Report on the national seminar on radioimmunoassays

REPORT ON THE NATIONAL SEMINARON RADIOIMMUNOASSAYS

JANUARY 16-20, 1978

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

DEPARTMENT OF ATOMIC ENERGYAND

WORLD HEALTH ORGANISATION

REPORT ON THE NATIONAL SEMINARON RADIOIMMUNOASSAYS

JANUARY 16—20, 1978

RADIATION MEDICINE CENTRE, B A R CTATA MEMORIAL HOSPITAL ANNEXE

JERBAI WADIA ROAD, PARELBOMBAY 400 012

ORGANISEDUNDER THE AUSPICES OF THE

DEPARTMENT OF ATOMIC ENERGY&

WORLD HEALTH ORGANISATION

C O N T E N T S

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 thesecond National Seminar jointly sponsored by Department ofAtomic Energy and World Health Organisation and organised atRadiation Medicine Centre. The first one on 'Nuclear Medicine inIndia' was held in December 1976. The present Seminar onRadioimmunoassays was distinguished by the participation ofDr. Rosalyn Yalow, Nobel Laureate in Medicine for 1977. She,along with Dr. Solomon A. Berson, discovered the techniqueof Radioimmunoassay and nurtured it through the early yearswith hard and meticulous work to establish its usefulness inmedical science. Her presence at the Seminar was a continu-ous source of pride and inspiration to both the organisers andthe participants.

The Seminar was inaugurated by Dr. R. Ramanna, Director,Bhabha Atomic Research Centre on January 16, 1978. In hisinaugural address he said that the Bhabha Atomic ResearchCentre had been honoured to have Dr. Rosalyn Yalow, who wasspending three weeks at Radiation Medicine Centre as a visitingscientist. He outlined the usefulness of the technique, itssimplicity and applications in our country. This method iscomparatively cheaper than most nuclear medicine procedures.He suggested that Indian scientists could explore the possibilityof applying this technique in the diagnosis and epidemiologicalsurveys of infectious diseases.

This Seminar has provided a good opportunity in bringingtogether scientific workers in this field to review the work doneso far, to air their grievances, to describe the difficulties whichthey encounter and to try and find practical solutions to thejrproblems. This is an essential exercise of scientific progressespecially in this field as its application involves assistance fromseveral government and public institutions.

With the rapid and exponential growth of the medical ap-plications of this methodology, it is no longer considered asan esoteric or an exotic facility but an essential tool in the diag-nostic armamentarium to be used as commonly as a routineclinical chemistry examination.

The object of the Seminar was to evaluate : •

a. The application of this technique in the routine medicalcare of patients and its use in public health programmes.

b. To evaluate whether a Central or Regional Assay Servicecould effectively fulfil the needs of a region or district.

c. The logistics of establishing such a service and to studythe cost against the benefit to the public.

d. To arrive at some estimates for funds required, and tofind out which agencies could be asked to aid in organisingthese facilities.

The Seminar concerned itself towards answering some ofthese questions and arriving at some helpful solutions. Theinputs required to get an assay going are simple. It puts verylittle demand on technical knowledge and skill when kits forradioimmunoassays are available. Towards this progress, theIsotope Division of Bhabha Atomic Research Centre has takenpioneering steps in making available several useful "kits" at afairly economical price. The establishment of the IndianNational Pituitary Agency under the auspices of the IndianCouncil of Medical Research will provide and make availablereagents for the assays. This would relieve the burden onIndian scientists who at the present have to go out with a beg-ging bowl for the basic ingredients of the assay system. TheElectronics Division of Bhabha Atomic Research Centre andthe Electronics Corporation of India Ltd., would make availableeasy to use, efficient and what is very important cheap manualgamma counters which would be within the budget of mostmedical colleges and hospitals.

The Institute for Research in Reproduction, Bombay,Endocrinology Department of Post-graduate Institute, Chandi-garh and Radiation Medicine Centre, Bhabha Atomic ResearchCentre, have contributed a great deal towards the growth anddevelopment of these techniques in India. In addition program-mes for training technologists and physicians are offered by allthese Institutes. One of the problems for discussion at thisseminar was to evaluate how effective and useful these trainingprogrammes have been and whether any changes were neededin the duration and type of courses offered.

This Seminar has given not only members of RadiationMedicine Centre but a lot of other scientists a rare opportunityto speak, hear and discuss their problems and difficulties witha Nobel Laureate, for which the organisers would like to thankthe World Health Organisation for their generous help in mak-ing this Seminar a reality. Dr. M. Thangavelu, Advisor, Regional

Office of World Health Organisation, New Delhi, participatedin the Seminar and his contribution in the scientific deliberationsof the seminar was most worthwhile.

Dr. (Mrs.) A. M. Samuel

Administrative Responsibilities

1. Chairman of PlenarySessions

2. Scientific Co-ordination

3. Preview of Scientificpapers, their duplicationand distribution

4. Rapporteurs for plenarysessions and triggersessions

5. Accommodation andTransport

6. Catering

7. Editing of the reportof the proceedings

Dr. K. SundaramDirector,Bio-medical Group, BARC,Trombay, Bombay 400 085.

Dr. R. D. GanatraHead,Radiation Medicine Centre,BARC,Tata Memorial Centre, Parel,Bombay 400 012.

Shri G. V. Kadival

1. Dr. (Mrs.) A. M. Samuel2. Shri G. V. Kadival

1. Shri P. Ramanathan2. Shri M. C Patel

1. Mrs. L. S. Mani2. Shri A. V. Borkar

1. Dr. (Mrs.) A. M. Samuel2. 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 Educationand 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 andB.Y.L. Nair Charitable Hospital,Bombay 400 008.

5. Dr. S. D. Bhandarkar,Hon. Asst. Professor of Endocrinology andHon. 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. Ganatra2. Dr. (Mrs) A. M. Samuel3. Dr. (Miss) D. H. Shah4. Mr. K. B. Desai5. Mr. P. Ramanathan6. Mr. M. C. Patel7. Mr. M. N. Mehta8. Mr. G. V. Kadival

10

NATIONAL SEMINAR ON RADIOIMMUNOASSAYSPROGRAMME

MONDAY—JANUARY 16, 1978

Inauguration

Dr. R. RamannaDirector, B.A.R.C.

Session 1: RESOURCES

1. Radionuclides2. RIA kits3. Antibodies4. Pituitary hormones5. Counting equipment

R. S. ManiK. B. ShahK. B. DeasiA. R. ShethV. A. Pethe

6. Radiation Protection S. Somasundaram

Trigger Session 1

Quality Control of RIA—Introduction byB. B. GaitondeRECENT ADVANCES IN RIA—IROSALYN YALOW

TUESDAY—JANUARY 17, 1978Session 2 : RADIOIMMUNOASSAYS IN INDIA

1.2.3.4.5.

6.

7.8.9.0.1.

Thyroid hormonesDiabetesNutritionSex hormonesSterility andFertilityReceptor boundgonadotrophichormonesAustralia AntigenNucleotidesCircadian RhythmsEnzyme ImmunoassaysRadioimmunometry

N.G.K.A.S,

c.

A.C.R.U.D.

KochupillaiK. RastogiJaya RaoR. ShethM. Shahani

S. Sheela Ra

J. BaxiParijaD. KulkarniM. JoshiK. Hazra

11

Trigger Session 2

USEFULNESS AND LIMITATIONS OF RIA INCLINICAL DIAGNOSIS

Introduction by S. D. Bhandarkar

RECENT ADVANCES IN RIA—II

ROSALYN YALOW

WEDNESDAY—JANUARY 18, 1978

Session 3 : REVIEW OF WORK AT DIFFERENTCENTRES

1. PGI, Chandigarh G.K. Rastogi2. AIIMS, Delhi N. Kochupillai3. BHU, Benaras J. Chansouria4. IRR, Bombay S. S. Rao5. PGMER Calcutta K. K. Ghosh6. 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. SamuelD. H. Shah

Session 4. ORGANISATION SUPPORT

1. W.H.O. M. Thangavelu2. 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 ANDGUIDELINES

RECENT ADVANCES IN —RIA—V

ROSALYN YALOW

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BASIC REQUIREMENTS FOR RADIOIMMUNOASSAYSIN INDIA

This chapter deals with the inputs required to establishradioimmunoassay procedures. Problems encountered by thescientists like the difficulties in obtaining reagents within thecountry were discussed. Some solutions to these problems wasoffered.

