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B IODOSIMETRY AVA I LABLE METHODS AND R OLE IN DOSE ASSESMENT AND PROGNOS I S

B IODOSIMETRY AVA I LABLE METHODS AND R OLE IN DOSE ASSESMENT AND PROGNOS I S. Module X. Accidental dosimetry. BIOLOGICAL DOSIMETRY. PHYSICAL DOSIMETRY. CLINICAL DOSIMETRY. CYTOGENETIC DOSIMETRY Dicentrics, FISH, PCC, MN A . DOSE RECONSTRUCTION , Personal Dosimeters.

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B IODOSIMETRY AVA I LABLE METHODS AND R OLE IN DOSE ASSESMENT AND PROGNOS I S

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  1. BIODOSIMETRYAVAILABLEMETHODS AND ROLE IN DOSEASSESMENT AND PROGNOSIS Module X

  2. Accidental dosimetry BIOLOGICAL DOSIMETRY PHYSICAL DOSIMETRY CLINICAL DOSIMETRY CYTOGENETIC DOSIMETRY Dicentrics,FISH, PCC, MNA DOSE RECONSTRUCTION, Personal Dosimeters NAUSEA, VOMITING, BLOOD CELLS COUNTS, SKINREACTIONS... OTHER BIOINDICATORS Module Medical X.

  3. Physicaldosimetry Module Medical X.

  4. Clinicaldosimetry Crude estimate of absorbed dose obtainable from clinical presentation • Vomiting • Onset: 2 h after exposure or later • Onset: 1-2 h after exposure or later • Onset: earlier than 1 h after exposure • Onset: Earlier than 30 min after exposure MILD ARS (1-2 Gy) MODERATE ARS (2-4 Gy) SEVERE ARS (4-6 Gy) VERY SEVERE ARS (6-8 Gy) Module Medical X.

  5. Clinical dosimetry using early changes in lymphocyte counts Module Medical X.

  6. Clinical dosimetry using granulocyte counts Module Medical X.

  7. Cytogenetic dosimetry • Analysis of chromosomal aberrations in peripheral blood lymphocytes - widely used biologicaldosimetry method forassessing radiation dose, especially useful • in persons not wearing dosimeters while exposed to radiation • in cases of claims for compensation for radiation injuries not supported by unequivocal dosimetric evidence • for validation of occupational radioprotection cases involving suspected low-dose exposures Module Medical X.

  8. Biophysical background to chromosome damage High LET ******************************** * * ** * * * ** Low LET Module Medical X.

  9. DNA damage Module Medical X.

  10. Chromosomal structure Module Medical X.

  11. Human lymphocytes • Dose assessment predominantly based on data obtained from lymphocytes • Easily obtained in large quantities from peripheral blood • Vast majority of peripheral lymphocytes reside in Go phase of e cell cycle • Phytohaemagglutinin (PHA) converts resting lymphocytes into dividing cells allowing visualization of possible DNA lesions in methaphase chromosomes Module Medical X. LYMPHOCYTES

  12. Human karyotype Module Medical X.

  13. Classification of chromosomal aberrations Breaks Symmetrical (STABLE) Asymmetrical (UNSTABLE) Centric Ring Intrachange Inversion Interchange Translocation Dicentric Module Medical X.

  14. Biological dose assessment using standard dicentric analysis • Introduced by M. Bender in 1964 • Isolated lymphocytes stimulated by phytohaemagglutin (PHA) into mitosis • Arrest of metaphase using colchicine • Scoring of dicentric chromosome aberrations in metaphase spreads Module Medical X.

  15. Dicentric chromosome aberrations in metaphase spreads dic f dic f f f Module Medical X.

  16. Dose response curves Y = A+aD + bD2 Module Medical X.

  17. Relationship between RBE and LET RBE LET (keV/m) Module Medical X.

  18. Calibration curves Module Medical X.

  19. Dose estimation of acute vs chronic exposure  particles Fast neutrons (High LET) Gamma rays, X-rays acute exposure (Low LET) Y = c + aD Y = c + aD + bD2 Effect Dicentric yield Y = c + aD Gamma rays X-rays chronic exposure (Low LET) Dose Module Medical X.

  20. Methods for estimating radiation doses in partial bodyexposure:Sasaki-method Module Medical X.

  21. Dicentric assay • Most accurate method for dose estimation with sensitivity threshold of about 0.1 Gy for whole body low LET radiation • Especially useful • in cases where dosimeter not used, e.g. radiation accident • to support physical dosimetry results in radiation protection and safety practice • to determine partial body exposure not detected by locally placed dosimeter Module Medical X.

