|Year : 2013 | Volume
| Issue : 5 | Page : 29-35
|Date of Web Publication||29-Nov-2013|
Source of Support: None, Conflict of Interest: None
|How to cite this article:|
. Miscellaneous. Indian J Nucl Med 2013;28, Suppl S1:29-35
Development of radioimmunoassay procedure for rat insulin
Jayula Sunil Sarnaik, Inder Singh Rawat, Vijay Kadwad, Nagalingam Sivaprasad
Radiopharmaceuticals Programme, Board of Radiation and Isotope Technology, BRIT Vashi Complex, Navi Mumbai, Maharashtra, India
Objective: Type 2 diabetes is a common disorder presently affecting more than 100 million people worldwide. In diabetes research, rat models are extensively used for testing of various therapeutic agents. We at BRIT have modified the human insulin radioimmunoassay (RIA) kit intended for the measurement of insulin in human serum for the measurement of relative insulin concentration in rat serum samples. Materials and Methods: Guinea pig anti-insulin antibody to porcine insulin was from Fitzgerald, USA. 125 I was from Radiopharmaceuticals Division, BARC. Rat serum was obtained locally. Multi-well gamma counter from Startec, Germany was used. Rat serum depleted of insulin by charcoal treatment was used to prepare insulin standards. The tracer and antibody concentration were optimized to obtain an optimal standard curve. The modified assay based on sequential saturation employs insulin standards from concentrations of 3.75 μIU/mL to 200 μIU/mL and separation system was based on 22% PEG containing 0.1% Tween-20. Results: The method was validated for accuracy, precision, linearity and sensitivity. Percentage recovery ranged from 89% to 112%, Coefficients of variation for intra-assay and inter-assay variability for 6 different serum samples ranged from 2.4% to 10% and 5.6% to 13.9% respectively. Parallelism was only evident when insulin-depleted rat serum was used as a diluent. Additionally, the antibody used in this study cross-reacts well with rat insulin as determined by linear serum dilution curves. Insulin standards prepared with insulin-free serum along with separation system based on 22% PEG minimized the matrix effects in the assay. The developed assay can measure the relative insulin concentration from 2.0 μIU/mL to 200 μIU/mL. Conclusion: The modified insulin assay provides a reliable tool for the measurement of relative rat insulin concentration in studies using rats as animal models. The developed assay procedure is being extensively used by investigators engaged in diabetes related research at various Pharmacy Colleges and Research Institutes of India for drug development.
Development of two-step ria for the measurement of free triiodothyronine in human serum
Rani Gnanasekar, Jayula Sunil Sarnaik, Namperiel Chacko Joseph, Vijay Kadwad, Nagalingam Sivaprasad
Radiopharmaceuticals Programme, Board of Radiation and Isotope Technology, BRIT Vashi Complex, Navi Mumbai, Maharashtra, India
Objective: Development of a simple, user friendly and valid two-step radioimmunoassay format based on antibody coated tubes for the measurement of free triiodothyronine (FT3) in human serum. Materials and Methods: The system uses a polyclonal anti-T3 antibody with an affinity constant of 0.6 Χ 10 11 L/mol. Radiolabelled triiodothyronine was prepared by radiolabelling diiodothyronine with 125 I by chloramine-T oxidation method and purified over Sephadex G-25 column. FT3 standards of concentrations ranging from 0 to 30 pg/mL were prepared in T3 free human serum. 0.14 M Tris-saline buffer pH 7.4 and 0.035 M Tris-saline buffer pH 7.4 with 0.1% Tween-20 were used as assay buffer and wash buffer respectively. Results: Assay covers a range of 0-30 pg/mL with the sensitivity of 0.4 pg/mL. Anti-T3 antibody concentration, reaction time, reaction volume and sample volume were optimised to yield 1% extraction efficiency which is mandatory for a valid free hormone measurement. Analytical assay parameters such as intra assay variation and inter assay variation were within acceptable limits. Recoveries at FT3 concentrations 1.5, 2.8 and 4.8 pg/mL were 90, 102 and 92.5% respectively. Progressive dilution of hypo and euthyroid samples up to 1:96 times yielded virtually constant values validating the assay procedure, whereas a hyperthyroid sample with FT3 concentration of 9.9 pg/mL declined to 60% at 1:96 times dilution as expected. Euthyroid range established using the developed system is 1.6-4pg/mL. The FT3 concentration of clinical samples measured using the optimised procedure correlated well with the commercial kit value (r = 0.94). Conclusion: Simple, user friendly and valid immunoassay procedure developed as per the guidelines of American Thyroid Association can be used for the routine measurement of free triiodothyronine in human serum.