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RADIOISOTOPES FOR RADIOIMMUNOASSAYS

An important raw material required for a successful radio-immunoassay is the radionuclide for labelling the antigen orother ingredient sought to be measured. The principalcharacteristics 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 andpurity, (e) ready incorporation into the molecule without affect-ing the specific immunological properties of the compoundlabelled, and (f) radiation emission which ensures stability ofthe labelled compound and a reasonably long shelf-life.

Considering the structure and properties of varioushormones, antigens, vitamins, viral proteins, antibodies, drugsetc. of interest in radioimmunoassay, both "genuine" and"forced" labelling procedures can be adopted. The radioisotopeswhich are either already widely employed in radioimmunoassayor 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 targetfor the production of 1-125. For large scale production, xenongas is enclosed in special zircalloy containers at pressures rang-ing upto 200 atmospheres and is irradiated at neutron fluxes ofthe order of 1013n Cnr2 Sec.-1 The impurities which arise as aresult of side reactions are 1-126 (half life 13 days), Cs-137 andBa-137m and traces of 1-127 (stable). Post irradiation coolingof about 60 days reduce the 1-126 contaminatoin to less than 2%.

Under optimum irradiation conditions, 1-125 with specificactivities upto 17 Ci/mg representing an isotopic abundanceexceeding 90% can be obtained. Irradiation of natural Te con-taining 34.5% of Te-130 yields 1-131 with specific activities upto30 Ci/mg representing approximately 25% isotopic abundance.To attain the highest specific activities, however, special precau-tions are to be taken to minimise or eliminate stable iodineproduction/contamination at various stages. Fission of U-235can yield much higher specific activities ; but this method is lesspracticable for routine use. H-3 with almost 100% abundancecan be produced by reactor irradiation of lithium alloy targetsfollowed by processing and subsequent enrichment by chromato-

15

graphy or diffusion. These processes are practicable andeconomical only at the multikilocurie level of activity of H-3.

The Isotope Group of Bhabha Atomic Research Centre doesnot as yet produce 1-125, H-3 and C-14. Arrangements have,however, been made to import these isotopes in bulk from abroadand supply to users.

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KITS FOR RADIOIMMUNOASSAY

Radioimmunoassay techniques permit measurements ofminute concentrations of virtually any substance of biologicalinterest. The technique is used to trace peptide and non-peptidalhormones as well as to detect non-hormonal substances likeenzymes, viruses, tumour antigens and several drugs includingmorphine, LSD and barbiturates;

Three procedures are generally adopted for carrying outRIA by various institutions.

i. Some Institutions which have developed considerableexpertise in RIA prefer to develop and use their own lot ofreagents including the labelled antigens, antibodies and self-standardised techniques for separating bound and free fraction.

ii. Some other clinical and research centres, who also havedeveloped expertise in RIA and are interested in carrying outRIA more routinely and in large numbers, procure ready to useRIA kits. These kits are now available from a large number ofcommercial suppliers.

iii. Some centres where facilities and expertise for RIAhave 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 theirpotential importance in our country, Bhabha Atomic ResearchCentre—Isotope group has taken up a programme to developand provide reliable and ready to use kits for RIA. Kits forinsulin are available, while the kits for thyroid stimulatinghormone, human growth hormone, digoxin, digitoxin, humanchorionic gonadotrophin, hepatitis associated antigen, angio-tensin I and II are under development.

The kits consist of the following reagents:

i. Labelled hormoneii. Standard hormone

iii. High quality antibodyiv. Reagents to separate antibody bound antigen from free

antigen.

Whereas pure hormones are procured from abroad, effortsare being made to make them indigenously by collaboratingwith various institutions. It is heartening to note that, the

17

recent establishment of the National Pituitary Agency by theIndian Council of Medical Research at the Institute for Researchin Reproduction, Parel, Bombay will give a great impetus to thedevelopment of RIA programmes in our country.

For procurement of antisera, we either collaborate withthose institutions such as Institute for Research in Reproduc-tion, Bombay or Haffkine Institute, Bombay which have acquiredconsiderable expertise in the field or we ourselves undertakeimmunisation programmes and produce the antisera in ourCentre.

Various parameters such as stability and purity of thelabelled compound, sensitivity and specificity of the antisera arestudied before the formulation of the kits. The kits are subjectedto quality control tests such as non-specific binding, the bindingof labelled hormone with the antibody in the absence of un-labelled hormone, inter-assay and intra-assay variation in pooledserum samples, recovery etc. The kit performance is checkedat 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, havefacilities to carry out RIA; Bhabha Atomic Research Centre—Isotope Group offers a limited RIA service, for the analysis ofserum samples.

Future programmes include (i) development of new kitsfor hormones, and non-hormonal substances of biological inte-rest, (ii) introduction of newer and simpler techniques for theseparation of bound and free fractions for the existing kits.

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AVAILABILITY OF ANTIBODIES

Many centres in India have been successful in raising anti-bodies to a variety of substances. A common problem that onefaces is the difficult}' in acquiring specific antigens. Antibodiesto albumin, thyroglobulin. triiodothyronine, thyroxine, thyrotro-pin, growth hormone, insulin and transferrin are used at Radia-tion Medicine Centre in RIA or other investigations. These anti-gens except thyroglobulin and in many instances even antiserahave to be imported. This problem is common to most Indianworkers. 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 ofthese antigens or antiserum from abroad.

In this context it is imperative that a centre or centresshould be entrusted with the work of processing and acquiringstandards and raising antibodies for distribution to variouslaboratories 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 basicand clinical research in the country the requirements for hor-mones and the diagnostic reagents should be met from withinthe country and that less dependance should be placed on thegenerosity of the National Pituitary Agency for the supply ofreagents. It was therefore felt that a Central Agency be esta-blished in the country to undertake this work which would alsoensure the quality control of hormone assays.

The establishment of a National Pituitary Agency (NPA)under the aegis of the Indian Council of Medical Research, re-quires very close and active collaboration of a large number ofhospitals, institutes and their staff from all over the country.Prominent scientists from all over India have agreed to guidethe activities of NPA.

The major efforts of the* agency at this stage will be asfollows:

a) Collection of pituitaries on a national basis.

Following centres have started collecting pituitaries:

Bombay, Delhi, Chandigarh, Bangalore, Madras, Lucknowand Varanasi. About 1500 pituitaries were collected during aperiod of 4 months. With further organisation and co-operativeefforts it should not be difficult to collect more than 5000 pitui-taries per year.

b) Purified hormones and antiseras will be calibratedagainst International Standards and made available for distri-bution to different workers in India.

c) Requests for growth hormones and gonadotrophins fortherapeutic use shall be considered by Medical Advisory Boardof NPA.

d) Attempts will be made to improve existing RIAs anddevelop simpler assays for protein hormones.

e) National Pituitary Agency will offer laboratory facilitiesto other workers in India where such facilities cannot be esta-blished at their centres.

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WELL-COUNTER FOR RADIOIMMUNOASSAY

The most essential instrument required for radioimmuno-assay is a counter for the quantification of radioactivity. Gammacounters are used for gamma emitting isotopes such as the morecommonly used 1-125 and 1-131. The most widely used detec-tor for gamma emitters is the Nal well-counter.

The best geometry is a 4 TT geometry and highest efficiencyis obtained when all radiations are absorbed within the crystal.A small sample of low energy emitter positioned at the centreof the 2"x2" or 3"x3" Nal crystal through a hole will essen-tially detect all gamma radiations except minor losses escapingthrough the hole. Most well-counters presently in use employ Nalcrystals If" in diameter, 2" in height with f" diameter, ty'deep well.

Many variables affect the data obtained with a well-coun-ter. Therefore, the conditions of measurement such as samplevolume, the fraction of the pulse height spectrum included inthe measurement, and whether the efficiency refers to the num-ber of disintegrations or to the number of gamma radiations emitt-ed must be stated.

