New Trends for Radiation Applications in Medicine

Transcription

1 New Trends for Radiation Applications in Medicine C.B. Zamboni, S. Metaiorn Instituto de Pesquisas Energéticas e Nucleares, IPEN/CNEN, Brasil M.A. Rizzutto, S. Bernardes Universidade de São Paulo, IFUSP, Brasil M.R.A. Azevedo Universidade de Santo Amaro, UNISA, Brasil

2 Focus To show the advantages to use alternatives analytical techniques (NAA and XRF) for clinical practices Why use these techniques? The main advantage is the viability to use small quantities of blood (0.1mL) comparatively to conventional analyses performed in serum or plasma (using at least 0.5 to 10mL) using conventional procedure: anticoagulant and reagents To wait for coagulation To perform the centrifugation using BLOOD all theses steps are eliminated

3 Since 2000 NAA technique has been applied for clinical finality at IPEN/CNEN-SP, and more recently XRF ( good results ) (2004) in collaboration with Blood Banks and Research Centers from Brazil we started a Project: Determination of REFERENCE VALUES (RV) for elements of clinical relevance in human blood using alternatives analytical techniques (NAA and XRF) it is still in progress Why to determine Reference Values of inorganic elements in blood? Conventional analyses are performed in serum or plasma, so the limits (normal range) for clinical practice are well known for Brazilian population However, for blood these limits are not established Partners: UNISA, IFUSP, Banco de Sangue Paulista, UNIFESP, Instituto Butantan (SP), HEMOPE, UFPE, LABEX/UNICAMP, UFF, UNIBAN Financial Support: CNPq, FAPESP, CAPES, IPEN/CNEN

4 Reference Values in blood for Brazilian population Environment Physiological Pathological Geographic localization Gender Age: years Weight: above 50kg Life style Factors: smoking and/or drinking habits medicine intake physical exercise Blood banks perform screening tests: HIV, Sífilis, Hepatitis B and C and Chagas Health Donors

5 Br, gl -1 Na (gl -1 ) log S, g/l K, g/l Blood concentrations of Ca, K, Mg and S for inhabitants of Brazil by age and gender 0,8 0,6 Blood samples Female Male Ca, gl -1 0,4 0,3 Whole Blood Male Female 0,24 0,20 0,4 0,2 0,2 0,0 Mg, gl -1 0,050 0,025 0, Age (years) > 450 Age (years) 0, > 50 5 Age (years) Blood concentrations of Br and Na for inhabitants of Brazil by gender and geographic localization 0,03 1,92 0,02 1,76 0,01 1,60 0,00 Male Female São Paulo Male Female Recife Male Female São Paulo Male Female Recife

6 Sample Preparation blood is collected in a vacuum plastic tube (without anticoagulant or reagents) attached to the donor s arm immediately after the collection 100 L of blood is transferred to ~2.5 cm 2 pieces of filter paper (Whatman, n 41) and dried using an infrared lamp The sample can be storage without refrigeration (non destructive method)

7 Experimental Procedure using NAA The samples are collected and prepared at blood bank and after, they are irradiated at the IEA-R1 nuclear reactor (3-4.5 MW, pool type) at IPEN/CNEN-SP Blood sample 2. Detail of sample irradiation at the Automatic Pneumatic Station 3. Automatic Pneumatic station 4. Nuclear Reactor 5. HPGe detector (GEM ORTEC) 6. Nuclear instrumentation and data system (Amplifier - Ortec 671 and MCA - Ortec 919E) 7. Quantitative analysis using (in-house) Ativação software 8. Storage samples

8 S keV counts V keV K keV K keV Fe keV contagens Na keV Ca keV Cl keV 1657keV (SECl keV) Co keV Na keV 609keV Br keV 857keV (SE Na 24 ) Br keV Br keV counts I keV I keV Br keV Br keV Br keV Mg keV Cu keV 511keV Br keV Br keV Gamma ray spectrum of blood using NAA Considering the optimized conditions: Blood samples : 100 ± 5%µL Irradiation time : 240s Thermal neutron flux: ~ n cm -2 s -1 Counting Time : 900s Blood Bg blood Bg Mg keV + Mn keV Blood Bg keV (DE Cl 38 ) B channel B channel B channel Blood Bg 2500 Blood Bg 6000 Blood Bg channel B channel B channel Simultaneous determination of elements of clinical relevance (such as: Cl, Ca, I, K, Mg, Na, )

