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while the stars represent the 50 states of the Union. The colors of the flag are symbolic as well; red symbolizes hardiness and valor; white signifies purity and innocence; and blue represents vigilance, perseverance, and justice. Traditionally, a symbol of liberty, the American flag has carried the message of freedom, and inspired Americans, both at home and abroad. In 1814, Francis Scott Key was so moved at seeing the Stars and Stripes waving after the British shelling of Baltimore’s Fort McHenry that he wrote the words to The Star-Spangled Banner. In 1892 the flag inspired Francis Bellamy to write the “Pledge of Allegiance,” our most famous flag salute and patriotic oath. In July 1969 the American flag was flown in space when Neil Armstrong planted it on the surface of the moon. Today, our flag flies on constellations of Air Force satellites that circle our globe, and on the fin flash of our aircraft in harm’s way in every corner of the world. Indeed, it flies in the heart of every Airman who serves our great Nation. The sun never sets on our U.S. Air Force, nor on the flag we so proudly cherish. Since 1776, no generation of Americans has been spared the responsibility of defending freedom… Today’s Airmen remain committed to preserving the freedom that others won for us for generations to come. By displaying the flag and giving it a distinctive fold, we show respect to the flag and express our gratitude to those individuals who fought, and continue to fight for freedom, at home and abroad. Since the dawn of the 20th century, Airmen have proudly flown the flag in every major conflict, on lands and skies around the world. It is their responsibility…our responsibility…to continue to protect and preserve the rights, privileges, and freedoms we,
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as Americans, enjoy today. The United States flag represents who we are. It stands for the freedom we all share and the pride and patriotism we feel for our country. We cherish its legacy as a beacon of hope to one and all. Long may it wave. 24.12. Types of Flags There are specific sizes, types, and occasions for proper display of the United States flag. Understanding the significance of each of these types of flags will ensure its proper display. Installation Flag. The installation flag is lightweight nylon bunting material, 8 feet 11 3/8 inches by 17 feet, and is only displayed in fair weather from an installation flagstaff. This is the typical flag used at Air Force installations. All-Purpose Flag. The all-purpose flag is made of rayon bunting material, 3 feet by 4 feet. This size can be used for outdoor display with flags of friendly foreign nations, in arrival ceremonies for international dignitaries, or to indicate joint occupancy of a building by two or more countries. 243 All-Purpose (All-Weather) Storm Flag. The all-purpose, all-weather storm flag is a lightweight nylon bunting material, 5 feet by 9 feet 6 inches. Use this size as an alternate for the installation flag in inclement weather. Ceremonial Flag. The ceremonial flag is rayon or synthetic substitute material, 4 feet 4 inches by 5 feet 6 inches, trimmed on three edges with yellow rayon fringe 2 inches wide. Organizational Flag. The organizational flag is rayon or synthetic substitute material and is 3 feet by 4 feet. This flag is trimmed on three edges with rayon fringe 2 inches wide. Retirement Flag. The retirement flag may be either 3 feet by 4 feet or 3 feet by 5 feet. Members retiring from the Air Force are entitled to presentation of a United States
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flag. For details, refer to AFI 65-601, Volume 1, Budget Guidance and Procedures, on using Organization & Maintenance funds for this purchase. Garrison Flag. The garrison flag is 20 feet by 38 feet. This flag is flown on holidays and special occasions and can be substituted with the installation flag. Interment Flag. The interment flag is 5 feet by 9 feet 6 inches of any approved material. The interment flag is authorized for deceased military personnel and for deceased veterans. This is the size flag used to drape over a closed casket. Note: To receive an interment flag from the Department of Veterans Affairs, fill out VA Form 27-2008, Application for U.S. Flag for Burial Purposes. The form is available online at: . 24.13. Display of the United States Flag Sunrise to Sunset. The universal custom is to display the United States flag only from sunrise to sunset on buildings and on stationary flagstaffs in the open. However, when a patriotic effect is desired, the flag may be displayed 24 hours a day if properly illuminated during the hours of darkness. All other flags should also be illuminated when displayed with the United States flag. Locations. Air Force installations are authorized to fly one installation flag from reveille to retreat, normally on a flagstaff placed in front of the installation headquarters, and additional flagstaffs and flags are authorized adjacent to each dependent school on the installation. The flag should be displayed daily on or near the main administration building of every public institution; it should also be displayed during school days in or near every schoolhouse. Holidays. The United States flag should be displayed on all days as may be proclaimed by the U.S. President, the birthdays of states (date of admission), and on state holidays. It should also
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be displayed on the following national holidays. New Year's Day Father's Day Inauguration Day Independence Day Martin Luther King Jr.'s Birthday Nat’l Korean War Veterans Armistice Day Lincoln's Birthday Labor Day Washington's Birthday Constitution Day Easter Sunday (variable) Columbus Day 244 Mother's Day Navy Day Armed Forces Day Veteran s Day Memor ial Day (half -staff until noon) Thanksgiving Day Flag Day Christmas Day 24.14. Position and Manner of Display The United States flag is always displayed on a stage or in a parade on its own right. In other words, for an audience looking at a stage, the flag is on the audience’s left. When displaying the flag, the union (the white stars on the blue field) is displayed at the uppermost, right side of the flag itself. Figure 24.2. is provided as an example for proper display of the United States flag in various situations and configurations. Carried in Procession with Another Flag. As a rule of thumb, when the United States flag is displayed or carried in a procession with another flag or flags, it should be either on the right of all others, or in front of and centered ahead of other flags, if there is a line of other flags in the same procession. Displayed with Crossed Staffs. When the United States flag is displayed with another flag against a wall from crossed staffs, it should be on the right, the flag’s own right (the observer’s left), and the staff should be in front of the staff of the other flag. Radiating from a Central Point. When the United States flag is flown with a number of flags displayed from staffs radiating from a central point, and no foreign flags are in the display, the United States flag will be in the center and at
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the highest point of the group. Projecting from a Building. When the United States flag is displayed from a staff projecting horizontally or at an angle from the windowsill, balcony, or front of a building, the union of the flag should be placed at the peak of the staff. In a Row or Line with Equal Height. When the United States flag is flown with a number of flags displayed from staffs set in a line, all staffs will be of the same height and same finial. The United States flag will be on the right side of the group (the observer’s left). In a Row or Line with Elevated Height. When no foreign national flags are involved in the display, the United States flag may be placed at the center of the line and displayed at a higher level than the other flags in the display. Displayed with One or More Nations. When the United States flag is displayed with one or more other nations, they are flown from separate staffs of the same height. The flags should be of equal size. In most cases, member country flags are displayed in a line, alphabetically, with the United States flag at its own right (the observer’s left). Displayed on a Staff near a Speaker’s Platform. When displayed from a staff in a church or public auditorium, the United States flag should hold the position of superior prominence and the position of honor at the clergyman’s or speaker’s right as he or she faces the audience. Any other flag so displayed should be placed on the left of the clergyman or speaker, or to the right of the audience. 245 Displayed Vertically. When displayed from a building, a window, or over the middle of a street, the United States flag should
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be suspended vertically with the union to the uppermost and the flag’s own right, that is, to the observer’s left (north on an east and west street, or east on a north and south street). This also applies when the flag is suspended from the main entrance of a building or hangar. Displayed Horizontally. When displayed horizontally against a wall or when displayed behind a speaker’s platform, the union of the United States flag should be uppermost and to the flag’s own right (the observer’s left). When displayed in a window, the flag should be displayed in the same way, with the union to the observer’s left. Displayed on a Closed Casket. On a closed casket, place the United States flag lengthwise with the union at the head and over the left shoulder of the deceased. Do not lower the flag into the grave, and do not allow the flag to touch the ground. The interment flag may be given to the next of kin at the conclusion of the interment. Displayed at Half-Staff. The term “half-staff” means the position of the United States flag when it is one-half the distance between the top and bottom of the staff. All flags displayed with the United States flag are flown at half-staff when the United States flag is flown at half-staff, with the exception of foreign national flags, unless the foreign country has granted permission for their flag to also be at half-staff. Within the Air Force, the installation commander may direct that the United States flag be flown at half-staff on occasions when it is considered proper and appropriate. When flown at half-staff, the flag shall be first hoisted to the peak for an instant, and then lowered to the half-staff. The flag should be raised to the peak position
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before lowering at the end of the day. 246 Figure 24.2 . U.S. Flag Display Configurations . U.S. Flag Carried in Procession with Another Flag. U.S. Flag and Another Flag Displayed with Crossed Staffs. U.S. Flag with Other Flags Radiating from a Central Point. U.S. Flag Projecting from a Building. U.S. Flag Displayed in a Line with Equal Height. U.S. Flag Displayed in a Line with Elevated Height. U.S. Flag Displayed with One or More Nations. U.S. Flag Displayed from a Staff at Speaker’s Platform. U.S. Flag Positioned Vertically. U.S. Flag Displayed Flat at Speaker's Platform. U.S. Flag Draped Over a Closed Casket. U.S. Flag Flown at Half-Staff. 247 24.15. Care and Respect for the United States Flag Some acts for showing proper respect for the United States flag include: - When in uniform, salute the United States flag six paces before it passes in a procession or parade and hold the salute until it has passed six paces. - Regimental colors, state flags, and organizational or institutional flags are always dipped as a mark of respect to the United States flag. The Air Force flag and organizational flags may be dipped as appropriate. The United States flag will not be dipped to any person or thing, and at no time will a foreign national flag be dipped. - The United States flag should never be displayed with union down, except as a signal of dire distress in instances of extreme danger to life or property. - The United States flag should never touch anything beneath it, such as the ground, floor, water, or merchandise. The United States flag should never be used to cover for a statue or monument. - The United States flag should never be carried flat or horizontally, but always aloft and free. - The United
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States flag should never be used as wearing apparel, bedding, or drapery. The United States flag should never be festooned, drawn back or up, or in folds, but always allowed to fall freely. - The United States flag should never be fastened, displayed, used, or stored in such a manner as to permit it to be easily torn, soiled, or damaged. - The United States flag should never be used as a covering for a ceiling. - The United States flag should never have placed upon it, nor on any part of it, nor attached to it, any mark, insignia, letter, word, figure, design, picture, or drawing of any nature. - The United States flag should never be used as a receptacle for receiving, holding, carrying, or delivering anything. - The United States flag should never be used for advertising purposes. Advertising signs should not be fastened to a staff or halyard from which the United States flag is flown. - The United States flag should never be printed or embroidered on such articles as cushions, handkerchiefs, paper napkins, boxes, or anything that is designed for temporary use. - The United States flag should not be displayed on a float in a parade, except from a staff. - The United States flag should not be draped over the hood, top, sides, or back of a vehicle, railroad train, or boat. - No part of the United States flag should be used as a costume or athletic uniform. However, a United States flag patch may be affixed to the uniform of military personnel, firemen, policemen, and members of patriotic organizations. - A United States flag lapel pin, being a replica of the flag, should be worn on the left lapel near the heart. - No other flag or pennant should be
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placed above or, if on the same level, to the right (observer’s left) of the United States flag, except during church services conducted by naval chaplains at sea when the church pennant may be flown above the flag during church services for the personnel of the Navy. 248 - Exercise extreme care to ensure proper handling and cleaning of soiled flags. When the United States flag is in such condition that it is no longer a fitting emblem for display, it should be destroyed in a dignified way, preferably by burning. There may be instances when a flag is retired from service and preserved because of its historical significance. Disposition instructions should be obtained from the proper authority, such as the installation honor guard or protocol office. - A folded flag is considered cased; therefore, a salute is not necessary. 24.16. Service Flags In accordance with Title 10 United States Code, Armed Forces , service flags will be displayed by order of service date, with the most senior service flags being given the position of honor on the far right. Service flags will be displayed in the following order of precedence from their own right or the observer’s left: U.S. Army (11 July 1775), U.S. Marine (10 November 1775), U.S. Navy (13 October 1775), U.S. Air Force (18 September 1947), and U.S. Coast Guard (4 August 1790). Following the service flags, the order of precedence of flags is as follows: North American Aerospace Defense, U.S. Space Command, major commands (in alphabetical order), field operating agencies, Air National Guard, Air Force Reserve, direct reporting units, Numbered Air Forces and wings (in descending order), and personal/position (using branch appropriate flags). Section 24C—Respect for Individuals 24.17. Showing Respect for Individuals Respect, consideration, manners, common sense, and politeness are all ways of demonstrating common
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acts of courtesy. Common acts of courtesy that contribute to a positive, professional working environment include simple things like saying “please” and “thank you” and respecting other people’s time. When in the workplace, being helpful, taking and delivering messages, and offering assistance when possible, are demonstrations of consideration for others. All Air Force personnel should demonstrate common acts of courtesy while on- and off-duty. Distinguished Visitors. Certain individuals who are considered distinguished visitors (DV) are often afforded particular courtesies as a matter of respect, as well as tradition. A DV may be defined as any: (1) General or Flag Officer; (2) government official with rank equivalent to a Brigadier General or higher; (3) foreign military officer or civilian designated a DV by the Under Secretary of the Air Force for International Affairs; or (4) visitor or group designated by the commander. Persons in certain positions may be given DV status as designated by the commander. A DV visit is an important event and should be given close attention to detail Review AFI 34-1201, Protocol , and AFPAM 34-1202, Guide to Protocol , and contact the installation protocol office for further guidance on responsibilities and proper procedures for DVs. 24.18. Position of Honor Junior personnel shall employ a courteous and respectful bearing toward senior personnel. Give the senior person, enlisted or commissioned, the position of honor on the right when walking, riding, or sitting. The junior person takes the position to the senior’s left. Entering or Exiting an Area. Unless told otherwise or impractical, rise and stand at attention 249 when a senior official enters or departs a room. If more than one person is present, the person who first sees the officer calls the area to attention. An exception to this is when an officer is already in the room
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who is equal to or has a higher rank than the officer entering the room. In that case, do not call the room to attention. Entering or Exiting Vehicles. Military personnel enter automobiles and small boats in reverse order of rank. Juniors will enter a vehicle first and take their appropriate seat on the senior’s left. The senior officer will be the last to enter the vehicle and the first to exit. 24.19. Ranks, Titles, and Terms of Address Military personnel are addressed by the rank associated with their grade or title. While all Air Force personnel are Airmen, it is appropriate to address officers by their grade, such as Lieutenant Colonel, and enlisted members by their grade, such as Master Sergeant. It is also acceptable to address enlisted members relative to their tier, such as Airman, Sergeant, Chief, as appropriate. Air Force members may also be addressed as “Sir” or “Ma’am.” Chaplains may be addressed as Chaplain or by their ecclesiastical title. Respect for Civilians. Civilians and civil service employees should be addressed appropriately as “Mr,” “Mrs,” “Miss,” or “Ms,” and their last name. Also, using “Sir” or “Ma’am” is appropriate. Respect for Retirees. Retirees are entitled to the same respect and courtesies as active military members. They will be addressed by their retired grade on all official records and official correspondence, except for correspondence and other matters relating to a retiree’s civilian employment. Refer to AFI 36-3106, Retiree Activities Program , for additional details. Respect for Uniformed Forces and Other Services. Extend military courtesies to members Uniformed Forces, other services, and friendly foreign nations. Pay the same respect to the national anthems and flags of other nations as rendered the United States national anthem and flag. While not necessary to learn the identifying insignia of the military grades
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of all nations, you should learn the ranks, grades, and insignia of the most frequently contacted nations, particularly during an overseas assignment or deployment. 24.20. Rendering the Salute Saluting is a courtesy exchanged between members of the U.S. Armed Forces as both a greeting and a symbol of mutual respect. The salute is an expression of recognition for one another as members of the profession of arms; representing a personal commitment of self-sacrifice to preserve the American way of life. Salutes are appropriate to the U.S. President, Vice President, Secretary of Defense, Service Secretaries, all superior commissioned and warrant officers, all Medal of Honor recipients, and superior officers of friendly foreign nations. A salute is also rendered as a sign of respect to the United States flag and during official ceremonies, as covered in this chapter. Saluting Uniformed Forces and Other Services. Salutes will be exchanged between officers (commissioned and warrant), and enlisted personnel of the U.S. Armed Forces. Salutes will also be exchanged between U.S. Armed Forces personnel and the Uniformed Services of the National Oceanic and Atmospheric Administration and the U.S. Public Health Service, as appropriate. 250 Saluting Protocol. When a salute is exchanged among individuals, the junior member always salutes the senior member first. The junior member should initiate the salute in time to allow the senior officer to return it. To prescribe an exact distance for all circumstances is not practical; however, good judgment should dictate when salutes are exchanged. While any Airman (enlisted or officer) recognizing a need to salute or a need to return a salute may do so anywhere at any time, there are circumstances when saluting may or may not be practical or warranted. Indoors. Salutes are not rendered indoors, except for formal reporting. When reporting to an officer indoors, knock once,
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enter when told to do so, march to approximately two paces from the officer or desk, halt, salute, and report, “Sir (Ma’am), (rank and last name) reports as ordered,” or “Sir (Ma’am), (rank and last name) reports.” When the conversation is completed, execute a sharp salute, hold the salute until the officer acknowledges the salute, perform the appropriate facing movement, and depart. Outdoors. When outdoors (outside of a building, on a porch, a covered sidewalk, an entryway, a reviewing stand, or at a bus stop) the salute will be exchanged. This applies both on and off military installations. When Carrying Items. Individuals carrying articles in both hands (unable to be transferred to the left hand) need not initiate or return the salute when impractical, but should nod or offer a verbal greeting, acknowledging the appropriateness of a salute. In Formation. When in formation, members do not salute or return a salute unless given the command to do so. The person in charge of the formation salutes and acknowledges salutes for those in the formation. In a Work Detail. When in a work detail, individual workers do not salute. The person in charge of the detail salutes for those in the detail. In Groups. When in groups, when a senior officer approaches, the first individual noticing the officer calls the group to attention. All members face the officer and salute. If the officer addresses an individual or the group, all remain at attention (unless otherwise ordered) until the end of the conversation, at which time they salute the officer. Public Gatherings. When attending public gatherings, such as sporting events, meetings, or when a salute would be inappropriate or impractical, salutes between individuals are not required. In Vehicles. Exchange of salutes between members in moving military vehicles is not mandatory. For pedestrians,