Most of the tests use 1-125 which decays to Te-125 witha half life of 60 days. The decay occurs in all cases bya process of electron capture, after which the Te nucleushas excitation energy of 35.5 Kev and the atomic structure hasan inner shell vacancy. When this vacancy is filled there isemission of either an X-ray or an Auger electron. If the X-rayis from the K-shell it has an energy of 27.5 Kev and is easilydetected by a Nal (TI) crystal detector. If it is emitted fromany other shell it has insufficient energy to be detected. Augerelectrons are not detected due to absorption in the source mate-rial. The excited nuclear state of Te-125 decays either by emis-sion of a detectable 35.5. Kev gamma ray (in 7% of cases) or byinternal conversion (93%) which results again in an inner shellvacancy with the same possibilities for emission as above. Thevarious processes occur virtually simultaneously.

Not all detectable quanta successfully interact with thedetector so that some simultaneous double emission result inonly a single interaction. Those double quanta which aresuccessfully detected simultaneously produce a response which

21

is equivalent to an event of twice the normal energy. Thisproduces the second peak of the characteristic 1-125 pulse heightspectrum.

Because of low energy emitted by 1-125, the internal linershould have as low a thickness as possible. The liner could bepreferably made of Beryllium. Maximum efficiency is obtainedwhen plastic tubes are used. The setting up of the counter shouldbe done carefully to optimise the signal to noise ratio.

There is a large variety of equipment commercially avail-able, starting with simple portable low priced counter, to com-pletely automatic multiradionuclide counters with preset timeand count facilities and with background subtract arrange-ment. Many of them also have computing facilities which canbe fed to available computer systems.

Many of these systems use a through hole detector givinguniformity of counting efficiency regardless of varied samplevolumes. The detector is also operated in a controlled tempe-rature environment eliminating the inherent errors from changesin 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 intro-duced recently is unique in many respects. It employs amultiple detector system with sixteen detectors matched to eachother (within 1%). Each detector is surrounded by an effectiveshield of lead (350 micro) to ensure that no cross-talk can occureven with very highly active sources. The system has anexcellent 1-125 counting performance of 75% efficiency at lowbackground. With automation eliminated, there are no movingparts 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 ofa non-linear standard curve is difficult and inaccurate. Restric-tion of dose interpolation to a small linear segment of the curvemarkedly 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 inthis method is the need for computations involving logs andantilogs 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 decaysolely by electron capture to the first excited level (35.5 Kev)of Te-125 which in turn decays to the ground state either byemission" of 35.5 Kev gamma-ray (77o) or by internal conver-sion (93%). The vacancies in the inner electronic shells occur-ring due to electron capture and internal conversion are follow-ed by emission of x-rays or Auger electrons. Dillman (1) hascalculated the energies and intensities of electromagneticradiations and electrons emitted in the decay of 1-125. 1-125gives 1.43 photons per disintegration of which 96% are 27.4 KeVTe K x-rays and the resi 35.5 KeV gamma-rays. The mostenergetic electrons are of 32.4 KeV which have a range ofabout 21 um in tissue.

Radiation dose from 1-125

Gavron and Fiege (2) have calculated the radiation dosedue to 1-125, considering an exact model of normal thyroidfollicles and taking into account the distribution of radioiodine.The radiation dose consists of two components—the electroncomponent and the photon component. For 1 nCi of 1-125 pergram of thyroid, the electron dose-rate is 44 mrad/hr and thephoton dose-rate is 21 mrad/hr. Taking into account the pre-sence of small follicles, Gavron and Fiege have obtained atotal dose-rate of 93 mrad/hr due to 1 nCi of 1-125 per gramof thyroid. For a maximum permissible dose-rate of 600 mradper week, the maximum permissible thyroid burden of 1-125works out to 770 nCi. This is reduced by a factor of 2 to takeinto account the enhanced biological effects of 1-125 and amaximum permissible thyroid burden of 300 nCi has beensuggested. In Indian subjects the uptake of iodine is higherand the mass of thyroid is less than 20 g assumed for the ICRPReference Man and the maximum permissible thyroid burdenof 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 otherpublications from World Health Organisation, InternationalAtomic Energy Agency, ICRP (3-5). A few papers have appear-ed in the literature which deal specifically with radiation safetyaspects in RIA (6-8). The safety considerations would dependupon the amount of the radioisotope, its physical and radio-chemical properties, its radiotoxicity and the types of operationsperformed. Apart from ensuring general safety in handlingany radiochemical substance, work involving the labelling ofproteins with 1-125 should be carried out in specially designedsafety cabinets. Chesworth and Clarke (9) have described asafety cabinet suitable for this purpose. The safety cabinetshould accommodate special tongs or devices to handle highspecific activity solutions in vials. Area monitoring should becarried out in areas where 1-125 is handled. During and afteriodination, air samples should be collected using special filtersand the filters counted in a well-type scintillation counter. Theair concentration of 1-125 should be kept below 4 x 10-° uCi/cms

for 40 hr work week. Surface decontamination of concentratedfree idoine should be carried out by treating it with sodiumthiosulphate which renders it chemically stable.

Monitoring of 1-125

Despite all the precautions taken during handling, the possi-bility of internal contamination of workers with 1-125 shouldnot be overlooked, since the solutions are highly active. Suchcontamination may occur through inhalation of iodine vapouror via skin contamination. The seriousness of intake shouldbe assessed by a regular programme of thyroid monitoring.Weekly monitoring of workers is advisable. If required, thy-roid uptake of 1-125 iray be blocked by giving an oral dose ofstable iodine (Lugol's solution) to the workers, of course, underproper medical advice.

For monitoring 1-125 thyroid burdens in workers bioassay(urine analysis) may be done. In our laboratory we havedesigned a probe (10) which detects externally the 27.4 KeVX-rays and 35 KeV gamma-rays which are emitted in the decayof 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 ispositioned to view the neck region of the subject seated in anordinary chair. The probe has been calibrated by an in vitroprocedure using a REMCAL phantom, the thyroid cavity ofwhich 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 170cpm for an unexposed subject the corresponding minimumdetectable activity (3 times the standard deviation in back-ground) of T-125 in thyroid is about 0.2 nCi for a counting periodof 10 min. The probe is used for monitoring 1-125 thyroidburdens in workers. 1-125 thyroid burdens ranging up to 15nCi have been observed. In one subject a thyroid burden ofabout 100 nCi has been detected.

Work is in progress to develop a battery operated surveymater incorporating a thin Nal (Tl) detector for monitoring1-125 contamination on surfaces,

REFERENCES

1. L. T. Dillman, "Journal of Nuclear Medicine," 10, Supplement2 (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 ofHealth Physics (USA).

8. J. J. McLintock and J. L. Young, "Laboratory Contamination byRadioactive 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 aThin Nal (Tl) Detector," Paper presented at Ninth AnnualMeeting 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 tothe conditions in India were discussed.

1. Among the various radionuclides used for radioim-munoassays and competitive protein binding assays it was feltthat 1-125 was the isotope of choice. However since 1-125 iscurrently imported from abroad, the feasibility of its produc-tion in India was discussed. It was felt that 1-125 could beproduced by the Isotope Division of Bhabha Atomic ResearchCentre in two years time. A suggestion was made that if theamount of 1-125 required was small it would be more profitableto import it in bulk and distribute as required rather thanspend large amounts on production. It was also proposed thata central agency could supply labelled compounds on requestinstead of each centre trying to label its own antigens.

2. The feasibility of producing kits for various hormonesin addition to the kits already supplied was discussed. It wasstrongly felt that kit production and distribution should bepriority oriented. This should be based on the needs of thecountry, like community health problems, and for screening ofblood donors for the presence of Australia antigen. The decisionon priorities should be taken only after consultation with con-cerned scientific and health authorities. However it was also feltthat if kit preparations were limited to a single Centre then thestate of art would be limited by the capabilities of that Centre.Hence there should be room for improvement and quality con-trol by other Centres. It was also felt that the various reagentsused in radioimmunoassay could be supplied to the user onrequest.

It was pointed out that some of the kits already suppliedalso needed improvement in sensitivity.

3. The problems faced in producing antisera were dis-cussed. It was suggested that the second antisera used in radio-immunoassays which required immunisations in bigger animalsshould be produced and supplied by one Central agency. Theproduction of first antisera could be done in local laboratoriesand multiple antigens could be injected in the same animal.It was not also necessary to have a very pure preparation ofthe 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 PituitaryAgency which would supply the needs of the country. The firstbatch of human growth hormone has been ampouled and isavailable on request. FSH, LH and TSH would be shortlyavailable. The programme also envisages the supply of hormonesfor therapeutic use and also animal hormones and specific anti-sera to these in due course of time.

