Report on the National Seminar on Radioimmunoassays Radiation Medicine Centre, B.A.R.C

REPORT ON THE NATIONAL SEMINAR ON RADIOIMMUNOASSAYS JANUARY 16-20, 1978

RADIATION MEDICINE CENTRE, B.A.R.C.

DEPARTMENT OF ATOMIC ENERGY AND WORLD HEALTH ORGANISATION REPORT ON THE NATIONAL SEMINAR ON RADIOIMMUNOASSAYS

JANUARY 16—20, 1978

RADIATION MEDICINE CENTRE, BARC TATA MEMORIAL HOSPITAL ANNEXE JERBAI WADIA ROAD, PAREL BOMBAY 400 012

ORGANISED UNDER THE AUSPICES OF THE DEPARTMENT OF ATOMIC ENERGY & WORLD HEALTH ORGANISATION CONTENTS

Page Preface ...... 3 Administrative Responsibilities ...... 6 List of Participants 7 Programme ...... 11 Basic Requirements of RIA in India ...... 14 a. Radioisotopes for RIA's ...... 15 b. Kits for RIA 17 c. Availability of Antibodies 19 d. National Pituitary Agency ...... 20 e. Well-counter for RIA 21 f. Radiation Protection Aspects of RIA 23 Review of Discussion following the Session .. .. 26 State of Art of RIA's in India 28 Trigger Sessions 36 a. Quality Control of RIA's 37 b. Usefulness and limitations of RIA's in clinical diagnosis 39 c. RIA's in tropical diseases •...... 42 d. Centralised assay services ...... 44 Recommendations and Guidelines ...... 46 Backword 51 Bibliography ...... 53 PREFACE

This National Seminar on Radioimmunoassays was the second National Seminar jointly sponsored by Department of Atomic Energy and World Health Organisation and organised at Radiation Medicine Centre. The first one on 'Nuclear Medicine in India' was held in December 1976. The present Seminar on Radioimmunoassays was distinguished by the participation of Dr. Rosalyn Yalow, Nobel Laureate in Medicine for 1977. She, along with Dr. Solomon A. Berson, discovered the technique of Radioimmunoassay and nurtured it through the early years with hard and meticulous work to establish its usefulness in medical science. Her presence at the Seminar was a continu- ous source of pride and inspiration to both the organisers and the participants. The Seminar was inaugurated by Dr. R. Ramanna, Director, Bhabha Atomic Research Centre on January 16, 1978. In his inaugural address he said that the Bhabha Atomic Research Centre had been honoured to have Dr. Rosalyn Yalow, who was spending three weeks at Radiation Medicine Centre as a visiting scientist. He outlined the usefulness of the technique, its simplicity and applications in our country. This method is comparatively cheaper than most nuclear medicine procedures. He suggested that Indian scientists could explore the possibility of applying this technique in the diagnosis and epidemiological surveys of infectious diseases. This Seminar has provided a good opportunity in bringing together scientific workers in this field to review the work done so far, to air their grievances, to describe the difficulties which they encounter and to try and find practical solutions to thejr problems. This is an essential exercise of scientific progress especially in this field as its application involves assistance from several government and public institutions. With the rapid and exponential growth of the medical applications of this methodology, it is no longer considered as an esoteric or an exotic facility but an essential tool in the diagnostic armamentarium to be used as commonly as a routine clinical chemistry examination. The object of the Seminar was to evaluate : • a. The application of this technique in the routine medical care of patients and its use in public health programmes. b. To evaluate whether a Central or Regional Assay Service could effectively fulfil the needs of a region or district. c. The logistics of establishing such a service and to study the cost against the benefit to the public. d. To arrive at some estimates for funds required, and to find out which agencies could be asked to aid in organising these facilities. The Seminar concerned itself towards answering some of these questions and arriving at some helpful solutions. The inputs required to get an assay going are simple. It puts very little demand on technical knowledge and skill when kits for radioimmunoassays are available. Towards this progress, the Isotope Division of Bhabha Atomic Research Centre has taken pioneering steps in making available several useful "kits" at a fairly economical price. The establishment of the Indian National Pituitary Agency under the auspices of the Indian Council of Medical Research will provide and make available reagents for the assays. This would relieve the burden on Indian scientists who at the present have to go out with a begging bowl for the basic ingredients of the assay system. The Electronics Division of Bhabha Atomic Research Centre and the Electronics Corporation of India Ltd., would make available easy to use, efficient and what is very important cheap manual gamma counters which would be within the budget of most medical colleges and hospitals. The Institute for Research in Reproduction, Bombay, Endocrinology Department of Post-graduate Institute, Chandi- garh and Radiation Medicine Centre, Bhabha Atomic Research Centre, have contributed a great deal towards the growth and development of these techniques in India. In addition programmes for training technologists and physicians are offered by all these Institutes. One of the problems for discussion at this seminar was to evaluate how effective and useful these training programmes have been and whether any changes were needed in the duration and type of courses offered. This Seminar has given not only members of Radiation Medicine Centre but a lot of other scientists a rare opportunity to speak, hear and discuss their problems and difficulties with a Nobel Laureate, for which the organisers would like to thank the World Health Organisation for their generous help in making this Seminar a reality. Dr. M. Thangavelu, Advisor, Regional Office of World Health Organisation, New Delhi, participated in the Seminar and his contribution in the scientific deliberations of the seminar was most worthwhile.

Dr. (Mrs.) A. M. Samuel Administrative Responsibilities

1. Chairman of Plenary Dr. K. Sundaram Sessions Director, Bio-medical Group, BARC, Trombay, Bombay 400 085. 2. Scientific Co-ordination Dr. R. D. Ganatra Head, Radiation Medicine Centre, BARC, Tata Memorial Centre, Parel, Bombay 400 012. 3. Preview of Scientific Shri G. V. Kadival papers, their duplication and distribution 4. Rapporteurs for plenary 1. Dr. (Mrs.) A. M. Samuel sessions and trigger 2. Shri G. V. Kadival sessions 5. Accommodation and 1. Shri P. Ramanathan Transport 2. Shri M. C Patel 6. Catering 1. Mrs. L. S. Mani 2. Shri A. V. Borkar 7. Editing of the report 1. Dr. (Mrs.) A. M. Samuel of the proceedings 2. Shri G. V. Kadival List of Participants

From Outside Bombay 1. Dr. (Mrs.) Rosalyn S. Yalow, Ph.D. Senior Medical Investigator, VA, Veterans Administration Hospital, 130, West Kingsbridge Road, Bronx, New York 10468, USA. 2. Dr. M. Thangavelu, Regional Adviser in Non-communicable Diseases, World Health Organisation, World Health House, Indraprastha Estate, Ring Road, New Delhi 110 001. 3. Dr. J. P. N. Chansouria, Research Officer, Surgical Research Laboratory, Institute of Medical Sciences, Banaras Hindu University, Varanasi 221005. 4. Dr. K. K. Ghosh, Assistant Professor, Post-graduate Institute of Medical Education and Research, Calcutta. 5. Dr. D. K. Hazra, Reader in Medicine, P. G. Department of Medicine. S. N. Medical College, Agra. 6. Dr. K. S. Jaya Rao, Deputy Director, Endocrinology Unit, National Institute of Nutrition, Indian Council of Medical Research, Jamai-Osmania, P.O. Hyderabad 500 007 (AP) 7. Dr. N. Kochupillai, Dept. of Medicine, All India Institute of Medical Sciences, New Delhi. 8. Dr. C. Parija, Asst. Professor of Biochemistry, M.K.C.G. Medical College, Berhampur, Orissa. 9. Dr. G. K. Rastogi, Prof, and Head, Dept. of Endocrinology, Post-graduate Institute of Medical Education and Research, Chandigarh. 10. Dr. (Miss) S. Sheela Rani, Laboratory of Endocrinology, Dept. of Biochemistry, Indian Institute of Science, Bangalore 560 012. 11. Dr. Brij. Saxena, Visiting Scientist to National Pituitary Agency, Indian Council of Medical Research, Institute for Research in Reproduction, Parel, Bombay 400 012.

From. Bkabha Atomic Research Centre, Bombay 1. Dr. K. Sundaram, Director, Bio-medical Group, BARC, Modular Laboratories, Trombay, Bombay 400 085. 2. Dr. V. A. Pethe, Head, Electronics Division, BARC, Modular Laboratories, Trombay, Bombay 400 085. 3. Shri S. Somasundaram, Head, Body Burden, Measurement Section, Health Physics Division, BARC, . Trombay, Bombay 400 085. 4. Dr. R. S. Mani, Head, Radiopharmaceutical Section, Isotope Group, Radiological Laboratories, BARC, Trombay, Bombay 400 085.

8 5. Mr. K. B. Shah, Radiopharmaceutical Section, Isotope Group, BARC, Trombay, Bombay 400 085.

From Institute for Research in Reproduction (IRR), Bombay 1. Dr. (Mrs.) S. S. Rao, Director, Institute for Research in Reproduction, Parel, Bombay, 400 012. 2. Dr. A. B. Sheth, Assistant Director, Institute for Research in Reproduction, Parcl, Bombay 400 012. 3. Dr. (Mrs.) U. M. Joshi, Assistant Director, Institute for Research in Reproduction, Parel, Bombay 400 012.

From other Centres 1. Dr. B. B. Gaitonde, Director, Haffkine Institute, Parel, Bombay 400 012. 2. Dr. A. J. Baxi, Blood Group Reference Centre, Seth G. S. Medical College, Parel, Bombay 400 012. 3. Dr. R. D. Kulkami, Prof, of Pharmacology, Grant Medical College, Bombay 400 008. 4. Dr. (Mrs.) S. M. Shahani, Professor Endocrinology, T. N. Medical College and B.Y.L. Nair Charitable Hospital, Bombay 400 008. 5. Dr. S. D. Bhandarkar, Hon. Asst. Professor of Endocrinology and Hon. Physician, Dept. of Medicine, G c Medical College and K.E.M. Hospital, Pa. Bombay 400 012. 6. Dr. R. D. Lele, Chief Physician and Head, Dept. of Nuclear Medicine, Jaslok Hospital and Research Centre, 15, Dr. G. Deshmukh Marg, Bombay 400 026.

From Radiation Medicine Centre 1. Dr. R. D. Ganatra 2. Dr. (Mrs) A. M. Samuel 3. Dr. (Miss) D. H. Shah 4. Mr. K. B. Desai 5. Mr. P. Ramanathan 6. Mr. M. C. Patel 7. Mr. M. N. Mehta 8. Mr. G. V. Kadival

10 NATIONAL SEMINAR ON RADIOIMMUNOASSAYS PROGRAMME

MONDAY—JANUARY 16, 1978 Inauguration Dr. R. Ramanna Director, B.A.R.C.

Session 1: RESOURCES 1. Radionuclides R. S. Mani 2. RIA kits K. B. Shah 3. Antibodies K. B. Deasi 4. Pituitary hormones A. R. Sheth 5. Counting equipment V. A. Pethe 6. Radiation Protection S. Somasundaram

Trigger Session 1 Quality Control of RIA—Introduction by B. B. Gaitonde RECENT ADVANCES IN RIA—I ROSALYN YALOW TUESDAY—JANUARY 17, 1978 Session 2 : RADIOIMMUNOASSAYS IN INDIA 1. Thyroid hormones N. Kochupillai 2. Diabetes G. K. Rastogi 3. Nutrition K. Jaya Rao 4. Sex hormones A. R. Sheth 5. Sterility and S, M. Shahani Fertility 6. Receptor bound c. S. Sheela Ra gonadotrophic hormones 7. Australia Antigen A. J. Baxi 8. Nucleotides C. Parija 9. Circadian Rhythms R. D. Kulkarni 0. Enzyme Immunoassays U. M. Joshi 1. Radioimmunometry D. K. Hazra

11 Trigger Session 2 USEFULNESS AND LIMITATIONS OF RIA IN CLINICAL DIAGNOSIS Introduction by S. D. Bhandarkar RECENT ADVANCES IN RIA—II ROSALYN YALOW

WEDNESDAY—JANUARY 18, 1978 Session 3 : REVIEW OF WORK AT DIFFERENT CENTRES 1. PGI, Chandigarh G.K. Rastogi 2. AIIMS, Delhi N. Kochupillai 3. BHU, Benaras J. Chansouria 4. IRR, Bombay S. S. Rao 5. PGMER Calcutta K. K. Ghosh 6. INMAS, Delhi S. U. Anjaria

Trigger Session 3 : RIAs IN TROPICAL DISEASES Introduction by R. D. Ganatra RECENT ADVANCES IN RIA—III ROSALYN YALOW

THURSDAY—JANUARY 19, 1978 Session 3—Continued 1. R.M.C. Bombay A. M. Samuel D. H. Shah

Session 4. ORGANISATION SUPPORT 1. W.H.O. M. Thangavelu 2. B.A.R.C. K. Sundaram Discussion and Comments

12 Trigger Session 4: CENTRALISED ASSAY SERVICES- Introduction by R. D. Lele RECENT ADVANCES IN RIA—IV ROSALYN YALOW

FRIDAY—JANUARY 20, 1978 RECOMMENDATIONS AND GUIDELINES RECENT ADVANCES IN —RIA—V ROSALYN YALOW

13 BASIC REQUIREMENTS FOR RADIOIMMUNOASSAYS IN INDIA

This chapter deals with the inputs required to establish radioimmunoassay procedures. Problems encountered by the scientists like the difficulties in obtaining reagents within the country were discussed. Some solutions to these problems was offered.