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when officer passengers are readily identifiable (for example, officers in appropriately marked staff vehicles), the salute must be rendered. This includes the U.S. President, the Vice President, Secretary of Defense, Service Secretaries, and senior officers in vehicles when distinguished by vehicle plates and/or flags. In Civilian Attire. Persons in uniform may salute civilians or senior military members in civilian clothes upon recognition. At “No Salute” Areas. Saluting is not required in areas designated as “no salute” areas. In Physical Training Gear. Saluting individuals due to rank recognition is not required when wearing the physical training gear, but may be expected during specified academic training environments. When outdoors in physical training gear, Airmen are required to salute during reveille and retreat. 251 At Military Funerals / Memorials. When at a military funeral or memorial in uniform, salute the caisson or hearse as it passes and the casket as it is carried past. Also, salute during the firing of volleys and the playing of Taps. Note: Many installations across the Air Force play Taps to signify “lights out” at the end of the day. For these purposes, the salute is not required. Section 24D—Ceremonies and Events 24.21. Military Ceremonies The Air Force has many different types of ceremonies that are unique customs of our military profession, many of them held in honor of significant events throughout a member’s career. Official military ceremonies include: promotions, changes and assumptions of command, activations and in-activations, re-designations, enlistments and reenlistments, awards, decorations, arrivals, departures, reveille, retreat, building dedications, ribbon cuttings, retirements, and funerals. Some are very formal and elaborate, while others are quite simple and personal. 24.22. Event Planning and Preparation All events begin with planning. Consideration should always be given to the nature and sequence of the event, scheduling, guests, and budget. To give guests
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time to plan, aim at having details planned out at least three weeks in advance, or more. In such cases, planning committees will need to begin meeting and discussing details of the event far in advance of the invitations being sent out. This could mean, depending on the size and scope of the event, planning as early as several months to a year in advance. Because ceremonies are often steeped in tradition, there are almost always resources available for helping planners get started. Rather than starting from scratch, reach out to other organizations or review checklists from previous events to help get things started. 24.23. Parades and Honors Arrivals or Departures Ceremonies, such as parades, honor cordons, motorcades, and other ceremonies that involve large numbers of Airmen and resources, may be held when officials entitled to such honors visit military installations. Full honors are reserved for statutory appointees and General or Flag Officers, foreign dignitaries, and occasions when ceremonies promote international good will. The installation commander determines which types of honors are rendered. Award Ceremony. An award ceremony affords an opportunity to recognize a member’s accomplishments. The commander or other official determines whether to present an award at a formal ceremony or to present it informally. Many units present awards during commander’s call. Because there are no specific guidelines for an award presentation, commanders and supervisors must ensure the presentation method reflects the significance of the award. Decoration Ceremony. Decoration ceremonies formally recognize service members for meritorious service, outstanding achievement, or heroism. Formal and dignified decoration ceremonies preserve the integrity and value of decorations. When possible, commanders should personally present decorations. Regardless of where the presentation is conducted, the ceremony is conducted at the earliest possible date after approval of the decoration. All military participants and attendees should wear the
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uniform specified by the host. 252 Promotion Ceremony. Promotions are significant events in the lives of military people. Commanders and supervisors are responsible for ensuring their personnel receive proper recognition. Many of the guidelines for promotion ceremonies are the same as for decoration ceremonies. Because most promotions are effective the first day of the month, the promotion ceremony is customarily conducted on the last duty day before the promotion effective date. Some bases hold a base-wide promotion for all promotes, while other bases prefer to recognize promotes within their organizations. Reenlistment Ceremony. Unit commanders will honor all reenlistees through a dignified reenlistment ceremony. Airmen may request any commissioned officer to perform the ceremony, and may invite guests. The member’s immediate family should be invited to reinforce the recognition that when a member makes a commitment to the Air Force, the family is also making a commitment. The ceremony may be conducted in any place that lends dignity to the event. The United States flag has traditionally served, and should be used when available, as a backdrop for reenlistment ceremonies. Reenlistees and officers administering the oath must wear an authorized uniform for the ceremony, unless the officer performing the reenlistment is retired, then the uniform requirement for the reenlisting officer is optional. For additional information on reenlistments, refer to AFI 36-2606, Reenlistment and Extension of Enlistment in the United States Air Force . 24.24. Oaths At the core of the ceremony is the oath. The oath is recited by the officer and repeated by the reenlistee. The reenlistee and the officer administering the oath must be physically collocated during the ceremony. The officer, enlisted, and civilian oaths are very similar, but vary to some degree. If desired, the words “so help me God” may be omitted. Officer Oath I (state your
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name), /// having been appointed a (rank), in the United States Air Force /// do solemnly swear (or affirm) that I will support and defend /// the Constitution of the United States /// against all enemies, foreign and domestic, /// that I will bear true faith and allegiance to the same, /// that I take this obligation freely, /// without any mental reservation or purpose of evasion, /// and that I will well and faithfully discharge /// the duties of the office upon which I am about to enter, /// so help me God. Enlisted Oath I (state your name), /// do solemnly swear (or affirm) /// that I will support and defend /// the Constitution of the United States /// against all enemies, foreign and domestic, /// that I will bear true faith /// and allegiance to the same, /// and that I will obey the orders of the President of the United States /// and the orders of the officers appointed over me, /// according to regulations /// and the Uniform Code of Military Justice, /// so help me God. 253 Air National Guard Enlisted Oath I do hereby acknowledge to have voluntarily enlisted this ____ day of ________, 20____, in the ___________ National Guard of the State of ___________ for a period of ____ year(s) under the conditions prescribed by law, unless sooner discharged by proper authority. I (state your name), /// do solemnly swear (or affirm) /// that I will support and defend /// the constitution of the United States /// and of the State of ___________ /// against all enemies, foreign and domestic, /// that I will bear true faith /// and allegiance to them, /// and that I will obey the orders of the President of the United States /// and the Governor of
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__________, /// and the orders of the officers appointed over me, /// according to law and regulations, /// so help me God. Civilian Oath I, (state your name), /// do solemnly swear (or affirm) that I will support and defend /// the constitution of the United States /// against all enemies, foreign and domestic, /// that I will bear true faith and allegiance to the same, /// that I take this obligation freely, /// without any mental reservation or purpose of evasion, /// and that I will well and faithfully discharge /// the duties of the office upon which I am about to enter, /// so help me God. 24.25. Retirement Ceremony Recognition upon retirement is a longstanding tradition of military service with a tangible expression of appreciation for contributions to the Air Force mission, and with the assurance of continuation as a part of the Air Force family in retirement. Commanders are responsible for ensuring members have a retirement ceremony to recognize their contributions. They must offer the retiring member the courtesy of a formal ceremony in keeping with the customs and traditions of the service, unless the member prefers otherwise. Family members and friends should be invited and encouraged to attend the ceremony. During the retirement ceremony, the member receives a certificate of retirement, a United States flag, the Air Force retired lapel button, various certificates and letters of appreciation, as well as appropriate awards, decorations, and honors. Spouses also receive special recognition at a member’s retirement ceremony. Retirement ceremonies often combine official, long standing Air Force traditions with a member’s desire to personalize the ceremony for family and invited guests. Anyone involved in planning a retirement should consult AFI 36-3203, Service Retirements, for complete details. 24.26. Reveille Ceremony The signal for the start of the official duty
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day is the playing of reveille. Because the time for the start of the duty day varies among different locations, the commander designates the specified time for reveille. If the commander desires, a reveille ceremony may accompany the raising of the flag. This ceremony takes place after sunrise near the base flagstaff. Shortly before the specified time, Airmen march to a pre-designated position near the base flagstaff, halt, face toward the flagstaff, and dress. The flag security detail arrives at the flagstaff at this time and remains at attention. 254 A typical reveille ceremony will involve the following commands and procedures: - The unit commander (or senior participant) commands “Parade, REST.” - At the specified time, the unit commander commands “SOUND REVEILLE.” The flag detail assumes the position of attention, moves to the flagstaff, and attaches the flag to the halyards. - After reveille is played, the unit commander commands “Squadron, ATTENTION” and “Present, ARMS” and then faces the flagstaff and executes present arms. On this signal, the national anthem or To the Color is sounded. - On the first note of the national anthem or To the Color, the flag security detail begins to raise the flag briskly. The senior member of the detail holds the flag to keep it from touching the ground. - The unit commander holds the salute until the last note of the national anthem or To the Color is played, then executes order arms, faces about, and commands “Order, ARMS.” - The Airmen are then dismissed or marched to the dismissal area. Raising the Flag. When practical, a detail consisting of one senior member and two junior members hoists the flag. The detail forms in line with the senior member carrying the flag in the center. The detail then marches to the flagstaff, halts,
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and attaches the flag to the halyards. The two junior members attend the halyards, taking a position facing the staff to hoist the flag without entangling the halyards. The flag is always raised and lowered from the leeward side of the flagstaff. The senior member continues to hold the flag, taking particular care that no portion of the flag touches the ground. When the flag is clear of the senior member’s grasp, the senior member comes to attention and executes present arms. On the last note of the national anthem, To the Color, or after the flag has been hoisted to the staff head, all members of the detail execute order arms on command of the senior member. The halyards are then secured to the cleat of the staff or, if appropriate, the flag is lowered to half-staff before the halyards are secured. The detail is formed again and then marches to the dismissal area. 24.27. Retreat Ceremony The retreat ceremony serves a twofold purpose; it signals the end of the official duty day, and it serves as a ceremony for paying respect to the United States flag. Because the time for the end of the duty day varies among different locations, the commander designates the time for retreat ceremonies. The retreat ceremony may take place at the squadron area, on the base parade ground, or near the base flagstaff. If conducted at the base parade ground, retreat may be part of the parade ceremony. Shortly before the specified time for retreat, Airmen participating in the ceremony are positioned facing the flagstaff and dressed. If a band is present, the band precedes the Airmen participating in the ceremony. A typical reveille ceremony will involve the following commands and procedures: - If the band and Airmen march to the flagstaff, a
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flag security detail also marches to the flagstaff and halts, and the senior member gives the command “Parade, REST” to the security detail. - As soon as the Airmen are dressed, the commander commands “Parade, REST.” The commander 255 then faces the flagstaff, assumes parade rest, and waits for the specified time for retreat. - At the specified time, the commander orders the bandleader to sound retreat by commanding “SOUND RETREAT.” - During the playing of retreat (either by a band or over a loud speaker), junior members of the flag security detail assume the position of attention and move to the flagstaff to arrange the halyards for proper lowering of the flag. Once the halyards are arranged, the junior members of the flag security detail execute parade rest in unison. - After retreat has played, the commander faces about and commands “Squadron (Group, etc.), ATTENTION.” - The commander then commands “Present, ARMS.” The members of the flag security detail and members in formation execute present arms on command of the commander. The commander faces to the front and also assumes present arms. - The national anthem is played, or a bugler plays To the Color. The junior members of the flag security detail lower the flag slowly and with dignity. - The commander executes order arms when the last note of the national anthem or To the Color is played and the flag is securely grasped. The commander faces about, gives the Airmen in formation the command of “Order, ARMS,” and then faces to the front. - The flag security detail folds the flag. The senior member of the detail remains at attention while the flag is folded unless needed to control the flag. - When the flag is folded, the flag security detail, with the senior member on
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the right and the flag bearer in the center, marches to a position three paces from the commander (or officer of the day in an informal ceremony). The senior member salutes and reports “Sir (Ma’am), the flag is secured.” The commander returns the salute, and the flag security detail marches away. The Airmen in formation are then marched to their areas and dismissed. Note: Uniformed military members not assigned to a formation face the flag (if visible), or the music, and assume the position of parade rest on the first note of retreat. Upon completion of retreat, they should assume the position of attention and salute on the first note of the national anthem or To the Color. Lowering the Flag. When practical, the detail lowering the flag should be one senior member and three junior members for the all-purpose flag, and one senior member and five junior members for the installation flag. The detail is formed and marched to the flagstaff. The halyards are detached and attended from the leeward side. On the first note of the national anthem or To the Color, the members of the detail not lowering the flag execute present arms. The lowering of the flag is coordinated with the playing of the music so the two are completed at the same time. The senior member commands the detail “Order, ARMS” when the flag is low enough to be received. If at half-staff, briskly hoist the flag to the staff head while retreat is sounded and then lower on the first note of the national anthem or To the Color. The flag is detached from the halyards and folded. The halyards are secured to the staff. The correct method for folding the United States flag can be found in AFI 34-1201. 256 24.28. The Dining-In
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and Dining-Out Dining-ins and dining-outs are both formal events. The one significant difference is that nonmilitary spouses, friends, and civilians may attend a dining-out, but the dining-in is a formal dinner for military members only. The present dining-in format had its beginnings in the Air Corps when General Henry H. “Hap” Arnold held his famous wingdings. The association of Army Air Corps personnel with the British and their dining-ins during World War II also encouraged their popularity in the Air Force. Members now recognize the event as an occasion where ceremony, tradition, and good fellowship serve an important purpose and are effective in building and maintaining high morale and esprit de corps. Military members who attend these ceremonies must wear the mess dress or the semiformal uniform. Civilians wear the dress specified in the invitations. Note: The combat dining-in is an event similar to the dining-in because it maintains the traditional form; however, the difference is primarily in the dress and atmosphere. Combat dining-ins typically celebrate the evening in some form of utility uniform in a much more relaxed environment deliberately prepared to encourage camaraderie. 24.29. The Order of the Sword Induction Ceremony Induction into the order of the sword is an honor reserved for individuals who have provided outstanding leadership and support to enlisted members as a “Leader among Leaders and an Airman among Airmen.” The order of the sword event is conducted with the dignity that reflects its significance as the highest honor and tribute an enlisted member can bestow on anyone. Similar to the dining-in, this evening affair usually consists of a social period, formal dinner, and induction ceremony. The required dress is the mess dress, semiformal uniform, or equivalent. The only approved levels for award of the sword are the Air Force level and major command
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level. The Chief Master Sergeant of the Air Force and major command command chiefs are known as the “keepers of the sword,” and maintain the official lists of order of the sword recipients, respectively. History of the Order of the Sword. The first recorded order of the sword ceremony in the United States was in the 1860s when General Robert E. Lee was presented a sword by his command. The ceremony was revised, updated, and adopted by the Air Force in 1967 to recognize and honor military senior officers, Colonel or above, and civilian equivalents, for conspicuous and significant contributions to the welfare and prestige of the Air Force enlisted force mission effectiveness as well as the overall military establishment. 257 Chapter 25 PROFESSIONALISM # Chapter 25 PROFESSIONALISM Section 25A—Air Force Professional 25.1. Professionalism We are worthy of the Nation’s trust by integrating our core values of Integrity First, Service Before Self, and Excellence in All We Do into our mission and everything we do. Professionalism describes who we are as a service and how we conduct ourselves as Airmen and representatives of the U.S. Air Force. It sets the standards all Airmen are expected to adhere to - and exceed. Professionalism within the Air Force is framed by the requirements of trust, loyalty, dignity, and personal commitment. We must be dependable and responsible for our own actions while being good wingmen for fellow Airmen and co-workers. At the root of professionalism is respect. Respect is what bonds every Airman’s contribution to the mission with the collective understanding of what it means to serve with humility and deference for those we serve with. The Air Force is a Total Force that effectively leverages the unique capabilities of officer, enlisted, and civilian Airmen across Regular Air Force, Guard, Reserve, and Auxiliary
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Components. As a Total Force Air Force, we are a values-based, mission-focused, people-oriented air and space force. Professionalism is the heart and soul of who we are and who we aspire to be every day. Our sense of professionalism underlies the pride we feel when we say I am an American Airman . Professional Obligation and Status. Every Airman has an obligation to be the very best professional possible. Professional status comes to people at different times in their lives and careers. At what point can an individual claim or profess to have professional military status? As stated in AFI 1-1, Air Force Standards , an Air Force professional’s primary responsibility is to do our part to accomplish the mission; however, accomplishing the mission requires more than just technical proficiency. Our conduct and performance must be consistent with the safe and proper fulfillment of instructions, directives, technical orders, and other lawful orders. Quality and quantity of work are both important since they are the primary measures of efficiency and productivity. Professional status is expressed by attitudes and commitments, and by internalizing military values. Studying and understanding these factors are vital to Airmen and the future of the Air Force. Professional military members of today and tomorrow must accept responsibility for their actions, hold others accountable, and take appropriate action to never hide behind excuses. Focus must be directed toward devoted service to the Nation, not on pay, working conditions, or the next assignment. Our Air Force is a critical part of the greatest fi ghting force the world has ever known. It is powered by the greatest Airmen the world has ever seen. Values-Driven. We are one Air Force, uniformed and civilian. We, as Airmen, are warriors and professionals dedicated to service and living our values— Integrity, Service and Excellence
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—doing the right thing, even when no one is looking. We develop partnerships at home and around the world, grounded in integrity and trust. Our culture embraces diversity and fiercely protects character, respect, and leadership. Mission-Focused. As Airmen, we stand ready, performing selfless duty in defense of our Nation. We, and our families, are dedicated to answering our Nation’s call, making sacrifices for the good of the mission. We, as Airmen, are warriors with the courage to take risks when necessary. Our 258 heritage of breaking barriers—going faster, farther, first—drives us to see things differently, continually innovate, and improve our craft. People-Oriented. Our most important asset is the people who commit to serve as Air Force professionals. Taking care of our wingmen is our duty. We are an integrated force—strong, able, and ready. We, as Airmen, value the contribution of every member of our Air Force team and motivate each other to achieve excellence. We honor and respect all who are brave enough to serve, and we must strengthen our alliances—we are stronger together. Section 25B—Profession of Arms 25.2. The Profession of Arms The Air Force and Airmen wield our Nation’s most powerful and responsive weapon systems. Every member of the Air Force team is entrusted with the responsibility of preserving United States national security. We provide vital skills to ensure the Air Force is ready to answer our Nation’s call. The trust placed by the Nation in us rests upon confidence in the character and competency of the men and women who serve. To continue this trust, we must maintain and project power within the boundaries of a very sacred and honored Air Force ideal…one based on our core values to develop and inspire Airmen within the profession of arms. Whether in war or peace, at home or abroad,