Development of indigenous immunoradiometric assay kit for thyroid stimulating hormone based on magnetizable cellulose
Shubhangi Manoj Mirapurkar, Vijay Kadwad, Nagalingam Sivaprasad
Radiopharmaceuticals Programme, Board of Radiation and Isotope Technology, BRIT Vashi Complex, Navi Mumbai, Maharashtra, India
Objective: To develop user-friendly immunoradiometric assay (IRMA) kit formulation for human thyroid stimulating hormone (TSH). Estimation of circulating levels of TSH in human serum using ultrasensitive immunoassay procedure is very important in detection, differential diagnosis and management of thyroid disorders. The main function of TSH is the regulation of synthesis and release of the thyroid hormones; triiodothyronine (T 3 ) and thyroxine (T 4 ) by a negative feedback effect on the target organ, the thyroid. Hence, TSH estimation is the most sensitive indicator for the diagnosis of hypothyroidism and hyperthyroidism. We have developed a simple and user friendly IRMA kit formulation for TSH using antibody coupled magnetizable cellulose particles. Materials and Methods: In house produced magnetizable cellulose was used for the preparation of capture antibody. 125 I from Radiopharmaceuticals Division, BARC was used for the preparation of detector antibody. hTSH monoclonal antibodies from various commercial sources were screened and evaluated for identification of suitable detector - capture antibody pair. The identified detector antibody was radiolabeled with 125 I and capture antibody was covalently linked to magnetizable cellulose particles to form a stable solid support. Results: The optimised assay makes use of ready to use serum based standards with only three pipetting steps and has an incubation of 2 h at room temperature. Standard range of 0-100 μIU/mL with a sensitivity of 0.037 μIU/mL can cover most of the physiological and diagnostic range. The intra-assay and inter-assay coefficient of variations were found be less than 10% and 15% respectively. The recovery varied from 96% to 102%. The developed kit was compared with the commercial kit by way of analysing clinical samples with regression equation, Y (developed kit) = 0.992 Χ (commercial kit) + 0.041. Conclusion: This product has got Radiopharmaceuticals Committee (RPC) clearance and is now available with BRIT.
Operation and maintenance of a nuclear medicine information management system at radiation medicine centre: An experience
NS Baghel, SH Moghe, SU Awasare, MGR Rajan, YR Karunanidhi 1 , PV Subbalakshmi 1
Radiation Medicine Centre, BARC, Tata Memorial Centre Annexe, Parel, Mumbai, Maharashtra, 1 Electronics Corporation of India Limited, Hyderabad, Andhra Pradesh, India
Introduction: Information Technology (IT) is widely used in Hospital Information Management Systems (HIMS) for reliable and prompt patient information. At Radiation Medicine Centre (RMC) a Nuclear Medicine Information Management System (NMIMS) had been developed, and in use since 2010. Aims and Objectives: To assess the benefits, challenges and requirements for operation, maintenance and improvements of a NMIMS for smooth and trouble free online data access. Materials and Methods: A customised web based NMIMS had been developed with M/s ECIL, which is facilitated by an IBMX 3650 M3 Server, thin-clients/desktop PCs and Windows 2008; server operating system and MS-SQL 2005 server software using a dedicated LAN to avoid virus attacks. The various modules have been developed to meet the requirements of different activities pertaining to various stages of investigations. The NMIMS has been in operation since 2010 and has undergone various debugging and modification processes. The database backup is regularly taken automatically as well as manually in removable hard disks. Results : The initial experience of having NMIMS has been challenging as the routine activities were shifted to NMIMS from registration procedure onwards. More than 26,000 registrations are completed using NMIMS for various investigations till now. The server is maintained to keep maximum uptime as patient appointments etc is not possible without NMIMS now. The workflows have become convenient and smoother as the availability of resources are known online to accommodate extra patients immediately and also status of the investigations known online. The existing NMIMS provides only a text-based information and no images are stored with patient information, a Picture Archiving Communication System (PACS) integration to the existing database system is being procured to archive images online. Conclusion: The browser-based online database system NMIMS in RMC has been extremely useful. The patients' appointments and investigation procedures are managed very smoothly. A PACS integration is an essential requirement in order to facilitate complete information of patient studies including images for online prompt and reliable information and management.
Radiorespirometry evaluation of ethnomedicinal plant extracts for anti-tuberculosis properties
Bhavesh Tiwari, 1 Savita Kulkarni, Sunita Shailajan, 2 Sasikumar Menon, 1 Muktikanta Ray, 1 MGR Rajan
Herbal Research Lab, Ramnarain Ruia College, Matunga, 1 Radiation Medicine Center, BARC, C/O Tata Memorial Hospital, Parel, 2 Institute for Advanced Training and Research in Interdisciplinary Science, Sion, Mumbai, Maharashtra, India
Objectives: The emergence of Multi Drug Resistant Tuberculosis is as a major threat to the control of tuberculosis in India. Hence, discovery of new drug candidates is essential to combat this form of tuberculosis. Plants are a natures repository of various compounds which have bacteriostatic or bacteriocidal properties. Radiorespiromery procedure utilizes 14 C labeled carbohydrates to enumerate growth index of bacteria before the onset of visible colonies thereby making it a rapid method for detecting fastidious organisms like Mycobacterium tuberculosis. This study utilized radiorespirometry method to evaluate the effect of products from ethanomedicinal plants against the growth of M. tuberculosis. Materials and Methods: Ethyl acetate, hexane and hydroalcoholic extracts from Cuscuta reflexa, Caesalpinia bonduc, Mangifera Indica, Carissa carandas, Woodfordia fruticosa and Euphorbia hirta were prepared. Phytochemical fingerprint was developed using HPLC and/or HPTLC. Preliminary screening for anti-TB properties of extracts was performed by using REMA. Fractions exhibiting anti-TB activity were further evaluated by Radiorespirometry. In Radiorespirometry a dual biphasic vial system was used utilizing 14 C-actate as a carbon source and LJ media as the specific culture media. Results: Twelve of the 30 extracts demonstrated anti-TB properties by REMA at concentrations ranging from 12.5 to 25 PPM. These extracts were further evaluated for anti-TB properties by Radiorespirometry. Three compounds were found effective against 10 6 bacilli in radiorespirometry. Conclusion: Radiorespiromertry is a rapid method for screening of compounds for their anti-TB properties. Three extracts showing inhibition of growth for M. tuberculosis will be further processed to purify the active anti-TB principle and the enrichment factor will be monitored using radiorespirometry.
