AFRRI's Gamma-,Ray, X-Ray, and

Fission-'Ne6utron Calibration ,Curves for* the Lymphocyte, bic entric" Assay: Appliation of aK

IMetaphase Finder- System

PG.S. Pras~nna

H.M. Gerstenberg/

B.N.Torres

- C.W. Sheftata

K. L. Duffy

R.S. flowta

A.W. Khusen

W..Jackson

W.F Blakely -

DISTRIBUTION STATEMENTA

Distribution Unlimited 2002071JI2 129

Armed Forces Radiobiology Research Institute
AFRRI's Gamma-Ray, X-Ray, and

Fission-Neutron Calibration Curves for the

Lymphocyte Dicentric Assay: Application of

a 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. Khusen 1

'

3

W.E. Jackson1

W.F. BlakelyI

1

Armed Forces Radiobiology Research Institute, Applied Cellular

Radiobiology and Radiation Sciences Departments, 8901 Wisconsin

Avenue, Bethesda, MD 20889-5603

2

Loats Associates Inc., 201 East Main Street, Westminster, MD 21157

3

Current address: Center for Standardization and Radiological Safety

Research, National Atomic Energy Agency, Jakarta, Indonesia

Armed Forces Radiobiology Research Institute

8901 Wisconsin Avenue

Bethesda, Maryland 20889-5603

www.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 the

Defense 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 calibration

biology Research Institute (AFRRI) is the sole curves in order to achieve an acceptable perDepartment of Defense research laboratory for formance standard. Predictive value is enmedical radiological defense. Its primary mis- hanced further when each radiation type for

sion is to develop medical countermeasures which a calibration curve is generated is fully

against ionizing radiation. Developmental and characterized relative to microdosimetric paraapplied research focuses on prevention, assess- meters. These high standards are employed by

ment, and treatment of radiological injury, and the AFRRI Biological Dosimetry Team and are

on the confounding problems of combined in- described in this report.

jury involving radiation and other battlefield

stressors. A major research thrust of the team is to improve the performance characteristics of cyto- Precision and accuracy are hallmarks of effec- genetic tests. Through a collaborative effort untive and meaningful biological tests. This is es- der a cooperative research and development

pecially true for the lymphocyte-dicentric assayas o t boloica aplis dsimtryandtoagreement with Loats Associates Inc., West- say as it applies to biological dosimetry and to minster, Maryland, the team has developed an

the estimation of radiation doses in individuals. mihster, Marylandmthe am ha deveope an

Gamma rays, x rays, and fission neutrons induce enhancemdgialimage an al neto -

morphologic aberrations in lymphocyte chro- tm for to t ia aca sis. to

mosomes that can be quantifiably measured lection for this report was facilitated using the

using sophisticated cytogenetic techniques. The system's mature automated metaphase-finding

dicentric chromosome is one such aberration, component coupled with lymphocyte-dicentric

and it is recognized as a biomarker of exposures scoring at peripheral, or satellite, scoring stato ionizing radiation. Measuring the frequency tions.

of dicentric chromosomes in peripheral blood

lymphocytes gives a good approximation of ra- In addition to its core objective of developing,

diation dose. Accordingly, the lymphocyte di- testing, and validating deployable biodosimetry

centric assay finds utility in cases of accidental systems for military field operations, the team

or intentional exposures when there is a need to maintains one of the nation's few reference testdocument radiation doses in individuals, and the ing facilities for radiation dose assessment. This

assay's predictive value (precision and accura- resource responds to military, domestic, and incy) is of paramount importance when used to ternational nuclear or radiological emergenaide medical triage and manage the radiation cies involving human exposures to ionizing

injured, radiation. It is for this reason that the studies

reviewed in this report were undertaken.

The lymphocyte-dicentric assay is a technically

demanding and time-consuming procedure, re- The accomplishments documented herein point

quiring a highly trained technical or profession- to the critical role of radiobiological research in

al staff. Even with highly qualified individuals, defending our nation against current and future

controlling inter-laboratory variability is proble- threats through medical readiness, on both milimatic. Each laboratory must therefore develop tary and Homeland Security fronts.

ROBERT R. ENG, COL, MS, USA

DIRECTOR, 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-likelihood

Radiobiology 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. Whole

blood from healthy human volunteers was used Comparison of the data with other published

after obtaining informed consent. Peripheral studies is presented. The dose-response relablood 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 model distribution of the lineal energy, ydos. of aberration formation, Y = otD + PD 2

. An inkeV/pm, 1 Gy/min); x rays (250 kVp, FE=83 crease in YD resulted in an increase in dicentric

yield. As expected, fission neutrons induced a

keV, YD= 4 keV/pm, 1 Gy/min); or a fission- significantly higheryield ofdicentrics than that

spectrum neutron source (E =0.71 MeV, yl = 65 caused by low-LET sources. The linear comkeV/jim, 0.25 Gy/min). Distribution of radia- ponent of the model, corresponding to damage

tion-induced dicentrics among cells exhibited caused by single-tracks, is predominant with

