Monte Carlo study of in-field and out-of-field dose distributions from a linear accelerator operating with and without a flattening-filter.

PURPOSE: To compare dosimetric characteristics of 6 MV photon fields originating from a linear accelerator operating with (FF) and without (FFF) a flattening-filter. The main objective is to establish a FFF model that results in similar depth-dose and build-up profiles as the original FF model, and subsequently estimate and compare out-of-field dose distributions. METHODS: The EGSnrc Monte Carlo user codes BEAMnrc and DOSXYZnrc are used for photon beam simulations of an Elekta linear accelerator and dose calculations in a water phantom, respectively. Three beam models were analyzed: (1) the conventional linear accelerator with the flattening-filter in place and incident electron energy 6.45 MeV (FF 6.45 MeV), (2) similar flattening-filter-free model (FFF 6.45 MeV), and (3) as (2) but with increased electron energy (FFF 8.0 MeV). The field size 5 × 5 cm(2) was used for characterization of dose output, depth dose profiles, and photon spectrum. The field size 40 × 40 cm(2) was used for characterization of cross-field photon energy, photon fluence, and dose distributions. Out-of-field dose distributions were analyzed in both in-plane and cross-plane directions for 5 × 5 cm(2) and 10 × 10 cm(2) fields. RESULTS: Comparable depth dose distributions, including the build-up region, for FF and FFF fields were achieved by increasing the electron energy from 6.45 MeV to 8.0 MeV for the FFF beam. The FFF beams result in reduced out-of-field dose compared to the FF beam: the reduction was most apparent in the cross-plane direction and more pronounced by the FFF 8.0 MeV beam compared to the FFF 6.45 MeV beam. Differences in out-of-field dose due to direction (in-plane vs cross-plane) were up to 40% for the FF beam; this effect was significantly reduced for the FFF beams. As the flattening-filter is a major source of contaminating electrons, superficial out-of-field dose was expected, and was found to be, reduced for FFF beams. CONCLUSIONS: The build-up and depth-dose characteristics of a conventional "6 MV" beam can be maintained when changing to a flattening-filter-free modality by increasing the incident electron energy from 6.45 MeV to 8.0 MeV. This will at the same time reduce the out-of-field dose for regions up to 20 cm from the central axis by 10%-30% compared to the original FF situation.

Flow cytometry: a high-resolution instrument for everyone.

A new flow configuration for flow cytometry has been devised in which a flat, laminar stream of water, containing the stained cells in a narrow sector, is formed on a microscope cover slip by a pressurized jet of water directed onto the glass at low angle. The stream of cells is viewed by means of a fluorescence microscope with incident illumination and a pulse photometer. Coupled to a multichannel pulse height analyzer, the instrument constitutes a stable and easy-to-operate flow cytometer with a resolution equal to or better than a coefficient of variance of 1.4 percent in measurements of cellular DNA.

Contralateral breast doses measured by film dosimetry: tangential techniques and an optimized IMRT technique.

The contralateral breast (CLB) doses for three tangential techniques were characterized by using a female thorax phantom and GafChromic EBT film. Dose calculations by the pencil beam and collapsed cone algorithms were included for comparison. The film dosimetry reveals a highly inhomogeneous dose distribution within the CLB, and skin doses due to the medial fields that are several times higher than the interior dose. These phenomena are not correctly reproduced by the calculation algorithms. All tangential techniques were found to give a mean CLB dose of approximately 0.5 Gy. All wedged fields resulted in higher CLB doses than the corresponding open fields, and the lateral open fields resulted in higher CLB doses than the medial open fields. More than a twofold increase in the mean CLB dose from the medial open field was observed for a 90 degrees change of the collimator orientation. Replacing the physical wedge with a virtual wedge reduced the mean dose to the CLB by 35% and 16% for the medial and lateral fields, respectively. Lead shielding reduced the skin dose for a tangential technique by approximately 50%, but the mean CLB dose was only reduced by approximately 11%. Finally, a technique based on open medial fields in combination with several IMRT fields is proposed as a technique for minimizing the CLB dose. With and without lead shielding, the mean CLB dose using this technique was found to be 0.20 and 0.27 Gy, respectively.