14 RADIOISOTOPES FOR RADIOIMMUNOASSAYS

An important raw material required for a successful radioimmunoassay is the radionuclide for labelling the antigen or other ingredient sought to be measured. The principal characteristics required for the labelling radionuclide include (a) relatively long half-life, (b) high counting efficiency, (c) ready availability and low cost, (d) high specific activity and purity, (e) ready incorporation into the molecule without affect- ing the specific immunological properties of the compound labelled, and (f) radiation emission which ensures stability of the labelled compound and a reasonably long shelf-life. Considering the structure and properties of various hormones, antigens, vitamins, viral proteins, antibodies, drugs etc. of interest in radioimmunoassay, both "genuine" and "forced" labelling procedures can be adopted. The radioisotopes which are either already widely employed in radioimmunoassay or are of some potential interest are 1-125, 1-131, H-3, Co-58, Se-75, C-14, P-32 and S-35. The main interest is, however, centred round three isotopes, namely, 1-125, 1-131 and H-3. Pure natural gas containing 0.096% of Xe-124 is the target for the production of 1-125. For large scale production, xenon gas is enclosed in special zircalloy containers at pressures ranging upto 200 atmospheres and is irradiated at neutron fluxes of the order of 1013n Cnr2 Sec.-1 The impurities which arise as a result of side reactions are 1-126 (half life 13 days), Cs-137 and Ba-137m and traces of 1-127 (stable). Post irradiation cooling of about 60 days reduce the 1-126 contaminatoin to less than 2%. Under optimum irradiation conditions, 1-125 with specific activities upto 17 Ci/mg representing an isotopic abundance exceeding 90% can be obtained. Irradiation of natural Te containing 34.5% of Te-130 yields 1-131 with specific activities upto 30 Ci/mg representing approximately 25% isotopic abundance. To attain the highest specific activities, however, special precautions are to be taken to minimise or eliminate stable iodine production/contamination at various stages. Fission of U-235 can yield much higher specific activities ; but this method is less practicable for routine use. H-3 with almost 100% abundance can be produced by reactor irradiation of lithium alloy targets followed by processing and subsequent enrichment by chromato- 15 graphy or diffusion. These processes are practicable and economical only at the multikilocurie level of activity of H-3. The Isotope Group of Bhabha Atomic Research Centre does not as yet produce 1-125, H-3 and C-14. Arrangements have, however, been made to import these isotopes in bulk from abroad and supply to users.

16 KITS FOR RADIOIMMUNOASSAY

Radioimmunoassay techniques permit measurements of minute concentrations of virtually any substance of biological interest. The technique is used to trace peptide and non-peptidal hormones as well as to detect non-hormonal substances like enzymes, viruses, tumour antigens and several drugs including morphine, LSD and barbiturates; Three procedures are generally adopted for carrying out RIA by various institutions. i. Some Institutions which have developed considerable expertise in RIA prefer to develop and use their own lot of reagents including the labelled antigens, antibodies and self- standardised techniques for separating bound and free fraction. ii. Some other clinical and research centres, who also have developed expertise in RIA and are interested in carrying out RIA more routinely and in large numbers, procure ready to use RIA kits. These kits are now available from a large number of commercial suppliers. iii. Some centres where facilities and expertise for RIA have not been well established, get their samples analysed utili- sing such services offered by larger hospitals or institutions. Considering the large scale demand for the RIA and their potential importance in our country, Bhabha Atomic Research Centre—Isotope group has taken up a programme to develop and provide reliable and ready to use kits for RIA. Kits for insulin are available, while the kits for thyroid stimulating hormone, human growth hormone, digoxin, digitoxin, human chorionic gonadotrophin, hepatitis associated antigen, angiotensin I and II are under development. The kits consist of the following reagents: i. Labelled hormone ii. Standard hormone iii. High quality antibody iv. Reagents to separate antibody bound antigen from free antigen. Whereas pure hormones are procured from abroad, efforts are being made to make them indigenously by collaborating with various institutions. It is heartening to note that, the

17 recent establishment of the National Pituitary Agency by the Indian Council of Medical Research at the Institute for Research in Reproduction, Parel, Bombay will give a great impetus to the development of RIA programmes in our country. For procurement of antisera, we either collaborate with those institutions such as Institute for Research in Reproduc- tion, Bombay or Haffkine Institute, Bombay which have acquired considerable expertise in the field or we ourselves undertake immunisation programmes and produce the antisera in our Centre. Various parameters such as stability and purity of the labelled compound, sensitivity and specificity of the antisera are studied before the formulation of the kits. The kits are subjected to quality control tests such as non-specific binding, the binding of labelled hormone with the antibody in the absence of un- labelled hormone, inter-assay and intra-assay variation in pooled serum samples, recovery etc. The kit performance is checked at two to three intervals within the shelf-life of the kit, to as- certain its reliability. For the benefit of the institutions which do not, as yet, have facilities to carry out RIA; Bhabha Atomic Research Centre— Isotope Group offers a limited RIA service, for the analysis of serum samples. Future programmes include (i) development of new kits for hormones, and non-hormonal substances of biological interest, (ii) introduction of newer and simpler techniques for the separation of bound and free fractions for the existing kits.

18 AVAILABILITY OF ANTIBODIES

Many centres in India have been successful in raising antibodies to a variety of substances. A common problem that one faces is the difficult}' in acquiring specific antigens. Antibodies to albumin, thyroglobulin. triiodothyronine, thyroxine, thyrotropin, growth hormone, insulin and transferrin are used at Radia- tion Medicine Centre in RIA or other investigations. These antigens except thyroglobulin and in many instances even antisera have to be imported. This problem is common to most Indian workers. In fact a previous analysis (Hazra) in National Semi- nar on Nuclear Medicine (1976) revealed that all twenty Institu- tions engaged in doing RIA, had to depend upon imports of these antigens or antiserum from abroad. In this context it is imperative that a centre or centres should be entrusted with the work of processing and acquiring standards and raising antibodies for distribution to various laboratories in India. The advantages with respect to economy, quality control and administration are obvious.

19 NATIONAL PITUITARY AGENCY

A time has come that with the increased tempo of basic and clinical research in the country the requirements for hormones and the diagnostic reagents should be met from within the country and that less dependance should be placed on the generosity of the National Pituitary Agency for the supply of reagents. It was therefore felt that a Central Agency be established in the country to undertake this work which would also ensure the quality control of hormone assays. The establishment of a National Pituitary Agency (NPA) under the aegis of the Indian Council of Medical Research, requires very close and active collaboration of a large number of hospitals, institutes and their staff from all over the country. Prominent scientists from all over India have agreed to guide the activities of NPA. The major efforts of the* agency at this stage will be as follows: a) Collection of pituitaries on a national basis. Following centres have started collecting pituitaries: Bombay, Delhi, Chandigarh, Bangalore, Madras, Lucknow and Varanasi. About 1500 pituitaries were collected during a period of 4 months. With further organisation and co-operative efforts it should not be difficult to collect more than 5000 pituitaries per year. b) Purified hormones and antiseras will be calibrated against International Standards and made available for distribution to different workers in India. c) Requests for growth hormones and gonadotrophins for therapeutic use shall be considered by Medical Advisory Board of NPA. d) Attempts will be made to improve existing RIAs and develop simpler assays for protein hormones. e) National Pituitary Agency will offer laboratory facilities to other workers in India where such facilities cannot be established at their centres.

20 WELL-COUNTER FOR RADIOIMMUNOASSAY

The most essential instrument required for radioimmunoassay is a counter for the quantification of radioactivity. Gamma counters are used for gamma emitting isotopes such as the more commonly used 1-125 and 1-131. The most widely used detector for gamma emitters is the Nal well-counter. The best geometry is a 4 TT geometry and highest efficiency is obtained when all radiations are absorbed within the crystal. A small sample of low energy emitter positioned at the centre of the 2"x2" or 3"x3" Nal crystal through a hole will essen- tially detect all gamma radiations except minor losses escaping through the hole. Most well-counters presently in use employ Nal crystals If" in diameter, 2" in height with f" diameter, ty' deep well. Many variables affect the data obtained with a well-counter. Therefore, the conditions of measurement such as sample volume, the fraction of the pulse height spectrum included in the measurement, and whether the efficiency refers to the number of disintegrations or to the number of gamma radiations emitted must be stated. Most of the tests use 1-125 which decays to Te-125 with a half life of 60 days. The decay occurs in all cases by a process of electron capture, after which the Te nucleus has excitation energy of 35.5 Kev and the atomic structure has an inner shell vacancy. When this vacancy is filled there is emission of either an X-ray or an Auger electron. If the X-ray is from the K-shell it has an energy of 27.5 Kev and is easily detected by a Nal (TI) crystal detector. If it is emitted from any other shell it has insufficient energy to be detected. Auger electrons are not detected due to absorption in the source material. The excited nuclear state of Te-125 decays either by emission of a detectable 35.5. Kev gamma ray (in 7% of cases) or by internal conversion (93%) which results again in an inner shell vacancy with the same possibilities for emission as above. The various processes occur virtually simultaneously. Not all detectable quanta successfully interact with the detector so that some simultaneous double emission result in only a single interaction. Those double quanta which are successfully detected simultaneously produce a response which 21 is equivalent to an event of twice the normal energy. This produces the second peak of the characteristic 1-125 pulse height spectrum. Because of low energy emitted by 1-125, the internal liner should have as low a thickness as possible. The liner could be preferably made of Beryllium. Maximum efficiency is obtained when plastic tubes are used. The setting up of the counter should be done carefully to optimise the signal to noise ratio. There is a large variety of equipment commercially available, starting with simple portable low priced counter, to com- pletely automatic multiradionuclide counters with preset time and count facilities and with background subtract arrange- ment. Many of them also have computing facilities which can be fed to available computer systems. Many of these systems use a through hole detector giving uniformity of counting efficiency regardless of varied sample volumes. The detector is also operated in a controlled temperature environment eliminating the inherent errors from changes in the light output of the crystal and the gain of the photomulti- pliers, due to ambient temperature variations. A new concept high through-put counter for 1-125 introduced recently is unique in many respects. It employs a multiple detector system with sixteen detectors matched to each other (within 1%). Each detector is surrounded by an effective shield of lead (350 micro) to ensure that no cross-talk can occur even with very highly active sources. The system has an excellent 1-125 counting performance of 75% efficiency at low background. With automation eliminated, there are no moving parts and reliability is unsurpassed. RIA and Competitive Protein Binding Assay result in non- linear dose-response curves when either the bound over free (B/F) or its reciprocal are plotted versus arithmetic dose. Routine dose interpolation with the use of the entire range of a non-linear standard curve is difficult and inaccurate. Restric- tion of dose interpolation to a small linear segment of the curve markedly reduces the useful range of the assay. Perhaps point- to-point and logit-log are most popular methods for curve fitting. Logit-log is the most versatile method. The only draw back in this method is the need for computations involving logs and antilogs which are slow on some desk-top calculators.

(22 RADIATION PROTECTION ASPECTS OF RIA

Decay characteristics of 1-125 1-125 with a radioactive half-life of about 60 days decay solely by electron capture to the first excited level (35.5 Kev) of Te-125 which in turn decays to the ground state either by emission" of 35.5 Kev gamma-ray (77o) or by internal conversion (93%). The vacancies in the inner electronic shells occur- ring due to electron capture and internal conversion are followed by emission of x-rays or Auger electrons. Dillman (1) has calculated the energies and intensities of electromagnetic radiations and electrons emitted in the decay of 1-125. 1-125 gives 1.43 photons per disintegration of which 96% are 27.4 KeV Te K x-rays and the resi 35.5 KeV gamma-rays. The most energetic electrons are of 32.4 KeV which have a range of about 21 um in tissue. Radiation dose from 1-125 Gavron and Fiege (2) have calculated the radiation dose due to 1-125, considering an exact model of normal thyroid follicles and taking into account the distribution of radioiodine. The radiation dose consists of two components—the electron component and the photon component. For 1 nCi of 1-125 per gram of thyroid, the electron dose-rate is 44 mrad/hr and the photon dose-rate is 21 mrad/hr. Taking into account the presence of small follicles, Gavron and Fiege have obtained a total dose-rate of 93 mrad/hr due to 1 nCi of 1-125 per gram of thyroid. For a maximum permissible dose-rate of 600 mrad per week, the maximum permissible thyroid burden of 1-125 works out to 770 nCi. This is reduced by a factor of 2 to take into account the enhanced biological effects of 1-125 and a maximum permissible thyroid burden of 300 nCi has been suggested. In Indian subjects the uptake of iodine is higher and the mass of thyroid is less than 20 g assumed for the ICRP Reference Man and the maximum permissible thyroid burden of 1-125 would be less (200 nCi) for Indian subjects. Safety Consideration The safe handling of unsealed radioactive sources includ-

23 ing 1-125 is discussed in several codes of practice and other publications from World Health Organisation, International Atomic Energy Agency, ICRP (3-5). A few papers have appear- ed in the literature which deal specifically with radiation safety aspects in RIA (6-8). The safety considerations would depend upon the amount of the radioisotope, its physical and radiochemical properties, its radiotoxicity and the types of operations performed. Apart from ensuring general safety in handling any radiochemical substance, work involving the labelling of proteins with 1-125 should be carried out in specially designed safety cabinets. Chesworth and Clarke (9) have described a safety cabinet suitable for this purpose. The safety cabinet should accommodate special tongs or devices to handle high specific activity solutions in vials. Area monitoring should be carried out in areas where 1-125 is handled. During and after iodination, air samples should be collected using special filters and the filters counted in a well-type scintillation counter. The air concentration of 1-125 should be kept below 4 x 10-° uCi/cms for 40 hr work week. Surface decontamination of concentrated free idoine should be carried out by treating it with sodium thiosulphate which renders it chemically stable.

