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Automatic data acquisition and on-line analysis of trace element concentration in serum samples
N U C L E A R I'NSTRUMENTS AND METHODS 150 ( 1 9 7 8 )
289-299 ; ©
N O R T H - H O L L A N D PUBLISHING CO.
AUTOMATIC DATA ACQUISITION AND ON-LINE ANALYSIS OF TRACE ELEMENT CONCENTRATION IN SERUM SAMPLES R. LECOMTE, P. PARADIS, S. MONARO
Laboratoire de Physique Nucl~aire, Universit~ de Montreal, Montreal, Canada and M. BARRETTE, G. LAMOUREUX
D~partement de Radiobiologie et de M~decine Nucl~aire, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Canada and H. A. MI~NARD
Unit~ de Maladie Rhumatismale, D~partement de M~decine, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Canada Received 27 June 1977 and in revised form 12 September 1977 A completely automated system has been developed to determine the trace element concentration in biological samples by measuring charged particle induced X-rays. A CDC-3100 computer with ADC and CAMAC interface is employed to control the data collection apparatus, acquire data and perform simultaneously the analysis. The experimental set-up consists of a large square plexiglass chamber in which a commercially available 750H Kodak Carousel is suitably arranged as a computer controlled sample changer. A method of extracting trace element concentrations using reference spectra is presented and an on-line program has been developed to easily and conveniently obtain final results at the end of each
run.
1. Introduction Very recently, an experimental set-up and an analysis method to measure the concentration of trace elements in freeze-dried serum have been developed in this laboratoryl). The emphasis in that work was laid on obtaining maximum precision and reproducibility in the resulting concentration values of the trace elements present in the serum, particularly for Fe, Cu, Zn and Br. To this end, careful target preparation and target handling procedures were devised, together with a simple but reliable method of analysis. All this enabled us to achieve an overall precision of 10% or less in the absolute concentration values of the four elements mentioned above. Even though the reproducibility and precision in the absolute concentration values compared very favorably with those found in other studiesZ0, it became apparent to us that the data collection apparatus, the data acquisition and data analysis methods were somewhat "leisurely" conceived for practical applications. For instance, the old data collection apparatus was completely manually operated and could accomodate a maximum of only five targets. Furthermore, in order to obtain a " g o o d " spectrum, each specimen had to be bombarded for at
least 1.5 h for a total charge collection of 100/aC on the target. Finally, the analysis of the data was always performed off-line, which sometimes entailed a considerable delay in the interpretation of the results. It became clear that to overcome all these drawbacks one would need to develop a totally automated data collection and acquisition system capable of handling tens (or hundreds) of samples, and devise a simple but reliable on-line method of analysis. The present work is a considerable improvement over the previous methods. With the present system, the X-ray spectra from ~ 150 serum samples (with as good as, or better, statistics than those already reported t)) can be measured and analyzed in approximately 24 h. In section 2, the completely automated data acquisition will be described. The simultaneous on-line method of analysis is detailed in section 3.
2. Experimental procedure 2.1. SAMPLEANALYSISAPPARATUS The principal objective of a sample analysis system is to be efficient, rapid and handy at all times. In our case the apparatus is permanently mounted
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Fig. 2. General view of the beam line and peripheral equipment.
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A U T O M A T I C DATA A C Q U I S I T I O N AND ON-LINE ANALYSIS
291
Fig. 3. Detailed view of the plexiglass scattering chamber and Carousel slide changer.
° n ° n e of the beam lines attached to the EN Tandem Van de Graaff accelerator of the Universit6 de Montr6al. A lay-out of the beam line with beam collimation system, facility to handle the samples and detect the X-rays is shown in fig. 1. Actual pictures of the system and peripheral equipment are shown in figs. 2 and 3. The main part of the apparatus consists of a 2.5 cm thick plexiglass scattering chamber built in a box-like fashion. This chamber contains the chassis of a 750 H Carousel Kodak projector and its slide changer which can hold up to 80 slides. The choice of plexiglass was based on the fact that, with this transparent material, all problems related to the quality and position of the targets as well as the beam focusing or defocusing adjustments could be checked (and eventually corrected) at any time without opening the scattering chamber. In particular the possible deteriorating effects of the beam on the biological targets, employed in this work, could be easily monitored by means of a closed-circuit television network (see fig. 1). A
vacuum of the order of 10 -5 torr could be obtained in a very short time with the aid of an auxiliary pumping system. It is known that a better vacuum (of an order of magnitude or more) could have been attained by employing a metallic scattering chamber. However, the use of this type of chamber would have brought in too many hindrances which would have totally offset its only indisputable advantage. Furthermore, it was recognized (after careful checks) that a vacuum of the order of 10 -s torr was amply sufficient for the present and future experimental needs. The specimens, prepared in the same fashion as previously described1), are mounted on standard 110 plastic slide frames and fitted on 35 mm adapters. These samples are then placed in the slide changer of the Kodak projector whose chassis was suitably transformed to position the targets, one by one, in line with the beam. A complete target replacement is easily done by opening the chamber, removing the slide changer and inserting a new one containing a fresh batch of specimens. Since, in the scattering
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chamber there is a large quantity of metallic material (chassis, motor and slide changer) and organic material (slide frames), a very careful beam collimation system had to be devised in order to suppress entirely the possible scattering of the beam in the chamber itself. Two tantalum collimators, 0.472 cm in diameter, are placed in the beam line at 50 cm and 30 cm, respectively, before the scattering chamber. Another tantalum collimator, 0.635 cm in diameter, is then placed at 10 cm before the target to stop all the particles scattered by the two previous collimators (see fig. 1). A careful mapping of the beam position on targets whose elemental composition was exactly known, ensured us that in ordinary experimental conditions no beam scattering was present in the chamber. The X-ray detector is placed at 90° with respect to the beam axis and it is mounted directly inside the plexiglass chamber and as close as possible to the target. The target makes a 45 ° angle with respect to the beam axis and is oriented in such a way that the X-rays must go through the target itself to be detected by the counter. In such a way the low energy X-rays are attenuated before impinging on the detector. However, the filtering of
PLEXIGLASS CHAMBER
DETECTOR
TARGETROOM CONTROL ROOM START STOP RESET
TIM~G CONTROLLER
CDC
31OO
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I SYMBOLI NAME F Forword B I Bockword Fig. 4. Schematic of the automatic data acquisition system.
the low energy X-rays due to elements as Na, P, S, Cl, K and Ca (which are present in large quantities in biological samples as the ones presently studied), is mostly done by suitably thick mylar foils, placed on top of the detector. Finally, another tantalum collimator, 0.472 cm in diameter, is placed between the detector and the target (at 3.4 cm from the detector and 2.7 cm from the target) to ensure proper collimation of the X-rays reaching the detector. Details on the X-ray counter and electronic circuitry as well as on target preparation and determination of the relative X-ray yield curves have been already reported0 and will not be repeated here. 2.2. AUTOMATIC DATA ACQUISITION A schematic of the automatic data acquisition system is shown in fig. 4. The Carousel slide changer and data acquisition system are controlled by a CDC 3100 computer through two Camac interface modules. A "Latched Triac Output 3080" module is used to advance automatically the Carousel slide changer whereas the homemade "Timing controller" starts
ALITOMATIC
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ACQUISITION
or stops the accumulation and resets all scalers. Two manual control units (MC1 and MC2) one situated near the plexiglass chamber and the other in the control room can also be used to place the desired target before the beam. The manual controls and Triac module are used simply to initiate the target changing, the motor being mechanically turned off by a microswitch mounted on the projector mechanism when the target is in the proper position. Two magnetic tape drivers (T1 and T2) are needed to store the accumulated spectra and to
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Fig. 5. Flow-chart of the control program C O N T R I X and its
real-time routines (see text).