5. Training courses offered by I.C.M.R. and their useful-ness was also discussed. There was a general feeling that thetraining was not properly utilised by the participants due to thelack of assistance in supply of various reagents for radioimmuno-assay. It was felt that these training courses could be moreprofitably utilised if senior personnel who have access to fundsundergo this training. Funds were available to medical collegesfor research work if proposals could be sent to I.C.M.R. andDepartment of Science and Technology.

6. It was felt necessary that the country should make itsown counters. 1-125 being the isotope of choice for radioimmuno-assays it could be counted by a simple yet cheap equipmentwhich would cost from Rs. 10,000—12,000.

7. The regular radioimmunoassay procedures do not in-volve the handling of large amount of radioactivity and arerelatively safe. Iodination of proteins per se does not liberateiodine in the atmosphere and hence complicated fumehoods andlaminar flow systems are not essential. However, the area ofiodination 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 scientistsrepresenting some of the major centres in the country. TheseCentres use this technique for research and diagnostic purposes.This has been necessary in order to gather information on thevarious types of radioimmunoassays done, the availability ofmaterials and their clinical applications in research anddiagnosis.

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

The Institute h, j started doing radioimmunoassays since1969. Initially they had used radioimmunoassay kits from FarbeWerke Hoechst, Frankfurt, West Germany. Gradually the Insti-tute has acquired better counting equipment and additionaltechnical assistance. It was possible to procure several reagentsin the laboratory and to standardise various techniques. Wher-ever possible the Institute has tried to make its own antibodies orelse have obtained the reagents from National Pituitary Agency,National Institute of Health, USA. Occasionally antisera havebeen obtained by courtesy of friends. After initial failure withseveral separation techniques the double antibody has been used.The second antibody for guinea pig and rabbit sera have beenregularly prepared and titrated in the laboratory. Tracers forall hormones are also prepared in the laboratory. Initial attemptsto 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, wasnow commonly employed for labelling. In the past four yearsWorld Health Organisation grant as a collaborating researchcentre in reproduction studies has been used for procuringregular supplies of 1-125. Labelling of hormones generally didnot pose many problems.

Although regular diagnostic services have not yet been possi-ble, assays have been utilised to improve diagnostic capabilitiesat the centre. Samples from other centres are also received andprocessed. A large body of investigative data in various fieldshas been collected. For reasons of training and services, theinterests of the Institute have been fairly broad-based. Severalpeople have been trained at this Laboratory to carry out radio-immunoassays. A broad range of radioimmunoassays have beenperformed 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 UniversityRadioimmunoassays 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 Healthand 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 hasbeen to investigate response to stress in terms of various hor-monal and metabolic alterations. Assays of insulin, and growthhormone have been performed in a variety of stressful condi-tions like surgical trauma, burns injury and following electroconvulsive therapy. Insulin assays have also been done indisorders of myocardial infarction, heart failure, hypertensionafter tolbutamide provocation. Serum Digoxin assays havebeen done in case of digoxin toxicity. Besides this, studies havebeen done to evaluate associated changes in carbohydrate, lipidand protein metabolism in all the above mentioned conditions.Simultaneously other hormones like cortisol, catecholamineand certain enzymes have also been assayed. Presently theInstitute is trying to set up radioimmunoassays of other hor-mones useful in this research programme.

National Institute of Nutrition—HyderabadThis Institute has been one of the pioneers in establishing

radioimmunoassays in this country, and have attempted to startradioimmunoassays in 1967. However, they were only success-ful in doing so in 1970, when 1-125 became available in thiscountry. The first two hormones to be assayed were insulinand the growth hormone, levels of which were measured inprotein calorie malnutrition and diabetics. The other hormonesfor which radioimmunoassays were standardised were HPL,PTH and calcitonin. The latter two however, do not have agood sensitivity. PTH and calcitonin assays were set up to studythe hormonal profile in endemic fluorosis. Presently radio-immunoassays of prolactin and alphafetoprotein are also beingstandardised. Work at the Institute is for research and no dia-nostic services are offered. The Institute has been running train-ing courses in hormone assays with the aim of making cliniciansunderstand the indications, difficulties in establishing thesemethods and in the interpretation of results. The Institute col-laborates with Indian Council of Medical Research on the studyof bio-avaiiability of steroidal contraceptives and is presentlyengaged in procuring steroid conjugates and in standardisationof radioimmunoassays for steroidal substances. T-3 radio-immunoassays are also being performed. Presently non-availa-bility of 1-125 is the main limitation in carrying out these assays.

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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 establishthe method were made with kits. The unit caters to the needfor diagnostic methods for Eastern India and Bangladesh. Tillnow 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 hor-mones have been performed. The reagents have been obtainedfrom National Institute of Health and Indian Council of Medi-cal Research and through the courtesy of Dr K. Machiter ofRoyal Post-graduate Medical School, Hammersmith, Londonas a gift. An animal house has been established and the Insti-tute has been successful in the production of antisera againstT-3 and T-4. Attempts are being made to standardise the assaysof LH, FSH and HCG and Angiotensin II. Plasma cortisol andplasma renin have been performed with kits. The need forCentral Regional assay centre for the Eastern region is nowfelt acutely and the Institute feels that with an expanded staffthey could be entrusted to undertake these facilities. Somestaff members at present are under going training programmesabroad.. The main problem which the Institute faces is thenon-availability of 1-125, automatic gamma counters and simplelaboratory equipments. The Institute also feels that the radio-immunoassay service should be under the Nuclear MedicineDepartment 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 werespent in standardisations of labelling methods of HGH, produc-tion of antibodies and separations bound and free fractions.

Initial failures with 1-131 labelling was disappointing. Itwas demonstrated during Dr Berson's visit in 1970 that 1-131has a very poor isotopic abundance and is not very good forlabelling. 1-125 was then imported from Radiochemical CentreAmersham and since then there have seldom been any problemswith 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 andinfectious hepatitis.

c) Thyroglobulin levels in thyrodial disorders especiallythyroid 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—BombayThis is one of the largest and extensive centres in the coun-

try in this field. A very broad range of hormonal assays arebeing done both for diagnostic and research purposes in humansas well as in animals. The following assays are being doneat the Centre.

PROTEIN HORMONES

RIA OF PROTEIN HORMONES

HumanLH CGFSH PLPRLGHTSH

RADIORECEPTOR

LHPRLGH

RatLHFSHPRL

ASSAY

OvineLHFSHPRL

ENZYME-I

MonkeyLHFSH

MMUNOASSJ

HCGHPL

Iodination of about 20 hormones are done every month. About800 estimations are carried out per month.

Institute of Science — BangaloreThis group is carrying out experimental studies related to

reproduction. Their studies extend to assays of the followinghormones. Reagents are either prepared in their own labora-tory or obtained from National Institute of Health, NationalPituitary Agency.

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

LH and FSH Human sera 50- 60Monkey sera 400- 500Rat and Hamster sera 500- 600Testis and ovary 50- 60

Prolactin Human sera 10- 12Monkey sera 50- 100

Progesterone Human, monkey, rat andhamster serum and 500-1000ovarian tissue samples of each

Estradiol „ „Testosterone Monkey, rat and hamster 200- 300

sera and testis samples

All India Institute of Medical Sciences—New DelhiA review of the work done at the All India Institute of

Medical Sciences, Delhi, showed that there were several groupsdealing 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. Workis also done on the characterisation of proteins from plants inendemic and non-endemic zones. A problem was posed as towhether development of an assay for aflatoxin could be usefulin the study of epidemics of liver diseases like cryptogeniccirrhosis.

Endocrine Department, Topiwalla National Medical College,Bombay

At the endocrinology Department of the T. N. MedicalCollege, 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.

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Haffkine Institute — Bombay1. Corticosteroid.2. Australin Antigen.3. Cyclic AMP.

These are purely for research activities of this Institute andare not done as diagnostic tests. They also carry out some otherinvivo and invitro tests using radioisotopes for various researchprojects. The Institute is mainly involved in other types ofradiobiological studies on toxins, venoms and bacterial material.