Monitoring of 1-125 Despite all the precautions taken during handling, the possibility of internal contamination of workers with 1-125 should not be overlooked, since the solutions are highly active. Such contamination may occur through inhalation of iodine vapour or via skin contamination. The seriousness of intake should be assessed by a regular programme of thyroid monitoring. Weekly monitoring of workers is advisable. If required, thyroid uptake of 1-125 iray be blocked by giving an oral dose of stable iodine (Lugol's solution) to the workers, of course, under proper medical advice. For monitoring 1-125 thyroid burdens in workers bioassay (urine analysis) may be done. In our laboratory we have designed a probe (10) which detects externally the 27.4 KeV X-rays and 35 KeV gamma-rays which are emitted in the decay of 1-125. The probe consists of 7.6 cm diameter X 2 mm thick Nal (Tl) scintillation detector coupled to a low noise photomulti- plier. The detector is used without shield or collimator and is positioned to view the neck region of the subject seated in an ordinary chair. The probe has been calibrated by an in vitro procedure using a REMCAL phantom, the thyroid cavity of which was filled with a known activity of 1-125 solution. The

24 average sensitivity in the 18-39 KeV energy band for the un- collimated probe at 5 cm distance from the thyroid is 48 cpm/ nCi of 1-125. With an average background count-rate of 170 cpm for an unexposed subject the corresponding minimum detectable activity (3 times the standard deviation in background) of T-125 in thyroid is about 0.2 nCi for a counting period of 10 min. The probe is used for monitoring 1-125 thyroid burdens in workers. 1-125 thyroid burdens ranging up to 15 nCi have been observed. In one subject a thyroid burden of about 100 nCi has been detected. Work is in progress to develop a battery operated survey mater incorporating a thin Nal (Tl) detector for monitoring 1-125 contamination on surfaces,

REFERENCES 1. L. T. Dillman, "Journal of Nuclear Medicine," 10, Supplement 2 (1969). 2. A. Gavron and Y. Fiege, "Health Physics," 23 (1972), p. 401. 3. D. Frost and H. Jammet, "Manual on Radiation Protection in Hospitals and General Practice," Vol. 2, Unsealer. Sources, WHO, Geneva (1975). 4. "Safe Handling of Radioisotopes," Safet Series No. 1, IAEA, Vienna (1975). 5. "Handling, Disposal of Storage of Radioactive materials in "Hospitals and Medical Research Establishments,'' ICRP Publi- cation No. 25, Pergamon Press, Oxford (1977). 6. G. S. Linsley, "Atom," April 1976, pp. 108-109. 7. D. Dumas, A., "Practical Programme for the Safe Handling of 1-125," Paper presented at Twenty-second Annual Meeting of Health Physics (USA). 8. J. J. McLintock and J. L. Young, "Laboratory Contamination by Radioactive Iodine, Letter to Editor in "Lancet" (1977), pp. 769-770. 9. J. M. Chesworth and R. J. Clarke, "Laboratory Practice," (1977) p. 581. 10. S. P. Gar? et al, "In Vivo Monitoring of Iodine 125 Using a Thin Nal (Tl) Detector," Paper presented at Ninth Annual Meeting of Society of Nuclear Medicine (India), Trivandrum (November 1977).

25 Review of discussion following the session on inputs

The inputs for radioimmunoassays with special relation to the conditions in India were discussed. 1. Among the various radionuclides used for radioimmunoassays and competitive protein binding assays it was felt that 1-125 was the isotope of choice. However since 1-125 is currently imported from abroad, the feasibility of its production in India was discussed. It was felt that 1-125 could be produced by the Isotope Division of Bhabha Atomic Research Centre in two years time. A suggestion was made that if the amount of 1-125 required was small it would be more profitable to import it in bulk and distribute as required rather than spend large amounts on production. It was also proposed that a central agency could supply labelled compounds on request instead of each centre trying to label its own antigens. 2. The feasibility of producing kits for various hormones in addition to the kits already supplied was discussed. It was strongly felt that kit production and distribution should be priority oriented. This should be based on the needs of the country, like community health problems, and for screening of blood donors for the presence of Australia antigen. The decision on priorities should be taken only after consultation with concerned scientific and health authorities. However it was also felt that if kit preparations were limited to a single Centre then the state of art would be limited by the capabilities of that Centre. Hence there should be room for improvement and quality control by other Centres. It was also felt that the various reagents used in radioimmunoassay could be supplied to the user on request. It was pointed out that some of the kits already supplied also needed improvement in sensitivity. 3. The problems faced in producing antisera were discussed. It was suggested that the second antisera used in radioimmunoassays which required immunisations in bigger animals should be produced and supplied by one Central agency. The production of first antisera could be done in local laboratories and multiple antigens could be injected in the same animal. It was not also necessary to have a very pure preparation of the antigen for production of antisera and that randomly bred 26 animals gave the best antibodies. 4. It was heartening to note that the Indian Council of Medical Research (ICMR) has established a National Pituitary Agency which would supply the needs of the country. The first batch of human growth hormone has been ampouled and is available on request. FSH, LH and TSH would be shortly available. The programme also envisages the supply of hormones for therapeutic use and also animal hormones and specific antisera to these in due course of time. 5. Training courses offered by I.C.M.R. and their usefulness was also discussed. There was a general feeling that the training was not properly utilised by the participants due to the lack of assistance in supply of various reagents for radioimmunoassay. It was felt that these training courses could be more profitably utilised if senior personnel who have access to funds undergo this training. Funds were available to medical colleges for research work if proposals could be sent to I.C.M.R. and Department of Science and Technology. 6. It was felt necessary that the country should make its own counters. 1-125 being the isotope of choice for radioimmunoassays it could be counted by a simple yet cheap equipment which would cost from Rs. 10,000—12,000. 7. The regular radioimmunoassay procedures do not involve the handling of large amount of radioactivity and are relatively safe. Iodination of proteins per se does not liberate iodine in the atmosphere and hence complicated fumehoods and laminar flow systems are not essential. However, the area of iodination should be confined to one place as most of the con- taminations were due to poor handling techniques.

27 STATE OF ART OF RADIOIMMUNASSAYS IN INDIA

This chapter is a summary of presentations of scientists representing some of the major centres in the country. These Centres use this technique for research and diagnostic purposes. This has been necessary in order to gather information on the various types of radioimmunoassays done, the availability of materials and their clinical applications in research and diagnosis.

28 Institute of Post-graduate Medical Education and Research, Chandigarh.

The Institute h, j started doing radioimmunoassays since 1969. Initially they had used radioimmunoassay kits from Farbe Werke Hoechst, Frankfurt, West Germany. Gradually the Insti- tute has acquired better counting equipment and additional technical assistance. It was possible to procure several reagents in the laboratory and to standardise various techniques. Wher- ever possible the Institute has tried to make its own antibodies or else have obtained the reagents from National Pituitary Agency, National Institute of Health, USA. Occasionally antisera have been obtained by courtesy of friends. After initial failure with several separation techniques the double antibody has been used. The second antibody for guinea pig and rabbit sera have been regularly prepared and titrated in the laboratory. Tracers for all hormones are also prepared in the laboratory. Initial attempts to use 1-131 made at Bhabha Atomic Research Centre were un- successful. 1-125 from Radiochemical Centre, Amersham, U.K. either through Bhabha Atomic Research Centre or directly, was now commonly employed for labelling. In the past four years World Health Organisation grant as a collaborating research centre in reproduction studies has been used for procuring regular supplies of 1-125. Labelling of hormones generally did not pose many problems. Although regular diagnostic services have not yet been possible, assays have been utilised to improve diagnostic capabilities at the centre. Samples from other centres are also received and processed. A large body of investigative data in various fields has been collected. For reasons of training and services, the interests of the Institute have been fairly broad-based. Several people have been trained at this Laboratory to carry out radioimmunoassays. A broad range of radioimmunoassays have been performed at the Institute which include assays of insulin, glucogen, growth hormone, steroidal hormones, FSH, LH, HCG. T-4, HPL, PRL, Alphafeto protein and cAMP. Nuclear Medicine Department—Banaras Hindu University Radioimmunoassays were started in 1973 at the Surgical Research Laboratory. In the beginning, the problem was in 29 procuring the standardised antisera, tracers and other reagents. These reagents were obtained from National Institute of Health and other sources. Later kits obtained commercially were used. At present radioimmunoassays have been done with close co- operation of Department of Medicine. The main interest has been to investigate response to stress in terms of various hormonal and metabolic alterations. Assays of insulin, and growth hormone have been performed in a variety of stressful conditions like surgical trauma, burns injury and following electro convulsive therapy. Insulin assays have also been done in disorders of myocardial infarction, heart failure, hypertension after tolbutamide provocation. Serum Digoxin assays have been done in case of digoxin toxicity. Besides this, studies have been done to evaluate associated changes in carbohydrate, lipid and protein metabolism in all the above mentioned conditions. Simultaneously other hormones like cortisol, catecholamine and certain enzymes have also been assayed. Presently the Institute is trying to set up radioimmunoassays of other hormones useful in this research programme. National Institute of Nutrition—Hyderabad This Institute has been one of the pioneers in establishing radioimmunoassays in this country, and have attempted to start radioimmunoassays in 1967. However, they were only successful in doing so in 1970, when 1-125 became available in this country. The first two hormones to be assayed were insulin and the growth hormone, levels of which were measured in protein calorie malnutrition and diabetics. The other hormones for which radioimmunoassays were standardised were HPL, PTH and calcitonin. The latter two however, do not have a good sensitivity. PTH and calcitonin assays were set up to study the hormonal profile in endemic fluorosis. Presently radioimmunoassays of prolactin and alphafetoprotein are also being standardised. Work at the Institute is for research and no dia- nostic services are offered. The Institute has been running training courses in hormone assays with the aim of making clinicians understand the indications, difficulties in establishing these methods and in the interpretation of results. The Institute col- laborates with Indian Council of Medical Research on the study of bio-avaiiability of steroidal contraceptives and is presently engaged in procuring steroid conjugates and in standardisation of radioimmunoassays for steroidal substances. T-3 radioimmunoassays are also being performed. Presently non-availability of 1-125 is the main limitation in carrying out these assays.