AND ON-LINE
ANALYSIS
293
perform the on-line analysis (see section 3.3). The data acquisition system shown in fig. 4 is automatically driven by the control program CONTRIX whose flow-chart is presented in fig. 5 together with its real-time routines. Since the routines of the program CONTRIX are on a real-time basis the use of this program does not hamper any batch processing work (as, for instance, the simultaneous data analysis). The input parameters of CONTRIX are: MIN and MAX which represent the lower and upper limits of a chosen reference X-ray peak in the spectrum; NCOUNT which is the minimum number of counts to be accumulated in that peak; LIMT and IDEF which are the bombarding time limit for each target and the first spectrum identifier, respectively. Logical flags IF1 and IF2 are used to indicate that the target changing routine is under way (IF1 = 1) or that the data analysis program is looking for a spectrum on the data tape (IF2= 1). CONTRIX resets all scalers and the ADC, turns off all the switches on the Triac and Timing controller modules, starts the data acquisition system (START) and calls for the control routines (CAROUSEL). The subroutine CAROUSEL resets and starts a time counting program (CLOCKTM) that gives the exact elapsed time and sets the internal computer clock to call the real time routine INTEG after one second. When the preset time limit or preferably the preset number of counts (NCOUNT) in the X-ray peak is reached a change of target is carried out (NEXT). The real-time routine TEST verifies if NEXT is completed. The sequence of steps done by the subroutine NEXT is as follows: 1) The current integrator, timer and various scalers together with the ADC are disabled; 2) the spectrum is recorded on magnetic tape with the proper identifier; 3) everything is reset to zero and a new identifier is given;r 4) the slide changer is advanced by one step (or more if desired), placing a new target in the beam which is " o n " all the time; 5) the ADC and all the peripheral counting equipment are then enabled.
3. Spectrum analysis and results 3.1. GENERAL CONSIDERATIONS Once the X-ray spectra from the various samples are obtained, the experimenter is faced with
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the problem of how to determine which trace ele- resorted to a different approach in the method of ments are present in the spectra and calculate analysis of the X-ray spectra that does not require their respective amounts. To this end, it is neces- any previous knowledge of all the physical factors sary to locate all the peaks in the X-ray spectrum, which could have a bearing on the shape of the calculate their energies and determine their inten- spectra. These factors, however, are all implicitly sities. Usually the first part of the analysis (i.e. lo- considered and included in the analysis without calization and energy determination of the peaks involving any theoretical hypothesis on the X-ray in the spectrum) does not constitute a major prob- peak shapes. This method is essentially based on lem, nor does the intensity calculation when only the very simple fact that an X-ray spectrum can few peaks, well separated in energy, are present in be sometimes very complicated, but it is always the spectra. This situation, however, is the excep- formed by a finite number of elements giving imtion rather than the rule since, generally, multi- mutable peak shapes at all times once the experplets of varying complexity are present in many X- imental conditions, under which the measureray applications. In this case it is clear that reliable ments are carried out, are kept carefully uncomputer codes must be available and these codes changed. should be fast and possibly completely automated (and this point ought to be well emphasized!) 3.2. METHODOF ANALYS|S especially when a very large number of specimens The calculation of the amount of an element in are being processed. a sample is done by comparing the area of the The similarities in the physical conditions of 7- K~(or L~) peak of the element of interest with that ray spectroscopy (whether in radioactive decay stu- of a reference (or doping) element. The choice of dies or nuclear reaction work) and X-ray spectra such an internal standard is usually a matter of have led many workers to adapt computer codes convenience. Yttrium was chosen here since it is originally designed for ),-ray spectroscopy to the completely missing in the serum specimens, and it analysis of X-ray spectra4,5). These methods gen- does not interfere greatly with the elements under erally produce very good results but have the lim- study in the serum and can be easily manipulated. itation that the codes require the extensive use The relation giving the concentration for an eleof large computers and great manual manipula- ment i is: tion; therefore, they are not well suited for routine Si Cy work and even less for an eventual complete on- Ct - Sy Rir (1) line automation procedure, which is, usually, carwhere Ct and Cy are the concentrations of the eleried out with much smaller computers. A different approach in handling the X-ray ment i and yttrium, respectively; St and Sy are the spectrum analysis has been taken by Kaufmann areas of the K~(or L~) peaks of the element i and and Akselsson6), who employ a model which is yttrium and R~y is the yield of element i measured based on the physical processes involved in the X- relative to that of yttrium. This value can be obray production methods. All the model parameters tained from the properly constructed relative yield (approximately 12) are simultaneously adjusted to curves (see ref. 1, fig. 1). Since the concentration reproduce the background and the peaks in the X- Cy of the doping element is known exactly, the ray spectrum. The important feature of this code accuracy of Ci depends on how precisely the areas (which requires approximately a 24 K memory) is Si and Sy and the value of R~y can be obtained. A the possibility of processing a large number of correct determination of R~y usually does not presspectra without operator intervention. It is clear ent a great problem (see ref. l) whereas the calcuthat this method and those derived from y-ray lation of St, and to a lesser extent Sy, often can spectroscopy depend heavily on the step by step be very difficult due to the complexity of the Xreproduction procedure of the peak and back- ray spectra. It is at this point that specific peak ground shape. Thus several physical factors must and background shape functions are considered in be included in the programs; this leads to multi- the codes performing the appropriate analysis of parameter equations which are difficult to solve the X-ray spectra. Instead of employing such anand which converge slowly. On the other hand, if alytical methods, we have used the much simpler some factors are neglected, one could miss some non-analytical approach of building an assembly of important information. For this reason we have standard peak shapes from most of the elements
AUTOMATIC
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ACQUISITION
liable to be detected in the samples under investigation. These standard peak shapes are obtained from targets containing only the element of interest and bombarded in the same experimental conditions as for the specimens under study. Particular care was taken in obtaining the reference targets with the same physical inherent characteristics (i.e. target thickness, type of backing and its thickness and so on (see also ref. 1, sections 2.2-2.4) of the sample targets. This in order to avoid possible absorption differences of the emitted X-rays in both sets of targets. For the KXrays we did produce standard spectra from targets containing K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, Ag and Cd, for the K and L X-rays Sn, Sb and Te, and I have been used and finally for the L X-rays Ba, W, Au, Hg and Pb. In all these reference spectra the background is so negligible in comparison to the peak height that no peak shape distorsion is present. Let us consider now an X-ray spectrum obtained from a sample containing some elements. This spectrum can be considered as formed by a superposition of a number of standard peak shapes superimposed on a continuous background. Thus, the number of counts in each channel can be expressed as: N(x) =
Ji
+ B(x).