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on- or off-duty, Airmen must hold true to the sacred trust our institution requires. This level of trust demonstrates respect to all fellow Airmen, strives to bring out the best version of each of us, commits to a higher calling of service, and maintains the honor our standards demand. As Airmen, we continually seek to deepen and foster our commitment to high personal standards of conduct. Ultimately, we value who we are as Airmen within the profession of arms and demonstrate our commitment to service as our hallmark to shape and sustain the Air Force culture today and well into the future. 25.4. The Airman’s Perspective The Airman’s perspective is a broad encompassing framework for thinking about present and future warfare. The Airman’s perspective is shaped by what we know and believe about the use of military force in four dimensions - speed, range, altitude, and time - and in relation to the air and space operating environment. Airmen are essential to the Air Force institution and the successful execution of the mission. Understanding and actively advocating the Airman’s perspective on the use of airpower is important and gives Airmen a distinct advantage when performing the mission. Airmen not only bring knowledge, skills, and abilities to accomplish the mission, but also bring a unique manner of approaching mission accomplishment through Airmindedness. 25.5. Airmanship Mindset In the Air Force, enlisted members, officers, and civilians are all Airmen. As Airmen, we are part of a professional subculture, and we demonstrate various disciplines in defense of our Nation. What exactly is Airmindedness or an Airmanship mindset? First, mindset is a mental disposition or attitude that predetermines one’s responses and interpretations of situations. And, in the case of Airmanship, that mental disposition or attitude is what we think and how we feel about membership
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in the profession of arms, which is in turn reflected in our behavior and serves to 259 guide us in proudly exhibiting the highest levels of professional service to our country. Standards, responsibilities, and the readiness to perform perpetuate the Air Force culture and provide a clear picture of what is expected of Airmen. A genuine belief in the oath of enlistment, internalizing the Air Force core values as our own, committing to the profession of arms, and possessing an unstoppable determination known as warrior ethos are the hallmarks of the Airmanship mindset. 25.6. Strategic Roadmap As stated in the Strategic Roadmap: USAF Profession of Arms , the profession of arms requires unique expertise to fulfill our collective responsibility to the American people. Our profession is defined by our values, ethics, standards, skills, and attributes. Our expertise in the justified application of lethal military force and the willingness of those who do serve to die for our Nation distinguishes us as the Air Force profession of arms. Professionalism Vision: Airmen who do the RIGHT thing - the RIGHT way - for the RIGHT reason Professionalism Mission: Leaders forging professional Airmen who embody Integrity, Service , and Excellence Professionalism Goals: Aspirations and inspirations Vision. The Strategic Roadmap defines vision as a mental image of the future - the preferred end state - including how to approach the customer and satisfy the mission, how services are delivered, and how to organize and manage people and other resources. The future of the Air Force rests on the degree to which we can continue to attract, recruit, develop, and retain individuals committed to the profession of arms. Airmen must be trusted professionals with exemplary character, judgment, and competence, who hold themselves and their fellow Airmen accountable. Mission. The Strategic Roadmap defines mission as a
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fundamental reason for being, a purpose of the organization/effort, and why it exists beyond present day operations. Every Airman, those who are leaders and those who aspire to lead, will be vital to the process of developing our personnel. The Air Force will proactively develop each Airman within a professional culture requiring the highest degree of commitment toward institutional standards. Our standards require Airmen to make the right choices guided by our core values at all times. Goals. The Strategic Roadmap defines goals as an expression of the desired future state of the Air Force in a particular area or theme. Goals define and prioritize broad direction and are inherently long-term in nature, can be achieved by meeting objectives, and lead to desired effects that lead to achieving an expected outcome. - Goal 1: Inspire a strong commitment to the Profession of Arms. Professionalism is based on a shared commitment to standards and Air Force core values. Professionals fully understand and embrace the sacred trust the decision to join the profession of arms requires. On- and off-duty, in peace and in war, Airmen embrace and live by the standards our institution requires. - Goal 2: Promote the right mindset to enhance effectiveness and trust. Professionalism is based on one’s commitment to the organization and its shared objectives. Serving as an Airman, whether on Regular Air Force status, the Reserve, Guard, or as a civilian, is not just a job—it’s a 260 profession. We have been given the sacred trust of the American people, and that trust is maintained only when Airmen perform with integrity and character. To meet this expectation, all Airmen must build their lives and shape our service on the foundation of our Air Force core values. All Airmen must develop and sustain a positive attitude, enhance the
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understanding of airpower, and develop professional perspectives that will create and maintain the future force. - Goal 3: Foster relationships that strengthen an environment of trust. Trust is the foundation of the profession of arms. How we treat one another and how we strive to bring out the best version of our people will determine our ability to meet our shared objective of United States national security. As a service, providing opportunities to build healthy relationships throughout the force requires leadership to appropriately prioritize resources and provide clear expectations and guidance at all levels. - Goal 4: Enhance a culture of shared identity, dignity, and respect. The Air Force must strengthen its identity through Airmen first, and through occupational specialty second. Airmen must understand their role in the enduring connection between airpower and national security. Within this shared identity, we must embrace a culture that preserves human dignity as a mission imperative. Section 25C—Air Force Core Values 25.7. The Air Force Core Values At the heart and soul of our profession, the Air Force recognizes our core values as universal, consistent standards used to evaluate the ethical climate of all Air Force organizations. When needed in the cauldron of war, core values are the beacons that light the path of professional conduct and the highest ideals. # Integrity First , Service Before Self , and Excellence In All We Do Values represent enduring, guiding principles for which we stand. Values, such as the Air Force core values of integrity, service, and excellence , should motivate attitudes and actions on- and off-duty as essential moral principles or beliefs that are held in the highest regard. Our core values represent the Air Force’s firm convictions about the nature of our personal character, our commitment to each other and our Nation, and the
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manner in which we perform our service. Core values are so fundamental that they define our very identity through a common bond among all professional Airmen - past, and present. For those of us who join this proud community, being a part of the Air Force family requires a commitment to living by these values at all times. Reflecting the Air Force core values in one’s personal and professional lives is a challenge that must be faced every day. In doing so, we honor the heritage and continue the legacy of those who served before us and sacrificed so much. It is through this alignment of our actions with these values that we, as an Air Force, earn the public’s trust, strengthen our service, and accomplish our mission. This is the expectation of our profession, and is the standard that our fellow service members and the American public hold us to. 261 25.8. The Little Blue Book America’s Air Force: A Profession of Arms , has historically been recognized and referred to as, the little blue book. The little blue book is the document containing and prescribing the Air Force core values. An excerpt from America’s Air Force: A Profession of Arms states, “First, we must understand that our chosen profession is that of a higher calling in which we hold ourselves to higher standards. To serve proudly and capably, our commitment to our cause must be unbreakable; it must be bonded in our mutual respect for each other. Throughout our service we are guided and reminded of this awesome responsibility. The oaths we take remind us that we serve freely in support and defense of our Constitution. We abide by a code of conduct that captures our resolve, while our Airman’s creed highlights the strength of our diverse Airmen
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who fl y, fi ght, and win as one Air Force. We are the world’s greatest Air Force...powered by Airmen, fueled by innovation. We are surrounded by reminders on a daily basis of the meaning of service in our profession...the profession of arms.” 25.9. Core Values – Defined Professional Air Force ethics consist of three fundamental and enduring values (core values) of Integrity First, Service Before Self, and Excellence In All We Do . Success hinges on the incorporation of these values into the character of every Airman. The Air Force core values represent the commitment each Airman makes when joining the Air Force and provide a foundation for leadership, decision-making, and success in every level of assignment, regardless of difficulty or dangers presented by the mission. In today’s compressed, dynamic operational environment, an Airman does not have the luxury of examining each issue at leisure. He or she must fully internalize the core values to be able to expeditiously act in all situations while maintaining professional Air Force standards. In light of the demands placed upon our people to support security interests around the globe, each of these core values are essential. Integrity provides the bedrock for our military endeavors, and is fortified by service to country. This, in turn, fuels the drive for excellence . Each core value can be distinctly defined and described as an essential aspect of our service. Additionally, each core value is interwoven and interdependent on the unyielding, unwavering commitment to uphold our standards at all times. Integrity First. Integrity First is a character trait and the willingness to do what is right even when no one is looking. Being faithful to one’s convictions is part of integrity. A person of integrity acts on conviction, demonstrating impeccable self-control without acting rashly. Following principles, acting
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with honor, maintaining independent judgment, and performing duties with impartiality, help to maintain integrity and avoid conflicts of interest. Integrity encompasses many characteristics indispensable to Airmen and makes us who we are and what we stand for. Integrity is as much a part of a professional reputation as an ability to fly or fix jets, operate a computer network, repair a runway, or defend an airbase. Integrity is the ability to hold together and properly regulate all the elements of one’s personality. Integrity is the moral compass, the inner voice that keeps us on the right path when we are confronted with ethical challenges and personal temptations. An individual realizes integrity when thoughts and actions align with what he or she knows to be right. Virtues of Integrity. The virtues of integrity include honesty, courage, and accountability. - Honesty is the hallmark of integrity. Honesty means our words must be unquestionable so we preserve the trust that unites us through a common goal and purpose. Honesty requires us to 262 evaluate our performance against standards, and to conscientiously and accurately report fi ndings. This is the only way to preserve the trust we hold so dear with each other and with the population we serve. - Courage is not the absence of fear, but doing the right thing despite the fear. Courage empowers us to take necessary personal or professional risks, make decisions that may be unpopular, and admit to our mistakes. Having the courage to take these actions is crucial for the mission, the Air Force, and the Nation. - Accountability is responsibility with an audience. Accountability instills our responsibility while maintaining transparency and ownership for our actions. Our audience may be the American people, our units, our supervisors, our fellow Airmen, our families, our loved ones, and even
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ourselves. Accountable individuals maintain transparency, seek honest and constructive feedback, and take ownership of the outcomes of their actions and decisions. They are responsible to themselves and others, and refrain from actions which discredit themselves or our service. Service Before Self. As an Air Force core value, Service Before Self represents an abiding dedication to the age-old military virtue of selfless dedication to duty, including putting one’s life at risk if called to do so. Service Before Self tells us that professional duties take precedence over personal desires. Airmen are practitioners of the profession of arms, entrusted with the security of the Nation, the protection of its citizens, and the preservation of their way of life. In this capacity, Airmen serve as guardians of America’s future, and this responsibility requires the needs of service and country to be placed before our own. In today’s world, service to country requires not only a high degree of skill, but also a willingness to make personal sacrifices. Airmen work long hours to provide the most combat capability possible for the taxpayer dollar. Military duties require us to perform on temporary duty assignments, accept permanent changes of station, and deploy to the far corners of the globe without complaint, to execute the mission in extremely harsh conditions to meet national security needs. Having the heart and mindset for service allows us to embrace expectations and requirements not levied on the American public or other professions. The reasons professionals remain with the Air Force cannot be counted or measured. Military professionals gain satisfaction from doing something purposeful, gain pride in significantly contributing to an organization that lives by high standards, and gain a sense of accomplishment from defending the Nation and its people. Virtues of Service Before Self. The virtues of Service Before Self include
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duty, loyalty, and respect. - Duty. While duty is the obligation to perform what is required for the mission as determined by the law, the Department of Defense, and Air Force instructions, directives, and guidance, duty may also involve having to make sacrifices in ways that no other profession has or will. Our sense of duty is a personal one and bound by the oath of service we took as individuals. - Loyalty. Loyalty is an internal commitment to the Nation, to the values and commitments of our Air Force, and to the men and women with whom we serve. Loyalty to our leaders requires us to trust, follow, and execute their decisions; offer alternative solutions and innovative ideas most effectively through the chain of command; and ultimately help each other to always act with honor. - Respect. Respect is treating others with dignity and valuing them as individuals. We must always act knowing that all Airmen possess a fundamental worth as human beings and treat others 263 with the utmost dignity and respect, understanding that our diversity is a powerful source of strength. Excellence In All We Do. Excellence In All We Do directs us to develop a sustained passion for the continuous improvement and innovation that propels the Air Force, as well as ourselves, beyond the capabilities of our adversaries. This core value demands that Airmen constantly strive to perform at our best. Adherence of this core value means that Airmen seek out and complete developmental education; constantly work hard to stay in physical, mental, emotional, spiritual, and moral shape; continue to enhance professional competencies; and are diligent to maintain their job skills, knowledge, and personal readiness at the highest possible levels. We must have a passion for continuous improvement and innovation that propels America’s Air Force in
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quantum leaps towards accomplishment and performance. Virtues of Excellence In All We Do. The virtues of excellence include mission, discipline, and teamwork. - Mission. Having a mission focus encompasses operation, product, and resource excellence. The complex undertaking of the Air Force mission requires us to harness the ingenuity, expertise, and collective effort of all Airmen. We approach it with the mindset of stewardship, initiative, improvement, pride, and a continued commitment to anticipate and embrace change. Our people are the platform for delivering innovative ideas, strategies, and technologies to the fi ght. Our work areas, our processes, and our interpersonal interactions must be undeniably professional and positive. - Discipline. Discipline is an individual commitment to uphold the highest of personal and professional standards. We demonstrate it in attitude, work ethic, and effort directed at continuous improvement, whether pursuing professional military education or nurturing ourselves physically, intellectually, emotionally, or spiritually. The appearance, actions, and words of Airmen represent the Air Force and shape the culture of the Air Force and the reputation of the military profession. - Teamwork. Teamwork is essential at every level. Airmen recognize the interdependency of every member’s contribution toward the mission and strive for organizational excellence as a team. We not only give our personal best, but also challenge and motivate each other. We carry our own weight, and whenever necessary, help our wingmen carry theirs. We serve in the greatest Air Force in the world, and we embrace the idea that our part of the Air Force meets that world-class standard. Section 25D—Warrior Ethos 25.10. The Warrior Ethos Building warrior leaders requires employing Airmen who have the competencies and skills to understand the complexity of expeditionary operations in unilateral, joint, or coalition operations. Each Airman should understand and be able to articulate the full potential and application
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of Air Force capabilities required to support the Air Force mission and meet national security objectives. As Airmen, we proudly serve in the most lethal Air Force the world has ever seen. We have inherited an Air Force forged through the ingenuity, courage, and strength of Airmen who 264 preceded us. It is our duty to continue to provide the Nation and the next generation of Airmen an equally dominant force. Doing so requires a full understanding of the profession of arms, the commitment made by taking an oath of office, and the acceptance of living according to the Air Force’s core values. This understanding, commitment, and acceptance is the warrior ethos that builds the confidence and commitment necessary to shape professional Airmen who are able to work as a team to accomplish the mission. The warrior ethos is demonstrated through expeditionary service in garrison, during combat, through humanitarian response and disaster relief operations, and by the lessons learned from those experiences. The warrior ethos is also developed and sustained over the course of a career through a continuum of learning, focused training and education, associated developmental experiences, and a wide variety of assignments. No less important is the strengthening of the warrior ethos through exhibiting pride in the Air Force uniform, physical conditioning, and understanding of the Air Force symbols, history, and culture. 25.11. Code of Ethics As stated in AFI 1-1, Air Force Standards , as a member of the Air Force, the highest standards of conduct and integrity must be practiced, not only on the job, but also in relationships, in financial dealings, and in interaction with the civilian community. The code of ethics must be such that behavior and motives do not create even the appearance of impropriety. Personal values, such as happiness or stability, are
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almost always present, but they must not take precedence over ethical values. Ethical values relate to what is right and wrong, and thus take precedence over non-ethical values when making decisions. The key is to align ethical values with personal values, and enhance the commitment we have made to the dedicated service of our Nation. Our ethical code is prescribed in our core values, our oaths, the Airman’s Creed, Air Force instructions, and the Uniform Code of Military Justice. When faced with decisions related to mission, personal life, or the interest of peers, the choice can always be made with consideration for our ethical code. Principles and Guidelines. Embedded in our code of ethics, and driven by our competence and character, are key guidelines that help clarify acceptable and unacceptable behavior. Principles and ethical guidelines can be used to help identify what right looks like and continue to fortify our Air Force culture. Title 5, CFR, Part 2635, Standards of Ethical Conduct for Employees of the Executive Branch, establishes the basic ethical principles and guidelines that must be followed by every government employee. A few examples of ethical expectations outlined in the regulation are provided here. - Public service is a public trust, requiring employees to place loyalty to the Constitution, the laws, and ethical principles, above private gain. - Employees shall not hold financial interests that conflict with the conscientious performance of duty. - An employee shall not solicit or accept any gift or other item of monetary value from any person or entity seeking official action from, doing business with, or conducting activities regulated by the employee’s agency, or whose interests may be substantially affected by the performance or nonperformance of the employee’s duties. - Employees shall not knowingly make unauthorized commitments or promises of any kind purporting