Nuclear Medicine Department, S. N. Medical College — AgraThe 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. Theircapacity is 100 samples/month for FSH, LH, Prolactin and 75samples/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 kitseither obtained from Bhabha Atomic Research Centre or Radio-chemical Centre, Amersham. At the present all these assaysare done chiefly for research purposes, for specific projects andnot for routine diagnostic services.

M.K.C.G. Medical College, Berhampur—OrissaThe 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 eitherobtained from Bhabha Atomic Research Centre or RadiochemicalCentre, Amersham. The assays are research oriented forspecific projects and no routine diagnostic facilities are available.

SUMMARY

Radioimmunoassays of a variety of substances are beingdone 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 toindividuals. This situation is very tenuous and would be likely

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to break down easily. The instruments locally available at thepresent are manual gamma counters. This is turn restricts thecapacity of handling sample loads. The disadvantages ofimported equipment is reduced down time due to poor servicefacilities and difficult in obtaining spare parts.

As is seen from the presentations the use of the techniqueis confined to research projects and limited, restricted diagnosticfacilities. No broad based service facilities to a larger samplingarea is offered by any of the organisations engaged in the useof this technique.

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TRIGGER SESSIONS

This chapter includes the Trigger Sessions. These triggersessions were on subjects of interest, relevant to conditions pre-valent at the moment in the country. The discussions wereinformal and intended to assess the views of some of the leadingscientists in the field in India. A summary of the presentationsand the ensuing discussions is reproduced in the followingpages.

QUALITY CONTROL OF RADIOIMMUNOASSAYS

Quality control is the watchword of our modern era.Radioimmunoassays are now being done extensively to studya variety of substances- This has been possible because of theeasy availability of kits and developments of automation. InIndia the use of radioimmunoassays is limited to only those fewlaboratories which have facilities for radioisotope handling,processing and counting. The availability of kits made atBhabha Atomic Research Centre has enlarged the scope of usersin India. As in the case of all other types of assays and kits,the radioimmunoassays also necessitate quality control checkat various stages. For setting up a radioimmunoassay thefollowing 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 timeto time for routine laboratory assays. Such checks are mainlylimited to testing for sensitivity, specificity, precision, repro-ducibility and validity of the assay components. This type ofa routine check or intralaboratory check is mandatory for eachlaboratory. Routine intrassay and interassy variability checksshould be done consistently and regularly.

A second type of quality control is an external or inter-laboratory control whereby comparisons between variouslaboratories can be made and one standard reference source canbe user'. Such interlaboratory comparative studies are beingconducted by the World Health Organisation. Some of thelaboratories like Institute for Research in Reproduction, Post-graduate Institute, Chandigarh and Agra Medical College whohad participated in the programme commended World HealthOraganisation on their efforts to maintain a good quality controlof reagents and standards. However opinion was expressed thatvarious laboratories reported such wide variations in valuesthat although the mean value obtained by the laboratories

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tallied, the variations around the mean were wide. It was feltthat although such interlaboratory checks are useful, what ismore important is regular intralaboratory quality control asmentioned before.

Definite standard rules for quality control of radioimmu-noassays in general are very difficult to define. Radioimmu-noassays are being performed of such varied and diverse groupof substances that each assay system would require its owndefinition of quality control. Rigorous quality control is notessential wherever stimulation tests are performed and theseare more informative of dynamic function than single, random,spot evaluations. Each group of substances to be assayed shouldhave their own quality control requirements and no generalguidelines could be given when such a diverse group of sub-stances are being examined.

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USEFULNESS AND LIMITATIONS OFRADIOIMMUNOASSAYS IN CLINICAL DIAGNOSIS

There is no doubt that radioimmunoassays have gone com-merial in the western world. Most of the laboratories there doradioimmunoassays with kits readily available from more thanone company. There is lot of sales pressure to generate amarket for these kits and each company extolls the virtue ofone or more radioimmunoassay for which they supply kits. Ifone 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 physicianin his day to day decision making ?

There is no doubt that a vast number of substances can beestimated by immunoassay techniques. Their wide applicationhas brought forth immensely helpful information in elucidatingthe nature and progress of a disease e.g., our present knowledgeof interplay of various hormones in the pathophysiology ofdiabetes has been possible only because of radioimmunoassays.However, no one would say that radioimmunoassay of insulinis a 'must' for the diagnosis and management of diabetes.

Where a referring physician has nothing to do but send afew ccs of patient's blood to the laboratory for estimation ofone or more hormones, a large number of radioimmunoassayswill be carried out regardless of their information content forpatient diagnosis. With increasing availability of automatedradioimmunoassay procedures with on line computer to carryout all the * wnplex curve fitting and calculations, large numberof radioimmunoassays will be carried out first, because it is EQeasy to get them and secondly, because it is so fashionable toask for them.

In India, where radioimmunoassays have not gone commer-cial and berserk like the western world we shall have to identifyradioimmunoassays which are clinically useful. Very few kitsare available, 1-125 is required to be imported, many of theantisera remains to be cultivated by the user—with all theseodds against setting up a radioimmunoassay, a hospital labora-tory will have to decide discreetly what they should set upas 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 understandingof pathophysiological processes.

Radioimmunoassays are extremely useful in diagnosis whereno substitute tests are available e.g. T-3 in T-3 toxicosis, prolac-tin in prolactin producing adenomas, HGH in dwarfism. Theyare 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 thatthe results are not available immediately within a day or so.A batch of samples is to be collected before they can be proces-sed. Where results are required before a clinical decision canbe taken, such waiting periods of several weeks would be in-tolerable. One way to solve this problem would be to establishcentralised assay services which can easily collect a largenumber of samples and process them batch by batch in rapidsuccession. With such type of service indiscriminate referralwould be unavoidable.

Where clinical decisions are critical and requires a quickresult, for example, HCG, HPL, Estradiol, Digoxin, it is possibleto shorten the assay time by what is called as the "quickie"assay test where serum samples with low medium and highlevels of the substance to be measured are incubated with largerconcentrations of antibody for shorter periods of time alongwithunknown and results compared to such plasma values insteadof standards.

The abuse of radioimmunoassay in western world is due tothe fact that neither the institute nor the patient pays for thetest. In our counry, the problems of cost either to the instituteor to the patient is important especially where multiple testsneed to be done e.g. TSH. T-3, T-4 or where tests need to berepeated often like HCG, and HPL. It is essential then thatin a public hospital there should be some screening of therequests made for radioimmunoassays to prevent its misuse.Referring physicians should not over-load laboratories withrequests for assays unless and until there has been a thoroughinvestigation of the patient by other methods.

Radioimmunoassay is the cheapest and the simplest tool forclinical research. Notwithstanding what has been said above,one hopes that radioimmunoassays are available widely in thiscountry 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 techniqueslike Elisa depending on color reactions for assays may be doneby paper strip in the clinician's backroom laboratory cubicle.Till such time, we in India, will have to move cautiously andgenerate our own experience to find out what is useful, whatis needed, what is elegant and what is a fad as far as radio-immunoassays are concerned.

RADIOIMMUNOASSAYS IN TROPICAL DISEASES

Usefulness of radioimmunoassays in clinical diagnosis islimited. Its greatest impact has been on the understanding ofpathophysiology of many of the diseases. However, applicationof radioimmunoassays in diagnosis or understanding of infec-tious diseases has been sadly neglected. If radioimmunoassayshave to thrive in tropical countries they must become relevantto problems of medical care in these countries. When we wantfunds for radioimmunoassays in developing countries, the ques-tion that would be asked is do they help to resolve in any waythe problems of population, pestilence or nutrition ?

Application of radioimmunoassays so far recognised intropical diseases are in the following two directions:

1. Study of endocrine status in various tropical diseases.2. Study of endocrine status in various nutritional dis-

orders like malnutrition, undernutrition, starvation, malabsorp-tion, dietary oddities, etc.

In both above cases, the radioimmunoassays as techniquesare 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 dif-ficult exercise as such, even in this imitative way. It needsextending the begging" bowl all along. Request for hormonesfrom National Institute of Health, USA or Medical ResearchCouncil, UK. beg for antisera from this or that lab. Indentready-made kits from abroad or write for protocols of assaysto friendly scientists abroad.