(2)
i=1
where f is the scalar factor giving the ratio between the areas of the peaks due to the element i in the spectrum under investigations and in the reference spectrum, respectively; F~(x) is the standard spectrum shape in channel x of element i; B(x) is the corresponding background in the spectrum under study. The summation is carried out on the n elements present in the target. Thus if one wants to know how an element is contributing to the total spectrum, this element must be stripped from the spectrum itself with the "ad hoc" standard shape until a continuous and regular background shape is obtained under the peaks of the element of interest. It is clear that the accuracy in the calculations depends a great deal also on the choice of a suitable background shape under the peaks to be analysed. This point will be discussed in more detail in the next section. Now the area of a peak due to an element i present in the sample is given by: s, = f, x
(3)
AND ON-LINE
ANALYSIS
295
where S~ is the area of the same peak in the reference spectrum. By inserting eq. (3) in eq. (1) one obtains: Ci = --~-~ , (4) fr Sy Riy where fr and S~ have the usual meaning and are referred to the doping element (yttrium in our case). The calculation of (7,. can be simplified if the standard spectra are built so that SiR=S~, then one obtains: A cy
C~ = fy Riy"
(5)
It is evident from the last expression that in order to obtain the concentration Ci of the element i present in the sample only the following items are needed: a) the standard shape (or reference spectrum) of the elements; b) the standard shape (or reference spectrum) of the doping element Y added with concentration Cy in the sample; c) the value of Riy deducible from the relative X-ray yield curve pertinent to the experimental conditions under which the experiment is carried out (see ref. 1, fig. 1). The method described above is certainly striking for its simplicity and effectiveness since no very sophisticated hypotheses are necessary to carry out the analysis which can be done on-line (and completely automatically) in relatively small computers. The only possible disadvantage could be that the assembly of standard spectra must be obtained rigorously under the same experimental conditions used in the study of the samples. In fact, every change would entail the determination of a new assembly of standard peak shapes as well as relative x-ray yield curves. It is felt, however, that this kind of applied research, where hundreds of samples must be measured and analysed in a short time, should be performed with a set-up sufficiently supple to meet a large variety of experimental needs of the researchers. Thus eventual time consuming changes in the experimental conditions would be avoided as much as possible. 3.3. AUTOMATIONOF THE ANALYSIS The calculations described in the previous section are automatically performed on-line by a CDC 3100 computer. As has been already mentioned the precision in the calculation is affected by the choice of the background shape under the peaks of interest. Primarily the background in Xray work is due to the bremsstrahlung produced
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by electron scattering. This kind of background is characterized by a more or less pronounced bump with a sharp cut-off, the end-point of which depends on the type of beam employed and its energy (see ref. 1). Above this cut-off the remaining background, which is due to Compton scattering of ),-rays and projectile bremsstrahlung is usually linear and its amount depends essentially on the quantity of material intercepted by the beam in the specimen. Thus, depending on the position of the peaks in the spectrum, the background shape is fitted by a parabolic or linear function. The fit is done in a region around the peak sufficiently large (usually seven times the width of the peak at half-maximum) and it is minimized by a X2 analysis. At this point the scalar factor f is obtained and subsequently the concentration of the element i in the sample is calculated. In figs. 6a, b and c the results obtained in the analysis of a spectrum with well resolved peaks (iron in this case) lying (ai
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Fig. 6. Typical analysis of the well resolved Fe peaks lying on a high background (see text).
on a high background (heavily loaded sample) are shown as typical example. However, as already mentioned above, the physical situation presented in fig. 6b is somewhat exceptional since usually multiplets of varying complexity are present in the spectra. For instance, in the serum specimens the K, copper peak is overlapped by the stronger K~ peak of zinc and the KB potassium peak is completely masked by the K~ peak of calcium. Furthermore in the case of energy multiplets it is also much more difficult to fit the functional form of the background under them. Thus it is quite evident that the analysis performed by the program would yield large errors in the elemental concentration values if these problems were not tackled and resolved with appropriate procedures. The, ideal situation would be to obtain elemental spectra of the type shown in fig. 6b, i.e. spectra with well resolved peaks, lying on a more or less pronounced background, which could be easily analysed by the respective reference spectra. To this end we have devised a completely automated program which performs by iterative methods a "peak stripping" of the X-ray spectra. As a final result only the peaks due to the element, the concentration of which has to be calculated, are left in the spectrum. The calculations are carried out following the prescriptions described above and are performed simultaneously for all the elements present in the specimen regardless of their stronger or weaker concentration levels. The program has been devised so that the number of input data is reduced to the indispensable minimum and all human "fumbling" is eliminated during the calculations. The required information are stated in the start up procedure or are automatically accessible during the running of the program. To give a better description of the program TOURIX employed in the present analysis, its flow-chart is shown in fig. 7. The chemical symbols of the elements under analysis (IREF(N)), the concentration of the doping element (CONCY) and the preset maximum number of iterations (NRAF) are the input data. At this point the spectrum searching routine is " o n " and the spectra are automatically analysed at the end of each data accumulation. The reference spectrum (YY(X)) as well as all the information relative to the position (YY(1024)), full-width at half-maximum (YY(1023)) and detection efficiency (EFF(N)) of the principal peak of the same element are read from an auxiliary magnetic tape
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AND ON-LINE ANALYSIS
iNPUT: e~ementstobeanalysed IREF(N), I EFF(N) ; CONCY,NRAF; spectrumtobeanalysedYA(X) =YB(X) t
I NN .ol
NNN: 2 ) I SCAL : R[N,J-I DECAL=D{N,J-I) PERC =0 02/J
SCAL: DECAL: O.O PERC: 0.02 | JTTAPE(IREF(NI;YY(X) ) I
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[ TTAPE(IREF(NI;YY(XI) [ |
[MAA:YY(IO24)-35XYY('O23) I MAB=YY(IO24)÷ 35 X YY(IO23) [ NSUB(YY(X),YB(X),MAA,MAB,SCAL,DECAL,PERC,NNN;YC(X)) ] I [ IR(N,J) = SCAL 1 D(N,J) =DECAL
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OUTPUT: N,R(N,NRAF),D(N,NRAF), CONC(N),ERR(N),SENS(N), NJ(N) final s~ectrum: YC(X)
Fig. 7. Flow-chart of the program TOURIX used for the automatic analysis of X-ray spectra (see text).
YES
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R. LECOMTE et al.
