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Channels ratio quench correction using Čerenkov radiation for the assay of 42K in biological samples
T~lmical notes International Journal of Applied Radiation and Isotopes, 1971, Vol. 22,
p. 213. Pergamon Press. Printed in Northern Ireland
A Rapid Method for Preparation of asP-Applicators (Received 10 October 1970) APPLICATORS containing ssp have been very often used in the treatment of hemangioma. Several methods have been reported in the literature3x-l°) We tried to find a simple method using commercially available products for clinical purposes with minimum radiation hazard. We use a plastic anion exchange resin on which the 8sp activity is adsorbed. A Zerolith FF foil immersed into the sodium phosphate-SSP solution adsorbed only part of the activity. The maximum adsorption can be obtained from a carrier-free solution; using a phosphate cartier leads to decrease in activity sorption. I n all cases maximum absorption is reached after 10 hr. Autoradiography of a foil prepared with carrier-free phosphate-8*P showed the distribution of 3sp to be rather inhomogeneous, a greater part of the activity was adsorbed on the rim of the applicator. Homogeneous distribution was, however, reached in solutions containing 3 nag P/ml as a carrier. I n this case the activity adsorbed after 10 hr is about 60 per cent. The effect of the area of the surface of the applicator and the volume of the solution on the sorbed amount was also studied. These factors seems to be of no significance.
Recommended procedure One hundred #Ci of carrier-free phosphate-ssP or phosphoric acid-asP per each ems of the applicator is placed into a glass beaker. Distilled water (2.5 ml) and 0-075 ml of sodium phosphate (10 mg of P per ml) for each cm s is then added. Plastic anion exchange resin foll (Zerolith FF) is transformed into the O H ' form by washing it in I N sodium hydroxide for 24 hr. After 2-3 washings with distilled water the required shape is cut out and immersed into the solution of asP for I0 hr. The foils are removed, washed with distilled water and dried. The homogeneity of a2p activity distribution in the applicator is determined by autoradiography. Applicators prepared by our method are quite suitable for clinical use and have been successfully tested in clinical practice. The only disadvantage is the loss ofthe part ofa~P activity in the preparation.
213
However, this does not play any significant role from the point of view of economy. J. SVOBODOv.~ L. KRONa/~ J. KontmgX
Research Institute for Medical Use of Radioisotopes Prague Nuclear Research Institute Cxechoslovak Academy of Sciences v v Rez u Prague, Czechoslovakia
References 1. Low-BEER B. V.A. Radialo~ 47, 213 (1946). 2. N O H ~ t ~ B. A. Acta Soc. raed. Ups. 62, 152 (1957). 3. Gtmxa~xJ. d. radiol. Elect. 45, 589 (1964). 4. NARTSlSSOVAK. P. Vest. Rentgenol. 64, (1954). 5. Lzol~rrj~vx A. A. and VOROm'SOv D. V. Vest. RentgenoL Radial. 34, 83 (1959). 6. V~NSRONXG. Minerva Chit. 20, 241 (1965). 7. RoED. S.A. Lamet269, I l l l (1959). 8. HEmLAW P. S., SUmER R. S. and RILEY E. P. Radiology 52, 401 (1949). 9. ZADUnAU M. and KtmSTAnT E. Radiobial. Radiother. (Berlin) 3, 153 (1962). 10. BRJANCEVA. V., Vxmxag A. D., SALmKOVE. T. and Rooovm Z A., Med. radial. (Moscow) 8, 25 (1967).
International Journal of Applied Radiation and Isotopes, 1971,Voi. 22, pp. 213-216. Pergamon Preu. Printed in Northern Ireland
Channels Ratio Quench Correction Using Cerenkov Radiation for the Assay of ~K in Biological Santples (Received 29 April 1969; /n revisedform 1 October 1969) Introduction HIOH Em~ROY p-emitting nuclides may be measured by the detection of (~erenkov radiation emitted in aqueous solution.~1-s) It has also been shown{0 that the channels ratio method (5) could be applied to this method of estimating ssp to correct for the efficiency of its detection in coloured solutions. I n a recent review of the use of ~erenkov radiation as a means of detecting t-emitting nuclides(sl it was concluded that the colour attenuation of ~erenkov radiation was too small to be of use for accurate correction of counting efficiency. This is contrary to the author's experience in estimating ~ in samples of biological fluid.