Role of technetium-99m ethyl cysteinate dimer perfusion single-photon emission computed tomography in medically refractory epilepsy: A pilot study
Ashmi Manglunia, Shwetal Uday Pawar, Gundu Hari Tilve
Department of Nuclear Medicine, KEM Hospital and Seth G.S. Medical College, Mumbai, Maharashtra, India
Objective: Epilepsy is a chronic neurological disorder that can be managed by medical treatment or in refractory cases, by surgery. The success of surgery relies on accurate pre-surgical localization of the epileptogenic focus, which remains a diagnostic challenge. Functional imaging modalities are promising tools for this as they reflect the functional pathology associated with the seizure. This study evaluated the patients referred for interictal and ictal Technetium-99m (Tc-99m) Ethyl Cysteinate Dimer (ECD) SPECT scintigraphy for localizing the epileptogenic foci by comparing with MRI, video-EEG and post-surgical outcome. Materials and Methods: 27 patients who were referred to our Nuclear Medicine department for ictal and interictal brain Tc-99m ECD SPECT scintigraphy were assessed. These were also subjected to video-electroencephalographic (V-EEG) monitoring and magnetic resonance imaging (MRI). The patients who underwent surgery were then followed up for a period of 1 year to assess seizure free status. Results: The mean age of the included patients was 24.2 years (SD 10.5 years). Out of 27 patients, 13 underwent surgery and remained seizure-free on 1 year follow-up. 8 refused surgery due to bilateral changes and 6 were excluded because of neuropsychological risks. MRI showed corresponding abnormality as SPECT in 5 out of 27 patients (18.5%). EEG failed to localize the seizure focus in 16 patients (59.2%). Tc-99m ECD SPECT findings correlated with post-surgical outcome in all 13 operated patients. Conclusion: In cases with bilateral structural changes on MRI or an essentially normal MRI, Tc-99m ECD Brain Perfusion SPECT plays an important role during the pre-surgical evaluation of patients with refractory partial epilepsy.
Development of a prototype automated module for synthesis of 177 Lu-DOTA-Peptides using a microcontroller and human machine interface software
YR Nitin, HH Shimpi, MGR Rajan
Radiation Medicine Centre, Bhabha Atomic Research Centre, TMH Annexe, Parel, Mumbai, Maharashtra, India
Objective: To develop a prototype automated modular system for synthesis of Lu-177 DOTA-peptides. Materials and Methods: The prototype automated system for the routine production of 177 Lu-DOTA peptides is based on ATMega 8, 8 bit AVR Microcontroller with 8 k Bytes In-System Programmable Flash Memory, which is controlled by HMI software that sends commands to the hardware controller board through the USB port. The control signal then drives the solid state relays (SSRs) that control the hardware (solenoid valves, heaters etc.) as per the time-list created to carry out the steps required for the synthesis. The hardware is mounted on an aluminum frame with the reaction vial and reagent vials connected through various valves by suitable inert and radiation resisitant tubing. The reaction vessel is housed in a lead pot for radiation shielding and is heated by way of a copper tube to provide a conductive jacket for the reaction vial. The HMI software was developed using the Microsoft Visual Basic. NET which also provides an active Graphical User Interface which shows all the connections like the heater and inert gas lines along with reactant and product vials. The software provides PC based control of the Lu-177 DOTA-Peptide module. Nitrogen pressure of 4 Bar facilitates fluid transfer. The graphics change according to the events to depict the flow of reactants and products making it easier for the operator to understand which step of the synthesis is in progress. This software also provides graphical monitoring of process state and temperature while keeping it constant at required limits. The software also features temperature logging, final report generation and printing capabilities. Results: The prototype synthesis module has been tested with non-radioactive solutions and performs as expected. It will be tested in trial operations after radiological safety testing. Conclusion: The prototype automated module is easy to operate and serves the purpose of reducing the radiation dose to the radio pharmacist. It provides provision for automated wash and purification of the product without or with minimal manual intervention.