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Fig. 8. Example of analysis of an X-ray spectrum from the serum of a rheumatoid arthritis affected patient. (a) X-ray spectrum; (b) resulting background after analysis with the program TOURIX.
during the calculations, by means of the subroutine TTAPE. These information, together with the standard spectra, are previously written on the magnetic tape by means of the program RESPEC and constitute the "data bank".
With the first iteration (J= 1) the program TOURIX obtains an approximate value of the scalar factor f~(R(N, J= 1) = SCAL) for all elements present in the spectrum, without taking into account interferences due to the existence of multiplets. In this preliminary analysis, which is carried out with the subroutine (NSUB), a simple linear function ( N N N = 1) is used to adjust the background shape under and around the peaks. The background fit is done in a smaller region (from MAA to MAB) than that previously described (usually 3 times the full-width at halfmaximum) since it is advisable to exclude, as much as possible, close-by peaks. The calculation of the scalar factors is stopped when a precision (PERC) of 2% is attained. It is clear that at this point the results are approximate but, in general, they give a fair idea of the order of magnitude of the area of the peaks. These results are then employed during the following iterations (J> 1). All the elements present in the specimen with the exception of that under analysis are subtracted until the peaks of interest are well isolated in the spectrum (in other words one obtains a spectrum of the type shown in fig. 6b). From this "clean" spectrum the final scalar factor (SCAL).~ of the element i is calculated using again the subroutine NSUB and considering also the full background shape treatment previously described, i.e. employing a linear function or a parabolic one (NNN = 2) depending on the case in point. The subroutine POLFIT carries out the X 2 analysis for the minimization of the background fit. The same procedure is then followed for all the n trace elements present in the specimen (R(N, J)) and subsequent-
TABLE 1 Results of the analysis of the spectrum shown in fig. 8a. These results, extracted with the program TOURIX, are obtained as a direct print-out from the CDC 3100 computer at the end of the calculations. PAU2 is the spectrum identifier; DECAL means SHIFT. The other headlines are self-explanatory. Analysis of spectrum PAU2. Element SCAL
K Ca Fe Cu Zn Br Y Au
0.026567 0.074739 0.010320 0.012883 0.011592 0.013550 0.063787 0.010319
DECAL
- 0.10 -0.06 -0.13 -0.06 -0.01 0.03 - 0.01 0.21
Concentration (mg/I) 126.210957 66.573588 0.728781 1.216645 1.340214 4.335204 50.130(0)00 5.151798
Stat. error (%)
Sensibility (mg/l)
Number of iterations
2.257 1.703 3.086 2.855 2.962 2.806 1.771 3.087
2.577622 0.861085 0.071617 0.019938 0.045958 0.041469 0.035138 0.157226
5 5 4 4 4 5 3 4
AUTOMATIC DATA ACQUISITION AND ON-LINE ANALYSIS
ly their concentration values (CONC(N)) are calculated from eq. (5). The attainable precision with this analytical method depends essentially on the accuracy one wishes to achieve. In turn the accuracy limits have a bearing on the number of iterations necessary to carry out the calculations. Direct experience on hundreds of specimens bombarded under the same experimental conditions has shown that in order to achieve a precision of 0.5 or 10% (thus below or, at the most, of the same order as the statistical errors) 3 or 4 iterations are sufficient in the case of well separated peaks (as in fig. 6b) whereas for complicated multiplets between 5 to 8 iterations are necessary. Finally with the program TOURIX the statistical errors (ERR(N)) in the areas of the peaks can be obtained as well as the detectability limil~s (SENS(N)) of each element (see ref. 1) together with the number of iterations (NJ(N)) required to calculate the area of each peak. Furthermore, if desired, it is possible to plot the final spectrum (YC(X)) at the end of the calculations. An example of the analysis carried out by the program TOURIX is presented in figs. 8a and 8b. Fig. 8a shows the typical spectrum from the serum of a patient with rheumatoid arthritis under chrysotherapy treatment with gold salts. The specimen has been doped with yttrium having an exactly known concentration (50rag/l). Fig. 8b shows the background shape attained at the end of the calculations. The results of the analysis, directly obtained from the CDC 3100 computer at the end of the calculations, are presented in table 1. 4. Conclusion A sample control set-up and an automatic data acquisition system have been described in detail together with a spectrum analysis program (TOU-
299
RIX) totally automated. The data accumulation and its analysis can be carried out simultaneously on a CDC 3100 having 32 K memory. This is an important feature since it greatly minimizes the time lag usually existing between the data acquisition and its physical interpretation. The capabilities of the program TOURIX have been thoroughly checked from a plethora of specimens containing several trace elements with known and widely different concentrations. In every instance the program TOURIX could detect the elements present and measure precisely their concentrations even in the case of very weak peaks partially (or totally) masked by stronger close-by peaks. On the other hand if the element was not present, the program gave a concentration value either equal to zero or smaller than the detectability limits (see column "Sensibility" in table 1). The authors wish to thank Dr. J.-P. Martin for his helpful advice concerning the development of the programs and Dr. G. R. Rao for the critical reading of the manuscript. The financial support of the National Research Council and Medical Research Council of Canada is gratefully acknowledged. References l) M. Barrette, G. Lamoureux, E. Lebel, R. Lecomte, P. Paradis and S. Monaro, Nucl. Instr. and Meth. 134 (1976) 189. 2) V. Valkovic, R. B. Liebert, T. Zabel, H. T. Larson, D. Miljanic, R. M. Wheeler and G. C. Phillips, Nucl. Instr. and Meth. 114 (1974) 573. 3) p, S. Ong. P. K. Lund, C. E. Litton and B. A. Mitchell, Adv. X-ray Anal. 16 (1974) 834. 4) M. A. Mariscotti, Nucl. Instr. and Meth. 50 (1967) 309. 5) j. T. Routti and S. G. Prussin, Nucl. Instr. and Meth. 72 (1969) 125. 6) H. C. Kaufmann and R. Akselsson, Advan. X-ray Anal. 18 (1975) 353.
289-299 ; ©
N O R T H - H O L L A N D PUBLISHING CO.
AUTOMATIC DATA ACQUISITION AND ON-LINE ANALYSIS OF TRACE ELEMENT CONCENTRATION IN SERUM SAMPLES R. LECOMTE, P. PARADIS, S. MONARO
Laboratoire de Physique Nucl~aire, Universit~ de Montreal, Montreal, Canada and M. BARRETTE, G. LAMOUREUX
D~partement de Radiobiologie et de M~decine Nucl~aire, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Canada and H. A. MI~NARD
Unit~ de Maladie Rhumatismale, D~partement de M~decine, Centre Hospitalier Universitaire de Sherbrooke, Sherbrooke, Canada Received 27 June 1977 and in revised form 12 September 1977 A completely automated system has been developed to determine the trace element concentration in biological samples by measuring charged particle induced X-rays. A CDC-3100 computer with ADC and CAMAC interface is employed to control the data collection apparatus, acquire data and perform simultaneously the analysis. The experimental set-up consists of a large square plexiglass chamber in which a commercially available 750H Kodak Carousel is suitably arranged as a computer controlled sample changer. A method of extracting trace element concentrations using reference spectra is presented and an on-line program has been developed to easily and conveniently obtain final results at the end of each
run.
