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Year : 2016  |  Volume : 39  |  Issue : 3  |  Page : 149-154  

Methodology for estimation of phosphorus-32 in bioassay samples by Cerenkov counting

Radiation Safety Systems Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra, India

Date of Web Publication30-Nov-2016

Correspondence Address:
Sonal M Wankhede
Radiation Safety Systems Division, Bhabha Atomic Research Centre, Trombay, Mumbai, Maharashtra
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/0972-0464.194964

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Bioassay is the preferred individual monitoring technique for radiation workers handling phosphorus-32 (32 P), a pure beta emitter (βmax = 1.71 MeV) with 14.3 day half-life. The method standardized at Bioassay Laboratory, Trombay and in use for this purpose includes estimation of 32 P in urine by coprecipitation with ammonium phosphomolybdate followed by gross beta counting. In this study, the feasibility of Cerenkov counting for detection of 32 P in bioassay samples was explored, and the results obtained were compared with the conventional gross beta technique.

Keywords: Ammonium phosphomolybdate, Cerenkov, liquid scintillation spectrometry, phosphorus-32, triple to double coincidence ratio, urine

How to cite this article:
Wankhede SM, Sawant PD, Yadav RB, Rao D D. Methodology for estimation of phosphorus-32 in bioassay samples by Cerenkov counting. Radiat Prot Environ 2016;39:149-54

How to cite this URL:
Wankhede SM, Sawant PD, Yadav RB, Rao D D. Methodology for estimation of phosphorus-32 in bioassay samples by Cerenkov counting. Radiat Prot Environ [serial online] 2016 [cited 2023 May 30];39:149-54. Available from: https://www.rpe.org.in/text.asp?2016/39/3/149/194964

  Introduction Top

Phosphorus-32 (32 P) has applications in medicine, industry, and tracer studies.32 P as phosphate is used to effectively reduce bone pain in terminal cancer patients. Several hospitals including Radiation Medicine Centre, Mumbai apply this treatment method and to meet the requirement, production, as well as synthesis of 32 P compounds, has increased over the years. Monitoring of radiation workers for possible 32 P internal contamination is in place for providing radiological protection at 32 P processing facility. Implementation of radiological protection program at these facilities includes quantification of 32 P intake as well as assessment of internal dose received by the radiation workers. Following inhalation,32 P gets deposited in the respiratory tract and based on its solubility (Type F or Type M) [Table 1][1] is cleared from lungs into the body fluids. From the plasma, it is removed with a biological half-time of 0.5 days and distributed to cell fluids (15%), other soft tissue components (40%), bone (30%), and excreta (15%). Phosphorus returns to plasma from cell fluids and other soft tissues with half-times of 2 days and 19 days, respectively. Based on the default assumption used by International Commission on Radiological Protection [1],[2] for bone-volume-seekers, the bone deposit of P isotope with half-life <15 days is assigned to bone surface and for P isotope with longer half-life is assigned to bone volume.32 P that enters the bone is assumed to decay on the bone surface, and 33 P that enters bone is assumed to decay in bone volume. Ninety percent of Premoved from a tissue goes to the urinary bladder contents and is subsequently excreted in urine, and 10% gets excreted in feces. The urine to fecal excretion ratio (fu) observed is 9:1, indicating that removal of deposited 32 P from the body occurs principally through urine [Figure 1]. Hence, bioassay is the preferred individual monitoring technique for radiation workers handling 32 P. The conventional method followed at Bioassay Laboratory, Trombay, includes estimation of 32 P in urine by coprecipitation along with ammonium phosphomolybdate (AMP) formed in-situ followed by gross beta counting [Figure 2]. The method though sensitive, has limitations on the amount of urine sample (25 mL) that can be initially taken up for analysis to avoid self-absorption of some of the β particles emitted by 32 P in AMP. Thus, to overcome this limitation, a feasibility study for detection of 32 P in bioassay samples by Cerenkov counting was carried out in this study.
Table 1: Solubility types, compounds, gut absorption factor (f1) for 32P

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Figure 1: Biokinetic model for phosphorus (ICRP Publication 68, 1994)

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Figure 2: Flow chart for the estimation of phosphorus-32 in bioassay samples

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Cerenkov radiations are emitted when a charged particle (such as an electron) passes through a transparent dielectric medium (e.g., water) at a velocity greater than the speed of light in that medium due to localized polarization of molecules in the medium.[3] Minimum energy (Emin) required for the production of Cerenkov radiation is a function of the refractive index (η) of the medium and β, ratio of velocity (v) of the particle in the medium to speed of light (c) in that medium. For the production of Cerenkov radiation in any medium, “η × β” should be >1. Thus, if the refractive index, η, of a medium is known, it is possible to calculate the β and then Emin using Equation 1:

Emin, required by beta particle to produce Cerenkov radiation in water (η =1.33) is ~0.263 MeV [4] and 32 P with average β energy of 690 keV can, therefore, produce Cerenkov radiation in an aqueous medium. The present study reports the Cerenkov counting technique standardized for detection and estimation of 32 P in bioassay samples using liquid scintillation spectrometry (LSS) system.

