Determination of Iodine Uptake by Cancer Thyroid Cells with Μ-AMS and Gamma Detector

Determination of iodine uptake by cancer thyroid cells with µ-AMS and gamma detector

Ana Sofia Gusmão Gomes Thesis to obtain the Master of Science Degree in Biomedical Technologies Supervisor: Dr. Maria de Lurdes Gano Dr. Hélio Fernandes Luís

Examination Committee Chairperson: Prof. Maria Margarida Fonseca Rodrigues Diogo Supervisor: Dr. Hélio Fernandes Luís Members of the committee: Prof. Maria Micaela Leal da Fonseca

November of 2016

Abstract: Compared with the normal thyroid cells, thyroid cancer cells have a reduced iodine uptake.

Nevertheless, radioactive iodine is the principal treatment for thyroid cancer. The aim of this dissertation is to evaluate the possibility of the use of µ-AMS as an alternative to gamma counting techniques in studies of iodine uptake in thyroid cells. For the determination of the precision and accuracy of the gamma counter used in bioassay laboratory of radiopharmaceutical group in CTN, 3 tubes with 125I solution and an activity of 0,9 µCi were prepared. These were used for the determination of the geometric effect, the reproducibility of the measurements, detection efficiency and the detection limit for 125I. In order to determine the detection limit for 129I with LATR/CTN µ-AMS system, an AgI pellet was made and measured. Another pellet, using carcinoma cells, was made to mimic the extraction of 129I by thyroid. The study allowed us to conclude that, the Berthold LB2111 can detect a minimum of 9,6x107 125I particles (4%uncertainity) compared with 2,8x105 127I particles (1% uncertainty) by µ-AMS. Since cancer cells have a lower iodine uptake than healthy cells, the better detection limit of µ-AMS is a valuable tool in the study of iodine uptake of thyroid cancer cells. This study was the first to measure iodine by µ-AMS and demonstrated the possibility of this technique to decrease the sample mass by 2 orders of magnitude when compared with conventional AMS. This will compensate the difference in primary beam intensity between the two techniques.

Key-words: thyroid cancer, cells, iodine, µ-AMS, gamma detection

1. Introduction the type of counter used in this dissertation. The Well counters have a well shape, are Thyroid, the biggest endocrine organ of the extremely sensitive and commonly used for body, has a butterfly shape with two lobes, blood and urine samples, and they allow for these are connected by a strip of thyroid multiple measurements at the same time [9] tissues called isthmus. This organ releases [10]. the hormones triiodothyronine (T3) and These counters have a scintillation detector, thyroxine (T4) which control the metabolism, a photomultiplier, a pre-amplifier, an growth and maturation of our body [1] [2] [3]. amplifier, a pulse height analyser and a Differentiated thyroid cancer is the most temporizer. These are connected to a common endocrine cancer and is increasing computer which allow the visualization of the in incidence. For unclear reasons thyroid results [11] [12]. cancers occur about 3 times more often in The interaction of the incident radiation with women that in men, the risk pick is often in the detector results in the raising of the their 40-50 years. A diet low in iodine and atoms in detection material to excited states. radiation exposure are other proven risk When the atoms get back to their state level, factors of this disease [4] [5] [6]. they emit energy in the light form. These Compared with normal thyroid cells, thyroid photons are then transformed into electrical cancer cells have a reduced iodine uptake. pulses that are counted [12]. Nevertheless, beyond surgery, radioactive The Well counter has a strong geometric iodine is the principal treatment for thyroid dependence. The geometric efficiency is cancer. The main administrated radionuclide defined as the number of radiation incident is 131-odine (131I), with 8.04 days of half-life, on the detector in a given interval divided by an emission of a 606keV (maximum energy) the number emitted by the radiation source β particles and a 365 keV  radiation in the same time. The geometric efficiency emission [4] [7]. decreases with increasing source-to- Because of this duality, studies of iodine detector distance. Therefore, if the sample is uptake by thyroid cells are extremely not positioned correctly in the Well detector relevant. The most used protocols for these the overall efficiency will decrease, so the studies are based on 125 iodine (125I) and on size of the sample will influence the gamma detection [8]. efficiency of the detection. Since the detector 125I has a 54.49 days half-life, 35 keV -ray has a well shape, as the sample gets bigger, emission and a 4 keV /31 keV β particles the proportion of the radiation detected by emission. It has one-third of the 131I dose, is the detector gets smaller. The ideal sample cheaper than 131I and has a longer half-life volume for the best geometric efficiency is than 131I. These are some of the reasons why 1mL [13] [14]. 125I is the main radionuclide used in thyroid On the other hand, it is not possible to uptake studies. measure samples with an activity higher Gamma counting is based on the radioactive than 37 kBq, due to the increase of the dead proprieties of the particles. Well counter is time [15].