1. Introduction Very recently, an experimental set-up and an analysis method to measure the concentration of trace elements in freeze-dried serum have been developed in this laboratoryl). The emphasis in that work was laid on obtaining maximum precision and reproducibility in the resulting concentration values of the trace elements present in the serum, particularly for Fe, Cu, Zn and Br. To this end, careful target preparation and target handling procedures were devised, together with a simple but reliable method of analysis. All this enabled us to achieve an overall precision of 10% or less in the absolute concentration values of the four elements mentioned above. Even though the reproducibility and precision in the absolute concentration values compared very favorably with those found in other studiesZ0, it became apparent to us that the data collection apparatus, the data acquisition and data analysis methods were somewhat "leisurely" conceived for practical applications. For instance, the old data collection apparatus was completely manually operated and could accomodate a maximum of only five targets. Furthermore, in order to obtain a " g o o d " spectrum, each specimen had to be bombarded for at
least 1.5 h for a total charge collection of 100/aC on the target. Finally, the analysis of the data was always performed off-line, which sometimes entailed a considerable delay in the interpretation of the results. It became clear that to overcome all these drawbacks one would need to develop a totally automated data collection and acquisition system capable of handling tens (or hundreds) of samples, and devise a simple but reliable on-line method of analysis. The present work is a considerable improvement over the previous methods. With the present system, the X-ray spectra from ~ 150 serum samples (with as good as, or better, statistics than those already reported t)) can be measured and analyzed in approximately 24 h. In section 2, the completely automated data acquisition will be described. The simultaneous on-line method of analysis is detailed in section 3.
2. Experimental procedure 2.1. SAMPLEANALYSISAPPARATUS The principal objective of a sample analysis system is to be efficient, rapid and handy at all times. In our case the apparatus is permanently mounted
290
R. L E C O M T E Valve
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I
~..
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/s
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,
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Fig. 1. Schematic diagram of the experimental set-up.
Fig. 2. General view of the beam line and peripheral equipment.
IOcm
To diffusionpump
I
A U T O M A T I C DATA A C Q U I S I T I O N AND ON-LINE ANALYSIS
291
Fig. 3. Detailed view of the plexiglass scattering chamber and Carousel slide changer.
° n ° n e of the beam lines attached to the EN Tandem Van de Graaff accelerator of the Universit6 de Montr6al. A lay-out of the beam line with beam collimation system, facility to handle the samples and detect the X-rays is shown in fig. 1. Actual pictures of the system and peripheral equipment are shown in figs. 2 and 3. The main part of the apparatus consists of a 2.5 cm thick plexiglass scattering chamber built in a box-like fashion. This chamber contains the chassis of a 750 H Carousel Kodak projector and its slide changer which can hold up to 80 slides. The choice of plexiglass was based on the fact that, with this transparent material, all problems related to the quality and position of the targets as well as the beam focusing or defocusing adjustments could be checked (and eventually corrected) at any time without opening the scattering chamber. In particular the possible deteriorating effects of the beam on the biological targets, employed in this work, could be easily monitored by means of a closed-circuit television network (see fig. 1). A
vacuum of the order of 10 -5 torr could be obtained in a very short time with the aid of an auxiliary pumping system. It is known that a better vacuum (of an order of magnitude or more) could have been attained by employing a metallic scattering chamber. However, the use of this type of chamber would have brought in too many hindrances which would have totally offset its only indisputable advantage. Furthermore, it was recognized (after careful checks) that a vacuum of the order of 10 -s torr was amply sufficient for the present and future experimental needs. The specimens, prepared in the same fashion as previously described1), are mounted on standard 110 plastic slide frames and fitted on 35 mm adapters. These samples are then placed in the slide changer of the Kodak projector whose chassis was suitably transformed to position the targets, one by one, in line with the beam. A complete target replacement is easily done by opening the chamber, removing the slide changer and inserting a new one containing a fresh batch of specimens. Since, in the scattering
292
R. LECOMTE et al.
chamber there is a large quantity of metallic material (chassis, motor and slide changer) and organic material (slide frames), a very careful beam collimation system had to be devised in order to suppress entirely the possible scattering of the beam in the chamber itself. Two tantalum collimators, 0.472 cm in diameter, are placed in the beam line at 50 cm and 30 cm, respectively, before the scattering chamber. Another tantalum collimator, 0.635 cm in diameter, is then placed at 10 cm before the target to stop all the particles scattered by the two previous collimators (see fig. 1). A careful mapping of the beam position on targets whose elemental composition was exactly known, ensured us that in ordinary experimental conditions no beam scattering was present in the chamber. The X-ray detector is placed at 90° with respect to the beam axis and it is mounted directly inside the plexiglass chamber and as close as possible to the target. The target makes a 45 ° angle with respect to the beam axis and is oriented in such a way that the X-rays must go through the target itself to be detected by the counter. In such a way the low energy X-rays are attenuated before impinging on the detector. However, the filtering of
PLEXIGLASS CHAMBER
DETECTOR
TARGETROOM CONTROL ROOM START STOP RESET
TIM~G CONTROLLER
CDC
31OO
CAMAC
I SYMBOLI NAME F Forword B I Bockword Fig. 4. Schematic of the automatic data acquisition system.
the low energy X-rays due to elements as Na, P, S, Cl, K and Ca (which are present in large quantities in biological samples as the ones presently studied), is mostly done by suitably thick mylar foils, placed on top of the detector. Finally, another tantalum collimator, 0.472 cm in diameter, is placed between the detector and the target (at 3.4 cm from the detector and 2.7 cm from the target) to ensure proper collimation of the X-rays reaching the detector. Details on the X-ray counter and electronic circuitry as well as on target preparation and determination of the relative X-ray yield curves have been already reported0 and will not be repeated here. 2.2. AUTOMATIC DATA ACQUISITION A schematic of the automatic data acquisition system is shown in fig. 4. The Carousel slide changer and data acquisition system are controlled by a CDC 3100 computer through two Camac interface modules. A "Latched Triac Output 3080" module is used to advance automatically the Carousel slide changer whereas the homemade "Timing controller" starts
ALITOMATIC
DATA
ACQUISITION
or stops the accumulation and resets all scalers. Two manual control units (MC1 and MC2) one situated near the plexiglass chamber and the other in the control room can also be used to place the desired target before the beam. The manual controls and Triac module are used simply to initiate the target changing, the motor being mechanically turned off by a microswitch mounted on the projector mechanism when the target is in the proper position. Two magnetic tape drivers (T1 and T2) are needed to store the accumulated spectra and to
ICONTRIX I
| I IN'PUT: MIN,MAX,NCOUNT~LIMT, I DEF I
[ TURN OFF TRIAC AND TIMINGCONTROLERI ICAROUSELIlFI ,I F2,MIN,MAX,NCOUNT, L IMT) I
ICAROUSEL I I
ICLOCKTM=OI
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...........
