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Liquid scintillation counting in nuclear medicine
Liquid Scintillation C o u n t i n g in Nuclear Medicine E d w i n D. Bransome, Jr. M a n y of the radionuclides used in nuclear medicine can be measured by liquid scintillation (LS) counting, and the technique is the only practical approach to counting low-energy/]emissions. This review is intended to be a brief exposition of the capabilities of LS counting and of some precautions that should be observed. The liquid scintillation process is basically simple: Electrons a n d / o r photons from radioactive disintegrations are absorbed by a solvent, w i t h the resulting energy being transferred to the 7r electrons of an organic scintillator. The scintillator emits fluorescence in its return to its leastexcited ground state; the emitted photoelectron spectrum is proportionate to the energy of the photons emitted in the original radioactive disintegration. The photons are absorbed by the cathode of a photomultiplier tube, w i t h the resulting pulse in voltage being amplified and resolved in nanoseconds before being recorded as a count. In modern LS counters, coincidence circuitry eliminates much of the background inherent in the counting system. Errors that must be avoided or assessed include quenching: impurity or chemical quenching that increases w i t h the atomic number of the impurities and color quenching that distorts the photoelectron spectrum in inverse proportion to the e m i t t e d energy. Use of quench correction curves and methods used to determine the loss of efficiency or pulse height are
necessary in LS counting if there is any significant variation in sample preparation. If there are t w o radionuclides to be counted in a sample, a quench correction must always be applied because the pulse height shifts to a l o w e r energy region w h e n e v e r quenching occurs. Thus the spillover or overlap of t w o isotope spectra w i l l vary w i t h the energy of the radionuclides and the degree and type of quenching. None of the three methods of determining the extent of quenching (internal standardization, sample channels ratios, or external standardization) is ideal for all situations or is by any means foolproof. For example, if the scintillator solution and sample are not mixed to near homogeneity, counts may be lost because of self-absorption. Excitation of the scintillation solvent is decreased and cannot adequately be comprehended by any of the quench correction techniques. Because most scintillation solvents will tolerate only minimal amounts of aqueous samples of the sort usually of interest in nuclear medicine, oxygen flask combustion (for 14C or 3H), hydrolysis, addition of surfactants (solubilizers), or thixotropic gels--all relatively expensive procedures--may have to be used for sample preparation. The cost of sample preparation is frequently the only significant unfavorable feature of LS counting w h e n compared to g a m m a (Nalcrystal) scintillation counting.
HE USE of liquid scintillation counting in nuclear medicine laboratories has increased significantly in recent years as the use of radio-immunoassays and radioligand (protein-binding) assays of drugs, hormones, and other biologically important molecules have proliferated. Tritium (3 H) labeling of antigens or ligands displaced from binding proteins has become more common, inasmuch as labeling small molecules with 12sI or 13~I is often impossible. Liquid scintillation counting also lends itself to routine quantitation of radionuclides that decay by gamma emission or by electron capture.
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From the Medical College of Georgia, Augusta, Ga. Supported in part by NIH grant CA 12455. Edwin D. Bransome, Jr., M.D.: Professor o f Medicine and Chief of the Division of Metabolic and Endocrine Disease, Medical College of Georgia, Augusta, Ga. 01973 by Grune & Stratton, Inc. Seminars in Nuclear Medicine, Vol. 3, No. 4 (October), 1973
389
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EDWIN D. BRANSOME, JR.
Efficiencies are high, often greater than in well (NaI-crystal) counters. The resolution of photoelectron spectra, although less than in 3' counters, is adequate for the simultaneous counting of many isotope pairs, including i2Sl and 131I (Fig. 1). Excellent discussions of the physics of the processes outlined in the abstract are available in compilations of recent symposia 2-4 on liquid scintillation counting. These are volumes that anyone really interested in LS counting should take the trouble to look at. The principles of LS counting instrumentation are reviewed by Birks, s and the features of specific LS counters, including equipment currently marketed, have been presented in historical perspective by Rapkin. 6'7 CAPABILITIES
OF
LS C O U N T I N G
Although the principal use of LS counting has involved the measurement offl emissions, other charged particles such as a particles and protons can also be counted.
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Fig. 1. Spectra of 12Sl and 1311 obtained by counting 2% windows in a Beckman LS-150 with the amplifier gain set for counting 3H. The curves for each isotope were obtained under four conditions (A-D) on replicate samples containing 1311 (33,500 DPM) or 1251 (220,000 DPM); counting efficiencies were obtained in a wide (0-1000 division) window. Condition
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Radionuclides that decay by electron capture and by 7 emission can be counted with high detection efficiency. Radioactive decay of some isotopes (e.g., 12sI) creates a vacancy in one of the inner electron shells of an atom (K, L, or M); the shell is then filled by an outer electron and the release of X-rays and low-energy (Auger) electrons results. If energetic enough, K X-rays, L X-rays, and Auger electrons are all capable of exciting dissolved scintillators. Quenching by impurities of the photoelectron spectra from K and L X-ray interactions is similar to that seen in ~ counting, but the photoelectron spectra and therefore quench correction curves may be somewhat different than those for/3 emissions of similar energy. 8 The interaction of 3' rays with scintillators s is principally a result of the Compton effect, a yield of electrons with 0 to 75% of the 3' energy. The photoelectric effect of 7 interaction usually does not occur above 30 keV in liquid scintillators, but may be evident at higher 7 energies in the vial walls or if the scintillator solution is loaded with heavy elements. Ashcroft 9 has suggested that addition to the scintillation mixture of a compound of high electron density (e.g., tetrabutyl tin, tetrabutyl lead) will increase the counting efficiency of 125I. Ting 1~ has recently suggested that ~2Sl-labeled samples in microcentrifuge tubes suspended in a scintillation mixture containing 5% tetrabutyl lead can be counted at 70 percent efficiency. Both Ashcroft and Ting have ascribed their results (not fully documented in the latter case) to an attenuation of the low-energy 3' of 12s I, so that fewer photons escape and more interact. The fact that this approach is not suitable for 3' emitters of higher energy suggests that their interpretation may be incorrect. The 70% detection efficiency reported is most probably a result of attenuation of the ~2Sl K X-rays and of the photoelectric effect. Since the effects of quenching and self-absorption of the photoelectric effect have not been worked out and since the counting efficiencies are no better than can be obtained in a normal liquid scintillation mixture, ~ I would suggest avoiding this approach. With high-energy 3' emissions, there may be another effect of interaction-the Cerenkov effect (see below)-if Compton electron energies are above 256 keV. Pair production, which occurs at 3' energies above 1.02 meV, is not a phenomenon occurring in the usual situation of LS counting. SAMPLE P R E P A R A T I O N
Each of the components of the sample being counted by liquid scintillation plays a role in determining the counting efficiency.