Technical notes
214 Experimental
The method to be described for the estimation of 12K was part of a general experimental schedule ¢7) which allowed the subsequent measurement of other radioisotopes and permitted the recovery of the samples for chemical analysis. Because this method required a deliberate exclusion of the lower energy radiation during counting, the gain and discriminator settings given are not quite optimal for the estimation of 4aK in the absence of other nuclides. Samples, each of 6 ml, were prepared in polythene tubes (60 mm x 15 mm dia., Xlon). These tubes were held in standard 20 ml glass counting vials during counting in a Packard Series 3003 Tri-Carb liquid scintillation counter fitted with a 2SSRa external standard. Figure 1 shows the effect of varying amplification on the count rate in a channel between discriminator settings of 100 and 1000 scale divisions for a sample of 42KC1 in distilled deionised water. The effectiveness of correcting for quenching by the channels ratio method cs) and by the use of an external standard was examined for amplification factors between 20-40 per cent of maximum with a series of standard samples of 42K quenched with varying quantities of whole blood, urine or plasma. Throughout the experiments the lower discriminators of channels A and B were set at I00 scale divisions and the upper discriminator of channel B at 1000
scale divisions. The amplification factors of both channels were varied in a similar manner over the range studied and the counts in channel B and the ratio of counts in channel A/counts in channel B, (A/B) were recorded at each setting of amplification with the upper discriminator of channel A set at values varying from 200-500 scale divisions. The ratio of A/B was plotted against the efficiency of counting in channel B. A linear plot over the greatest range of efficiency was found for an ampllfication factor of 25 per cent with the upper discriminator of channel A set at 300 scale divisions. These results are shown in Fig. 2 in comparison with the results obtained for the same samples when the efficiency of counting 42K was plotted against the counts from the external ~26Ra standard detected in channel B. No significant differences were observed for individual quench curves for whole blood, urine or plasma and Fig. 2 shows the limits within which the results of all the samples lay. The marked effect of whole blood as a colour quenching agent is also illustrated. For 4~K, in the counting efficiency range of 37-20 per cent, quenching which caused a decrease in the efficiency by I0 per cent of the original value resulted in a decrease of approximately 16 per cent in the counts due to the external standard. The sources of error involved in using external standardisation as a means of correcting for colour quenching have
L,0-
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20-
10-
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L'o AHPLIFICATiON~/~
Fzo. I. Variation of counting efficiency with amplification.
;o
Technical notes
215
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// \\ \\
~20.=_.
,/ \\/
/ / /
• Whole Blood • Urine
II II ~
.--.
IIii 7/
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10-
11
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I
I
!
1
2
3 I, 5 6 ,7 O - - - Externol Stondord(x 10~cpm}
0'.4
~
/ 1
olf
I
I
0'.6
--ChonneL Rotio(A/B)
!
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9
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FIG. 2. EFficiency of detection of 4OK by Cerenkov radiation in samples of whole blood, urine or plasma. Limits (range) of c~cicncy plotted against (a) channels ratio A/B and (b)counts from the external standard in channel B. Numbers shown on graph indlcatc the dilutions the biologicalfluidsrequired for various counting c~ciencies: I. undiluted, 2. I[I0, 3. 1/100, 4. I/I000, 5. 1/10000. already been well reviewed,(e) and our present results are in accord with these previous estimates of its accuracy. It has been reported(s)that a I0 per cent change in counting c~ciency produced only a 2 per cent change in channel ratio. However, throughout the range of counting e~clency 37-20 per cent alterations in counting efficiency produced inversely proportional changes in the channel ratio which should not bc compared dlrcctIy as percentage changes due to the reciprocal nature of the linear plot. The present results (Fig. 2) show that the channel ratio method
of quench correction, when used for aK, is at least four times as sensitive as the approximate estimate given previously,co) While alteration of counting ciTiciency causes a smaller proportional change in the channel ratio than in the counts from the external standar~ the channel ratio method gave lessvariable estimates, as can be seen from the limits of the results in Fig. 2. Thus it would appear that for U K at least,the most accurate estimate of the cmciency of detcctlng (~ercnkov radiation at high counting efficicncics is given by using the channels ratio method.