Changes in antigen expression on human FME melanoma cells after exposure to photoactivated hematoporphyrin derivative.

Exponentially growing melanoma cells of the line FME were incubated with hematoporphyrin derivative (HpD) for 1 and 18 h and subsequently exposed to light in the presence of HpD. Quantitative changes in the expression of the melanoma-associated surface antigen p250 recognized by the monoclonal antibody 9.2.27 were studied by flow cytometry. Treatment with HpD and light resulted in no immediate changes in the antigen expression. However, a few hours after light exposure a significant reduction in antigen expression was observed. For cells incubated with HpD for 1 h, the minimum expression of the antigen was observed 6 h after the irradiation, and the duration of the reduced expression was almost dose independent. On the other hand, the duration of the reduced antigen expression increased strongly with light dose for cells incubated with HpD for 18 h. In both cases antigen expression decreased exponentially with the product of drug concentration and light dose, indicating that there is no rapid mechanism by which the cells can repair the damage which leads to reduced antigen expression. Days were needed before the cells expressed a normal level of the antigen. A slight overshoot of the level of antigen expression above that for untreated cells was observed 2-5 days after light exposure depending on the incubation conditions with HpD and the light dose. At a given cell survival level (greater than 0.1), the decrease in antigen expression was more pronounced on cells incubated with HpD for 1 h than on cells incubated with the drug for 18 h.

Hyperthermia-induced changes in antigen expression on human FME melanoma cells.

Quantitative changes in the expression of three melanoma-associated antigens on human FME cells were studied by means of flow cytometry as a function of time after exposure to hyperthermia (42 degrees C for 1, 2, and 3 h; 43.5 degrees C for 1 and 2 h; 45 degrees C for 20 min). The expression of the three different surface antigens p250, p210, and p97a recognized by the monoclonal antibodies 9.2.27, 5.1, and 4.1, respectively, underwent qualitatively similar changes after hyperthermia. The antigen expression was reduced immediately after end of the treatment and decreased further to reach a minimum 1 day after treatment. Then the antigen expression gradually increased and reached a maximum above the level for unheated cells before it returned to this level about 1 wk after hyperthermia. The magnitude of these effects increased with increasing temperature and with increasing heating time at a given temperature. Quantitatively there were individual variations for the three different antigens. A positive correlation was found between the surviving fraction of the cells and the minimum level of antigen expression, indicating that different heat treatments which inactivated the same number of cells induced the same reduction in antigen expression. The demonstrated therapy-induced changes in antigen expression may be of importance for the use of monoclonal antibodies in diagnostic imaging of tumor tissue after hyperthermia or as therapeutic agents in combination treatments involving hyperthermia.

Specific killing of human melanoma cells by 125I-labeled 9.2.27 monoclonal antibody.

The anti-melanoma antibody 9.2.27 localizes to melanoma cells when administered i.v. to melanoma patients, but high doses of this antibody alone have no specific cytotoxic effect in vivo. To determine whether radiolabeled antibodies would exhibit specific antimelanoma cytotoxicity in vitro, cell survival curves were established for NCl-N892 human melanoma cells treated with 125I-labeled 9.2.27 monoclonal antibody. The binding capacity per cell was 5 X 10(5) molecules of 9.2.27 immunoglobulin G, and the association constant of binding was 10(10) M-1. Antibody preparations with specific radioactivities of 9-80 microCi/micrograms were used. Colony-forming ability after in vitro exposure to 125I-9.2.27 was determined by a 1-h antibody incubation at saturating concentrations, washing, and cell freezing for various exposure durations. Colony survival was dose dependent, varying with the radioactivity per cell and the exposure time. The survival curves demonstrated no shoulder effect and had a 37% incremental survival dose of 0.5-0.9 X 10(5) decays/cell. Selective killing of melanoma cells was demonstrated in experiments where NCl-N417 lung cancer cells were mixed with the melanoma cells prior to antibody treatment. The NCl-N417 cells did not express the melanoma-associated antigen, were more sensitive to conventional external irradiation than were the melanoma cells, and could easily be distinguished from them by different growth morphology. In spite of a growth advantage for the melanoma cells in the clonogenic assay, the antigen-negative lung cancer cells selectively survived the treatment and were the only surviving cells after 15 days of exposure.