Poisson statistics as characterized by the Pap- fission neutrons so that the dose-effect

worth method (Papworth 1970). Dose-response relationship is essentially linear. An automated

relationships for the yield of dicentrics for metaphase finder system with a satellite scoring

photon 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 effectiveness

contributions in both accidental and occupation- (RBE) are inadequate, if not irrelevant, paraal overexposures. This biological dosimeter is meters for the quantification of biological

the 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 its

relatively radiation specific; only a few chem- energy can be dissipated in terms of discrete

icals are known to interfere with this assay. Low packets, e.g., spurs and blobs, the number and

background levels (about 1 dicentric in 2000 magnitude of which can be determined by

cells), high sensitivity (a threshold dose of 0.05 microdosimetry (I.C.R.U. 1993). While the

Gy), and known dose dependency up to 4 Gy (for absorbed dose reflects the macroscopic depolow-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 lineal

ation quality and dose rate are well character- energy y and its dose- and frequency-weighted

ized (Edwards 1997). The influence of time be- mean values YD and YF that describes the

tween radiation exposure and analysis for a radiation energy interactions at the microscopic

broad dose range is not critical for at least the level. Bauchinger reviewed the importance of

first 2 weeks after exposure (I.A.E.A. 2001). microdosimetry on the classical and alternative

However, published reports show that differ- mechanisms of chromosome-aberration formaences 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 calibration

tory should establish its own calibration curves curves of measured dicentric yields following

for 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. In

Dicentric yield from radiation exposure is de- addition, we compare these dose-response calpendent not only on the dose, but also on radi- ibration curves with similar studies from other

ation quality. Radiation quality depends on mi- laboratories. Estimating radiation dose by chrocroscopic energy deposition events that are char- mosome aberration analysis requires time-deacterized by temporal, spatial, and energy dis- manding and labor-intensive scoring by expert

tributions of the radiation fields within the ir- cytogeneticists. Our attempt to decrease cytoradiated volume. There is evidence that radio- genetic scoring time in biodosimetric assessbiological effects are more closely related to ment for radiation accidents is addressed by the

lineal energy than to neutron energy (I.C.R.U. use of satellite scoring stations used in con1980). 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 YD

man donors was collected into vacutainers Sorce YD neutrons

containing ethylenediamine tetraacetic acid y rays 1.9 ne

0.4 xrays 4.0

(EDTA) (Becton-Dickinson, Rutherford, NJ). nurons .0

The informed consent form used in this study x y *1. 

was approved by the Uniformed Services >, 0.2

University of the Health Sciences, Human Use y ry "

Committee (Bethesda, MD). Lymphocytes were lp 0.° I 

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 1000

washed 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 distribuLife Technologies, Rockville, MD) at a con- tions for AFRRI's gamma rays, x rays, and fission

centration 1-1.5 x 1 06/ml for exposure to neutrons. The measurements were made using a

different radiation types. TEPC detector with a gas filling made using a

pressure corresponding to a 1-pim diameter. The

Radiation sources and dosimetry. Dosimetry dose distributions, d(y), are normalized to unit dose

procedures 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 enerrays (Redpath et al. 1995; Blakely et al. 1995; gies in this range. This is the standard represenPrasanna 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 is

Prasanna 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 distributions

rays, and fission neutrons are shown in Figure 1. may vary depending on experimental arrangements and measurement parameters.

Gamma-ray exposures were performed in the

bilateral field of the 60

Co 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 Al

dose 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 chambers

ing 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 the

2%. Cells in suspension were placed in 15-ml International Commission on Radiation Units

polypropylene 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 susyF where yF = LET., (Turner 1992; Rossi 1959), pension in 25-cm2 tissue-culture flasks were

measured using a 1-pm diameter tissue- placed on a rotating Plexiglas holder for

equivalent proportional counter (TEPC), has irradiation and exposed at room temperature at

been previously described (Stankus et al. 1995). a dose rate of 1 Gy/min. The YF for this x-ray

X-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 et

Hamburg, 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 in

AFRRI's training, research, isotope, General excellent agreement with our value determined

Atomic (TRIGA) Mark-F, nuclear reactor. Sam- at the same diameter.

ples for irradiation were placed in a lead box

with 5-cm thick walls. Additional 15 cm of lead The number of neutron hits per cell nucleus was

shielding was placed in front of the reactor tank determined from the dose and fluence relationwall, 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 cell

projected 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 assuming

a track to allow the array to be placed at a re- a Poisson distribution of hits (Fisher and Harty

producible distance from the reactor core. An 1982). Similar calculations were performed for

extractor system was used for placing and the 60Co gamma-ray source using a LET value

retrieving 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 assumption

15-ml polypropylene centrifuge tubes, placed in that the literature value of 1.7 keV/jim for 200

a Plexiglas holder, and exposed at room keV x rays held for the 250 kVp x-ray source.