Technical notes
216
The method currently used is to dilute the samples so that channel ratio A[B falls between 0.35 and 0.55. The efficiency of detection is then determined from the linear portion of the channels ratio quench curve. If the ratio of channel A[B is greater than 0.55 and subsequent analytical determinations do not permit a portion of the sample to be diluted and recounted, a fairly good estimate of the efficiency of detection of 4~K can be obtained from the external standard quench curve. A. T. B. MOIR
Medical Research Coundl Unit for Research in Brain Metabolism Department of Pharmacology University of Edinburgh Medical School Edinburgh, Scotland References I. HA~ERER K. and K6LLE W. Atompro~xis I I , 664 (1965). 2. DE VoLPI A. and PoRouS K. G. A. Int. J. appl. Radiat. Isotopes 16, 496 (1965). 3. P~.CER R. P. and EtRIeK R. H. Int. J. appl. Radiat. Isotopes 17, 361 (1966). 4. Sa~OBBSR. D. and JACKSON A. Int..1.. appl. Radiat. Isotopes 18, 857 (1967). 5. BAILLIE L. A. Int. J. appl. Radiat. Isotopes 8, 1 (1960). 6. ELRIC_~ R. H. and PARKER R. P. Int. J. appl. Radiat. Isotopes 19, 263 (1968). 7. MOIR A. T. B. and HALLmAYJ . I n preparation.
International Journal of Applied Radiation and Isotopes,1971,Vol. 22p pp. 216-218. Pergamon Preu. Printed m Northern Ireland
Population Longevity in G~mm~. Irradiated Collembola* (Received 20 July 1970; in revisedform 2 September 1970) A POPULATIONexplosion of Hypogastrura meridionalis Steiner~" (Collembola Arthropleona, Poduridae), occurred on decaying plant material, as often happens for
other species of the same genus (~VIAYNARD(1)), on the grounds of the Euratom Joint Research Centre at Ispra, Italy. This made it possible to test the radiosensitivity of these primitive Apterygota Insects.
Materlals mad Methods The collected insects were maintained at 24°C in boxes of plexiglass in which was placed the same decaying material on which they lived. The material which was periodically renewed was dampened daily. One thousand individuals of uniform size (about 1.8 mm) were used for each radiation dose. The treated springtails and controls were checked daily with a stereoscopic binocular microscope in order to remove dead individuals and to keep the culture healthy. The insects were irradiated in plexiglass specimen tubes, placed in the centre of the irradiation chamber of a 2480-Ci cobalt-60 Gammacell 220 at a rate of 3670 R/min and a temperature of 22°C. The dosimetry was calculated with a cavity ionization chamber P T W N. 16204 calibrated with a Radium standard. The accuracy was calculated to be approximately 2.5 per cent.
Results and (~oncluslons As can be seen in Table 1, no significant difference in mortality was found between the controls and the irradiated springtails up to 10 days, with doses inferior to 100 krad. After this period, the percentage of mortality increased with increasing doses above 100 krad. Immediate effects which provoked rapid mortality were seen at doses above 100 krad., probably due to irreparable damages to the central nervous system of the insects. When the values for STs0 (survival time of 50 per cent of the insects) are plotted against time, the curve shows (Fig. 1) that at low doses (up to 20 krad.) longevity is greater than the unirradiated controls as reported by GROSOH(2) for other insect t a x a . However, at doses above 30 krad., STs0 decreases linearly with the dose. With reference to the controls, ST60 is reduced to one-half at 150 krad. and to one-fourth at 200 krad. As one can deduce from the above results, the springtails showed a very high radioresistance with respect to what is known for Pterygota Insects (N6THZL(a)). RAFFAELE CAVALLORO
* Contribution N. 606 of the Euratom Biology Division. t We wish to thank Professor V. Parisi, Institute of Zoology, University of Parma, for the identification of this species.