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I NEXT(IDEFTI F I ) I t
[
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@
,o o
1 i SETTO,NCALL TERNAL CLOC I_, INTEG
L_ SET INTERNAL CLOCK TO CALL TEST
ICLOCKTM= O I | I
@
Fig. 5. Flow-chart of the control program C O N T R I X and its
real-time routines (see text).
AND ON-LINE
ANALYSIS
293
perform the on-line analysis (see section 3.3). The data acquisition system shown in fig. 4 is automatically driven by the control program CONTRIX whose flow-chart is presented in fig. 5 together with its real-time routines. Since the routines of the program CONTRIX are on a real-time basis the use of this program does not hamper any batch processing work (as, for instance, the simultaneous data analysis). The input parameters of CONTRIX are: MIN and MAX which represent the lower and upper limits of a chosen reference X-ray peak in the spectrum; NCOUNT which is the minimum number of counts to be accumulated in that peak; LIMT and IDEF which are the bombarding time limit for each target and the first spectrum identifier, respectively. Logical flags IF1 and IF2 are used to indicate that the target changing routine is under way (IF1 = 1) or that the data analysis program is looking for a spectrum on the data tape (IF2= 1). CONTRIX resets all scalers and the ADC, turns off all the switches on the Triac and Timing controller modules, starts the data acquisition system (START) and calls for the control routines (CAROUSEL). The subroutine CAROUSEL resets and starts a time counting program (CLOCKTM) that gives the exact elapsed time and sets the internal computer clock to call the real time routine INTEG after one second. When the preset time limit or preferably the preset number of counts (NCOUNT) in the X-ray peak is reached a change of target is carried out (NEXT). The real-time routine TEST verifies if NEXT is completed. The sequence of steps done by the subroutine NEXT is as follows: 1) The current integrator, timer and various scalers together with the ADC are disabled; 2) the spectrum is recorded on magnetic tape with the proper identifier; 3) everything is reset to zero and a new identifier is given;r 4) the slide changer is advanced by one step (or more if desired), placing a new target in the beam which is " o n " all the time; 5) the ADC and all the peripheral counting equipment are then enabled.
3. Spectrum analysis and results 3.1. GENERAL CONSIDERATIONS Once the X-ray spectra from the various samples are obtained, the experimenter is faced with
294
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et al.
the problem of how to determine which trace ele- resorted to a different approach in the method of ments are present in the spectra and calculate analysis of the X-ray spectra that does not require their respective amounts. To this end, it is neces- any previous knowledge of all the physical factors sary to locate all the peaks in the X-ray spectrum, which could have a bearing on the shape of the calculate their energies and determine their inten- spectra. These factors, however, are all implicitly sities. Usually the first part of the analysis (i.e. lo- considered and included in the analysis without calization and energy determination of the peaks involving any theoretical hypothesis on the X-ray in the spectrum) does not constitute a major prob- peak shapes. This method is essentially based on lem, nor does the intensity calculation when only the very simple fact that an X-ray spectrum can few peaks, well separated in energy, are present in be sometimes very complicated, but it is always the spectra. This situation, however, is the excep- formed by a finite number of elements giving imtion rather than the rule since, generally, multi- mutable peak shapes at all times once the experplets of varying complexity are present in many X- imental conditions, under which the measureray applications. In this case it is clear that reliable ments are carried out, are kept carefully uncomputer codes must be available and these codes changed. should be fast and possibly completely automated (and this point ought to be well emphasized!) 3.2. METHODOF ANALYS|S especially when a very large number of specimens The calculation of the amount of an element in are being processed. a sample is done by comparing the area of the The similarities in the physical conditions of 7- K~(or L~) peak of the element of interest with that ray spectroscopy (whether in radioactive decay stu- of a reference (or doping) element. The choice of dies or nuclear reaction work) and X-ray spectra such an internal standard is usually a matter of have led many workers to adapt computer codes convenience. Yttrium was chosen here since it is originally designed for ),-ray spectroscopy to the completely missing in the serum specimens, and it analysis of X-ray spectra4,5). These methods gen- does not interfere greatly with the elements under erally produce very good results but have the lim- study in the serum and can be easily manipulated. itation that the codes require the extensive use The relation giving the concentration for an eleof large computers and great manual manipula- ment i is: tion; therefore, they are not well suited for routine Si Cy work and even less for an eventual complete on- Ct - Sy Rir (1) line automation procedure, which is, usually, carwhere Ct and Cy are the concentrations of the eleried out with much smaller computers. A different approach in handling the X-ray ment i and yttrium, respectively; St and Sy are the spectrum analysis has been taken by Kaufmann areas of the K~(or L~) peaks of the element i and and Akselsson6), who employ a model which is yttrium and R~y is the yield of element i measured based on the physical processes involved in the X- relative to that of yttrium. This value can be obray production methods. All the model parameters tained from the properly constructed relative yield (approximately 12) are simultaneously adjusted to curves (see ref. 1, fig. 1). Since the concentration reproduce the background and the peaks in the X- Cy of the doping element is known exactly, the ray spectrum. The important feature of this code accuracy of Ci depends on how precisely the areas (which requires approximately a 24 K memory) is Si and Sy and the value of R~y can be obtained. A the possibility of processing a large number of correct determination of R~y usually does not presspectra without operator intervention. It is clear ent a great problem (see ref. l) whereas the calcuthat this method and those derived from y-ray lation of St, and to a lesser extent Sy, often can spectroscopy depend heavily on the step by step be very difficult due to the complexity of the Xreproduction procedure of the peak and back- ray spectra. It is at this point that specific peak ground shape. Thus several physical factors must and background shape functions are considered in be included in the programs; this leads to multi- the codes performing the appropriate analysis of parameter equations which are difficult to solve the X-ray spectra. Instead of employing such anand which converge slowly. On the other hand, if alytical methods, we have used the much simpler some factors are neglected, one could miss some non-analytical approach of building an assembly of important information. For this reason we have standard peak shapes from most of the elements
AUTOMATIC
DATA
ACQUISITION
liable to be detected in the samples under investigation. These standard peak shapes are obtained from targets containing only the element of interest and bombarded in the same experimental conditions as for the specimens under study. Particular care was taken in obtaining the reference targets with the same physical inherent characteristics (i.e. target thickness, type of backing and its thickness and so on (see also ref. 1, sections 2.2-2.4) of the sample targets. This in order to avoid possible absorption differences of the emitted X-rays in both sets of targets. For the KXrays we did produce standard spectra from targets containing K, Ca, Ti, Cr, Mn, Fe, Co, Ni, Cu, Zn, As, Se, Br, Rb, Sr, Y, Zr, Nb, Mo, Ag and Cd, for the K and L X-rays Sn, Sb and Te, and I have been used and finally for the L X-rays Ba, W, Au, Hg and Pb. In all these reference spectra the background is so negligible in comparison to the peak height that no peak shape distorsion is present. Let us consider now an X-ray spectrum obtained from a sample containing some elements. This spectrum can be considered as formed by a superposition of a number of standard peak shapes superimposed on a continuous background. Thus, the number of counts in each channel can be expressed as: N(x) =
Ji
+ B(x).
