Microdosimetry of diagnostic X rays: applications of the variance-covariance method.

Microdosimetric measurements in beams of diagnostic X rays (between 30 and 125 kV) have been performed. In these pulsed radiation fields, microdosimetric measurements are possible only by application of the variance-covariance technique. The dose mean lineal energy, yD, is determined for various simulated diameters, at different depths in the absorber, and at different points within the pulse intervals. From the measured temporal dependences one can also obtain values of yD for different X-ray pulse generators. The results demonstrate the potential of the variance-covariance method for a diversity of microdosimetric measurements in radiation protection and in the quality control of radiation beams.     

Measurements of neutron energy using a recoil-proton telescope and a high-pressure ionization chamber

Two very different techniques for measuring the energy of neutrons in the energy range 0.1-10 MeV are presented and compared. A recoil-proton spectrometer is used to determine the energy spectra of neutrons produced by the d(4)-Be and p(4)-Be reactions down to the low-energy threshold of 0.7 MeV. The same radiation fields are also measured with a recently developed method using a high-pressure ionization chamber that can be used to determine the mean energy of the neutrons in a mixed neutron-gamma radiation field provided the gamma-ray absorbed dose fraction is determined independently. An intercomparison of the two methods shows that the high-pressure ionization chamber compares well and supplements the established recoil-proton spectrometer technique. The almost isotropic response of the chamber has enabled measurements to be made of the variation of mean neutron energy with depth in water for the two radiation fields.     

Proportional counter dosimetry and microdosimetry for radiotherapy with multiple pion beams

At the Swiss Institute for Nuclear Research (SIN) cancer patients are irradiated with negatively charged pi mesons using a 60-beam medical pion generator, the Piotron. A low-pressure tissue-equivalent proportional counter was used to measure absorbed dose and microdosimetric spectra. A method was developed to allow discrimination of events from different beam components, i.e., beam contamination (electrons and muons), pions in flight, and stopping pions. Measurements were performed along the axis and at lateral distances off one of these identical pion beams. The marked changes of total microdosimetric spectra with depth in phantom detected in earlier measurements are mainly due to large variations in the dose contributions of the beam components and much less to changes in the shapes of the individual microdosimetric spectra. The single beam measurements were used to calculate three-dimensional distributions of absorbed dose and of dose mean lineal energy, yD, for dynamic patient irradiations. Within the whole target volume yD remains nearly constant when irradiated with all 60 beams, whereas considerable changes were found for irradiations with 31 beams coming from a semicircle. Both size and shape of target volumes influence yD, the maximum values ranging from 30 to 45 keV/micron.     

Derivation of radiation quality average parameters in neutron-gamma radiation fields with the high-pressure ionization chamber: theory and practice

The established radiation quality parameters in mixed neutron-gamma radiation fields may be measured by applying the initial (columnar) recombination of ions in tissue-equivalent (TE) high-pressure ionization chambers (recombination chambers). The mean quality factor can be determined to within 10-15% for mixed fields with neutrons ranging from thermal to 10 MeV, and the dose mean LET of the proton component can be determined to within 10-15% if the gamma-ray absorbed dose fraction is known. These average parameters are derived by measuring the ratio of the ionization currents collected at two high-field strengths and constant gas pressure applied to the ionization chamber. By utilizing approximate correlations between physical parameters in the neutron energy region from thermal to 10 MeV, the dose mean LET of the heavy ion component, the overall dose mean LET, and the microdosimetric parameter y0,D of the mixed field can also be derived. Experimental verification of the method is presented for various neutron-gamma radiation spectra in air and in water by comparison to theoretical calculations and results from low-pressure proportional counter measurements. Good agreement is shown. The TE high-pressure ionization chamber appears to have wide potential for use as a dose-equivalent meter in radiation protection or as a beam characterization device in radiobiology.     