A sequential binding assay with a working range extending beyond seven orders of magnitude.

A new immunometric sequential binding assay has been developed in which the sample is first reacted with a solid phase binding partner in low concentration, and subsequently with a second binding partner at a higher concentration. The amounts of analyte bound to the two solid phase binding partners are separately measured, thus establishing a double standard curve. There is a shift between the two standard curves along the concentration axis. Thus an unambiguous determination of analyte concentration is obtained, even in the descending region of the curves where the 'hook' effect causes decreasing signal with increasing analyte concentration. A two-particle immunofluorometric assay for AFP based on this principle measured by flow cytometry, resulted in an assay with rapid binding (approximately 2 h), a detection limit of 0.1 kIU/l and a working range (0.3 to > 3 x 10(6) kIU/l) in excess of 7 log10 orders. Assay results compared well with those of an immunoradiometric assay.

Immunometric assay by flow cytometry using mixtures of two particle types of different affinity.

An improved dynamic range in a particle based flow cytometric immunoassay for carcinoembryonic antigen (CEA) was obtained using a binary mixture of two distinguishable particle types, namely particles of 7 and 10 microns diameter that were distinguishable by their light scattering characteristics in the flow cytometer. The two particle types were coated with antibody of the same specificity but different affinity. The association constants were 3.2 x 10(10) and 3.3 x 10(9) for the antibodies on the 7 and 10 micron particles, respectively. A dilution series of CEA samples was incubated with aliquots of the particle mixture and secondary biotin-streptavidin-phycoerythrin-conjugated antibody directed against a different epitope on the CEA molecule. The fluorescence intensity of the two particle types was measured flow cytometrically, and a double standard curve plotted from the mean logarithmic fluorescence values. The precision profile derived from the standard curve demonstrated that an increase in the dynamic range of about 50% (from 2 to 3 log) was obtained by using a mixture of high and low affinity particles, compared to using the high affinity particles alone.

Dual analyte assay based on particle types of different size measured by flow cytometry.

Simultaneous flow cytometric assays have been developed for alpha-fetoprotein (AFP) and human chorionic gonadotropin (hCG), with internal determination of sample related non-specific binding (NSB). The assays use particles of 7.5, 6.5 and 5.5 microns diameter coated with, respectively, monoclonal antibodies specific for AFP, hCG or an epitope normally not present in serum. The different particle types were identified simultaneously by light-scatter measurements as their specific immunofluorometric responses were determined. The NSB in the simultaneous assay of AFP and hCG was increased by approximately 30% compared to corresponding single analyte assays. The working range of the dual analyte assays was 0.6-2000 kIU/l for AFP and 6-10,000 IU/l for hCG. No significant interference from the presence of the other analyte was observed in the measurement of either AFP or hCG. The 95% confidence interval for the ratio of dual over single analyte assay results was [0.81, 1.11] for AFP and [0.88, 1.16] for hCG.

Determination of the immunoreactive fraction of radiolabeled monoclonal antibodies by linear extrapolation to binding at infinite antigen excess.