30 Institute of Post-graduate Medical Education and Research— Calcutta The radioimmunoassay unit at Calcutta started function- ing only from April 1976. Earlier sporadic attempts to establish the method were made with kits. The unit caters to the need for diagnostic methods for Eastern India and Bangladesh. Till now radioimmunoassays of T-3, T-4, TSH, growth hormone, insulin and prolactin have been established on a regular basis. Upto 31st December 1977, about 16,000 assays of the above hormones have been performed. The reagents have been obtained from National Institute of Health and Indian Council of Medi- cal Research and through the courtesy of Dr K. Machiter of Royal Post-graduate Medical School, Hammersmith, London as a gift. An animal house has been established and the Insti- tute has been successful in the production of antisera against T-3 and T-4. Attempts are being made to standardise the assays of LH, FSH and HCG and Angiotensin II. Plasma cortisol and plasma renin have been performed with kits. The need for Central Regional assay centre for the Eastern region is now felt acutely and the Institute feels that with an expanded staff they could be entrusted to undertake these facilities. Some staff members at present are under going training programmes abroad.. The main problem which the Institute faces is the non-availability of 1-125, automatic gamma counters and simple laboratory equipments. The Institute also feels that the radioimmunoassay service should be under the Nuclear Medicine Department rather than be distributed in several other depart- ments in the Institute. Radiation Medicine Centre—Bombay The use of radioimmunoassays in Radiation Medicine Cen- tre started in 1969. The initial couple of months were spent in standardisations of labelling methods of HGH, production of antibodies and separations bound and free fractions. Initial failures with 1-131 labelling was disappointing. It was demonstrated during Dr Berson's visit in 1970 that 1-131 has a very poor isotopic abundance and is not very good for labelling. 1-125 was then imported from Radiochemical Centre Amersham and since then there have seldom been any problems with labelling. The activities of the department fall into two groups. 1. Routine diagnostic assays of T-3, T-4, TSH HGH, Cor- ticosteroids,

31 2. Research Projects like : a) Hormonal status in protein calorie malnutrition both in children and experimental animals. b) TSH and thyroid hormones in various disease states like non-toxic goitres, Down's Syndrome, renal diseases and infectious hepatitis. c) Thyroglobulin levels in thyrodial disorders especially thyroid cancer. d) Thyroidal disorders and its effects on corticosteroids. e) Effect of topical applications of steroidal preparations on the plasma cortisol levels. f) Evaluation of insulin in prediabetics. Institute for Research in Reproduction—Bombay This is one of the largest and extensive centres in the country in this field. A very broad range of hormonal assays are being done both for diagnostic and research purposes in humans as well as in animals. The following assays are being done at the Centre. PROTEIN HORMONES RIA OF PROTEIN HORMONES Human Rat Ovine Monkey LH CG LH LH LH FSH PL FSH FSH FSH PRLGH PRL PRL TSH

RADIORECEPTOR ASSAY ENZYME-IMMUNOASSJ LH HCG PRL HPL GH Iodination of about 20 hormones are done every month. About 800 estimations are carried out per month. Institute of Science — Bangalore This group is carrying out experimental studies related to reproduction. Their studies extend to assays of the following hormones. Reagents are either prepared in their own laboratory or obtained from National Institute of Health, National Pituitary Agency.

32 Hormone assayed Source material Approximate No. of samples assayed/year

LH and FSH Human sera 50- 60 Monkey sera 400- 500 Rat and Hamster sera 500- 600 Testis and ovary 50- 60 Prolactin Human sera 10- 12 Monkey sera 50- 100 Progesterone Human, monkey, rat and hamster serum and 500-1000 ovarian tissue samples of each Estradiol „ „ Testosterone Monkey, rat and hamster 200- 300 sera and testis samples

All India Institute of Medical Sciences—New Delhi A review of the work done at the All India Institute of Medical Sciences, Delhi, showed that there were several groups dealing with different assays of pituitary hormones and steroids. Extensive work is being done in fertility control with HCG. Insulin and HGH are also studied in relation to diabetics. Work is also done on the characterisation of proteins from plants in endemic and non-endemic zones. A problem was posed as to whether development of an assay for aflatoxin could be useful in the study of epidemics of liver diseases like cryptogenic cirrhosis. Endocrine Department, Topiwalla National Medical College, Bombay At the endocrinology Department of the T. N. Medical College, Bombay, the following essays are carried out. 1. Serum FSH-RIA by double antibody method. 2. Serum LH-RIA by double antibody method. 3. Prolactin-RIA by double antibody method. 4. HCG by Radio Receptor Test. Number of samples assayed in 1977. 1. FSH-about 100 samples. 2. LH~about 300 samples. 3. HCG-about 400 samples. 4. Prolactin started in 1978-50 samples assayed so far.

33 Haffkine Institute — Bombay 1. Corticosteroid. 2. Australin Antigen. 3. Cyclic AMP. These are purely for research activities of this Institute and are not done as diagnostic tests. They also carry out some other invivo and invitro tests using radioisotopes for various research projects. The Institute is mainly involved in other types of radiobiological studies on toxins, venoms and bacterial material. Nuclear Medicine Department, S. N. Medical College — Agra The Nuclear Medicine Department of the S. N. Medical Col- lege Agra have started in November 1977 with renin assays. They will be performing the following assays regularly. 1. FSH, LH, and prolactin. 2. Cortisol, Progesterone oestradiol and testosterone. These assays are being done with reagents received under World Health Organisation matched reagent programme. Their capacity is 100 samples/month for FSH, LH, Prolactin and 75 samples/month for the rest. The limitations are due to the non- availability of liquid scintillators. In addition they do insulin, HPL, T-3 and T-4, digoxin, renin and Mate assays with kits either obtained from Bhabha Atomic Research Centre or Radio- chemical Centre, Amersham. At the present all these assays are done chiefly for research purposes, for specific projects and not for routine diagnostic services. M.K.C.G. Medical College, Berhampur—Orissa The Nuclear Medicine Department at M.K.C.G. Medical Col- lege, Berhampur is engaged in the assays of Insulin, T-3, T-4, CAMP and HPL. All these are performed with kits either obtained from Bhabha Atomic Research Centre or Radiochemical Centre, Amersham. The assays are research oriented for specific projects and no routine diagnostic facilities are available.

SUMMARY Radioimmunoassays of a variety of substances are being done in India. The situation at the present moment is the dif- ficulty in obtaining 1-125, reagents and in certain institutes, equipment. The reagents are either imported or gifted to individuals. This situation is very tenuous and would be likely

34 to break down easily. The instruments locally available at the present are manual gamma counters. This is turn restricts the capacity of handling sample loads. The disadvantages of imported equipment is reduced down time due to poor service facilities and difficult in obtaining spare parts. As is seen from the presentations the use of the technique is confined to research projects and limited, restricted diagnostic facilities. No broad based service facilities to a larger sampling area is offered by any of the organisations engaged in the use of this technique.

35 TRIGGER SESSIONS

This chapter includes the Trigger Sessions. These trigger sessions were on subjects of interest, relevant to conditions prevalent at the moment in the country. The discussions were informal and intended to assess the views of some of the leading scientists in the field in India. A summary of the presentations and the ensuing discussions is reproduced in the following pages. QUALITY CONTROL OF RADIOIMMUNOASSAYS

Quality control is the watchword of our modern era. Radioimmunoassays are now being done extensively to study a variety of substances- This has been possible because of the easy availability of kits and developments of automation. In India the use of radioimmunoassays is limited to only those few laboratories which have facilities for radioisotope handling, processing and counting. The availability of kits made at Bhabha Atomic Research Centre has enlarged the scope of users in India. As in the case of all other types of assays and kits, the radioimmunoassays also necessitate quality control check at various stages. For setting up a radioimmunoassay the following steps are essential. a) Production of first and second antibodies. b) Labelling of antigens. c) Extraction of antigen from sample wherever necessary. d) The control of incubation reaction. e) An effective separation of bound and free antigen. f) Optimal counting statistics. All these steps require appropriate quality control from time to time for routine laboratory assays. Such checks are mainly limited to testing for sensitivity, specificity, precision, repro- ducibility and validity of the assay components. This type of a routine check or intralaboratory check is mandatory for each laboratory. Routine intrassay and interassy variability checks should be done consistently and regularly. A second type of quality control is an external or interlaboratory control whereby comparisons between various laboratories can be made and one standard reference source can be user'. Such interlaboratory comparative studies are being conducted by the World Health Organisation. Some of the laboratories like Institute for Research in Reproduction, Post- graduate Institute, Chandigarh and Agra Medical College who had participated in the programme commended World Health Oraganisation on their efforts to maintain a good quality control of reagents and standards. However opinion was expressed that various laboratories reported such wide variations in values that although the mean value obtained by the laboratories

37 tallied, the variations around the mean were wide. It was felt that although such interlaboratory checks are useful, what is more important is regular intralaboratory quality control as mentioned before. Definite standard rules for quality control of radioimmunoassays in general are very difficult to define. Radioimmu- noassays are being performed of such varied and diverse group of substances that each assay system would require its own definition of quality control. Rigorous quality control is not essential wherever stimulation tests are performed and these are more informative of dynamic function than single, random, spot evaluations. Each group of substances to be assayed should have their own quality control requirements and no general guidelines could be given when such a diverse group of substances are being examined.

38 USEFULNESS AND LIMITATIONS OF RADIOIMMUNOASSAYS IN CLINICAL DIAGNOSIS

There is no doubt that radioimmunoassays have gone com- merial in the western world. Most of the laboratories there do radioimmunoassays with kits readily available from more than one company. There is lot of sales pressure to generate a market for these kits and each company extolls the virtue of one or more radioimmunoassay for which they supply kits. If one believes all the advertising literature, one would be con- vinced that radioimmunoassays are the simplest, most-easy-to- perform, clinically most useful, indispensable diagnostic test. Are they really that useful for a practising clinical physician in his day to day decision making ? There is no doubt that a vast number of substances can be estimated by immunoassay techniques. Their wide application has brought forth immensely helpful information in elucidating the nature and progress of a disease e.g., our present knowledge of interplay of various hormones in the pathophysiology of diabetes has been possible only because of radioimmunoassays. However, no one would say that radioimmunoassay of insulin is a 'must' for the diagnosis and management of diabetes. Where a referring physician has nothing to do but send a few ccs of patient's blood to the laboratory for estimation of one or more hormones, a large number of radioimmunoassays will be carried out regardless of their information content for patient diagnosis. With increasing availability of automated radioimmunoassay procedures with on line computer to carry out all the * wnplex curve fitting and calculations, large number of radioimmunoassays will be carried out first, because it is EQ easy to get them and secondly, because it is so fashionable to ask for them. In India, where radioimmunoassays have not gone commercial and berserk like the western world we shall have to identify radioimmunoassays which are clinically useful. Very few kits are available, 1-125 is required to be imported, many of the antisera remains to be cultivated by the user—with all these odds against setting up a radioimmunoassay, a hospital laboratory will have to decide discreetly what they should set up as a diagnostic service and what they should leave for research

39 laboratories. The assays which are of diagnostic importance are Australia antigen, T-4, T-3, TSH, HCG, FSH, LH, HPL, prolactin, digoxin, cortisol, testosterone and gastrin while insulin, GH, angio- stensin, ACTH and others are more useful in the understanding of pathophysiological processes. Radioimmunoassays are extremely useful in diagnosis where no substitute tests are available e.g. T-3 in T-3 toxicosis, prolactin in prolactin producing adenomas, HGH in dwarfism. They are less critical where substitute methods are possible like HPL, HCG, T-4, T-3, Digoxin and Australia antigen. The greatest single limitation of radioimmunoassay is that the results are not available immediately within a day or so. A batch of samples is to be collected before they can be processed. Where results are required before a clinical decision can be taken, such waiting periods of several weeks would be in- tolerable. One way to solve this problem would be to establish centralised assay services which can easily collect a large number of samples and process them batch by batch in rapid succession. With such type of service indiscriminate referral would be unavoidable. Where clinical decisions are critical and requires a quick result, for example, HCG, HPL, Estradiol, Digoxin, it is possible to shorten the assay time by what is called as the "quickie" assay test where serum samples with low medium and high levels of the substance to be measured are incubated with larger concentrations of antibody for shorter periods of time alongwith unknown and results compared to such plasma values instead of standards. The abuse of radioimmunoassay in western world is due to the fact that neither the institute nor the patient pays for the test. In our counry, the problems of cost either to the institute or to the patient is important especially where multiple tests need to be done e.g. TSH. T-3, T-4 or where tests need to be repeated often like HCG, and HPL. It is essential then that in a public hospital there should be some screening of the requests made for radioimmunoassays to prevent its misuse. Referring physicians should not over-load laboratories with requests for assays unless and until there has been a thorough investigation of the patient by other methods. Radioimmunoassay is the cheapest and the simplest tool for clinical research. Notwithstanding what has been said above, one hopes that radioimmunoassays are available widely in this country for clinical research. There are many of our tropical diseases where application of radioimmunoasay would bring u. information yet unavailable so as to change our thinking com- pletely about the management of these diseases. If I look far into the future, I can envisage that techniques like Elisa depending on color reactions for assays may be done by paper strip in the clinician's backroom laboratory cubicle. Till such time, we in India, will have to move cautiously and generate our own experience to find out what is useful, what is needed, what is elegant and what is a fad as far as radioimmunoassays are concerned. RADIOIMMUNOASSAYS IN TROPICAL DISEASES

Usefulness of radioimmunoassays in clinical diagnosis is limited. Its greatest impact has been on the understanding of pathophysiology of many of the diseases. However, application of radioimmunoassays in diagnosis or understanding of infectious diseases has been sadly neglected. If radioimmunoassays have to thrive in tropical countries they must become relevant to problems of medical care in these countries. When we want funds for radioimmunoassays in developing countries, the ques- tion that would be asked is do they help to resolve in any way the problems of population, pestilence or nutrition ? Application of radioimmunoassays so far recognised in tropical diseases are in the following two directions: 1. Study of endocrine status in various tropical diseases. 2. Study of endocrine status in various nutritional disorders like malnutrition, undernutrition, starvation, malabsorp- tion, dietary oddities, etc. In both above cases, the radioimmunoassays as techniques are the same as developed and applied in western countries. The application has been mostly imitative and not innovative. To develop radioimmunoassays in our country has been a difficult exercise as such, even in this imitative way. It needs extending the begging" bowl all along. Request for hormones from National Institute of Health, USA or Medical Research Council, UK. beg for antisera from this or that lab. Indent ready-made kits from abroad or write for protocols of assays to friendly scientists abroad. There has been hardly any effort towards developing radioimmunoassays for actual detection of antigen or antibodies in infectious diseases. This is surprising because most of our knowledge of antigens and antibodies is derived from the study of infectious diseases but we have been singularly lacking in enterprise in applying the radioimmunoassay principle to detect- tion of antigens and antibodies in infectious diseases themselves. There are few reports in literature of using C-14, 1-125 labelled antigens or antibodies for diagnosis of infectious diseases but they are too few, too preliminary and widely scattered. In hardly any case, they are convincingly better than serodiag-