Aspects of fast-ion dosimetry

A first step in the dosimetry of fast-ion beams is the determination of accurate Bragg (ionization) functions. Bragg functions for several substances have been measured and calculated for 3480 MeV carbon ions. In the measurements, the ions first traverse an absorber in which the energy is reduced to either 1900 or 1200 MeV, then a "range gauge" followed by a thin ionization chamber. Functions are calculated with an analytical method using convolutions of straggling functions. This approach gives results without the stochastic variations implicit in Monte Carlo methods. The comparison of measured and calculated functions shows how reliable the calculations are. An important part of the calculations is the determination of the total range of the ions. The range can be determined from the Bragg function. The measured range is given by the sum of the thickness of the absorber and the residual range measured with the range gauge. For water, the range is about 150 mm, and the precision of the measurements is +/-0.05 mm. Because the ion energy at the surface of the absorber fluctuates with time, measurements with water are used to define this energy. Thus the ranges (or average stopping powers) in absorbers are obtained relative to those in water. Measured ranges R(m) are compared with ranges R(0) calculated with a current version of the Bethe theory. For light absorbers (atomic number Z < 20), differences between R(m) and R(0) are less than +/-0.3 mm; for Z > 20 differences are between 0 and +/-0.6 mm. This agreement between calculated and measured ranges confirms the value I = 80 eV for water measured earlier for protons. The ionization by nuclear fragments is obtained from the difference between measured and calculated ionization functions, and has little influence on the ranges of the primary ions.     

Techniques for boron determination and their application to the analysis of plant and soil samples

This paper reviews techniques for determining B concentration and isotopic ratio and their application to soil and plant samples. Boron concentration has been determined utilising spectrophotometry, potentiometry, chromatography, flame atomic emission and absorption spectrometry, inductively coupled plasma (ICP) optical emission (OES) and mass spectrometry (MS), and neutron activation analysis using neutron radiography and prompt- activation analysis. Isotopic ratios of B have been measured by ICP–MS, thermal ionisation mass spectrometry (TIMS) and secondary ion mass spectrometry (SIMS). For isotopic measurements, TIMS and SIMS are more sensitive and provide higher degrees of accuracy and resolution than ICP–MS, however, extensive sample preparation and purification, and time-consuming measurements limit their usefulness for routine analyses.While the spectrophotometric technique using a colorimetric reaction of B with azomethine-H has been the most extensively applied B determination method for soil and plant samples, colorimetric methods, in general, suffer from numerous interferences and have poor sensitivity and precision. The prompt- method can determine B concentration in intact samples which enables this method to be especially useful for some applications in agriculture. Research involving B behaviour in plant and soil environments would benefit from this technology. In recent years, the use of ICP–OES and ICP–MS for B determination in plant and soil samples has grown tremendously. The application of ICP–OES brought a significant improvement in B analysis because of its simplicity, sensitivity and multielement detection capability. However, besides matrix interferences, the two most sensitive emission lines for B suffer strong spectral interference from Fe. The ICP–OES is not adequately sensitive for some nutritional work involving low B concentrations and B translocation studies using the isotope tracer technique.Plasma is one of the most effective analyte ionisers and MS is the most sensitive ion detector. Coupling of plasma with MS resulted in the development of plasma source MS technology (ICP–MS) which has outperformed all previous analytical methods for trace element determination. Boron determination by ICP–MS suffers no spectroscopic interferences, and is considered the most practical and convenient technique for B isotope determination. The ability of ICP–MS to measure isotopic ratios as well as B concentration enables: (1) B concentration determination by the isotope dilution method, (2) verification of B concentration by isotope fingerprinting in routine analysis and (3) determination of total B concentration as well as B isotope ratio in the same run for biological tracer studies. Therefore, ICP–MS is the method of choice among the present-day technologies for determining B concentration and a convenient method for B isotope determination. In recent years, new generations of plasma-source MS instruments have been developed using alternative plasma generation methods and high-resolution mass spectrometers. These instruments are expected to bring further improvements in accuracy, sensitivity and precision of B determination.     