temperature. The dose rate and neutron and

gamma portions of the mixed field radiation Lymphocyte culture and metaphase spread

configuration were determined using the pairedion chamber technique (I.C.R.U. 1977) and applying previously determined spectral inform- the lymphocytes were washed and re-suspendation for this radiation configuration (Verbin- ed in media, stimulated to grow by adding

ski et al. 1981). The dose rate was 0.25 Gy/min. phytohemagglutinin (0.5 jig/ml; Murex DiagThe neutron to total dose ratio was 0.95+0.07. nostics Ltd, Dartford, England), and incubated

Fluence-weighted mean energies (E) for this at 37 C. After 44 h of stimulation, colcemid

configuration 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 in

was uniform to within 2.5%. The YF for this 235U first division metaphases and then incubated for

reactor produced the degraded fission-neutron an additional 4 h. Less than 3% of the metaspectra that have been previously described phases were in second division metaphases as

(Blakely, Benevides and Gerstenberg 1995; Pra- determined by the fluorescence plus Giemsa

sanna et al. 1997). technique at this culture time (data not shown).

Previous studies have estimated YF for spherical Following hypotonic treatment in 1% sodiumvolumes 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 standard

qualities (Blakely, Benevides and Gerstenberg method (Preist 1977). The slides were stained in

1995; Prasanna et al. 1997). The 6°Co- 4% Giemsa in PBS for dicentric analysis.

gamma-ray YF was measured to be 0.39 keV/jim

using a TEPC detector with gas pressure Automated metaphase-finding and dicentric

corresponding to a 1-jim diameter. The YF for 10 analysis. Figure 2 illustrates the automated

jim, 0.53 keV/pim was obtained by linear metaphase finder system, software utilities, and

interpolation 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 automated

having equivalent diameters from 0.5 to 20 jim. metaphase finder (LAI Metafind, Loats AssoBiavati and Boer's measured value at 1-jim ciates Inc., Westminster, MD). This system

4
Materials and Methods

A B ! control of image recognition parameters and

S,relocation of metaphase spreads on the 16-

Miri:LJ slide capacity microscope and stand-alone mi1 croscopes, here referred to as satellite scoring stations. Images of metaphase spreads were

I: :acquired using a color camera and a colorc- digitizer board. Images were displayed on a

computer monitor. The metaphase spreads were

located using a 10-x magnification objective

\ - lens and were relocated with a 100-x

magnification 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 of

automated metaphase finder (A) consists of a mi- the collected spreads were transferred to

croscope equipped with a 16-slide capacity stage, satellite scoring stations, and the relocation of

motorized x-, y-, and z-axis computer-controlled spreads using a 100-x magnification objective

positioning with specially adapted auto-focal cap- was done for manual dicentric analysis by

abilities. 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 a

three-chip RGB color camera and color-digitizer Data Analysis. Dose-response relationships for

board. The system automatically locates scorable the yield of dicentrics for photon sources were

metaphase spreads at low magnification, and saves

image 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 and

location coordinates. Software utilities were devel- for the neutron source by the weighted linear reoped to permit the metaphase finder system to re- gression model Y = aD. Weights were based

locate a spread for analysis either in the metaphase on the reciprocal of the standard error (SE) of

finder 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 metaphases

capacity stage and motorized x-, y-, and z-axis included the determination of the mean±SE and

computer-controlled positioning with specially the evaluation of the frequency distribution

adapted auto-focal capabilities. The system in- using the G2/y and ji test ofPapworth (Papworth

cludes specialized software utilities (Loats As- 1970). Using the Papworth test, a t value besociates 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 0.06

contrast to the common low-LET photon sources o.o 5

(250-kVp x rays, Cogamma rays), the quality 04.04 -

of high-LET neutron sources can vary o

considerably. Neutrons are classified according 0

to their energies (Attix 1986). Thermal neutrons W 0.02

have energies less than 0.5 keV (Attix 1986). o.ol -

Intermediate energy neutrons, sometimes re-_ o.ooferred to as "slow," "intermediate," "reso- -

nance," or "epithermal" neutrons, have energies 0.021

from 0.5 keV up to 10 keV. Neutrons with -1 0o 2 3 4 5 6 7 8

energies above 10 keV but below 20 MeV are -rime, min

called "fast" neutrons, and those with energies

above 20 MeV are called "relativistic" neutrons Figure 3. Dose-rate and time-course for neutron

(Turner 1992) . 235

U-fission reactor neutrons exposure. This figure illustrates dose and dose-rate

measurements from a typical experiment where produce energies in the range from above 0.1 samples were exposed to fission neutrons. Each

keV to over 10 MeV (I.C.R.U. 1977) and hence neutron run was selectively monitored using fission

include mostly slow and fast neutrons. Degraded and ionizing chambers in the exposure room. The

fission spectrum neutrons, commonly used in time course of dose measurements detected with a

radiobiology studies, are often referred to as 0.5 cm3 ionizing chamber for a 1.5-Gy dose at 0.25

fission spectrum neutrons. Gy min- is illustrated. Data measured in units of

nC/10-sec interval is integrated for each run and

In these studies, the dose rate for the photon analyzed to determine dose and dose rate. The

sources was 1 Gy/min. A fourfold lower dose sample placed in the lead box was extracted from

rate (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 re8 min in these studies. spectively (Fig. 1).