9 Na meq/l Cl meq/l K meq/l Na meq/l Cl meq/l K meq/l Comparison (serum): NAA & conventional techniques Serum analyses using NAA Serum sample Soro Análise por Ativação com nêutrons 68% 95% Soro Análise por Ativação com nêutrons 68% 95% 10 8 Soro Análise por Ativação com Neutrons 68% 95% Amostras Amostras Amostras Serum analyses using Ion Specific Electrode (Na and K) and Titrymetry (Cl) Soro Eletrodo Íon Seletivo 68% 95% Soro Eletrodo Íon Seletivo 68% 95% 10 8 Soro Eletrodo Íon Seletivo 68% 95% Amostras Amostras Amostras The results are in agreement considering ±1SD

10 Limitation of using NAA: The necessity of nuclear reactor or accelerator We decided to check the viability of using XRF technique Experimental Procedure The measurements were carried out an EDXRF Spectrometer SHIMADZU Co., model Rany 720 (50kV, 100μA-variable, Rh target) at Centro de Quimica e Meio Ambiente/IPEN Blood and Serum samples fixed on filter paper were analyzed using: Si(Li) detector Fixed time: 100s Line emission Kα for all elements: Ca, Cl, Cu, Fe, K, Mg, Na, P, S, Zn Quantitative Analysis was performed using Fundamental Parameters method software from Shimadzu The results (NAA, XRF and Conventional) are in a good agreement This alternative procedure can be used for clinical finalities

11 In progress To optimize the performance of the (PXRFS) for clinical analyses Portable X-Ray Fluorescence Spectrometry Experimental Conditions using PXRFS The XRF analysis was performed using MINI X spectrometer from Amptek, with Ag X-Ray tube at Universidade de São Paulo / IFUSP The characteristics X-Ray fluorescent intensities (K lines) were measured with a Si Drift detector (25 mm 2 x 500μm / 0.5 mil) with Be window (1.5") The blood samples (100μL) were irradiated for 300s (fixed time) using 30kV and 5µA excitation) The quantitative analysis (using K lines) was performed using WINAXIL software program.

12 Counts Si drift detector (US$9,900.00) Mini X-Ray tube with Silver target (US$6,500.00) Blood spectrum using portable spectrometer (PXRFS) Whole Blood 3500 Ar S Cl Ar K Ca Fe Fe B Channels

13 Fe, g/l Iron concentration on blood samples by NAA and XRF (using PXRFS) 1,0 0,8 Confidence Interval: 68% 95% Experimental Data: : NAA : XRF 0,6 upper limit 0,4 using the same blood sample 0,2 lower limit whole blood samples The portable apparatus can be an used in clinical laboratories

14 Conventional Analysis/ Technique Brometo (Br - ) / Colorimetry Conventional Analyses Biological material/ Quantities (ml) Execution time Days (*) Cost US$ Blood / Conventional Analyses Serum: Ca, Cl, Fe, K, Mg, K, Na, Zn Blood: Br Execution Time: 1-15days Cost: US$ 394,26 Calcemia (Ca)/ Colorimetry Serum / Alternative techniques Quantity: 100 µl Cloremia (Cl)/ Dry Chemistry Iron/ Dry Chemistry Magnesemia (Mg)/ Colorimetry Calemia (K)/ Dry Chemistry Natremia (Na)/ Dry Chemistry Zinc (Zn)/ Atomic Absorption Spectrophotometry * Laboratório Fleury S/C Ltda. São Paulo Actualized: Serum / Serum / Serum / Serum / Serum / Serum / NAA (IEA-R1 nuclear reactor) Blood: Br, Ca, Cl, Fe, I, K, Mg, Mn, Na, Rb, S and Zn Cost (irradiation) : US$ 23,00 EDXRF (Rh X-Ray tube) Blood: Al, Br, Ca, Cl, Fe, K, Mg, Mn, Na, P, S and Zn Cost: US$ 25,00 PSXRF (Ag X-Ray tube) Blood: Ca, Cl, Fe, K and S Cost: US$ 25,00