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to bind the government. 265 - Employees shall act impartially and not give preferential treatment to any private organization or individual. - Employees shall protect and conserve federal property and shall not use it for other than authorized activities. - Employees shall satisfy, in good faith, their obligations as citizens, including all just financial obligations, especially federal, state, or local taxes that are imposed by law. - Employees may generally not accept gifts from subordinates or employees that make less pay than themselves. - Employees may not solicit a donation or a contribution from other personnel for a gift to a superior, make a donation for a gift to a superior official, or accept a gift from subordinate personnel, except for voluntary gifts or contributions of nominal value (not to exceed $10), on occasions of special personal significance (such as marriage or birth of a child), or occasions that terminate the superior-subordinate relationship, such as retirement, permanent change of station or assignment. 25.12. Ethical Dilemma An ethical dilemma is a situation where one is forced to choose between at least two alternatives. Three general causes or sources of ethical dilemmas are: uncertainty, competing values, and potential harm. Uncertainty is the result of not having all the facts pertaining to a situation; not having enough experience for dealing with a situation; or not having a clearly established policy, procedure, or rules for deciding how to make an optimal decision. Competing values occur when our personal values conflict with those of our institution, subordinates, peers, or supervisors; however, the mark of a true professional is maintaining high professional standards despite conflicting values. Potential harm relates to the intentional and unintentional consequences caused by actions. Decisions and Actions. Airmen should always think through second and third order effects of our actions. We must
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apply a sense of order to our priorities so we are able to overcome temptation to stray from our military norms and values. When contemplating what to do, consider possible courses of action by listing to the best options and quality checking ideas to take the right path. When possible, take the decision process to the next level and put each course of action to the test. Dr. Robert M. Hicks, former Deputy Director of the Civil Air Patrol, Chaplain Services, identified three tests we can use to check the morality of our actions and decisions. - The Network Test. The network test consists of asking yourself, “How would this decision look if it was aired on the news?” If your actions were broadcast on the evening news, would you be proud of your actions or ashamed of your actions? Would your actions bring credit to yourself and the Air Force or would they discredit yourself or those we owe? If you find yourself leaning toward a negative response to these questions, then your decision doesn’t pass the network test. - The United States of America Test. The United States of America test focuses on asking yourself, “Is this decision good for the United States? Is this decision good for the U.S. Air Force? Is this decision good for my unit (us)? Is this decision good for me?” If you take this course of action, are you properly ordering your priorities? If you can’t answer with a resounding yes, this might not be the best decision. 266 - The Devine Test. The devine test deals with asking yourself, “Would I feel good about the decision when I give account for my life?” When telling the story of your proud and honorable service to our country, would you include conversation about
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this decision? Would you feel guilt or loss of trust from this action? If you can’t confidently provide a positive response, the course of action fails the divine test. 25.13. Honorable Characteristics Airmen share a history of valor, courage, and sacrifice. From the earliest days of airpower to the heights of space, Airmen have built an extraordinary heritage that forms the foundation for a boundless horizon. We are technology focused, we embrace change, and through transformation and innovation, we ensure a viable Air Force for the future. Always keep focus on demonstrating honorable service and commitment to the profession of arms. Through skills, knowledge, and experience developed in the Air Force, listen to your internal compass while fostering the same in your peers. Remind yourself and your peers of the reason you do what you do. Declare the importance of serving for a higher cause, adhering to established ethical codes, and embracing an Air Force culture steeped in honor and tradition. Rely on what you know is true and what is right. Be the Airman who makes decisions and leads in a way you can be proud of. Airmen firmly grounded in the core values and ingrained with the warrior ethos react to combat stresses, operational deployment pressures, and daily home station demands with valor, courage, and sacrifice. While many acts of valor, courage, or sacrifice go unseen, they should be recognized not only as part of Air Force culture, but also to illustrate that any Airman may be called upon at any time to perform above and beyond in the profession of arms. Valor. Valor is the ability to face danger or hardship in a determined and resolute manner. Valor is commonly and rightly recognized as bravery, fearlessness, fortitude, gallantry, heart, and nerve. When acting with valor, one expresses
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the willingness to step outside the comfort zone to deal with unexpected situations. Such situations can happen almost anywhere. In addition to demonstrating valor on the battlefield, an Airman can exhibit valor when presented with unusual circumstances in the daily routine of life. Consider the demonstration of valor in the following quotation from the Air Force Memorial in Washington, D.C. In the summer of 2005, Senior Airman Shea Dodson wanted to do more than his assigned administrative duties inside of Baghdad’s Green Zone. The call was out for volunteers to provide security for ongoing convoys, so Airman Dodson raised his hand. After some intense just-in-time training, he was performing security detail for his first convoy. On this mission, Airman Dodson put that training to good use. When a suspected vehicle-borne suicide bomber raced toward the convoy, he fired .50 caliber rounds into the engine block no fewer than four times, disabling the vehicle. During the same mission, his unit became mired in traffic near a high-rise development. He noticed movement above and saw an Iraqi armed with an AK-47 creeping toward the edge of a balcony overlooking the convoy. Airman Dodson immediately engaged with indirect warning fire from his M-16, hitting the wall next to the suspected insurgent’s head. The armed Iraqi dove for cover and never returned. When the convoy arrived at its final destination, a children’s school, he continued with a complete security sweep of the perimeter houses to ensure it was clear. Airman 267 Dodson remained on armed watch as his team handed out school supplies to the kids in the open courtyard. By two o’clock that same day, Airman Dodson was back at his desk, keeping track of critical data for the Commanding General of the Multinational Security Transition Command– Iraq. It was all in a
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day’s work for this dedicated Airman. Courage. Courage is about the ability to face fear, danger, or adversity. Three types of courage are critical in the profession of arms: personal, physical, and moral. Personal courage is about doing what’s right even when risking one’s career. Physical courage is the ability to overcome fears of bodily harm to get the job done, or willingness to risk harm to yourself for someone else’s sake in battle or the course of everyday life. Finally, moral courage is the ability to stand by the core values when moral courage may not be the popular thing to do. Integrity breeds courage when and where the behavior is most needed. More often than not, courage is manifested as an act of bravery on the battlefield when Airmen face the challenges present in combat. Demonstration of Courage. Consider the demonstration of courage in the following quotation from the Air Force Memorial in Washington, D.C. While on a special mission in Southwest Asia in 2005, Technical Sergeant Corey Clewley was loading cargo on his aircraft when he saw a Romanian C-130 experience a hard landing. Unbeknownst to the Romanian crew, the aircraft brakes caused a fire, causing Sergeant Clewley to spring into action. He instructed a fellow loadmaster to inform his aircraft commander of the situation and to ensure that someone contacted the control tower of the fire while he and a crew chief grabbed fire extinguishers and ran toward the burning aircraft. The Romanian C-130 fire intensified as it spread to the aircraft’s fuselage and ruptured the hydraulic brake line. Despite the danger to himself, Sergeant Clewley got within a few feet of the flames and attempted to suppress the fire. His sense of urgency tripled when he realized the C-130 crew was still inside the aircraft
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and was unable to get out of the burning aircraft. He saw a member of the crew mouthing ‘please, please’ and pointing to the troop exit door. Sergeant Clewley refocused his attention to that area and began suppressing the fire, enabling the crew to safely exit the aircraft. He continued to keep the fire under control until the fire department arrived. Sergeant Clewley credits the team effort that kept the incident from becoming a deadly event and never considered the risk to his own life as he worked to save a crew and aircraft that was not part of his responsibility, his service, or even his Nation. He noted that saving the lives of the people on board was more important than who owned the aircraft. Sacrifice. Sacrifice involves a willingness to give your life, time, or comfort to meet others' needs. Personal sacrifice occurs on many levels, but is commonly evident in the heroic actions of Airmen in combat. Day-to-day deployed garrison activities also present opportunities to put others' needs before individual wants. Demonstration of Sacrifice. Consider the demonstrations of sacrifice as quoted in the words of those who have served before us. The following quote is from a letter written by Sergeant Carl Goldman to his parents. Sergeant Goldman was a U.S. Army Air Forces B-17 gunner who was killed in Western Europe during World War II. His parents had the quote inscribed at the American Cemetery and Memorial in Cambridge, England, in his honor. …Am going on a raid this afternoon…there is a possibility I won’t return…do not worry about me as everyone has to leave this earth one way or another, and this is the way I have selected. If after 268 this terrible war is over, the world emerges a saner place…pogroms and persecutions halted,
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then I’m glad I gave my efforts with thousands of others for such a cause. - Sergeant Carl Goldman, U.S. Army Air Forces, WWII This next quote is from a letter written by Sergeant Arnold Rahe to his parents. Sergeant Rahe was in the U.S. Army Air Forces and was killed in France during World War II. As I prepare for this…mission, I am a bit homesick… Mother and Dad, you are very close to me, and I long so to talk to you. America has asked much of our generation, but I’m glad to give her all I have because she has given me so much. - Sergeant Arnold Rahe, U.S. Army Air Forces, WWII Call to Duty. Airmen are wingmen, leaders, and warriors with backgrounds and skills as diverse as our Nation. When America’s sons and daughters commit to service, the Air Force takes on the charge to develop them into Airmen. The Air Force culture is one that embraces diversity and fiercely protects its foundational attributes. Over the next 30 years, the Air Force’s ability to continue to adapt and respond faster than our potential adversaries will depend on the flexibility and adaptability of our current and next generation Airmen. We will recruit, develop, and retain exceptional Airmen through strategies and programs designed to develop and care for our Total Force, strengthen the Air Force culture, and leverage development opportunities that employ creative concepts across the force. When faced with the call to duty, we must remember that we are Airmen. As Airmen, we understand the price that is paid for freedom and the sacrifices that come from willingly serving our country. We understand the meaning of belonging to the profession of arms. 25.14. The Airman’s Creed The Airman's Creed was presented to the Air Force in
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2007 by General T. Michael Moseley, Chief of Staff of the U.S. Air Force. Moseley introduced the creed as an aspect of one of his top priorities to reinvigorate the Total Force. The intent of the creed was to enhance the building of a warrior ethos among Airmen and establish a coherent bond between the members of the U.S. Air Force. 269 # THE AIRMAN’S CREED # I am an American Airman. # I am a Warrior. # I have answered my Nation’s call. # I am an American Airman. # My mission is to Fly, Fight, and Win. # I am faithful to a Proud Heritage, # A Tradition of Honor, # And a Legacy of Valor. # I am an American Airman. # Guardian of Freedom and Justice, # My Nation’s Sword and Shield, # Its Sentry and Avenger. # I defend my Country with my Life. # I am an American Airman. # Wingman, Leader, Warrior. # I will never leave an Airman behind, # I will never falter, # And I will not fail.
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Title: EPR-Biodosimetry URL Source: Markdown Content: # Cytogenetic Dosimetry: Applications in Preparedness for and Response to Radiation Emergencies P u b l i C A t i o n D At E : S E P t E m b E R 2 0 1 1 E m E R g E n C y P R E PA R E D n E S S > A n D R E S P o n S E > E P R - > bi o DoSi m EtRy # 2 0 1 1 IAEA SAFETY STANDARDS AND RELATED PUBLICATIONS IAEA SAFETY STANDARDS Under the terms of Article III of its Statute, the IAEA is authorized to establish or adopt standards of safety for protection of health and minimization of danger to life and property, and to provide for the application of these standards. The publications by means of which the IAEA establishes standards are issued in the IAEA Safety Standards Series . This series covers nuclear safety, radiation safety, transport safety and waste safety. The publication categories in the series are Safety Fundamentals , Safety Requirements and Safety Guides .Information on the IAEAs safety standards programme is available at the IAEA Internet site The site provides the texts in English of published and draft safety standards. The texts of safety standards issued in Arabic, Chinese, French, Russian and Spanish, the IAEA Safety Glossary and a status report for safety standards under development are also available. For further information, please contact the IAEA at PO Box 100, 1400 Vienna, Austria. All users of IAEA safety standards are invited to inform the IAEA of experience in their use (e.g. as a basis for national regulations, for safety reviews and for training courses) for the purpose of ensuring that they
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continue to meet users needs. Information may be provided via the IAEA Internet site or by post, as above, or by email to Official.Mail@iaea.org. RELATED PUBLICATIONS The IAEA provides for the application of the standards and, under the terms of Articles III and VIII.C of its Statute, makes available and fosters the exchange of information relating to peaceful nuclear activities and serves as an intermediary among its Member States for this purpose. Reports on safety and protection in nuclear activities are issued as Safety Reports ,which provide practical examples and detailed methods that can be used in support of the safety standards. Other safety related IAEA publications are issued as Radiological Assessment Reports , the International Nuclear Safety Groups INSAG Reports , Technical Reports and TECDOCs . The IAEA also issues reports on radiological accidents, training manuals and practical manuals, and other special safety related publications. Security related publications are issued in the IAEA Nuclear Security Series .The IAEA Nuclear Energy Series consists of reports designed to encourage and assist research on, and development and practical application of, nuclear energy for peaceful uses. The information is presented in guides, reports on the status of technology and advances, and best practices for peaceful uses of nuclear energy. The series complements the IAEAs safety standards, and provides detailed guidance, experience, good practices and examples in the areas of nuclear power, the nuclear fuel cycle, radioactive waste management and decommissioning. CYTOGENETIC DOSIMETRY: APPLICATIONS IN PREPAREDNESS FOR AND RESPONSE TO RADIATION EMERGENCIES AFGHANISTAN ALBANIA ALGERIA ANGOLA ARGENTINA ARMENIA AUSTRALIA AUSTRIA AZERBAIJAN BAHRAIN BANGLADESH BELARUS BELGIUM BELIZE BENIN BOLIVIA BOSNIA AND HERZEGOVINA BOTSWANA BRAZIL BULGARIA BURKINA FASO BURUNDI CAMBODIA CAMEROON CANADA CENTRAL AFRICAN REPUBLIC CHAD CHILE CHINA COLOMBIA CONGO COSTA RICA CÔTE DIVOIRE CROATIA CUBA CYPRUS CZECH REPUBLIC DEMOCRATIC REPUBLIC OF THE CONGO DENMARK DOMINICAN
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REPUBLIC ECUADOR EGYPT EL SALVADOR ERITREA ESTONIA ETHIOPIA FINLAND FRANCE GABON GEORGIA GERMANY GHANA GREECE GUATEMALA HAITI HOLY SEE HONDURAS HUNGARY ICELAND INDIA INDONESIA IRAN, ISLAMIC REPUBLIC OF IRAQ IRELAND ISRAEL ITALY JAMAICA JAPAN JORDAN KAZAKHSTAN KENYA KOREA, REPUBLIC OF KUWAIT KYRGYZSTAN LATVIA LEBANON LESOTHO LIBERIA LIBYA LIECHTENSTEIN LITHUANIA LUXEMBOURG MADAGASCAR MALAWI MALAYSIA MALI MALTA MARSHALL ISLANDS MAURITANIA MAURITIUS MEXICO MONACO MONGOLIA MONTENEGRO MOROCCO MOZAMBIQUE MYANMAR NAMIBIA NEPAL NETHERLANDS NEW ZEALAND NICARAGUA NIGER NIGERIA NORWAY OMAN PAKISTAN PALAU PANAMA PARAGUAY PERU PHILIPPINES POLAND PORTUGAL QATAR REPUBLIC OF MOLDOVA ROMANIA RUSSIAN FEDERATION SAUDI ARABIA SENEGAL SERBIA SEYCHELLES SIERRA LEONE SINGAPORE SLOVAKIA SLOVENIA SOUTH AFRICA SPAIN SRI LANKA SUDAN SWEDEN SWITZERLAND SYRIAN ARAB REPUBLIC TAJIKISTAN THAILAND THE FORMER YUGOSLAV REPUBLIC OF MACEDONIA TUNISIA TURKEY UGANDA UKRAINE UNITED ARAB EMIRATES UNITED KINGDOM OF GREAT BRITAIN AND NORTHERN IRELAND UNITED REPUBLIC OF TANZANIA UNITED STATES OF AMERICA URUGUAY UZBEKISTAN VENEZUELA VIETNAM YEMEN ZAMBIA 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’’. The following States are Members of the International Atomic Energy Agency: CYTOGENETIC DOSIMETRY: APPLICATIONS IN PREPAREDNESS FOR AND RESPONSE TO RADIATION EMERGENCIES ## INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 2011 # Emergency Preparedness and Response > E P R -b i o D o S i m E t R y # 2 0 1 1 COPYRIGHT NOTICE All IAEA scientific and technical publications are protected by the terms of the Universal Copyright Convention as adopted in 1952 (Berne) and as revised in 1972 (Paris). The copyright has
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since been extended by the World Intellectual Property Organization (Geneva) to include electronic and virtual intellectual property. Permission to use whole or parts of texts contained in IAEA publications in printed or electronic form must be obtained and is usually subject to royalty agreements. Proposals for non-commercial reproductions and translations are welcomed and considered on a case-by-case basis. Enquiries should be addressed to the IAEA Publishing Section at: Sales and Promotion, Publishing Section International Atomic Energy Agency Vienna International Centre PO Box 100 1400 Vienna, Austria fax: +43 1 2600 29302 tel.: +43 1 2600 22417 email: sales.publications@iaea.org For further information on this publication, please contact: Incident and Emergency Centre Department of Nuclear Safety and Security International Atomic Energy Agency Vienna International Centre PO Box 100 1400 Vienna, Austria email: official.mail@iaea.org CYTOGENETIC DOSIMETRY: APPLICATIONS IN PREPAREDNESS FOR AND RESPONSE TO RADIATION EMERGENCIES IAEA, VIENNA, 2011 IAEA-EPR © IAEA, 2011 Printed by the IAEA in Austria September 2011 FOREWORD Cytogenetic dosimetry 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 association has been through a sequence of coordinated research programmes (CRPs), the running of regional and national training courses, the sponsorship of individual training fellowships, and the provision of equipment to laboratories in Member States, establishing capabilities in biological dosimetry. From this has arisen the provision to Member States of advice regarding the best focus
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for research and suggestions for the most suitable techniques for future practice in biological dosimetry. One CRP resulted in the publication in 1986 of a manual, entitled Biological Dosimetry: Chromosomal Aberration Analysis for Dose Assessment (Technical Reports Series No. 260). This was superseded in 2001 by a revised second edition, Technical Reports Series No. 405. This present publication constitutes a third edition, with extensive updating to reflect the considerable advances that have been made in cytogenetic biological dosimetry during the past decade. The IAEA wishes to express its thanks to all authors and reviewers of this publication. The major contributions of Dr. D. Lloyd are especially acknowledged. This publication has been co-sponsored by the Pan American Health Organization and the World Health Organization. The IAEA officer responsible for this publication was E. Buglova of the Department of Nuclear Safety and Security. EDITORIAL NOTE 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. CONTENTS 1. INTRODUCTION .............................................................................................................1 1.1. Background ..............................................................................................................1 1.2. Objective ..................................................................................................................4 1.3. Scope and History of Development .........................................................................4 1.4. Structure ...................................................................................................................5 2. APPLICATION OF DOSE CONCEPTS IN BIOLOGICAL DOSIMETRY ...................7 3. BIOPHYSICAL BACKGROUND TO CHROMOSOME DAMAGE .............................9 4. HUMAN LYMPHOCYTES............................................................................................15 5. CHROMOSOMAL STRUCTURE .................................................................................19 5.1. Chromatin Packing .................................................................................................19 5.2. Human Karyotype and DNA Content of Chromosomes........................................19 5.3. Cell Cycle ...............................................................................................................23 6. RADIATION INDUCED CHROMOSOMAL ALTERATIONS ...................................25 6.1. Radiation Induced DNA Lesions ..............................................................................25 6.2. Chromosome-Type Aberrations ...............................................................................27 6.3.