Radiation qualities for the sources used in this AFRRI's fission neutron facility produces a

study were extensively characterized (Fig. I). radiation quality that is qualitatively similar to

Table I lists the radiation dosimetric parameters other 235

U-reactor fission-neutron facilities, infor the gamma-ray, x-ray, and degraded fission- cluding Janus located at Argonne National Labspectrum neutron sources used to irradiate hu- oratory (ANL, Argonne, IL) (Marshall and

man lymphocytes in vitro. Measured values for Williamson 1985), British Experimental Pile

they YD were determined for 1 -pm diameter (BEPO) located at the National Radiological

volumes 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 neutron

volumes 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 simiposed to these sources over dose ranges as larities and differences in the radiation qualities

shown in Table 1. Cell fractions receiving no of these sources. The TRIGA and Janus microhits were negligible (less than 1 x 10-3

) at these dosimetry spectra have been compared and

doses, 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/GY

6

°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 __ 289

Fission 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 use

higher mean neutron energy (1.6 MeV) com- of an automated metaphase finder. The metapared to the neutron energy (0.7 MeV) for the phase spreads were either automatically reTRIGA or Janus sources. This difference can be located by the system or the digital data on loattributed to the thickness of the high atomic cation of spreads and slides were transferred to

number (Z) material that the beam transverses, two satellite scoring stations for manual analRENT's neutron beam is filtered by 2.5 cm of ysis. The use of multiple scoring stations exlead, while the TRIGA neutron bean transverses pedited the analysis (Fig. 2).

20 cm of lead. It should be noted that for a

typical fission spectrum, when filtered through There have been significant previous efforts to

lead, the neutron spectrum peak would be shifted use automated metaphase finders to detect and

down in energy. This shift is due to the energy score cytogenetic biodosimetry endpoints

dependence 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 metaphase neutrons below MeV. These neutrons c 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 facilitate Eisnhaer' cacultio (Esenaue 191) hatdata acquisition (Prasanna et al. 1998). This

an 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 metaphase

value from 50 to almost 90 keV/jim when no finder in another microscope station and using

lead is present compared with 20 cm of filtered the computer-assisted scoring sheets in the relead (Gerstenberg 1989). The mean value of the mote station. In this approach, a single metaspectrum YD also shifts, but not so dramatically phase finder can support simultaneous scoring

because 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 thousand metaphases from numerous healthy do- Lymphocyte-dicentric calibration curves.

nors were analyzed to establish radiation-cali- Since the introduction by Bender and Gooch

bration curves for the induction of dicentric for- (Bender and Gooch 1966), the lymphocytemation. Collection of this data from slides was dicentric assay has been the generally accepted

8
Results and Discussion

method for biodosimetric dose assessment in 2.0

cases of accidental and occupational overexpo- * 0 250-kVp x rays

sures. This approach is based on the use of in . A Fission neutrons

vitro-generated calibration curves for various 1 * C0Co gamma rays

radiation qualities. Experiments were per- c

formed at AFRRI to produce lymphocyte- 1.0 -

dicentric calibration curves using an established 0

protocol (I.A.E.A. 2001). The number of cells (

scored, the mean, and the frequency distribution E 0.5

of dicentrics per cell are presented for 6°Co Z

gamma rays, 250-kVp x rays, and fission 0.0

neutrons and are shown in Tables 2-4. 0 1 2 3 4 5 6

Progressive increases in radiation doses result in Radiation dose (Gy)

decreases in the fraction of cells with no dicentrics and increases in the fraction with Figure 4. Dose-response calibration curves for the

dicentrics. These dose-response data for di- induction of dicentrics in human lymphocytes

centric 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 mean

distribution as determined by the &2/y and number of dicentrics per cell as a function of radiation dose was fitted to a linear-quadratic equation Papworth 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 the

lished findings from similar experiments by yield was fitted to a straight line (Y = aD) by the

others (Edwards et al. 1979). weighted least squares regression method.

Weights were based on the reciprocal of the SE of

The classical hypothesis of aberration induction the mean squared. These results represent the

is 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 these

lesions may arise from one or two independent cy for dicentric yields as described for lowionizing tracks. Dicentrics produced by single LET sources spanning a broad range of energy

track events are proportional to the dose of radi- (Straume 1995). Fitted data for low-LET

ation (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 in

radiation, such as 250-kVp x rays or 6 °Co good general agreement with published

gamma rays, the dicentric yield (Y) has been findings of others. For example, AFRRI's

shown to best fit to a linear quadratic model. dicentric 60Co gamma-ray (Fig. 5A) and x-ray

(Fig. 5B) dose-response data are compared

Dose responses for the mean number of dicen- with similar published studies from other

trics 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 -037

0.50 329 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 -04

1.0 103 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- -0.3-9

0.50 _ 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 B 1.5 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 F

0 V

$ 0.5 - E -~ B ,.".. E Z .Z •"...

