AFRRI's Gamma-,Ray, X-Ray, andFission-'Ne6utron Calibration ,Curvesfor* the Lymphocyte, bic entric" Assay:

Appliation of aKIMetaphase Finder- System

PG.S. Pras~nna

H.M. Gerstenberg/B.N.Torres

- C.W. SheftataK. L. DuffyR.S. flowta

A.W. KhusenW..Jackson

W.F Blakely -

DISTRIBUTION STATEMENTA

Distribution Unlimited 2002071JI2 129

Armed Forces Radiobiology Research Institute

AFRRI's Gamma-Ray, X-Ray, andFission-Neutron Calibration Curves for the

Lymphocyte Dicentric Assay: Application ofa Metaphase Finder System

P.G.S. Prasanna1

H. Loats2

H.M. Gerstenberg'B.N. TorresI

C.W. ShehataI

K.L. Duffy1

R.S. Floura1

A.W. Khusen1' 3

W.E. Jackson1

W.F. BlakelyI

1Armed Forces Radiobiology Research Institute, Applied CellularRadiobiology and Radiation Sciences Departments, 8901 WisconsinAvenue, Bethesda, MD 20889-5603

2Loats Associates Inc., 201 East Main Street, Westminster, MD 211573Current address: Center for Standardization and Radiological Safety

Research, National Atomic Energy Agency, Jakarta, Indonesia

Armed Forces Radiobiology Research Institute8901 Wisconsin Avenue

Bethesda, Maryland 20889-5603www.afrri.usuhs.mil

Cleared for public release; distribution unlimited.]

AFRRI Special Publication 02-1

Printed May 2002

This and other AFRRI publications are available to qualified users from theDefense Technical Information Center, Attention: OCP, 8725 John J.Kingman Road, Suite 0944, Fort Belvoir, VA 22060-6218; telephone (703)767-8274. Others may contact the National Technical Information Service,5285 Port Royal Road, Springfield, VA 22161; telephone (703) 487-4650.

Contents

Foreword ................. v

Precis .................. vi

Introduction ............... 1

Materials and Methods ......... 3

Results and Discussion ......... 7

References ................ 13

Appendix ................. 15

,..11

iv

Foreword

Established in 1961, the Armed Forces Radio- and periodically update its own calibrationbiology Research Institute (AFRRI) is the sole curves in order to achieve an acceptable per-Department of Defense research laboratory for formance standard. Predictive value is en-medical radiological defense. Its primary mis- hanced further when each radiation type forsion is to develop medical countermeasures which a calibration curve is generated is fullyagainst ionizing radiation. Developmental and characterized relative to microdosimetric para-applied research focuses on prevention, assess- meters. These high standards are employed byment, and treatment of radiological injury, and the AFRRI Biological Dosimetry Team and areon the confounding problems of combined in- described in this report.jury involving radiation and other battlefieldstressors. A major research thrust of the team is to im-

prove the performance characteristics of cyto-Precision and accuracy are hallmarks of effec- genetic tests. Through a collaborative effort un-tive and meaningful biological tests. This is es- der a cooperative research and developmentpecially true for the lymphocyte-dicentric as-

sayas t aplis o boloica dsimtryandtoagreement with Loats Associates Inc., West-say as it applies to biological dosimetry and to minster, Maryland, the team has developed anthe estimation of radiation doses in individuals. mihster, Marylandmthe am ha deveope anGamma rays, x rays, and fission neutrons induce enhancemdgialimage an al neto -morphologic aberrations in lymphocyte chro- tm for to t ia aca sis. tomosomes that can be quantifiably measured lection for this report was facilitated using theusing sophisticated cytogenetic techniques. The system's mature automated metaphase-findingdicentric chromosome is one such aberration, component coupled with lymphocyte-dicentricand it is recognized as a biomarker of exposures scoring at peripheral, or satellite, scoring sta-to ionizing radiation. Measuring the frequency tions.of dicentric chromosomes in peripheral bloodlymphocytes gives a good approximation of ra- In addition to its core objective of developing,diation dose. Accordingly, the lymphocyte di- testing, and validating deployable biodosimetrycentric assay finds utility in cases of accidental systems for military field operations, the teamor intentional exposures when there is a need to maintains one of the nation's few reference test-document radiation doses in individuals, and the ing facilities for radiation dose assessment. Thisassay's predictive value (precision and accura- resource responds to military, domestic, and in-cy) is of paramount importance when used to ternational nuclear or radiological emergen-aide medical triage and manage the radiation cies involving human exposures to ionizinginjured, radiation. It is for this reason that the studies

reviewed in this report were undertaken.The lymphocyte-dicentric assay is a technicallydemanding and time-consuming procedure, re- The accomplishments documented herein pointquiring a highly trained technical or profession- to the critical role of radiobiological research inal staff. Even with highly qualified individuals, defending our nation against current and futurecontrolling inter-laboratory variability is proble- threats through medical readiness, on both mili-matic. Each laboratory must therefore develop tary and Homeland Security fronts.

ROBERT R. ENG, COL, MS, USADIRECTOR, ARMED FORCES RADIOBIOLOGY

RESEARCH INSTITUTE

v

Dose Response Relationships for Dicentric Yield

Precis

Facilities are established at the Armed Forces ratic model using the maximum-likelihoodRadiobiology Research Institute (AFRRI) to method for the neutron source by a weighted

perform radiation-induced chromosome aber- linear regression method.

ration analysis for biological dosimetry. Wholeblood from healthy human volunteers was used Comparison of the data with other published

after obtaining informed consent. Peripheral studies is presented. The dose-response rela-

blood lymphocytes were exposed in vitro to tionships for dicentric induction by low- and

different types of radiation; 60Co gamma rays high-linear energy transfer (LET) radiation are(1y,=1 .25 MeV, mean of the absorbed dose consistent with the single- and two-track modeldistribution of the lineal energy, ydos. of aberration formation, Y = otD + PD 2. An in-

keV/pm, 1 Gy/min); x rays (250 kVp, FE=83 crease in YD resulted in an increase in dicentricyield. As expected, fission neutrons induced a

keV, YD= 4 keV/pm, 1 Gy/min); or a fission- significantly higheryield ofdicentrics than thatspectrum neutron source (E =0.71 MeV, yl = 65 caused by low-LET sources. The linear com-keV/jim, 0.25 Gy/min). Distribution of radia- ponent of the model, corresponding to damagetion-induced dicentrics among cells exhibited caused by single-tracks, is predominant withPoisson statistics as characterized by the Pap- fission neutrons so that the dose-effectworth method (Papworth 1970). Dose-response relationship is essentially linear. An automatedrelationships for the yield of dicentrics for metaphase finder system with a satellite scoringphoton sources were fitted with a linear-quad- utility was used to improve data collection.