So, radioactive decay is a random process, from the sample surface. These particles emitted radiation measurements are form a secondary beam, and will be subjected to statistical fluctuations that are a analysed by the system. source of imprecision, furthermore, as said After being generated in the target chamber before Well counters are strongly dependent the secondary beam passes through the low on the sample’s size [16] [17]. energy transport system. This system transports the beam with a 10keV energy to µ-AMS is highly sensitive technique based the accelerator. on isotopic ratios measurements and not on On this system the beam passes through a radionuclides decay, which doesn’t restrict secondary Einzel, three sets of Faraday us to short half-life isotopes and gamma Cups (FC)’s and slits, deflectors plates X emission isotopes. µ-AMS uses 129I instead and Y, a low energy ESA and a low energy of 125I, a long lived isotope with 1,57x107 magnet. years of half-life [18] [19]. The LE ESA allows in one hand the vertical 129I decays with the emission of β particles and horizontal focus of the beam and on the with an energy of 154 keV and -ray other the deflection of the same. This device emission with 39keV. This isotope is usually will act as an energy filter. used to mimic 131I due to its lower dose. The LE magnet will allow the mass selection. µ-AMS measures 129I by measuring the The selected mass will be injected into the 129I/127I isotopic ratio. 127I is the only stable accelerator, which is a 3 MV Tandem iodine isotope. accelerator. This technique is a combination of two In the accelerator the beam changes its previous techniques, SIMS and AMS. It was polarity and will be accelerated. The polarity developed with the goal of combining the change process is called stripping. The capabilities of the SIMS technique in terms stripping process is the physical process by of in depth and spatial analyses of samples which the electrons in the outer shells of the with the AMS’s ability to resolve molecular atoms in the beam will be removed by the and isobaric interferences that limit the SIMS interaction with a gas, situated inside a tube technique. µ-AMS is basically a SIMS in the center of the accelerator, called the sample chamber connected to an AMS stripping channel. This process will system [20] [21]. dissociate all the molecules in the beam, µ-AMS is generally composed by an ion resolving all molecular interferences. After source, a target chamber, a beam transport the accelerator the beam will be monoatomic system, a Tandem accelerator and by a and composed of several positive charge detection system [20] [23] [24] [25]. states. The measurement in µ-AMS system starts After the accelerator, in the high energy with a negative beam production by the transport system, the beam will be analysed sputtering process. The sputtering process, again by a magnet and ESA like in the low in the µ-AMS technique, consists in energy side. This system transports the sputtering a sample with a positive beam, usually a Cs beam, extracting the particles

3 beam to the detector system, which will µL, keeping the same amount of 125I in each measure 129I. sample. The activity of each volume of the The detector system is composed by two sample was measured by the counter. different chambers. The first chamber is 2. Reproducibility composed by a FC, a PIPS detector and an ETP. The second, which is used when there Tube number 2 was measured in each of the is an isobaric interference, is an ionization detector’s channels, with the aim of verifying chamber. In this study we don’t have an the measurement’s reproducibility in all of isobaric interference, so 129I will be them. Each channel was measured 20 measured in the first chamber. times. So, since the sensitivity of the gamma 3. Linearity of the detector’s counting techniques is not enough to measure the uptake of some carcinoma response thyroid cells, the goal of this dissertation is to In order to understand the counter’s 129 measure the µ-AMS detection limit for I in reliability, and in order to calculate its order to see if it can be an alternative to the detection limit, dilutions were performed with gamma counting technique. the following dilution factors: 1, 1/2, 1/4, 1/10, 1/50,1/100, 1/500, 1/1000, 1/2000, 2. Materials and Methods 1/3000, 1/4000, 1/5000, 1/10000 and 1/20000. These solutions were measured in Determination of 125I by gamma detector the gamma counter. Each dilution was

