Laboratory Training Manual on Radioimmunoassay in Animal Reproduction

TECHNICAL REPORTS SERIES No. 233

Laboratory Training Manual on Radioimmunoassay in Animal Reproduction JOINT FAO/IAEA DIVISION OF ISOTOPE AND RADIATION APPLICATIONS OF ATOMIC ENERGY FOR FOOD AND AGRICULTURAL DEVELOPMENT

INTERNATIONAL ATOMIC ENERGY AGENCY, VIENNA, 1984

LABORATORY TRAINING MANUAL ON RADIOIMMUNOASSAY IN ANIMAL REPRODUCTION

TECHNICAL REPORTS SERIES No.233

LABORATORY TRAINING MANUAL ON RADIOIMMUNOASSAY IN ANIMAL REPRODUCTION

A JOINT UNDERTAKING BY THE FOOD AND AGRICULTURE ORGANIZATION OF THE UNITED NATIONS AND THE INTERNATIONAL ATOMIC ENERGY AGENCY

INTERNATIONAL ATOMIC ENERGY AGENCY VIENNA, 1984 LABORATORY TRAINING MANUAL ON RADIOIMMUNOASSAY IN ANIMAL REPRODUCTION IAEA, VIENNA, 1984 STI/DOC/10/233 ISBN 92-0-115084-9

Permission to reproduce or translate the information contained in this publication may be obtained by writing to the International Atomic Energy Agency, Wagramerstrasse 5, P.O. Box 100, A-1400 Vienna, Austria.

Printed by the IAEA in Austria January 1984 FOREWORD

Since the development of the radioligand assay some twenty years ago the whole field of endocrinology in both humans and animals has been revolutionized. The ability to measure the extremely low quantities of hormones that exist in blood and tissues has increased our knowledge of the reproductive function in domestic animals to an enormous extent, and is now coming to be used at the farm level. Reproduction must always be regarded as one of the major limiting factors in animal production and many of the modern methods for improving reproduction rely heavily on the ability to measure hormone levels in blood and milk. This has produced a world-wide demand for laboratory facilities to carry out hormone assays and the need for specialist training to allow these assays to be undertaken. The need to measure nanogram and picogram quantities and the use of radionuclides requires a good deal of skill and care and this Manual has been prepared to aid training and provide the sort of information that rarely appears in scientific papers. It represents a further step in the Joint FAO/IAEA Division's series of Laboratory Training Manuals, and has been designed to aid training programmes of the type carried out during the Joint FAO/IAEA Interregional Training Course on Radioimmunoassay and its Application in Research on Animal Reproduction at Cornell University in July 1982. Many of the laboratory exercises described in this Manual are based on those conducted during the course. This Manual has been produced on similar lines to the Joint FAO/IAEA Division's Laboratory Training Manual on the Use of Nuclear Techniques in Animal Parasitology (Technical Reports Series No. 219). Indeed, Part I and the Basic Exercises in Part V are largely intact, but sections on the biology of reproduction and a general introduction to the radioimmunoassay have been incorporated. The Applied Exercises have been replaced by a series of exercises describing all aspects of the radioimmunoassay of steroid and protein hormones involved in the regulation of reproduction. The difficulties, particularly in developing countries, of measuring the soft beta emissions from 3H-labelled materials have been borne in mind, and emphasis has been placed on methods based on radioiodinated steroids (125 I) so that relatively simple gamma counting procedures may be used. For these techniques, and for much helpful advice, FAO and the IAEA are grateful to Dr. W.M. Hunter and Dr. J.E.T. Corrie of the MRC Immunoassay Team, Edinburgh, UK, and to Dr. B.A. Morris of the University of Surrey, UK. FAO and the IAEA would also like to thank the scientists who contributed to the success of the course at Cornell University, particularly Drs T.J. Reimers and F.W. Lengemann, and those responsible for preparing the Manual: Dr. W.R. Carr of the ARC Animal Breeding Research Organisation, Roslin, UK; Dr. J.D. Dargie of the Animal Production and Health Section, Joint FAO/IAEA Division; Dr. L.-E. Edqvist of the Department of Clinical Chemistry of the Swedish University of Agricultural Sciences, Uppsala, Sweden; Dr. T.J. Reimers of the Diagnostic Laboratory, New York State College of Veterinary Medicine, Cornell University, Ithaca, USA; and Dr. H.A. Robertson of the Animal Research Centre, Agriculture Canada, Ottawa, Canada. CONTENTS