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Chromatid-Type Aberrations ....................................................................................32 6.4. Premature Chromosome Condensation ....................................................................35 6.5. Micronuclei ...............................................................................................................37 7. BLOOD SAMPLING ......................................................................................................41 7.1. Timing ....................................................................................................................41 7.2. Anticoagulant .........................................................................................................41 7.3. Containers ..............................................................................................................42 7.4. Transport ................................................................................................................42 8. PRODUCTION OF AN IN VITRO DOSE–RESPONSE CURVE ................................45 8.1. General Considerations ..........................................................................................45 8.2. Physical Considerations .........................................................................................45 8.3. Statistical Considerations .......................................................................................48 9. DICENTRIC ANALYSIS ...............................................................................................53 9.1. Culturing ................................................................................................................53 9.2. Fixation Procedure .................................................................................................57 9.3. Staining ..................................................................................................................58 9.4. Analysis of Slides ...................................................................................................60 9.5. Recording of Data ..................................................................................................61 9.6. Storage of Information and Slides..........................................................................62 9.7. Dose Assessment ....................................................................................................63 10. TRANSLOCATION ANALYSIS ...................................................................................81 10.1. Cell Culture and Fixing Procedures .......................................................................83 10.2. Painting the Chromosomes ....................................................................................84 10.3. Scoring Criteria ......................................................................................................86 10.4. Data Handling ........................................................................................................87 10.5 The Control Level of Translocations .....................................................................90 10.6. Persistence of Translocations .................................................................................93 10.7. Calibration Curves .................................................................................................94 10.8. Examples of Fish Being Used for Retrospective Biological Dosimetry ................95 11. PREMATURE CHROMOSOME CONDENSATION (PCC) ANALYSIS .................103 11.1. PCC by Mitotic Fusion.........................................................................................103 11.2. PCC by Chemical Induction.................................................................................108 11.3. A Radiation Accident Investigated by the PCC Rings Method ...........................111 12. THE CYTOKINESIS-BLOCK MICRONUCLEUS (CBMN) ASSAY .......................113 12.1. Background ..........................................................................................................113 12.2. Lymphocite Culture for CBMN Assay ................................................................114 12.3. CBMN Assay Scoring Criteria.............................................................................114 12.4. CBMN Assay Data Handling ...............................................................................116 12.5. Application of the CBMN Assay for Biological Dosimetry ................................120 13. AUTOMATION OF CHROMOSOMAL ASSAYS .....................................................125 13.1. Automated Sample Processing .............................................................................125 13.2. Automated Image Analysis ..................................................................................126 13.3. Laboratory Information Management System (LIMS) ........................................130 14. MASS CASUALTY EVENTS ......................................................................................133 14.1. Potential Radiation Exposure Scenarios ..............................................................133 14.2. Historical Experience ...........................................................................................134 14.3. Role of Biological Dosimetry ..............................................................................135 14.4. Existing Mass Casualty Strategies .......................................................................137 15. QUALITY PROGRAMMES AND THE ISO STANDARDS .........................................141 15.1. The Rationale for a Quality Assurance and Quality Control Programme ...........141 15.2. Current Structure of the ISO 19238 Document....................................................142 15.3. Application to Population Triage .........................................................................143 16. SAFETY OF LABORATORY STAFF .........................................................................147 16.1. Infection ...............................................................................................................147 16.2. Optical ..................................................................................................................147 16.3. Chemical ..............................................................................................................148 REFERENCES........................................................................................................................151 ANNEX
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I: DICENTRIC ASSAY......................................................................................173 ANNEX II: FISH BASED TRANSLOCATION ASSAY..................................................177 ANNEX III: PREMATURE CHROMOSOME CONDENSATION ...................................181 ANNEX IV: CYTOKINESIS-BLOCK MICRONUCLEUS ASSAY .................................187 ANNEX V: CRITERIA FOR DETERMINING MITOTIC INDEX ..................................193 ANNEX VI: STATISTICAL ANALYSIS ...........................................................................195 Annex VII: AN EXAMPLE OF AN INTERLABORATORY COMPARISON EXERCISE FOR QUALITY ASSURANCE..................................................203 REFERENCES TO THE ANNEXES .....................................................................................207 LIST OF ABBREVIATIONS .................................................................................................209 DEFINITIONS ........................................................................................................................213 CONTRIBUTORS TO DRAFTING AND REVIEW ............................................................227 ACKNOWLEDGEMENTS OF COMMENTS RECEIVED .................................................229 1. INTRODUCTION 1.1. BACKGROUND Biological dosimetry, based on the analysis of solid stained dicentric chromosomes, has been used since the mid-1960s. The intervening years have seen great improvements bringing the technique to a point where dicentric analysis has become a routine component of the radiation protection programmes of many Member States . Experience of its application in thousands of cases of actual or suspected overexposures has proved the worth of the method and also helped to define its limitations. Biological dosimetry using chromosome damage biomarkers is particularly important because, unlike physical measurement of dose, it takes into account interindividual variation in susceptibility. It should be emphasized that chromosomal aberrations are used as a dosimeter and provide one input, frequently a very important one, into the compendium of information that needs to be collected and considered when a nuclear or radiological emergency is investigated 1 . Diagnostic sources of information may come from other biologically-based radiation biomarkers as well as clinical signs and symptoms that persons might display, and also from physical measurements such as those made on personal monitoring badges and thermoluminescence, optically stimulated luminescence or electron spin resonance on solid matrix components from (i.e. teeth dental enamel, fingernails, extracted bone, etc.) or associated (i.e. watch or eye glasses, etc.) with the irradiated persons. Information from questioning of patients and witnesses on basic facts such as time in the locality and distance
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from the radiation source may also assist with dose calculations. All these sources of information may be combined with biological dosimetry to obtain a clearer evaluation of the case. For many years the dicentric assay using blood lymphocytes was the only method of biological dosimetry available, and still today it is the technique most frequently used. The dicentric and other aberrations may also be observed in other cells such as skin fibroblasts and buccal epithelial cells but this is beyond the scope of this publication which is confined to assays with blood lymphocytes. However there are now a number of other biological endpoints: micronuclei, translocations and aberrations in prematurely condensed chromosomes (Fig. 1 and Table 1) that can also be assayed in lymphocytes and these are described. 1 In this context, a ‘radiation emergency’ means the same as a ‘nuclear or radiological emergency’. 1FIG. 1. Schematic for sample accession of peripheral blood lymphocytes for various cytogenetic-chromosome aberration assays i.e., Premature chromosome condensation (PCC) assay, metaphase spread dicentric (and ring) chromosome aberration assay (DCA), metaphase spread fluorescence in situ hybridation (FISH) translocation assay, and cytokinesis-block micronuclei (CBMN) assay used for dose assessment. 2TABLE 1. COMPARISON OF CYTOGENETIC ABERRATION ASSAYS USED FOR DOSE ASSESSMENT aOther assays, beyond the scope of this publication, use molecular endpoints that measure DNA breakage, changes in the regulation of some sentinel genes or the presence of protein biomarkers that may be detected within cells or in blood plasma/serum. This is an area of rapidly emerging technologies with a number of assays at differing stages in development and verification. The range of biological dosimetry options now available have led to proposals for a multiparametric approach to investigating an overexposed person and having a variety of assays available may be particularly useful if a laboratory has to deal
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with an event involving many casualties. In the investigation of radiation emergencies it is important to estimate the dose to exposed persons for several reasons. In the case of high exposures (>1 Gy acute), information on doses assists in the planning of therapy and in alerting physicians to likely deterministic (tissue injury) health consequences that could arise in the following weeks and months. For exposures below the level where treatment is needed, dosimetric information is important for the physician in counselling irradiated persons on the risk of their developing stochastic consequences — i.e. cancer. For persons whose exposure is very low, the knowledge that no significant elevation in chromosomal damage could be found is frequently Cytogenetic Aberration Assays Premature chromosome condensation (PCC) Dicentric (and ring) (DCA) Fluorescent in situ hybridization (FISH) Cytokinesis-block micronucleus (CBMN) Typical aberrations scored for biological dosimetry applications excess chromosome fragments; dicentrics b and rings dicentrics b(and rings) dicentrics b (and rings) micronuclei translocations b nucleoplasmic bridges translocations bTypical radiation scenario applications acute acute acute acute protracted protracted protracted recent exposure recent exposure old exposure recent exposure Photon equivalent, acute dose range (Gy) for whole-body dose assessment 0.2 to 20 0.1 to 5 0.25 to 4 0.3 to 4 Useful for partial-body exposure applications Yes Yes NA c NA Useful for triage dose assessment Yes Yes NA Yes Status of assay standardization NA ISO standards [3, 4] NA ISO standard — pending, and a Table modified from TMT Handbook . b Specific chromosome aberrations typically detected by use of centromeric and whole-chromosome specific DNA hybridization probes. c NA: not applicable/not available. 3very reassuring. This is particularly the case where details of events are poorly known and no physical dose measurements or calculations are available. Then biological dosimetry may be the only means of quantifying dose, although,
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as will be discussed, there are quantification problems associated with factors such as non-uniform exposures, intake of radionuclides and delayed blood sampling. Biological dosimetry also has a valuable role to contribute in the early period after a radiation emergency or terrorist attack where many persons may have been exposed. At this time, triage of casualties using biological and clinical endpoints that initially and rapidly can identify individuals suspected of exposure to life-threatening doses as well as to provide a triage dose is needed. 1.2. OBJECTIVE The primary objective of this publication is to provide the user with technical information for selecting and implementing, in a standardized manner, the appropriate cytogenetic technique to ensure comparable dose assessment following accidental exposure to ionizing radiation. The publication describes the four possible cytogenetic methods (Fig. 1 and Table 1) currently available for biological dosimetry. It is appropriate to have all these techniques readily available in main geographical regions, but, given a degree of international cooperation and networking, it is not necessary to have all of them available in each national biological dosimetry laboratory. 1.3. SCOPE AND HISTORY OF DEVELOPMENT The first manual in this series concentrated exclusively on the dicentric assay. That timely publication provided a valuable and frequently cited landmark in biological dosimetry. It was written to be read at two levels. First, it was to serve as a laboratory manual, providing a convenient and comprehensive source of information at the technical level. In addition, it was intended to provide a concise summary of the technical background of the subject, for use in teaching radiobiology or for persons such as health physicists, lawyers or policy makers who may require some professional understanding of biological dosimetry. In a revised edition of this manual , published 15 years later, FISH chromosome painting, premature chromosome
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condensation (PCC) and micronuclei (CBMN) assays were included. Now, a further 10 years later, this document has been produced. Much of the original text on the metaphase spread dicentric (and ring) chromosome aberration assay (DCA) from the earlier editions is still valid and has been retained although where appropriate, updated. The FISH, PCC and CBMN assays have been considerably revised in the light of recent research and experience in using them. It was inevitable that, over the 25 years spanned by these three editions, the topic of cytogenetic dosimetry would expand considerably and become more technically complex. However the present revision is arranged so that a minimally equipped laboratory newly entering the field should not find the publication too daunting. It is possible still to extract material from just those sections that relate to the two most important core assays that should be established, namely the dicentric and micronucleus assays. Clear advice is given on how to apply them in practice, by constructing basic dose response curves and interpreting the data from overexposure case investigations. A major new development in recent years, reflected in this revision, has been the arrangements to perform triage in radiation mass casualty events. Considerations are provided of how a biological dosimetry laboratory can respond to a sudden surge in cases by using assays in a triage mode, speeding up analyses with computer assisted microscopy and by networking with other laboratories. Coincidentally with this increased provision for 4collaborative working in emergency response have come international guidelines on quality assurance, quality control and participation in interlaboratory comparison exercises. These topics are now covered in this revision. Multiple cytogenetic assays are useful for biological dosimetry since no one single assay is sufficiently robust for all potential radiation scenarios including early-phase acute-exposures, partial-body exposures, retrospective or prior exposure (e.g.
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biosampling years after exposure) as well as applications involving triage cytogenetics for radiation mass casualty events. 1.4. STRUCTURE This publication is arranged as follows following this Introduction (Section 1), in Section 2 consideration is given to what is meant by ‘dose’ as determined from chromosome damage and how this relates to the values of personal dose derived by physical methods and the concept of equivalent dose as defined by the International Commission of Radiological Protection (ICRP). In Section 3 some biophysical and microdosimetric background to the induction of chromosomal damage is described. This is followed, in Section 4, by a brief description of the human lymphocytes from which the T types are the cells used for biological dosimetry. In Section 5 the chromosomal structure is outlined. In Section 6 the types of DNA lesion induced by interactions with ionizing radiation, together with a description and classification of those chromosomal alterations that can be observed in lymphocytes after irradiation are discussed. In Section 7 the requirements of blood sampling are described and Section 8 considers physical, biological and statistical requirements for constructing dose response curves. Sections 9, 10, 11 and 12 then describe the techniques for performing biological dosimetry with, respectively, the four cytogenetic endpoints of dicentrics, fluorescence in situ hybridization based translocations, prematurely condensed chromosomes and micronuclei. In Section 13 the considerable advances that have been made in recent years in developing automatic analysis of the chromosomal assays are described and in Section 14 another recent development, how the chromosomal dosimetry community can respond most effectively to mass casualty events, is considered. In Section 15 the guidance and procedures for quality assurance are described and the final Section 16 discusses safety of laboratory staff carrying out cytogenetic analysis. The comprehensive up to date reference list is followed by seven Annexes,
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the first four describing reproducible working protocols for DCA, FISH based translocation, PCC and CBMN assays. Annex V describes the criteria for measuring the mitotic index and Annex VI contains a guide to a number of statistical tests that are commonly employed in biological dosimetry data analysis and its underlying research. The final Annex, VII, presents a worked example of an interlaboratory quality assurance exercise on dicentric scoring and dose estimation. The publication concludes with a list of abbreviations used, a glossary of important technical terms, and finally a list of contributors to the drafting and peer review of this much revised third edition of the publication. 52. APPLICATION OF DOSE CONCEPTS IN BIOLOGICAL DOSIMETRY This section provides brief information on dosimetric terms, on the physical meaning of absorbed dose and on its interpretation for biological (cytogenetic) assessment of the dose from accidental exposure to different types of ionizing radiation. Chromosome aberrations in lymphocytes are used to estimate absorbed dose to overexposed persons. The aberrations scored in the lymphocytes are interpreted in terms of absorbed dose by reference to a dose response calibration curve. This curve will have been produced by exposure of blood in vitro to doses of the appropriate quality of radiation. The doses given to the specimens should be traceable via a physical instrument such as an ionization chamber, to a primary or secondary standard. Physical devices that measure photons and neutrons are usually calibrated in terms of air kerma, and therefore when considering doses delivered to tissue (or blood specimens) correction factors need to be applied. For photons, these are derived from the ratio of mass energy absorption coefficients, and the values to be used may be obtained from standard tables . For neutrons, instruments may be made of tissue equivalent material and thus indi-cate dose
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to tissue. Alternatively, some primary or secondary dosimetry laboratories calibrate in terms of neutron fluence, which may be converted to dose to tissue. As the biological endpoint being scored is chromosomal aberrations, strictly speaking these reflect dose to the cells’ nuclei. For photons and neutrons, dose to soft tissue is a very good approximation to the dose to the nucleus. This is because the lymphocyte nucleus diameter is small, ~6 μm, compared with the ranges of secondary particles produced by both photons and neutrons. Thus the Bragg-Gray cavity theory can be applied . There are, however, a few exceptions. For example, with exposure to tritiated water, the distances travelled by the beta particles lie in the range 0–7 μm. Therefore, most of the dose to a cell nucleus is due to emissions from tritium contained within that nucleus. In this case, the dose to the lymphocyte nucleus forms the basis of calibration, and this depends on the water content of the nucleus, with respect to that of blood . Another example could be exposure to low energy neutrons of less than about 100 keV, where the recoil protons have a range of less than 2 μm. In this case the dose to the lymphocyte nucleus would relate to its hydrogen content. It is, however, unlikely that an accident would involve exposure to neutrons predominantly in this energy range. The dose value obtained by referring a measured yield of aberrations, such as dicentrics, to a calibration curve represents an averaged absorbed dose to the lymphocytes. This would approximate to an averaged out whole body dose because lymphocytes are widely distributed around the body and are mobile. By methods to be described later in this publication, it is sometimes possible to refine the whole body dose estimate for situations where non-uniform or
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partial body irradiations from external sources have occurred. Most internally incorporated radionuclides also result in non-uniform irradiations but here the dose of concern is not that to the lymphocytes but rather that to the specific organs and tissues where the radioactivity deposits. The usefulness of chromosomal analysis is often somewhat limited because, for example, following an intake of radioiodine, aberrations will be induced in lymphocytes but these cannot be interpreted in terms of dose to the thyroid. Exceptions to this are those nuclides that have a wide distribution in the body such as tritiated water or radiocaesium where experience has shown that lymphocyte aberration analysis provides meaningful dose estimates. 7For retrospective biological dosimetry, a decade or more after exposure, where translocations are measured by the FISH method, the dose estimate represents average dose to the active bone marrow. This is because the original exposure was to the stem cell precursors of the lymphocytes that are scored. For shorter times the translocations will be observed in a mixture of long-lived lymphocytes and descendants of irradiated stem cells. Often it is the result of a routine measurement of dose recorded by an individual dosimeter that triggers an investigation. Individual dosimeters are normally calibrated to measure the personal dose equivalent at a specified depth. This operational quantity provides a reasonable estimate of effective or equivalent dose in most radiation fields encountered in practice. Effective dose and equivalent dose are intended for use in radiological protection. They are not suitable for determining the effects of high absorbed doses. It is therefore recommended that laboratories undertaking biological dosimetry should calibrate their procedures in terms of absorbed dose (Gy) specifying, where appropriate, sufficient details to characterize the radiation type and quality [12–15]. 83. BIOPHYSICAL BACKGROUND TO CHROMOSOME DAMAGE This section provides information that is intended to
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aid in understanding and interpreting the principles that underlie the methodology presented in the later sections. Refs [16, 17] should be consulted for additional information. When ionizing radiation passes through an object, it ejects electrons from the atoms through which it travels, leaving positively charged ions. The distribution of primary events, ionizations and excitations along the track of an ionizing particle will vary according to the type of radiation. The average separation of these primary events decreases with increasing charge and mass of particles (neutrons or alpha particles). As will be discussed below, it is necessary to define a particular radiation in terms of the amount of energy deposited per unit of track length, because this characteristic alters the effectiveness of the particular radiation type in inducing various biological endpoints. A useful comparative term to describe the deposition of energy by different types of radiation is linear energy transfer (LET). For radiations with a wide range of LET, e.g. neutrons, an average LET may be derived. This may be obtained by weighting each LET interval according to the energy imparted (or dose) or according to the length of the track travelled. These give, respectively, dose average and track average LET. Track average appears to be the better quantity to describe the relative biological effectiveness (RBE) variations for chromosomal damage . The track average LET for 250 kVp (kilovolts peak) X rays is about 2 keV/ μm, as compared with heavy charged particles that have track average LET values of 100–2000 keV/ μm or greater. The important point to consider is that the same and various types of radiation can differ considerably in the quantity of energy deposited per micrometre of track, and this can clearly alter the biological effectiveness of different types of radiation. One consequence of the distribution of
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ionization for radiation of different LET is in the frequency distribution of chromosome aberrations between cells. With low LET, or sparsely ionizing radiation, the ionization at any particular dose will be randomly distributed between cells, particularly since there will be a very large number of tracks. The DNA damage will also be randomly distributed between cells and, on the assumption that there is an equal probability that any damage can potentially be converted into an aberration, therefore, the aberrations will be also randomly distributed between cells. This has been shown to be the case following X or γ irradiation, where the induced chromosome aberrations fit a Poisson distribution. With high LET, or densely-ionizing radiation, the ionization tracks will be non-randomly distributed between cells, with the energy being deposited in more ‘discrete packets’. The number of tracks will be much lower than with low LET radiation at equivalent doses. The result, making the same assumptions as for low LET radiation, is that the induced aberrations will be non-randomly distributed between cells. At any observed mean aberration frequency, there will be more cells with multiple aberrations and with zero aberrations than expected from a Poisson distribution. These features can be of use in biological dosimetry, as will be discussed in Section 9.7.4.3 particularly with regard to non-uniform or partial body exposures. The effectiveness of different types of radiation for inducing a particular biological endpoint is commonly represented by the term ‘relative biological effectiveness’ (RBE). The RBE is defined as the ratio of the dose of the reference radiation (usually, orthovoltage X rays) to the dose of the particular radiation being studied that produces the same biological effect. That is, 9fect Z oducing ef diation pr dose of ra g effect Z s producin kVp X ray -of dose RBM 250 200 =