In order to determine the Berthold LB2111 measured 20 times. detector’s accuracy and precision, 3 tubes 4. Efficiency with 500 µL and 0,9 µCi were prepared. Tube number 1 was used to study the To determine the detector’s efficiency, the geometric effect. Secondly, with tube absolute efficiency of a 125I source was number 2 we analysed measurement’s measured by a Camberra 2020, and reproducibility in the detector’s different compared with our detector measurements. positions. Finally, tube number 3 was used The Camberra 2020 was calibrated with a to assess the linearity of the detector’s 131Ba source. response. In the end, the gamma counter Determination of 129I by µ-AMS efficiency was calculated. Aiming to determine the 129I detection limit 1. Influence of the geometric effect for the LATR/CTN µ-AMS system, an AgI in the measured sample activity pellet was developed and measured.

Aiming to evaluate the sample’s geometric Then a breast carcinoma cells pellet was effect on the detector’s measurements, prepared in order to mimic the extraction of 129 water was progressively added to tube 1, I by thyroid cells. with the following values: 500 µL, 750 µL, 1. AgI’s pellet 1000 µL,1500 µL, 2000 µL, 2500 µL e 3000

This pellet was based on the precipitation of A 0,54 T magnetic field was applied by the

AgI following a mixture of AgNO3 and NaI low energy magnet. The value was solutions calculated based on the information bellow (table1). 퐴푔푁푂3 + 푁푎퐼 → 푁푎푁푂3 + 퐴푔퐼 (1) Energy 1,6x10-15 J Charge 1 The solution was prepared by adding 0,350 Mass 2x10-25 kg

5 g of AgNO3 to 9,5 mL of water and 0,308 g Velocity 1,2x10 m/s of NaI to 217,5 mL of water. Both solutions Magnet’s radius 0,3 m were mixed, and the new compound was left Table 1. Parameters used to calculate the magnetic for 15 minutes. Afterwards the precipitated field for the low energy magnet was removed and transferred to an oven at Just like in the low energy system, devices 40ºC for 24 hours. Finally, it was pressed in were adjusted in the high energy system as to a pellet using a Specac press. well, using the computer program. A magnet scan was performed in order to identify all 2. Measurement of the AgI pellet the different 127I charges states. Afterwards, The pellet was mounted on the µ-AMS’s a 0,84 T magnetic field was applied to the sample holder and loaded into the sample high energy magnet, in order to select the chamber (figure 1). The µ-AMS source was 127I3+ beam. This value was calculate using turned on and the ionizer was slowly heated the information bellow (table2). to around 1000ºC, followed by the heating of Energy 1,28x10-12 J the reservoir to 200ºC. Charge 3 Next the accelerator was turned on and the Mass 2x10-25 kg voltage increased to 1600 kV. Velocity 3,6x106 m/s Magnet’s radius 1,3m The low energy beam transport system was tuned using the Tandem Control Computer Table 2. Parameters used to calculate the magnetic field for the high energy magnet Program. The beam used was composed of 127I- ions. This program allows the selection The beam was conducted to the detection and adjustment of the different system chamber and measured using a FC. devices. Afterwards the magnetic field was set for 129I3+, and the detector was put in place. No counts were registered during a period of 15 minutes, which was expected since there was no 129I in the sample.