vi

Introduction

Application of the lymphocyte-dicentric assay macroscopic radiation descriptors such as dose,for biological dosimetry has made significant LET, and relative biological effectivenesscontributions in both accidental and occupation- (RBE) are inadequate, if not irrelevant, para-al overexposures. This biological dosimeter is meters for the quantification of biologicalthe most thoroughly investigated system (Muller effects of ionizing radiation (Watt et al. 1994).and Streffer 1991). Dicentrics are considered A characteristic of ionizing radiation is that itsrelatively radiation specific; only a few chem- energy can be dissipated in terms of discreteicals are known to interfere with this assay. Low packets, e.g., spurs and blobs, the number andbackground levels (about 1 dicentric in 2000 magnitude of which can be determined bycells), high sensitivity (a threshold dose of 0.05 microdosimetry (I.C.R.U. 1993). While theGy), and known dose dependency up to 4 Gy (for absorbed dose reflects the macroscopic depo-low-LET radiation) make this assay quite robust sition within a given material, it is micro-(Greenstock and Trivedi 1994). Effects of radi- dosimetry, with parameters such as linealation quality and dose rate are well character- energy y and its dose- and frequency-weightedized (Edwards 1997). The influence of time be- mean values YD and YF that describes thetween radiation exposure and analysis for a radiation energy interactions at the microscopicbroad dose range is not critical for at least the level. Bauchinger reviewed the importance offirst 2 weeks after exposure (I.A.E.A. 2001). microdosimetry on the classical and alternativeHowever, published reports show that differ- mechanisms of chromosome-aberration forma-ences exist in the measured yield of dicentrics tion (Bauchinger 1983).per Gy among several laboratories (Lloyd et al.1987). Therefore, it is advised that each labora- This paper reports dose-response or calibrationtory should establish its own calibration curves curves of measured dicentric yields followingfor the induction of dicentrics by different radi- exposure to 250-kVp x rays, 60 gamma rays,ation types over a range of doses and dose rates and fission neutrons, whose radiation qualities(I.A.E.A. 2001). have been measured at AFRRI (Bethesda, MD)

in terms of their microdosimetric parameters. InDicentric yield from radiation exposure is de- addition, we compare these dose-response cal-pendent not only on the dose, but also on radi- ibration curves with similar studies from otheration quality. Radiation quality depends on mi- laboratories. Estimating radiation dose by chro-croscopic energy deposition events that are char- mosome aberration analysis requires time-de-acterized by temporal, spatial, and energy dis- manding and labor-intensive scoring by experttributions of the radiation fields within the ir- cytogeneticists. Our attempt to decrease cyto-radiated volume. There is evidence that radio- genetic scoring time in biodosimetric assess-biological effects are more closely related to ment for radiation accidents is addressed by thelineal energy than to neutron energy (I.C.R.U. use of satellite scoring stations used in con-1980). Furthermore, it has been stated that the junction with an automated metaphase finder.

Dose Response Relationships for Dicentric Yield

2

Materials and Methods

Lymphocytes. Whole blood from healthy hu- 0.6 SSource YDman donors was collected into vacutainers Sorce YD neutronscontaining ethylenediamine tetraacetic acid

y rays 1.9 ne

0.4 xrays 4.0(EDTA) (Becton-Dickinson, Rutherford, NJ). nurons .0The informed consent form used in this study x *1. ywas approved by the Uniformed Services >, 0.2University of the Health Sciences, Human Use y ry "Committee (Bethesda, MD). Lymphocytes were lp I 0.°isolated using a density gradient (Histopaque 0.0 I , a "-:" ' .1..1077, Sigma Chemical Co., St. Louis, MO), 0.01 0.1 1.0 10 100 1000washed with phosphate buffered saline (PBS), y(keV/tm) Stankus etal. (1995)and resuspended in complete growth medium(Karyomax, bone marrow karyotyping medium, Figure 1. Measured lineal-energy dose distribu-Life Technologies, Rockville, MD) at a con- tions for AFRRI's gamma rays, x rays, and fissioncentration 1-1.5 x 1 06/ml for exposure to neutrons. The measurements were made using adifferent radiation types. TEPC detector with a gas filling made using a

pressure corresponding to a 1-pim diameter. TheRadiation sources and dosimetry. Dosimetry dose distributions, d(y), are normalized to unit doseprocedures and radiation sources used in these and plotted as y*d(y). In a semi-logarithmic repre-studies were previously described for 7 rays sentation such as this, the area under a curve de-(studies we previou5;sly descred for 1998rays limited by any two values of y proportional to the(Stankus et al. 1995; Prasanna et al. 1998), x fraction of dose delivered by events with lineal ener-rays (Redpath et al. 1995; Blakely et al. 1995; gies in this range. This is the standard represen-Prasanna et al. 1997), and fission neutrons tation of microdosimetric spectra. The region of the(Redpath et al. 1995; Blakely et al. 1995; gamma-ray spectrum below 0.1 keV tm-1 isPrasanna et al. 1997). Measured lineal energy explained in Stankus et al. (Stankus et al. 1995).dose distributions for AFRRI's gamma rays, x The definitions and references for the published

data are in the text. However, these distributionsrays, and fission neutrons are shown in Figure 1. may vary depending on experimental arrange-ments and measurement parameters.

Gamma-ray exposures were performed in the

bilateral field of the 60Co facility at AFRRI as 83 keV (source to sample distance = 55.2 cm,described earlier (Carter and Verrelli 1973). The 250 kVp at 12.5 mA, 0.2-mm Cu and 1-mm Aldose rate was measured with a tissue-equivalent filtration) at doses between 0.5 and 3.5 Gy.ionization chamber before irradiation follow- Dosimetry was performed using ion chambersing a well-established dosimetry protocol placed in tissue-culture flasks filled with(A.A.P.M. 1983). The field was uniform within tissue-equivalent plastic as described by the2%. Cells in suspension were placed in 15-ml International Commission on Radiation Unitspolypropylene centrifuge tubes and irradiated at and Measurements (ICRU) (I.C.R.U. 1973).room temperature at a dose rate of 1 Gy/min. The Field uniformity was within 2%. Cells in sus-yF where yF = LET., (Turner 1992; Rossi 1959), pension in 25-cm 2 tissue-culture flasks weremeasured using a 1-pm diameter tissue- placed on a rotating Plexiglas holder forequivalent proportional counter (TEPC), has irradiation and exposed at room temperature atbeen previously described (Stankus et al. 1995). a dose rate of 1 Gy/min. The YF for this x-rayX-ray irradiation was performed using a 320- source has been previously described (Blakely,kVp Philips industrial x-ray machine (GMBH, Benevides and Gerstenberg 1995; Prasanna etHamburg, Germany), with an effective energy of al. 1997).