(2)
i=1
where f is the scalar factor giving the ratio between the areas of the peaks due to the element i in the spectrum under investigations and in the reference spectrum, respectively; F~(x) is the standard spectrum shape in channel x of element i; B(x) is the corresponding background in the spectrum under study. The summation is carried out on the n elements present in the target. Thus if one wants to know how an element is contributing to the total spectrum, this element must be stripped from the spectrum itself with the "ad hoc" standard shape until a continuous and regular background shape is obtained under the peaks of the element of interest. It is clear that the accuracy in the calculations depends a great deal also on the choice of a suitable background shape under the peaks to be analysed. This point will be discussed in more detail in the next section. Now the area of a peak due to an element i present in the sample is given by: s, = f, x
(3)
AND ON-LINE
ANALYSIS
295
where S~ is the area of the same peak in the reference spectrum. By inserting eq. (3) in eq. (1) one obtains: Ci = --~-~ , (4) fr Sy Riy where fr and S~ have the usual meaning and are referred to the doping element (yttrium in our case). The calculation of (7,. can be simplified if the standard spectra are built so that SiR=S~, then one obtains: A cy
C~ = fy Riy"
(5)
It is evident from the last expression that in order to obtain the concentration Ci of the element i present in the sample only the following items are needed: a) the standard shape (or reference spectrum) of the elements; b) the standard shape (or reference spectrum) of the doping element Y added with concentration Cy in the sample; c) the value of Riy deducible from the relative X-ray yield curve pertinent to the experimental conditions under which the experiment is carried out (see ref. 1, fig. 1). The method described above is certainly striking for its simplicity and effectiveness since no very sophisticated hypotheses are necessary to carry out the analysis which can be done on-line (and completely automatically) in relatively small computers. The only possible disadvantage could be that the assembly of standard spectra must be obtained rigorously under the same experimental conditions used in the study of the samples. In fact, every change would entail the determination of a new assembly of standard peak shapes as well as relative x-ray yield curves. It is felt, however, that this kind of applied research, where hundreds of samples must be measured and analysed in a short time, should be performed with a set-up sufficiently supple to meet a large variety of experimental needs of the researchers. Thus eventual time consuming changes in the experimental conditions would be avoided as much as possible. 3.3. AUTOMATIONOF THE ANALYSIS The calculations described in the previous section are automatically performed on-line by a CDC 3100 computer. As has been already mentioned the precision in the calculation is affected by the choice of the background shape under the peaks of interest. Primarily the background in Xray work is due to the bremsstrahlung produced
296
R. LECOMTE et al.
by electron scattering. This kind of background is characterized by a more or less pronounced bump with a sharp cut-off, the end-point of which depends on the type of beam employed and its energy (see ref. 1). Above this cut-off the remaining background, which is due to Compton scattering of ),-rays and projectile bremsstrahlung is usually linear and its amount depends essentially on the quantity of material intercepted by the beam in the specimen. Thus, depending on the position of the peaks in the spectrum, the background shape is fitted by a parabolic or linear function. The fit is done in a region around the peak sufficiently large (usually seven times the width of the peak at half-maximum) and it is minimized by a X2 analysis. At this point the scalar factor f is obtained and subsequently the concentration of the element i in the sample is calculated. In figs. 6a, b and c the results obtained in the analysis of a spectrum with well resolved peaks (iron in this case) lying (ai
R E F E R E N C E SPECTRUM l]
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I
I
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5000
2000 " O IO O I
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I
180
I
190 CHANNEL
I
I
200
210
NUMBER
Fig. 6. Typical analysis of the well resolved Fe peaks lying on a high background (see text).
on a high background (heavily loaded sample) are shown as typical example. However, as already mentioned above, the physical situation presented in fig. 6b is somewhat exceptional since usually multiplets of varying complexity are present in the spectra. For instance, in the serum specimens the K, copper peak is overlapped by the stronger K~ peak of zinc and the KB potassium peak is completely masked by the K~ peak of calcium. Furthermore in the case of energy multiplets it is also much more difficult to fit the functional form of the background under them. Thus it is quite evident that the analysis performed by the program would yield large errors in the elemental concentration values if these problems were not tackled and resolved with appropriate procedures. The, ideal situation would be to obtain elemental spectra of the type shown in fig. 6b, i.e. spectra with well resolved peaks, lying on a more or less pronounced background, which could be easily analysed by the respective reference spectra. To this end we have devised a completely automated program which performs by iterative methods a "peak stripping" of the X-ray spectra. As a final result only the peaks due to the element, the concentration of which has to be calculated, are left in the spectrum. The calculations are carried out following the prescriptions described above and are performed simultaneously for all the elements present in the specimen regardless of their stronger or weaker concentration levels. The program has been devised so that the number of input data is reduced to the indispensable minimum and all human "fumbling" is eliminated during the calculations. The required information are stated in the start up procedure or are automatically accessible during the running of the program. To give a better description of the program TOURIX employed in the present analysis, its flow-chart is shown in fig. 7. The chemical symbols of the elements under analysis (IREF(N)), the concentration of the doping element (CONCY) and the preset maximum number of iterations (NRAF) are the input data. At this point the spectrum searching routine is " o n " and the spectra are automatically analysed at the end of each data accumulation. The reference spectrum (YY(X)) as well as all the information relative to the position (YY(1024)), full-width at half-maximum (YY(1023)) and detection efficiency (EFF(N)) of the principal peak of the same element are read from an auxiliary magnetic tape
AUTOMATIC
DATA ACQUISITION
297
AND ON-LINE ANALYSIS
iNPUT: e~ementstobeanalysed IREF(N), I EFF(N) ; CONCY,NRAF; spectrumtobeanalysedYA(X) =YB(X) t
I NN .ol
NNN: 2 ) I SCAL : R[N,J-I DECAL=D{N,J-I) PERC =0 02/J
SCAL: DECAL: O.O PERC: 0.02 | JTTAPE(IREF(NI;YY(X) ) I
YES
R(N ,JJ) =R(N.J-I),JJ=J. NRAF D(N,JJ ) =D(N,J-I),JJ= J, NRAF ]
NO YES
t
MAA=YY(IO24)-I 5 X YY(IO23) MAB=YY(IO24)-I 5 X YY(IO23
NO I YB(X) =YA(X) J
~
- R ~ Y E S
F---F ~F'7 FFT- ~
[ TTAPE ([REF(I),YY(X) ) I I
YB(X)=YB(X)-R(I J-I)X YY(X'H) (I,J-I)) I
[ TTAPE(IREF(NI;YY(XI) [ |
[MAA:YY(IO24)-35XYY('O23) I MAB=YY(IO24)÷ 35 X YY(IO23) [ NSUB(YY(X),YB(X),MAA,MAB,SCAL,DECAL,PERC,NNN;YC(X)) ] I [ IR(N,J) = SCAL 1 D(N,J) =DECAL
NO YES [ YC(X)~YA(X) J
l TTAPE(IRERN)iYY(X)) I I
I YC(X}=YC(X)-R(N,NRAF)XYY(X*EXN,NRAF))I
I ONC(N),N=I,NELI ERR IN), N=I,NEL SENS(N , N: I,NEL
I
OUTPUT: N,R(N,NRAF),D(N,NRAF), CONC(N),ERR(N),SENS(N), NJ(N) final s~ectrum: YC(X)
Fig. 7. Flow-chart of the program TOURIX used for the automatic analysis of X-ray spectra (see text).
