-
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
15
-
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
Effects.
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
16
-
References
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
18
-
Distribution List
DEPARTMENT OF DEFENSE U.S. ARMY MEDICAL RESEARCH INSTITUTE OF
CHEMICALDEFENSE
ARMED FORCES RADIOBIOLOGY RESEARCH INSTITUTE ATTN:
MCMR-UV-RATTN: INFORMATION SERVICES DIVISIONATTN: TECHNICAL LIBRARY
U.S. ARMY NUCLEAR AND CHEMICAL AGENCY
ATTN: MONA-NUARMY/AIR FORCE JOINT MEDICAL LIBRARY
ATTN: DASG-AAFJML U.S. ARMY RESEARCH INSTITUTE OF
ENVIRONMENTALMEDICINE
ASSISTANT TO THE SECRETARY OF DEFENSE ATTN: DIRECTOR OF
RESEARCHATTN: AEATTN: HA(IA) U.S. ARMY RESEARCH LABORATORY
ATTN: DIRECTORDEFENSE SPECIAL WEAPONS AGENCY
ATTN: TITL WALTER REED ARMY INSTITUTE OF RESEARCHATTN: DDIR
ATTN: DIVISION OF EXPERIMENTALATTN: RAEM THERAPEUTICSATITN: MID
DEFENSE TECHNICAL INFORMATION CENTERATTN: ACQUISITIONATTN:
ADMINISTRATOR DEPARTMENT OF THE NAVY
ALBUQUERQUE OPERATION, DEFENSE THREAT BUREAU OF MEDICINE &
SURGERYREDUCTION AGENCY ATTN: CHIEF
ATTN: DASIACATTN: AORSE NAVAL AEROSPACE MEDICAL RESEARCH
LABORATORY
ATTN: COMMANDING OFFICERINTERSERVICE NUCLEAR WEAPONS SCHOOL
ATTN: DIRECTOR NAVAL MEDICAL RESEARCH AND DEVELOPMENTCOMMAND
LAWRENCE LIVERMORE NATIONAL LABORATORY ATITN: CODE 42ATTN:
LIBRARY
NAVAL MEDICAL RESEARCH INSTITUTEUNDER SECRETARY OF DEFENSE
(ACQUISITION) ATTN: LIBRARY
AT-TN: OUSD(A)/R&ENAVAL RESEARCH LABORATORY
UNIFORMED SERVICES UNIVERSITY ATTN: LIBRARYATTN: LIBRARY
OFFICE OF NAVAL RESEARCHDEPARTMENT OF THE ARMY ATTN: BIOLOGICAL
& BIOMEDICAL S&T
HARRY DIAMOND LABORATORIESATTN: SLCSM-SE
OFFICE OF THE SURGEON GENERAL DEPARTMENT OF THE AIR FORCE
ATTN: MEDDH-NBROOKS AIR FORCE BASE
U.S. ARMY AEROMEDICAL RESEARCH LABORATORY ATTN: AL/OEBZATTN:
SCIENCE SUPPORT CENTER
OFFICE OF AEROSPACE STUDIESU.S. ARMY CHEMICAL RESEARCH,
DEVELOPMENT, & ATTN: OAS/XRSENGINEERING CENTER
ATTN: SMCCR-RST OFFICE OF THE SURGEON GENERALATTN: HQ
AFMOA/SGPT
U.S. ARMY INSTITUTE OF SURGICAL RESEARCH ATTN: HQ
USAF/SGESAT-TN: COMMANDER
U.S. AIR FORCE ACADEMYU.S. ARMY MEDICAL DEPARTMENT CENTER AND
SCHOOL ATTN: HQ USAFA/DFBL
ATTN: MCCS-FCMU.S. AIR FORCE OFFICE OF SCIENTIFIC RESEARCH
U.S. ARMY MEDICAL RESEARCH AND MATERIEL COMMAND ATTN: DIRECTOR
OF CHEMISTRY & LIFEATTN: COMMANDER SCIENCES
-
OTHER FEDERAL GOVERNMENT BRITISH LIBRARY
ATTN: ACQUISITIONS UNITARGONNE NATIONAL LABORATORY
A'TTN: ACQUISITIONS CENTRE DE RECHERCHES DU SERVICE DE SANTE
DESARMEES
BROOKHAVEN NATIONAL LABORATORY ATTN: DIRECTORATTN: RESEARCH
LIBRARY, REPORTS SECTION
FEDERAL ARMED FORCES DEFENSE SCIENCE AGENCY FORCENTER FOR
DEVICES AND RADIOLOGICAL HEALTH NBC PROTECTION
ATTN: DIRECTOR ATTN: LIBRARY
GOVERNMENT PRINTING OFFICE FOA NBC DEFENCEATTN: DEPOSITORY
ADMINISTRATION BRANCH ATTN: LIBRARYATTN: CONSIGNED BRANCH
INHALATION TOXICOLOGY RESEARCH INSTITUTELIBRARY OF CONGRESS
ATTN: LIBRARY
ATTN: UNIT XINSTITUTE OF NUCLEAR MEDICINE AND ALLIED
SCIENCES
LOS ALAMOS NATIONAL LABORATORY ATTN: DIRECTORATTN: REPORT
LIBRARY
INSTITUTE OF RADIOBIOLOGY, ARMED FORCES MEDICALNATIONAL