1. Cell’s pellet

In order to optimize the sample preparation

method for µ-AMS, iodine was extracted

from the cells samples and transformed into

Figure 1. Vacuum lock with and without sample AgI sample. Since no thyroid cells were holder. Below is a sample holder with the sample. 5 available adenocarcinoma breast cells, MDA Later 1 mL of KIO3 (1000µg/mL), 0,5 mL of

MB-231 were used instead. HNO3 and 200 µL of AgNO3 5% (m/v) were The Cell line was grown under sterile added to the funnel. conditions, supplemented with fetal bovine The solution was left for 18 hours in a dark serum 10% (v/v), inactive by the heat and place in order to precipitate. Finally, the with penicillin antibiotic solution 1%. Cells mixture was centrifuged at 2000 rpm for 5 were left in a humidified atmosphere minutes and was placed for 2 hours in an incubator (with 95% air and 5% CO2) at oven at 70°C. After that the funnel was 37ºC. The medium was changed each to 2 placed at room temperature for 48 hours. days. Cells were counted by an optical microscope Results with a Neubauer chamber. Counting and Determination of 125I by gamma detector cells’ viability were determined by exclusion criteria with trypan blue dye. Because of Radioactive decay is a random process; the membrane damage, this method is based on measurement of the radiation is subject to a the penetration of the solution only in the fluctuation that often causes a measurement non-viable cells. Finally, we counted the error. cells that didn’t manifest blue colour. Cells These fluctuations can be compared with were counted in the 4 squares of the theoretical models, if they are not consistent Neubauer chamber. The number of cells we will conclude an anomaly is present in the present in the solution was calculated by the measurement system. Oscillations in the following expression: results must not exceed ±5% of uncertain and ±10% of error. (2) 푁푢푚푏푒푟 표푓 푐푒푙푙 = Uncertainty was calculated through the ratio 푠푢푚 표푓 푚푒푎푛 𝑖푛 푒푎푐ℎ 푠푞푢푎푟푒 × 푑𝑖푙푢푡𝑖표푛 between the standard deviation and the × 104 average multiplied by 100%. The error was

estimated through the ratio between the Results were expressed by number of difference of the first measurement and the cells/mL. Then, for the pellet we used average, multiplied by 100%. 8,7x106 cells (11mL).

In one funnel an acid solution with 40 mL of 1. Influence of the geometric effect water, 2 mL of 4% C5H10O, 2 mL of H2O2 and in the measured sample activity 4 mL of 5M H2SO4, was prepared.

6 In another funnel the 8,7x10 lysed cells (11 Measurements show an error higher than mL) were placed. This funnel was 10% for sample volumes higher than 1500 centrifuged at 2000 rpm for 5 minutes. µL (22,1% of error). Consequently, we can Then, the medium was aspirated and it was conclude an acceptable accuracy for values added 11 mL of acidified solution to the until 1000 µL. For volumes higher than 1000 funnel with cells. Afterwards, the funnel was µL volume, there is no more linearity in the agitated by a vortex with an ultrasonic bath detector’s response (table 3). for 5-10 minutes.

Volume (µL) Ativity (cpm) Error (%) Regarding the detector’s detection limit, we 250 1 088 095,2 -1,5 considered it the lowest value that maintains 500 1 068 742,6 -3,3 the linearity between the measured activity 750 1 040 947,6 5,8 and the dilution factor. 1000 996 480,4 -9,8 From the table above (table4) we can 1500 860 882,4 -22,1 conclude that the value 567,3 cpm (4,4% 2000 719 294,0 -34,9 uncertainty and 1,88 error) for 1/2000

2500 606 103,8 -45,1 dilution was the minimum value measured

3000 512 244,2 -53,6 with uncertainty and acceptable error

values. Table 3. Volume vs measurement activity and corresponding error.

3. Efficiency 1. Reproducibility The absolute activity of the source in day 1

The variability of the measurements measured by Camberra 2020 equipment between the detector channels was was 15115349 cpm, 6,8 µCi. The average of considered acceptable, since both the the measurements of LB2111 detector in imprecision (0,45%) and the error (-0,9%) day 6 was 943111 cpm (0,5% uncertainty - were lower than ±0,5% and ±10% 0,28 error). The decay of the measurement respectively. Therefore, we can conclude by Camberra 2020 was calculated, so the that there is a good reproducibility of the value for the day 6 is 1414390 cpm. measurements between all the channels. Then, efficiency is,

2. Linearity of the detector’s 943111 퐸푓𝑖푐𝑖푒푛푐푦 = × 100 = 66,6 % (3) 1414390 response

Average Standard Uncertainty Error Dilution deviation (%) (%) factor (cpm) This value allowed us conclude that the