YES
298
R. LECOMTE et al.
(o) INITIAL SPECTRUM 2500
X I/2
:!
'
2000
1500
If!
I ~
2'
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1500
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Fig. 8. Example of analysis of an X-ray spectrum from the serum of a rheumatoid arthritis affected patient. (a) X-ray spectrum; (b) resulting background after analysis with the program TOURIX.
during the calculations, by means of the subroutine TTAPE. These information, together with the standard spectra, are previously written on the magnetic tape by means of the program RESPEC and constitute the "data bank".
With the first iteration (J= 1) the program TOURIX obtains an approximate value of the scalar factor f~(R(N, J= 1) = SCAL) for all elements present in the spectrum, without taking into account interferences due to the existence of multiplets. In this preliminary analysis, which is carried out with the subroutine (NSUB), a simple linear function ( N N N = 1) is used to adjust the background shape under and around the peaks. The background fit is done in a smaller region (from MAA to MAB) than that previously described (usually 3 times the full-width at halfmaximum) since it is advisable to exclude, as much as possible, close-by peaks. The calculation of the scalar factors is stopped when a precision (PERC) of 2% is attained. It is clear that at this point the results are approximate but, in general, they give a fair idea of the order of magnitude of the area of the peaks. These results are then employed during the following iterations (J> 1). All the elements present in the specimen with the exception of that under analysis are subtracted until the peaks of interest are well isolated in the spectrum (in other words one obtains a spectrum of the type shown in fig. 6b). From this "clean" spectrum the final scalar factor (SCAL).~ of the element i is calculated using again the subroutine NSUB and considering also the full background shape treatment previously described, i.e. employing a linear function or a parabolic one (NNN = 2) depending on the case in point. The subroutine POLFIT carries out the X 2 analysis for the minimization of the background fit. The same procedure is then followed for all the n trace elements present in the specimen (R(N, J)) and subsequent-
TABLE 1 Results of the analysis of the spectrum shown in fig. 8a. These results, extracted with the program TOURIX, are obtained as a direct print-out from the CDC 3100 computer at the end of the calculations. PAU2 is the spectrum identifier; DECAL means SHIFT. The other headlines are self-explanatory. Analysis of spectrum PAU2. Element SCAL
K Ca Fe Cu Zn Br Y Au
0.026567 0.074739 0.010320 0.012883 0.011592 0.013550 0.063787 0.010319
DECAL
- 0.10 -0.06 -0.13 -0.06 -0.01 0.03 - 0.01 0.21
Concentration (mg/I) 126.210957 66.573588 0.728781 1.216645 1.340214 4.335204 50.130(0)00 5.151798
Stat. error (%)
Sensibility (mg/l)
Number of iterations
2.257 1.703 3.086 2.855 2.962 2.806 1.771 3.087
2.577622 0.861085 0.071617 0.019938 0.045958 0.041469 0.035138 0.157226
5 5 4 4 4 5 3 4
AUTOMATIC DATA ACQUISITION AND ON-LINE ANALYSIS
ly their concentration values (CONC(N)) are calculated from eq. (5). The attainable precision with this analytical method depends essentially on the accuracy one wishes to achieve. In turn the accuracy limits have a bearing on the number of iterations necessary to carry out the calculations. Direct experience on hundreds of specimens bombarded under the same experimental conditions has shown that in order to achieve a precision of 0.5 or 10% (thus below or, at the most, of the same order as the statistical errors) 3 or 4 iterations are sufficient in the case of well separated peaks (as in fig. 6b) whereas for complicated multiplets between 5 to 8 iterations are necessary. Finally with the program TOURIX the statistical errors (ERR(N)) in the areas of the peaks can be obtained as well as the detectability limil~s (SENS(N)) of each element (see ref. 1) together with the number of iterations (NJ(N)) required to calculate the area of each peak. Furthermore, if desired, it is possible to plot the final spectrum (YC(X)) at the end of the calculations. An example of the analysis carried out by the program TOURIX is presented in figs. 8a and 8b. Fig. 8a shows the typical spectrum from the serum of a patient with rheumatoid arthritis under chrysotherapy treatment with gold salts. The specimen has been doped with yttrium having an exactly known concentration (50rag/l). Fig. 8b shows the background shape attained at the end of the calculations. The results of the analysis, directly obtained from the CDC 3100 computer at the end of the calculations, are presented in table 1. 4. Conclusion A sample control set-up and an automatic data acquisition system have been described in detail together with a spectrum analysis program (TOU-
299
RIX) totally automated. The data accumulation and its analysis can be carried out simultaneously on a CDC 3100 having 32 K memory. This is an important feature since it greatly minimizes the time lag usually existing between the data acquisition and its physical interpretation. The capabilities of the program TOURIX have been thoroughly checked from a plethora of specimens containing several trace elements with known and widely different concentrations. In every instance the program TOURIX could detect the elements present and measure precisely their concentrations even in the case of very weak peaks partially (or totally) masked by stronger close-by peaks. On the other hand if the element was not present, the program gave a concentration value either equal to zero or smaller than the detectability limits (see column "Sensibility" in table 1). The authors wish to thank Dr. J.-P. Martin for his helpful advice concerning the development of the programs and Dr. G. R. Rao for the critical reading of the manuscript. The financial support of the National Research Council and Medical Research Council of Canada is gratefully acknowledged. References l) M. Barrette, G. Lamoureux, E. Lebel, R. Lecomte, P. Paradis and S. Monaro, Nucl. Instr. and Meth. 134 (1976) 189. 2) V. Valkovic, R. B. Liebert, T. Zabel, H. T. Larson, D. Miljanic, R. M. Wheeler and G. C. Phillips, Nucl. Instr. and Meth. 114 (1974) 573. 3) p, S. Ong. P. K. Lund, C. E. Litton and B. A. Mitchell, Adv. X-ray Anal. 16 (1974) 834. 4) M. A. Mariscotti, Nucl. Instr. and Meth. 50 (1967) 309. 5) j. T. Routti and S. G. Prussin, Nucl. Instr. and Meth. 72 (1969) 125. 6) H. C. Kaufmann and R. Akselsson, Advan. X-ray Anal. 18 (1975) 353.