GAvr~o D~LZXO
Biology Division---EntomoloO Section Euratom: European Atomic Energy Community Joint Nuclear Research Centre, Ispra Italy
p. 213. Pergamon Press. Printed in Northern Ireland
A Rapid Method for Preparation of asP-Applicators (Received 10 October 1970) APPLICATORS containing ssp have been very often used in the treatment of hemangioma. Several methods have been reported in the literature3x-l°) We tried to find a simple method using commercially available products for clinical purposes with minimum radiation hazard. We use a plastic anion exchange resin on which the 8sp activity is adsorbed. A Zerolith FF foil immersed into the sodium phosphate-SSP solution adsorbed only part of the activity. The maximum adsorption can be obtained from a carrier-free solution; using a phosphate cartier leads to decrease in activity sorption. I n all cases maximum absorption is reached after 10 hr. Autoradiography of a foil prepared with carrier-free phosphate-8*P showed the distribution of 3sp to be rather inhomogeneous, a greater part of the activity was adsorbed on the rim of the applicator. Homogeneous distribution was, however, reached in solutions containing 3 nag P/ml as a carrier. I n this case the activity adsorbed after 10 hr is about 60 per cent. The effect of the area of the surface of the applicator and the volume of the solution on the sorbed amount was also studied. These factors seems to be of no significance.
Recommended procedure One hundred #Ci of carrier-free phosphate-ssP or phosphoric acid-asP per each ems of the applicator is placed into a glass beaker. Distilled water (2.5 ml) and 0-075 ml of sodium phosphate (10 mg of P per ml) for each cm s is then added. Plastic anion exchange resin foll (Zerolith FF) is transformed into the O H ' form by washing it in I N sodium hydroxide for 24 hr. After 2-3 washings with distilled water the required shape is cut out and immersed into the solution of asP for I0 hr. The foils are removed, washed with distilled water and dried. The homogeneity of a2p activity distribution in the applicator is determined by autoradiography. Applicators prepared by our method are quite suitable for clinical use and have been successfully tested in clinical practice. The only disadvantage is the loss ofthe part ofa~P activity in the preparation.
213
However, this does not play any significant role from the point of view of economy. J. SVOBODOv.~ L. KRONa/~ J. KontmgX
Research Institute for Medical Use of Radioisotopes Prague Nuclear Research Institute Cxechoslovak Academy of Sciences v v Rez u Prague, Czechoslovakia
References 1. Low-BEER B. V.A. Radialo~ 47, 213 (1946). 2. N O H ~ t ~ B. A. Acta Soc. raed. Ups. 62, 152 (1957). 3. Gtmxa~xJ. d. radiol. Elect. 45, 589 (1964). 4. NARTSlSSOVAK. P. Vest. Rentgenol. 64, (1954). 5. Lzol~rrj~vx A. A. and VOROm'SOv D. V. Vest. RentgenoL Radial. 34, 83 (1959). 6. V~NSRONXG. Minerva Chit. 20, 241 (1965). 7. RoED. S.A. Lamet269, I l l l (1959). 8. HEmLAW P. S., SUmER R. S. and RILEY E. P. Radiology 52, 401 (1949). 9. ZADUnAU M. and KtmSTAnT E. Radiobial. Radiother. (Berlin) 3, 153 (1962). 10. BRJANCEVA. V., Vxmxag A. D., SALmKOVE. T. and Rooovm Z A., Med. radial. (Moscow) 8, 25 (1967).
International Journal of Applied Radiation and Isotopes, 1971,Voi. 22, pp. 213-216. Pergamon Preu. Printed in Northern Ireland
Channels Ratio Quench Correction Using Cerenkov Radiation for the Assay of ~K in Biological Santples (Received 29 April 1969; /n revisedform 1 October 1969) Introduction HIOH Em~ROY p-emitting nuclides may be measured by the detection of (~erenkov radiation emitted in aqueous solution.~1-s) It has also been shown{0 that the channels ratio method (5) could be applied to this method of estimating ssp to correct for the efficiency of its detection in coloured solutions. I n a recent review of the use of ~erenkov radiation as a means of detecting t-emitting nuclides(sl it was concluded that the colour attenuation of ~erenkov radiation was too small to be of use for accurate correction of counting efficiency. This is contrary to the author's experience in estimating ~ in samples of biological fluid.