Generalization of the variance-covariance method for microdosimetric measurements

The variance method of microdosimetric measurements and its extension, the variance-covariance method, permit the determination of an essential parameter of radiation quality, the dose mean event size,y _d. The methods have — among other advantages — the feature that they permit measurements for smaller simulated sites than the conventional single-event technique. It is, therefore, desirable to employ them also for the determination of further moments of the distribution ofy. The formulae for the first three moments are here derived both for the case of constant dose rate and of fluctuating dose rates. A second article will use the same mathematical approach to deduce formulae that remain valid even if there are slow changes of the ratio of dose rates in the two detectors for the variance-covariance method. A third article will explore — in terms of microdosimetric data — the applicability of the formulae.     

Quantification of DNA and protein adsorption by optical phase shift

A primary advantage of label-free detection methods over fluorescent measurements is its quantitative detection capability, since an absolute measure of adsorbed material facilitates kinetic characterization of biomolecular interactions. Interferometric techniques relate the optical phase to biomolecular layer density on the surface, but the conversion factor has not previously been accurately determined. We present a calibration method for phase shift measurements and apply it to surface-bound bovine serum albumin, immunoglobulin G, and single-stranded DNA.Biomolecules with known concentrations dissolved in salt-free water were spotted with precise volumes on the array surface and upon evaporation of the water, left a readily calculated mass. Using our label-free technique, the calculated mass of the biolayer was compared with the measured thickness, and we observed a linear dependence over 4 orders of magnitude. We determined that the widely accepted conversion of 1 nm of thickness corresponds to 1 ng/mm² surface density held reasonably well for these substances and through our experiments can now be further specified for different types of biomolecules. Through accurate calibration of the dependence of thickness on surface density, we have established a relation allowing precise determination of the absolute number of molecules for single-stranded DNA and two different proteins.     

Comparative risk assessment of secondary cancer incidence after treatment of Hodgkin's disease with photon and proton radiation

Probabilities for secondary cancer incidence have been estimated for a patient with Hodgkin's disease for whom treatment has been planned with different radiation modalities using photons and protons. The ICRP calculation scheme has been used to calculate cancer incidence from dose distributions. For this purpose, target volumes as well as critical structures have been outlined in the CT set of a patient with Hodgkin's disease. Dose distributions have been calculated using conventional as well as intensity-modulated treatment techniques using photon and proton radiation. The cancer incidence has been derived from the mean doses for each organ. The results of this work are: (a) Intensity-modulated treatment of Hodgkin's disease using nine photon fields (15 MV) results in nearly the same cancer incidence as treating with two opposed photon fields (6 MV). (b) Intensity-modulated treatment using nine proton fields (maximum energy 177.25 MeV) results in nearly the same cancer incidence as treating with one proton field (160 MeV). (c) Irradiation with protons using the spot scanning technique decreases the avoidable cancer incidence compared to photon treatment by a factor of about two. This result is independent of the number of beams used. Our work suggests that there are radiotherapy indications in which intensity-modulated treatments will result in little or no reduction of cancer incidence compared to conventional treatments. However, proton treatment can result in a lower cancer incidence than photon treatment.     

How Informative Is Wright's Estimator of the Number of Genes Affecting a Quantitative Character?