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(1) It should be noted that X-rays are 2–3 times more effective than gamma rays and therefore the reference radiation should always be defined . Fig. 2 shows the typically shaped linear and linear quadratic dicentric dose response curves obtained with, respectively, high and low LET radiations. FIG. 2. Typical linear and linear quadratic dose response curves, showing how RBE changes with yield . The reasons for the shapes are discussed later in this Section. The RBE at a high dicentric yield that would be associated with high doses is illustrated by the upper horizontal dashed line which intercepts the two curves at 1.0 and 3.5 Gy. The RBE is the ratio of the two doses which is 3.5/1.0 = 3.5. The lower horizontal dashed line intercepts at 0.1 and 1.0 Gy, resulting in a higher RBE: 1.0/0.1 = 10. The maximum RBE, which describes the situation at low doses, usually designated as RBE m , would be the ratio of the linear coefficients of the two curves’ yield equations. It has been shown for many endpoints (including mutations, cell killing and chromosome aberrations) that the RBE varies with LET such that a hump-shaped response curve is obtained (Fig. 3). 10 FIG. 3. Generalized relationship between RBE and LET . This curve shows that the RBE increases up to an optimum value of about 100 keV/ μm and then decreases at higher values of LET. The interpretation of the curve is best considered here for the induction of chromosome aberrations. For illustrative purposes, the dicentric aberration is used as an example, partly because it clearly involves an interaction (or exchange) between two chromosomes, and also because it is the aberration type that is most frequently used in biological dosimetry. In order to produce a dicentric aberration, DNA damage must be
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induced in the two unreplicated chromosomes involved such that the damaged chromosomes can undergo exchange. This exchange can occur either as a result of the misrepair of DNA strand breaks induced directly by the radiation, or as a result of misrepair during the excision repair of base damage. Thus it can be seen that the lesions in the two chromosomes must be close together, within what is called ‘rejoining distance’, for misrepair to be able to take place. This defined region can be considered as the target. Two lesions, one in the DNA double helix of each unreplicated chromosome, need to be produced within this target. This target, or zone of interaction, is small, generally considered to be less than 1.0 μm diameter. X rays have low LET, with low frequencies of ionization per unit track length. Thus there is a low probability that two ionizing events from a single track will occur within the target. Two ionizations, at a minimum, are necessary to produce damage in the two chromosomes involved in a dicentric. There is a much higher probability that the two lesions will be produced by ionization from two independent tracks. Dicentrics produced by one track will have a frequency that is proportional to a linear function of dose, whereas dicentrics induced by two tracks will have a frequency proportional to the square of the dose. At doses below 0.5 Gy, the probability of two tracks traversing a target is sufficiently low that dicentrics will be produced almost exclusively by one track and at a low frequency. As the dose increases, the contribution of two track induced dicentrics will also increase. Thus the dose–response curve for low LET induced dicentrics (Fig. 2) will be a combination of one- and two-track events, with the former being more frequent at
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low and the latter much more frequent at high doses. The dose– response curve is generally assumed to fit Eq. (2) 11 2 DDCY βα ++= (2) where: Y is the yield of dicentrics, D is the dose, C is the control (background frequency), α is the linear coefficient, and β is the dose squared coefficient. The ratio of α/β can be referred to as the cross-over dose. It is equal to the dose at which the linear and the quadratic components contribute equally to the formation of dicentrics. As the LET of the radiation increases up to a maximum, there is a greater probability that two lesions within the target will be induced by two ionizing events along the same track, resulting in two consequences. The dose–response curve at linear energy transfers above approximately 20 keV/ μm will be linear (Fig. 2). Also, the efficiency, or RBE, of the higher LET radiation for inducing dicentrics increases with increasing LET as a result of the increasing probability that the two lesions will be produced by one track. Producing the two required lesions by one track is much more efficient than the random process of producing a lesion by a second track close to a lesion already produced by another track, particularly at lower doses, where the track density is low. The maximum RBE will be at a LET value where ionization is optimally spaced to produce damage in each of the two DNA helices involved in the formation of dicentrics without ‘wasting’ energy, that is, depositing more ionization in the target than is needed. However, as LET increases above this optimum value, more energy will be deposited in the target than is necessary, and under these circumstances the RBE will decrease as LET increases, as shown in the plot of
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RBE versus LET in Fig. 3. Summarizing this discussion, the dose–response curve (Fig. 2) for low LET radiation, high energy protons and fast neutrons will be non-linear and best fit a linear-quadratic model; the dose–response curve for high LET radiation (fission neutrons and alpha particles) will be linear, or close to linear; the RBE increases with increasing LET to a maximum of around 100 keV/ μm and decreases at higher LET values (Fig. 3). How does the dose rate affect the yield of cytogenetic alterations? For the purpose of this discussion, it is easier to refer to dicentrics although the principles also apply to micronuclei and translocations. It is known that those lesions induced in the DNA that can be converted into dicentrics can be repaired, taking from a few minutes up to several hours, depending on the particular lesion. If the two lesions needed for inducing a dicentric are produced by separate tracks, and the dose rate is reduced, there is a probability that the lesion produced by the first track will be repaired before the target is traversed by a second track, forming the second lesion. Although two lesions have been produced within the target, they cannot interact to produce a dicentric. The probability of the two lesions being able to interact will decrease with decreasing dose rate, the lower the dose rate, the lower the frequency of ionization tracks per unit time and thus the longer the time available for repair of the first lesion before the second can be formed. Thus the situation for low LET radiation is the following: lowering the dose rate decreases the dicentric frequency per unit dose. The dose– response curve for dicentrics at very low dose rates, where the probability of two-track aberrations is essentially zero, will be linear, with a
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slope equal to that of the linear portion of the linear-quadratic curve for acute exposures. The same argument holds true for fractionated or split doses. If two or more doses are received, lesions from the first can interact with lesions produced by the second, or subsequent, dose, provided that the time interval between the first dose and the subsequent dose fraction is not longer than the time it takes to repair the lesions induced by the first or previous dose. Thus, if doses are separated 12 by times long enough to allow repair between dose fractions, the frequency of dicentrics produced by the total dose (the sum of the fractions) will be less than that from the total dose delivered at one time. The situation can be different with regard to high LET radiation, since both lesions involved in the induction of dicentrics are produced by a single track. Thus, lowering the dose rate does not alter the frequency of dicentrics, because repair of the lesions during longer exposures will not be an influencing factor. The same argument applies to fractionated exposures; the repair of lesions between the fractions does not have much influence since both are produced concurrently by a single track. The points discussed in this section indicate factors that should be considered in the practice of biological dosimetry. The shape of the dose–response curve is influenced by the radiation quality (LET). Therefore, when estimating dose, the standard curve to be used should be that of a radiation quality which is the same as, or very similar to, that of the particular type of radiation involved in the emergency. This is an important requirement because there are demonstrable differences in RBE for induced chromosome damage by various low LET radiations even though for radiological protection purposes, they are
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weighted identically (W R = 1) . For low LET radiation, decreasing the dose rate also decreases the dicentric frequency per unit dose, such that at very low dose rates the curve is linear and is the same as the linear component of the dose–response curve for acute exposure. A linear curve can be produced from a standard acute curve for X and/or γ rays and could possibly be used as a standard curve for chronic exposures, with appropriate corrections for the duration of the exposure and the lifetimes of lymphocytes. With high LET radiation, changes in dose rate do not affect dicentric frequency, and so the curve obtained for acute exposures can be used for chronic or fractionated exposures, again taking into consideration the duration of exposure and the lifetimes of peripheral lymphocytes. 13 4. HUMAN LYMPHOCYTES Human peripheral lymphocytes represent a cell population which is predominantly in a DNA presynthetic stage of the cell cycle (i.e. the G 0 phase). Only 0.2% or less of the peripheral lymphocytes are in the autosynthetic cell cycle, and these probably come from the pool of large lymphoid cells representing stimulated lymphocytes or immature plasma cells. Cells from this group may give rise to the rare mitoses found occasionally in peripheral blood. Nowell was the first to show that peripheral ‘human leukocytes’ can be stimulated to undergo in vitro mitoses by phytohaemagglutinin (PHA), a protein derived from the bean plant Phaseolus vulgaris. While Carstairs showed that ‘small lymphocytes’ are the target cells for mitogenic initiation by PHA. Peripheral small lymphocytes when observed in a blood smear have large dense nuclei surrounded by relatively little cytoplasm (Figs 4 and 5). They have a diameter of around 6 μm, and the volume is estimated to be around 110 μm 3 . FIG.
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4. A typical blood smear with a small lymphocyte and some red cells shown enlarged. FIG. 5. A small lymphocyte as seen in an electron microscope. 15 Two main types of lymphocytes can be distinguished, i.e. T and B cells. Both types originate from immunologically incompetent stem cells in the yolk sac and eventually settle in the bone marrow. These undifferentiated stem cells migrate into the thymus and other primary lymphoid organs, multiply there, undergoing somatic mutations and give rise to a pool of long lived lymphocytes that circulate. On the basis of their surface markers, T and B cells comprise a mixture of naïve and memory cells with differing life spans and differing roles in the immunological processes . It is the T cells, mostly of the CD4 + and CD8 + subtypes, which are stimulated in vitro by phytohaemagglutinin and are used for biological dosimetry. Lymphocyte concentrations in the peripheral blood vary as a function of age, ethnicity, presence of pathogens and environmental factors (i.e. smoking, obesity, alcohol use, etc.). For example, certain ethnical populations (i.e. in East Africa) exhibit lower baseline values of lymphocyte counts compared with the overall population reference levels. A trend towards a decrease of the lymphocyte count is observed with age. This is particularly visible during childhood when a continuous drop is observed to reach around 2x10 9 / L at 15 years old. The decrease tendency is also observed for adults but the drop is slower and at 75 years old and more, the lymphocyte number is below 2x10 9 / L . In general for a healthy adult the normal range of lymphocytes in peripheral blood is 1.5-4.0 x10 9 / L . However, in the case of irradiation to high doses of a few Gy to much of the body,
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one of the early deterministic reactions is a rapid fall in the peripheral blood lymphocyte count. These factors should be borne in mind for early blood sampling of radiation casualties for biological dosimetry . The total number of lymphocytes in a healthy young adult has been estimated to be approximately 500 x 10 9 . Only about 2% (10 x 10 9 ) of these are present in the peripheral blood, the others being located generally throughout other tissues, with particular concentrations in the thymus, lymph nodes, tonsils, the lymphatic tissues of the intestines, the spleen and in bone marrow. The lifetimes of lymphocytes are variable and the definition of lifespan can mean either that the cell dies or that it divides. T cells of the CD4 + and CD8 +subtypes can be further divided into subsets based on the expression of different isoforms of the CD45 antigen. At birth, >90% of T cells express CD45RA isoform, and these have been called unprimed or naïve cells. By adulthood, this falls to about 50% by conversion to a CD45RO subset of primed or memory cells. Chromosomal damage induced by radiotherapy has been studied in PHA stimulated T cells of both RA and RO forms . The persistence of unstable damage has shown that the naïve RA cells divide on average once every 3.5 years, whilst the memory RO cells divide more frequently, on average every 22 weeks. Memory cells may also revert to the naïve phenotype but only, on average, after about 3.5 years in the memory class. For interpreting in vivo induced chromosome aberrations in humans, it is of great importance that the bulk of the peripheral lymphocytes belongs to the ‘redistributional pool’. That is, the lymphocytes should be able to leave the peripheral blood, pass through the spleen, the
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lymph nodes and other tissues, and re-enter the circulation. The mean time that a given lymphocyte of the redistributional pool is present in the peripheral blood is about 30 min. It has been estimated that about 80%, that is, 400 x10 9 lymphocytes, belong to the redistributional pool and that the overall recirculation time is about 12 hours. This means that lymphocytes with chromosome aberrations that have been induced anywhere in the body will eventually be present in the peripheral blood. Thus, with the human lymphocyte test system, not only can chromosome aberrations that have been induced in lymphocytes in the peripheral 16 blood itself be detected, but also those that have been induced in lymphocytes distributed in different organs throughout the body . Most of the peripheral lymphocytes are in a ‘resting’ stage of the cell cycle (G 0 ) and have a diploid DNA content of about 5.6 pg. These cells can be initiated to undergo in vitro mitotic divisions by the introduction of phytohaemagglutinin (PHA). PHA is an extremely comprehensive mitogen that stimulates a broad spectrum of T cells. Under the influence of PHA, the lymphocytes are transformed into blastoid cells, and the volumes of the nucleus and of the whole cells increase. Peripheral lymphocytes 48 hours after stimulation have a cell volume of about 500 μm 3 , as compared with ~110 μm 3 before stimulation. The cytoplasmic volumes are ~50 μm 3 before and ~350 μm 3 after stimulation. Nuclear volume increases from about ~50 μm 3 to 170 μm 3 following stimulation. The cell cycle progression of lymphocytes following stimulation with PHA can be quite different depending on the culture conditions using different culture media such as Ham’s F-10, RPMI (Roswell Park Memorial Institute, RPMI-1640), medium TC-199, or minimum essential medium (MEM). For example,
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in Ham’s F-10 medium, the DNA synthesis starts about 26 hours after culture initiation and the first mitoses are found after about another 10 hours. There are two peaks of DNA synthesis measured by tritiated thymidine treatment, one at 34 hours and a second at 40 hours, and two peaks of mitotic activity, one at around 44 hours and a second at around 49 hours. This may represent two subpopulations of cells which show different stimulation patterns in a culture set up with Ham’s F-10 and PHA . However, in lymphocytes grown in TC-199 medium, the tritium labelled interphases, as well as the mitotic indices, follow an irregular pattern, thus making it difficult to draw any conclusions about the subpopulations. 17 5. CHROMOSOMAL STRUCTURE 5.1. CHROMATIN PACKING The association of DNA and histones in a nucleosome structure has been demonstrated in considerable detail, although the association of the non-histone proteins with the nucleosome assembly is not yet fully understood. In addition, it is clear that DNA is external to the histone core of the nucleosome. Some studies support the existence of an axial core structure formed by non-histone proteins or a non-histone protein scaffold [29, 30] in a metaphase chromosome. The involvement of such core structures in the formation of chromosome aberrations has not yet been elucidated. Core structures can also be demonstrated in a light microscope as silver stainable regions in the chromosome of different mitotic stages. Although the existence of an organized nuclear protein matrix in interphase is well documented, the existence of a scaffold in metaphase chromosomes is probably an artefact. A model of the organization of a metaphase chromosome is shown in Fig. 6. FIG. 6. Schematic illustration of the many different orders of chromatin packing that give rise to the highly condensed metaphase chromosome (courtesy
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REAC/TS, USA). 5.2. HUMAN KARYOTYPE AND DNA CONTENT OF CHROMOSOMES The human karyotype (Fig. 7) is the characteristic chromosome complement for humans, and consists of 23 pairs of large linear chromosomes of different sizes, giving a total of 46 chromosomes in every diploid cell. Human chromosomes are normally combined into seven 19 groups from A to G plus a pair of sex chromosomes X and Y . The chromosomal groups are: A:1–3, B: 4 and 5, C: 6 –12, D: 13–15, E: 16–18, F: 19 and 20 and G: 21 and 22. Male 20 Female FIG. 7. A banded chromosome/karyotype preparation from a normal male, 46, XY and a normal female 46, XX (courtesy Mayo Clinic, USA). The relative DNA contents of the human chromosomes for either gender are shown in Tables 2 and 3. These data have been calculated from Morton, 1991 . 21 TABLE 2. PER CENT DNA CONTENT OF THE HUMAN MALE GENOME OCCUPIED BY EACH PAIR OF AUTOSOMES AND EACH SEX CHROMOSOME Chromosome No. p arm q arm Both arms Chromosome No. p arm q arm Both arms 1 4.03 4.25 8.28 13 0.50 3.09 3.59 2 3.12 4.92 8.04 14 0.50 2.93 3.43 3 3.12 3.62 6.74 15 0.54 2.80 3.34 4 1.76 4.63 6.39 16 1.23 1.86 3.09 5 1.64 4.47 6.11 17 0.88 2.02 2.90 6 2.05 3.72 5.77 18 0.63 2.05 2.68 7 2.05 3.34 5.39 19 0.94 1.17 2.11 8 1.57 3.31 4.88 20 0.98 1.29 2.27 9 1.61 2.96 4.57 21 0.35 1.23 1.58 10 1.38 3.15 4.53 22 0.41 1.35 1.76 11 1.83 2.71 4.54 X 0.97 1.61 2.58 12 1.23 3.27 4.50 Y 0.20 0.73 0.93 Total 100 TABLE 3. PER CENT DNA CONTENT OF THE HUMAN FEMALE GENOME OCCUPIED BY EACH PAIR OF CHROMOSOMES Chromosome No. p
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arm q arm Both arms Chromosome No. p arm q arm Both arms 1 3.97 4.18 8.15 13 0.49 3.04 3.53 2 3.07 4.83 7.90 14 0.50 2.88 3.38 3 3.07 3.56 6.63 15 0.53 2.76 3.29 4 1.74 4.55 6.29 16 1.21 1.83 3.04 5 1.61 4.40 6.01 17 0.87 1.98 2.85 6 2.02 3.66 5.68 18 0.62 2.01 2.63 7 2.01 3.29 5.30 19 0.93 1.15 2.08 8 1.55 3.25 4.80 20 0.96 1.27 2.23 9 1.58 2.91 4.49 21 0.34 1.21 1.55 22 Chromosome No. p arm q arm Both arms Chromosome No. p arm q arm Both arms 10 1.36 3.10 4.46 22 0.40 1.34 1.74 11 1.80 2.66 4.46 X 1.92 3.16 5.08 12 1.21 3.22 4.43 Total 100 5.3. CELL CYCLE Important information on the clastogenic effects of physical or chemical agents on interphase cells can be obtained by examining the chromosomes at the point of ensuing cell division which for somatic cells is at mitosis. The cell cycle has a number of stages which can be distinguished by their appearance and function (Fig. 8). FIG. 8. The cell cycle (courtesy REAC/TS, USA). During mitosis, stages such as prophase, metaphase, anaphase and telophase are recognized. During the interphase, the chromosome material (that is, DNA and associated proteins) duplicates. This is called the ‘S’ (synthetic) period and is preceded by a period 23 called G 1 (a presynthetic gap) and is followed by G 2 (a postsynthetic gap) within the interphase. In cells which are not cycling, for example peripheral lymphocytes, the cell remains in G 0 .For cycling cells, interphase is metabolically the most active part of the cell cycle, and most of the energy requiring reactions in the nucleus takes place at this stage. The duration of each stage in the cycle varies
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with the type of cell and the conditions of growth. One can determine the lengths of the stages by using radiolabelled DNA precursors such as tritiated thymidine. In lymphocytes, the first cell cycle following stimulation is nearly synchronized, and these cells are especially convenient for radiobiological studies. Cycling mammalian cells in cultures are, of course, not synchronized, but can be brought to synchrony by several techniques. Different stages in the cell cycle vary in their sensitivity to the action of chemicals or radiation, and the types of chromosomal aberration produced vary depending on the cell stage that was treated . Thus, it is important in such studies to work with a synchronized population, or at least to have an estimate of the proportions of cells at the different stages that are present at the time of treatment. The progression through the cell cycle is controlled at different checkpoints to ensure maximum fidelity in the DNA integrity and proper chromosome segregation to daughter cells. The main checkpoints act at the end of G 1, prior to replication, at the end of G 2 , prior to mitosis and at the metaphase/anaphase transition, prior to chromosome segregation and cell division. At these checkpoints, the cell cycle progression can be blocked if DNA damage, incomplete replication or abnormal spindle structure are detected. 24 6. RADIATION INDUCED CHROMOSOMAL ALTERATIONS The first reported evidence that X rays could induce chromosomal aberrations came from the genetic studies by Müller of Drosophila. This was confirmed by the cytological studies of Painter and Müller . Sax later developed his ‘breakage first’ hypothesis on the origin of X ray induced chromosome aberrations, followed by Revell who proposed the alternative exchange hypothesis. In essence, Sax proposed that damaged regions of separate chromosomes come into contact after
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complete breaks have been induced and the ends move about and eventually combine to form exchanges. Alternatively, Revell envisaged that the points of damage are not complete severances but are unstable sites which can interact with similar sites to form pairwise exchanges. There is a third possibility, introduced later by Chadwick and Leenhouts , of a lesion/non-lesion interaction whereby a damaged site, in the Revell sense, may interact with an undamaged chromosome to form an exchange. 6.1. RADIATION INDUCED DNA LESIONS Ionizing radiation is characterized by the production of discrete energy deposition events (i.e. spurs, blobs, and tracks) in time and space that damage DNA directly and indirectly by the generation of reactive species mainly produced by the radiolysis of water . Biophysical studies of track structure show that low-LET radiation can produce localized clusters of ionizations within a single electron track. High-LET radiation produces asomewhat larger number of ionizations that are close in spatial extent (Fig. 9A). Ionizing radiation induces a wide range of damage in DNA including base damage (BD), single strand breaks (SSB), abasic sites (AS), DNA-protein cross-links (DPC), and double strand breaks (DSB) (Fig. 9B). 25 FIG. 9. Ionization pattern for low- and high-LET radiation (A) and radiation induced DNA lesions (B) [40, 41]. The energy to form an ion pair (H 2 O+ + e -) from the radiolysis of water is ~20 eV, and ionizing radiation deposits energy in events that range in energies up to hundreds of eV, with the average amount being 60 eV. Because this energy is sufficient to produce approximately three ion pairs, the radicals formed will react in the vicinity of a concrete region. The resultant DNA lesions for all types of radiation can be single DNA lesions involving SSB, AS, or BD as well as Multiple
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Damage Sites (MDS) or clustered DNA lesions . MDS lesions produced in DNA might involve one or more DSB, several SSB as well as BD. Complex clustered DNA lesions may be more difficult to repair or indeed fail to repair and hence potentially lead to the generation of lethal chromosome aberrations . The cell has complex signal transduction, cell-cycle checkpoint and repair pathways to respond to the DNA damage. BD, AS, and SSB are repaired by different processes like base excision repair (BER), nucleotide excision repair (NER), and single strand break repair (SSBR) [44, 45]. DPC are repaired by NER and homologous recombination repair (HRR) . DSB are critical lesions and their misrepair or non-repair are involved in the formation of chromosome aberrations like dicentrics or translocations . The HRR and DNA non-homologous end-joining (NHEJ) are the two major DSB repair mechanisms [48, 49]. These two mechanisms act at different phases of the cell cycle. Whereas NHEJ contributes substantially to DSB repair in all cell cycle phases, HRR contributes modestly in G 1 and progressively more as cells move through the cycle into G 2 . A comprehensive review of the biophysical and molecular processes leading to the formation of chromosomal aberrations by radiation has been published by Sasaki . 26 6.2. CHROMOSOME-TYPE ABERRATIONS Schemes for the classification of chromosomal aberrations have been presented [52, 53]. The peripheral lymphocyte population that is mitogenically stimulated is normally non-cycling and resides in the G 0 stage of the cell cycle. The chromosome aberrations induced by radiation will consequently be of the chromosome-type, i.e. they involve both chromatids of a chromosome. It is well known that ionizing radiation is an S independent clastogen, unlike UV radiation and chemical mutagens, which are S dependent agents. Therefore, with ionizing radiation, chromosome and chromatid-type aberrations are
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induced following treatment of G0 /G 1 and G 2/S cells, respectively. However, UV and chemicals induce mostly chromatid-type aberrations at all stages of the cell cycle. If chromatid-type aberrations are observed in G 0 /G 1cells that have been exposed to ionizing radiation, it can be assumed that these are either not radiation induced or have already passed through a second in vitro cell cycle. Chromatid-type aberrations therefore have little place in biological dosimetry because they are not induced by irradiation of G 0 lymphocytes. Nevertheless they do occur as part of the overall background frequency of chromosomal damage and may be present in excess if the person being investigated for suspected irradiation also has history of exposure to chemical clastogens. It is therefore important for the microscope scorers to be fully cognisant of chromatid-types and not to confuse them with the chromosome-types. In addition, with the increasing research interest in delayed chromosomal instability and bystander phenomena there is renewed interest in the chromatid aberrations. Chromosome-type aberrations are therefore covered in this Section and chromatid-type aberrations are addressed in Section 6.4. 6.2.1. Unstable aberrations Dicentrics The dicentric (Fig. 10) is the main aberration used for biological dosimetry. 27 FIG. 10. A dicentric chromosome with its accompanying acentric fragment, (Giemsa staining). It is an exchange between the centromeric pieces of two broken chromosomes which in its complete form is accompanied by an acentric fragment composed of the acentric pieces of these chromosomes. Particularly after high doses, multicentric configurations can be formed. Tricentrics are accompanied by two fragments, quadricentrics by three fragments, etc. The dicentric assay is covered in detail in Section 9. Centric rings In human lymphocytes, centric rings are much rarer than the dicentrics. Some researchers combine them with dicentrics while others choose to ignore them for dose estimation.