3

Dose Response Relationships for Dicentric Yield

Neutron irradiation was performed using diameter (Biavati and Boer 1996) was inAFRRI's training, research, isotope, General excellent agreement with our value determinedAtomic (TRIGA) Mark-F, nuclear reactor. Sam- at the same diameter.ples for irradiation were placed in a lead boxwith 5-cm thick walls. Additional 15 cm of lead The number of neutron hits per cell nucleus wasshielding was placed in front of the reactor tank determined from the dose and fluence relation-wall, and borated polyethylene slabs were ship as previously described (Keifer 1990)placed around the sides of the tank wall, which and the assumption that LET= YF, a mean cellprojected into the exposure room. The lead box diameter of 10 jm, and the designated dose.was mounted on a wooden table and rolled along The hit frequency was then calculated assuminga track to allow the array to be placed at a re- a Poisson distribution of hits (Fisher and Hartyproducible distance from the reactor core. An 1982). Similar calculations were performed forextractor system was used for placing and the 60Co gamma-ray source using a LET valueretrieving the samples within the lead box of 0.23 keV/jim (I.C.R.U. 1980). The same was(Redpath et al. 1995). Cells were suspended in done for the x-ray source, with the assumption15-ml polypropylene centrifuge tubes, placed in that the literature value of 1.7 keV/jim for 200a Plexiglas holder, and exposed at room keV x rays held for the 250 kVp x-ray source.temperature. The dose rate and neutron andgamma portions of the mixed field radiation Lymphocyte culture and metaphase spreadconfiguration were determined using the paired-ion chamber technique (I.C.R.U. 1977) and ap-plying previously determined spectral inform- the lymphocytes were washed and re-suspend-ation for this radiation configuration (Verbin- ed in media, stimulated to grow by addingski et al. 1981). The dose rate was 0.25 Gy/min. phytohemagglutinin (0.5 jig/ml; Murex Diag-The neutron to total dose ratio was 0.95+0.07. nostics Ltd, Dartford, England), and incubatedFluence-weighted mean energies (E) for this at 37 C. After 44 h of stimulation, colcemidconfiguration are 0.71 MeV for neutrons(N.I.S.T. 1991) and 1.80 MeV for gamma rays was ade (1 st/p Sigm cemicalsCo St(Zeman and Ferlic 1984). The radiation field Louis, MO) to stop cell cycle progression inwas uniform to within 2.5%. The YF for this 235U first division metaphases and then incubated forreactor produced the degraded fission-neutron an additional 4 h. Less than 3% of the meta-spectra that have been previously described phases were in second division metaphases as(Blakely, Benevides and Gerstenberg 1995; Pra- determined by the fluorescence plus Giemsasanna et al. 1997). technique at this culture time (data not shown).

Previous studies have estimated YF for spherical Following hypotonic treatment in 1% sodium-

volumes with I 0-jim diameters (the mean diam- citrate solution, cells were fixed in 1:3 acetic

eter of the human lymphocytes used in this methanol. Metaphase spreads were prepared on

work) for x-ray and fission neutron radiation acid-cleaned glass slides by the standardqualities (Blakely, Benevides and Gerstenberg method (Preist 1977). The slides were stained in1995; Prasanna et al. 1997). The 6°Co- 4% Giemsa in PBS for dicentric analysis.gamma-ray YF was measured to be 0.39 keV/jimusing a TEPC detector with gas pressure Automated metaphase-finding and dicentriccorresponding to a 1-jim diameter. The YF for 10 analysis. Figure 2 illustrates the automatedjim, 0.53 keV/pim was obtained by linear metaphase finder system, software utilities, andinterpolation of Co YF data (Biavati and Boer satellite-scoring concept used in these studies.1996) determined with a walled TEPC detector Slides were placed on the stage of an automatedhaving equivalent diameters from 0.5 to 20 jim. metaphase finder (LAI Metafind, Loats Asso-Biavati and Boer's measured value at 1-jim ciates Inc., Westminster, MD). This system

4

Materials and Methods

control of image recognition parameters andA B !S,relocation of metaphase spreads on the 16-

Miri:LJ slide capacity microscope and stand-alone mi-1 croscopes, here referred to as satellite scor-

ing stations. Images of metaphase spreads wereI: :acquired using a color camera and a color-

c- digitizer board. Images were displayed on acomputer monitor. The metaphase spreads werelocated using a 10-x magnification objective

\ - lens and were relocated with a 100-xmagnification objective on slides by the system

Figure 2. Automated metaphase-finding and anal- for manual chromosome aberration analysis.ysis of aberrations in satellite-scoring stations. The Alternatively, the slide and vernier locations ofautomated metaphase finder (A) consists of a mi- the collected spreads were transferred tocroscope equipped with a 16-slide capacity stage, satellite scoring stations, and the relocation ofmotorized x-, y-, and z-axis computer-controlled spreads using a 100-x magnification objectivepositioning with specially adapted auto-focal cap- was done for manual dicentric analysis byabilities. Accuracy of position in the x-, y-, and z- several investigators.axes are within 0.5 tm, 0.5 htm, and 0.05 Jpm,respectively. Images of spreads are acquired using athree-chip RGB color camera and color-digitizer Data Analysis. Dose-response relationships forboard. The system automatically locates scorable the yield of dicentrics for photon sources weremetaphase spreads at low magnification, and savesimage and location of each spread on a slide (B). An fitted by the linear-quadratic model Y = D +England finder slide (C) is used to calibrate precise PD2 using the maximum-likelihood method andlocation coordinates. Software utilities were devel- for the neutron source by the weighted linear re-oped to permit the metaphase finder system to re- gression model Y = aD. Weights were basedlocate a spread for analysis either in the metaphase on the reciprocal of the standard error (SE) offinder or in the satellite-scoring station (D). the mean squared. Correlation coefficients (r)consists of a standard binocular microscope of the fitted models were also determined. The(Olympus, Japan) equipped with a 16-slide analysis of the yield of dicentrics in metaphasescapacity stage and motorized x-, y-, and z-axis included the determination of the mean±SE andcomputer-controlled positioning with specially the evaluation of the frequency distributionadapted auto-focal capabilities. The system in- using the G2/y and ji test ofPapworth (Papworthcludes specialized software utilities (Loats As- 1970). Using the Papworth test, a t value be-sociates Inc., Westminster, MD) that permit user tween -1.96 and 1.96 indicates overdispersion.