15 Conventional Analyses Conventional Analysis/ Technique Calcemia (Ca)/ Colorimetry Cloremia (Cl)/ Colorimetry Iron/ Plasma Emission Spectroscopy Magnesemia (Mg)/ Colorimetry Urine/ Quantities (ml) Execution Time Days (*) Cost US$ Conventional Analyses Urine: Ca, Cl, Mg, Fe, K, Na and Zn Cost: US$ Alternative techniques Quantity: 100 µl Urine: Br, Ca, Cl, Cu, Fe, I, K, Mg, Na, S and Zn Cost (irradiation): US$ Calemia (K)/ Ion-Selective Electrode Natremia (Na)/ Ion-Selective Electrode Zinc (Zn)/ Plasma Emission Spectroscopy Perspective the cost of Iron analysis can be reduced using these alternative techniques *Laboratório Fleury S/C Ltda. São Paulo Actualized: 13/06/13

16 Conventional Clinical Analysis Alternative Analytical techniques Serum and Plasma samples Blood sample 2ml serum 4 ml blood 0.5m serum 0.5m serum 1 ml serum 3 ml plasma 2 ml serum 0.1 ml Colorimetry De Lauber Inductively Coupled Emission Spectrophotometry NAA and XRF Ion Specific Electrode Br* Dry Chemistry Titrymetry Cl Fe Calorimetry Ca Zn* Simultaneous determination: Br, Cl, Ca, Fe*, I**, K, Mg, Mn, Na, P, S, Zn* K, Na Mg Execution Time FRX: <30 minutes NAA:1 hour Execution time: 1-3 days; * execution time ~ 7-10 days I: not determined in serum, plasma or blood **I determined by NAA small quantity of blood, simplicity in the sample preparation, simultaneous determination and reduced cost

17 Economic Viability According to the last ProEx (Programme External Quality Assessment of clinical laboratories in Brazil) carried out in billion clinical examinations were performed: average of 8 exams per inhabitant/brazil average cost of R $ 13,00/exam estimated an increase ~2% clinical examinations in 2013 average of 3% of biochemistry exams (Na, Cl, K, Fe) average of ~ 1% other (Mg, Ca, Br and Zn) ~56 million of clinical examinations can be done using these alternatives techniques

18 Others applications: The use of these alternatives procedures (NAA and XRF) are very useful when the biological material is scarce in veterinary medicine to perform clinical analyses in small size animal (mice, rabbits, hamsters, ) For analyses of other body fluids (saliva, urine, serum ) Quality control of biological products developed for applications in public health area (antivenom, new reagents, ) To give an idea I will shown some applications

19 Mg gl -1 Na gl -1 K gl -1 Br gl -1 Ca, gl -1 Cl gl -1 patients (dialysis treatment) PATIENTS WITH CHRONIC KIDNEY DISEASE Situation in Brazil: Percentile variation (2000/11) increase of 114% PUBLIC HEALTH PROBLEM Nowadays there are 687 units of Hemodialysis Census / SBN (Nephrology Brazilian Society) B According to ANVISA the treatment evaluation is performed by clinical analysis of: Na, Cl, K and Mg in serum (before and after dialysis) and Ca and Fe monthly During 2004/2006 NAA was applied to investigate blood of patients submitted a dialysis (before and after) and the results were compared with conventional (good agreement) 0,04 blood collection: before HD after HD upper limit 1,0 0,8 5 upper limit 0,6 4 0,02 0, patients lower limit 0,4 0,2 0,0 upper limit lower limit patients lower limit patients These alternative procedures can be used in the units dialysis treatment 3,0 0,12 3 2,5 0,08 upper limit 2 upper limit 2,0 1,5 upper limit Collaboration: IPEN, HEMOPE, UNISA, UFPe 0,04 1,0 lower limit 0,00 lower limit 1 lower limit 0, patients patients patients

20 concentration Diagnostic: Saliva of patients with periodontal disease PERIODONTAL DISEASE Submandibular and small glands: 1-5% Parotid: 20-25% Sublingual: 70-75% It is an infectious inflammatory disease that affects the gum tissue and support the teeth (bone loss). According to Public Health Service (ANVISA, 2009) severe forms affects between 5 to 20% of the Brazilian population. Whole saliva is the mixture of these glandular secretions, bacterias and epithelias cells 1 0,8 0,6 0,4 Control Disease C Ca /C S 0,4 0,3 0,2 Control Group Periodontal Patients Ca and S in whole saliva are good biomarkers 0,2 0,1 0 Cl, g/l K, g/l Br, mg/l S, g/l Mg, g/l Na, g/l Ca x100 I, ug/l Collaboration: IPEN, UNIBAN/Brazil mixed non-stimulated saliva samples The use saliva (100μL) and the PXRFS can be used in ontological office