  Experimental Top

The standard

32 P standard solution (4.2 mCi, IGANo: 777Z) procured from Board of Radiation and Isotope Technology was further diluted with 0.1M HCl to make a working standard (S1) of 300 Bq/mL. S1 standard was used to prepare quality control spike samples.


HCl, HNO3 and ammonium nitrate (AR grade), ammonium molybdate (AR grade), ammonia (AR grade), and distilled water.

The liquid scintillation spectrometry system

In the present study, Hidex 300SL triple to double coincidence ratio (TDCR) LSS system was used for detection of Cerenkov radiation produced by 32 P in aqueous samples. This system consists of three photomultiplier tubes placed at an angle of 120° in a highly reflective chamber with 7 cm thick lead shielding in all directions. Thus, unlike the conventional double coincidence counting techniques, the TDCR technique uses a triple coincidence counting method. Interferences from quenching and other factors are reflected in triple coincidence counts and ultimately expressed in the ratio of TDCR. Thus, TDCR is directly proportional to Cerenkov counting efficiency (εCerenkov) and can be used to correct for the effects of any experimental variations on εCerenkov.[5] The relationship between TDCR and εCerenkov for 32 P in plastic vial is shown in [Figure 3].
Figure 3: εCerenkovand triple to double coincidence ratio for known amount of phosphorus-32 activity spiked in 20 mL of distilled water

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Standardization of Cerenkov technique

Various parameters such as effect of vial material, sample volume, molarity of NH3 used for dissolution of AMP, weight of AMP, color and turbidity of the sample were studied for standardizing the procedure.

Set 1: Effect of vial material

To study the effect of vial material on Cerenkov counting efficiency, 30–150 Bq of 32 P was added to plastic and glass vials, mixed with 20 mL of distilled water, and counted in LSS system for 30 min.

Set 2: Effect of sample volume

Known activity (30 Bq) of 32 P was added to plastic as well as glass vials and evaporated to near dryness under infrared (IR) lamp. The volume of distilled water added to these vials was then increased from 1 to 20 mL, and the values of TDCR with an increase in sample volume were recorded.

Set 3: Effect of molarity of NH3 used for dissolution of AMP

150 Bq of 32 P was transferred to glass and plastic vials along with 0.5 g of AMP. Ammonia (NH3) solution of varying molarity (1–5M) was added to these vials to dissolve AMP and then counted in LSS system.

Set 4: Effect of weight of AMP

To observe the effect of higher amount of AMP on TDCR, Set 4 was prepared by adding known amount of 32 P (150 Bq) to plastic and glass vials, however, the amount of AMP added was varied from 0.5 to 3.5 g and dissolved in 2M NH3. This experiment provided important information regarding the feasibility of increasing the initial sample volume that could be taken up for analysis of 32 P.

Set 5: Effect of color and turbidity

To study the effect of color as well as turbidity, a series of brown colored solutions made by dissolving food grade brown color in distilled water were spiked with known activity of 32 P (30–150 Bq) and counted in LSS system. These samples were then made turbid by the addition of 100 µL of milk and recounted in LSS system. Experiments were also conducted in triplicates by adding known activity of 32 P (660–1320 Bq) to actual urine samples (20 mL each).

Set 6: Comparison of gross beta and Cerenkov counting technique for estimation of phosphorus-32 in bioassay samples

Twenty-four hours urine sample was collected from a radiation worker not handling 32 P and was divided into two sets (Set 6a and Set 6b), each set consisted of 25 aliquots (25 mL, each) which were spiked with 32 P activity (30–150 Bq). These aliquots were processed as per the procedure given in [Figure 2]. However, in case of Set 6a, the AMP formed in-situ was dissolved in 2M ammonia and taken for Cerenkov counting using LSS system and for Set 6b, the precipitate was further washed with 90% alcohol and then transferred to Al-planchettes. These planchetts were dried under IR lamp, cooled and counted in alpha beta radiometer UMF-2000 (make Doza, Russia).

Procedural blank samples (10 numbers for each set) were prepared using urine samples except for the addition of 32 P activity and processed through the entire procedure [Figure 2].