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The ring chromosome is an exchange between two breaks on separate arms of the same chromosome and is also accompanied by an acentric fragment (Fig. 11). 28 FIG. 11. A metaphase spread with two rings (arrowed), a dicentric and acentric fragments (Giemsa staining). Acentrics Acentric aberrations can be formed independently of the exchanges described above and as such are usually referred to as excess acentrics. They can be terminal or interstitial deletions of varying sizes but it is not always possible to determine their origin and so they are combined. Acentric rings where clear spaces may be seen within the dots are normally considered to be interstitial deletions whereas minutes which appear as double dots are mostly terminal deletions [54, 55]. Rogue cells Rogue cells are defined as metaphase cells prepared from cultured blood lymphocytes which exhibit extremely high levels of chromosome damage in the absence of an overt cause. An example is shown in Fig. 12 where chromosome breakage and rearrangements are so extensive that it is difficult to identify more than one or two normally appearing monocentric chromosomes. 29 FIG. 12. A rogue cell observed among 500 otherwise normal metaphases taken from a healthy, non-smoker, control person with no occupational or medical history of radiation exposure and living in a low radon area. The metaphase displays the characteristic features of many polycentric chromosomes and acentric fragments including a large number of double minutes. In contrast, the cell has numerous polycentric chromosomes, acentric fragments and double minutes. Double minutes are tiny bodies of chromatin containing a few megabases of DNA and can be defined as cytogenetic equivalents of amplified DNA sequences . These unique cells have been observed in all races and ethnic groups throughout the world. For example, rogue cells were first observed in 1968 in blood samples
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collected from the Yanomami Indian tribes that inhabit the rainforests of Venezuela . Subsequently they have been reported in the inhabitants of many countries including England, Japan, Ukraine, Lithuania and the Russian Federation. The term ‘rogue’ cells was coined by Awa and Neel who described these cells in the offspring of both irradiated and non-irradiated control subjects from the bombing of Hiroshima. Similar to the atomic bomb survivor studies, cytogenetic evaluations performed on exposed and unexposed populations living near Chernobyl showed that rogue cells did not associate with radiation exposure since they were also found in the non-exposed control groups [59, 60]. Although the worldwide occurrence of rogue cells in the human population has been firmly established, their clinical significance, if any, is unknown. In studies where serial sampling was performed rogue cells have been found to be transitory appearing intermittently in brief bursts simultaneously in certain individuals of discrete populations. It is noteworthy that rogue cells have not been reported in the clinical cytogenetics literature which is likely due to the longer three-day culture allowing 2 or 3 cell divisions by which time rogue cells would probably be lost. In addition, clinical cytogeneticists analyse relatively few metaphase cells (i.e.15–20) to reach a diagnosis on the normal or abnormal state of an individual’s karyotype. In contrast, radiation cytogeneticists routinely analyse hundreds even a thousand or more first-division metaphase cells from an individual which greatly increases the probability of detecting rogue cells which are known to occur at low frequency. In conclusion, the 30 etiology and medical significance of rogue cells in human lymphocytes remains an enigma although some evidence suggests that viruses such as the JC human polyoma virus may play a role in their expression . In view of the occurrence of rogue cells, the recommendation is therefore
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that, for most biological dosimetry purposes, isolated metaphases having the appearance of rogue cells should be excluded from the dose evaluation. An exception to this could be where there is additional evidence of exposure to high LET radiations and then, ideally, several multiply damaged cells present with a continuous spectrum of damage. 6.2.2. Stable aberrations Reciprocal translocations The reciprocal translocation is the exchange of terminal portions of two separate chromosomes. The various types of translocation were originally described by using the G banding technique and karyotyping, but this procedure is too laborious for routine biological dosimetry. With solid Giemsa staining, translocations are not observed so reliably. Their application to dosimetry is now possible with the FISH method (see Section 10). By the FISH method these are visualized as bicoloured monocentric chromosomes (Fig. 13). FIG. 13. A metaphase illustrating FISH-based chromosome ‘painting’ to detect translocations. Chromosome pairs 1, 2 and 4 are ‘painted’ red and chromosome pairs 3, 5 and 6 are ‘painted’ green. A reciprocal translocation is illustrated by the two bicolored chromosomes (2 and 5) which have exchanged segments at the ends of their long arms (courtesy Ramsey and Tucker, LLNL, USA). Non-reciprocal translocations When only one bicoloured chromosome can be seen, this has often been called a terminal, or incomplete, or one-way translocation. However, using a combination of whole chromosome, centromere and telomere probes, a number of translocations designated as terminal or 31 incomplete were found to be in reality reciprocal. It is very likely that the signal of the missing counterpart is below the limit of visual resolution, and it has therefore been suggested to designate such patterns as one-way exchanges or translocations. The current view is that true terminal translocations do exist but they form a small percentage of the total, e.g. at 4 Gy they
|
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are about 5% . Interstitial translocations (insertions) This is a bicoloured chromosome where an acentric piece of one chromosome has been inserted within an arm of another chromosome. An example is shown in Fig. 14. FIG. 14. A human metaphase spread with an insertion. Chromosome pair 1 is painted yellow and all other chromosomes are counterstained with propidium iodide. Stable and unstable cells Retrospective FISH biological dosimetry is possible because stable aberrations such as a reciprocal translocation will pass successfully through mitosis and into the daughter cells. However for this to succeed the complete genome needs to be stable. A translocation can still fail to negotiate division if there is an unrelated and unstable structure such as a dicentric or an excess acentric also present in the same cell. This has led to the need to consider stability not only of individual types of aberrations but of the cell as a whole. This is a concept recognized many years ago by Buckton et al. 1967 who introduced the designations Cs and Cu for stable and unstable cells respectively. The concept has again come to prominence with the development of retrospective biological dosimetry by FISH where it has been demonstrated that reciprocal (two-way) translocations seem to be more stable than incomplete (one-way) ones [64, 65]. 6.3. CHROMATID-TYPE ABERRATIONS Chromatid-type aberrations are generally classified in the same way as chromosome-type aberrations; the apparent unit of involvement in a chromatid-type aberration is, in most cases, 32 the single chromatid and not the whole chromosome, as is the case for chromosome-type aberrations. Terminal and interstitial deletions A terminal deletion is a distinct displacement of the chromatid fragment distal to the lesion (Fig. 15). FIG. 15. A metaphase spread with chromatid breaks (b) and gaps (g). If there is no displacement, the non-staining region
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between the centric and acentric regions must be of a width greater than the width of a chromatid to be considered a terminal deletion. This latter definition is used to distinguish between terminal deletions (chromatid breaks) and achromatic lesions (gaps). Chromatid-type interstitial deletions are not as readily observable as their chromosome-type counterparts, in part due to the fact that the small deleted fragment is often separated from the deleted chromosome and is not observed. 33 Achromatic lesions Achromatic lesions (or gaps), Fig. 15, are non-staining or very lightly stained regions of chromosomes present in one chromatid (single) or in both sister chromatids at apparently identical loci (double). If the non-staining region is of a width less than that of a chromatid, the event is recorded as an achromatic lesion. This is clearly only a working definition. It is generally suggested that achromatic lesions be recorded, but always separately from chromatid deletions. Their frequency should not be included in the totals for aberrations per cell since their significance and relationship to other ‘true’ aberration types is at present unclear. Isochromatid deletions Isochromatid deletions appear as exceptions to the class of chromatid-type aberrations, since they involve both chromatids, apparently with ‘breaks’ at the same position on both. However, in suitable material they can be shown to be induced by radiation in the S and G 2phases of the cell cycle, as is the case for other chromatid-type aberrations. There are several possible types depending on the nature of the sister unions that occur. If a sister union occurs, it is possible to distinguish isochromatid aberrations from chromosome-type terminal deletions. In mammalian cells, however, a sister union is a rare event and most of the isochromatid deletions are of the non-union proximal and distal types. The acentric fragment is most often not associated
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with the deleted centric part of the chromosome. The convention for analysis stipulates that since the radiation-induced aberrations in G 0lymphocytes are of the chromosome-type, all paired acentric fragments are to be classified as chromosome-type terminal deletions. Since the frequency of isochromatid deletions will in any case be low in lymphocytes, this convention is not unreasonable. Asymmetrical interchanges Asymmetrical interchanges (interarm interchanges and asymmetrical chromatid exchanges) are the chromatid-type equivalents of chromosome-type dicentrics. Symmetrical interchanges Symmetrical interchanges (symmetrical chromatid exchanges), Fig. 16, are the chromatid-type equivalents of chromosome-type reciprocal translocations. 34 FIG. 16. A metaphase spread with a chromatid symmetrical exchange, Giemsa stained. In the case of chromatid-type symmetrical exchanges, somatic pairing maintains an association between the chromosomes involved in the exchange and thus they can be readily observed in the absence of any chromosome-banding procedures. Asymmetrical and symmetrical interchanges There are two forms of symmetrical and asymmetrical interarm interchanges, but when analysing metaphase cells only one of each is distinguishable. Somatic pairing allows the symmetrical interchange to be observed. Triradials A triradial (three-armed configuration) can be described as the interaction between one chromosome having an isochromatid deletion and a second having a chromatid deletion. This classification scheme is clearly not exhaustive, since there are many types of complex aberrations that can be produced. The ones described are by far the most commonly observed. A more complete classification is given by Savage . 6.4. PREMATURE CHROMOSOME CONDENSATION When cycling cells enter mitosis, the chromatin condenses into the familiar shaped chromosomes. Techniques have been developed to cause chromatin also to condense when it is not in mitosis and this is termed premature chromosome condensation (PCC). Premature condensation can be induced by fusing interphase cells to mitotic Chinese hamster ovary (CHO) or HeLa cells using Sendai virus or polyethylene glycol (PEG) as
|
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the fusing agent . However, fusion by means of Sendai virus requires cells with membranes especially receptive to the virus particles and it has been reported that G 0 lymphocytes cannot be satisfactorily fused using Sendai virus. This difficulty was overcome for the purpose of biological dosimetry with the use of PEG for PCC induction . 35 Chemical methods of inducing PCC, using inhibitors of DNA phosphorylation such as okadaic acid or calyculin A, have also been developed. Most of these methods require the cells to be cycling in culture [68, 69]. The PCC technique, which is described in detail in Section 11, is a very useful research tool to probe the immediate post-irradiation processes and kinetics of chromosomal break restitution and/or misrepair to form aberrations (i.e. dicentrics and translocations) [70–72]. These studies demonstrate that the dicentrics, complete and incomplete translocations and acentric fragments that one sees eventually at metaphase are formed in G 0 at differing times that are dependent on the dose. In human lymphocytes, at low doses of X rays (1–2 Gy), both dicentrics and translocations are formed rapidly. However, at higher doses of 4 and 6 Gy, the frequencies of chromosome exchanges increase proportionally to the restitution of chromosome breaks (repair). FIG. 17. Premature chromosome condensation induced by PEG-mediated fusion in an unirradiated human lymphocyte fused with a mitotic CHO cell. Forty-six distinct single chromatid PCCs can be seen. 6.4.1. PCC techniques The different PCC techniques can be divided as follow: Fusion-PCC assay is the first that has been described in the literature in 1974 [73, 74]. In this assay, lymphocytes are fused with mitotic cells, often CHO cells are used, in order to induce premature condensation of the human chromosomes . By this approach, it is possible to score the number of human chromosomal pieces
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and therefore, the number of radiation induced chromosomal fragments in excess of background frequency. It has also been used to 36 estimate non-uniform exposure . The major advantage of this method is that damage can be observed shortly after blood sampling. Rapid Interphase Chromosome Assay (RICA) allows the visualisation of radiation-induced damage using FISH probes. DNA of chromosomes is artificially condensed in order to identify the chromosome domains and to detect exchanges between two different domains [76–78]. Dic-PCC assay allows the observation of dicentrics in other phases of the cell cycle (mainly in the G 2 phase) than the classical M phase and therefore to visualize cells that would not have been seen with the conventional dicentric assay . This is particularly interesting when the lymphocyte count has dropped following exposure and when it is difficult to obtain classical mitoses. Using the fusion-PCC method the time between sampling and dose estimation can be reduced , however using the chemical techniques for inducing PCC most laboratories culture for 48 hours and hence there is no reduction in time. Ring-PCC assay corresponds to the visualisation of radiation-induced rings in cells in different phases of the cell cycle (Fig. 18). FIG. 18. PCC rings (arrows) in a lymphocyte taken from patient A of the Tokai-mura accident (see Section 11.4). The major advantage of this approach is the measurement of much higher doses than with the classical dicentric assay as the saturation of ring number appears only at doses above 20 Gy for low LET radiation [70, 80, 81]. 6.5. MICRONUCLEI Micronuclei (MN) are formed from lagging chromosomal fragments or whole chromosomes at anaphase which are not included in the nuclei of daughter cells (Fig. 19A, B). They are therefore seen as distinctly separate small spherical objects that have the same morphology and staining
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properties of nuclei, within the cytoplasm of the daughter cells . 37 A > B > C FIG. 19. (A) Schematic diagram showing the mechanism of origin of micronuclei and nucleoplasmic bridges in the cytokinesis-block micronucleus assay. (B) Examples of binucleated cells without and with 1 and 2 micronuclei. (C) Examples of binucleated cells with 1 and with 2 nucleoplasmic bridges; in each case the nucleoplasmic bridge is accompanied by a micronucleus. 38 In the mid-1980s a major technical innovation was introduced. This was the method for blocking cytokinesis in cultured lymphocytes by adding cytochalasin B (Cyt-B) to the medium without inhibiting nuclear division. The cytokinesis block [83, 84] produces binucleate (BN) cells rather than permitting the two daughter cells to separate. With this protocol it is therefore possible to distinguish between proliferating (following the first mitosis) and non-proliferating cells and to specifically score MN in those cells capable of expressing MN i.e. BN cells. The modified assay allows identification and quantification of MN in binucleate cells with preserved cytoplasm (Fig. 19B). Measurement of micronuclei within BN cells can be further refined with the use of centromere probes which allows micronuclei originating from acentric chromosome fragments to be distinguished from those originating from whole chromosomes [85, 86]. Current developments in the automation of MN scoring, give new prospectives for the use of the assay in mass radiation casualties and routine biomonitoring (Section 13.3.3). The cytokinesis-block micronucleus assay has now also evolved into a cytome assay where a spectrum of chromosomal damage can be assessed including breakage, asymmetric chromosome rearrangement, chromosome loss and non-disjunction as well as necrosis, apoptosis and cytostasis . This method is also specifically used to measure nucleoplasmic bridges (NPB), (Fig. 19A, C) a surrogate biomarker of dicentric chromosomes which result from either telomere end-fusions or misrepair
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of DNA double strand breaks . NPB measured in the cytokinesis-block cytome assay are thus also applicable for biological dosimetry of ionizing radiation exposure . A strong correlation and similar dose-response curves were observed between NPB, and dicentric chromosomes and centric rings . Detailed information on MN analysis and the cytome assay is given in Section 12. 39 7. BLOOD SAMPLING 7.1. TIMING A venipuncture blood sample, preferably 10 mL, could be taken within a few hours of a whole body radiation exposure. However, in the case of a partial-body or non-uniform exposure the lymphocytes in the circulating and extravascular pools will not have reached equilibrium until about 24 hours . This could result in an unrepresentative proportion of irradiated cells in the specimen and therefore delaying sampling until at least the next day is advisable. An effort should be made to ensure that the sample is obtained promptly because even if the patient’s haematological parameters are within normal limits after about four weeks have elapsed, aberration yields begin to fall, causing greater uncertainty in any estimates of the radiation dose . In the event of a serious overexposure, where there is the likelihood of severe depletion of the white cell count, a ‘time window’ of possibly only a few hours or days exists before the lymphocyte count drops to a level where insufficient cells can be obtained for cytogenetic analysis. If medical treatment includes whole-blood or blood fraction transfusions, it is important to obtain a specimen of the patient’s blood before this treatment commences. For purposes of scientific interest, the laboratory should endeavour to obtain a sequence of blood samples at frequent intervals. This is ethically acceptable as such sampling would be undertaken to monitor changes in the differential white cell count. It may not always be possible to
|