5

Dose Response Relationships for Dicentric Yield

6

Results and Discussion

Radiation quality and microdosimetry. In 0.06contrast to the common low-LET photon sources o.o 5(250-kVp x rays, Cogamma rays), the quality 040.04 -of high-LET neutron sources can vary oconsiderably. Neutrons are classified according 0

to their energies (Attix 1986). Thermal neutrons W 0.02have energies less than 0.5 keV (Attix 1986). o.ol -Intermediate energy neutrons, sometimes re-_ o.oo-ferred to as "slow," "intermediate," "reso- -nance," or "epithermal" neutrons, have energies 0.021from 0.5 keV up to 10 keV. Neutrons with -1 0o 2 3 4 5 6 7 8energies above 10 keV but below 20 MeV are -rime, min

called "fast" neutrons, and those with energiesabove 20 MeV are called "relativistic" neutrons Figure 3. Dose-rate and time-course for neutron(Turner 1992) . 235U-fission reactor neutrons exposure. This figure illustrates dose and dose-ratemeasurements from a typical experiment whereproduce energies in the range from above 0.1 samples were exposed to fission neutrons. EachkeV to over 10 MeV (I.C.R.U. 1977) and hence neutron run was selectively monitored using fissioninclude mostly slow and fast neutrons. Degraded and ionizing chambers in the exposure room. Thefission spectrum neutrons, commonly used in time course of dose measurements detected with aradiobiology studies, are often referred to as 0.5 cm3 ionizing chamber for a 1.5-Gy dose at 0.25fission spectrum neutrons. Gy min- is illustrated. Data measured in units of

nC/10-sec interval is integrated for each run andIn these studies, the dose rate for the photon analyzed to determine dose and dose rate. Thesources was 1 Gy/min. A fourfold lower dose sample placed in the lead box was extracted fromrate (25 cGy/min) was used for the fission- the exposure room just before the fall in the relative

neutron studies. Figure 3 illustrates the typical dose rate after 6 min, indicated by the arrow(,-).

time versus dose-rate profile from a single run.Steady state conditions were obtained after 1 with relative progressive increases for neutrons,min. Neutron exposure intervals spanned 1.9 to x rays, and gamma rays, 1:11:79-fold re-8 min in these studies. spectively (Fig. 1).

Radiation qualities for the sources used in this AFRRI's fission neutron facility produces astudy were extensively characterized (Fig. I). radiation quality that is qualitatively similar toTable I lists the radiation dosimetric parameters other 235U-reactor fission-neutron facilities, in-for the gamma-ray, x-ray, and degraded fission- cluding Janus located at Argonne National Lab-spectrum neutron sources used to irradiate hu- oratory (ANL, Argonne, IL) (Marshall andman lymphocytes in vitro. Measured values for Williamson 1985), British Experimental Pilethey YD were determined for 1 -pm diameter (BEPO) located at the National Radiologicalvolumes and ranged from 1.9 to 65 keV/jtm. Protection Board, Harwell, UK (Lloyd et al.The YF values are shown for 1 0-jim diameter 1976; Scott et aL 1969), and the reactor neutronvolumes and span approximately a 50-fold range therapy converter (RENT) located in Germany(0.35 to 18 keV/jim). Lymphocytes were ex- (Bauchinger et al. 1984). There are both simi-posed to these sources over dose ranges as larities and differences in the radiation qualitiesshown in Table 1. Cell fractions receiving no of these sources. The TRIGA and Janus micro-hits were negligible (less than 1 x 10-3) at these dosimetry spectra have been compared anddoses, but the hit frequency per nucleus varied found nearly identical (Gerstenberg 1991). In

7

Dose Response Relationships for Dicentric Yield

Table 1. Radiation dosimetry parameters used to irradiate human lymphocytes in vitro.Radiation F YF YD d, Dose rate Dose range NMean

type (MeV)a (keVI!tm)b.c (keVpm)e (Gy/min) (Gy) hitslnuceus/GY6°Co gamma rays 1.25 0.35 1.9 1.0 ___ 0.25- 5.0 __2134

250-kVp x rays 0.083 1.53 4.0 1.0 0.25-3.5 __ 289Fission neutrons 0.71 18.0 __ 65.0 0.25 0.75- 2.5 1 27

a. E is the mean energy.b. YF is the frequency-weighted mean of the lineal energy.c. Equivalent detector diameter of 10 pm.d. YD is the dose-weighted mean of the lineal energy.e. Equivalent detector diameter of 1 pm.f. Cell fractions receiving no hits were negligible (less than 1 x 103 ) in samples exposed to designated doses of

anyof these radiation sources... . . . . . . . ...

contrast, the RENT source has significantly efficiently and rapidly accomplished by the usehigher mean neutron energy (1.6 MeV) com- of an automated metaphase finder. The meta-pared to the neutron energy (0.7 MeV) for the phase spreads were either automatically re-TRIGA or Janus sources. This difference can be located by the system or the digital data on lo-attributed to the thickness of the high atomic cation of spreads and slides were transferred tonumber (Z) material that the beam transverses, two satellite scoring stations for manual anal-RENT's neutron beam is filtered by 2.5 cm of ysis. The use of multiple scoring stations ex-lead, while the TRIGA neutron bean transverses pedited the analysis (Fig. 2).20 cm of lead. It should be noted that for atypical fission spectrum, when filtered through There have been significant previous efforts tolead, the neutron spectrum peak would be shifted use automated metaphase finders to detect anddown in energy. This shift is due to the energy score cytogenetic biodosimetry endpointsdependence of the inelastic neutron cross-sec- (Lloyd 1984; Rutovitz 1992; Blakely et al.tion for lead or any high Z-material, creating 1995). In these instances, automated metaphaseneutrons below c MeV. These neutrons are finders were used alone. In this work, a new

built-up by the higher-energy neutrons scat- concept of digital transfer of data and slides to

tering to a lower energy. This is consistent with multiple satellite scoring stations for analysis

Eisenhauer's calculation (Eisenhauer 1991) that emerged and was used successfully to facilitateEisnhaer' cacultio (Esenaue 191) hatdata acquisition (Prasanna et al. 1998). Thisan increase in the thickness of lead at AFRRI's conceptqisotin in Figue 2.

s

reactor results in a progressive decrease in the scoring station consists of a microscope with a

mean neutron energy. An opposite shift occurs vernier stage and a computer with Metafind

in the lineal-energy spectrum where calculations satellite scoring software. Analysis at the

show that the peak moves up in lineal energy; the satellite scoring station involves recalling the

resulting spectrum peak will be shifted up in y originally detected spreads by a metaphasevalue from 50 to almost 90 keV/jim when no finder in another microscope station and usinglead is present compared with 20 cm of filtered the computer-assisted scoring sheets in the re-lead (Gerstenberg 1989). The mean value of the mote station. In this approach, a single meta-spectrum YD also shifts, but not so dramatically phase finder can support simultaneous scoringbecause of the change in the shape of the y at multiple stations by different investigators;spectra. this results in saved time and an increase in

effective throughput.Automated metaphase-finding. Several thou-sand metaphases from numerous healthy do- Lymphocyte-dicentric calibration curves.nors were analyzed to establish radiation-cali- Since the introduction by Bender and Goochbration curves for the induction of dicentric for- (Bender and Gooch 1966), the lymphocyte-mation. Collection of this data from slides was dicentric assay has been the generally accepted