Conjugates of monoclonal antibodies with radioactive isotopes, drugs or toxins have great potential for specific radiolocalization and inactivation of tumor cells. Because the conjugation procedure may adversely alter the antibody, quality control procedures must be applied to determine important characteristics of the conjugated antibody. One such property is how much of the conjugated antibody is able to bind to the relevant antigen. Based on theoretical considerations, we have developed a binding assay for radiolabeled monoclonal antibodies in which the fraction of immunoreactive antibody is determined by linear extrapolation to conditions representing infinite antigen excess. This ensures that the true value of the immunoreactive fraction is obtained, as opposed to the apparent immunoreactive fraction determined under conditions of limited antigen excess. The described assay is based on a double-inverse plot of the binding data which may be considered a modification of the Lineweaver-Burk plot. We established the method using 125I- and 111In-labeling of the 2 monoclonal antibodies T101 and 9.2.27 which currently are undergoing radioimaging trials at the National Cancer Institute. For properly performed conjugation procedures, immunoreactive fractions of about 0.9 were obtained, but a prolonged chloramine-T reaction for 125I-labeling resulted in an immunoreactive fraction of only 0.6. Due to its principle of determining binding at infinite antigen excess, the present method is quite insensitive to variation in the actual amounts of cells and antibody used, as well as the incubation time. We therefore recommend it as a quality control procedure for radiolabeled antibodies. Under certain conditions, this procedure is also applicable for quality control of drug- and toxin-conjugated monoclonal antibodies.

Selection of optimal model for the DNA histogram by analysis of error of estimated parameters.

The ability of four different mathematical models of the DNA histogram to give accurate estimates for the fractions of cells in G1, S, and G2 + M has been investigated. The models studied differ in the form and number of parameters of the function used to represent cells in S-phase. Results obtained from simulated DNA histograms suggest that the standard deviations of the model parameters increase exponentially with the width of the G1 and G2 + M peaks of the histogram. Error analysis is presented as a method to select a model of optimal complexity in relation to the resolution provided by the data in a given set of DNA histograms. Introduction of additional parameters improves the agreement between model and data but may result in a less well-posed model. A model with an optimal number of parameters can therefore be found that will yield parameter estimates with the smallest possible standard deviations.

A flow cytometric DNA analysis of medullary thyroid carcinoma.

Flow cytometric DNA analysis of biopsy specimens from 10 patients with medullary carcinoma of the thyroid (MCT) was performed to evaluate the possible value of such data as indicators of the biologic behavior of these tumors. Some of the tumors had a unimodal distribution of nuclear DNA content, whereas others were bimodal. The mean modal DNA content in the cells of unimodal medullary carcinomas was not higher than that in the cells of thyroid parenchyma with a normal histologic appearance. This finding contradicts previous reports based on conventional cytophotometry. Bimodal DNA histograms were found in biopsy specimens from four patients, and the two patients with predominantly spindle-shaped cells belonged to this group. The total of nine biopsy specimens with bimodal DNA distributions were all from metastatic foci. Bimodal patterns were found, however, to co-exist with unimodal patterns, when several samples were examined from the same case. Bimodality or more severe aneuploidy did not seem to be related to shorter survival of the patients. The mean value of the percentage of cells in S-phase in our MCT series was higher than in follicular but lower than in anaplastic carcinomas. This fits well with clinical studies, where medullary carcinoma patients have been found to live longer than patients with follicular carcinomas but for a shorter time than those having anaplastic carcinomas.

The diagnostic value of flow cytometric DNA measurements in selected disorders of the human thyroid.

A flow cytometric (FCM) analysis of biopsies from 19 patients with thyroid lesions (Hashimoto's thyroiditis, hyperplastic goiter, colloid and adenomatous nodular goiter, follicular adenoma, follicular carcinoma, anaplastic carcinoma) was performed to evaluate the relevance of such results for the individual diagnosis and prognosis and prognosis of such diseases. All cases of Hashimoto's thyroiditis, the colloid nodular goiters, and the hyperplastic goiter had a unimodal diploid pattern, while the anaplastic carcinomas were aneuploid. These lesions are, however, easily diagnosed on morphological grounds and their FCM classification would only be redundant. Follicular adenomas and follicular carcinomas may be either unimodal diploid or aneuploid, and our single case of adenomatous colloid goiter showed a bimodal DNA distribution. This means that a diploid modal DNA content in a follicular tumor does not rule out malignancy, while aneuploidy is not an unequivocal sign on malignancy. The evaluation of the percentage of cells in S-phase did not seem to contribute valuable information for the individual diagnosis of malignancy within the group of thyroid lesions studied. Our study indicates a rather limited clinical value of FCM measurements of DNA in the individual case of thyroid disease.