Technical notes
214 Experimental
The method to be described for the estimation of 12K was part of a general experimental schedule ¢7) which allowed the subsequent measurement of other radioisotopes and permitted the recovery of the samples for chemical analysis. Because this method required a deliberate exclusion of the lower energy radiation during counting, the gain and discriminator settings given are not quite optimal for the estimation of 4aK in the absence of other nuclides. Samples, each of 6 ml, were prepared in polythene tubes (60 mm x 15 mm dia., Xlon). These tubes were held in standard 20 ml glass counting vials during counting in a Packard Series 3003 Tri-Carb liquid scintillation counter fitted with a 2SSRa external standard. Figure 1 shows the effect of varying amplification on the count rate in a channel between discriminator settings of 100 and 1000 scale divisions for a sample of 42KC1 in distilled deionised water. The effectiveness of correcting for quenching by the channels ratio method cs) and by the use of an external standard was examined for amplification factors between 20-40 per cent of maximum with a series of standard samples of 42K quenched with varying quantities of whole blood, urine or plasma. Throughout the experiments the lower discriminators of channels A and B were set at I00 scale divisions and the upper discriminator of channel B at 1000
scale divisions. The amplification factors of both channels were varied in a similar manner over the range studied and the counts in channel B and the ratio of counts in channel A/counts in channel B, (A/B) were recorded at each setting of amplification with the upper discriminator of channel A set at values varying from 200-500 scale divisions. The ratio of A/B was plotted against the efficiency of counting in channel B. A linear plot over the greatest range of efficiency was found for an ampllfication factor of 25 per cent with the upper discriminator of channel A set at 300 scale divisions. These results are shown in Fig. 2 in comparison with the results obtained for the same samples when the efficiency of counting 42K was plotted against the counts from the external ~26Ra standard detected in channel B. No significant differences were observed for individual quench curves for whole blood, urine or plasma and Fig. 2 shows the limits within which the results of all the samples lay. The marked effect of whole blood as a colour quenching agent is also illustrated. For 4~K, in the counting efficiency range of 37-20 per cent, quenching which caused a decrease in the efficiency by I0 per cent of the original value resulted in a decrease of approximately 16 per cent in the counts due to the external standard. The sources of error involved in using external standardisation as a means of correcting for colour quenching have
L,0-
30-
w
20-
10-
1'o
L'o AHPLIFICATiON~/~
Fzo. I. Variation of counting efficiency with amplification.
;o
Technical notes
215
ii / //i/ 30-
\\
// \\ \\
~20.=_.
,/ \\/
/ / /
• Whole Blood • Urine
II II ~
.--.
IIii 7/
// // // i/ // //
10-
11
o',3
I
I
!
1
2
3 I, 5 6 ,7 O - - - Externol Stondord(x 10~cpm}
0'.4
~
/ 1
olf
I
I
0'.6
--ChonneL Rotio(A/B)
!
I
!
9
0'.7
FIG. 2. EFficiency of detection of 4OK by Cerenkov radiation in samples of whole blood, urine or plasma. Limits (range) of c~cicncy plotted against (a) channels ratio A/B and (b)counts from the external standard in channel B. Numbers shown on graph indlcatc the dilutions the biologicalfluidsrequired for various counting c~ciencies: I. undiluted, 2. I[I0, 3. 1/100, 4. I/I000, 5. 1/10000. already been well reviewed,(e) and our present results are in accord with these previous estimates of its accuracy. It has been reported(s)that a I0 per cent change in counting c~ciency produced only a 2 per cent change in channel ratio. However, throughout the range of counting e~clency 37-20 per cent alterations in counting efficiency produced inversely proportional changes in the channel ratio which should not bc compared dlrcctIy as percentage changes due to the reciprocal nature of the linear plot. The present results (Fig. 2) show that the channel ratio method
of quench correction, when used for aK, is at least four times as sensitive as the approximate estimate given previously,co) While alteration of counting ciTiciency causes a smaller proportional change in the channel ratio than in the counts from the external standar~ the channel ratio method gave lessvariable estimates, as can be seen from the limits of the results in Fig. 2. Thus it would appear that for U K at least,the most accurate estimate of the cmciency of detcctlng (~ercnkov radiation at high counting efficicncics is given by using the channels ratio method.
