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Cytogenetic Analysis for Radiation Dose Assessment : a Manual. — Vienna : International Atomic Energy Agency, 2001

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Cytogenetic Analysis for Radiation Dose Assessment : a Manual. — Vienna : International Atomic Energy Agency, 2001 TECHNICAL REPORTS SERIES No. 405 Cytogenetic Analysis for Radiation Dose Assessment A Manual INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 2001 CYTOGENETIC ANALYSIS FOR RADIATION DOSE ASSESSMENT A Manual The following States are Members of the International Atomic Energy Agency: AFGHANISTAN GHANA PAKISTAN ALBANIA GREECE PANAMA ALGERIA GUATEMALA PARAGUAY ANGOLA HAITI PERU ARGENTINA HOLY SEE PHILIPPINES ARMENIA HUNGARY POLAND AUSTRALIA ICELAND PORTUGAL AUSTRIA INDIA QATAR AZERBAIJAN, REPUBLIC OF INDONESIA REPUBLIC OF MOLDOVA BANGLADESH IRAN, ISLAMIC REPUBLIC OF ROMANIA BELARUS IRAQ RUSSIAN FEDERATION BELGIUM IRELAND SAUDI ARABIA BENIN ISRAEL SENEGAL BOLIVIA ITALY SIERRA LEONE BOSNIA AND HERZEGOVINA JAMAICA SINGAPORE BRAZIL JAPAN SLOVAKIA BULGARIA JORDAN SLOVENIA BURKINA FASO KAZAKHSTAN SOUTH AFRICA CAMBODIA KENYA SPAIN CAMEROON KOREA, REPUBLIC OF SRI LANKA CANADA KUWAIT SUDAN CENTRAL AFRICAN LATVIA SWEDEN REPUBLIC LEBANON SWITZERLAND CHILE LIBERIA SYRIAN ARAB REPUBLIC CHINA LIBYAN ARAB JAMAHIRIYA THAILAND COLOMBIA LIECHTENSTEIN THE FORMER YUGOSLAV COSTA RICA LITHUANIA REPUBLIC OF MACEDONIA COTE D’IVOIRE LUXEMBOURG TUNISIA CROATIA MADAGASCAR TURKEY CUBA MALAYSIA UGANDA CYPRUS MALI UKRAINE CZECH REPUBLIC MALTA UNITED ARAB EMIRATES DEMOCRATIC REPUBLIC MARSHALL ISLANDS UNITED KINGDOM OF OF THE CONGO MAURITIUS GREAT BRITAIN AND DENMARK MEXICO NORTHERN IRELAND DOMINICAN REPUBLIC MONACO UNITED REPUBLIC ECUADOR MONGOLIA OF TANZANIA EGYPT MOROCCO UNITED STATES OF AMERICA EL SALVADOR MYANMAR URUGUAY ESTONIA NAMIBIA UZBEKISTAN ETHIOPIA NETHERLANDS VENEZUELA FINLAND NEW ZEALAND VIET NAM FRANCE NICARAGUA YEMEN GABON NIGER YUGOSLAVIA GEORGIA NIGERIA ZAMBIA GERMANY NORWAY ZIMBABWE The Agency’s Statute was approved on 23 October 1956 by the Conference on the Statute of the IAEA held at United Nations Headquarters, New York; it entered into force on 29 July 1957. The Headquarters of the Agency are situated in Vienna. Its principal objective is “to accelerate and enlarge the contribution of atomic energy to peace, health and prosperity throughout the world’’. © IAEA, 2001 Permission to reproduce or translate the information contained in this publication may be obtained by writing to the International Atomic Energy Agency, Wagramer Strasse 5, P.O. Box 100, A-1400 Vienna, Austria. Printed by the IAEA in Austria November 2001 STI/DOC/010/405 TECHNICAL REPORTS SERIES No. 405 CYTOGENETIC ANALYSIS FOR RADIATION DOSE ASSESSMENT A Manual INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 2001 VIC Library Cataloguing in Publication Data Cytogenetic analysis for radiation dose assessment : a manual. — Vienna : International Atomic Energy Agency, 2001. p. ; 24 cm. — (Technical reports series, ISSN 0074–1914 ; no. 405) STI/DOC/010/405 ISBN 92–0–102101–1 Includes bibliographical references. 1. Human cytogenetics. 