"I I 0 I t ti

0.0 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- C

response calibration curves with different 1.0

biodosimetry laboratories. A(gamma rays), B(220-

to 250-kVp x rays), C (neutrons). For neutrons the _ 0.8

a coefficients (per cell per Gy) of the linear fit for .!

TRIGA, BEPO, and Janus reactors were compared. _0.6

The abscissa indicates the laboratory acronyms

where measurements were made. Fast neutrons 0.4

with an estimated mean energy of 0.7 MeV were .

produced in the BEPO reactor in Atomic Energy c

Research Establishment by bombarding a uranium 0. 0.2

converter plate with 14.7 MeV thermal neutrons. The <

gamma contamination was 10% of the fast neutron 0.0 - Scott

dose (Lloyd et aL 1976; Scott et aL 1969). Fission TRIGA Lloyd Scott

neutrons of 0.85 MeV were produced at the JANUS AFRRI NRPB ANL

reactor of Argonne National Laboratory. Gamma ray

contribution was approximately 3% of the neutron

dose. another laboratory (I.A.E.A. 2001). The formation of dicentric aberrations by high-LET

However, significant differences exist between irradiation are dominated by single-track elaboratories. Inter-laboratory variations in dose- vents, hence their yield is proportional to the

response curves, aberration yields, and dose dose of radiation (aD). Dose-response relaestimates for simulated accidents were noted by tionships for dicentric yields following expoLloyd et al. (Lloyd et al. 1987) in a collaborative sure to AFRRI fission neutrons were fitted with

biodosimetry exercise conducted with the sup- the mathematical function Y =(X D over a dose

port 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 is

curves and aberration yields may be overcome comparable to similar studies performed at

by adopting centromere painting with a pan- 235U-reactor fission- neutron facilities (Lloyd et

centromeric 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 the

al. 1995; Roy et al. 1996). We are currently LET dependency seen for dicentric yields as destudying the influence of centromere painting on scribed for particle sources spanning a broad

the detection of dicentrics. In order to avoid

uncertainty in dose assessment, it is advised that

each laboratory use its own calibration curve Irradiation of blood lymphocytes in vitro or in

rather 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 Energyof dicentrics in an exposed person's blood Agency, Vienna. The views expressed are those

lymphocytes may be used to assess previous of the authors; no endorsement by AFRRI has

radiation exposure by comparison with an in been given or inferred. The expert contribuvitro- produced dose-response calibration curve. tions of the Radiation Sciences Department

The influence of sample size on the uncertainties staff members, who provided technical

on the estimated dose is discussed in the assistance in repeated radiation exposures and

appendix. 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 design

layout by A. Ward are also greatly appreciated.

Acknowledgements We also wish to thank, for their assistance and

helpful 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, The

research. A.W. Khusen was supported by a fel- Netherlands).

12
Appendix

Estimation of Radiation Dose: Influence of An example of increasing the number of metaSample Size on Uncertainties. Radiation dose is phases analyzed from 50 to 500 for varying

estimated without any difficulty by comparing number of dicentrics observed on the 95% conthe measured yield of dicentrics in an exposed fidence limits for estimated radiation doses

individual'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 cothere is no unified way of deriving the un- efficients of our calibration curve for gamma

certainty on the estimated dose, which is radiation. Generally, analysis of 200 metanormally expressed as a confidence interval. By phases is sufficient to estimate a dose with reaconvention, a 95% confidence limit is chosen as sonable confidence in accidental exposure

the standard, meaning that the estimated dose is levels of military relevance.

accurate 95 out of 100 times. The uncertainty on

the estimated dose arises from uncertainties The 95% confidence limits for our calibration

associated with two factors: (1) the Poisson curves for different radiation qualities are

nature of the yield of dicentrics and (2) the shown in Figure Al. The coefficients of these

calibration curve. The nature of the distribution calibration curves are used to determine

of dicentrics after exposure to different radiation radiation doses in accidental exposures of

qualities 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 on

the estimated whole-body equivalent after acute exposure using the AFRRI 60Co gamma-ray

calibration curve.

Number of Mean Lower confidence limit (cGy) Upper confidence limit (cGy)

dicentrics dose Sample size Sample size

per 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 B

2.0 1.5

.2 o 1.0-

S 1.0

o 0.5 7 0.5-

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 C

for dicentric yields in human lymphocytes with upper

and lower 95% confidence limits for (A) 60o gamma

rays, (B) x rays, and (C) neutrons. 1.5 //

.)1.0

0.5

z

0.0 I I I I

0 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, Pyle

in 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 chromination of absorbed dose from high-energy mosome condensation assay in simulated parphoton and electron beams. Medical Physics, tial-body radiation exposures: Evaluation of the

10:741-747. use of an automated metaphase finder. Stem

Cells, 13:223-230.