AERONAUTICS AND SPACE ADMINISTRATION ACADEMY
ATTN: RADLAB ATTN: DIRECTOR
NATIONAL AERONAUTICS AND SPACE ADMINISTRATION OAK RIDGE
ASSOCIATED UNIVERSITIESGODDARD SPACE FLIGHT CENTER ATTN: MEDICAL
LIBRARY
ATTN: LIBRARYRESEARCH CENTER OF SPACECRAFT RADIATION SAFETY
NATIONAL CANCER INSTITUTE ATTN: DIRECTORATTN: RADIATION RESEARCH
PROGRAM
RUTGERS UNIVERSITYU.S. DEPARTMENT OF ENERGY ATTN: LIBRARY OF
SCIENCE AND MEDICINE
AT'IN: LIBRARYUNIVERSITY OF CALIFORNIA
U.S. FOOD AND DRUG ADMINISTRATION AT-N: DIRECTOR, INSTITUTE OF
TOXICOLOGYATTN: WINCHESTER ENGINEERING AND & ENVIRONMENTAL
HEALTH
ANALYTICAL CENTER ATTN: LIBRARY, LAWRENCE BERKELEYLABORATORY
U.S. NUCLEAR REGULATORY COMMISSIONATTN: LIBRARY UNIVERSITY OF
CINCINNATI
ATTN: UNIVERSITY HOSPITAL, RADIOISOTOPELABORATORY
RESEARCH AND OTHER ORGANIZATIONS XAVIER UNIVERSITY OF
LOUISIANA
ATTN: COLLEGE OF PHARMACY
AUSTRALIAN DEFENCE FORCEATTN: SURGEON GENERAL AUTORIDAD
REGULATORIA NUCLEAR
CENTRO DE INFORMACIONAUTRE, INC.
ATTN: PRESIDENT
-
REPORT DOCUMENTATION PAGE Form Approved0MB No. 0704-0 188
Public reporting burden fpr this collection of information is
estimated to average 1 hour per responte including the time for
reviewingjnstlutions searching existing datasources a anderi
anImaintainmg the data needed, and completing and reviewing the
colection 9fnlormation. Send cormnents garng this burden estimate
or any othe
pect oithiscolection of information, includin sugestions for
reducing teburden, to Wasngton Headquarters Services. Directorae
for Information Operations andReports, 115 Jeflerson Davis Highway,
Suite I 204, Aington, VA 2220T-4302, and to the Office of
Management and Budget, Paperwork Reduction
ProjectI74-0188),Washington, DC 20503.
1. AGENCY USE ONLY (Leave blank) 2.REPORTDATE 3. REPORT TYPE AND
DATES COVERED
May 2002 Special Publication4. TITLE AND SUBTITLE 5. FUNDING
NUMBERS
AFFRI's Gamma-Ray, X-Ray, and Fission-Neutron Calibration Curves
forthe Lymphocyte Dicentric Assay: Application of a Metaphase
FinderSystem
6. AUTHOR(S)
Prasanna PGS, Loats H, Gerstenberg HM, Torres BN, Shehata
CW,Duffy KL, Floura RS, Khusen AW, Jackson WE, Blakely WF
7. PERFORMING ORGANIZATION NAME(S) AND ADDRESS(ES) 8.PERFORMING
ORGANIZATIONREPORT NUMBER
Armed Forces Radiobiology Research Institute8901 Wisconsin
AvenueBethesda, MD 20889-5603
9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10.
SPONSORING/MONITORINGAGENCY REPORT NUMBER
11. SUPPLEMENTARY NOTES
12 a. DISTRIBUTION/AVAILABILITY STATEMENT 12 b. DISTRIBUTION
CODE
Approved for public release; distribution unlimited.
13. ABSTRACT (Maximum 200 words)
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.
14. SUBJECT TERMS 15. NUMBER OF PAGES
3616. PRICE CODE
17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19.
SECURITY CLASSIFICATION 20. LIMITATION OFOF REPORT OF THIS PAGE OF
ABSTRACT ABSTRACT
UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED ULNSN 7540-01-280-5500
Standard Form 298 (Rev.2-89)
Prescribed By ANSI Sta 239-18
-
SECURITY CLASSIFICATION OF THIS PAGE
CLASSIFIED BY:
DECLASSIFIED ON:
SECURITY CLASSIFICATION OF THIS PAGE