8

Results and Discussion

method for biodosimetric dose assessment in 2.0cases of accidental and occupational overexpo- 0 * 250-kVp x rayssures. This approach is based on the use of in A . Fission neutronsvitro-generated calibration curves for various 1 * C0Co gamma raysradiation qualities. Experiments were per- cformed at AFRRI to produce lymphocyte- 1.0 -dicentric calibration curves using an established 0protocol (I.A.E.A. 2001). The number of cells (scored, the mean, and the frequency distribution E 0.5of dicentrics per cell are presented for 6°Co Zgamma rays, 250-kVp x rays, and fission 0.0neutrons and are shown in Tables 2-4. 0 1 2 3 4 5 6Progressive increases in radiation doses result in Radiation dose (Gy)

decreases in the fraction of cells with no di-centrics and increases in the fraction with Figure 4. Dose-response calibration curves for thedicentrics. These dose-response data for di- induction of dicentrics in human lymphocytescentric yields, with the one exception of 60Co following in vitro exposure to 60Co gamma rays,gamma rays at a dose of 2 Gy, fit a Poisson 250-kVp x rays, and fission neutrons. The meandistribution as determined by the &2/y and number of dicentrics per cell as a function of radi-

ation dose was fitted to a linear-quadratic equationPapworth test (Papworth 1970). These findings =aD + aD2 for low-LET radiation, 250-kVp x rays,of Poisson statistics are consistent with pub- and 60°o gamma rays; for fission-neutrons thelished findings from similar experiments by yield was fitted to a straight line (Y = aD) by theothers (Edwards et al. 1979). weighted least squares regression method.

Weights were based on the reciprocal of the SE ofThe classical hypothesis of aberration induction the mean squared. These results represent theis used for the quantitative derivation of dose- pooled mean from 3 independent experiments.effect relationships. In this model, two lesions Error bars represent SE of the mean.are required for producing a dicentric, and theselesions may arise from one or two independent cy for dicentric yields as described for low-ionizing tracks. Dicentrics produced by single LET sources spanning a broad range of energytrack events are proportional to the dose of radi- (Straume 1995). Fitted data for low-LETation (aD), while the yield of dicentrics in- radiation sources were (0.098 ± 0.0209) D +duced by two separate track events are propor- (0.044 ±0.0093) D2 for 60Co gamma rays r =tional to the square of the dose (OD2). 0.999)and (0.059±0.0136) D + (0.029±0.0046)Following exposure of lymphocytes to low-LET D2 for x rays r= 0.995). These findings are inradiation, such as 250-kVp x rays or 6 °Co good general agreement with publishedgamma rays, the dicentric yield (Y) has been findings of others. For example, AFRRI'sshown to best fit to a linear quadratic model. dicentric 60Co gamma-ray (Fig. 5A) and x-ray

(Fig. 5B) dose-response data are comparedDose responses for the mean number of dicen- with similar published studies from othertrics per cell for the three radiation sources are laboratories (Edwards 1997; Lloyd et al. 1987;shown in Figure 4. The data at these two photon Bauchinger et al. 1984; Bauchinger et al. 1979;energies are consistent with the LET dependen- N.C.R.P. 1990; Schmid et al. 1984).

9

Dose Response Relationships for Dicentric Yield

Table 2. Distribution of dicentrics in human lymphocytes exposed in vitro to 60CO gamma rays.*______Frequency of dicentrics

Dose Number of 01 2 3 4 Totallmeta- aF2(Gy) metaphases ____ _ _4phase ±SE ratioA~E

0 395 1.00 - - - ----

0.25 332 0.9698 0.0301 - - - 0.0301±0.0094 0.97±0.08 -0370.50 329 0.9640 0.0365 - - - 0.0365±0.,01,04 -0.97±0.08--- -0.4-5

0.75 51 0.9020 0.0980 - - - 0.0980±0.0421 0.92±0.20 -041.0 103 0.9610 0.0390 - - - 0.0390±0.0190 0.97±0.14 -0.24

1.5 191 0.8482 0.1466 0.0052 - - 0.1570±0.0274 0.91±0.10 -0.85

2.0 - 80 0.9125 0.0500 0.0375 - - 0.1250±0.0483 1.49±0.16 3.27

2.5 65 0.6615 0.2923 0.0308 0.0154 - 0.4001±0.0785 _ 1.00±0.18 0.00

3.0 108 0.6852 0.2500 0.0648 - - 0.3796±0.0584 0.97±0.14 -0.22

3.5 40 0.5250 0.3500 0.0750 0.0500 - 0.6500±0.1318 1.07±0.23 0.31

4.0 173 0.4913 0.3757 0.0983 0.0289 0.0058 0.6822±0.0618 0.97±0.14 -0.30

5.0 91 0.2967 0.4286 0.1758 0.0550 0.0440 1.1208±0.1092 -- 0.97±0.15 --0.21--

Table 3. Distribution of dicentrics in human lymphocytes exposed in vitro to 250 kVp x-rays.-*______Frequency of dicentrics

Dose Number of 01 2 3 5 Total/meta- o2 /Y,(Gy) metaphases ________ _phase ±SE ratio±S E

0 395 1.00 -- - -_---

0.25 235 0.9617 0.0383 - - - 0.0383±0.0125 -0.97±0.-09- -0.3-90.50 _ 185 0.9405 0.0595 - - - 0.0595±0.0174 0.95±0.10 -0.55

0.75 153 0.9020 0.0980 _- - - 0.0980±0.0240 0.91±0.11 -0.83

1.0 216 0.8657 0.1296 0.0046 - - 0.1388±0.0245 0.93±0.10 -0.72

2.0 201 0.6970 0.254 0.0500 - - 0.3540±0.0410 0.93±0.10 -0.67

3.0 202 0.5149 0.3614 -0. 1089 0.0149 - 0.6239±0.0519 0.87±0.10 -1.28-3.5 87 _ 0.3448 0.3563 0.2184 0.0690 0.0115 1.0575±0.1089 0.98±0.15 -0.16

Table 4. Distribution of dicentrics in human lymphcye exposed in vitro to fission neutrons._*_______ ____ Frequency of dicentrics___

Dose Number of 012 3 4 Totallmeta- Ci /y,(Gy) metaphases phase±SE ratio±SE ~

0 395 1.00 - ------ --0.75 100 0.8100 0. 1400 - 0.0500 - - 0.2400±0.0534 1. 19±0-.14-- 1.36

1.0 138 0.6377 0.2826 0.0797 - - 0.4420±0.0544 0.93±0.12 - -0.62-1.5 100 0.5000 0.3500 0.1200 0.0300 - 0.6800±0.0803 0.95±0.14 -0.37

2.0 149 0.3154 0.3624 0.2349 0.0604 0.0269 1. 12 10±0.0830 0.92±0.12 -0.73

2.5 72 0.2778 0.3056 0.2222 0.1250 0.0694 1.4026±0.1435 1.06±0.17 0.34

*Note: Distribution analysis of the number of dicentrics was analyzed as described by Papworth (Papworth 1970) using 02/y and the

overdispersion parameter (p). A p value between -1.96 and 1.96 indicates a Poisson distribution.