A duplex microarray immunoassay for thyroid hormones using track-etched membranes
Bharti Jain, MGR Rajan
Radiation Medicine Centre, Bhabha Atomic Research Centre, C/o T.M.H Annexe, Parel, Mumbai, Maharashtra, India
Objective: The aim of the study was the development and validation of a duplexed microarray immunoassay (MI) for the measurement of serum T4 and TSH. Materials and Methods: The MI was performed by immobilizing antibodies on polycarbonate track-etched membranes. The T4 was tested in competitive immunoassay format and TSH was tested in sandwich immunoassay format. 125 I-T4 and 125 I-TSH monoclonal antibodies were used for detection and quantification. Membranes were imaged using PhosphorImager (Typhoon Trio) and the images obtained were analyzed using ImageJ analysis software to estimate the mean signal intensity of each spot. The assays were validated and compared to established methods. Results: In the duplex MI, there was no cross reactivity between the capture antibodies. The MI was sensitive (T4 - 0.12 μg/dl, TSH - 0.03 μIU/ml) and reproducible. The inter-assay %CV was 22.8%, 17.2% and 12.4% for T4 concentration of 3.8, 9.3 and 13.5 μg/dl and 7.8%, 13.3% and 15.4 % for TSH concentration of 1.6, 3.4 and 9.7 μIU/ml. The intra-assay %CV was 8.1% and 9% for T4 concentration of 3.5 and 12.1 μg/dl and 10.5% and 7% for TSH concentration of 2.2 and 8 μIU/ml. MI correlate significantly to established assays (MI =0.86 RIA −0.04, r = 0.92, P < 0.001, n = 35 for T4 and MI =1.01IRMA −0.70, r = 0.995, P < 0.001, n = 35 for TSH). Observed to the expected ratios for dilutional parallelism ranged from 77% and 143% for T4 and 80% and 126% for TSH. Conclusion: These results describe the development of a sensitive, specific and cost-effective MI that is economical on sample volume and produces values similar to traditional RIA. By multiplexing immunoassays for multiple targets of interest, increased amounts of data can be acquired from a particular sample with a corresponding reduction in the time and cost required for each analysis.
Estimation of microalbuminuria and advanced glycation end products by immunoassays in diabetes mellitus, hypertension and pregnancy induced hypertension for the early diagnosis of nephropathy
Smita N Gawandi, MGR Rajan, Meera Venkatesh1, AV Kulkarni2, S Gangawane2, A Chakrabarti2, SV Kedare2, Vaishali Patke2
Radiation Medicine Centre, 1 RPHD and 2 BARC Hospital, Mumbai, India
Objectives: The prevalence of non-communicable diseases like diabetes mellitus (DM) and hypertension (HTN) is growing worldwide. Both the diseases are associated with development of nephropathy if not controlled effectively. Microalbuminuria (MAU) is recognised as an excellent predictor for nephropathy in abovementioned diseases. Additionally, the timely detection of Advanced Glycation End Products (AGEs) is also emerging as an important prognostic factor for diabetic and non-diabetic nephropathies. The early screening for MAU and AGEs by immunoassays represents a useful and relatively inexpensive clinical tool to control the nephropathy incidence in such circumstances. Materials and Methods: The study subjects comprised of DM (n = 182), HTN (n = 50) and PIH (n = 70) groups. Diabetic and hypertensive patients were selected from B.A.R.C. hospital and PIH subjects were selected from K.E.M and Vashi Municipal Hospital. The early morning urine samples and blood samples were collected from all the patients for MAU and serum AGEs estimation respectively. MAU levels were estimated by in-house RIA kit. MAU-RIA kit consisted of HSA standards ranging from 0 to 200 μg/ml, 125 I labeled HSA and anti-albumin antibodies coupled to magnetic particles. The serum CML levels were estimated by in-house RIA. The CML-RIA consisted of CML-BSA standards covering a range of 0-0.5 μg/ml, 125 I labeled CML-BSA and anti CML-KLH antibodies. The serum total AGEs levels were determined by commercial ELISA kit. Results: The levels of MAU, CML and AGE-BSA were significantly higher (P < 0.001) in DM, HTN and PIH patients than controls. The prevalence of MAU was 30%, 22% and 70% in DM, HTN and PIH subjects studied in this study indicating that these subjects were at risk to develop nephropathy. The median levels of MAU in normal, diabetic, hypertensive and PIH subjects were 0.8, 15.7, 17.5 and 39 μg/ml respectively. The MAU levels ranged from 0-30, 0-281, 0-276 and 12-91 μg/ml in normal, DM, HTN and PIH subjects respectively. The mean CML levels were 0.10 0.06, 0.38 0.12, 0.31 0.14 and 0.25 0.06 μg/ml in normal, DM, HTN and PIH subjects respectively and the mean AGE-BSA levels were 1.03 0.23, 1.95 0.81, 1.4 0.36 and 1.76 0.59 μg/ml in normal, DM, HTN and PIH subjects respectively. Upon comparison, MAU and AGE-BSA levels showed reasonably better correlation when compared to MAU and CML levels. Conclusion: Increased MAU and AGEs levels in DM, HTN and HTN subjects indicated that they were susceptible to develop renal complications. Early diagnosis of nephropathy risk by MAU and AGEs-detection by immunoassays would be more practicable than using tedious analytical techniques. It will help clinicians in identifying and regular screening of high-risk subjects. Eventually, the timely renoprotective measures would certainly reduce incidence of nephropathy in diabetes as well as in hypertension and PIH.