21 concentration, g/l concentration proportions Analysis of saliva tick (Amblyomma cajennense species) from Brazil It has medical applications Saliva collection The saliva was collected into capillary tubes (75 length x 1.5 mm diameter) attached to the hypostome Each tick produces ~40µL of saliva ~30 tick were necessary for extraction of ~ 500μL 100 µl was fixed on filter paper The elements concentrations determined in saliva samples 6 Saliva comparison of different Ambyamma species cajannense americanum variegatun % 3 75% 2 50% 1 25% 0 Cl Na S Br x100 K P Ca Mg x10 I x samples % Na K Collaboration: IPEN/ Instituto Butuntan, Brazil Cl

22 Veterinary Medicine: to perform clinical analyses in small size animal Test of new vaccines and medicines Br, gl -1 0,024 0,012 0,000 A/J MDX Experimental Data Human Reference Value SJL H III Human NZB B 10 I max CB57 Ca, gl -1 0,8 0,6 0,4 0,2 SJL Experimental Data Human Reference Value A/J H III Humano Dmd CB57 Mg, gl -1 0,15 0,10 0,05 H III Dmd Experimental Data Human Reference Value B 10 Human CB57 I min SJL A/J 0, whole blood samples whole blood samples 0, whole blood samples 5 4 Experimental Data Human Reference Value 0,15 0,10 Experimental Data Human Reference Value 2,5 Experimental Data Human Reference Value CB57 Cl, gl -1 3 NZB SJL A/J L III H I III BALB/ c max I min Human MDX B 10 CB56 Mg, gl -1 0,05 H III Dmd B 10 Human CB57 I min SJL A/J Na, gl -1 2,0 1,5 BALB/ H c III Imax NZB MDX A/J B I SJL min 10 Human L III whole blood samples 0, whole blood samples whole blood samples Collaboration: IPEN/ Instituto Butuntan

23 Potential for Future applications

24 Clinical practice performed in premature infants A premature baby (~1kg) has ~100mL of blood circulating in the body and depending on the dysfunction/treatment the child may need two to three blood samples/day. Consequently, blood samples for laboratory tests in pediatric practice are the main causes of transfusions in premature infants According to the Children's Institute at Brazil are performed ~ blood collections/month. The use Blood sample (100μL) fixed on paper and the Portable Spectrometer can be a promissory alternative procedure

25 concentration ratio concentartion ratio concnetration ratio Pre diagnostic of Acute Renal Failure Analysis of elements in serum, urine and kidney in Wistar rats with Acute Renal Insufficiency using NAA 2,5 2 1,5 Serum Before During After 1 - kidney with acute renal failure 2 - normal kidney 3 - kidney with chronic renal failure Control Kidney IRA 1 0,5 2 0 Br Ca Cl Cu K Mg Na S 1,5 1 Urine 0,5 8 Before During After 0 Br Ca Cl K Mg Na S Br Ca Cl I K Mg Na S Collaboration: IPEN/ UNIFESP, Brazil The correlation analyses between kidney & urine and kidney & serum suggest that: I and Cu can be used as biological markers for ARF

26 Laboratório de Espectroscopia e Espectrometria das Radiações- LEER IPEN/CNEN-SP C. B. Zamboni; S. Metairon; J. A. G. de Medeiros; L. C. Oliveira; L. Kovacs and I. M. M. A. Medeiros czamboni@ipen.br and collaborators: Maria Regina A. Azevedo (UNISA) Márcia A. Rizzutto (IFUSP) Suene Bernardes (IFUSP) Ivone Mulato Sato (CQMA/IPEN) Denise Vaz de Macedo (UNICAMP) Lázaro Alessandro Soares Nunes (UNICAMP) Thiago F. Lourenço (UNICAMP) Hugo R. Lewgoy (UNIBAN) Osvaldo Augusto Sant Anna (Instituto Butantan) Simone. M. Simons (Instituto Butantan) Daniella G. L. Oliveira (Instituto Butantan) Roberto Meigikos dos Anjos (UFF) Fernanda T. Borges (UNIFESP) Miriam F. Suzuki (CBT/IPEN) Carlos R. Bueno Junior (Instituto de Biociências/USP) MUCHS GRACIAS!!!!!