S. Wright suggested an estimator, m, of the number of loci, m, contributing to the difference in a quantitative character between two differentiated populations, which is calculated from the phenotypic means and variances in the two parental populations and their F1 and F2 hybrids. The same method can also be used to estimate m contributing to the genetic variance within a single population, by using divergent selection to create differentiated lines from the base population. In this paper we systematically examine the utility and problems of this technique under the influences of unequal allelic effects and initial allele frequencies, and linkage, which are known to lead m to underestimate m. In addition, we examine the effects of population size and selection intensity during the generations of selection. During selection, the estimator m rapidly approaches its expected value at the selection limit. With reasonable assumptions about unequal allelic effects and initial allele frequencies, the expected value of m without linkage is likely to be on the order of one-third of the number of genes. The estimates suffer most seriously from linkage. The practical maximum expectation of m is just about the number of chromosomes, considerably less than the "recombination index" which has been assumed to be the upper limit. The estimates are also associated with large sampling variances. An estimator of the variance of m derived by R. Lande substantially underestimates the actual variance. Modifications to the method can ameliorate some of the problems. These include using F3 or later generation variances or the genetic variance in the base population, and replicating the experiments and estimation procedure. However, even in the best of circumstances, information from m is very limited and can be misleading.     

Small-sample reproducibility estimates: an example using rebreathing measurements

We developed a statistical technique to estimate the reproducibility of a parameter from a population in which only two repeated measurements can be made in a single individual. The following data were analyzed: acetylene cardiac output (Qc), lung tissue volume (Vti), and carbon monoxide diffusing capacity (DLCO) measured by rebreathing techniques in a population of 86 healthy subjects (51 men and 35 women). Each subject was measured twice with a computerized rebreathing system using a test gas of 10% He-0.3% C18O-0.7% C2H2-25% O2-balance N2 while sitting at rest. The estimated coefficients of variation for repeated measurements were 6.8, 10.3, and 5.7% for Qc, Vti, and DLCO, respectively. Chebyshev's inequality was used to estimate the imprecision for a single measurement of these parameters and for averages of two or more repeated values. A single measurement of Qc would be within 14.2% of a "true" mean 90% of the time, whereas an average of three consecutive measurements would be within 8.2% of the true mean 90% of the time. Single measurements of Vti and DLCO were found to be within 21.7 and 12.0%, respectively, of the true mean 90% of the time. When three consecutive measurements are averaged, Vti is within 12.6% and DLCO is within 6.9% of the true mean 90% of the time. We conclude that 1) rebreathing Qc is as reproducible as other measurements of cardiac output, 2) rebreathing measurements of DLCO are as reproducible as those made by the single-breath technique, and 3) an average of two to three measurements of Vti should be made to obtain values with a reasonable degree of precision.     

Integrating NOE and RDC using sum-of-squares relaxation for protein structure determination

We revisit the problem of protein structure determination from geometrical restraints from NMR, using convex optimization. It is well-known that the NP-hard distance geometry problem of determining atomic positions from pairwise distance restraints can be relaxed into a convex semidefinite program (SDP). However, often the NOE distance restraints are too imprecise and sparse for accurate structure determination. Residual dipolar coupling (RDC) measurements provide additional geometric information on the angles between atom-pair directions and axes of the principal-axis-frame. The optimization problem involving RDC is highly non-convex and requires a good initialization even within the simulated annealing framework. In this paper, we model the protein backbone as an articulated structure composed of rigid units. Determining the rotation of each rigid unit gives the full protein structure. We propose solving the non-convex optimization problems using the sum-of-squares (SOS) hierarchy, a hierarchy of convex relaxations with increasing complexity and approximation power. Unlike classical global optimization approaches, SOS optimization returns a certificate of optimality if the global optimum is found. Based on the SOS method, we proposed two algorithms—RDC-SOS and RDC–NOE-SOS, that have polynomial time complexity in the number of amino-acid residues and run efficiently on a standard desktop. In many instances, the proposed methods exactly recover the solution to the original non-convex optimization problem. To the best of our knowledge this is the first time SOS relaxation is introduced to solve non-convex optimization problems in structural biology. We further introduce a statistical tool, the Cramér–Rao bound (CRB), to provide an information theoretic bound on the highest resolution one can hope to achieve when determining protein structure from noisy measurements using any unbiased estimator. Our simulation results show that when the RDC measurements are corrupted by Gaussian noise of realistic variance, both SOS based algorithms attain the CRB. We successfully apply our method in a divide-and-conquer fashion to determine the structure of ubiquitin from experimental NOE and RDC measurements obtained in two alignment media, achieving more accurate and faster reconstructions compared to the current state of the art.     