42 nosis. The best of talents in this field are not applied to nurturing this kind of assays. The single most successful radioimmunoassay for an infective agent is that for Australia antigen. Even here, what we need urgently is an assay for hepatitis-A antigen. Some work was done at Haffkine Institute in collaboration with Radiation Medicine Centre towards developing an immu- noprecipitation test for typhoid antibodies by using C-14 labelled antigen. A technique for labelling polysaccharide typhoid antigen with Cr-51 has been also published from Radia- tion Medicine Centre. Radioimmunoassays for infectious diseases can be for two purposes: 1. Detection of antibodies. This may be useful in studying the antibody profile against a specific disease in a community, for finding circulating antibody levels at various stages of of diseases and to find out success or failure of an immunisation programme. 2. Detection of antigens. Presence of infective organism is difficult to detect in many infectious diseases, e.g. tubercle bacilli in blood, urine, C.S.F. or other body fluids. This kind of assays can be of great help in early detection of extrapulmonary tuberculosis, leprosy, carriers of various infective agents, etc. There would be lot of difficulties in setting up these assays. Any single assay would be a challenging proposition by itself. There would be either antigen excess or antibody excess at various stages of the disease making it difficult to set up a sensitive assay system. In most cases antigen is crude, mixed and altogether an unknown commodity. There may be a lot of cross reactivity between antigens. Who can take up the gauntlet of ploughing the unprospective land ? This task can be best taken up in this country. Antigen is available indigenously, there are vast reservoirs of it and can be cultivated easily. The antibodies are in a prodigious supply in humans. Unlike other radioimmunoassays, the situation is favourable here in one respect. There is no need to buy or beg for antigen or antibody. If we can take up the challenge the next decade in radioimmunoassays will be of infectious diseases just as the last decade was of endocrinology.

43 CENTRALISED ASSAY SERVICES

A central or regional assay service would offer the diagnostic facility of multiple radioimmunoassays to regional and district hospitals and clinics. The point which needs to be established is whether there is a genuine need to establish such centres to do radioimmunoassays or is this technique a prero- gative of selected research oriented institutes ? A look at the current scene reveals that at the present only nine cities in the country have centres engaged in radioimmunoassay techniques. They are Agra, Bangalore, Bombay, Calcutta, Chandigarh, Cuttack, Delhi, Hyderabad and Varanasi and hope- fully Lucknow, Madras, Trivendrum and Vellore will soon be initiating steps to establish these methods. Out of the 108 medical colleges in the country, only eight have introduced a limited facility for routine diagnostic service of this type. How much of this can be attributed to lack of initiative and poverty of ideas and how much to financial and other constraints ? The concept of a "Centralised Service" is not well defined. Should these Centres offer service for clinical diagnosis only or support and offer service facilities for research work ? Should these central assay services opt to supply only reagents to users ? The logistics of sample collection, transport, storage of sera and time taken for despatching the results are highly complex. If such centres are to be set up, then there should be peripheral centres which could cater to the district levels, another at the level of Medical Colleges and the larger Regional Reference Centre at well established Institutes. Each of these centres would need to have personnel trained in handling radioisotopes and multiple gamma counters. The services offered for assays should be need based. The im- mediate diagnostic potentials of radioimmunoassays are limited. They are powerful tools in the pathophysiological understanding of diseases. To come to brass-tacks, the diagnostic utility is chiefly in the evaluation of thyroidal disorders, disorders of growth, adrenocortical tumours, insulinomas, pituitary micro- adenomas and a few others. AH except thyroidal disorders are uncommon diseases. Considering the magnitude of the problems in logistics and the corresponding poor returns, in the present context central assay services would not serve a significant

44 diagnostic role in our country. However, in future when epidemiological surveys for detection of conditions like neonatal hypothyroidism, Australia Antigen and infectious diseases are started, then such a service could be more profitably utilised. The general concensus of opinion was that for the present a central assay service was premature. What was important was : a) There should be a liaison between various international agencies and the creation of a national pool of expertise. b) The feasibility of Bhabha Atomic Research Centre sup- plying 1-125 and labelled reagents to users in the country. c) The feasibility of a cheap, simple counting system to be made locally. d) Liaison with large centres like Institute for Research in Reproduction, Post-graduate Institute and Radiation Medicine Centre etc. for steady supply of antibody, standard and other reagents. e) Monitoring of various centres requesting these reagents and finally a quility control protocol of each procedure was essential. f) Easy availability of "kits".

45 RECOMMENDATIONS AND GUIDELINES

1 Introduction Radioimmunoassay and related assay techniques constitute a powerful tool for resolving health problems of the country. Apart from its well recognised role in the diagnosis of diseases, it is likely to be of positive help in elucidating epidemiologieal and pathophysiological mechanisms of disease widely prevalent in our country. Its versatility, sensitivity, low cost and capability of processing speedily large number of samples at the same time make this technique especially suitable for studying various problems of community health, because it does not require transportation of a patient to the hospital but involves only a collection of a blood sample from the patient and arrange- ment for transport of these samples to a centralised investigational facility. This technology can be applied to the following health problems : i. For elucidation of the epidemiology and pathophysiology of diseases which pose national public health problems. ii. As rapid diagnostic techniques to help early diagnosis and to monitor the effectiveness of various therapeutic and preventive measures adopted for these public health problems. iii. Early detection of various diseases leading to their timely treatment. Certain public health problems where these techniques may be applicable were identified. Apart from the obvious applications in the field of reproductive biology and family welfare, the study of communicable diseases was identified as a priority area. It may be possible to screen for humoral antibodies and/ or for antigens in biological material. The former might define the infection incidence and antibody profile in the population and help to assess the success/failure of immunisation programme. Detection of antigens or bacterial products could be extremely useful in finding contacts, carriers and early cases, and determine appropriate management. The feasibility of such studies in leprosy, tuberculosis, rabies, tetanus, typhoid, hepatitis, rubella, encephalitis, malaria, kalazar, amoebiasis, salmonelosis brucella etc. and their relative merits in comparison 46 to conventional diagnostic methods need to be defined. Other such areas were the study of endemic goitres, neonatal hypothyroidism and malnutrition, especially the role of food contaminants in the genesis of diseases like lathyrism, pancreatitis, toxic neuritis and hepatic disorders. 2. Resources The 1976 Seminar on Nuclear Medicine, jointly organised by the World Health Organization and Department of Atomic Energy had identified various advantages that could accrue from centralisation of certain facets of this radioassay technology. It is noted that a beginning has been made with a National Pitui- tary Agency having been established under the aegis of the Indian Council of Medical Research. This is a most commendable effort and would greatly augment the radioimmunoassay programme in this country once purified pituitary hormones become available from this source. Material for labelling, labelled materials and purified matched standards and antisera could be supplied through this central service. Centralisation and subsidised or even free supply of imported 1-125 was another area where costs could be reduced and time saved. Currently, this is a significant bottleneck. It is estimated that the approximate national requirement at current prices would involve a relatively small annual expenditure and Bhabha Atomic Research Centre was urged to provide this radionuclide through its resources as early as possible. The supply of bulk matched reagents could also be centralised. The Haffkine Institute and its sister organisation could initiate the manufacture and supply of second antibody. Later various institutes and laboratories could undertake the supply of first antibodies for selected assays. Requests for materials mentioned above would need to be justified by the investigator and evaluated by an appropriate committee in terms of genuine needs and relevance to national problems. However, it is strongly felt that the existance of these centralised supplies should not preclude individual investigators from obtaining independent supplies for use in areas in which they were particularly interested. The structure, functions and requisite equipment for radioassay services at the national, regional and medical college hospitals' levels have already been detailed in 1976 report and is not being reconsidered here. It was felt that indigenous equipment is far more acceptable than forf-'«n equipment because it minimises problems of

47 repairs, spares and maintenance. Single sample manual well- counter is presently available in the country. Provision of inte- grated circuitry, use of smaller crystals and perhaps omission of spectrometer by providing pre-set 125-1 counting facility may further reduce the cost of such a counter. The relative merits of 'through hole' detector needs to be evaluated in such a counting system. The Committee notes that both Electronics Division of Bhabha Atomic Research Centre and Electronics Corporation of India Ltd., have capabilities of making 50 to 100 samples automatic counters. Such counters would be more suitable for centralised assay service. It was emphasised that having multiple low cost manual or automatic sample changers is more desi- rable than having a large sample capacity single sample changer which might have longer 'down time' because of difficulties in repairs and spares. The various laboratories in the country could pool scarce financial, technical and material resources by co-operating in areas such as liaison with international agencies, equipment manufactures, Division of Radiological Protection, quality control, establishment of Indian norms, and minimising duplication of effort in assays for antigens for which demand is limited. International agencies like the World Health Organisation and allied organisations should support the above activities with financial and technical assistance. The pioneering efforts of the Radiopharmaceutical Section of the Isotope Group, Bhabha Atomic Research Centre, in providing kits and saving foreign exchange were commended. Kit production should be priority oriented specially to deal with community health problems. However, it was also felt that excessive centralisation whether in kit production or reagent supply could limit individual initiative if the state of the art became limited by the capabilities of a particular centre. There should be room for improvement and quality control of kits and bulk reagents by other centres. 3. Research It was felt that so far the development of Radioimmunoassay in the country, though heartening, had relied heavily on borrow- ed technology and established assay techniques. It was recognised that in order to apply the radioimmunoassay techniques to our national problems, the creation of technology which is cheap, robust and more suited to our conditions as regards sample collection, sample transport and assay methods should be

48 encouraged. The latter would include filter paper, solid phase and labelled antibody techniques. The creation of assay techniques relevant to our public health problems for hormones, infective antigens, antibodies, food contaminants is vitally necessary. 4. Service In areas where diagnostic role of radioimmunoassay is well- established, its day-to-day application and medical practice should continue. These include radioimmunoassays for thyroid hormones, TSH, HCG, Australia antigen and HPL. Information about availability and usefulness of these assays should be widely disseminated in the appropriate medical forum. Later as research identifies uses in other areas such as infectious diseases or diseases possibly related to food contaminants the diagnostic role could be extended. Until such time as assay facilities become generally available, institutes with facilities for various assays could provide referral service on the basis of limited justified clinical request irrespective of geographical distances. The development of assay facilities in teaching hospitals or other medical centres could be supported by the existing centres as well as appropriate national agencies like Indian Council of Medical Research, Ministry of Health, Department of Atomic Energy and International Atomic Energy Agency. 5. Training The existance of training facilities at various centres was noted. The training of senior persons in adequate depth was emphasised. Training of technicians would also be obligatory. It was felt that the expertise available in these and other centres should be pooled and used in a co-ordinated manner so that the courses proved more useful. The size and the extent of the training programme should be reviewed periodically so as to be commensurate with needs of the country. After the training the trainees should be assisted in setting up these techniques in their own laboratories. 6. Information Exchange and Dissemination The exchange of information regarding newer developments in this country and abroad can be facilitated by a News- letter. The Society of Nuclear Medicine and Endocrinology Society of India should hold special radioassay sessions so that further

49 developments in these techniques is encouraged. There should be a periodic review of the national activity in the field of radioassay so as to record the capability of the various centres and define areas for further work. It may be worthwhile to publish a 'method's manual' detail- ing the various techniques of radioassay as being practised in the country and their applications.