Scintillation Solvent An alkylbenzene or p-dioxane (lO0 g naphthalene/liter) is used as the scintillation solvent. Of the alkylbenzenes, toluene is usually chosen because of its high scintillation yield, intermediate susceptibility to impurity quenching, and relatively low price.
Primary Scintillator An aromatic compound with a high fluorescence quantum efficiency is used as the primary scintillator. Those in common use are listed in Table 1 along with their lowest concentrations for optimum efficiency in toluene as determined by Birks and Poullis. n The absorption and emission spectra of the scintillators are also important, the
EDWIN D. BRANSOME, JR.
392
Table 1. Common Organic Scintillators
Primary PPO PBO PBD butyl PBD BBOT BIBUQ TP
2,5-diphenvloxazole 7 g/liter* 5-phenyl-2-(4-biphenylyl)-oxazole 7.5 g/liter 2-phenyl, 5-(4-biphenylyl)-l,3,4-oxadiazole 12 g/liter 2.(4t-tert-butylphenyl), 5-(4"-biph enylyl)- 1,3,4-oxadiazole 12 g/liter 2,5-bis(5'-tert-butyl-2-benzoxazolyl)-thiophene 8 g/liter 4,4-bis(2-butyloctyloxy-p-quaterphenyl) 24 g/liter p-terphenyl 7 g/liter Secondary
POPOP dimethyl POPOP BBO PBBO bis-MSB NPO
1,4-bis [2-(5-phenyloxazolyl)]-benzene 1 g/liter* 1,4-bis [2-(4-methyl-5-phenyloxazolyl)] -benzene 0.02 g/liter 2,5-di(4-biphenylyl)-oxazole 0.02 g/liter 2-(4-biphenylyl)-6-phenylbenzoxazole 1 g/liter p-bis (o-methylstyryl)-benzene 0.2 g/liter 2-(1-naphthyl), 5-phenyl-oxazole 1 g/liter
*Optimum concentrations in toluene. Taken in part from the data of Birks and Poullis. l l
former for the solvent-to-scintillator energy transfer and the latter for the match of the emitted fluorescence spectrum to the spectral response of the photomultiplier tubes of the scintillation counter.
Secondary Scintillator With some primary scintillators, which emit a fluorescence spectrum mismatched to the spectral response of the phototubes of the specific scintillation counter being used (e.g., most of the spectrum of p-terphenyl is absorbed by glass vials), it is necessary to add another scintillator that will shift the spectrum toward the optimum phototube response. Secondary scintillators in common use are listed in Table 1. The experiments of Birks and Poullis ~ suggest that BBO, PBBO, POPOP, and bis-MSB have the best overall characteristics.
Nature of the Radiation If t3 or Compton electrons are at or above a 0.l-meV energy endpoint, counting efficiency is directly proportional to photon energy; for lower energy electrons and for other ionizing radiation (a, 3', protons, etc.) there is less efficiency of detection per keV of Ema x .
Scintillation Counting Vial The portion of a fluorescence spectrum presented to the photonmltiplier tube is partially dependent on the band-pass characteristics of the vial wall. Polyethylene, for example, will transmit considerable light over the uv range, whereas glass will not.
Physical and Chemical Properties of Sample The physical and chemical properties of a sample are of great importance. Will a sample (although labeled radioisotopically) be a significant impurity quencher or a color quencher? If the sample is not homogeneously mixed with the solvent, or if the sample is precipitated (trapped) onto a solid support, there may be significant problems in the assessment of the absolute radioactivity of the sample.
LIQUID S C I N T I L L A T I O N COUNTING
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Most of the important considerations of sample preparation for LS counting can conveniently be reviewed in chapters 16 to 27 of reference 2 and in reference 12. It should be emphasized that for calculations of the activity of a sample to be reliable, the radioactivity must be uniformly distributed in the sample.
Emulsion Counting Translucent emulsions with microscopic micelles ~2'1a may be counted as easily as true solutions provided some precautions are observed. If two isotopes are being counted, particularly if one of the isotope pair is 3H or 12sI, they must be in the same phase. This might seem to be a trivial problem, since emulsion counting is of aqueous samples, but it is not-there may be differential extraction of the labeled solutes into the organic solvent. Absorption (of the lower energy isotope in particular) onto the vial surface may greatly alter detection efficiency)4 If the Emax of an isotope is equivalent to or greater than that of ~4C and a commercial solubilizer of aqueous samples has been included in the sample, the surfactant itself may fluoresce in response to radioactivity. This renders quench correction curves derived from sealed commercial standards invalid. 1s:6 All of these proscriptions may be summarized in the cardinal and frequently neglected general rule for scintillation counting: standards must be of the same geometry as the unknown samples.
Counting on Solid Supports The problems of inhomogeneity and geometry are even greater. If samples are first collected on Solid supports, put in a scintillation vial, and covered with solvent, etc., the first question to be asked is whether the sample is either completely or partly eluted from the support. Self-absorption (attenuation of the particle path) reduces the chance of interaction with the scintillation solvent and is very significant, especially for low-energy/3 emissions 17:8 (Table 2). None of the quench correction techniques, which are reviewed below, can estimate adequately the decrease in efficiency when such heterogenous systems are employed.
QUENCH CORRECTION Quench correction techniques by Neary and Budd, 19 Peng, 2~ Cavanaugh, 21 and Wang 22 have been thoroughly reviewed in reference 2, and those by Klein and Eisler 23 and Ten Haaf~ are covered in references 3 and 4, respectively. Table 2. Effects of Sample Geometry on Relative Detection Efficiency* Relative Efficiency (%)
Isotope
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*The CPM in a 0.1-ml aqueous solution of 1251-Nal, 1311-Nal, 14C-adenine, or 3H-cycJic AMP were taken as 1.00 and compared to replicates air-dried onto discs 20 mm in diameter. Liquid scintillation counting was with a Beckman LS-150 system with a wide channel encompassing the total spectrum of each isotope. The aqueous samples were counted in toluene 7.0 g/liter PPO (2,5-diphenyloxazole), 10% Biosolv BBS-3 (Beckman) solubilizer. The samples dried onto the filters were counted in totuene-PPO without Biosolv.
394
EDWIN D. BRANSOME, JR.