2. Radiation dosimetry. I. International Atomic Energy Agency. II. Series: Technical reports series (International Atomic Energy Agency) ; 405. VICL 01–00273 FOREWORD Chromosome aberration analysis is recognized as a valuable dose assessment method which fills a gap in dosimetric technology, particularly when there are difficulties in interpreting the data, in cases where there is reason to believe that persons not wearing dosimeters have been exposed to radiation, in cases of claims for compensation for radiation injuries that are not supported by unequivocal dosimetric evidence, or in cases of exposure over an individual’s working lifetime. The IAEA has maintained a long standing involvement in biological dosimetry commencing in 1978. This has been via a sequence of Co-ordinated Research Programmes (CRPs), the running of Regional Training Courses, the sponsorship of individual training fellowships and the provision of necessary equipment to laboratories in developing Member States. The CRP on the “Use of Chromosome Aberration Analysis in Radiation Protection” was initiated by IAEA in 1982. It ended with the publication of the IAEA Technical Report Series No. 260, titled “Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment”, in 1986. The overall objective of the CRP (1998–2000) on “Radiation Dosimetry through Biological Indicators” is to review and standardize the available methods and amend the above mentioned previous IAEA publication with current techniques on cytogenetic bioindicators which may be of practical use in biological dosimetry worldwide. An additional objective is to identify promising cytogenetic techniques to provide Member States with up to date and generally agreed advice regarding the best focus for research and suggestions for the most suitable techniques for near future practice in biodosimetry. This activity is in accordance with the International Basic Safety Standards (BSS) published in 1996. To pursue this task the IAEA has conducted a Research Co-ordination Meeting (Budapest, Hungary, June 1998) with the participation of senior scientists of 24 biodosimetry laboratories to discuss the methods and their modifications used for biological dose assessment worldwide, as well as the need and the possible ways of standardization of the available and promising cytogenetic techniques to evaluate the dose of accidental acute or cumulative chronic exposure to ionizing radiation. The contribution of the chief scientific investigators of the IAEA CRP on “Radiation Dosimetry through Biological Indicators” is sincerely acknowledged (the list of the participating biodosimetry laboratories is given at the end of the Manual). Four Consultancy Services Meetings were held in 1999–2000 to prepare this Manual on standardized conventional methods and amend them with newly available, proven techniques such as fluorescence in situ hybridization (FISH), premature chromosomal condensation (PCC) and study of micronuclei (MN) frequency. The IAEA wishes to express its thanks to all authors and reviewers of this Manual as listed at the end of the book. Primary contributions of D.C. Lloyd (United Kingdom), F. Darroudi (Netherlands), M. Fenech (Australia) and G.J. Köteles (Hungary) are especially acknowledged. I. Turai of the Division of Radiation and Waste Safety was the IAEA Scientific Secretary responsible for the preparation of this Manual and for the CRP on “Radiation Dosimetry through Biological Indicators” in 1998–2000. EDITORIAL NOTE Although great care has been taken to maintain the accuracy of information contained in this publication, neither the IAEA nor its Member States assume any responsibility for consequences which may arise from its use. The use of particular designations of countries or territories does not imply any judgement by the publisher, the IAEA, as to the legal status of such countries or territories, of their authorities and institutions or of the delimitation of their boundaries. The mention of names of specific companies or products (whether or not indicated as registered) does not imply any intention to infringe proprietary rights, nor should it be construed as an endorsement or recommendation on the part of the IAEA. Reference to standards of other organizations is not to be construed as an endorsement on the part of the IAEA. CONTENTS 1. INTRODUCTION . 