There has been hardly any effort towards developing radio-immunoassays for actual detection of antigen or antibodies ininfectious diseases. This is surprising because most of ourknowledge of antigens and antibodies is derived from the studyof infectious diseases but we have been singularly lacking inenterprise 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 labelledantigens or antibodies for diagnosis of infectious diseases butthey are too few, too preliminary and widely scattered. Inhardly any case, they are convincingly better than serodiag-

42

nosis. The best of talents in this field are not applied tonurturing this kind of assays. The single most successfulradioimmunoassay for an infective agent is that for Australiaantigen. Even here, what we need urgently is an assay forhepatitis-A antigen.

Some work was done at Haffkine Institute in collaborationwith Radiation Medicine Centre towards developing an immu-noprecipitation test for typhoid antibodies by using C-14labelled antigen. A technique for labelling polysaccharidetyphoid antigen with Cr-51 has been also published from Radia-tion Medicine Centre.

Radioimmunoassays for infectious diseases can be for twopurposes:

1. Detection of antibodies. This may be useful in studyingthe antibody profile against a specific disease in a community,for finding circulating antibody levels at various stages ofof diseases and to find out success or failure of an immunisationprogramme.

2. Detection of antigens. Presence of infective organism isdifficult to detect in many infectious diseases, e.g. tubercle bacilliin blood, urine, C.S.F. or other body fluids. This kind of assayscan be of great help in early detection of extrapulmonarytuberculosis, 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 atvarious stages of the disease making it difficult to set up asensitive assay system. In most cases antigen is crude, mixedand altogether an unknown commodity. There may be a lotof cross reactivity between antigens. Who can take up thegauntlet of ploughing the unprospective land ?

This task can be best taken up in this country. Antigen isavailable indigenously, there are vast reservoirs of it and canbe cultivated easily. The antibodies are in a prodigious supplyin humans. Unlike other radioimmunoassays, the situation isfavourable here in one respect. There is no need to buy or begfor antigen or antibody. If we can take up the challenge thenext decade in radioimmunoassays will be of infectious diseasesjust as the last decade was of endocrinology.

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CENTRALISED ASSAY SERVICES

A central or regional assay service would offer the diagno-stic facility of multiple radioimmunoassays to regional anddistrict hospitals and clinics. The point which needs to beestablished is whether there is a genuine need to establish suchcentres 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 onlynine cities in the country have centres engaged in radioimmuno-assay techniques. They are Agra, Bangalore, Bombay, Calcutta,Chandigarh, Cuttack, Delhi, Hyderabad and Varanasi and hope-fully Lucknow, Madras, Trivendrum and Vellore will soon beinitiating steps to establish these methods. Out of the 108medical colleges in the country, only eight have introduced alimited facility for routine diagnostic service of this type. Howmuch of this can be attributed to lack of initiative and povertyof 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 orsupport and offer service facilities for research work ? Shouldthese central assay services opt to supply only reagents to users ?The logistics of sample collection, transport, storage of seraand time taken for despatching the results are highly complex.If such centres are to be set up, then there should be peripheralcentres which could cater to the district levels, another at thelevel of Medical Colleges and the larger Regional ReferenceCentre at well established Institutes. Each of these centreswould need to have personnel trained in handling radioisotopesand 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 understand-ing of diseases. To come to brass-tacks, the diagnostic utilityis chiefly in the evaluation of thyroidal disorders, disorders ofgrowth, adrenocortical tumours, insulinomas, pituitary micro-adenomas and a few others. AH except thyroidal disorders areuncommon diseases. Considering the magnitude of the problemsin logistics and the corresponding poor returns, in the presentcontext central assay services would not serve a significant

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diagnostic role in our country. However, in future when epi-demiological surveys for detection of conditions like neonatalhypothyroidism, Australia Antigen and infectious diseases arestarted, then such a service could be more profitably utilised.

The general concensus of opinion was that for the presenta central assay service was premature. What was importantwas :

a) There should be a liaison between various internationalagencies 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 bemade locally.

d) Liaison with large centres like Institute for Researchin Reproduction, Post-graduate Institute and Radiation MedicineCentre etc. for steady supply of antibody, standard and otherreagents.

e) Monitoring of various centres requesting these reagentsand finally a quility control protocol of each procedure wasessential.

f) Easy availability of "kits".

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RECOMMENDATIONS AND GUIDELINES

1 Introduction

Radioimmunoassay and related assay techniques constitutea 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 epidemiologiealand pathophysiological mechanisms of disease widely prevalentin our country. Its versatility, sensitivity, low cost and capa-bility of processing speedily large number of samples at thesame time make this technique especially suitable for studyingvarious problems of community health, because it does notrequire transportation of a patient to the hospital but involvesonly a collection of a blood sample from the patient and arrange-ment for transport of these samples to a centralisedinvestigational facility.

This technology can be applied to the following healthproblems :

i. For elucidation of the epidemiology and pathophysiologyof diseases which pose national public health problems.

ii. As rapid diagnostic techniques to help early diagnosisand to monitor the effectiveness of various therapeutic andpreventive measures adopted for these public health problems.

iii. Early detection of various diseases leading to theirtimely treatment.

Certain public health problems where these techniquesmay be applicable were identified. Apart from the obvious appli-cations in the field of reproductive biology and family welfare,the study of communicable diseases was identified as a priorityarea. It may be possible to screen for humoral antibodies and/or for antigens in biological material. The former might definethe infection incidence and antibody profile in the populationand help to assess the success/failure of immunisation pro-gramme. Detection of antigens or bacterial products could beextremely useful in finding contacts, carriers and early cases,and determine appropriate management. The feasibility ofsuch 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. Othersuch areas were the study of endemic goitres, neonatal hypo-thyroidism and malnutrition, especially the role of food conta-minants in the genesis of diseases like lathyrism, pancreatitis,toxic neuritis and hepatic disorders.

2. ResourcesThe 1976 Seminar on Nuclear Medicine, jointly organised

by the World Health Organization and Department of AtomicEnergy had identified various advantages that could accrue fromcentralisation of certain facets of this radioassay technology. Itis noted that a beginning has been made with a National Pitui-tary Agency having been established under the aegis of theIndian Council of Medical Research. This is a most commendableeffort and would greatly augment the radioimmunoassay pro-gramme in this country once purified pituitary hormones becomeavailable from this source. Material for labelling, labelled mate-rials and purified matched standards and antisera could be sup-plied through this central service.

Centralisation and subsidised or even free supply of imported1-125 was another area where costs could be reduced and timesaved. Currently, this is a significant bottleneck. It is estimatedthat the approximate national requirement at current priceswould involve a relatively small annual expenditure andBhabha Atomic Research Centre was urged to provide this radio-nuclide through its resources as early as possible.

The supply of bulk matched reagents could also be centra-lised. The Haffkine Institute and its sister organisation couldinitiate the manufacture and supply of second antibody. Latervarious institutes and laboratories could undertake the supplyof first antibodies for selected assays.

Requests for materials mentioned above would need to bejustified by the investigator and evaluated by an appropriatecommittee in terms of genuine needs and relevance to nationalproblems. However, it is strongly felt that the existance of thesecentralised supplies should not preclude individual investigatorsfrom obtaining independent supplies for use in areas in whichthey were particularly interested.

The structure, functions and requisite equipment for radio-assay services at the national, regional and medical collegehospitals' levels have already been detailed in 1976 report andis not being reconsidered here.

It was felt that indigenous equipment is far more acceptablethan 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 omissionof spectrometer by providing pre-set 125-1 counting facility mayfurther reduce the cost of such a counter. The relative meritsof 'through hole' detector needs to be evaluated in such acounting system.

The Committee notes that both Electronics Division ofBhabha Atomic Research Centre and Electronics Corporationof India Ltd., have capabilities of making 50 to 100 samplesautomatic counters. Such counters would be more suitable forcentralised assay service. It was emphasised that having multi-ple low cost manual or automatic sample changers is more desi-rable than having a large sample capacity single sample changerwhich might have longer 'down time' because of difficulties inrepairs and spares.