Inactivation of human osteosarcoma cells in vitro by 211At-TP-3 monoclonal antibody: comparison with astatine-211-labeled bovine serum albumin, free astatine-211 and external-beam X rays.

The potential usefulness of alpha-particle radioimmunotherapy in the treatment of osteosarcoma was studied in vitro by using the monoclonal antibody TP-3 and cells of three human osteosarcoma cell lines (OHS, SAOS and KPDX) differing in antigen expression. Cell survival curves were established after treatment with (a) 211At-TP-3 of different specific activities, (b) 211At-labeled bovine serum albumin (BSA), (c) free 211At and (d) external-beam X rays. The three osteosarcoma cell lines showed similar survival curves, whether treated with external-beam X rays, 211At-BSA or free 211At. The D0's were lower for free 211At than for 211At-BSA. The survival curves for 211At-TP-3 treatment, on the other hand, differed significantly among the cell lines, suggesting that sensitivity to 211At-TP-3 treatment was governed by cellular properties other than sensitivity to external-beam X rays. The cellular property most important for sensitivity to 211At-TP-3 treatment was the antigen expression. Cell inactivation after 211At-TP-3 treatment increased substantially with increasing specific activity of the 211At-TP-3. At high specific activities, the cytotoxic effect of 211At-TP-3 was significantly higher than that of 211At-BSA. In conclusion, 211At-TP-3 has the potential to give clinically favorable therapeutic ratios in the treatment of osteosarcoma.

Signal attenuation and localization in optical coherence tomography studied by Monte Carlo simulation.

A Monte Carlo model has been developed for optical coherence tomography (OCT). A geometrical optics implementation of the OCT probe with low-coherence interferometric detection was combined with three-dimensional stochastic Monte Carlo modelling of photon propagation in the homogeneous sample medium. Optical properties of the sample were selected to simulate intralipid and blood, representing moderately (g = 0.7) and highly (g = 0.99) anisotropic scattering respectively. For shallow optical depths in simulated intralipid (<3 scattering mean free path (mfp) units), the number of detected backscattered photons followed the extinction-single-backscatter model, and OCT was found to detect only minimally scattered photons. Within this depth range the backscatter positions of detected photons corresponded well with the nominal focus position of the probe. For propagation to deeper positions in intralipid, localization of backscattering was quickly lost due to detection of stray photons, and the number of detected photons remained constant with increasing depth in the non-absorbing medium. For strongly forward-directed scattering in simulated blood, the number of detected photons approached the extinction-single-backscatter model only for very shallow depths (<2 mfp units). However, backscattering positions for detected photons correlated well with the nominal focus position of the probe even for optical depths greater than 40 mfp units.

Accuracy and noise in optical Doppler tomography studied by Monte Carlo simulation.

A Monte Carlo model has been developed for optical Doppler tomography (ODT) within the framework of a model for optical coherence tomography (OCT). A phantom situation represented by blood flowing in a horizontal 100 microm diameter vessel placed at 250 microm axial depth in 2% intralipid solution was implemented for the Monte Carlo simulation, and a similar configuration used for experimental ODT measurements in the laboratory. Simulated depth profiles through the centre of the vessel of average Doppler frequency demonstrated an accuracy of 3-4% deviation in frequency values and position localization of flow borders, compared with true values. Stochastic Doppler frequency noise was experimentally observed as a shadowing in regions underneath the vessel and also seen in simulated Doppler frequency depth profiles. By Monte Carlo simulation, this Doppler noise was shown to represent a nearly constant level over an investigated 100 microm interval of depth underneath the vessel. The noise level was essentially independent of the numerical aperture of the detector and angle between the flow velocity and the direction of observation, as long as this angle was larger than 60 degrees. Since this angle determines the magnitude of the Doppler frequency for backscattering from the flow region, this means that the signal-to-noise ratio between Doppler signal from the flow region to Doppler noise from regions underneath the flow is improved by decreasing the angle between the flow direction and direction of observation. Doppler noise values from Monte Carlo simulations were compared with values from statistical analysis.