10

Results and Discussion

A B1.5 3 A. ARRI

-- B. NRPB, UK [6] " *B. NRPB, UK [4].C. Leiden, The Netherlands [6] - E. REACT/S, USA [45]

- - D. Germany [44] - T A...... E. REACT/S, Oak Ridge [45] A -0 2 -F . AAEC, Australia [6]

"Z E~i Q ', ,

0 F0 V

$ 0.5 --~ EE B ,."..

Z .Z •"...

0"I I I t ti0.0 0 1 2 3 4 5 60 1 2 3 4 5 6 7

Radiation dose (Gy) Radiation dose (Gy)

Figure 5. Intercomparison of AFRRI's dose- Cresponse calibration curves with different 1.0biodosimetry laboratories. A(gamma rays), B(220-to 250-kVp x rays), C (neutrons). For neutrons the _ 0.8a coefficients (per cell per Gy) of the linear fit for .!TRIGA, BEPO, and Janus reactors were compared. _0.6The abscissa indicates the laboratory acronymswhere measurements were made. Fast neutrons 0.4with an estimated mean energy of 0.7 MeV were .produced in the BEPO reactor in Atomic Energy cResearch Establishment by bombarding a uranium 0. 0.2converter plate with 14.7 MeV thermal neutrons. The <gamma contamination was 10% of the fast neutron 0.0 - Scottdose (Lloyd et aL 1976; Scott et aL 1969). Fission TRIGA Lloyd Scottneutrons of 0.85 MeV were produced at the JANUS AFRRI NRPB ANLreactor of Argonne National Laboratory. Gamma raycontribution was approximately 3% of the neutrondose. another laboratory (I.A.E.A. 2001). The for-

mation of dicentric aberrations by high-LETHowever, significant differences exist between irradiation are dominated by single-track e-laboratories. Inter-laboratory variations in dose- vents, hence their yield is proportional to theresponse curves, aberration yields, and dose dose of radiation (aD). Dose-response rela-estimates for simulated accidents were noted by tionships for dicentric yields following expo-Lloyd et al. (Lloyd et al. 1987) in a collaborative sure to AFRRI fission neutrons were fitted withbiodosimetry exercise conducted with the sup- the mathematical function Y =(X D over a doseport of International Atomic Energy Agency range from 0.75 to 2.5 Gy. The a coefficient(IAEA). Discrepancies related to dose-response was 0.677-0.0003 r = 0.996). This finding iscurves and aberration yields may be overcome comparable to similar studies performed atby adopting centromere painting with a pan- 235U-reactor fission- neutron facilities (Lloyd etcentromeric DNA-hybridization probe for aber- al 1976; Scoff et al 1969; Carrano 1975) (Fig.ration analysis (Kolanko et aL 1993; Schmid et 5C). These data are also consistent with theal. 1995; Roy et al. 1996). We are currently LET dependency seen for dicentric yields as de-studying the influence of centromere painting on scribed for particle sources spanning a broadthe detection of dicentrics. In order to avoiduncertainty in dose assessment, it is advised thateach laboratory use its own calibration curve Irradiation of blood lymphocytes in vitro or inrather than using calibration curves produced by vivo produces similar levels of dicentrics per

11

Dose Response Relationships for Dicentric Yield

cGy (I.A.E.A.2001). Therefore, observed yields lowship from the International Atomic Energy-of dicentrics in an exposed person's blood Agency, Vienna. The views expressed are thoselymphocytes may be used to assess previous of the authors; no endorsement by AFRRI hasradiation exposure by comparison with an in been given or inferred. The expert contribu-vitro- produced dose-response calibration curve. tions of the Radiation Sciences DepartmentThe influence of sample size on the uncertainties staff members, who provided technicalon the estimated dose is discussed in the assistance in repeated radiation exposures andappendix. Chromosome aberration analysis dosimetry support, is gratefully acknowledged.remains a valuable radiation dose assessment The expert assistance of T.D. Roberge, S.M.method for biological dosimetry in accidental Gribben, M.D. Pyle, and J. Sanders is

and occupational radiation exposures. appreciated. The editorial assistance of M.Greenville and E. Pirrung, and desktop designlayout by A. Ward are also greatly appreciated.

Acknowledgements We also wish to thank, for their assistance andhelpful discussions, E.E. Kearsley, Ph.D.

The Armed Forces Radiobiology Research (NCRP, Bethesda, MD), K.S. Kumar, Ph.D.Institute, under work unit AFRRI-98-3 and (AFRRI, Bethesda, MD), L. Gayle Littlefield,cooperative research and development agree- Ph.D. (ORISE, Oak Ridge, TN), and A.T.ment (CRADA) AFRRI/LAI-95 supported this Natarajan, Ph.D. (Leiden University, Theresearch. A.W. Khusen was supported by a fel- Netherlands).

12

Appendix

Estimation of Radiation Dose: Influence of An example of increasing the number of meta-Sample Size on Uncertainties. Radiation dose is phases analyzed from 50 to 500 for varyingestimated without any difficulty by comparing number of dicentrics observed on the 95% con-the measured yield of dicentrics in an exposed fidence limits for estimated radiation dosesindividual's blood lymphocytes with an in- between 0.08 and 4.93 Gy is shown (Table Al).vitro-generated calibration curve. However, These estimations were derived from the co-there is no unified way of deriving the un- efficients of our calibration curve for gammacertainty on the estimated dose, which is radiation. Generally, analysis of 200 meta-normally expressed as a confidence interval. By phases is sufficient to estimate a dose with rea-convention, a 95% confidence limit is chosen as sonable confidence in accidental exposurethe standard, meaning that the estimated dose is levels of military relevance.accurate 95 out of 100 times. The uncertainty onthe estimated dose arises from uncertainties The 95% confidence limits for our calibrationassociated with two factors: (1) the Poisson curves for different radiation qualities arenature of the yield of dicentrics and (2) the shown in Figure Al. The coefficients of thesecalibration curve. The nature of the distribution calibration curves are used to determineof dicentrics after exposure to different radiation radiation doses in accidental exposures ofqualities is shown in Tables 2-4. military personnel.

Table Al. An example of the effect of the sample size on lower and upper 95% confidence limits onthe estimated whole-body equivalent after acute exposure using the AFRRI 60Co gamma-raycalibration curve.