Comparative evaluation of enzyme linked immunoadsorbent assay for detection of microalbuminuria with existing radioimmunoassay technique
Smita Gawandi, Vanita Gadagkar 1 , MGR Rajan
RIA Services, Radiation Medicine Centre, BARC, TMH Annexe, Parel, 1 Ruia College, Matunga, Mumbai University, Mumbai, India
Objectives: The minimally increased urinary albumin levels are referred to as Microalbuminuria (MAU) in the absence of any urinary tract infection and acute illnesses. MAU is predictor of Diabetic nephropathy and indicator of cardiovascular disease in Hypertension and Pre-eclampsia in pregnancy induced hypertension. MAU is expressed as Urinary Albumin Excretion Rate (UAER) in the range of 30-300 mg/day which is equivalent to 30-200 μg/ml assuming average urine output of 1 L/day. MAU is not detected by conventional dipstick-methods and requires a sensitive assay like immunoassay for its estimation. RIA for MAU detection was standardized previously and well validated. RIA is robust immunoassay with convenient detection step; however, many of the laboratories do not have equipments for setting up of RIA, license to handle radioactivity or proper radioactivity disposal system. Hence, alternative immunoassay methods (for example ELISA) are preferred more often in these circumstances. Therefore, ELISA was standardized for MAU detection and its performance was compared with existing MAU-RIA. Materials and Methods: MAU-RIA was previously standardized using Human Serum Albumin (HSA) from Sigma Chemicals for preparation of standards. The anti-HSA antibodies were raised in rabbits and HSA was labeled with 125 I by using iodogen coated tubes. The performance of MAU-RIA was validated by performing various Q.C. parameters and correlating the results with commercial DPC kit. A protocol for MAU-ELISA was recently standardized by using HSA standards, anti-HSA antibodies raised in rabbits, HRP labeled anti-rabbit antibodies. The optimization of different parameters viz. the quantity of HSA to be coated on the ELISA plate wells, volume of sample, titer of primary and secondary antibody, volume of the substrate and the incubation conditions was carried out. Quality Control parameters like sensitivity, intra- and inter-assay variation, recovery test and parallelism test were analyzed. The performance of the developed MAU-ELISA was compared with the existing MAU-RIA Results: The sensitivity of the assay was around 3 μg/ml. The intra- and inter-assay variation was within 10 and 15% respectively. The results of the recovery and parallelism tests were within acceptable limits. Excellent correlation was observed between developed MAU-ELISA and existing MAU-RIA procedures (r = 0.97, P < 0.001, n = 135). Conclusion: The standardized ELISA for MAU detection was comparable with the existing standardized RIA. MAU-ELISA can be opted over MAU-RIA in primary health care centers where RIA processing set-up is not available.
Quality assurance tests of dual positron emission tomography-computed tomography systems using National Electrical Manufacturers Association NU-2 2007 standards
Deepti Rathod, Swasti Dixit, Sujith Rai, Anand Zade,B Rajashekharrao, A Velumani
Nuclear Healthcare Limited, Kopar Khairane, Navi Mumbai, Maharashtra, India
Objectives: To assess the performance of two numbers of PET-CT Systems installed at our centre, using National Electrical Manufacturers Association (NEMA) NU-2 2007 standards. Materials and Methods: The following tests were performed on two GE Discovery 600 PET-CT scanners using recommended phantoms, as per NEMA NU 2-2007 standards. Results:
Conclusion: The QA tests undertaken on both the scanners at our institute demonstrated an excellent performance characteristics meeting the NEMA NU-2 2007 standards and requirements of AERB.
Interfacing radioimmunoassay laboratory with hospital information system for efficient patient service
MK Ray, J Kumarsamy, Chandrakala Gholve, Smita Gawandi, NS Baghel, R Asopa, MGR Rajan
RIA Services, Radiation Medicine Centre BARC, TMH Annexe, Parel, Mumbai, Maharashtra, India
Objectives: Non availability of automation in radioimmunoassay (RIA) labs has been one of the biggest reasons for commercial establishment not adapting RIA methods, which is otherwise is a, truly open-equilibrium immunoassay system, where trouble shooting by the user is possible. Recently we have upgraded our RIA services by introducing a suitable interface for 2 way communication between RMC data base (DB) and RIA lab. We report our experience in fully automating RIA Services at RMC. Materials and Methods: Automation of RIA LAB was carried out in three steps: (i) We procured a Stratec SR - 300 in 2005 and successfully transferred our magnetic particle RIAs and coated tube IRMA to it and (ii) automated the patient data entry and reporting by installing a total laboratory management system (TLM), which established a two-way communications between the SR-300 and the lab staff for scheduling the assays and transfer the assay results in an error free format for reporting (iii) we installed an interface custom made for us connecting TLM with the RMC DB as a client. Results: (1) The interface allows to download patient details such as name, hospital ID, Test requisition to the TLM by click of mouse. (2) TLM assigns unique autogenerated Lab Number to each sample. TLM prepares acquisition list for different test and communicates the same to SR 300 for processing RIA. (4) After the assay is over, processed data from Sr300 is communicated to TLM electronically. (5) Only results processed within limits of Quality Control flag is accepted and patient values is electronically exported to RMC DB. (7) The lab turn around time (LTAT) for receiving and reporting 75 patient values has dramatically decreased from 80 to 90 min to less than a minute. (8) The TLM auto generates the Shewhart chart which saves user from manual updating of QC. Conclusion: Automation of RIA Services by interfacing TLM and RMC DB has (i) enhanced patient services, (ii) made RIA cost-effective comparable to commercial chemiluminescent systems, (iii) improved laboratory audit, (iv) because lesser manual intervention, human induced error was avoided. RIA Lab automation decreased the time of stay of users in radioactive lab.