  22. Limitations of dicentric analysis for dose estimation • Dicentrics are unstable and lymphocytes carryingaberration elimininated with time (average lifetime 150-220 days, depending on dose), hence can underestimate magnitude of dose • Method useful only within few months of irradiation Module Medical X.

  23. Translocation assay • In retrospective dosimetry and chronic exposurereciprocal translocations used for dose assessment • Translocations considered stable in cell division so yield should not fall with time • Typically detected using specific whole chromosome DNA hybridization probes and FISH methodology Module Medical X.

  24. Stable chromosome aberration analysis with G-banding A normal G banded male karyotype An idiogram showing the banding patterns of individual chromosomes by fluorescent and Giemsa staining Module Medical X.

  25. Stable chromosome aberration analysis with FISH Translocation Deletion Module Medical X.

  26. Painting chromosomes Pancentromeric and telomeric probes Module Medical X.

  27. Applicability of stable chromosome aberrationanalysis for biological dosimetry • Method based on scoring stable chromosome aberrations (translocations and insertions) detected with fluorescent in-situ hybridization of whole chromosomes • Requires complex procedures and technical equipment • May be use decades after exposure • Sensitivitythreshold a few cGy but method not feasible for doses less than 0.2 Gy because of expense and time needed for analysis • Spontaneous level of stable chromosome aberrations not well established Module Medical X.

  28. Premature chromosome condensation (PCC) assay • Initially introduced by Johnson and Rao (1970) • Mitotic-inducer cells (i.e. CHO) isolated using chemical (colcemid) and physical (rapid shaking of flask) technique • Test cells (i.e. human lymphocytes) fused with CHO cells using polyethylene glycol (PEG) • Interphase DNA of test cells condense into chromatid/chromosome-like structures (46 for non-irradiated human cells) Module Medical X.

  29. PCC technique CHINESE HAMSTER OVARY (CHO) CELLS (Grown in BrdU) COLCEMID MITOTIC SHAKE OFF (METAPHASE CELLS) FUSE IN PEG PERIPHERAL BLOOD CHO LYMPHOCYTES FICOL SEPARATION Incubate 1 h (Medium+PHA+Colcemid) PCC Module Medical X.

  30. Evaluation criteria for scoring PCCs Module Medical X.

  31. PCCs and FISH Irradiated cells with excess break Unirradiated control Module Medical X.

  32. Estimation of irradiated body fractions Module Medical X.

  33. Applicability of PCC assay for biological dosimetry • Dose estimates obtainable within 48 hours of receipt of blood inlaboratory • Radiation induced mitotic delay does not interfere with assay since performed on interphase nuclei and does not require cell division • Method envisioned applicable after partial-body/supra-lethal exposure &improves detection level of lower doses Module Medical X.

  34. Micronucleus (MN) assay Cytochalasin B Module Medical X.

  35. MN and nucleoplasmic bridges in binucleated cells(Giemsa stained) B A Module Medical X.

  36. MN assay with pancentromeric probe A B centromere positive centromere negative Module Medical X.

  37. Application of MNassay for biological dosimetry • Micronuclei not specific toradiation exposure • Discrimination between total and partial body exposure moredifficult • High doses of radiation interfere with cell division • High baseline frequencyand age dependency make reliability of assay questionable Module Medical X.

  38. Glycophorin A (GPA) somatic cell mutation assay • Performed by two-color immunofluorescence flow cytometry on peripheral blood erythrocytes • Based of measuring N/0 variants of erythrocytes, which display phenotype consistent with loss of expression of GPA (M) allele • Can be performed only on individuals heterozygous at this locus that codes for the N/M blood group antigens (approximately half of population) • Prompt but requires complex and expensive equipment • Sensitivity threshold about 0.2-0.25 Gy Module Medical X.

  39. Application of GPAassay for biological dosimetry Relationship between glycophorin A mutant frequency in red blood cells and radiation dose for about 1200 A-bomb survivors Module Medical X.

  40. Biophysical assays - ESR(electron spin resonance) • Persistent free radicals formed in solid matrix biomaterial (e.g. dental enamel, nailclippings, hair) from accidentally exposed victim can be detected via ESR • Measurements provide reliable biophysical dose estimates & partial body exposure information • In some circumstances, certain clothing material, particularly hard plasticsand buttons, may be measured and absorbed dose estimated Module Medical X.

  41. Characterization of biological dosimetry methods Module Medical X.

  42. Review points • In radiation accidents, important to estimate the absorbed doses in victims to plan appropriate medical treatment • In most accidents, physical dosimetry of absorbed dose is not possible. Even where possible, important to confirm the estimates by other methods • Most commonly used method cytogenetic analysis of chromosomal aberration in peripheral blood lymphocytes using dicentrics, translocations, PCC and micronuclei assays Module Medical X.

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