The various laboratories in the country could pool scarcefinancial, technical and material resources by co-operating inareas such as liaison with international agencies, equipmentmanufactures, Division of Radiological Protection, quality con-trol, establishment of Indian norms, and minimising duplicationof effort in assays for antigens for which demand is limited.International agencies like the World Health Organisation andallied organisations should support the above activities withfinancial and technical assistance.

The pioneering efforts of the Radiopharmaceutical Sectionof the Isotope Group, Bhabha Atomic Research Centre, in pro-viding kits and saving foreign exchange were commended. Kitproduction should be priority oriented specially to deal withcommunity health problems. However, it was also felt thatexcessive centralisation whether in kit production or reagentsupply could limit individual initiative if the state of the artbecame limited by the capabilities of a particular centre. Thereshould be room for improvement and quality control of kits andbulk reagents by other centres.

3. ResearchIt 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 recog-nised that in order to apply the radioimmunoassay techniquesto our national problems, the creation of technology which ischeap, robust and more suited to our conditions as regards sam-ple collection, sample transport and assay methods should be

48

encouraged. The latter would include filter paper, solid phaseand labelled antibody techniques. The creation of assay tech-niques relevant to our public health problems for hormones,infective antigens, antibodies, food contaminants is vitallynecessary.

4. Service

In areas where diagnostic role of radioimmunoassay is well-established, its day-to-day application and medical practiceshould continue. These include radioimmunoassays for thyroidhormones, TSH, HCG, Australia antigen and HPL. Informationabout availability and usefulness of these assays should bewidely disseminated in the appropriate medical forum.

Later as research identifies uses in other areas such as infec-tious diseases or diseases possibly related to food contaminantsthe diagnostic role could be extended.

Until such time as assay facilities become generally availa-ble, institutes with facilities for various assays could providereferral service on the basis of limited justified clinical requestirrespective of geographical distances.

The development of assay facilities in teaching hospitals orother medical centres could be supported by the existing centresas well as appropriate national agencies like Indian Council ofMedical Research, Ministry of Health, Department of AtomicEnergy and International Atomic Energy Agency.

5. Training

The existance of training facilities at various centres wasnoted. The training of senior persons in adequate depth wasemphasised. Training of technicians would also be obligatory.It was felt that the expertise available in these and other cen-tres should be pooled and used in a co-ordinated manner so thatthe courses proved more useful. The size and the extent of thetraining programme should be reviewed periodically so as tobe commensurate with needs of the country. After the trainingthe trainees should be assisted in setting up these techniquesin their own laboratories.

6. Information Exchange and Dissemination

The exchange of information regarding newer develop-ments in this country and abroad can be facilitated by a News-letter.

The Society of Nuclear Medicine and Endocrinology Societyof 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 thevarious 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 inthe country and their applications.

50

BACKWORD

Usually, such reports of the proceedings have a "foreword"glistening, hopeful and inspiring, setting the right tone for aponderous report to follow.

Nobody writes the 'backword', probably because nobodyexpects the reader to last till the end.

Nobody writes the 'backword', probably because it alsosounds so much like 'backward'. And that we certainly are not.How can we be backward after such an erudite conference andafter 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 humantalents, intelligence and ingenuity.

She said many such things at the conference. She gave aseries of five marvellous lectures reviewing her entire work inthe field of radioimmunoassays starting from the concept of theidea to its ever burgeoning applications.

The first 'backword' is that the Editors feel sorry that thisreport does not contain all that glory. Difficulties of transcrib-ing taped talks, and problems of reproducing a large numberof slides have made it impossible to put her lectures in thisreport.

So then, as far as this report is concerned, her role remainsthat of a catalyst. All that has been recorded here was discuss-ed in her presence. Her comments, criticism, praise, disdain allhave contributed in moulding the trends of what transpired atthe 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 whatwas the purpose of this report. It was a good meeting. Lots ofnice discussion. Gems of recommendations. And at the end of it,a well-edited report, something of a souvenir, like a fork thatyou 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 ofrecommendations, something like a staged play so that at theend of a fantasy, you rush out of the theatre to hunt for themost difficult-to-get taxi ? The similie is not inept. The confer-

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ence provided a panoramic vista but now let us hurry back towriting requests for ever elusive antigens and antibodies to ourforeign friends and benefactors !

Who puts the recommendations in practice ? The very-word implies that we expect some-one else to follow them. Andthat 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 theythought of was worthwhile. Each of the scientist has to beginhis struggle after the conference. Scientists have to mould theinstitutions where they work and institutions have to put pres-sure on various national and international agencies. But thefirst 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 kindof introspective meditative code of conduct for scientiststhemselves.

And therefore, as a 'backword', let us state few facts thatemerged at the conference and which apply to all of us.

Radioimmunoassays offer the cheapest possible investiga-tive tool for clinical research.

In the context of our country, the role of radioimmunoassaysfor a day to day clinical diagnosis is limited.

Indian scientists should stop imitating western science andstart applying these techniques to the study of infectious diseasesfor which antigen and antibody are available in abundance.

The resources are not hopeless, what is needed is the willto apply them for purposeful project so that more can beobtained out of little at hand.

These few 'backwords' should be enough to show that con-ference 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 haveto come out to push the car. But the car is new, engine is capableand Radioimmunoassays should roll forward with a big roar.

R. D. Ganatra

52

BIBLIOGRAPHY

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

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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 estimationSNM/220/81. Under publication in Proc. of IAEA Symp.Nov. 1977. West Berlin.

3. Hazra, D. K.Radioimmunoassays and related techniques in clinicalmedicine. Jr. Ass. Phys. Ind. 26:95, 1978.

4. Joshi, U. M., Raghavan, V. and Sheth, A. R.Development of an enzyme linked immunoassay forhuman 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 hormonesusing 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 levelsof 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-ovineLH serum Endocrinology, 99 :1554, 1976.

10. S. G. Nandini, H. Lipper and N. R. MoudgalA model system for studying inhibin. Endocrinology 98 :1460, 1976.

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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 FSHand 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-pitui-tary 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 prolactinlevels 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 onserum LH and prolactin levels in adult female rats. FertSteril. 26:847, 1975.

5. Dash, R. J., Vasista, K., Gupta, S. K., Lata, V. andSharma, B. R.Horrr \ne in reproduction IV. Application of beta sub-

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unit of human chorionic gonadotropin in evaluatin ofearly pregnancy. Bull Postgrad Med Inst. (Chandigarh)10:53, 1976.

6. Dash, R. J., Sharma, B. B., Lata, V. and Devi, P. KHormones in reproduction V. Radioimmunoassy of HCG.betasubunit in normal human pregnancy. Bull PostgradMed Inst 11:128, 1977.

7. Dash, R. J., Gupta, S. K., Vasista, K and Devi, P. K.Serum HCG and endometrial histology in early preg-nancy. 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 nonedocrinemalignancy. 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 tumormorphology, 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 gonado-tropin 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 humanmenstrual 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 normalpregnancy. Ind. J. Med. Res. 64 :963, 1972.

13. Dattatreyamurty, B., Sheth, A. R., Joshi, L. R., andRao, S. S.Changes in the ratio between serum HCG and 'specificHCG' levels in different trimesters of pregnancy. Amer.J. Obstet. Gynec. 121: 300, 1975.

14. Dattatreyamurty, B., Raghavan, V. P., Sheth, A. R. andRao, S. S.Synergistic action of prolactin with human chorionic

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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 ofimmunoreactive 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 andmedroxyprogesterone acetate on glucose, insulin growthhormone 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 hypotha-lamic LH-RH, pituitary LH and plasma LH in developingmale rats. Endocrinology 69 :195, 1977.

19. Goomer, N., Saxena, B. N. and Sheth, A. B.Effect of neonatal testosterone and estradiol treatmenton the development of hypothalamo-hypophysial axis inthe 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 pro-lactin 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 andplasma 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 pitui-tary to luteinizing hormone releating hormone LH-RH inthe male and female rats. Endocrinologie Experimentals.(In Press).

23. Gupta, P. B., Sinha, M. K., Dash, E. J., Wahi, P. L. andBastogi, G. K.Plasma insulin, Free fatty acid and blood sugar responseto intravenous glucogen in healthy subjects and patientsw.i'> myocardial infarction Circulation 49 :357, 1974.

