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Do-it-yourself external standardization for a liquid scintillation counter
Technical notes
486
for 5 rain, any uncomplexed Tc 99m is removed by passing the solution through a column orion-exchange resin 1 cm in diameter, containing 4 g of Amberlite IRA-400(C1), overlaid with 1 g ofDowex 1 × 2 (C1) 50-100 mesh, prepared as described by STEV~ et al. ta) both resins being of analytical grade. The first 2 ml of eluate are discarded. The column is then washed with physiological saline and to the subsequent 15 ml of eluate are added 1 ml of 1% albumin in physiological saline, as "carrier" and the pH is adjusted to 4.5-5 with N-NaOH. 1 ml of 0"2 M acetate buffer pH 5"0 is added and the protein solution is re-sterilized by filtration through sterile 0.45 # porosity "Millipore"membranes. Thealbumin is then aggregated by mechanically shaking the solution in a water-bath at 75 ° for 15 rain. After cooling, the aggregate is centrifuged for 5 rain at 180 G, the supernatant is discarded and the aggregate resuspended in a volume of sterile physiological saline equal to that of the supernatant. By this method, 70-80 per cent of Tc 99m is complexed with protein and 90-95 per cent of the complex is recovered in the form of aggregate. Particle sizes range from 50-100 # in diameter.
Discussion Method The main modification to the method described by STERN et al. cs), necessitated by the comparatively large volume in which the Tc 99m is eluated from the Mo 99 column, was to increase tenfold the concentrations both of ferric chloride and of ascorbie acid added. With lower concentrations of these reagents, the proportion of Tc 99m complexed fell rapidly. Temperature, pH and albumin concentration are critical in producing uniform aggregates of the required size: with higher temperatures or values of pH, or with lower albumin concentration, bulky non-uniform aggregates were obtained. Occasionally, for reasons yet obscure, a few massive particles may appear among aggregates that are largely of the correct size. These may be removed by filtration through stainless steel grids of 150 kt mesh.
Uptake in lungs Figures 1 and 2 show anterior and posterior lung scintiscans obtained with 2 mc of Tc99m-labelled albumin aggregates in a patient with partial collapse of the right lower lobe and a tumour in the left mid-zone, seen in the radiograph (Fig. 3). Particles too small to be trapped in the lungs are taken up by the liver, and the absence of activity over the liver (Fig. 1) is an indication of the uniformity of particle size produced by this technique. The value of Tc99m-labelled aggregates for studies
of lung pathology is emphasized by comparison of the anterior and posterior seanss. The lower left lung field, obscured by the heart in the anterior view is; visualized in the posterior view and shows an even distribution of isotope. The indentation of the lateral border of the lung by the tumour in the left mid-zone, seen in the anterior view, does not appear in the posterior view, thus indicating that the tumour is located anteriorly. These precise anatomical interpretations are made possible by the low y-energy of the isotope.
Acknowledgments--We are grateful to Professor D. W. SMITHER3, Director of the Radiotherapy Department, for encouraging us to develop isotope techniques for studying lung pathology. We also wish to thank Miss ROSE~ARY FRENCH of the Physics Department for her help with the preparation of Tc 99m and Dr. W D'A. MAYCOCKof the Lister Institute for supplies of dried human serum albumin. MYFANWY M . GWYTHER E. O . FIELD
Radiotherapy Research Unit Royal Marsden Hospital Sutton Surrey References l. HARPER P. V., LATHROP K. A., JIMINEZ F., FINE R. and GOTTSCHALKA. Radiology 85, 101 (1965). 2. STERN H. S., ZOLLE I. and McAFEE J. G. Int. J. appl. Radiat. Isotopes 16, 283 (1965). 3. WAGNERH. N., SAmSTOND. C., IIo M., McAFEE J. G., MEYERJ. K. and LANGANJ. K. J. Am. med. A~s. 187, 601 (1964).
