat victims. People that have received a moderate dosage (up to 7 Gy)

Why use T Lymphocytes? White Blood Cells Easy to acquire Radiosensitive T cells are lymphocytes that aid in the immune system’s protection against pathogens. Regardless of where aberrations are induced, due to the recirculation of lymphocytes, they will be present in peripheral blood within 24 hours. So, a sample of cells exhibiting these alterations can be obtained by drawing blood from a peripheral source. Samples must be obtained as close to 24 hours after exposure as possible due to the destruction and resulting drop in lymphocytes when overexposed to ionizing radiation.

Dicentric Chromosones Dicentric chromosomes can form as a result of incorrect DNA repair process Double Stranded Breaks in two chromosomes DNA replication Acentric fragment Dicentric chromosome Radiation exposure Centromere Fusion Double stranded breaks are quickly mended by DNA repair mechanisms. In some cases, when these breaks occur in adjacent chromosomes, they rejoin incorrectly. If each chromosome contains a centromere, the result is a chromosomes with 2 centromeres, or a dicentric chromosome. There are also fragments without centromeres that are connected and these are referred to as acentric fragments. Figure from REAC/TS

Dicentric Chromosome Analysis Only occur as a result of radiation exposure Used in Biodosimetry-considered the “gold standard” Sensitive from 0.2 to 5.0 Gray (Gy) Reproducible Dicentric chromosomes have routinely been used to estimate radiation dose since the mid 60s (IAEA). Using biological markers as a measure of radiation absorption is more accurate than physical dosimeters because lymphocytes circulate throughout the whole body whereas the physical dosimeters are located on only one part of the body and can over or under-represent absorption. The sensitivity of lymphocytes to chromosomal aberrations allows this process to be accurate for a broad range of exposure levels. The number of dicentric chromosomes that appear in a blood sample due to everyday or more common exposure methods is only 1 in 1000 cells, so damage form radiation is much more evident when the number increases. Lastly, because the dicentrics are relatively easy to identify, the results are reliably reproducible in different labs and with different screeners. Centromere Centromere reac/ts DCA presentation

Methods Collecting and Incubating Cells Growth medium added to blood samples Incubated to encourage mitosis Colcemid added to stop mitosis in metaphase Cell Harvesting and Preparation Lymphocytes were harvested by centrifugation Hypotonic salt solution spread chromosomes Cells fixed and stained Lymphocyte capture and imaging Metaphase chromosome spreads located and imaged with Metafer 4 The whole blood sample obtained was cultured or the lymphocytes can be removed and cultured separately. The growth medium contained necessary nutrients, fetal calf serum, and a mitogen that causes the normally non-dividing cells to undergo mitosis. We used PHA. Cells are incubated at 37 degrees Celsius under sterile conditions for at least 48 hours. Colcemid was added to disrupt the formation of spindle fibers during metaphase so sister chromatids do not separate and chromosomes are “frozen” in place. Adding Potassium Chloride separates the chromosomes so they are evenly spread out on a slide. After washing the cells with a fixative of methanol and acetic acid, they were dropped onto a slide and stained with giemsa stain. The Metafer 4 is an automated microscope that is equipped with an algorithm to locate cells in metaphase and capture images of the chromosomes.

Counting Dicentrics Dicentric chromosomes are located and counted in a minimum of 500 metaphase cells. This slide contains 4 dicentric chromosomes as the arrows indicate. There are also 2 fragments remaining. Dicentric formation always results in fragments. Many times, the fragments are lost in processing. So even though they are formed they may not show on the slide.

Dosage Calculations The frequency of dicentrics per cells is calculated A calibration curve is used to estimate dosage Calibration curves for each lab are created by analyzing samples exposed to known amounts of inonizing radiation. Then the dicentrics are counted and plotted using Chromosomal Aberration Calculation Software. The number of dicentrics in an experimental sample is counted and used to determine the dose by locating it on the curve. If there are 0.6 pabberrations/cell counted by an evaluator, that indicates that the patient absorbed 2.5 Gy +/- the confidence interval.

Can people with minimal training accurately complete dosimetry? Yes! 102 chromosomal spreads were analyzed All results within 95% confidence level A study is being undertaken to ascertain the answer to this question. When 102 slides with pictures of metaphase spreads were analyzed to identify the number of dicentric chromosomes by 5 minimally trained evaluators, us!, the results varied but they were all within the margin of error. They are not all the same due to chromosome morphology and personal interpretation. Professionally, 57 disentrics were identified, but all of the data is within the allowable error.

Significance of this research Allows a large number of people to estimate dosage in the case of a nuclear catastrophe This research has great significance. With an increasing nuclear presence in the world, there is also an increased risk of a nuclear catastrophe. In that instance, there is a need to have more people trained to estimate dosage of radiation. This allows for faster counting of dicentrics and dosage estimation to get people triaged as necessary.

Further Research FISH- Fluorescence In Situ Hybridization More accurate since the centromere can be seen Fluorescence In Situ Hybridization uses fluorescent probes to bind to particular sections of chromosomes. Using the metafer microscope, the metaphase spreads are imaged with a fluorescent filter and the telomeres, seen here in red, and centromeres, seen in green, are clearly different colors than the chromosome, in blue. This process makes it much easier to identify centromeres which reduces the amount of error in counting dicentrics; 2 green sparks= a dicentric chromosome.

Thank you Thank you for this opportunity to expand our minds!