1 2. APPLICATION OF DOSE CONCEPTS IN BIOLOGICAL DOSIMETRY . 3 3. BIOPHYSICAL BACKGROUND TO CHROMOSOME DAMAGE . 4 4. HUMAN LYMPHOCYTES . 10 5. CHROMOSOMAL STRUCTURE . 12 5.1. Chromatin packing . 12 5.2. Human karyotype . 12 5.3. DNA contents of chromosomes . 13 5.4. Cell cycle . 13 6. RADIATION INDUCED CHROMOSOMAL ALTERATIONS . 14 6.1. Introduction . 14 6.2. Radiation induced DNA lesions . 18 6.3. Chromosome type aberrations . 19 6.3.1. Unstable aberrations . 19 6.3.2. Stable aberrations . 20 6.4. Premature chromosome condensation (PCC) . 22 6.5. Micronuclei (MN) . 24 7. BLOOD SAMPLING . 24 7.1. Timing . 24 7.2. Anticoagulant . 25 7.3. Containers . 26 7.4. Transport . 26 8. DICENTRIC ANALYSIS . 27 8.1. Culturing . 27 8.1.1. Choice of culture medium . 27 8.1.2. Choice of serum . 28 8.1.3. Bromodeoxyuridine . 28 8.1.4. Mitogens . 28 8.1.5. The cultures . 29 8.1.5.1. Whole blood . 29 8.1.5.2. Separated lymphocytes . 29 8.1.6. Mitotic arrest . 30 8.2. Fixation procedure . 30 8.3. Staining . 32 8.3.1. Pretreatment . 33 8.3.2. Fluorescence plus Giemsa (FPG) staining . 33 8.3.3. Conventional Giemsa staining . 34 8.4. Analysis of slides . 34 8.5. Recording of data . 35 8.6. Storage of information and slides . 37 8.7. Production of an in vitro dose-response curve . 37 8.7.1. Physical considerations . 37 8.7.2. Statistical considerations . 39 8.8. Dose assessment in whole body exposure . 40 8.8.1. Choice of curve . 40 8.8.2. Number of cells to be analysed . 40 8.8.3. Uncertainty on dose estimates . 41 8.8.4. Actual examples of dose estimations . 46 8.8.4.1. Acute whole body exposure: An example . 47 8.8.4.2. Criticality . 48 8.8.4.3. Dealing with low dose overexposure cases . 49 8.8.4.4. Dose assessment in partial body exposure . 51 8.8.4.5. Dose assessment after delayed blood sampling . 54 8.8.4.6. Dose assessment after protracted and fractionated exposure . 56 8.8.4.7. Internal incorporation of radionuclides . 58 9. TRANSLOCATION ANALYSIS . 60 9.1. Introduction . 60 9.2. Cell culture and fixing procedures . 60 9.3. Painting the chromosomes . 61 9.3.1. What should be painted . 61 9.3.2. Choice of chromosome specific DNA libraries . 61 9.4. Scoring criteria . 62 9.4.1. Selection of scorable cells . 62 9.4.2. Nomenclature . 62 9.4.3. Recording data . 63 9.5. Data handling . 64 9.5.1. Single colour painting . 65 9.5.2. Two colour painting . 66 9.5.3. More than two colours . 67 9.6. Contemporary and retrospective biodosimetry following occupational and accidental exposure . 67 9.6.1. Retrospective biological dosimetry in population groups without prior personal dosimetry . 67 9.6.2. Retrospective biological dosimetry in population or occupational exposure groups with physical dose estimates . 68 9.6.3. Retrospective biological dosimetry in persons with known biological dose estimates made shortly after accidents, using conventional dicentric analyses . 69 9.7. Persistence of translocations with post-exposure time in animals . 72 9.8. Persistence of stable translocations in In vitro experiments with human lymphocytes .
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