50 BACKWORD

Usually, such reports of the proceedings have a "foreword" glistening, hopeful and inspiring, setting the right tone for a ponderous report to follow. Nobody writes the 'backword', probably because nobody expects the reader to last till the end. Nobody writes the 'backword', probably because it also sounds so much like 'backward'. And that we certainly are not. How can we be backward after such an erudite conference and after drawing such imposing recommendations and guidelines? Remember, Rosalyn Yalow said that India is not backward. We may be short of funds but there never is a dearth of human talents, intelligence and ingenuity. She said many such things at the conference. She gave a series of five marvellous lectures reviewing her entire work in the field of radioimmunoassays starting from the concept of the idea to its ever burgeoning applications. The first 'backword' is that the Editors feel sorry that this report does not contain all that glory. Difficulties of transcrib- ing taped talks, and problems of reproducing a large number of slides have made it impossible to put her lectures in this report. So then, as far as this report is concerned, her role remains that of a catalyst. All that has been recorded here was discussed in her presence. Her comments, criticism, praise, disdain all have contributed in moulding the trends of what transpired at the conference. Like a true catalyst she was inexhaustible, providing refreshing candour throughout the five days of inter- minable discussions. Now that, we have finished reading it, let us think what was the purpose of this report. It was a good meeting. Lots of nice discussion. Gems of recommendations. And at the end of it, a well-edited report, something of a souvenir, like a fork that you pinch from a hotel after a pleasant party. Both are free, the report and the fork: both full of sweet memories. Does the conference end with the report and statement of recommendations, something like a staged play so that at the end of a fantasy, you rush out of the theatre to hunt for the most difficult-to-get taxi ? The similie is not inept. The confer- 51 ence provided a panoramic vista but now let us hurry back to writing requests for ever elusive antigens and antibodies to our foreign friends and benefactors ! Who puts the recommendations in practice ? The very- word implies that we expect some-one else to follow them. And that is where the 'backword' remains to be said. Science is carried forward by individuals. Yalow and Ber- son had to struggle hard to convince the world that what they thought of was worthwhile. Each of the scientist has to begin his struggle after the conference. Scientists have to mould the institutions where they work and institutions have to put pressure on various national and international agencies. But the first push has to come from the scientist, and from his planned, purposeful, imaginative and innovative scientific work. The recommendations are not as admonishments but a kind of introspective meditative code of conduct for scientists themselves. And therefore, as a 'backword', let us state few facts that emerged at the conference and which apply to all of us. Radioimmunoassays offer the cheapest possible investigative tool for clinical research. In the context of our country, the role of radioimmunoassays for a day to day clinical diagnosis is limited. Indian scientists should stop imitating western science and start applying these techniques to the study of infectious diseases for which antigen and antibody are available in abundance. The resources are not hopeless, what is needed is the will to apply them for purposeful project so that more can be obtained out of little at hand. These few 'backwords' should be enough to show that conference has not ended with this report. It has just begun. In a way then this is a 'foreword'. A stalled car needs a push from backwards. There are so few passengers in this car that the scientist himself may have to come out to push the car. But the car is new, engine is capable and Radioimmunoassays should roll forward with a big roar.

R. D. Ganatra

52 BIBLIOGRAPHY

A list of papers on Radioimmunoassays originating from India was compiled from information given by the participants. This list is not necessarily complete. It would serve merely as an inkling as to what is going on in the country.

53 General Methodology

1. Hazra, D. K. Immuno Radiometric Methods in the study of glyco- protein hormones. Proc. of the I st Asia and Oceania Cong, of Uuclear Medicine, Sydney, 1:411, 1974. 2. Hazra, D. K. Immunoradiometric method in glycorprotein estimation SNM/220/81. Under publication in Proc. of IAEA Symp. Nov. 1977. West Berlin. 3. Hazra, D. K. Radioimmunoassays and related techniques in clinical medicine. Jr. Ass. Phys. Ind. 26:95, 1978. 4. Joshi, U. M., Raghavan, V. and Sheth, A. R. Development of an enzyme linked immunoassay for human chorionic gonadotropin. Ind. J. Med. Res. 65 :807, 1977. 5. Kadival, G. V. and Samuel, A. M. Polyethylene glycol in RIA of TSH. Ind. J. Med. Res., 65:1537, 1976. 6- Moodbidri, S. B., Joshi, L. B. and Sheth, A. R. A procedure for radioiodination of peptide hormones using lactoperoxidase isolated from buffalo milk. Ind. J. Expt. Biol. 14 : 572, 1976. 7. Moodbidri, S. B., Johi, L. R. and Sheth, A. R. Isolation of an inhibin—like substance from ram testis, IRCS Medical Science 4 :217, 1976. 8. Moodbidri, S. B., Joshi, L. R., Sheth, A. R. and Rao, S.S. Development of radioimmunoassay for circulating levels of gonadotropin releasing hormone. Ind. J. Med. Res., 64:1649, 1976. 9. Murty, B. D., Sheth, A. R., Purandare, T. V. and Rao, S. S. Nature of cross-reaction between HCG and anti-ovine LH serum Endocrinology, 99 :1554, 1976. 10. S. G. Nandini, H. Lipper and N. R. Moudgal A model system for studying inhibin. Endocrinology 98 : 1460, 1976.

54 11. Rao, A. J. and Moudgal, N. R. An immunochemical study of ovine FSH Arch. Biochem. Biophys. 138 :189, 1970. 12. Ramasarma, K., Murlidhar, K. and Moudgal, N. R. Heterologous radioimmunoassay for measurement of FSH and LH in the bonnet monkey. Ind. J. Exp. Biol. 1978. (In Press). 13. Rastogi, G. K. and Sawhney, R. C. Radioimmunoassay of Triiodothronine. Ind. J. Med. Res., 62:225, 1974. 14. Samuel, A. M. and Deshpande, U. R. Modification of RIA of HGH estimation. Ind. J. Med. Res. 61: 98, 1973. 15. Sawhney, R. C. and Rastogi, G. K. Direct radioimmunoassay of thyroxine in the serum. Ind. J. Med. Res. 62 :1233, 1974. 16. Sawhney, R. C. and Rastogi, G. K. Estimation of thyroxine binding globulin in the serum. Horm. Net. Res., 6 :436, 1974.

Peptide Hormones 1. Arbatfii, N. J., Sheth, A. R. and Vaidya, R. A. Mode of action of Centchroman on Hypothalamo-pituitary axis in male rats. Ind. J. Expt. Biol. (Accepted for publication) 2. Arbatti, N. J., Sheth, A. R. and Vaidya, R. A. The effect of L-dopa on Centchroman induced prolactin levels in female rats. Ind. J. Expt. Biol. (Accepted for publication). 3. Beg, A. A., Varma, V. K. and Dash, R. J. Effect of chlorpromazine on human growth hormone. Am. J. Psychiatry 1978 (In press). 4. Chakravarty, I., Sheth, A. R., Ghosh, J. J. Effect of acute D9-Tetrahydrocannabinol treatment on serum LH and prolactin levels in adult female rats. Fert Steril. 26:847, 1975. 5. Dash, R. J., Vasista, K., Gupta, S. K., Lata, V. and Sharma, B. R. Horrr \ne in reproduction IV. Application of beta sub-

55 unit of human chorionic gonadotropin in evaluatin of early pregnancy. Bull Postgrad Med Inst. (Chandigarh) 10:53, 1976. 6. Dash, R. J., Sharma, B. B., Lata, V. and Devi, P. K Hormones in reproduction V. Radioimmunoassy of HCG. betasubunit in normal human pregnancy. Bull Postgrad Med Inst 11:128, 1977. 7. Dash, R. J., Gupta, S. K., Vasista, K and Devi, P. K. Serum HCG and endometrial histology in early pregnancy. Ind. J. Med. Res. 1978 (In press). 8. Dash, R. J., Datta, T. K., Purohit, O. P., Jayakumar, R. V. and Gupta, B. D. Prevalence of ectopic HCG production in nonedocrine malignancy. Ind. J. Cancer 1978 (In press). 9. Dash, R. J., Datta, T. K., Gupta, S. K., Jayakumar, R. V. Purohit, O. P., Joshi, V. V. and Datta, B. N. Ectopic HCG in non-endocrine malignancies. With tumor morphology, Ind. J. Path & Microbiol. 1978. (In press). 10. Dash, R. J., Vasista, K., Sharma, B. R., Rastogi, G. K. and Devi, P. K. Hormones in Reproduction II, Human Chorionic gonadotropin in normal pregnancy, a cross sectional study. Bull. Postgrad. Med. Inst. Chandigarh 9 :139, 1976. 11. Dash, R. J., Vasista, K., Sialy, R., Sharma, B. R., Rastogi, G. K. and Devi, P. K. Hormones in Reproduction I—Hormonal profile of human menstrual cycle. Bull. Postgrad. Med. Inst. Chandigarh, 9:134, 1976. 12. Dash, R. J., Sharma, B. R., Rastogi, G.K. Measurement of serum chorionic gonadotropin in normal pregnancy. Ind. J. Med. Res. 64 :963, 1972. 13. Dattatreyamurty, B., Sheth, A. R., Joshi, L. R., and Rao, S. S. Changes in the ratio between serum HCG and 'specific HCG' levels in different trimesters of pregnancy. Amer. J. Obstet. Gynec. 121: 300, 1975. 14. Dattatreyamurty, B., Raghavan, V. P., Sheth, A. R. and Rao, S. S. Synergistic action of prolactin with human chorionic

56 gonadotropin on rat ventral prostate. J. Reprod Fert. 44: 556, 1975. 15. Dattatreyamurty, B. and Sheth, A. R. Size of heterogenity and specific binding property of immunoreactive prolactin in human seminal plasma. Molecular and Cellular Endocrinology, 7 :263, 1977. 16. Devi, P. K., Joshi, U. M., Moodbidri, S. B., Naik, V. K., Susheela, P. S. and Sheth, A. R. Long term effect of vasectomy on pituitary gonadal axis. Ind. J. Med. Res. 66 :591, 1977. 17 Dhal, K., Kumar, M., Bastogi, G. K. and Devi, P. K. Short term effects of Noresthisterone cenenthate and medroxyprogesterone acetate on glucose, insulin growth hormone and serum lipids. Fert. Ster. 28 :159, 1977. 18. Goomer, N., Saxena, R. N. and Sheth, A. B. Effect of neonatal castration on the content of hypothalamic LH-RH, pituitary LH and plasma LH in developing male rats. Endocrinology 69 :195, 1977. 19. Goomer, N., Saxena, B. N. and Sheth, A. B. Effect of neonatal testosterone and estradiol treatment on the development of hypothalamo-hypophysial axis in the female rat. J. Reprod. Fertil, 50:239, 1977. 20. Goomer, N., Saxena, B. N. and Sheth A. B. Effect of estradiol and testosterone on the pituitary prolactin levels in developing male and female rats. Indian. J. Expt. Biol. 15 :647, 1977. 21. Goomer, N., Saxena, B. N. and Sheh, A. B. Correlation of hypothalamic LH-RH, pituitary LH and plasma LH in developing rats. Endocrinology (In Press). 22. Goomer, N., Saxena, B. N. and Sheth, A. B. Development changes in the in vitro responses of pituitary to luteinizing hormone releating hormone LH-RH in the male and female rats. Endocrinologie Experimentals. (In Press). 23. Gupta, P. B., Sinha, M. K., Dash, E. J., Wahi, P. L. and Bastogi, G. K. Plasma insulin, Free fatty acid and blood sugar response to intravenous glucogen in healthy subjects and patients w.i'> myocardial infarction Circulation 49 :357, 1974.

57 24. Haji, H. K., Vaidya, R. A., Meherji, P. K., Joshi, L. Sheth, A. R., Motashaw, N. D. and Devi, P. K. Scope of laparoscopic ovarian biopsy versus multiple serum gonadotropin estimations in the diagnosis of secondary amenorrhoea. Ind. J. Obst. Gyn. 26: 269, 1977. 25. Jayakumar, R. V., Desh, R. J., Purohit, O. P., Dutta, T. K. and Gupta B. D. Hormones in reproduction VII. Hepatic production of HCG in non-endocrine malignancies. Bull Postgrad. Med. Inst. (Chandigarh) 11: 197, 1977. 26. Jayaraman, S., Sinha, M. K., Raghavan, K. S., Mathur, V. S., Rastogi, G. K. and Devi, P. K. Hormonal and enzymatic changes during the induction of mid-trimester abortion with prostaglandin F2 hypertonic saline. Proc V Asia and Oceania Conference of Endocrinology, Ed. G. K. Rostogi, Endocrine Society of India 1, 93, 1974. 27. Jayaraman, S., Sinha, M. K., Raghavan, K. S., Mathur, V. S., Rastogi, G. K. and Devi, P. K. Hormonal and enzymatic changes during the induction of mid-trimester abortion with prostaglandin F2 and lypertonic saline. J. Obstet. and Gynec. 121: 528-530, 1975. 28. Jaya Rao, K. S. and N. Raghuramulu. Insulin Secretion in Kwashiorkor. J. Clin. Endocrin. Metab. 35: 63, 1972. 29. Joshi, L. R., Seth, A. R., Raghavan, Y. P. and Rao, S. S. Serum FSH and LH levels in fertile and subfertile men. Ind. J. Med. Res. 61:1308, 1973. 30. Kannan, Sinha, M. K. and Rastogi, G. K. Rebound thyrotropin rise after tri-iodothyronine. A test to predict remission in Graves' Disease. Bull. Post- grad. Med. Inst., Chandigarh, 6:186, 1972. 31. Kannan, V., Sinha, M. K. and Rastogi, G. K. Plasma thyrotropin and its response to thyrotropin releasing hormone in normal pregnancy. Obstetrics and Gynaecology 42 :547, 1973. 32. Kulkarni, R. D. Hypothalamous and growth hormone release. Ind. J. Physiol. Pharma. 13: 99, 1969.