Early experience with Sm-153 EDTMP for bone pain palliation at a tertiary cancer centre in the Hilly state of Uttarakhand
Vandana Kumar Dhingra, Nisha Bhatia
Department of Nuclear Medicine, Cancer Research Institute, HIHT, Dehradun, Uttarakhand, India
Objective: In a cancer centre located in the periphery of the hilly state like Uttarakhand using isotopes with short half life is challenging. We describe our early experience for bone pain palliation in patients with Sm-153 EDTMP. Materials and Methods: Initial patients treated with Sm-153 EDTMP over first 6 months of the current year were included in the study. A dose of 1 mg/kg of Sm-153EDTMP was injected in all patients. Hematological, biochemical parameters were kept in check for the first month. Clinical assessment was done at 2 weeks, 4 weeks and therafter monthly.We also studied feasability of isotope procurement. Results: 19 patients were included in the study. Sm-153 was ordered as per requirement and transported as per guidelines. Patients had primary in breast (N = 6), prostate (N = 10) and at other sites viz. gall bladder (N = 1), oral (N = 1), unknown primary (N = 1). None of the patients developed severe bone marrow suppression. 13 patients experienced significant relief in pain within 2-4 weeks. These patients continued to have relief upto their last follow-up. Performance status doing routine activities on their own improved in all the patients who experienced pain relief. 2 patients expired due to other complications (brain metastasis and disease progression) within 1 month of therapy. 4 patients did not experience significant improvement in pain. Conclusion: We saw an overall response of 68.4% of pain relief in metastatic cancer of the bones. This is in agreement with literature. In our experience, though procurement being logistically demanding due to its short half life, Sm-153 is safe and can be used in a well selected group of patients even in peripheral centers like ours.
Clinical outcome of samarium-153-edtmp therapy in patients with clinically aggressive malignancies excluding prostate and breast cancer
Barai Sukanta, Murthy Siddegowda, Gambhir Sanjay
Department of Nuclear Medicine, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
Objective: Painful skeletal metastases are common in advanced cancers. We report the clinical outcome of Samarium-153-EDTMP therapy in patients with clinically aggressive malignancies excluding prostate and breast cancer having multiple painful skeletal metastases. Materials and Methods: One hundred twenty-two patients with a mean age of 63.2 years with metastatic cancer other than prostate and breast primaries were treated with a single bolus infusion of Sm153-EDTMP (37 MBq/kg). All patients had multiple painful bone metastases and most had inadequate pain relief from conventional analgesics regime. Visual analog Pain score, Karnofsky, ECOG performance status, and blood count were evaluated before and up to 24 weeks after treatment. Results: Mean duration of follow-up was 18.7 weeks. Eighty-nine patients died within the 24 week observation period from terminal disease. Mean pain score at baseline was 8.3 2.18 and 1.6 1.13 after therapy. Responders maintained the pain palliation obtained till the end of 16 th week post therapy when effect starts waning with complete waning by 24 th week as documented by pain score of the 8.1 2.72 which is identical to initial pretreatment VAS score. Hematological toxicity was mild and reversible in all cases. Conclusion: Therapy with Sm153-EDTMP in cancer patients even with clinically aggressive malignancies with painful bone metastases offers clinical relevant pain relief with favorable safety profile.
Doxorubicin enhances 131 I-rituximab induced apoptotic celldeath in Raji cells
Chandan Kumar, BN Pandey 1 , Grace Samuel, Meera Venkatesh 2
Isotope Applications and Radiopharmaceuticals Division, 1 Radiation Biology and Health Sciences Division, Bhabha Atomic Research Centre, Mumbai, Maharashtra, India, 2 Division of Physical and Chemical Sciences, International Atomic Energy Agency, Vienna, Austria
Objective: Non-Hodgkin's lymphoma (NHL) is a group of hematological malignancies of which >90% is caused due to B cells. Although there are several therapeutic agents for NHL, they have their limitations for complete treatment. The objective of the study was to evaluate the enhancement in cell death when combining radioimmunotherapy with chemotherapy at optimum conditions thereby mitigating the limitations of each therapeutic modality. Raji cells which are CD20 positive Burkitt lymphoma, were treated with doxorubicin in combination with 131 I-rituximab (anti CD20) and cell toxicity and underlying signaling pathways were elucidated. Materials and Methods: 131 I-rituximab was prepared by iodogen method and purified by PD-10 column. Raji cells were grown in RPMI1640 complete media and seeded in 24 well plates at a density of 1 Χ 10 6 cells per well. Doxorubicin (0.5-10 μg/mL) was added in cultured cells and incubated for 4 h followed by addition of 1.85 MBq of 131 I-rituximab for 2 h. Cells were harvested after 6 h, washed with PBS and further incubated for 12 h. Harvested cells were counted for cell death in trypan blue dye. Extent of apoptosis was evaluated by ELISA method and cell death signaling pathways were established. Results: It was found that cellular toxicity was highest in Raji cells treated with doxorubicin (10 μg/mL) in combination with 131 I-rituximab. However, apoptosis study showed that DNA fragmentation and PARP cleavage were highest in Raji cells treated with doxorubicin (2 μg/mL) in combination with 131 I-rituximab. Western blotting showed that downregulation of bcl-xl and cleavage of PARP protein was highest in the Raji cells treated with doxorubicin in combination with 131 I-rituximab. Conclusion: It is concluded that doxorubicin has the potential to sensitize Raji cells by inducing cellular toxicity and apoptotic cell death induced by 131 I-rituximab.