Technical notes
216
The method currently used is to dilute the samples so that channel ratio A[B falls between 0.35 and 0.55. The efficiency of detection is then determined from the linear portion of the channels ratio quench curve. If the ratio of channel A[B is greater than 0.55 and subsequent analytical determinations do not permit a portion of the sample to be diluted and recounted, a fairly good estimate of the efficiency of detection of 4~K can be obtained from the external standard quench curve. A. T. B. MOIR
Medical Research Coundl Unit for Research in Brain Metabolism Department of Pharmacology University of Edinburgh Medical School Edinburgh, Scotland References I. HA~ERER K. and K6LLE W. Atompro~xis I I , 664 (1965). 2. DE VoLPI A. and PoRouS K. G. A. Int. J. appl. Radiat. Isotopes 16, 496 (1965). 3. P~.CER R. P. and EtRIeK R. H. Int. J. appl. Radiat. Isotopes 17, 361 (1966). 4. Sa~OBBSR. D. and JACKSON A. Int..1.. appl. Radiat. Isotopes 18, 857 (1967). 5. BAILLIE L. A. Int. J. appl. Radiat. Isotopes 8, 1 (1960). 6. ELRIC_~ R. H. and PARKER R. P. Int. J. appl. Radiat. Isotopes 19, 263 (1968). 7. MOIR A. T. B. and HALLmAYJ . I n preparation.
International Journal of Applied Radiation and Isotopes,1971,Vol. 22p pp. 216-218. Pergamon Preu. Printed m Northern Ireland
Population Longevity in G~mm~. Irradiated Collembola* (Received 20 July 1970; in revisedform 2 September 1970) A POPULATIONexplosion of Hypogastrura meridionalis Steiner~" (Collembola Arthropleona, Poduridae), occurred on decaying plant material, as often happens for
other species of the same genus (~VIAYNARD(1)), on the grounds of the Euratom Joint Research Centre at Ispra, Italy. This made it possible to test the radiosensitivity of these primitive Apterygota Insects.
Materlals mad Methods The collected insects were maintained at 24°C in boxes of plexiglass in which was placed the same decaying material on which they lived. The material which was periodically renewed was dampened daily. One thousand individuals of uniform size (about 1.8 mm) were used for each radiation dose. The treated springtails and controls were checked daily with a stereoscopic binocular microscope in order to remove dead individuals and to keep the culture healthy. The insects were irradiated in plexiglass specimen tubes, placed in the centre of the irradiation chamber of a 2480-Ci cobalt-60 Gammacell 220 at a rate of 3670 R/min and a temperature of 22°C. The dosimetry was calculated with a cavity ionization chamber P T W N. 16204 calibrated with a Radium standard. The accuracy was calculated to be approximately 2.5 per cent.
Results and (~oncluslons As can be seen in Table 1, no significant difference in mortality was found between the controls and the irradiated springtails up to 10 days, with doses inferior to 100 krad. After this period, the percentage of mortality increased with increasing doses above 100 krad. Immediate effects which provoked rapid mortality were seen at doses above 100 krad., probably due to irreparable damages to the central nervous system of the insects. When the values for STs0 (survival time of 50 per cent of the insects) are plotted against time, the curve shows (Fig. 1) that at low doses (up to 20 krad.) longevity is greater than the unirradiated controls as reported by GROSOH(2) for other insect t a x a . However, at doses above 30 krad., STs0 decreases linearly with the dose. With reference to the controls, ST60 is reduced to one-half at 150 krad. and to one-fourth at 200 krad. As one can deduce from the above results, the springtails showed a very high radioresistance with respect to what is known for Pterygota Insects (N6THZL(a)). RAFFAELE CAVALLORO
* Contribution N. 606 of the Euratom Biology Division. t We wish to thank Professor V. Parisi, Institute of Zoology, University of Parma, for the identification of this species.
GAvr~o D~LZXO
Biology Division---EntomoloO Section Euratom: European Atomic Energy Community Joint Nuclear Research Centre, Ispra Italy