Experimental limitations in microdosimetry measurements using the variance technique

Summary Microdosimetry measurements give information about the energy imparted to a volume of given size. With the most frequent method only data for object sizes above 0.2 µm are obtainable. Although restricted to the dose mean values, the variance technique can, however, be applied to object sizes an order of magnitude less. In this report some limiting parameters for this technique are discussed and results down to about 20 nm in a⁶⁰co-ray beam are reported. The resulting data exceed earlier reported values at object sizes below 0.1 µm, but are in good agreement for larger object sizes.     

Determination of Nitrogen in Living Patients

THE technique of in vivo activation analysis has been used in the sequential study of whole-body calcium¹. Neutrons are produced by bombarding a lithium target with 10 MeV protons from the 1.5 m Nuffield cyclotron. The patients, after being irradiated to a dose of about 1.5 rem, are transferred to a whole-body counter in which the activities induced in calcium, sodium and chlorine are monitored. We briefly describe here a new technique for making in vivo measurements on line by counting during the period of irradiation. In particular we have examined the feasibility of measuring the whole-body nitrogen content and of detecting trace amounts of the toxic element cadmium. Liquid phantoms simulating the conditions in the human body are used for these studies.     

“Broadbeam” irradiation of mammalian cells using a vertical microbeam facility

A “broadbeam” facility is demonstrated for the vertical microbeam at Surrey’s Ion Beam Centre, validating the new technique used by Barazzuol et al. (Radiat Res 177:651–662, 2012). Here, droplets with a diameter of about 4 mm of 15,000 mammalian cells in suspension were pipetted onto defined locations on a 42-mm-diameter cell dish with each droplet individually irradiated in “broadbeam” mode with 2 MeV protons and 4 MeV alpha particles and assayed for clonogenicity. This method enables multiple experimental data points to be rapidly collected from the same cell dish. Initially, the Surrey vertical beamline was designed for the targeted irradiation of single cells with single counted ions. Here, the benefits of both targeted single-cell and broadbeam irradiations being available at the same facility are discussed: in particular, high-throughput cell irradiation experiments can be conducted on the same system as time-intensive focused-beam experiments with the added benefits of fluorescent microscopy, cell recognition and time-lapse capabilities. The limitations of the system based on a 2 MV tandem accelerator are also discussed, including the uncertainties associated with particle Poisson counting statistics, spread of linear energy transfer in the nucleus and a timed dose delivery. These uncertainties are calculated with Monte Carlo methods. An analysis of how this uncertainty affects relative biological effect measurements is made and discussed.     

Histologic estimation of age at death using the anterior cortex of the femur

This report describes a new histologic method for determination of age at death, the latest in a series of studies that began with Kerley's pioneer presentation in 1965. The study population was collected from 328 documented individuals from an anatomy dissecting room in the United States, from two modern cemeteries in the Dominican Republic, and from autopsies performed in a Chilean hospital. Undecalcified thin sections 1.0 cm wide were made from specimens taken from the femoral midshaft directly opposite the linea aspera. Five 0.886 mm2 fields were located at the periosteal edge and photographed, mainly for purposes of defining the fields and providing a permanent record. Secondary osteons, type II osteons, osteon fragments, resorption spaces, and non-Haversian canals were recorded as number/mm2, and a 100-space grid was used to measure average percent of unremodeled, osteonal, and fragmental bone. Stepwise regression analysis of the measurements produced a series of regression equations for age estimation for females, males, and sexes combined. Most equations have a standard error of estimate of about 10 years, but the coefficients of determination (r2) range from 0.48 to 0.72. In practice, sex-specific equations gave better results than opposite-sex or nonspecific equations, mainly because males and females differed in the pattern of relations between osteons and osteon fragments with advancing age.     