International Journal of Applied Radiation and Isotopes. 1966, Vol. 17, pp. 486487. Pergamon Press Ltd. Printed in Northern Ireland
Do-It-Yourself External Standardization for a Liquid Scintillation Counter (Received 25 January 1966) THE external-standard method for estimating efficiency in liquid scintillation counting has been widely accepted in recent years by instrument manufacturers and presumably by users also. This note describes a simple alteration which allows external standardization to be used with all Packard counters up to the 314 series. The only modification needed is the drilling of a hole in the baseplate of the
Technical notes
487
TABLE 1. Effect of water quenching on Ba 1~ count
Water, (ml) 0 0.5 1.0
Ba 1~ c p m 3553 2454 1460
Relative counting rate 1.0 0.69 0.41
Efficiency for counting H s in same sample (approximate) o/ /O
Relative counting efficiency 1.0 0.66 0.39
11 7.2 4.3
H z was counted in a window from 40 to 500 pulse-height units at amplifier gain 250. Ba xzs was counted in a window from 500 to I000 pulse-height units at amplifier gain 100.
sample changing assembly vertically above the position of the vial being counted. A 7-source may be placed through this hole on to the top of the detector unit when required. W h e n a vial is being counted there is virtually no y-ray shielding between it and a source so placed. Hence the external source generates counts in the sample after the usual fashion of an external standard. T h e source used consists of about 10/zc of Ba laa sealed with epoxy resin into a short blind hole at one end of a brass plug about 2"5 cm long by 1"2 cm dia. This plug is a loose fit into the hole in the baseplate. Ba 1as was chosen chiefly because of its long half-life and moderate y-energy. T h e amount used (which could readily be increased if desired) will produce about 3000 cpm in a counting window suitable for the purpose when a 10 mi sample is present. T h e procedure for using this device is convenient although not fully automatic. T h e whole series of samples is counted in the ordinary way, the source is then placed in position and the series re-counted. Both series of counts are done with one of the instrument's channels set to suit the high-energy end of the Compton spectrum generated by the external source. T h e difference between the first and second count in this channel is then, after calibration, an indication of the efficiency of the first count, as the following example shows (see T a b l e 1). The sample was 10 ml of BRAY(1) scintillator in a polythene vial. W a t e r was added with withdrawal after mixing to preserve constant volume. T h e reproducibility of the scheme was investigated by making up ten identical blanks each comprising 10.0ml of BRAY scintillator in a polythene vial (Packard brand) and counting each one with the external source in place. T h e m e a n count and standard deviation were 9684 ± 136 i.e. 1.4~o (expected value 1"0 ~o).
By way of comparison the counts generated in the same ten blanks by the built-in external-standardizing system of a new instrument were observed; each vial was counted to 15,000 counts and the group often was passed through the instrument three times. T h e observed standard deviation of the count on each of the three passes was 0 . 8 ~ , 1"65~o and 1 . 6 0 ~ (expected value 0"8 ~o). Thus (assuming a normal distribution) the 9 5 ~ confidence interval for an observed count with the home-made system is -4-2.8~/oo. T h e corresponding confidence interval for an estimated HS-counting efficiency of 10~o is about ±0"4~o efficiency. T h e ease with which this modification was m a d e was due to the particular design of the loading system, which allowed a source to be placed outside the detector and clear of the loading mechanism, yet in a position to irradiate the sample being counted. A similar method could be adopted for most other designs of detector with a little ingenuity; it is only necessary to position the external source so that it irradiates the sample whilst being counted. T h e procedure has drawbacks common to most arrangements for external standardization. The calibration is upset by change of sample volume or marked change of composition (such as by addition of a gel to allow suspension counting). It is also upset by change of the type of vial used. Nevertheless it remains a usefuI alternative to the addition of an internal standard.