  Results and Discussion Top

The experimental parameters, such as (a) vial material (Set 1); (b) volume of sample (Set 2); (c) molarity of ammonia (Set 3); (d) weight of AMP precipitate formed (Set 4); and (e) presence of color and turbidity (Set 5), which influence the TDCR were studied. In Set 1 experiments, it was observed that counting efficiency was higher for plastic vials (range 0.41–0.44, average 0.43) as compared to glass vials (range 0.36–0.40, average 0.38) [Figure 4]. Cerenkov radiation is anisotropic as light photons are emitted as a cone of radiation at a specific angle to the direction of travel of β particles. Cerenkov light that cannot pass through polyethylene plastic is reflected away from the vial. When reflected away, the Cerenkov light may be seen as an isotropic source and therefore, can be detected more effectively.[6] Thus, use of a plastic vial changes the directional nature of the Cerenkov photons to an isotropic emission which improves the efficiency and thereby higher TDCR. Variation in TDCR between plastic and glass vials may also be partly due to the difference in refractive index, η, of the counting media. For plastic medium, with η =1.52, the Emin for the production of Cerenkov radiation is 0.167 MeV whereas for borosilicate glass medium (η =1.48), it is 0.183 MeV. Another reason for lower TDCR observed for glass vials may be because a significant portion of Cerenkov radiation occurs in the ultraviolet region which would be blocked to a large extent by the glass of counting vials.
Figure 4: Variation in triple to double coincidence ratio for glass and plastic vials for phosphorus-32 activity spiked in 20 mL of distilled water

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Sample volume (geometry) also influences the Cerenkov counting efficiency for glass as well as plastic vials. Variation in TDCR with different types of vials and varying sample volume is given in [Figure 5]. TDCR values using plastic vials were observed to reduce from 0.61 to 0.41 with the increase in sample volume whereas for glass vials the TDCR increased initially up to 8 mL and thereafter remained constant up to 20 mL. The difference in TDCR between plastic and glass vial was significant at lower volumes. However, for practical purposes, the sample volume in the present study was maintained at 20 mL to obviate any variability due to sample geometry.
Figure 5: Effect of sample volume on triple to double coincidence ratio

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Variation of TDCR was also studied for various molarities of ammonia solution used for dissolution of AMP (0.5 g) precipitate with known amount of 32 P activity (150 Bq). The experiments indicated not much variation in TDCR with different molarities of ammonia solution [Figure 6] using glass or plastic scintillation vials. However, the dissolution of AMP was slow in 1M and 1.5M and therefore, 2M NH3 was selected for dissolution of AMP in all further experiments.
Figure 6: Effect of molarity of NH3on triple to double coincidence ratio

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Quenching of signal is one of the main factors that can tremendously suppress TDCR of a sample. Two types of quenching are observed in LSS counting, namely, color and chemical quenching. Color quenching is the attenuation of light photons by the color in the sample whereas, in chemical quenching, the excitation energy of the excited molecule is dissipated as heat. As a result of both these quenching, the overall number of photons produced in the sample is reduced, which in turn reduces the TDCR. In case of Cerenkov technique, only color quenching can affect the TDCR. In this study, it was also observed that with an increase in the amount of AMP (0.5–3.5 g), the TDCR for 32 P reduced [Figure 7]. However, the color quenching reflected by a reduction in TDCR did not affect Cerenkov counting adversely and hence, the initial volume taken up for 32 P estimation could be easily increased by four folds (100 mL) which was not possible by a conventional method.
Figure 7: Effect of increasing amount of ammonium phosphomolybdate on triple to double coincidence ratio

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[Figure 8]a gives the comparison of the Cerenkov spectrum obtained for 32 P-AMP dissolved in 2M NH3 and for 32 P added to the actual urine sample. A similar comparison is shown for 32 P-AMP dissolved in 2M NH3 and turbid brown colored aqueous solution in [Figure 8]b. The Cerenkov spectrum for brown colored turbid solution was completely quenched and the TDCR observed, in this case, is 0.08 as compared to 0.33 observed for AMP dissolved in 2M ammonia. When 20 mL urine samples were spiked with 32 P activity in the range of 660–1320 Bq, the TDCR for plastic vials was observed to be 0.26 [Figure 9]. This experiment provided important information regarding the feasibility of direct counting of urine samples as a screening method in case of monitoring of individuals exposed during criticality accidents.
Figure 8: Compares the variation in TDCR between AMP dissolved in ammonia and direct urine or turbid brown solution. This experiment was done to observe the feasibility of direct urine sample counting for P-32 in case of criticality accidents. The figure indicates that urine samples can be directly counted in LSS but for turbid urine samples there is significant quenching

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Figure 9: Triple to double coincidence ratio for 20 mL urine sample spiked with phosphorus-32 activity in plastic vial

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After standardizing the Cerenkov counting procedure, urine samples spiked with 32 P (Set 6a and Set 6b) were analyzed by Cerenkov counting as well as by conventional gross beta method. The radiochemical recovery observed for Cerenkov technique was ~93% whereas for gross beta counting it was ~79% [Figure 10]. The reason for lower recovery in gross beta technique may be due to absorption of few β-particles by AMP precipitate formed in-situ.
Figure 10: Comparison of phosphorus-32 recovery observed in Cerenkov and conventional gross beta technique

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The bias and precision in measured activities of spiked samples were assessed to delineate the performance of the method. Method accuracy and relative precision assessed using relative bias as defined by ANSI N13.30[7] was found to be − 6.6% and 2.7% respectively (acceptance criterion for radiobioassay as − 25% to + 50% for relative bias and 40% for relative precision).