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culture cells promptly if, for example, sampling occurs in a remote region with poor communications. Blood samples may be kept refrigerated but loss of lymphocyte viability soon becomes a major problem . The problem is overcome by stimulating the lymphocytes with phytohaemagglutinin (PHA) immediately after venipuncture and keeping them cold (below 20°C) so that the lymphocytes do not transform and progress through the cell cycle until the cells are warmed up to 37°C. The following method devised by M.S. Sasaki (personal communication) has enabled cells to be cultured up to two weeks later: (1) Prepare in advance 10 mL sterile tubes containing 5 mL of Leibovitz’s L-15 medium with 20% fetal bovine serum and 4% dehydrated PHA (Leibovitz’s L-15 medium is essential for long term transportation because it is buffered by 10 times more amino acids than other common culture media and the pH is stable for a long time). (2) Take a blood sample into a conventional heparinized tube. (3) Put 5 mL of heparinized blood into the tube with L-15 medium and mix. (4) Keep the tubes cool (<20°C); in this condition they may be stored or despatched to the laboratory without a significant reduction in viability. (5) The cells are then washed in conventional medium and processed following the same steps described later in Section 9.1 for setting up conventional cultures. If the Leibovitz medium procedure is used, it will need to be validated with a dose response curve produced under the same conditions. 7.2. ANTICOAGULANT Preservative free lithium heparin is the most commonly used anticoagulant for lymphocyte cultures, although it is possible to use sodium or ammonium heparin. Other commonly available anticoagulants, e.g. ethylenediaminetetraacetic acid (EDTA), often result 41 in poor cell growth and should not be used. If a sample is received in the wrong
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anticoagulant it is preferable to request a fresh specimen. However this may not always be possible and in such a situation it is possible to ‘rescue’ the sample by washing. The procedure is to take 4 mL of the blood, add 6 mL of a balanced salt solution (Hank’s or Earle’s) or culture medium and spin at 600g for 3 to 5 min. Remove the supernatant and add a fresh 10 mL of the washing liquid to the cell pellet and spin again. After final removal of the supernatant the washed cells can be restored to the original volume of blood by adding culture medium containing 10% foetal calf serum. Cultures can then be set up as described later in Section 9.1, treating the washed specimen as if it were a normal blood sample. 7.3. CONTAINERS Disposable glass or plastic specimen tubes containing the correct amounts of lithium heparin are available from several manufacturers. Both the older style screw-top tubes and vacuum tubes can be used. They must be sterile and many manufacturers routinely supply them sterile but this should be confirmed. Tubes containing glass or plastic beads or gels should be avoided. If dried heparin is used, it is important that the blood be properly mixed by inverting the tube several times. It is preferable if the cytogenetics laboratory can supply the specimen tube from its own stock. This, incidentally, provides an opportunity to include a detailed set of instructions for the doctor and correctly addressed proper packaging for the return of the sample. 7.4. TRANSPORT Blood specimens should be maintained ideally between 18 and 24°C during transportation. If temperatures well outside this range are likely to be experienced, the provision of coolant or room temperature packs and temperature loggers is advisable. In any case, freezing during transportation should
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be avoided. Transport of specimens should comply with applicable national and/or international regulations for the transport of infectious substances, as outlined in the current WHO guidance on regulations for the transport of infectious substances . This document also explains to shippers how to classify, document, mark, label and package infectious or potentially infectious substances such as diagnostic blood samples. Standard glass or plastic lithium heparin tubes can be used. They should be placed in a rigid, crushproof and watertight secondary container. This container should also include cushioning material and sufficient absorbent material to be able to absorb the entire contents, but it must not contain cooling packs. The secondary container should then be placed in outer packaging, e.g. a sturdy cardboard box, with suitable labelling. Shipping of blood samples, not known to contain pathogens, for diagnostic purposes is characterized as ‘UN 3373. BIOLOGICAL SUBSTANCE, CATEGORY B’. The labelling should therefore include this phrase together with a white diamond label with black letters ‘UN 3373’. In addition the package should be marked with the sender’s name, address and telephone number; the receiver’s name, address and telephone number; and the telephone number of a responsible person, knowledgeable about the shipment . If it is felt that cooling or room temperature packs are needed they should be outside the secondary container, and the outer packaging should be of thermal insulation material such as an expanded polystyrene box. Packaging kits that conform to the regulations are commercially available. For international shipments, shippers need to obtain any necessary export or import permits and the receiving laboratory should be notified before shipment of the specimens, in order to arrange for an import license if required. It is often convenient to use an international 42 courier company that provides a rapid ‘door to door’ service and deals
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{
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with all customs paperwork, etc. Transit times of two or three days can be tolerated; however, blood samples need special delivery services to avoid long delays, such as around national holidays. During air transport the blood should not be X rayed in security checks. If this is likely, a piece of X ray film or a standard Thermoluminescent Dosimeter (TLD) or Optically Stimulated Luminescence (OSL) monitoring badge could be included in the package. A ‘DO NOT NOT X-RAY’ label should be placed on the package. This condition should be also written on accompanying paperwork. 43 8. PRODUCTION OF AN IN VITRO DOSE–RESPONSE CURVE 8.1. GENERAL CONSIDERATIONS Despite improvements in techniques and the adoption by different workers of more comparable statistical programs for data analysis, differences between laboratories’ calibration curves still remain. The interpretation of dose using a calibration curve produced elsewhere may introduce extra uncertainty, and therefore it must be recommended that any laboratory intending to carry out biological dosimetry should establish its own dose–response data . Most accidental overexposures involve gamma radiation sources but there are also an appreciable number of events involving X rays. It is well established that the calibration for these two low LET radiations are different particularly at low doses. Therefore, for a labo-ratory embarking upon a programme of biological dosimetry these are the qualities of radiation for which the dose response should be established first. Events involving exposure to neutrons are thankfully rare but the possibility should be considered that a laboratory may be requested to respond to a criticality accident. If so, a calibration curve for fission spectrum neutrons will be required. Lymphocytes should be irradiated in vitro to approximate as closely as possible the in vivo situation and when this is done the same dose–response relationship is obtained . Freshly taken blood
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specimens in lithium heparin tubes should be used and irradiated as whole blood at 37°C. After irradiation they should be held for a further 2 hours at 37 oC and then cultured by the standard method identical to that used for assaying dicentrics, translocations or micronuclei on specimens from suspected overexposure patients. 8.2. PHYSICAL CONSIDERATIONS The preparation of a dose–response curve must be supported by reliable and accurate physical dosimetry, and there are a number of points requiring consideration. The blood needs to be positioned such that the dose can be easily inferred, and it should be exposed far enough away from the source so that the irradiation can be regarded as uniform. For example, if the sample is 1 cm thick, then it needs to be at least 1 m from the source for the difference in dose between front and back to be less than 2%. There must be sufficient material surrounding the blood for charged particle equilibrium to exist. For 60 Co γ rays, 4 mm of unit dense material is sufficient; for 250 kVp X rays, only 1 mm is necessary. For neutrons, 1 mm is usually also sufficient. The surrounding materials should be reduced to a minimum to avoid the complications of scattered radiation. The materials should have atomic compositions similar to blood because the dose to blood close to the specimen container wall will be caused by electrons arising from interactions within the wall. A serious mismatch of atomic composition will result in a non-uniform irradiation of the cells. For X and γ rays, electron density is the main factor when considering mismatch, while for neutrons the atomic constituents are important because neutrons interact with the nuclei of the target atoms. The exposure set up should be calibrated by physical measurements and most commonly
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an ionization chamber is used but other methods are possible. Fig. 20 for example illustrates measurements using alanine. 45 FIG. 20. Exposure array used to hold and position 15 mL test tubes and 10 mL blood vacutainers for exposures to irradiation with γ-ray sources. The box container of the exposure array is constructed of plexiglass with 6 mm wall thickness as part of its design to assure charged particle equilibrium. The box container is also equipped with access ports at both ends to permit water flow with a circulating water bath (not shown) to maintain the contents at 37°C during irradiation. Test tubes and vacutainers filled either with water or vials of alanine used for dosimetry measurements are also illustrated (courtesy AFRRI, USA). The detector of a physical dosimeter should be surrounded by material equivalent to that which surrounds the blood. If possible it should have similar dimensions to the blood sample so that it can replace the sample for dosimetry purposes. The physical dosimeter would normally be calibrated in air kerma with the unit of Gy and be traceable to a national standard. The conversion factor to Gy in soft tissue is the ratio of mass energy absorption coefficients. Numerically it is obtained by multiplying the air kerma value with a factor of 1.09 for 250 kVp X rays and 1.10 for 60 Co γ rays. The factor is therefore energy dependent and is lower at lower energies. There is also a difference between the conversion factors for soft tissue and for blood, but for low LET radiation this is small enough to be ignored. For neutrons it may approach 5%. The calibration factor includes any absorption by the wall of the ionization chamber, but it will often be necessary to correct the dose rate owing to self-absorption
|
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by the blood. The usual method of determining doses is to convert the measured air kerma into absorbed dose in tissue or blood and then to convert as necessary for distance (the inverse square law), absorption and mismatch of material at the blood interface. The size and general geometry of the apparatus are a compromise between these factors because the smaller the blood specimen, the smaller the absorption correction and the larger the mismatch correction. Nevertheless, geometry and materials should be chosen to minimize the necessary corrections. In order to produce an in vitro calibration curve applicable to cases of acute accidental exposure, the dose rate should be chosen such that all doses are given in less than 15 min. The differences in delivery times between the different doses are then sufficiently small that the β or dose squared coefficient of yield will be influenced by no more than about 4%. Additionally, some researchers choose to produce non-acute calibrations in order to have a better understanding of how the β coefficient should be modified for interpreting aberration yields from accidents involving protracted irradiation. It is even more essential that such calibrations should be carried out at 37 oC. If done at room temperature there will be little or no repair during the exposures so that the resulting dose response curve will be the same as that from acute irradiations. An important point to remember is that exposure time, not dose rate, is the critical factor with protraction calibration. Therefore each data point should be from blood irradiated for the same time. This is achieved by varying the distance from the 46 source which of course requires a lot more physics calibration measurements. The easier alternative, using a constant dose rate and therefore a single irradiation position, requires different delivery times
|
{
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for each dose and the resultant data will not fit so well to the linear quadratic dose response equation . Some laboratories prefer to place the blood sample in a phantom for calibration purposes on the grounds that scatter in a human body is to some extent taken into account (Figs 20 and 21). FIG. 21. A water bath heated to 37 o C placed in front of a cobalt-60 gamma ray source. In order to achieve electronic equilibrium the blood sample is located inside a plexiglass holder. However rather more consideration needs to be given to the dosimetric correction factors above and whether an ionization chamber can be placed beside the blood sample. Water is generally used as the phantom and, it should be maintained at 37 oC. If the ionization chamber is placed in the same geometry as the blood sample this will take account of the dose due to the scattered radiation. Using warm water will require significant temperature and pressure corrections to be applied and of course the chamber must not get wet. Neutron calibrations performed in a phantom are particularly problematic. Rather than water, a tissue equivalent material for the phantom is preferable. This produces a radiation spectrum akin to that in the body with possibly a considerable enhancement of the low LET component of dose to the lymphocytes. Specifying the spectrum of the components to the total absorbed dose can be very difficult. 47 8.3. STATISTICAL CONSIDERATIONS As discussed in Section 3, there is very strong evidence that the yields of chromosome aberrations or micronuclei (Y) are related to dose (D) by the linear quadratic equation 2 DDCY βα ++= (3) or, for high LET radiation, the α-term becomes large and eventually the β-term becomes biologically less relevant and also statistically ‘masked’ and the
|
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dose response is approximated by the linear equation DCY α+= (4) The objective of curve fitting is to determine those values of the coefficients C, α and β which best fit the data points. For dicentrics, irradiation with X or gamma rays produces a distribution of damage which is very well represented by the Poisson distribution . In contrast, neutrons and other types of high LET radiation produce distributions which display overdispersion, where the variance ( σ2 ) exceeds the mean (y). Whether the ratio of variance to mean ( σ2 /y) is a function of dose is at present an open question. For micronuclei the data tend to overdispersion at all doses even with photon irradiation. Because curve fitting methods are based on Poisson statistics, the dicentric cell distribution should be tested for compliance with the Poisson distribution for each dose used to construct the calibration curve. Nowadays, the most widely used test is the u test [99, 100]. The u test statistic is a normalized unit of the dispersion index ( σ2 /y), which for a Poisson distribution should be unity. u values higher than 1.96 indicate overdispersion (with a two-sided significance level, α = 0.025). )11(21)1( 2 XNyu −−−=σ (5) where: N indicates the number of cells analysed, and X the number of dicentrics (or dicentrics plus rings) detected. u values of < -1.96 indicate underdispersion. Biologically, underdispersion is very unlikely to occur so values of u lower than -1.96 may be indicative of a problem in data sampling. Adequate curve fitting requires a sufficient number of degrees of freedom to minimize the error on the curve. Ideally, 10 or more doses should be used in the range 0.25–5.0 Gy. For low LET radiation it is not necessary to have data higher than approximately 5.0 Gy and,
|
{
"page_id": null,
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indeed, beyond this dose there is evidence of saturation of the aberration yield which will lead to a distortion of the β coefficient . For high LET radiation a maximum of 2.0 Gy is suggested. As most radiation accidents involve doses of less than 1.0 Gy, the lower end of the curve is of particular importance in estimating doses. A significant effort should therefore be made to reduce the statistical uncertainty associated with the α coefficient of yield. It is suggested that several of the calibration doses, certainly a minimum of four, should be in the range of 0.25–1.0 Gy. If the laboratory is capable of obtaining data at doses below 0.25 Gy, this is very desirable. At higher doses, scoring should aim to detect 100 dicentrics at each dose. However at lower doses this is difficult to achieve and instead several thousand cells per point should be scored; a number between 3000 and 5000 is suggested. In all cases, the actual number of cells scored should be dependent on the number of dose points in the low dose region, with the focus on 48 minimizing the error on the fitted curve. Table 4 gives example data used to construct dose-effect curves for low LET γ-radiation and high LET α radiation. TABLE 4. CYTOGENETIC RESULTS OBTAINED FROM BLOOD SAMPLES IRRADIATED WITH γ-RAYS AND HELIUM-4 PARTICLES [102, 103] > γ-rays (Cobalt-60) dose (Gy) NXcell distribution of dicentrics σ2/y u01234560.000 5000 84992 81.00 -0.07 0.100 5002 14 4988 14 1.00 -0.13 0.250 2008 22 1987 20 11.08 2.61 0.500 2002 55 1947 55 0.97 -0.86 0.750 1832 100 1736 92 41.03 0.79 1.000 1168 109 1064 99 51.00 -0.02 1.500 562 100 474 76 12 1.06 1.08 2.000 332 103 251 63 17 21.14 1.82 3.000 193 108 104 72 15 20.83
|
{
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-1.64 4.000 103 103 35 41 21 420.88 -0.84 5.000 59 107 11 19 11 9631.15 0.81 Average 1.0 20 MeV 4He particles dose (Gy) NXcell distribution of dicentrics σ2/y u012345670.000 2000 31997 31.00 -0.04 0.051 900 19 881 19 0.98 -0.44 0.104 1029 27 1004 23 21.12 2.84 0.511 1136 199 960 154 21 11.07 1.60 1.010 304 108 217 69 15 31.09 1.15 1.536 142 96 75 40 25 20.98 -0.20 2.050 137 120 63 44 16 12 21.20 1.65 2.526 144 148 66 34 25 14 321.40 3.40 3.029 98 108 47 16 17 17 011.56 3.93 Average 1.19 For each dose analysed, total number of cells scored (N), total number of dicentrics observed (X), cell distribution of dicentrics and dispersion index ( σ2 /y) and u-test (u) are presented. u values greater than 1.96 indicate overdispersion. The technique suggested for determining the best fit coefficients is that of maximum likelihood [104, 105]. Using this method, the best fit value for each coefficient is achieved by assuming a Poisson distribution and maximizing the likelihood of the observations by the method of iteratively reweighted least squares. For overdispersed (non-Poisson) distributions, as obtained after high LET radiation, the weights must take into account the overdispersion. If the data show a statistically significant trend of σ2 /y with dose, then that trend should be used. Otherwise, the Poisson weight on each data point should be divided by the average value of σ2 /y. The goodness of fit of the curve and significance of fitted α and β coefficients should then be tested, for instance using the Chi-squared ( χ2 ) test and an appropriate form of the F-test (e.g. F-test, z-test or t-test) respectively. These tests are detailed in Annex VI. If there is evidence of a lack of fit
|
{
"page_id": null,
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