Attix FH (1986) Introduction to Radiological

Physics and Radiation Dosimetry. New York: Carrano AV (1975) Induction of chromosomal

John 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 Cobalt

Chromosome Damage in Man. New York: Alan Whole-body Irradiator (AFRRI Technical ReR. 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 chromosomal

human lymphocytes induced by neutrons. Inter- aberrations in human lymphocytes for biolognational 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 and

dicentric yield after Co Y-irradiation of human the Poisson distribution. Radiation and Envirlymphocytes. 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 Instimosome aberrations induced by human whole- tute of Standards and Technology, 1-58.

body irradiation: The "Recuplex" criticality accident. Radiation Research, 29:568-582. Fisher DR, Harty R (1982) The microdosimetry

Biavati MH, Biavti Boer oerE H, E (1996) D(Y) spec- of lymphocytes irradiated by alpha particles. (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) (1995) Fission-neutron Fsion-neutonees effects on on chromosome hro er RSG-5 In: Proceedings Physical of Dosimetry the 1989 Workshop Subcommittee. of the

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, Streffer C (eds) Radiation Research 1895-1995, Gerstenberg HM (1991) Comparison ofYspecCongress Proceedings, Vol. 2: Congress tra from the TRIGA and Janus reactors: The

Lectures. Wurzburg, Germany: 10 ICRR implication for dose rate studies. In: Chapman

Society, 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 MamVol. 1: Congress Abstracts. San Diego: Aca- malian Systems. New York: Springer and

demic Press Inc., 110. Verlag, 3-14.

Greenstock CL, Trivedi A (1994) Biological Lloyd DC, Edwards AA, Prosser JS, Barjaktaand 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) A

I.A.E.A. (2001) Cytogenetic Analysis for collaborative exercise on cytogenetic dosimetry

Radiation 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, Edwards

I.C.R.U. (1973) Measurement of Absorbed Dose AA (1976) Chromosome aberrations induced in

in 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 indiI.C.R.U. (1980) Linear Energy Transfer (Report cators of radiation damage. International Jour16). Washington DC: International Commission nal of Radiation Biology, 59:863-873.

on Radiological Units.

N.C.R.P. (1990) The Relative Biological EffecI.C.R.U. (1993) Quantities and Units in Radi- tiveness of Radiations of Different Quality (Reation Protection Dosimetry (Report 51). Bethes- port 104). Bethesda, MD: Recommendations of

da, MD: International Commission on Radio- the National Council on Radiation Protection

logical Units. and Measurements, National Radiological Protection Board, 28-48.

Keifer J (1990) Biological Radiation Effects.

New York: Springer-Verlag, 444. N.I.S.T. (1991) Annual Progress Report in SupKolanko 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 Technomeric DNA hybridization probe and detection of logy.

in situ probe hybridization using color pig- apworth DG (1970) Appendix. In: Savage

ment/immunostaining. In: Proceedings of

NATO Workshop on Biomedical Aspects of JRK, Sites of radiation induced chromosome

Nuclear 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, Loats

simetry by conventional cytogenetic methods. H, Shehata CW, Gerstenberg HM, Blakely WF

In: 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 in

forces. In: Proceedings of the 1996 Workshop of human lymphocytes measured by conventional

the 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 chroPrasanna PGS, Kolanko CJ, Gerstenberg HM, mosome damage in human peripheral blood

Blakely WF (1997) Premature chromosome lymphocytes in vitro 1. RBE and dose-rate

condensation assay for biodosimetry: Studies studies with fission neutrons. Mutation Rewith fission neutrons. Health Physics, 72: search, 8:367-381.

594-600.

Preist JH (1977) Medical Cytogenetics and Cell Stankus AA, Xapsos MA, Kolanko CJ, GerCulture. Philadelphia: Lea and Febiger. stenberg HM, Blakely WF (1995) Energy deposition events produced by fission neutrons in

Redpath JL, Antoniono RJ, Sun C, Gerstenberg aqueous solutions of plasmid DNA. InterHM, Blakely WF (1995) Late mitosis/early GI national Journal of Radiation Biology, 68:1-9.

phase and mid-G1 phase are not hypersensitive

cell cycle phases for neoplastic transformation Straume T (1995) High energy gamma rays in

of HeLa x skin fibroblast human hybrid cells Hiroshima and Nagasaki: Implications for risk

induced by fission-spectrum neutrons. Radiation and WR. Health Physics, 69:954-956.

Research, 141:37-43.

Turner JE (1992) An introduction to microRossi 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 the

tion and conventional cytogenetics for dicen- Neutron and Gamma-ray Environment in and

tric scoring: A first-step validation for the use of arond the AFRRI TRIGA Reactor (DNA

FISH in biological dosimetry. International arou

Journal of Radiation Biology, 70:665-669. 5793F-2). Washington, DC: Defense Nuclear

Agency, 1-262.