Preparation of patient dose of 68 Ga-DOTA-NOC using 68 Ga eluted from novel 68 Ge/ 68 Ga BARC generator
Usha Pandey, Archana Mukherjee, Beena Shetye 1 , Rubel Chakravarty, Venkatesh Rangarajan 1 , Ashutosh Dash
Isotope Applications and Radiopharmaceuticals Division, Bhabha Atomic Research Centre, 1 Tata Memorial Hospital, Parel, Mumbai, Maharashtra, India
Objective: Gallium-68 is being widely used for PET imaging due to its favorable nuclear characteristics as well as its availability from a 68 Ge/ 68 Ga generator system. A novel 68 Ge/ 68 Ga generator using nanoceria-PAN composite as sorbent was developed indigenously at our end in BARC. Freeze dried kits of the somatostatin receptor-avid peptide DOTA-NOC were formulated for preparation of 68 Ga-DOTA-NOC using 68 Ga eluted from the 68 Ge/ 68 Ga BARC generator. Patient doses of 68 Ga-DOTA-NOC using 68 Ga eluted from 68 Ge/ 68 Ga BARC generator were prepared and imaging studies were carried out. Materials and Methods: 92.5-148 MBq (2.5-4.0 mCi) of 68 Ga from the 68 Ge/ 68 Ga BARC generator was eluted directly into the freeze dried DOTA-NOC kit vial using sterile 0.1 N HCl. The reaction was allowed to proceed for 10 min at 90C. Radiochemical yield of preparation was estimated by using paper chromatography using 50% aqueous acetonitrile. Before injection in patient for imaging, 10 mM EDTA was added to the kit vial and the radiolabeled preparation was filtered through 0.22 μm filter. Results: Under the optimized conditions, 68 Ga-DOTA-NOC could be prepared in >95% radiochemical yields. Consistency in 68 Ga labeling using kit vials was achieved when tested up to four months. Patient doses 55.5-92.5 MBq (1.5-2.5 mCi) were prepared and injected into patients for imaging studies. Conclusions: Successful preparation of patient doses of 68 Ga-DOTA-NOC using 68 Ga eluted from 68 Ge/ 68 Ga BARC generator using ready to use kit formulation of the peptide was acheived. Preparation of 68 Ga radiopharmaceuticals using kit based formulations is a cost effective and efficient method, especially for a hospital radiopharmacy.
Synthesis and biological evaluation of DTPA-chalcone derivative as single photon emission tomography/positron emission tomography tracer for imaging Aβ plaques
Kanchan Chauhan, K Ganesh Kadiyala, Anupriya Adhikari, Krishna Chuttani, Anil k Mishra, Anupama Datta
Division of Cyclotron and Radiopharmaceutical Sciences, Institute of Nuclear Medicine and Allied Sciences, DRDO, Delhi, India
Objective : Alzheimer's disease (AD) is a chronic neurologic brain disorder that impacts daily living through the progressive deterioration of cognitive processes. Accumulation of β-amyloid plaques in the brain is strongly associated with the pathogenesis of AD. Therefore, early detection of these accumulated plaques is important for the understanding and clinical treatment of the disease. Non-invasive techniques like positron emission tomography (PET) and single photon emission tomography (SPECT) may serve as a helpful tool in the diagnosis of AD in pre-symptomatic stages. So far many radiolabeled flavones have been reported for their high binding affinity to the Aβ- aggregates in in vitro studies. Towards the same direction, we have synthesised 68 Ga- and 99m Tc-labeled chalcone derivative as a PET and SPECT tracer for the diagnosis of β-amyloid plaques in the brain. Chalcone is a member of the class of flavonoids containing flavones. This derivative contains electron donating groups like dimethylamino, methoxy groups which play a critical role in the binding affinity to the Aβ-aggregates. Materials and Methods: Synthesis was done using chalcone derivative (E)-3-(4-(dimethylamino)phenyl)-1-(4-hydroxyphenyl)prop-2-en-1-one (Chal) obtained by Claisen-Schmidth condensation of 4-hydroxyacetophenone and N,N-dimethylbenzaldehyde in the presence aq. NaOH which was reacted with bromoethylamine-boc followed by boc cleavage to give Chal-NH 2 ((E)-1-(4-(2-aminoethoxy)phenyl)-3-(4-(dimethylamino)phenyl)prop-2-en-1-one). Its conjugation with chelate, diethylenetriaminepentaacetic acid gave (Chal) 2 -DT. Complexation of this DTPA analogue with cold Ga(III)chloride was carried out in water. Radiolabeling was done using 68 Ga and 99m Tc for biological evaluation. Results: (Chal) 2 -DT was synthesized in the high yield and characterized by spectroscopic studies. Molecular docking of derivatised compound with Aβ fibrils (2BEG) showed excellent binding with binding score as −8.8. Radiolabeling efficiency of 99m Tc-(Chal) 2 -DT, 68 Ga-(Chal) 2 -DT was >95% and >80% respectively. In vitro binding assay using Aβ aggregates (1-42) suggested high binding affinity with K d value in nanomolar range, transformation of the saturation binding to Scatchard plots gave linear plots suggestive of one binding site. Initial investigation of blood clearance showed a quick wash out from the circulation and biological half life were determined . Initial In vivo brain penetration was tested by injecting 68 Ga- and 99m Tc-(Chal) 2 -DT in normal rats, cortex and cerebellum regional brain tissue uptakes were determined at different time points which showed a good initial brain penetration and a rapid washout from the healthy brain. Conclusion : 68 Ga- and 99m Tc-(Chal) 2 -DT can be prepared in high radiochemical yield. High binding affinity to Aβ aggregates (1-42) by docking and in vitro binding studies and a good initial brain penetration along with a fast washout from the brain by in vivo biodistribution study in rats suggest that 68 Ga- and 99m Tc-(Chal) 2 -DT may be a useful PET tracer for imaging Aβ plaques in the brain of patients with Alzheimer's disease. The ability of the tracer to bind to Aβ plaques in the AD model is in progress.