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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 multipleserum gonadotropin estimations in the diagnosis ofsecondary 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 ofHCG 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 inductionof mid-trimester abortion with prostaglandin F2 hyper-tonic saline. Proc V Asia and Oceania Conference ofEndocrinology, Ed. G. K. Rostogi, Endocrine Society ofIndia 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 inductionof mid-trimester abortion with prostaglandin F2 andlypertonic 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. Atest 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 thyrotropinreleasing hormone in normal pregnancy. Obstetrics andGynaecology 42 :547, 1973.

32. Kulkarni, R. D.Hypothalamous and growth hormone release.Ind. J. Physiol. Pharma. 13: 99, 1969.

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33. Maneckjee R., Srinath, B. R. and Moudgal, N. R.Prolactin suppresses release of LH during lactation inthe 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 LHin 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 inbuffalo 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 HormoneRadioimmunoassay Ed. B. M. Jaffe and N. R. BehrmanP. 57, 1974, Academie Press, New York.

38. Moudgal, N. R. and Sheela Rani.Role of FSH in ovarian follicular maturation. Regulationof 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 onsterol 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 differentdays of pregnancy of LH deprivation in the rat and thehamster. 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—Factorsinfluencing binding and response. Biochem. J. 160:607,1976.

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43. Muralidhar, K. and Moudgal, N. R.Studies on rat ovarian receptors for lutropin. Interactionwith 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 lactatingrats 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. andHanda, 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 chronicmalnutrition. 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 monkeyantiserum to ovine LH. Contraception 12:137, 1975.

49. Raghavan, Vijaya, Purandare, Tarala, Sheth, A. R. andMunshi, S.Circulating levels of gonadotrophins in immature micetreated 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 malnutri-tion. J. Clin. Endocrin. Metab. 38 : 176, 1974.

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53. Raghuramulu, N.Plasma placental lactogen in pregnancy. Nutr. Metab. (InPress).

54. Raheja, B. S., Kulkarni, B. D. and the Talwalkar, N. A.Immunoreactive Insulin in serum of various groups ofdiabetices 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 inInsulin 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 fromHealthy Mothers. Diabetologia. 6 :445, 1970.

57. Rastogi, G. K., Letarte, J. and Fraser R.Proinsulin content of pancreases from human foetusesof healthy mothers. Lancet 1:7, 1970.

58. Rastogi, G. K., Sinha, M. K. and Dash, R. J.Long Term experience with glibenclamide in the treat-ment of diabetes mellitus with studies on insulinogeniceffect 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 thyroidfunction. Bull. Postgrad. Med. Inst, Chandigarh. 6:49,1972.

62. Rastogi, G. K., Dash, R. J., Sinha. M. K., Kannan, V. andSingh, T. M. TSH induced rise in plasma Tsh. An impor-tant 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 dis-orders. J. Ass. Phys. India. 21 : 183, 1973.

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64. Rastogi, G. K. and Kannan, V.Radioimmunoassay of plasma thyrotropin, an importantadvance in study of pituitary thyroid axis (Leadingarticle). 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 thyrotropinreleasing 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 fromdiabetic 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 gliben-clamide in diabetes mellitus. Hormone and MetabolicResearch, 5 : 303, 1973.

6P Bastogi, G. K., Dash, B. J. and Sinha, M. K.Radioimmunoasay of human gonadotropins and serumlevels 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 thosewith 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 andpatients 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 plasmainsulin, growth hormone, free fatty acid and blood sugarresponse to I. V. glibenclamide. J. Ind. Med. Ass. 61: 60-1973.

72. Bastogi, G. K.Utility of Plasma thyrotropin estimation and its responseto thyrotropin releasing hormone in diagnosis andresearch. Ann. Ind. Med. Sci. 9:97, 1973.

73. Bastogi, G. K., Chakraborty, J. and Sinha, M. K.Serum gonadotropins and their response to LHRH indiabetic men with and without importance. Horm. Metab.Res. 6 : 355, 1974.

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74. Rastogi, G. K., Sinha, M. K., Dash, R. J. and Datta, B. N.Serum levels of gonadotropins in male hypogonodism andresponse to Mesterolone in Ligozoospermis. Proceedingsof 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 responseto 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 pro-insulin like component (PLC) and serum IRI and PLCresponse to different stimuli in normal subjects andorganic 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 stagesof normal pregnancy. Ind. Obstet. Gynec. 26 : 65, 1976.

78. Rastogi, G. K., Dash, R. J. and Sinha, M. K.Thyrotropin releasing hormone induced thyrotropinrelease in euthyroid men: Effect of ethinyl estradiolpriming. 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 Societyof India, 1976. Circadian responsiveness of endocrineglands. 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 infectivefever. Horm. Met. Res. 8 : 409, 1976.

82. Rastogi, G. K., Saily, R. and Sawhney, R. C.Serum prolactin and thyrotropin response to thyrotropinreleasing 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 LHRHalone is postpartum lactating and non-lactating women.Ind. J. Med. Res. 65. 105, 1977.

84. Kastogi, G. K., Malhotra, M. S., Srivastava, M. C, SawhneyR. 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 seminalplasma 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 growthhormone 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. ClinEndo. 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 andTSH 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 : Alterationsin 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. Alterationsin 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.

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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 andSterility 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 levelsin 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 testosteroneceanantate on constituents of human seminal plasma andsrum 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 andpossible 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 pro-lactin 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 LHlevels 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 lac-togen to C3H (Jax) mice mammary gland. Europ. J. Cancer10 : 653, 1974.

102. Sheth, N. A., Saraiya, J. N., Ranadive, K. J. and Sheth,A. R.

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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 prolactinto murine mammary gland by polyamines. IRCS MedicalSciences 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 withgonadotropin deficiency. Proc. V Asia and Oceania Cenf.of Endocrinology, Ed. G. K. Rastogi, Endocrine Society ofIndia, 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 andmice. Proceedings of V Asia and Oceania Congress ofEndocrinology.

110. Sheela Rani, C. S. and Moudgal N. R.Examination of the role of FSH in the periovolatoryevents 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 initiationof follicular maturation in the hamster. A study usingantisera 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 developmentin 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. AnnalsInd. Acad, Med. Sci. 12 : 124, 1976.

114. Saily, R., Rastogi, G. K. and Gupta, A. N.Serum prolactin and its response to thyrotropin releasinghormone in normal pregnancy. Ind. J. Med. Res. 65 : 520-1977.

115. Sinha, M. K., Gupta, P. R., Dash, R. J., Wahi, P. L. andRastogi, G. K.Change in glucose, FFA and insulin following intravenousglucogon in normal subjects and patients following myo-cardial 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 endemicgenu valgum. Horm. Metab, Res. 8 : 317, 1976.

120. Siva Kumar, B.Circulating levels of growth hormone in endemic genuvalgum. Horm. Metab. Res. 9 : 436, 1977.

121. Sood, S., Katriya, R. N., Chari, P., Chopra, J. S. andDash, 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 theirresponse to thyrotropin releasing hormones in infectiveFebrile 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.

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Antisera to gonadotropins and plancental function in miceI. 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 intraamnioticinjection 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 andFSH 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 afterlabor. Ind. J. Ob. Gyn. 26 : 680, 1976.

128. Vijaya, E., Sankaran, M. S. and Sheth, A. R.Correlation between pituitary sialic acid and FSH concen-trations during .development afjter castration and testos-terone 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<tf; R> 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 inGraves' 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-tropicalregion. Metabolism 25 : 903, 1976.

7. Rastogi, G. K. and Sawhney, R. C.Value of urinary excretion of triiodothyronine (T-3) andthyroxine (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 inGraves' disease. Proc V Asia and Oceania Cong, of Endo-crinology Ed. G. K. Rastogi, Endocrine Society of India2 : 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 withdifferentiated 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 serumprogesterone in normal pregnancy. Relation to chorionicgonadotropin. 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. andJoshi, V. V.Clinical hormonal and ultrastructural studies of virilisinghepatoblastoma. Acta. Paediat. Scandisevica: In press.,1978 (In Press).

5. Mukku, V., Prahalada, S. and Moudgal, N. R.Effect of constant light on nychthemeral variations inserum 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 pro-tein 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 hepatocellularcancer. 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 HumanPlasma Protein fractions using Radioimmunoassay. BullHaffkine Inst. 3 : 156, 1975.

70