There is recent agreement that whereas quench correction curves for 3 H (Ema x = ! 8 keV) are not significantly different whether the quenching is from impurity or color, the curves diverge significantly if ~4C (Emax = 156 keV) or other more energetic nuclides are counted, particularly when samples are highly quenched. It may therefore be quite misleading to apply a quench correction curve obtained with commercially available sealed quenched standards to unknown samples of different composition. There are three techniques of quench correction in general use: Internal Standardization Internal standardization, or spiking, the addition of a Standard of known radioactivity to each sample, is the most accurate of any method if the addition of the known standard to the sample is careful and reproducible. Differences in the effects of impurity and color quenching (which may be important with 14C and more energetic nuclides) are obviated. The samples must be homogeneous, and the standard must be similar chemically to the sample solute if there is any question of an incomplete solution. Handling errors in adding the standard will be significant if the procedure is careless; ideally this procedure should be done at least in duplicate. Once a sample is spiked, it cannot be recounted. The procedure is extremely time-consuming. In double-label experiments, it is a laborious and statistically uncertain approach. Hendee et al. ~s have recently emphasized the problem of homogeneity in internal standardization. They found that addition of even very small volumes of 3H-H20 (a quencher) to toluene- or dioxane-based liquid scintillation mixtures resulted in a progressive decrease of 3H counting efficiency. In other words, internal standardization should be performed by the addition of a known amount of a labeled standard that will not act as a nonhomogeneous impurity. Ideally, labeled scintillation solvent should be used, but if radionuclides other than 3H or 14C are to be counted, this is impossible. Then the effect of adding the proposed volume of standard to the samples must be determined experimentally. Sample Channels Ratios An isotope is counted in two channels. One should be a wide window, and the other should encompass the lower energy range of the spectrum. As an isotope is quenched, a greater proportion of the total cpm will appear in the second window. Data may be obtained in two channels simultaneously; thus counting the sample only once is sufficient: The Sample itself is not altered. This approach is accurate for moderate and high count rates of single isotopes that are quenched slightly to moderately, and it is independent of sample volume over a wide range. One series of quenched standards will correct for both sorts of quenching of 3H. Nonhomogeneous samples (in suspension, in emulsion, on solid supports) may be quench-corrected, as long as standard curves are derived from known samples of the same composition and geometry. The procedure is inaccurate, however, for highly quenched samples or for samples with 10w count rates. Strov.g color quenching of ~4C or more energetic isotopes is not adequately corrected for. With two overlapping isotopes, corrections for spillover must first be made, with a significant additional statistical error. External Standard Channels Ratios This procedure, involving exposure of the unknown sample to an external 7 source, takes advantage of the secondary Compton electrons generated within the sample and
LIOUID SCINTILLATION COUNTING
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the resulting broad fluorescence spectrum. Modern instrumentation monitors and subtracts sample cpm from two separate channels and then sets the proper Compton cpm. Single-channel external standardization is undesirable, inasmuch as the dependence of cpm on sample volume is considerable. External standardization can be applied to samples of low activity. Statistically it is the most suitable for double-isotope quench correction, but it is the least accurate for highly quenched samples in which differences between the photoelectron spectra of sample and Compton electrons are magnified. Samples must be homogeneous. This technique is not applicable to samples in visible suspension, on solid supports, etc, CH EM I LUMI N ESCE NCE
One of the greatest concerns in utilizing LS counting, particularly if the sample radioactivity is low, is the background count rate: a combination of decay of radioisotopes in the system (e.g., 4~ in scintillation vial glass), cosmic radiation, thermal activity in the photomultiplier tubes, and optical cross-talk between the photomultipliers. 26 There may also be low-energy fluorescence as a result of chemical reactions 27 in the scintillation vial: chemiluminescence may be encountered if samples have been subjected to alkali base and the pH of the scintillation mixture is high, or if peroxides are present. The answers to these problems are relatively simple: lower the pH by acidification, or for peroxides add the enzyme catalaSe, which is effective even in the presence of toluene, z8,29 Scales 3~ has recently reviewed some more sporadic causes of chemiluminescence in LS counting: the reaction of vial caps of a particular composition to uv light or temperature, an impurity in some batches of Triton X-100 (a Rohm and Haas product used in emulsion counting), and an impurity accumulating as a breakdown product of reagent-grade dioxane. Obviously, if chemiluminescence is detected it can be avoided. Since chemiluminescence due to light or temperature activation wanes quickly, rapidly decreasing count rates in the same sample counted minutes or hours apart should be sufficient to alert one. When the luminescence is longer lived, the difference of the luminescence photoelectron spectrum from that of the radionuclide of interest may serve as an indicator, z9 Several LS counters of recent manufacture incorporate a delay line that allows monitoring of noncoincident single-photon events and thus of chemiluminescence. 7 DOUBLE-LABEL COUNTING
In most double-label experiments, two counting channels are used and the photoelectron spectra are only partially separated. In liquid scintillation counting there is too great a loss of detection efficiency if both isotopes are eliminated entirely from the converse channel: the photoelectron spectra of the fluorescence emitted from the organic scintillators are too broad. Indeed, if the photon energies are not .videly separated, it is statistically impractical to eliminate the spillover of either isotope from the other channel. The extent of quenching in a homogeneous solution of sample and phosphor in solvent will cause pulse-height shifts of fluorescence spectra to lower photoelectron energies as an inverse function of the energy of the emitted photons. Quenching in samples will thus affect the detection efficiencies of two isotopes variably, the spectral shift of the lower energy isotope being greater. There is then no single set of optimum instrument settings for specific isotope pairs. Correction of the overlap of radioactivities must be carried out before quench correction. Two questions that deserve attention are: How can the cpm of one isotope
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EDWIN D. BRANSOME, JR.
best be separated mathematically from the cpm of another? What are the optimum instrument settings for a specific isotope pair? A more extensive discussion of the answers than can be provided here may be found in references 1,31, and 32. One approach to double-isotope counting is to continue to use the optimum discriminator settings for each of the two isotopes. Since each channel will count contributions from both isotopes, simultaneous equations will be necessary for the calculation of the absolute radioactivity of each sample. Both isotopes are counted at maximum efficiency unless there is severe quenching, but a considerable accumulation of statistical error results from the repeated application of calculated efficiencies. If possible, the lower energy isotope should be eliminated from the channel used to count the higher energy isotope. If H is the isotope of higher energy and L the lower, then (with background subtracted) dpm H -
cpm channel 1 efficiency of H in channel 1
dpm L = (cpm channel 2 ) - (cpm channel 1 • efficiency of H in channel 2) efficiency of L in channel 2
(1) (2)
The cumulative error is greatly decreased by such a simplification. In our work, analysis of the variance occasioned by the use of simultaneous equations often results in unacceptably large errors, particularly when count rates for one or both isotopes are low and the overlap is large. There have been several suggestions in the recent literature that simultaneous equations may reliably differentiate two isotopes with considerable spectral overlap. Such advice is dangerous; if simultaneous equations are employed, exclusion of the lower energy isotope from the channel used to count the higher energy isotope should still be maximal. The spillover fraction (the percentage of the net cpm of an isotope counted in its own channel that falls into the channel used for the other isotope) is a constant in gamma counting, but will of course increase in liquid scintillation counting as the spectrum of isotope H is quenched and shifted toward lower energies. Progressively more H cpm fall in channel 2. In Beckman scintillation counters with automatic quench correction (A.Q.C.), this fraction can be held relatively constant, 22,33,a4 but in others a quench correction curve for this fraction must be obtained, just as it is obtained for the detection efficiencies of the two isotopes in 1heir optimum channels (see Fig. 2). Occasionally, workers have attempted to use simultaneous equations to solve for the activities of three isotopes in the same sample, using three or more channels. The statistical errors implicit in such procedures are unacceptably large. This concern about statistical variation is quite practical and is particularly appropriate for double-label counting, since the standard deviation of the determination of the radioactivity of a sample accumulates as the square root of the sums of the squared standard deviations of each component measurement (cpm, background, spillover, and efficiericy). There are good up-to-date discussions of this aspect of data handling in LS counting by Spratt as and Assailly et all 36 in reference 4. It is Sometimes possible to separate one overlapping isotope from another by combining different counting methods: 131I may be separated from 14C and aSS, while 12sI may be separated from 3H, by counting {he samples in a gamma counter and then by liquid scintillation alone. It is important that 13~1 or 1251 efficiency be determined
LIQUID SCINTILLATION
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in both counters so that an accurate spillover curve of either iodine isotope into the 14C, 3s S, or 3H channels can be calculated from standards. Energetic/3 emissions may also be counted in solution in the absence of phosphors by virtue of the Cerenkov effect. 37 The Cerenkov threshold in standard scintillation mixtures is approximately 256 keV; 32p and 131I may therefore be counted at reasonable efficiencies (without any impurity quenching), but Cerenkov photoelectron spectra are broad-band and are poorly resolved, so that double-isotope counting is not practical.