Prince Henry Hospital Little bay, N.S. W. Australia
D. P m x
Reference
1. BRAv G. A. Analyt. Biochem. 1~ 279 (1960).
486
for 5 rain, any uncomplexed Tc 99m is removed by passing the solution through a column orion-exchange resin 1 cm in diameter, containing 4 g of Amberlite IRA-400(C1), overlaid with 1 g ofDowex 1 × 2 (C1) 50-100 mesh, prepared as described by STEV~ et al. ta) both resins being of analytical grade. The first 2 ml of eluate are discarded. The column is then washed with physiological saline and to the subsequent 15 ml of eluate are added 1 ml of 1% albumin in physiological saline, as "carrier" and the pH is adjusted to 4.5-5 with N-NaOH. 1 ml of 0"2 M acetate buffer pH 5"0 is added and the protein solution is re-sterilized by filtration through sterile 0.45 # porosity "Millipore"membranes. Thealbumin is then aggregated by mechanically shaking the solution in a water-bath at 75 ° for 15 rain. After cooling, the aggregate is centrifuged for 5 rain at 180 G, the supernatant is discarded and the aggregate resuspended in a volume of sterile physiological saline equal to that of the supernatant. By this method, 70-80 per cent of Tc 99m is complexed with protein and 90-95 per cent of the complex is recovered in the form of aggregate. Particle sizes range from 50-100 # in diameter.
Discussion Method The main modification to the method described by STERN et al. cs), necessitated by the comparatively large volume in which the Tc 99m is eluated from the Mo 99 column, was to increase tenfold the concentrations both of ferric chloride and of ascorbie acid added. With lower concentrations of these reagents, the proportion of Tc 99m complexed fell rapidly. Temperature, pH and albumin concentration are critical in producing uniform aggregates of the required size: with higher temperatures or values of pH, or with lower albumin concentration, bulky non-uniform aggregates were obtained. Occasionally, for reasons yet obscure, a few massive particles may appear among aggregates that are largely of the correct size. These may be removed by filtration through stainless steel grids of 150 kt mesh.
Uptake in lungs Figures 1 and 2 show anterior and posterior lung scintiscans obtained with 2 mc of Tc99m-labelled albumin aggregates in a patient with partial collapse of the right lower lobe and a tumour in the left mid-zone, seen in the radiograph (Fig. 3). Particles too small to be trapped in the lungs are taken up by the liver, and the absence of activity over the liver (Fig. 1) is an indication of the uniformity of particle size produced by this technique. The value of Tc99m-labelled aggregates for studies
of lung pathology is emphasized by comparison of the anterior and posterior seanss. The lower left lung field, obscured by the heart in the anterior view is; visualized in the posterior view and shows an even distribution of isotope. The indentation of the lateral border of the lung by the tumour in the left mid-zone, seen in the anterior view, does not appear in the posterior view, thus indicating that the tumour is located anteriorly. These precise anatomical interpretations are made possible by the low y-energy of the isotope.
Acknowledgments--We are grateful to Professor D. W. SMITHER3, Director of the Radiotherapy Department, for encouraging us to develop isotope techniques for studying lung pathology. We also wish to thank Miss ROSE~ARY FRENCH of the Physics Department for her help with the preparation of Tc 99m and Dr. W D'A. MAYCOCKof the Lister Institute for supplies of dried human serum albumin. MYFANWY M . GWYTHER E. O . FIELD
Radiotherapy Research Unit Royal Marsden Hospital Sutton Surrey References l. HARPER P. V., LATHROP K. A., JIMINEZ F., FINE R. and GOTTSCHALKA. Radiology 85, 101 (1965). 2. STERN H. S., ZOLLE I. and McAFEE J. G. Int. J. appl. Radiat. Isotopes 16, 283 (1965). 3. WAGNERH. N., SAmSTOND. C., IIo M., McAFEE J. G., MEYERJ. K. and LANGANJ. K. J. Am. med. A~s. 187, 601 (1964).