Minimum detectable activity

The minimum detectable activity (MDA) of the present method using 25 mL urine sample was computed based on the analysis of the 10 blank sample aliquots processed in a similar way as the spiked samples and counted in Hidex 300 SL TDCR LSS. The average background counts of the blanks were used to compute the MDA [8] of the method standardized. The MDA of the present method standardized is ~9 Bq/day for 60 min counting time and assuming total daily urine output of 1.4 L/day [9] which is comparable with the conventional technique. In case of Cerenkov technique, the volume of sample taken up can be easily increased to 100 mL which would result in further reduction of MDA. The minimum detectable dose (MDD) computed using Integrated Modules for Bioassay Analysis [10] Professional Plus software for assuming intake at mid-point of monitoring interval (T = 30 days) and for 5 µm AMAD particles of Type F and M compounds is 5.3 and 16 µSv, respectively.

Similarly, MDA computed for direct Cerenkov measurement of 20 mL urine samples without any chemical processing was found to be ~16 Bq/day and the corresponding MDD within a day of exposure is 0.4 µSv for Type F and 1.3 µSv for Type M compounds. Thus, the direct Cerenkov counting technique proves to be rapid as well as sensitive to detect doses well below the recommended levels for routine as well as emergency monitoring.

  Conclusions Top

Cerenkov counting technique was successfully standardized and applied for estimation of 32 P in bioassay samples. The main advantage of the TDCR Cerenkov counting is its ability to automatically correct for interferences such as color quenching and variation in sample geometry, which needs external calibration in case of conventional LSS systems. The Cerenkov counting technique is cost-effective, environment-friendly, and an additional advantage is more number of samples (40 nos) can be counted automatically using this system.

Further, the initial volume of sample required for analysis can be increased in Cerenkov counting without any interference in 32 P activity measurements which is a limitation for conventional technique. The method's detection limit was found to be ~9 Bq/day using Hidex 300 SL LSS.

The present study also revealed the measurement of Cerenkov radiation in direct urine sample without any chemical processing. Further standardization of direct TDCR Cerenkov technique assures a fast and an inexpensive method for monitoring of individuals exposed during criticality accidents that can help in screening and segregating high- and low-level bioassay samples.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

International Commission on Radiological Protection. Limits for intakes of radionuclides by workers. ICRP Publication 30. Part 1. 1979; Annals of the ICRP 2 (3/4):69-70.  Back to cited text no. 1
International Commission on Radiological Protection. Dose coefficients for intakes of radionuclides by workers. ICRP Publication 68. 1994; Annals of the ICRP 24 (4):26.   Back to cited text no. 2
Scarpitta SC, Fisenne IM. Cerenkov counting as a complement to liquid scintillation counting. Appl Radiat Isot 1996;47:795-800.  Back to cited text no. 3
Ginkel GV. An investigation of various wavelength-shifting compounds for improving counting efficiency when 32 P Cerenkov radiation is measured in aqueous samples. Int J Appl Radiat Isot 1979;31:307-12.  Back to cited text no. 4
Kossert K. Activity standardization by means of a new TDCR-Cerenkov counting technique. Appl Radiat Isot 2010;68:1116-20.  Back to cited text no. 5
Simonnet F, Combe J, Gerard S. Detection of 32 P scintillating plastic vials (Technical note). Appl Radiat Isot 1987;38:311-2.  Back to cited text no. 6
Health Physics Society. Performance Criteria for Radio-bioassay: An American National Standard ANSI/HPS N 13.30. New York: Health Physics Society; 1996.  Back to cited text no. 7
Boecker B, Hall R, Inn K, Lawrence J, Ziemer P, Eisele G, et al. Current status of bioassay procedures to detect and quantify previous exposures to radioactive materials. Bioassay Procedures Working Group. Health Phys 1991;60 Suppl 1:45-100.  Back to cited text no. 8
International Commission on Radiological Protection. Reference Man: Anatomical, Physiological and Metabolic Characteristics. ICRP Publication 23, 1975;354.  Back to cited text no. 9
Birchall A, Puncher M, James AC, Marsh JW, Jarvis NS, Peace MS, et al. IMBA expert: Internal dosimetry made simple. Radiat Prot Dosimetry 2003;105:421-5.  Back to cited text no. 10


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7], [Figure 8], [Figure 9], [Figure 10]

  [Table 1]

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