Instrument Settings The method of selecting discriminator settings for the single-channel counting of a single radionuclide is straightforward. Since background cpm increase with channel width, the upper potentiometer setting should be decreased to the point where the count rate of a sample of the radionuclide begins to become significantly diminished. The lower discriminator should be set close to zero and raised only if there is a major contribution of instrument noise to background in the low-energy range, a problem not significant in instruments of recent manufacture. In double-label counting there is an obvious conflict between desires for minimum spillover and maximum efficiency. Mathematical solutions to the determination of optimum settings have been proposed; the most satisfactory is that of Davies and Deterding, 38 but it is quite complex. We have found that graphic methods are suf-
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EDWIN D. BRANSOME, JR.
ficient for double-isotope channel selection: a simple plot of upper discriminator settings (with the lower discriminator fixed) versus percent of the total cpm of a standard in the channel is appropriate for instruments with logarithmic or quasilogarithmic amplification of photomultiplier o u t p u t ) ' 3 2 For LS spectrometers with linear amplification, the Engberg plot, introduced by Kobayashi and Maudsley 31 will reveal the regions of optimum settings (see also references 1 and 32). D A T A REDUCTION
The subject of automatic data reduction is avoided in this review for several reasons. The foregoing paragraphs have been written in agreement with Spratt's contention that "excellent data handling is often performed on inappropriate data." In references 2-5 recommended above as good source materials for liquid scintillation counting, there are a number of discussions of off-line and on-line data reduction with large digital computers, minicomputers, and desk-top computer calculators. 33'34 Spratt's philosophical discussion3s is an excellent introduction. There are also discussions of computer applications elsewhere in this seminar. Computer programs have the advantage that they can include explicit or implicit error analysis (unacceptable deviations of data may be flagged). Description of specific programs here seems inappropriate, since they are machine-dependent and the array of computation equipment is rapidly changing. Indeed, most manufacturers of LS counters are offering peripherals and software for LS counting data reduction. REFERENCES
1. Bransome ED Jr, Sharpe SE II1: Mea- tion counting of I-125. Clinical brief 6. Fullersurement of 131I and 125I by liquid scintilla- ton, Calif., Beckman Scientific Instruments tion counting. Anal Biochem 49:343, 1972 Div., 1973 2. Bransome ED Jr (ed): The Current Status 11. Birks JB, Poullis GC: Liquid scintillators, of Liquid Scintillation Counting. New York, in Crook MA, Johnson P, Scales B (eds): Grune & Stratton, 1970 Liquid Scintillation Counting, p 1 3. Horrocks DL, Peng ET (eds): Organic 12. Turner JC: Sample Preparation for Liquid Scintillators and Liquid Scintillation Counting. Scintillation Counting, Review 6. The RadioNew York, Academic Press, 1971 chemical Center, Amersham, England, 1967; 4. Crook MA, Johnson P, Scales B (eds): reprinted in Wang U (ed): Handbook of RadioLiquid Scintillation Counting, vols. l and 2. active Nuclides. The Chemical Rubber Co., London, Heyden and Son, 1972 Cleveland, 1969, p 256 5. Birks JB: The Theory and Practice of 13. Greene RC: Heterogeneous systems: SusScintillation Counting. New York, Macmillan, pensions, in Bransome ED Jr (ed): reference 2, 1964 p 189 6. Rapkin E: Development of the modern 14 Litt GJ, Carter H: Sample absorption scintillation counter, in Bransome ED Jr (ed): problems in liquid scintillation counting, in reference 2, p 45 Bransome ED Jr (ed): reference 2, p 156 7. Rapkin E: in Crook MA, Johnson P, Scales 15. Sharpe SE III, Bransome ED Jr: The B (eds): Liquid Scintillation Counting, p 61 surfactant Biosolv-BBS-3 as a scintillator in 8. Horrocks DL" Obtaining the possible max- liquid scintillation counting. Anal Biochem (in imum of 90 percent efficiency for counting of press) 55Fe in liquid scintillator solutions, lnt J Appl 16. Sharpe SE I11, Bransome ED Jr: SurRadiat lsot 22:258, 1971 factants behave as scintillators in liquid scintil9. Ashcroft J: Gamma counting of iodine- lation counting, in: Proceedings of the Inter125 using a metal-loaded liquid scintillator. national Congress on Liquid Scintillation Anal Biochem 37:208, 1970 Counting, Sydney, Australia, Aug. 1972. New 10. Ting P: Further studies on liquid scintilla- York, Academic Press (in press)
LIQUID SCINTILLATION COUNTING
17. Furlong NB: Liquid scintillation counting of samples on solid supports, in Bransome ED Jr (ed): reference 2, p 201 18. Grower MF, Bransome ED Jr: Liquid scintillation counting of 3H and 14C on solid supports: A warning. Anal Biochem 38:401, 1970 19. Neary MP, Budd AL: Color and chemical quenching, in Bransome ED Jr (ed): reference 2, p 273 20. Peng CT: A review of methods of quench correction in liquid scintillation counting, in Bransome ED Jr (ed): reference 2, p 283 21. Cavanaugh R: Statistical considerations in external standardization, in Bransome ED Jr (ed): reference 2, p 293 22. Wang CH: Quench compensation by means of gain restoration, in Bransome ED Jr (ed): reference 2, p 305 23. Klein PD, Eisler WJ: Through darkest quench with analyser and camera, in Horrocks DL, Peng ET (eds); Organic Scintillators, p 395 24. Ten Haaf FEL: Color quenching in liquid scintillation coincidence counters, in Crook MA, Johnson P, Scales B (eds): Liquid Scintillation Counting, p 39 25. Hendee WR, Ibbott GS, Crusha KL: 3Htoluene, 3H-water and 3H-hexadecane as internal standards in toluene and dioxane-based liquid scintillation cocktails. Int J Appl Radiat Isot 23:90-95, 1972 26. Laney BH: Electronic rejection of optical crosstalk in a twin phototube scintillation counter, in Horrocks DL, Peng ET (ed): Organic Scintillators, p 991 27. Hercules DM: Physical basis of chemiluminescence, in Bransome ED Jr (ed): reference 2, p 315 28. Kalbhen DA: Chemiluminescence as a problem in liquid scintillation counting, in Bransome ED Jr (ed): reference 2, p 337
399