1 1021444,3 913,2 0,1 0,1 value 567,3 cpm is just 66,6% of the activity 1/2 515312,3 776,8 0,2 5,82E- of the sample. Then the sample’s activity is 05 1/4 251391,6 530,3 0,2 0,2 12,9 Bq. 1/10 100713,3 376,6 0,4 0,1 1/50 21664,6 204,8 0,9 -1,3 Formula (4) allows us to calculate the 1/100 10659,7 134,5 1,2 3,6 number of particles of 125I present in the 1/500 2107,9 40,9 1,9 2,4 sample with 12,9 Bq, 1/1000 1027,9 37,7 3,6 -0,1

1/2000 513,5 22,9 4,4 1,9 퐴 = 휆푁 (4) 1/3000 253,5 14,9 5,8 1,4

1/4000 240,5 15,5 6,4 -3,3 1/5000 200,5 125,8 62,5 -15,8 where, 휆 1/10000 90,2 16,1 17,8 -21,6 퐿푛[2] (5) 1/20000 34,1 11,2 33,1 -40,3 푡 = 1/2 휆

Table 4. Measurements relate to a fix volume of the sample and a variable concentration of the 125I solution’s.

Thus, A is 12,9 Bq and 휆 is 1,34x10-7 so N The equation bellow enables us to know the is equal to 9,6x107 particles, the minimum number of 127I particles related to this number of 127I particles detect by Berthold current. LB2111. 푄 푁푞푒 Determination of 129I by µ-AMS 퐼 = = (6) 푡 푡

1. Measurement of AgI’s pellet where I is the beam current, Q is the charge (where e is related The scan magnet program controls the low energy and high energy magnets. The plot in to electron charge and q to particle charge figure (2) was made using this program, and state, in this case 3+) and t is the time. shows the different charges state of 127I. Then, As can be seen in the figure, the beam 5,1 × 10−10 = 3 × 1,6 × 10−19 × N (127I) (7) intensities for each charge state of 127I were as follows; 3,6x10-10 A for 127I3+, 2,5x10-10 A 127 4+ -10 127 5+ -11 5,1×10−10 (8) for I , 1,8x10 A for I , 8,5x10 A for N (127I) = = 3×1.6×10−19 127I6+ and 3x10-12 A for 127I7+. 1,06푥109푝푎푟푡𝑖푐푙푒푠/푠푒푐표푛푑 Since 127I3+ had a higher current it was the state chosen to calculate the detection limit. This value allows us to calculate the Thus, after the beam transport devices were detection limit for 129I. optimized for 127I3+, the current measured in When determining µ-AMS’s sensitivity for a the FC of the chamber detector for 127I3+ was certain isotope the measurement time and 5,1x10-10 A. the size of the sample have to be taken into account.

127 -10 127 3+ Figure 2. The beam intensities for each charge state of I were as follows; 3,6x10 A for I , 2,5x10-10 A for 127I4+, 1,8x10-10 A for 127I5+, 8,5x10-11 A for 127I6+ and 3x10-12 A for 127I7+.

In terms of the measurement time, the higher detector chamber, 104 seconds the time of the time the higher the number of the the measurement, 8% the percentage of particles we can count, but also higher the ionization’s efficiency and 45% of 127I3+ uncertainty in the measurement associated particles in the high energy beam, we can with the beam’s instability. conclude that 2,8x1014 particles (6x10-8g) of So, to obtain an 1% statistical uncertainty a 127I are extracted from the sample. Since the minimum of 104 counts is needed. stoichiometric ratio of Ag and I in the sample Considering a minimum of 1 count per is 1:1, it will be necessary 4x10-8g of Ag, so second, this means a measurement time of the minimum size of the pellet would be 104 seconds. So, taken into a count this 1x10-7g. minimum of 1 count per second, which is By multiplication the R (formula 10) for the reasonable given the fact that a too high minimum number of 127I particles present in measurement time will impact the the pellet we can know that 2,8x105 is the uncertainty in the measurement due to the minimum number of 129I particles detectable beam instability in time, by using the isotopic by the system. ratio definition: Other important factor is the pellet’s volume. The volume is relevant because it is not R= n(129I)/n(127I) (9) advisable that the depth of the sputtered well should be greater than the lateral dimension, thus, the cube root of the volume represents It is possible to calculate the detection limit: the smaller lateral dimension of the beam. In 1 (10) this case, since we know the mass of the 푅 = = 1 × 10−9 1 × 109 sample we can know its volume, so the