58 33. Maneckjee R., Srinath, B. R. and Moudgal, N. R. Prolactin suppresses release of LH during lactation in the monkey. Nature 262 :507, 1976. 34. Mantri, A„, Sheth, A. R., Ajinkya, S. M., Raikar, R. S. and Wadadekar, K. B. Effect of synthetic LH-RH on the levels of serum LH in domestic hen. Ind. J. Anim. Sci. (In Press). 35. Moodbidri, S. B., Sheh, A. R. and Rao, Shanta S. In vitro binding of radioiodinated placental lactogen in buffalo corpus luteum. J. Reprod. Fert. 35: 455, 1973. 36. Moodbidri, S. B., Sheh, A. R., Rao, Shanta S., Penkar, S. J. and D'souza Mary. Binding of prolactin by myomas and normal mometrium. Indian J. Exp. Biol. 12 : 566, 1974. 37. Moudgal, N. R. and Madhwa Raj, H. G. Pituitary Gonadotrophins. In: Methods of Hormone Radioimmunoassay Ed. B. M. Jaffe and N. R. Behrman P. 57, 1974, Academie Press, New York. 38. Moudgal, N. R. and Sheela Rani. Role of FSH in ovarian follicular maturation. Regulation of growth and differentiated function in eukaryote cells. Ed. G P. Talwar, P. 431, 1975, Raven Press. New York. 39. Mukku, V. and Moudgal, N. R. Studies on luteolysis—Effect of antiserum to LH on sterol and Steroid levels in pregnant hamster. Endo- crinology 97 :1455, 1975. 40. Mukku, V. and Moudgal, N. R. Relative sensitivity of the corpus luteum of different days of pregnancy of LH deprivation in the rat and the hamster. Mol. Cell. Endocrinol, 6:71, 1976. 41. Muralidhar, K. and Moudgal, N. R. Studies on rat ovarian receptors for lutropin (LH)—Ap- plicability of RIA to measure lutropin bound to receptors. Biochem. J. 160 : 603, 1976. 42. Muralidhar, K. and Moudgal, N. R. Studies on rat ovarian receptors for lutropin—Factors influencing binding and response. Biochem. J. 160:607, 1976.

59 43. Muralidhar, K. and Moudgal, N. R. Studies on rat ovarian receptors for lutropin. Interaction with submit of sheep lutropin. Biochem. J. 160 :615, 1976. 44. Muralidhar, K., Maneckjee, B. and Moudgal, N. R. Inhibition of in vivo pituitary release of LH in lactating rats by exogenous Prolactin-Endocrinology 100:1137, 1977. 45. Naik, V. K., Thakur, A. N., Sheth, A. R., Josfei, U. M., Rao, S. S., Pardanani, D. S., Kulshreestha, J. K. and Handa, R. K. Effect of vasectomy on pituitary-gonadal function. J. Reprod. Fertil. 48:441, 1976. 46. Panda, N. C, Tripahy, B. B., Parija, C. R., Swain, N., Pairah, N., Sinha, M. K. and Rastogi, G. K. Observation on endocrine function in adults with chronic malnutrition. Proc V Asia and Oceania Conference Endo- crinology ed. G. K. Rastogi, Endocrine Society of India, 2:318, 1974. 47. Phadke, A. M., Sheth, A. R. and Joshi, L. R. Serum gonadotropin levels in necrospermica. Fertil. Steril, 1978 (In Press). 48. Prahalada, V., Mukku, V., Rao, A. J. and Moudgal, N. R. Termination of pregnancy in macaques using monkey antiserum to ovine LH. Contraception 12:137, 1975. 49. Raghavan, Vijaya, Purandare, Tarala, Sheth, A. R. and Munshi, S. Circulating levels of gonadotrophins in immature mice treated neonatally with antisera to gonadotropins. J. Reprod. Fertil. 49:401, 1977. 50. Raghavan, V. P., Sheth, A. R., Rao. S. S., Dave, So, Purandare, M. and Purandare, B. N. HCG levels in Indian Women, J. Ob. Gyn. India, 27: 675. 1977. 51. Raghavan, V. P., Sheth, A. R., Rao, S. S., Dave S., Purandare, M. and Purandare. B. N. Levels of HPL in Indian women. J. Ob. Gyn. India. 37: 363, 1977. 52. Raghuramulu, N. and Jaya Rao, K. S. Growth hormone secretion in protein-calorie malnutrition. J. Clin. Endocrin. Metab. 38 : 176, 1974. 60 53. Raghuramulu, N. Plasma placental lactogen in pregnancy. Nutr. Metab. (In Press). 54. Raheja, B. S., Kulkarni, B. D. and the Talwalkar, N. A. Immunoreactive Insulin in serum of various groups of diabetices and non-diabetics. J. Assoc. Phys. Ind. 19: 550, 1971. 55. Rastogi, G. K., Birdwel, C. and Fraser, R. Immuno-reactivity of Human and Bovine Proinsulin in Insulin Immunoassay System. J. Ass. Phys. India, 18 : 604, 1970. 56. Rastogi, G. K., Letarte, J. and Fraser, R. Pancreatic Insulin content of 203 Human Foetuses from Healthy Mothers. Diabetologia. 6 :445, 1970. 57. Rastogi, G. K., Letarte, J. and Fraser R. Proinsulin content of pancreases from human foetuses of healthy mothers. Lancet 1:7, 1970. 58. Rastogi, G. K., Sinha, M. K. and Dash, R. J. Long Term experience with glibenclamide in the treatment of diabetes mellitus with studies on insulinogenic effect of i.v. glibenclamide. Madhumeha 11:72, 1971. 59. Rastogi, G. K. Proinsulin "In Insulin and Metabolism" Ed. Bajaj, J. S. Diab. As. India, Bombay, p. 120-129, 1972. 60. Rastogi, G. K. (Boehringer Knoll Annual Lecture) Interaction of glucagon and insulin on the liver. J. Ass. Phys. India. 20 :859, 1972. 61. Rastogi, G. K., Sinha, M. K., Dash, R. J. and Kannan, V. Radioimmunoassay of plasma TSH. A test of thyroid function. Bull. Postgrad. Med. Inst, Chandigarh. 6:49, 1972. 62. Rastogi, G. K., Dash, R. J., Sinha. M. K., Kannan, V. and Singh, T. M. TSH induced rise in plasma Tsh. An important advance in thyroid function study. Bull. Postgrad. Med. Inst. Chandigarh 6 : 53, 1972. 63. Rastogi, G. K., Sinha, M. K., Dash, R. J. and Kannan V. Plasma thyrotropin levels in health and thyroid disorders. J. Ass. Phys. India. 21 : 183, 1973.

61 64. Rastogi, G. K. and Kannan, V. Radioimmunoassay of plasma thyrotropin, an important advance in study of pituitary thyroid axis (Leading article). J. Ass. Phys. India. 21: 235, 1973. 65. Bastogli, G. K., Dash, B. J., Sinha, M. K. and Kannan, V. Plasma thyrotropin and its response to thyrotropin releasing hormone in endemic goitre, Clinical Endocri- nology (London) 2 : 143, 1973. 66. Bastogi, G. K., Sinha, M. K. and Dash B. J. Insulin and proinsulin content of pancreases from diabetic and nondiabetic subjects. Diabetes 22: 804- 1973. 67. Bastogi, G. K., Dash, B. J. and Sinha, M. K. Glucose, IRI and FFA response to intravenous glibenclamide in diabetes mellitus. Hormone and Metabolic Research, 5 : 303, 1973. 6P Bastogi, G. K., Dash, B. J. and Sinha, M. K. Radioimmunoasay of human gonadotropins and serum levels in healthy women and those with gonadal dys- function. J. Ass. Phys. India. 21: 643, 1973. 69. Bastogi, G. K., Dash, B. J. and Sinha, M. K. Serum Gonadotropin levels in healthy men and those with gonadal disorders. J. Ass. Phys. India. 21: 639, 1973. 70. Bastogi, G. K., Sinha, M. K., Dash, B. J. and Kannan V. TRH stimulated TSH response in normal subjects and patients with thyroid disoders. J. Ass. Phys. India. 21: . 627, 1973. 71. Bastogi, G. K., Dash, B. J. and Sinha, M. K. Long term clinical trial of glibenclamide and plasma insulin, growth hormone, free fatty acid and blood sugar response to I. V. glibenclamide. J. Ind. Med. Ass. 61: 60- 1973. 72. Bastogi, G. K. Utility of Plasma thyrotropin estimation and its response to thyrotropin releasing hormone in diagnosis and research. Ann. Ind. Med. Sci. 9:97, 1973. 73. Bastogi, G. K., Chakraborty, J. and Sinha, M. K. Serum gonadotropins and their response to LHRH in diabetic men with and without importance. Horm. Metab. Res. 6 : 355, 1974.

62 74. Rastogi, G. K., Sinha, M. K., Dash, R. J. and Datta, B. N. Serum levels of gonadotropins in male hypogonodism and response to Mesterolone in Ligozoospermis. Proceedings of the International Symposium on Gonadotropins. Ed. Moudgal, N. R. Academie Press, New York U.S.A. pp. 54, 1974. 75. Rastogi, G. K., Chakravarti, J. and Sinha, M. K. Serum gonadotropins (LH and FSH) and their response to LHRH in diabetic men with and without impotence. Proc. of V Asia and Oceania Conf. of Endocrinology, Ed. G. K. Rostogi, Endocrine Society of India 1: 28, 1974. 76. Rastogi, G. K., Saily, R., Sinha, M. K., Dash, R. J., Chopra, J. S. and Kataria, R. N. Serum and pancreatic immunoreactive insulin and proinsulin like component (PLC) and serum IRI and PLC response to different stimuli in normal subjects and organic hyperinsulinism. Acta Diabetologica Latina 12 : 309, 1975. 77. Rastogi, G. K., Sinha, M. K., Jayaraman, S. and Devi, P. K. Serum human placental lactogen levels at different stages of normal pregnancy. Ind. Obstet. Gynec. 26 : 65, 1976. 78. Rastogi, G. K., Dash, R. J. and Sinha, M. K. Thyrotropin releasing hormone induced thyrotropin release in euthyroid men: Effect of ethinyl estradiol priming. Ind. J. Med. Sc. 28 : 529-531, 1974. 79. Rastogi, G. K., Sialy, R., Sinha, M. K., Dash, R. J., Chopra, First Annual Scienific Oration of the Endocrine Society of India, 1976. Circadian responsiveness of endocrine glands. J. Asso. Phys. India 24: 439, 1976. 80. Rastogi, G. K., Dash, R. J., Sharma, B. R., Sawhney, R. C. Saily, R. Circadian responsiveness of hypothalamic-pituitary exis. J. Clin. Endo. and Metab. 42: 798-803, 1976. 81. Rastogi, G. K., Sawhney, R. C. and Talwar, K. K. Serum and urinary thyroid hormones during infective fever. Horm. Met. Res. 8 : 409, 1976. 82. Rastogi, G. K., Saily, R. and Sawhney, R. C. Serum prolactin and thyrotropin response to thyrotropin releasing hormone during different phases of mentrual

63 cycle in healthy women. J. Ass. Phys. India. 24 : 491, 1976. 83. Kastogi, G. K., Saily, R., Thomas, Z., and Vasista, K. Hormonal response to combined LHRH-TRH and LHRH alone is postpartum lactating and non-lactating women. Ind. J. Med. Res. 65. 105, 1977. 84. Kastogi, G. K., Malhotra, M. S., Srivastava, M. C, Sawhney R. C, Dua, Gt. L., Sridharan, K., Hoon, R. S. and Singh, I. Study of pituitary thyroid function at high altitude. J. Clin. Endo. Metab. 44 : 447, 1977. 85. Bazdan, M. N., Siaghal, S. P., Sheth, A. R. and Wadade- kar, K. B. LH and prolactin levels in blood serum and seminal plasma of cross-bred bulls. Ind. J. Expt. Biol., 15 : 235, 1977. 86. Samal, K. C, Dash, R. J., Sinha, M. K. and Rastogi, G. K. Blood glucose, serum immunoreactive insulin and growth hormone responses to intravenous infusion of an ami- noacid mixture in healthy subjects and diabetics. J. Assoc. Phys. India. 25 : 175, 1977. 87. Samuel, A. M. and Deshpande, U. R. Growth Hormone in protein calorie malnutrition. J. Clin Endo. Metab. 35 : 865, 1972. 88. Sawhney, B. B., Chopra, J. S., Rastogi, G. K. and Kataria, R. N. Hyperinsulinism and neuroglycopenic syndromes. Neuro- logy India, 23 : 121, 1975. 89. Sawhney, R. C, Rastogi, I. and Rastogi, G. K. Evaluation of hypothalamic-pituitary-thyroid axis and TSH metabolism after estrogens. Annals Ind. Acad. Med. Sc. 1978. (In Press) 90. Sawhney, R. C, Rastogi, I. and Rastogi, G. K. Effect of Estrogens on Thyroid function. I: Alterations in plasma thyrotropin and its kinetics. Endocrinology : 1978 (In press). 91. Sawhney, R. C, Rastogi, I. and Rastogi, G. K. Effect of Estrogens on thyroid function II. Alterations in plasma thyroid hormone levels and their metabolism. Metabolism. 1978 (In press). 92. Seth, A. R., Joshi, L. R., Moodbidri, S. B. and Rao, S. S.