29. Bransome ED Jr, Grower MF: Detection and correction of chemiluminescence in liquid scintillation counting, in Bransome ED Jr (ed): reference 2, p 342 30. Scales B: Questions regarding the occurrence of unwanted luminescence in liquid scintillation samples, in Crook MA, Johnson P, Scales B (eds): Liquid Scintillation Counting, p 101 31. Kobayashi Y, Maudsley DV: Practical aspects of double isotope counting, in Bransome ED Jr (ed): reference 2, p 76 32. Bransome ED Jr: The design of double label radioisotope experiments, in: Methods in Enzymology: Hormones and Cyclic Nucleotides. New York, Academic Press (in press) 33. Grower MF, Bransome ED Jr: Off-line data reduction with small desk-top computercalculators, in Bransome ED Jr (ed): reference 2, p 356 34. Grower MF, Bransome ED Jr: Handling liquid scintillation counting data with small desk-top computers. Anal Biochem 31:159, 1969 35. Spratt JL: Acquisition and handling of liquid scintillation counting data, in Crook MA, Johnson P, Scales B (eds): Liquid Scintillation Counting, p 245 36. Assailly J, Bader C, Funck-Brentano JL, Pavel D: Determination of statistical precision of tritium d.p.m, in dual labelled samples with variable isotope ratios and quenching, in Crook MA, Johnson P, Scales B (eds): Liquid Scintillation Counting, p 293 37. Parker RP, Elrick RH: Cerenkov counting as a means of assaying /~-emitting radionuclides, in Bransome ED Jr (ed): reference 2, p 110 38. Davies PT, Deterding JH: Optimization of counting of samples with double radioactive labelling. Int J Appl Radiat Isot 23:293, 1972
necessary in LS counting if there is any significant variation in sample preparation. If there are t w o radionuclides to be counted in a sample, a quench correction must always be applied because the pulse height shifts to a l o w e r energy region w h e n e v e r quenching occurs. Thus the spillover or overlap of t w o isotope spectra w i l l vary w i t h the energy of the radionuclides and the degree and type of quenching. None of the three methods of determining the extent of quenching (internal standardization, sample channels ratios, or external standardization) is ideal for all situations or is by any means foolproof. For example, if the scintillator solution and sample are not mixed to near homogeneity, counts may be lost because of self-absorption. Excitation of the scintillation solvent is decreased and cannot adequately be comprehended by any of the quench correction techniques. Because most scintillation solvents will tolerate only minimal amounts of aqueous samples of the sort usually of interest in nuclear medicine, oxygen flask combustion (for 14C or 3H), hydrolysis, addition of surfactants (solubilizers), or thixotropic gels--all relatively expensive procedures--may have to be used for sample preparation. The cost of sample preparation is frequently the only significant unfavorable feature of LS counting w h e n compared to g a m m a (Nalcrystal) scintillation counting.
HE USE of liquid scintillation counting in nuclear medicine laboratories has increased significantly in recent years as the use of radio-immunoassays and radioligand (protein-binding) assays of drugs, hormones, and other biologically important molecules have proliferated. Tritium (3 H) labeling of antigens or ligands displaced from binding proteins has become more common, inasmuch as labeling small molecules with 12sI or 13~I is often impossible. Liquid scintillation counting also lends itself to routine quantitation of radionuclides that decay by gamma emission or by electron capture.
T
From the Medical College of Georgia, Augusta, Ga. Supported in part by NIH grant CA 12455. Edwin D. Bransome, Jr., M.D.: Professor o f Medicine and Chief of the Division of Metabolic and Endocrine Disease, Medical College of Georgia, Augusta, Ga. 01973 by Grune & Stratton, Inc. Seminars in Nuclear Medicine, Vol. 3, No. 4 (October), 1973
389
390
EDWIN D. BRANSOME, JR.
Efficiencies are high, often greater than in well (NaI-crystal) counters. The resolution of photoelectron spectra, although less than in 3' counters, is adequate for the simultaneous counting of many isotope pairs, including i2Sl and 131I (Fig. 1). Excellent discussions of the physics of the processes outlined in the abstract are available in compilations of recent symposia 2-4 on liquid scintillation counting. These are volumes that anyone really interested in LS counting should take the trouble to look at. The principles of LS counting instrumentation are reviewed by Birks, s and the features of specific LS counters, including equipment currently marketed, have been presented in historical perspective by Rapkin. 6'7 CAPABILITIES
OF
LS C O U N T I N G
Although the principal use of LS counting has involved the measurement offl emissions, other charged particles such as a particles and protons can also be counted.
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Radionuclides that decay by electron capture and by 7 emission can be counted with high detection efficiency. Radioactive decay of some isotopes (e.g., 12sI) creates a vacancy in one of the inner electron shells of an atom (K, L, or M); the shell is then filled by an outer electron and the release of X-rays and low-energy (Auger) electrons results. If energetic enough, K X-rays, L X-rays, and Auger electrons are all capable of exciting dissolved scintillators. Quenching by impurities of the photoelectron spectra from K and L X-ray interactions is similar to that seen in ~ counting, but the photoelectron spectra and therefore quench correction curves may be somewhat different than those for/3 emissions of similar energy. 8 The interaction of 3' rays with scintillators s is principally a result of the Compton effect, a yield of electrons with 0 to 75% of the 3' energy. The photoelectric effect of 7 interaction usually does not occur above 30 keV in liquid scintillators, but may be evident at higher 7 energies in the vial walls or if the scintillator solution is loaded with heavy elements. Ashcroft 9 has suggested that addition to the scintillation mixture of a compound of high electron density (e.g., tetrabutyl tin, tetrabutyl lead) will increase the counting efficiency of 125I. Ting 1~ has recently suggested that ~2Sl-labeled samples in microcentrifuge tubes suspended in a scintillation mixture containing 5% tetrabutyl lead can be counted at 70 percent efficiency. Both Ashcroft and Ting have ascribed their results (not fully documented in the latter case) to an attenuation of the low-energy 3' of 12s I, so that fewer photons escape and more interact. The fact that this approach is not suitable for 3' emitters of higher energy suggests that their interpretation may be incorrect. The 70% detection efficiency reported is most probably a result of attenuation of the ~2Sl K X-rays and of the photoelectric effect. Since the effects of quenching and self-absorption of the photoelectric effect have not been worked out and since the counting efficiencies are no better than can be obtained in a normal liquid scintillation mixture, ~ I would suggest avoiding this approach. With high-energy 3' emissions, there may be another effect of interaction-the Cerenkov effect (see below)-if Compton electron energies are above 256 keV. Pair production, which occurs at 3' energies above 1.02 meV, is not a phenomenon occurring in the usual situation of LS counting. SAMPLE P R E P A R A T I O N
Each of the components of the sample being counted by liquid scintillation plays a role in determining the counting efficiency.