Rutovitz D (1992) Reflections on the past, present and future of automated aberration scoring Watt DE, Alkharam AS, Child MB, Salikin MS

systems for radiation dosimetry. Journal of (1994) Dose as a damage specifier in radioRadiation Research, 33 (Supplement): 1-30. biology for radiation protection. Radiation Research, 139:249-251.

Schmid E, Bauchinger M, Streng S, Nahrstedt

U (1984) The effect of 220-kVp x rays with Zeman GH, Ferlic KP (1984) Paired ion chamdifferent spectra on the dose response of ber constants for fission gamma-neutron fields

chromosome aberrations in human lympho- (Technical Report). Bethesda, MD: Armed

cytes. 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 CHEMICAL

DEFENSE

ARMED FORCES RADIOBIOLOGY RESEARCH INSTITUTE ATTN: MCMR-UV-R

ATTN: INFORMATION SERVICES DIVISION

ATTN: TECHNICAL LIBRARY U.S. ARMY NUCLEAR AND CHEMICAL AGENCY

ATTN: MONA-NU

ARMY/AIR FORCE JOINT MEDICAL LIBRARY

ATTN: DASG-AAFJML U.S. ARMY RESEARCH INSTITUTE OF ENVIRONMENTAL

MEDICINE

ASSISTANT TO THE SECRETARY OF DEFENSE ATTN: DIRECTOR OF RESEARCH

ATTN: AE

ATTN: HA(IA) U.S. ARMY RESEARCH LABORATORY

ATTN: DIRECTOR

DEFENSE SPECIAL WEAPONS AGENCY

ATTN: TITL WALTER REED ARMY INSTITUTE OF RESEARCH

ATTN: DDIR ATTN: DIVISION OF EXPERIMENTAL

ATTN: RAEM THERAPEUTICS

ATITN: MID

DEFENSE TECHNICAL INFORMATION CENTER

ATTN: ACQUISITION

ATTN: ADMINISTRATOR DEPARTMENT OF THE NAVY

ALBUQUERQUE OPERATION, DEFENSE THREAT BUREAU OF MEDICINE & SURGERY

REDUCTION AGENCY ATTN: CHIEF

ATTN: DASIAC

ATTN: AORSE NAVAL AEROSPACE MEDICAL RESEARCH LABORATORY

ATTN: COMMANDING OFFICER

INTERSERVICE NUCLEAR WEAPONS SCHOOL

ATTN: DIRECTOR NAVAL MEDICAL RESEARCH AND DEVELOPMENT

COMMAND

LAWRENCE LIVERMORE NATIONAL LABORATORY ATITN: CODE 42

ATTN: LIBRARY

NAVAL MEDICAL RESEARCH INSTITUTE

UNDER SECRETARY OF DEFENSE (ACQUISITION) ATTN: LIBRARY

AT-TN: OUSD(A)/R&E

NAVAL RESEARCH LABORATORY

UNIFORMED SERVICES UNIVERSITY ATTN: LIBRARY

ATTN: LIBRARY

OFFICE OF NAVAL RESEARCH

DEPARTMENT OF THE ARMY ATTN: BIOLOGICAL & BIOMEDICAL S&T

HARRY DIAMOND LABORATORIES

ATTN: SLCSM-SE

OFFICE OF THE SURGEON GENERAL DEPARTMENT OF THE AIR FORCE

ATTN: MEDDH-N

BROOKS AIR FORCE BASE

U.S. ARMY AEROMEDICAL RESEARCH LABORATORY ATTN: AL/OEBZ

ATTN: SCIENCE SUPPORT CENTER

OFFICE OF AEROSPACE STUDIES

U.S. ARMY CHEMICAL RESEARCH, DEVELOPMENT, & ATTN: OAS/XRS

ENGINEERING CENTER

ATTN: SMCCR-RST OFFICE OF THE SURGEON GENERAL

ATTN: HQ AFMOA/SGPT

U.S. ARMY INSTITUTE OF SURGICAL RESEARCH ATTN: HQ USAF/SGES

AT-TN: COMMANDER

U.S. AIR FORCE ACADEMY

U.S. ARMY MEDICAL DEPARTMENT CENTER AND SCHOOL ATTN: HQ USAFA/DFBL

ATTN: MCCS-FCM

U.S. AIR FORCE OFFICE OF SCIENTIFIC RESEARCH

U.S. ARMY MEDICAL RESEARCH AND MATERIEL COMMAND ATTN: DIRECTOR OF CHEMISTRY & LIFE

ATTN: COMMANDER SCIENCES
OTHER FEDERAL GOVERNMENT BRITISH LIBRARY

ATTN: ACQUISITIONS UNIT

ARGONNE NATIONAL LABORATORY

A'TTN: ACQUISITIONS CENTRE DE RECHERCHES DU SERVICE DE SANTE DES

ARMEES

BROOKHAVEN NATIONAL LABORATORY ATTN: DIRECTOR

ATTN: RESEARCH LIBRARY, REPORTS SECTION

FEDERAL ARMED FORCES DEFENSE SCIENCE AGENCY FOR

CENTER FOR DEVICES AND RADIOLOGICAL HEALTH NBC PROTECTION

ATTN: DIRECTOR ATTN: LIBRARY

GOVERNMENT PRINTING OFFICE FOA NBC DEFENCE

ATTN: DEPOSITORY ADMINISTRATION BRANCH ATTN: LIBRARY

ATTN: CONSIGNED BRANCH

INHALATION TOXICOLOGY RESEARCH INSTITUTE

LIBRARY OF CONGRESS ATTN: LIBRARY

ATTN: UNIT X

INSTITUTE OF NUCLEAR MEDICINE AND ALLIED SCIENCES

LOS ALAMOS NATIONAL LABORATORY ATTN: DIRECTOR

ATTN: REPORT LIBRARY

INSTITUTE OF RADIOBIOLOGY, ARMED FORCES MEDICAL