International Journal of Applied Radiation and Isotopes. 1966, Vol. 17, pp. 486487. Pergamon Press Ltd. Printed in Northern Ireland
Do-It-Yourself External Standardization for a Liquid Scintillation Counter (Received 25 January 1966) THE external-standard method for estimating efficiency in liquid scintillation counting has been widely accepted in recent years by instrument manufacturers and presumably by users also. This note describes a simple alteration which allows external standardization to be used with all Packard counters up to the 314 series. The only modification needed is the drilling of a hole in the baseplate of the
Technical notes
487
TABLE 1. Effect of water quenching on Ba 1~ count
Water, (ml) 0 0.5 1.0
Ba 1~ c p m 3553 2454 1460
Relative counting rate 1.0 0.69 0.41
Efficiency for counting H s in same sample (approximate) o/ /O
Relative counting efficiency 1.0 0.66 0.39
11 7.2 4.3
H z was counted in a window from 40 to 500 pulse-height units at amplifier gain 250. Ba xzs was counted in a window from 500 to I000 pulse-height units at amplifier gain 100.
sample changing assembly vertically above the position of the vial being counted. A 7-source may be placed through this hole on to the top of the detector unit when required. W h e n a vial is being counted there is virtually no y-ray shielding between it and a source so placed. Hence the external source generates counts in the sample after the usual fashion of an external standard. T h e source used consists of about 10/zc of Ba laa sealed with epoxy resin into a short blind hole at one end of a brass plug about 2"5 cm long by 1"2 cm dia. This plug is a loose fit into the hole in the baseplate. Ba 1as was chosen chiefly because of its long half-life and moderate y-energy. T h e amount used (which could readily be increased if desired) will produce about 3000 cpm in a counting window suitable for the purpose when a 10 mi sample is present. T h e procedure for using this device is convenient although not fully automatic. T h e whole series of samples is counted in the ordinary way, the source is then placed in position and the series re-counted. Both series of counts are done with one of the instrument's channels set to suit the high-energy end of the Compton spectrum generated by the external source. T h e difference between the first and second count in this channel is then, after calibration, an indication of the efficiency of the first count, as the following example shows (see T a b l e 1). The sample was 10 ml of BRAY(1) scintillator in a polythene vial. W a t e r was added with withdrawal after mixing to preserve constant volume. T h e reproducibility of the scheme was investigated by making up ten identical blanks each comprising 10.0ml of BRAY scintillator in a polythene vial (Packard brand) and counting each one with the external source in place. T h e m e a n count and standard deviation were 9684 ± 136 i.e. 1.4~o (expected value 1"0 ~o).
By way of comparison the counts generated in the same ten blanks by the built-in external-standardizing system of a new instrument were observed; each vial was counted to 15,000 counts and the group often was passed through the instrument three times. T h e observed standard deviation of the count on each of the three passes was 0 . 8 ~ , 1"65~o and 1 . 6 0 ~ (expected value 0"8 ~o). Thus (assuming a normal distribution) the 9 5 ~ confidence interval for an observed count with the home-made system is -4-2.8~/oo. T h e corresponding confidence interval for an estimated HS-counting efficiency of 10~o is about ±0"4~o efficiency. T h e ease with which this modification was m a d e was due to the particular design of the loading system, which allowed a source to be placed outside the detector and clear of the loading mechanism, yet in a position to irradiate the sample being counted. A similar method could be adopted for most other designs of detector with a little ingenuity; it is only necessary to position the external source so that it irradiates the sample whilst being counted. T h e procedure has drawbacks common to most arrangements for external standardization. The calibration is upset by change of sample volume or marked change of composition (such as by addition of a gel to allow suspension counting). It is also upset by change of the type of vial used. Nevertheless it remains a usefuI alternative to the addition of an internal standard.
Prince Henry Hospital Little bay, N.S. W. Australia
D. P m x
Reference
1. BRAv G. A. Analyt. Biochem. 1~ 279 (1960).