Scintillation Solvent An alkylbenzene or p-dioxane (lO0 g naphthalene/liter) is used as the scintillation solvent. Of the alkylbenzenes, toluene is usually chosen because of its high scintillation yield, intermediate susceptibility to impurity quenching, and relatively low price.
Primary Scintillator An aromatic compound with a high fluorescence quantum efficiency is used as the primary scintillator. Those in common use are listed in Table 1 along with their lowest concentrations for optimum efficiency in toluene as determined by Birks and Poullis. n The absorption and emission spectra of the scintillators are also important, the
EDWIN D. BRANSOME, JR.
392
Table 1. Common Organic Scintillators
Primary PPO PBO PBD butyl PBD BBOT BIBUQ TP
2,5-diphenvloxazole 7 g/liter* 5-phenyl-2-(4-biphenylyl)-oxazole 7.5 g/liter 2-phenyl, 5-(4-biphenylyl)-l,3,4-oxadiazole 12 g/liter 2.(4t-tert-butylphenyl), 5-(4"-biph enylyl)- 1,3,4-oxadiazole 12 g/liter 2,5-bis(5'-tert-butyl-2-benzoxazolyl)-thiophene 8 g/liter 4,4-bis(2-butyloctyloxy-p-quaterphenyl) 24 g/liter p-terphenyl 7 g/liter Secondary
POPOP dimethyl POPOP BBO PBBO bis-MSB NPO
1,4-bis [2-(5-phenyloxazolyl)]-benzene 1 g/liter* 1,4-bis [2-(4-methyl-5-phenyloxazolyl)] -benzene 0.02 g/liter 2,5-di(4-biphenylyl)-oxazole 0.02 g/liter 2-(4-biphenylyl)-6-phenylbenzoxazole 1 g/liter p-bis (o-methylstyryl)-benzene 0.2 g/liter 2-(1-naphthyl), 5-phenyl-oxazole 1 g/liter
*Optimum concentrations in toluene. Taken in part from the data of Birks and Poullis. l l
former for the solvent-to-scintillator energy transfer and the latter for the match of the emitted fluorescence spectrum to the spectral response of the photomultiplier tubes of the scintillation counter.
Secondary Scintillator With some primary scintillators, which emit a fluorescence spectrum mismatched to the spectral response of the phototubes of the specific scintillation counter being used (e.g., most of the spectrum of p-terphenyl is absorbed by glass vials), it is necessary to add another scintillator that will shift the spectrum toward the optimum phototube response. Secondary scintillators in common use are listed in Table 1. The experiments of Birks and Poullis ~ suggest that BBO, PBBO, POPOP, and bis-MSB have the best overall characteristics.
Nature of the Radiation If t3 or Compton electrons are at or above a 0.l-meV energy endpoint, counting efficiency is directly proportional to photon energy; for lower energy electrons and for other ionizing radiation (a, 3', protons, etc.) there is less efficiency of detection per keV of Ema x .
Scintillation Counting Vial The portion of a fluorescence spectrum presented to the photonmltiplier tube is partially dependent on the band-pass characteristics of the vial wall. Polyethylene, for example, will transmit considerable light over the uv range, whereas glass will not.
Physical and Chemical Properties of Sample The physical and chemical properties of a sample are of great importance. Will a sample (although labeled radioisotopically) be a significant impurity quencher or a color quencher? If the sample is not homogeneously mixed with the solvent, or if the sample is precipitated (trapped) onto a solid support, there may be significant problems in the assessment of the absolute radioactivity of the sample.
LIQUID S C I N T I L L A T I O N COUNTING
393
Most of the important considerations of sample preparation for LS counting can conveniently be reviewed in chapters 16 to 27 of reference 2 and in reference 12. It should be emphasized that for calculations of the activity of a sample to be reliable, the radioactivity must be uniformly distributed in the sample.
Emulsion Counting Translucent emulsions with microscopic micelles ~2'1a may be counted as easily as true solutions provided some precautions are observed. If two isotopes are being counted, particularly if one of the isotope pair is 3H or 12sI, they must be in the same phase. This might seem to be a trivial problem, since emulsion counting is of aqueous samples, but it is not-there may be differential extraction of the labeled solutes into the organic solvent. Absorption (of the lower energy isotope in particular) onto the vial surface may greatly alter detection efficiency)4 If the Emax of an isotope is equivalent to or greater than that of ~4C and a commercial solubilizer of aqueous samples has been included in the sample, the surfactant itself may fluoresce in response to radioactivity. This renders quench correction curves derived from sealed commercial standards invalid. 1s:6 All of these proscriptions may be summarized in the cardinal and frequently neglected general rule for scintillation counting: standards must be of the same geometry as the unknown samples.
Counting on Solid Supports The problems of inhomogeneity and geometry are even greater. If samples are first collected on Solid supports, put in a scintillation vial, and covered with solvent, etc., the first question to be asked is whether the sample is either completely or partly eluted from the support. Self-absorption (attenuation of the particle path) reduces the chance of interaction with the scintillation solvent and is very significant, especially for low-energy/3 emissions 17:8 (Table 2). None of the quench correction techniques, which are reviewed below, can estimate adequately the decrease in efficiency when such heterogenous systems are employed.
QUENCH CORRECTION Quench correction techniques by Neary and Budd, 19 Peng, 2~ Cavanaugh, 21 and Wang 22 have been thoroughly reviewed in reference 2, and those by Klein and Eisler 23 and Ten Haaf~ are covered in references 3 and 4, respectively. Table 2. Effects of Sample Geometry on Relative Detection Efficiency* Relative Efficiency (%)
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*The CPM in a 0.1-ml aqueous solution of 1251-Nal, 1311-Nal, 14C-adenine, or 3H-cycJic AMP were taken as 1.00 and compared to replicates air-dried onto discs 20 mm in diameter. Liquid scintillation counting was with a Beckman LS-150 system with a wide channel encompassing the total spectrum of each isotope. The aqueous samples were counted in toluene 7.0 g/liter PPO (2,5-diphenyloxazole), 10% Biosolv BBS-3 (Beckman) solubilizer. The samples dried onto the filters were counted in totuene-PPO without Biosolv.