minimum volume of the pellet is 1,6x104 µm3, Regarding the sample’s size, it is defined by the smaller lateral dimension of the beam is the amount of 127I and Ag added to the 25 µm. Since this technique has a focus original sample extracted from the cells. So capacity until 30 µm, the measured of this the size of the sample will define the amount pellet is feasible. of 127I added, which will define the isotopic It was also verified the existence of isobaric ratio. In conventional AMS the minimum interferences. No counts were registered for amount of AgI needed for a successful 129I3+, during a period of 15 minutes, which measurement is 1 mg [4]. Since µ-AMS uses was expected since there was no 129I in the a µ-beam, it is possible to successfully sample. This confirmed that there was no measure a much smaller sample, which contamination present in the system, namely means that the isotopic ratio 129I/127I will be by the 129Xe isobar. higher for µ-AMS than conventional AMS for the same analysis. This will in part 3. Discussion and compensate for the higher sensitivity of conventional AMS. conclusion So, if we considered a beam current of 1x109 127I3+ particles per second measured in the

Through the secretion of the thyroid’s can alternatively be used instated of the hormones, thyroid controls all the organs of gamma counter. Another advantage of the the human body. For the secretion of these µ-AMS technique is that it detects 129I hormones it is necessary a high iodine instead of 125I, thus presenting a lower risk concentration in the thyroid follicle. for the user, due to the much longer half-life It was demonstrated that thyroid cancer of 129I. tissues have a lower iodine uptake than The disadvantages of the µ-AMS technique healthy tissues. Nevertheless, beyond are the following: the measurement process thyroidectomy, 131I is the principal treatment is longer and more complex than in gamma of thyroid cancer. So, to evaluate treatment spectrometry and sample preparation is efficacy it is important to know whether cells more complicated. take in iodine. Even though iodine AMS studies are Gamma spectrometry is the most common common, this study was, to our knowledge, method for iodine uptake in vitro studies by the first to measure iodine with the µ-AMS thyroid cells. This method is based on the 125I technique. decay’s proprieties. The larger beam diameter of AMS enables a The study performed with the Berthold higher beam intensity than in µ-AMS, but on LB2111 gamma counter present at bioassay the other hand it needs a larger sample size. laboratory of radiopharmaceutical group in This usually means better detection limits by CTN allowed us to conclude that the 3 orders of magnitude when compared with maximum volume for samples in the well µ-AMS. detector is 1 mL. From this value onwards a Regarding sample size, literature defines distortion of the activity measurements is 1mg as the minimum mass of a sample that verified. This distortion is a consequence of can be measured by AMS. This study the solid angle decrease. Regarding the allowed us to conclude that the minimum detection limit, it was determined that it was amount of iodine for an µ-AMS 9,6x107 125I particles with 4% of uncertainty. measurement is 1x10-7 g, which represents For the µ-AMS technique the detection limit 1,6x104 µm3 of sample volume. This volume was determined to be 2,8x105 127I particles does not permit the handling of the sample. with 1% of uncertainty. This 1% uncertainty This led us to consider the minimum amount is the statistical uncertainty associated with that enables handling and mounting the the spectrum accumulation. The overall sample to be about 5x10-5 g. The 2 orders of uncertainty will have to take into account magnitude difference between the minimum beam instabilities in the 104 measurement usable sample masses for µ-AMS and AMS time. However, this uncertainty should be means that for the same analyses the below 4%. amount of 127I added to the sample will be 2 Then, as the thyroid does not distinguish orders less and therefore the isotopic ratio in iodine isotopes, cancer cells have a lower a µ-AMS AgI sample will be 2 orders of uptake than healthy cells and since µ-AMS magnitude higher, which will somewhat has a better detection limit, this technique compensate for the better sensitivity of AMS.

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