64 Serum prolactin levels in fertile and subfertile men. Andrologie 5 : 297, 1973 93. Sheth, A. R., Mugatwala, P., Shah, G. V. and Rao Shanta, S. Occurrence of prolactin in human semen. Fertility and Sterility 26, 905, 1975. 94. Sheth, A. R., Gadgil, B. A., Joshi, L. R. and Rao, S. S. Occurrence of immunoreactive prolactin in bovine semen. Ind. J. An. Sc. 46 : 42, 1976. 95. Sheth, A. R., Vaidya, R. A. and Raikar, R. S. Presence of prolactin in human cervical mucus. Fert. Steril. 27 : 397, 1976. 96. Sheth, A. R., Shah. G. V., Gadgil, B. A. and Swamy, K. R. Effect of LH/FSH-RH on circulating gonadotropin levels in bonnet monkeps Ind. J. Expt. Biol. 14 : 272, 1976. 97. Sheth, A. R., Jayatilak, P. G., Thakur, A. N., Mugatwala, P. and Pardanani, D. S. Effect of administration of a single dose of testosterone ceanantate on constituents of human seminal plasma and srum gonadotropins. Andrologie 3, 259, 1976. 98. Sheth, A. R., Shah, G. V. and Mugatwala, P. Levels of LH in semen of fertile and infertile men and possible significance of LH in sperm matabolism. Fertil. Steril. 27, 933, 1976. 99. Sheth, A. R., Vaidya, R. A., Arbatti, N. J. and Devi, P. K. Effect of Centchroman on serum gonadotropins and prolactin in rats. Ind. J. Expt. Biol. 1978 (In Press). 100. Sheth, A. R., Wadadekar, K. B., Moodbidri, S. B., Janaki- raman, K. and Parameshwaran, M. Seasonal alteration in the serum of prolactin and LH levels in the water buffaloes. Current Science, Bangalore. (In Press) 101. Sheth, N. A., Ranadive, K. J. and Sheth, A. R. In vitro binding of radioiodinated human placental lactogen to C3H (Jax) mice mammary gland. Europ. J. Cancer 10 : 653, 1974. 102. Sheth, N. A., Saraiya, J. N., Ranadive, K. J. and Sheth, A. R.

65 Ectopic production of HCG by the human breast tumours. Brit. J. Cancer 30 : 566, 1974. 103. Sheth, N. A., Saraiya, J. N., Ranadive, K. J. and Sheh, A. R. Circulating levels of prolactin in breast cancer. Brit. J. Cancer 32: 160, 1975. 104. Sheth, N. A., Tikekar, S. S., Ranadive, K. J. and Sheth, A. R. Enhancement of invitro binding of rat prolactin to murine mammary gland by polyamines. IRCS Medical Sciences 4: 323, 1976. 105. Sheth, N. A., Saraiya, J. N., Sheth, A. R. and Ranadive, K. J. Ectopic production of HPL by human breast tumours. Cancer, Vol. 39, 1693, 1977. 106. Sekhuja, V., Rastogi, G. K. and Sialy, R. Growth hormone response to L-Dopa in Diabetes Mellitus. Acta Diabetologica Latina 12 : 242, 1976. 107. Shah, G. V., Desai, R. B. and Sheth, A. R. Effect of prolactin on the metabolism of human sperma- tozoa. Fert. Steril. 27, 1292, 1976. 108. Shah, P. N., Rastogi, G. K. and Rajendran, K. G. Eesponse of plasma FSH and LH to L-Dopa in males with gonadotropin deficiency. Proc. V Asia and Oceania Cenf. of Endocrinology, Ed. G. K. Rastogi, Endocrine Society of India, 1: 32, 1979. 109. Shahani, S. K., Sheth, A. R. and Rao, Shanta S. In vitro binding 125-1 rat growth hormone by adrenals, gonads and accessory reproductive organs of rats and mice. Proceedings of V Asia and Oceania Congress of Endocrinology. 110. Sheela Rani, C. S. and Moudgal N. R. Examination of the role of FSH in the periovolatory events in the hamster. J. Raprod. Fert. 50, 37, 1977. 111. Sheela Rani, C. S. and Moudgal, N. R. Role of the proestrus surge of gonadotropins in initiation of follicular maturation in the hamster. A study using antisera to FSH and LH. Endocrinology 101: 1484, 1977. 112. Sheela Rani, C. S. and Moudgal N. R. Measurement of FSH in the ovarian tissue by RIA-Cor-

66 relation to serum FSH levels and follicular development in hamster. (Communicated to Mol. Cell Endocrinol). 113. Sialy, R. and Rastogi, G. K. and Gupta, A. N. Serum prolactin levels in healthy men and women. Annals Ind. Acad, Med. Sci. 12 : 124, 1976. 114. Saily, R., Rastogi, G. K. and Gupta, A. N. Serum prolactin and its response to thyrotropin releasing hormone in normal pregnancy. Ind. J. Med. Res. 65 : 520- 1977. 115. Sinha, M. K., Gupta, P. R., Dash, R. J., Wahi, P. L. and Rastogi, G. K. Change in glucose, FFA and insulin following intravenous glucogon in normal subjects and patients following myocardial inferction. Bull. Postgrad. Med. Inst. Chandigarh, 7 : 58, 1973. 116. Sinha, M. K. and Rastogi, G. K. Radioimmunoassay of insulin. Madhumeh. 13 : 131, 1973. 117. Sinha, M. K., Mondal, A. N. and Rastogtf, G. K. Influence of age on the glucose tolerance text. Acta Dia- betologia Latina 11 : 78, 1974. 118. Sinha, M. K., Malik, G. P. and Rastogi, G. K. (1974). Growth hormone secretion in Diabetes mellitus. Acta. Diabetologica Latina. 11 : 89, 1974. 119. Siva Kumar, B. and Krüshnamachari, K. A. V. R. Circulating levels of parathyroid hormone in endemic genu valgum. Horm. Metab, Res. 8 : 317, 1976. 120. Siva Kumar, B. Circulating levels of growth hormone in endemic genu valgum. Horm. Metab. Res. 9 : 436, 1977. 121. Sood, S., Katriya, R. N., Chari, P., Chopra, J. S. and Dash, R. J. Surgical management of pancreatic hyperinsulinism Ind. J. Surg. 39 : 469, 1977. 122. Talwar, K. K., Sawhney, R. C. and Rastogi, G. K. Serum levels of thyrotropin, thyorid hormones and their response to thyrotropin releasing hormones in infective Febrile illness. J. Clin. Endoor, and Metab. 44: 398, 1977. 123. Thakiur, A. N., Sheth, A. R., Purandare, T. V., Munshi, S. R. and Rao, Shanta, S.

67 Antisera to gonadotropins and plancental function in mice I. Effect of polyamine content. J. Reprod. Fert. 43 : 221,1979. 124. Vaidya, P. R, Sheth, A. R., Raikar, R. S., Thakur, S. S. and Rao, Shanta, S. Serum levels of HPL and HCG following intraamniotic injection of hypertonic saline. Ind. J. Obst. Gynec 25 : 419, 1975. 125. Vaidya, R. A., Sheth, A R., Joshi, L. R. and Devi, P. K. Effect of Centchroman (non-steroidal) on serum LH and FSH levels in man. Fertil. Steril, 27, 459-462, 1976. 126. Vaidya, P; R,, Thakur, S. S., Sheth, A. R. and Raikar, R. S. *- Serum levels' of gonadotropins during lactational amenor- ''- rhea. Ind. J. Ob. Gyn. 26 : 727, 1976. 127. Vaidya, P. R., Sheth, A. R., Raikar, R. S. and Thakur, S. S. Serum levels of LH, FSH and prolactin during and after labor. Ind. J. Ob. Gyn. 26 : 680, 1976. 128. Vijaya, E., Sankaran, M. S. and Sheth, A. R. Correlation between pituitary sialic acid and FSH concentrations during .development afjter castration and testosterone treatment" in the male rat. Neuroendocrinology ,15: 137, 1974. " > ' '••' . *-•?•«'*•- >•

•-•Thyraid;, Hornione f jVIeena, Sheth, A. R., Raikar, R. S. and Vishwanath,

Obstetrics &

f C.fTanda, $ï. C. and Trdpathy,

,.-/ Th"-fnYid %i|jncjne levels /.in- admit" protein calorie malnu- iri-ïion. •«4^. ^astopri', (ï; K?Jè>ji$Xm C. and Kannan, V. . v 'CoTrelaiion'of ^eH-i'hi LATS with serum levels of thyroxine "&'« and,, tri-ïodothyronine in Graves disease. J. Assi Phys. India. 23 : 111. 1.975. 5. Rastogi, G. K., Thomas, Z. and Sawhney, R. C. Critical evaluation of urinary excretion of T-3 and T-4 in Graves' disease. Proc V Asia and Oceania Cong, of Endo- crinology, ed. G. K. Rastogi, Endocrine Society of India, 2 : 1, 1974. 6. Rastogi, G. K. and Sawhney, R. C. Thyroid function in changing weather in a sub-tropical region. Metabolism 25 : 903, 1976. 7. Rastogi, G. K. and Sawhney, R. C. Value of urinary excretion of triiodothyronine (T-3) and thyroxine (T"4). Ind. J. Med. Res. 64: 1639, 1976. 8. Rastogi, G. K. and Jaykumar, R. V. Thyroid hormones and related compounds in circulation. Ann. Ind. Acad. Sc. 1978 (under publication) 9. Sawhney, R. C, Rastogi, G. K. and Kannan, V. Correlation of LATS with serum levels of T-3 and T-4 in Graves' disease. Proc V Asia and Oceania Cong, of Endo- crinology Ed. G. K. Rastogi, Endocrine Society of India 2 : 49, 1974. 10. Sawhney, R. C. and Rastogi, G. K. Diagnostic value of total serum T-3 and T-4 estimation. Proc V. Asia and Oceania Cong, of Endocrinology, Ed. G. K. Rastogi, Endocrine Society of India, 2 : 9, 1974. 11. Shah, D. H., Dandekar, S. R. and Ganatra R. D. Thyroglobulin levels in serum and saliva of patients with differentiated carcinoma. Proc. Ind. Acad. Sci. 87 B : 169, 1978.

Steroid Hormones 1. Dash, R. J., Lata, V., Sharma, B. R. and Gupta, A. N. Hormones in reproduction VI. Circulating maternal serum progesterone in normal pregnancy. Relation to chorionic gonadotropin. Bull. Postgrad. Med. Inst. (Chandigarh) 11 : 184, 1977. 2. Dash, R. J., Samal, K. C, Sharma, B. R. and Rastogi G. K. Radioimmunoassay of testoterone in human serum. Bull. Postgrad. Med. Institute, Chandigarh 9 : 42, 1975. 3. Kadival, G. V. and Samuel, A. M. Effect of experimental malnutrition on corticosteroids and

69 V

liver cytoplasmic receptors in rats. Biochem. Med. 18 : 353; 1977. 4. Kumar, E. V., Kumar, L., Pathak, I. C, Dash, R. J. and Joshi, V. V. Clinical hormonal and ultrastructural studies of virilising hepatoblastoma. Acta. Paediat. Scandisevica: In press., 1978 (In Press). 5. Mukku, V., Prahalada, S. and Moudgal, N. R. Effect of constant light on nychthemeral variations in serum testosterone in male Macaca radiata. Nature 260, 778, 1976. 6. Samuel, A. M., Kadival, G. V., Patel, B. D. and Desai, A. G. Corticosteroids and corticosteroid binding globulin in protein calorire malnutrition. Amer. J. Clin. Nutr. 29 : 889, 1976.

Non- Hormonal substances 1. Gopal Naüdu, N. B. Aikat, B. K., Dash, R. J. and Sehgal, S. Serum alphafetoproteins in experimental hepatocellular cancer. Eur. J. Cancer 1978 (In Press). 2. Kulkarni, S. K., Bapat, J. P., Baxi, A. J., Kulkarni, Chanderkar, N. G. and Gaitonde, B. B. Evaluation of Radio-immuno Detection Method for Hepa- titis. B. (Australia) Antigen. Bull. Haffkine Inst. 3, 107- 1975. 3. Muthe, A. V., Kulkarni, S. K., Bapat, J. P., Baxi, A. J., Kulkarni, K. V., Chandergar, N. G. and Gaitonde, B. B. Detection of Hepatitis B (Australia) Antigen in Human Plasma Protein fractions using Radioimmunoassay. Bull Haffkine Inst. 3: 156, 1975.