394
EDWIN D. BRANSOME, JR.
There is recent agreement that whereas quench correction curves for 3 H (Ema x = ! 8 keV) are not significantly different whether the quenching is from impurity or color, the curves diverge significantly if ~4C (Emax = 156 keV) or other more energetic nuclides are counted, particularly when samples are highly quenched. It may therefore be quite misleading to apply a quench correction curve obtained with commercially available sealed quenched standards to unknown samples of different composition. There are three techniques of quench correction in general use: Internal Standardization Internal standardization, or spiking, the addition of a Standard of known radioactivity to each sample, is the most accurate of any method if the addition of the known standard to the sample is careful and reproducible. Differences in the effects of impurity and color quenching (which may be important with 14C and more energetic nuclides) are obviated. The samples must be homogeneous, and the standard must be similar chemically to the sample solute if there is any question of an incomplete solution. Handling errors in adding the standard will be significant if the procedure is careless; ideally this procedure should be done at least in duplicate. Once a sample is spiked, it cannot be recounted. The procedure is extremely time-consuming. In double-label experiments, it is a laborious and statistically uncertain approach. Hendee et al. ~s have recently emphasized the problem of homogeneity in internal standardization. They found that addition of even very small volumes of 3H-H20 (a quencher) to toluene- or dioxane-based liquid scintillation mixtures resulted in a progressive decrease of 3H counting efficiency. In other words, internal standardization should be performed by the addition of a known amount of a labeled standard that will not act as a nonhomogeneous impurity. Ideally, labeled scintillation solvent should be used, but if radionuclides other than 3H or 14C are to be counted, this is impossible. Then the effect of adding the proposed volume of standard to the samples must be determined experimentally. Sample Channels Ratios An isotope is counted in two channels. One should be a wide window, and the other should encompass the lower energy range of the spectrum. As an isotope is quenched, a greater proportion of the total cpm will appear in the second window. Data may be obtained in two channels simultaneously; thus counting the sample only once is sufficient: The Sample itself is not altered. This approach is accurate for moderate and high count rates of single isotopes that are quenched slightly to moderately, and it is independent of sample volume over a wide range. One series of quenched standards will correct for both sorts of quenching of 3H. Nonhomogeneous samples (in suspension, in emulsion, on solid supports) may be quench-corrected, as long as standard curves are derived from known samples of the same composition and geometry. The procedure is inaccurate, however, for highly quenched samples or for samples with 10w count rates. Strov.g color quenching of ~4C or more energetic isotopes is not adequately corrected for. With two overlapping isotopes, corrections for spillover must first be made, with a significant additional statistical error. External Standard Channels Ratios This procedure, involving exposure of the unknown sample to an external 7 source, takes advantage of the secondary Compton electrons generated within the sample and
LIOUID SCINTILLATION COUNTING
395
the resulting broad fluorescence spectrum. Modern instrumentation monitors and subtracts sample cpm from two separate channels and then sets the proper Compton cpm. Single-channel external standardization is undesirable, inasmuch as the dependence of cpm on sample volume is considerable. External standardization can be applied to samples of low activity. Statistically it is the most suitable for double-isotope quench correction, but it is the least accurate for highly quenched samples in which differences between the photoelectron spectra of sample and Compton electrons are magnified. Samples must be homogeneous. This technique is not applicable to samples in visible suspension, on solid supports, etc, CH EM I LUMI N ESCE NCE
One of the greatest concerns in utilizing LS counting, particularly if the sample radioactivity is low, is the background count rate: a combination of decay of radioisotopes in the system (e.g., 4~ in scintillation vial glass), cosmic radiation, thermal activity in the photomultiplier tubes, and optical cross-talk between the photomultipliers. 26 There may also be low-energy fluorescence as a result of chemical reactions 27 in the scintillation vial: chemiluminescence may be encountered if samples have been subjected to alkali base and the pH of the scintillation mixture is high, or if peroxides are present. The answers to these problems are relatively simple: lower the pH by acidification, or for peroxides add the enzyme catalaSe, which is effective even in the presence of toluene, z8,29 Scales 3~ has recently reviewed some more sporadic causes of chemiluminescence in LS counting: the reaction of vial caps of a particular composition to uv light or temperature, an impurity in some batches of Triton X-100 (a Rohm and Haas product used in emulsion counting), and an impurity accumulating as a breakdown product of reagent-grade dioxane. Obviously, if chemiluminescence is detected it can be avoided. Since chemiluminescence due to light or temperature activation wanes quickly, rapidly decreasing count rates in the same sample counted minutes or hours apart should be sufficient to alert one. When the luminescence is longer lived, the difference of the luminescence photoelectron spectrum from that of the radionuclide of interest may serve as an indicator, z9 Several LS counters of recent manufacture incorporate a delay line that allows monitoring of noncoincident single-photon events and thus of chemiluminescence. 7 DOUBLE-LABEL COUNTING
In most double-label experiments, two counting channels are used and the photoelectron spectra are only partially separated. In liquid scintillation counting there is too great a loss of detection efficiency if both isotopes are eliminated entirely from the converse channel: the photoelectron spectra of the fluorescence emitted from the organic scintillators are too broad. Indeed, if the photon energies are not .videly separated, it is statistically impractical to eliminate the spillover of either isotope from the other channel. The extent of quenching in a homogeneous solution of sample and phosphor in solvent will cause pulse-height shifts of fluorescence spectra to lower photoelectron energies as an inverse function of the energy of the emitted photons. Quenching in samples will thus affect the detection efficiencies of two isotopes variably, the spectral shift of the lower energy isotope being greater. There is then no single set of optimum instrument settings for specific isotope pairs. Correction of the overlap of radioactivities must be carried out before quench correction. Two questions that deserve attention are: How can the cpm of one isotope
396
EDWIN D. BRANSOME, JR.