Dosimetry of total skin electron irradiation

Elaboration of such a simple technique for total skin electron irradiation which ensures good dose homogeneity and minimal x-ray background dose. MATERIALS AND METHODS: We started large electron field irradiations with the Neptun 10p linear accelerator in the National Institute of Oncology -Budapest in 1986. After the installation of the Siemens Mevatron KD linear accelerator it was possible to introduce the modified Stanford technique. This technique satisfies better the requirements given in the objective. The required field size of 200x75 cm is produced as a result of two fields with 30 degrees angular separation (dual field) at a source skin distance of 465 cm. The patient's body is exposed to six dual electron fields. The electron energy is 6 MeV. Despite the long source skin distance the treatment time is relatively short due to the high dose rate (940 mu/min) capability of our Mevatron KD. The in air dose profiles were measured in miniphantom with semiconductor detector. Depth dose curves were measured in water and in polystyrene phantom with semiconductor detector and with films. RESULTS: The measured dose homogeneity of the 6 MeV energy dual field with 30 degrees angular separation is within +/- 5%in a 200x75cm plane field. The depth of dose maximum of the resulting dose distribution of six dual field irradiation is between 2 mm and 5 mm, while the depth of 80% isodose curve is about 8 mm. The total body x-ray background dose is less than 1% of the skin dose. CONCLUSION: The modified Stanford technique adapted to our Mevatron KD linear accelerator is suitable for total skin electron beam therapy.     

Representativity and reproducibility of DNA malignancy grading in different carcinomas

The reproducibility of the determination of the "DNA malignancy grade" (DNA-MG) was tested in 56 carcinomas of the colon, breast and lung while its representativity was tested on 195 slides from 65 tumors of the colon, breast and lung. DNA measurements were performed on Feulgen-stained smears with the TAS Plus TV-based image analysis system combined with an automated microscope. The variance of the DNA values of tumor cells around the 2c peak, the "2c deviation index" (2cDI), was taken as a basis for the computation of the DNA-MG, which ranges on a continuous scale from 0.01 to 3.00. The representativity, analyzed by comparison of the DNA-MGs measured in three different areas of the same tumor greater than or equal to 1.5 cm apart from each other, yielded an 81% agreement. No significant differences between DNA-MGs of these areas were found. The intraobserver and interobserver reproducibilities of the DNA grading system, investigated by repeated DNA measurements, were 83.9% and 82.2%, respectively. In comparison, histopathologic grading of the 27 breast cancers studied yielded 65% intraobserver and 57% interobserver reproducibilities and 66% representativity.     

Reliability and short-term intra-individual variability of telomere length measurement using monochrome multiplexing quantitative PCR

Background

Studies examining the association between telomere length and cancer risk have often relied on measurement of telomere length from a single blood draw using a real-time PCR technique. We examined the reliability of telomere length measurement using sequential samples collected over a 9-month period.

Methods and Findings

Relative telomere length in peripheral blood was estimated using a single tube monochrome multiplex quantitative PCR assay in blood DNA samples from 27 non-pregnant adult women (aged 35 to 74 years) collected in 7 visits over a 9-month period. A linear mixed model was used to estimate the components of variance for telomere length measurements attributed to variation among women and variation between time points within women. Mean telomere length measurement at any single visit was not significantly different from the average of 7 visits. Plates had a significant systematic influence on telomere length measurements, although measurements between different plates were highly correlated. After controlling for plate effects, 64% of the remaining variance was estimated to be accounted for by variance due to subject. Variance explained by time of visit within a subject was minor, contributing 5% of the remaining variance.

Conclusion

Our data demonstrate good short-term reliability of telomere length measurement using blood from a single draw. However, the existence of technical variability, particularly plate effects, reinforces the need for technical replicates and balancing of case and control samples across plates.