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION ACADEMY

ATTN: RADLAB ATTN: DIRECTOR

NATIONAL AERONAUTICS AND SPACE ADMINISTRATION OAK RIDGE ASSOCIATED UNIVERSITIES

GODDARD SPACE FLIGHT CENTER ATTN: MEDICAL LIBRARY

ATTN: LIBRARY

RESEARCH CENTER OF SPACECRAFT RADIATION SAFETY

NATIONAL CANCER INSTITUTE ATTN: DIRECTOR

ATTN: RADIATION RESEARCH PROGRAM

RUTGERS UNIVERSITY

U.S. DEPARTMENT OF ENERGY ATTN: LIBRARY OF SCIENCE AND MEDICINE

AT'IN: LIBRARY

UNIVERSITY OF CALIFORNIA

U.S. FOOD AND DRUG ADMINISTRATION AT-N: DIRECTOR, INSTITUTE OF TOXICOLOGY

ATTN: WINCHESTER ENGINEERING AND & ENVIRONMENTAL HEALTH

ANALYTICAL CENTER ATTN: LIBRARY, LAWRENCE BERKELEY

LABORATORY

U.S. NUCLEAR REGULATORY COMMISSION

ATTN: LIBRARY UNIVERSITY OF CINCINNATI

ATTN: UNIVERSITY HOSPITAL, RADIOISOTOPE

LABORATORY

RESEARCH AND OTHER ORGANIZATIONS XAVIER UNIVERSITY OF LOUISIANA

ATTN: COLLEGE OF PHARMACY

AUSTRALIAN DEFENCE FORCE

ATTN: SURGEON GENERAL AUTORIDAD REGULATORIA NUCLEAR

CENTRO DE INFORMACION

AUTRE, INC.

ATTN: PRESIDENT
REPORT DOCUMENTATION PAGE Form Approved

0MB 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 data sources a anderiImaintainmg 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 and

Reports, 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 Publication

4. TITLE AND SUBTITLE 5. FUNDING NUMBERS

AFFRI's Gamma-Ray, X-Ray, and Fission-Neutron Calibration Curves for

the Lymphocyte Dicentric Assay: Application of a Metaphase Finder

System

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 ORGANIZATION

REPORT NUMBER

Armed Forces Radiobiology Research Institute

8901 Wisconsin Avenue

Bethesda, MD 20889-5603

9. SPONSORING/MONITORING AGENCY NAME(S) AND ADDRESS(ES) 10. SPONSORING/MONITORING

AGENCY 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 perform

radiation-induced chromosome aberration analysis for biological dosimetry. Whole blood from healthy

human volunteers was used after obtaining informed consent. Peripheral blood lymphocytes were exposed

in vitro to different types of radiation; 60CO gamma rays (E=1.25 MeV, mean of the absorbed dose

distribution 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). Distribution

of radiation-induced dicentrics among cells exhibited Poisson statistics as characterized by the Papworth

method (Papworth 1970). Dose-response relationships for the yield of dicentrics for photon sources were

fitted with a linear-quadratic model using the maximum-likelihood method and for the neutron source by a

weighted linear regression method. Comparison of the data with other published studies is presented. The

dose-response relationships for dicentric induction by low- and high-linear energy transfer (LET) radiation

are consistent with the single- and two-track model of aberration formation, Y = otD + PD2

. An increase in yD

resulted in an increase in dicentric yield. As expected, fission neutrons induced a significantly higher yield

of dicentrics than that caused by low-LET sources. The linear component of the model, corresponding to

damage caused by single-tracks, is predominant with fission neutrons so that the dose-effect relationship is

essentially linear. An automated metaphase finder system with a satellite scoring utility was used to improve

data collection.

14. SUBJECT TERMS 15. NUMBER OF PAGES

36

16. PRICE CODE

17. SECURITY CLASSIFICATION 18. SECURITY CLASSIFICATION 19. SECURITY CLASSIFICATION 20. LIMITATION OF

OF REPORT OF THIS PAGE OF ABSTRACT ABSTRACT

UNCLASSIFIED UNCLASSIFIED UNCLASSIFIED UL

NSN 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