Number of Mean Lower confidence limit (cGy) Upper confidence limit (cGy)dicentrics dose Sample size Sample sizeper cell (cGy) 50 200 500 50 200 500

0.005 8 < 2 < 2 < 2 118 58 39

0.010 16 < 2 < 2 4 126 69 52

0.025 36 < 2 9 14 148 95 78

0.050 64 9 25 33 176 126 110

0.100 110 33 59 70 221 172 157

0.250 209 119 150 163 329 273 254

0.500 325 227 265 278 454 401 381

0.750 416 316 348 362 566 504 488

1.000 493 383 417 431 655 598 581

13

Dose Response Relationships for Dicentric Yield

A B2.0 1.5

.2o 1.0-

S 1.0

o 7 0.50.5-

0.0I I ,___0.0_ __ ___ __ ___I _ ___ __I__ __0 1 2 3 4 5 6 0 1 2 3 4 5 6

Radiation dose (Gy) Radiation dose (Gy)

Figure Al. AFRRI dose-response calibration curves Cfor dicentric yields in human lymphocytes with upperand lower 95% confidence limits for (A) 60o gammarays, (B) x rays, and (C) neutrons. 1.5 //

.)1.0

0.5

z

0.0 I I I I0 1 2 3 4 5 6

Radiation dose (GY)

14

References

A.A.P.M. (American Association of Physicists Blakely WF, Prasanna PGS, Kolanko CJ, Pylein Medicine), Radiation Therapy Committee MD, Mosbrook DM, Loats AS, Rippeon TL,Task Group 21 (1983) A protocol for deter- Loats H (1995) Application of premature chro-mination of absorbed dose from high-energy mosome condensation assay in simulated par-photon and electron beams. Medical Physics, tial-body radiation exposures: Evaluation of the10:741-747. use of an automated metaphase finder. Stem

Cells, 13:223-230.Attix FH (1986) Introduction to RadiologicalPhysics and Radiation Dosimetry. New York: Carrano AV (1975) Induction of chromosomalJohn Wiley & Sons. aberrations human lymphocytes by x rays and

Bauchinger M (1983) Microdosimetric aspects fission neutrons: Dependence on cell cycle

of the induction of chromosome aberrations. In: stage. Radiation Research, 63:403-421.

Ishihara T, Sasaki MS (eds) Radiation Induced Carter RE, Verrelli DM (1973) AFRRI CobaltChromosome Damage in Man. New York: Alan Whole-body Irradiator (AFRRI Technical Re-R. Liss, Inc., 1-22. port 73-3). Bethesda, MD: Armed Forces

Bauchinger M, Koester L, Schmid E, Dresp J, Radiobiology Research Institute, 1-8.

Streng S (1984) Chromosome aberrations in Edwards AA (1997) The use of chromosomalhuman lymphocytes induced by neutrons. Inter- aberrations in human lymphocytes for biolog-national Journal of Radiation Biology, ical dosimetry. Radiation Research, 148:S39-45:449-457. S44.

Bauchinger M, Schmid E, Dresp J (1979) Edwards AA, Lloyd DC, Purrott RJ (1979)Calculation of the dose-rate dependence of the Radiation induced chromosome aberrations anddicentric yield after Co Y-irradiation of human the Poisson distribution. Radiation and Envir-lymphocytes. International Journal of Radiation onmental Biophysics, 16:89-100.

Biology, 35:229-233.Eisenhauer C (1991) AFRRI Neutron Spectrum

Bender MA, Gooch PC (1966) Somatic chro- Directory. Gaithersburg, MD: National Insti-mosome aberrations induced by human whole- tute of Standards and Technology, 1-58.

body irradiation: The "Recuplex" criticality ac-

cident. Radiation Research, 29:568-582. Fisher DR, Harty R (1982) The microdosimetry

Biavati MH, Boer E (1996) D(Y) spec- of lymphocytes irradiated by alpha particles.Biavti H, oerE (996 D() sec-International Journal of Radiation Biology. 41:tragamma rays. In: Annual Report on Research 315-324.

Project. New York: Radiological Research 315-324.

Laboratory, Columbia University, 87-101. Gerstenberg, H.M. (1989) Application of

Blakely WF, Benevides LA, Gerstenberg HM microdosimetry to battlefield neutron spectra.(199) Fsion-neutonees on hro er In: Proceedings of the 1989 Workshop of the(1995) Fission-neutron effects on chromosome RSG-5 Physical Dosimetry Subcommittee.

damage in Chinese hamster V79 cells: Use of rcuil Fra ETCA.

thedaughter-and granddaughter- microcolony Arcueil, France: ETCA.

micronuclei assay. In: Hagan U, Jung H, Stref-fer C (eds) Radiation Research 1895-1995, Gerstenberg HM (1991) Comparison ofYspec-Congress Proceedings, Vol. 2: Congress tra from the TRIGA and Janus reactors: TheLectures. Wurzburg, Germany: 10 ICRR implication for dose rate studies. In: ChapmanSociety, 344-347. JD, Dewey WC, Whitmore GF (eds) Radiation

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Dose Response Relationships for Dicentric Yield

Research: A Twentieth Century Perspective, Dosimetry: Cytogenetic Approaches to Mam-Vol. 1: Congress Abstracts. San Diego: Aca- malian Systems. New York: Springer anddemic Press Inc., 110. Verlag, 3-14.

Greenstock CL, Trivedi A (1994) Biological Lloyd DC, Edwards AA, Prosser JS, Barjakta-and biophysical techniques to assess radiation rovic N, Brown JK, Horvat D, Ismail SR,exposure: A perspective. Progress in Biophyics Koteles GJ, Almassy Z, Krepinsky A,and Molecular Biology, 61:81-130. Kucerova M, Littlefield LG, Mukherjee U,

Natarajan AT, Sasaki MS (1987) AI.A.E.A. (2001) Cytogenetic Analysis for collaborative exercise on cytogenetic dosimetryRadiation Dose Assessment: A Manual. for simulated whole and partial body accidental(Technical Report 405). Vienna: International irradiation. Mutation Research, 179:197-208.Atomic Energy Agency.

Lloyd DC, Purrott RJ, Dolphin GW, EdwardsI.C.R.U. (1973) Measurement of Absorbed Dose AA (1976) Chromosome aberrations induced inin a Phantom Irradiated by a Single Beam of X or human lymphocytes by neutron irradiation.Gamma Rays (Report 23). Washington DC International Journal of Radiation Biology, 29:International Commission on Radiation Units 169-82.and Measurements.

Marshall IR, Williamson FS (1985) Micro-I.C.R.U. (1977) Neutron Dosimetry for Biology dosimetric measurements of Janus neutrons.and Medicine (Report 26). Bethesda, MD:International Commission on Radiation Units Radiation Protection Dosimetry, 13:111-115.and Measurements.

Muller WU, Streffer C (1991) Biological indi-I.C.R.U. (1980) Linear Energy Transfer (Report cators of radiation damage. International Jour-16). Washington DC: International Commission nal of Radiation Biology, 59:863-873.on Radiological Units.