In-vitro effect of high concentrations of L-thyroxine on micronuclei frequency in peripheral blood lymphocytes
Yogita S Raut, Uma S Bhartiya, Lebana J Joseph,Rohini W Hawaldar 1 , Muktikant Ray, MGR Rajan
Radiation Medicine Centre, Biomedical Group, Bhabha Atomic Research Centre, C/O Tata Memorial Hospital Annexe, 1 Tata Memorial Centre, Parel, Mumbai, Maharashtra, India
Objective: Thyroid hormones play a major role in normal growth and development of the body. However higher levels of these hormones have shown to change the activity of mitochondrial respiratory chain enzymes which may result in increased reactive oxygen species (ROS) production. This may lead to a possible damaging effect on the DNA of circulating lymphocytes. The objective of the experiment was to assess the genotoxic effect of high concentration of thyroxine in vitro in peripheral blood lymphocytes using cytokinesis blocked micronucleus assay. Materials and Methods: We have conducted an in vitro experiment in which the whole blood sample from 3 healthy volunteers was treated with increasing T 4 concentrations i.e 0.125 μM, 0.25 μM, 0.5 μM and 1.0 μM and processed by Cytokinesis-blocked micronuclei assay. Results: Significant increase in peripheral blood lymphocyte micronuclei frequency at T 4 concentration of 0.5 μM and above was noted when compared with basal T 4 levels and lower T 4 concentration i.e 0.125 μM in the experiment (P < 0.001). Conclusions: These findings demonstrate the in vitro genotoxicity of T 4 at and above 0.5 μM concentration. This observation denotes the necessity of careful handling of the acute T 4 overdose in vivo.
One-step IRMA for serum thyroglobulin in the follow-up of differentiated thyroid cancer patients
Chandrakala Gholve, J Kumarsamy, Archana Damle, SK Ghorui1, NV Patil1, MGR Rajan
Radiation Medicine Centre, BARC, TMH Annexe, Parel, Mumbai, 1National Research Centre on Camel, Bikaner, Rajasthan, India
Objectives: Thyroglobulin (Tg), is used as a tumor marker in patients with differentiated thyroid cancer (DTC) following thyroidectomy. At RMC, we assay between 4500-5000 samples each year for s-Tg with commercial IRMA kits (Izotop ) in DTC patients who visit for follow-up. Aim of the study was development and validation of a single-step IRMA for estimation of serum Tg in DTC patients. Materials and Methods: Standardization of Tg IRMA was carried out using a combination of anti-Tg polyclonal capture antibody raised in camels and a murine 125I-monoclonal antibody produced in-house. The standardized assay can be completed overnight. To evaluate the interference of anti-Tg autoantibody (TgAb) in s-Tg estimations by IRMA system, recovery tests were also conducted. Results: RMC-Tg assay has a satisfactory Bmax at 16-20% and NSB is <1%. The RMC-Tg compared well with the Izotop kit in respect of assay performance, required sensitivity, precision and clinically targeted to monitor DTC patients. A good correlation was observed in samples from controls with a regression equation of RMC-Tg IRMA=1.431 Izotop -0.209 [r=0.97, n=48, P<0.001]. Following regression equations were obtained on comparing s-Tg in in-house IRMA and Izotop kit in samples with DTC.
- Std pools prepared from patients serum samples (0-250 ng/ml)
RMC-Tg IRMA = 0.9211 Izotop + 1.2712 [r=0.96, n=90, P <0.001]
- Stds prepared in human hormone free serum (0-350 ng/ml)
RMC-Tg IRMA = 0.7179 Izotop + 1.5958 [r=0.96, n=90, P <0.001]
- Stds prepared in kit matrix with 1% BSA (0-250 ng/ml)
RMC-Tg IRMA = 0.5566 Izotop + 1.1047 [r=0.97, n=90, P <0.001]
The % recovery was calculated for each sample and the results were compared with those obtained using Izotop kit. On comparison, 83/90 patients were negative by all the assays. Remaining 7 samples were Positive, Positive/Negative or Non-Reliable by one or more assays. Conclusions: Satisfactory results have been obtained and the recovery test can be used in complement with the in-house Tg IRMA assay in the follow-up of DTC patients which would help clinicians in better decision making.
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