best be separated mathematically from the cpm of another? What are the optimum instrument settings for a specific isotope pair? A more extensive discussion of the answers than can be provided here may be found in references 1,31, and 32. One approach to double-isotope counting is to continue to use the optimum discriminator settings for each of the two isotopes. Since each channel will count contributions from both isotopes, simultaneous equations will be necessary for the calculation of the absolute radioactivity of each sample. Both isotopes are counted at maximum efficiency unless there is severe quenching, but a considerable accumulation of statistical error results from the repeated application of calculated efficiencies. If possible, the lower energy isotope should be eliminated from the channel used to count the higher energy isotope. If H is the isotope of higher energy and L the lower, then (with background subtracted) dpm H -
cpm channel 1 efficiency of H in channel 1
dpm L = (cpm channel 2 ) - (cpm channel 1 • efficiency of H in channel 2) efficiency of L in channel 2
(1) (2)
The cumulative error is greatly decreased by such a simplification. In our work, analysis of the variance occasioned by the use of simultaneous equations often results in unacceptably large errors, particularly when count rates for one or both isotopes are low and the overlap is large. There have been several suggestions in the recent literature that simultaneous equations may reliably differentiate two isotopes with considerable spectral overlap. Such advice is dangerous; if simultaneous equations are employed, exclusion of the lower energy isotope from the channel used to count the higher energy isotope should still be maximal. The spillover fraction (the percentage of the net cpm of an isotope counted in its own channel that falls into the channel used for the other isotope) is a constant in gamma counting, but will of course increase in liquid scintillation counting as the spectrum of isotope H is quenched and shifted toward lower energies. Progressively more H cpm fall in channel 2. In Beckman scintillation counters with automatic quench correction (A.Q.C.), this fraction can be held relatively constant, 22,33,a4 but in others a quench correction curve for this fraction must be obtained, just as it is obtained for the detection efficiencies of the two isotopes in 1heir optimum channels (see Fig. 2). Occasionally, workers have attempted to use simultaneous equations to solve for the activities of three isotopes in the same sample, using three or more channels. The statistical errors implicit in such procedures are unacceptably large. This concern about statistical variation is quite practical and is particularly appropriate for double-label counting, since the standard deviation of the determination of the radioactivity of a sample accumulates as the square root of the sums of the squared standard deviations of each component measurement (cpm, background, spillover, and efficiericy). There are good up-to-date discussions of this aspect of data handling in LS counting by Spratt as and Assailly et all 36 in reference 4. It is Sometimes possible to separate one overlapping isotope from another by combining different counting methods: 131I may be separated from 14C and aSS, while 12sI may be separated from 3H, by counting {he samples in a gamma counter and then by liquid scintillation alone. It is important that 13~1 or 1251 efficiency be determined
LIQUID SCINTILLATION
COUNTING
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in both counters so that an accurate spillover curve of either iodine isotope into the 14C, 3s S, or 3H channels can be calculated from standards. Energetic/3 emissions may also be counted in solution in the absence of phosphors by virtue of the Cerenkov effect. 37 The Cerenkov threshold in standard scintillation mixtures is approximately 256 keV; 32p and 131I may therefore be counted at reasonable efficiencies (without any impurity quenching), but Cerenkov photoelectron spectra are broad-band and are poorly resolved, so that double-isotope counting is not practical.
Instrument Settings The method of selecting discriminator settings for the single-channel counting of a single radionuclide is straightforward. Since background cpm increase with channel width, the upper potentiometer setting should be decreased to the point where the count rate of a sample of the radionuclide begins to become significantly diminished. The lower discriminator should be set close to zero and raised only if there is a major contribution of instrument noise to background in the low-energy range, a problem not significant in instruments of recent manufacture. In double-label counting there is an obvious conflict between desires for minimum spillover and maximum efficiency. Mathematical solutions to the determination of optimum settings have been proposed; the most satisfactory is that of Davies and Deterding, 38 but it is quite complex. We have found that graphic methods are suf-
398
EDWIN D. BRANSOME, JR.
ficient for double-isotope channel selection: a simple plot of upper discriminator settings (with the lower discriminator fixed) versus percent of the total cpm of a standard in the channel is appropriate for instruments with logarithmic or quasilogarithmic amplification of photomultiplier o u t p u t ) ' 3 2 For LS spectrometers with linear amplification, the Engberg plot, introduced by Kobayashi and Maudsley 31 will reveal the regions of optimum settings (see also references 1 and 32). D A T A REDUCTION
The subject of automatic data reduction is avoided in this review for several reasons. The foregoing paragraphs have been written in agreement with Spratt's contention that "excellent data handling is often performed on inappropriate data." In references 2-5 recommended above as good source materials for liquid scintillation counting, there are a number of discussions of off-line and on-line data reduction with large digital computers, minicomputers, and desk-top computer calculators. 33'34 Spratt's philosophical discussion3s is an excellent introduction. There are also discussions of computer applications elsewhere in this seminar. Computer programs have the advantage that they can include explicit or implicit error analysis (unacceptable deviations of data may be flagged). Description of specific programs here seems inappropriate, since they are machine-dependent and the array of computation equipment is rapidly changing. Indeed, most manufacturers of LS counters are offering peripherals and software for LS counting data reduction. REFERENCES
1. Bransome ED Jr, Sharpe SE II1: Mea- tion counting of I-125. Clinical brief 6. Fullersurement of 131I and 125I by liquid scintilla- ton, Calif., Beckman Scientific Instruments tion counting. Anal Biochem 49:343, 1972 Div., 1973 2. Bransome ED Jr (ed): The Current Status 11. Birks JB, Poullis GC: Liquid scintillators, of Liquid Scintillation Counting. New York, in Crook MA, Johnson P, Scales B (eds): Grune & Stratton, 1970 Liquid Scintillation Counting, p 1 3. Horrocks DL, Peng ET (eds): Organic 12. Turner JC: Sample Preparation for Liquid Scintillators and Liquid Scintillation Counting. Scintillation Counting, Review 6. The RadioNew York, Academic Press, 1971 chemical Center, Amersham, England, 1967; 4. Crook MA, Johnson P, Scales B (eds): reprinted in Wang U (ed): Handbook of RadioLiquid Scintillation Counting, vols. l and 2. active Nuclides. The Chemical Rubber Co., London, Heyden and Son, 1972 Cleveland, 1969, p 256 5. Birks JB: The Theory and Practice of 13. Greene RC: Heterogeneous systems: SusScintillation Counting. New York, Macmillan, pensions, in Bransome ED Jr (ed): reference 2, 1964 p 189 6. Rapkin E: Development of the modern 14 Litt GJ, Carter H: Sample absorption scintillation counter, in Bransome ED Jr (ed): problems in liquid scintillation counting, in reference 2, p 45 Bransome ED Jr (ed): reference 2, p 156 7. Rapkin E: in Crook MA, Johnson P, Scales 15. Sharpe SE III, Bransome ED Jr: The B (eds): Liquid Scintillation Counting, p 61 surfactant Biosolv-BBS-3 as a scintillator in 8. Horrocks DL" Obtaining the possible max- liquid scintillation counting. Anal Biochem (in imum of 90 percent efficiency for counting of press) 55Fe in liquid scintillator solutions, lnt J Appl 16. Sharpe SE I11, Bransome ED Jr: SurRadiat lsot 22:258, 1971 factants behave as scintillators in liquid scintil9. Ashcroft J: Gamma counting of iodine- lation counting, in: Proceedings of the Inter125 using a metal-loaded liquid scintillator. national Congress on Liquid Scintillation Anal Biochem 37:208, 1970 Counting, Sydney, Australia, Aug. 1972. New 10. Ting P: Further studies on liquid scintilla- York, Academic Press (in press)
LIQUID SCINTILLATION COUNTING
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