N.C.R.P. (1990) The Relative Biological Effec-I.C.R.U. (1993) Quantities and Units in Radi- tiveness of Radiations of Different Quality (Re-ation Protection Dosimetry (Report 51). Bethes- port 104). Bethesda, MD: Recommendations ofda, MD: International Commission on Radio- the National Council on Radiation Protectionlogical Units. and Measurements, National Radiological Pro-

tection Board, 28-48.Keifer J (1990) Biological Radiation

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New York: Springer-Verlag, 444. N.I.S.T. (1991) Annual Progress Report in Sup-

Kolanko CJ, Prasanna PGS, Nath J, Blakely WF port of Neutron Dosimetry. Gaithersburg, MD:

(1993) PCR synthesis of a human pancentro- National Institute of Standards and Techno-

meric DNA hybridization probe and detection of logy.

in situ probe hybridization using color pig- apworth DG (1970) Appendix. In: Savagement/immunostaining. In: Proceedings ofNATO Workshop on Biomedical Aspects of JRK, Sites of radiation induced chromosomeNuclear Defense, Panel 8, Research Study ex- changes. Current Topics in Radiation, 6:Group 23 on Ionizing Radiation. Bethesda, MD. 129-194.

Lloyd DC (1984) An overview of radiation do- Prasanna PGS, Garner DC, Khusen AW, Loatssimetry by conventional cytogenetic methods. H, Shehata CW, Gerstenberg HM, Blakely WFIn: Eisert WG, Medelsohn ML (eds) Biological (1998) Chromosome aberration analysis for

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biological dosimetry: A real case scenario of Schmid E, Braselmann H, Nahrstedt U (1995)diagnostic service supporting the U.S. armed Comparison of y-ray induced dicentric yields inforces. In: Proceedings of the 1996 Workshop of human lymphocytes measured by conventionalthe Research Study Group on the Biomedical analysis and FISH. Mutation Research, 348:Aspects of Nuclear Defense, Panel 8, Research 125-130.Study Group 23 on Ionizing Radiation. Ottawa,Canada, 5.1-5.10. Scott D, Sharpe H, Batchelor AL, Evans HJ,

Papworth DG (1969) Radiation-induced chro-Prasanna PGS, Kolanko CJ, Gerstenberg HM, mosome damage in human peripheral bloodBlakely WF (1997) Premature chromosome lymphocytes in vitro 1. RBE and dose-ratecondensation assay for biodosimetry: Studies studies with fission neutrons. Mutation Re-with fission neutrons. Health Physics, 72: search, 8:367-381.594-600.

Preist JH (1977) Medical Cytogenetics and Cell Stankus AA, Xapsos MA, Kolanko CJ, Ger-

Culture. Philadelphia: Lea and Febiger. stenberg HM, Blakely WF (1995) Energy de-position events produced by fission neutrons in

Redpath JL, Antoniono RJ, Sun C, Gerstenberg aqueous solutions of plasmid DNA. Inter-HM, Blakely WF (1995) Late mitosis/early GI national Journal of Radiation Biology, 68:1-9.phase and mid-G1 phase are not hypersensitivecell cycle phases for neoplastic transformation Straume T (1995) High energy gamma rays inof HeLa x skin fibroblast human hybrid cells Hiroshima and Nagasaki: Implications for riskinduced by fission-spectrum neutrons. Radiation and WR. Health Physics, 69:954-956.Research, 141:37-43.

Turner JE (1992) An introduction to micro-Rossi HH (1959) Specification of radiation dosimety. Radiation Protection Management,quality. Radiation Research, 10:522-531. 9:25-58.

Roy L, Sorokine-Durm I, Voisin P (1996) Com- Verbinski VV, Cassapakis CG, Hagan WK,parison between fluorescence in situ hybridiza- Ferlic K, Daxon E (1981) Calculation of thetion and conventional cytogenetics for dicen- Neutron and Gamma-ray Environment in andtric scoring: A first-step validation for the use of arond the AFRRI TRIGA Reactor (DNAFISH in biological dosimetry. International arouJournal of Radiation Biology, 70:665-669. 5793F-2). Washington, DC: Defense Nuclear

Agency, 1-262.Rutovitz D (1992) Reflections on the past, pre-sent and future of automated aberration scoring Watt DE, Alkharam AS, Child MB, Salikin MSsystems for radiation dosimetry. Journal of (1994) Dose as a damage specifier in radio-Radiation Research, 33 (Supplement): 1-30. biology for radiation protection. Radiation Re-

search, 139:249-251.Schmid E, Bauchinger M, Streng S, NahrstedtU (1984) The effect of 220-kVp x rays with Zeman GH, Ferlic KP (1984) Paired ion cham-different spectra on the dose response of ber constants for fission gamma-neutron fieldschromosome aberrations in human lympho- (Technical Report). Bethesda, MD: Armedcytes. Radiation Environmental Biophysics, 23: Forces Radiobiology Research Institute, 84-8.305-309.

17

Dose Response Relationships for Dicentric Yield

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AFFRI's Gamma-Ray, X-Ray, and Fission-Neutron Calibration Curves forthe Lymphocyte Dicentric Assay: Application of a Metaphase FinderSystem

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Prasanna PGS, Loats H, Gerstenberg HM, Torres BN, Shehata CW,Duffy KL, Floura RS, Khusen AW, Jackson WE, Blakely WF

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Facilities are established at the Armed Forces Radiobiology Research Institute (AFRRI) to performradiation-induced chromosome aberration analysis for biological dosimetry. Whole blood from healthyhuman volunteers was used after obtaining informed consent. Peripheral blood lymphocytes were exposedin vitro to different types of radiation; 60CO gamma rays (E=1.25 MeV, mean of the absorbed dosedistribution of the lineal energy, yD=1.9 keV/[tm, 1 Gy/min); x rays (250 kVp, E = 83 keV, YD =4 keV/[tm,1 Gy/min); or a fission-spectrum neutron source (E=0.71 MeV, yD= 65 keV/[tm, 0.25 Gy/min). Distributionof radiation-induced dicentrics among cells exhibited Poisson statistics as characterized by the Papworthmethod (Papworth 1970). Dose-response relationships for the yield of dicentrics for photon sources werefitted with a linear-quadratic model using the maximum-likelihood method and for the neutron source by aweighted linear regression method. Comparison of the data with other published studies is presented. Thedose-response relationships for dicentric induction by low- and high-linear energy transfer (LET) radiationare consistent with the single- and two-track model of aberration formation, Y = otD + PD2. An increase in yDresulted in an increase in dicentric yield. As expected, fission neutrons induced a significantly higher yieldof dicentrics than that caused by low-LET sources. The linear component of the model, corresponding todamage caused by single-tracks, is predominant with fission neutrons so that the dose-effect relationship isessentially linear. An automated metaphase finder system with a satellite scoring utility was used to improvedata collection.

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