Histological changes in Pinus sylvestris L. in the proximal-zone around the Chernobyl power plant.

In September 1990, samples of wood and bark were collected from Pinus sylvestris L. at three locations exposed to different levels of radioactive fallout from the 1986 accident at the Chemobyl nuclear power plant (NPP). Cross-sections of wood from the most exposed location showed a distinct change in histology in the annual ring of 1986, a consequence of the accident on 26 April. The width of annual rings decreased after the accident, and the relative width of latewood in annual rings increased transiently in 1986 and subsequently decreased in 1987. In 1987, an increase in the number of vertical resin ducts was observed, related to contamination at the location, and the number of radial rays decreased at the two locations of higher contamination. The radionuclide content in the bark was found to correlate with the degree of damage in the wood. There are several hypotheses about the contribution from various types of radioactive contamination, but the results indicate that both 'cloud gamma' and deposited radioactivity (beta and gamma) were of importance. The present work suggests that detailed studies of dose-effect relationships after exposure to different dose rates and radiation qualities may establish the usefulness of pine trees as in situ, time-recording differential dosimeters of ionizing radiation.

High-sensitivity optical lymph flow-meter.

An optical flow-meter is described which allows precise and continuous registration of the flow of lymph from cannulated human lymph vessels. The cannula from the lymph vessel is connected to the measurement tubing of the instrument where the flow is measured by automatically monitoring the movement of an air bubble introduced into the flow at the beginning of the measurement. The limit of sensitivity of the instrument is about 0.1 microliter, allowing reliable registration of stroke volumes of about 1 microliter which typically occur in human leg lymphatics. The size and capacity of the instrument were chosen to be suitable for clinical use. A technical description of the instrument is given. Application of the instrument is illustrated with recording of lymph flow and lateral intralymphatic pressure in a prenodal lymph vessel of the human leg.

Reconstruction of doses and deposition in the western trace from the Chernobyl accident.

A model is presented for the explosive cloud of particulates that produced the western trace of high radioactive ground contamination in the Chernobyl accident on 26 April 1986. The model was developed to reproduce measured dose rates and nuclide contamination and to relate estimated doses to observed changes in: (1) infrared emission from the foliage and (2) morphological and histological structures of individual pines. Dominant factors involved in ground contamination were initial cloud shape, particle size distribution, and rate of particle fallout. At time of formation, the cloud was assumed to be parabolical and to contain a homogeneous distribution of spherically shaped fuel particulates having a log-normal size distribution. The particulates were dispersed by steady winds and diffusion that produced a straight line deposition path. The analysis indicates that two clouds, denoted by Cloud I and Cloud II, were involved. Fallout from the former dominated the far field region and fallout from latter the region near the reactor. At formation they had a full width at half maximum of 1800 m and 500 m, respectively. For wind velocities of 5-10 m s(-1) the particulates' radial distribution at formation had a standard deviation and mode of 1.8 microm and 0.5 microm, respectively. This distribution corresponds to a release of 390 GJ in the runaway explosion. The clouds' height and mass are not uniquely determined but are coupled together. For an initial height of 3,600 m, Cloud I contained about 400 kg fuel. For Cloud II the values were, respectively, 1,500 m and 850 kg. Loss of activities from the clouds is found to be small. Values are obtained for the rate of radionuclide migration from the deposit. Various types of biological damage to pines, as reported in the literature, are shown to be mainly due to ionizing radiation from the deposit by Cloud II. A formula is presented for the particulate size distribution in the trace area.