Some basic symbols and units frequently used in this Manual 1

PART I. GENERAL INTRODUCTION TO EXPERIMENTAL WORK WITH ISOTOPES AND RADIATION 3

I— 1. Properties of radionuclides and radiations 3 I-1.1. Atomic model. Radioactivity 3 1—1.2. The equation of Einstein 7 1—1.3. Radioactive decay law. Specific activity 8 I— 1.4. The energy of radiations 12 1-1.5. Interaction of radiation with matter 14 1-1.5.1. Alpha particles 1-1.5.2. Beta particles 1-1.5.3. Gamma and X-ray photons 1-2. Radiation detection and assay of radioactivity 19 1-2.1. Autoradiography 21 1-2.2. Ionization detectors 22 1-2.2.1. Electroscope 1-2.2.2. Gas-filled detectors with collector/cathode voltage bias 1-2.2.3. Ionization chamber 1—2.2.4. Proportional counter 1-2.2.5. Geiger-Müller (GM) counter 1-2.3. Solid scintillation counting 29 1-2.4. Liquid scintillation counting 32 1-2.5. Semiconductor radiation detectors 34 1-2.6. Inverse-square-law effect 36 1-2.7. Counting efficiency (counting yield) 37 1-2.8. Counting statistics (natural uncertainty) 39 1-3. Radiation protection 42 1—3.1. Basic considerations and units 42 1—3.2. Protection of personnel 45 1-3.2.1. External exposure 1-3.2.2. Internal exposure 1-3.3. Control of contamination 49 1—3.3.1. Decontamination 1-3.3.2. Special laboratory design features 1-3.4. Waste disposal 54 1-3.4.1. Disposal of radioactive animals References to Part I 54 Bibliography to Part I 55 Working notes to Part I 56

PART II. MENTAL EXERCISES 61

Working notes to Part II 62

PART III. INTRODUCTION TO PRACTICAL WORK 65 III— 1. Principles 65 III—2. Basic considerations 65 III—2.1. Chemical effects 66 III—2.2. Radiochemical purity 66 III—2.3. Isotope effects 67 III—2.4. Radiation effects 67 III—2.5. Exchange reactions 68 III—2.6. Radionuclide preparation and delivery of dose 68 III—2.7. Procedures with animals 69 III-2.8. Handling of samples 69 III—3. Reproductive endocrinology in mammals 71 III—3.1. Hormones 71 III—3.2. Puberty 73 III—3.3. Oestrous cycle 74 III—3.4. Control of the corpus luteum 74 III—3.5. Early pregnancy 75 III—3.6. Pregnancy and parturition 75 III—3.7. Applications of radioimmunoassay in assessing reproductive efficiency 77 III—3.8. Hormones during pregnancy in cattle 78 III—3.9. Hormones during pregnancy in sheep 80 III—3.10. Hormones during pregnancy in pigs 82 III—3.11. Hormones during pregnancy in mares 82 III—3.12. General comments 83 References to Part III 84 PART IV. GENERAL INTRODUCTION TO RADIOIMMUNOASSAY 85

IV—1. Principles of radioligand assays 85 IV-2. Radioimmunoassay 89 IV—2.1. Production of antibodies 89 IV-2.2. Production of labelled antigens 90 IV-2.3. Separation of antibody-bound from free hormone 93 IV—2.3.1. Chemical precipitation IV-2.3.2. Absorption with coated charcoal IV-2.3.3. Absorption with Protein A IV-2.3.4. Second antibody preparation IV—2.3.5. Solid phase separation IV—2.4. Optimizing the conditions for an assay 97 IV—2.5. Validation of RIA 100 IV—2.5.1. Specificity IV-2.5.2. Sensitivity IV-2.5.3. Accuracy IV-2.5.4. Precision IV-2.6. Quality control in RIA 107 IV-2.6.1. Internal quality control schemes References to Part IV 111

PART V. LABORATORY EXERCISES 113

SECTION A. BASIC EXERCISES 113

Exercise 1. The plateau and operating voltage of a GM counter 113 Exercise 2. The resolving time of a GM counter 115 Exercise 3. Counting and sampling statistics 118 Exercise 4. External absorption of beta particles 119 Exercise 5. Self-absorption and self-scattering of beta particles 121 Exercise 6. Solid integral scintillation counting 125 Exercise 7. Solid differential scintillation counting 127 Exercise 8. Estimating the efficiency of a gamma counter 129 Exercise 9. Rapid radioactive decay 130 Exercise 10. Inverse-square law and attenuation of gamma rays 133 Exercise 11. Liquid scintillation counting: determination of optimum counter settings 135 Exercise 12. Preparation of samples for liquid scintillation counting 137 Exercise 13. Quench correction 137 Exercise 14. Reducing the effects of quenching 140 Exercise 15. Cerenkov counting in a liquid scintillation counter 143 SECTION B. APPLIED EXERCISES 145

Exercise 16. Sampling and handling of assay data 145 16.1. Collection and storage of samples for radioimmunoassay 145 16.2. Construction of standard curves 146 Exercise 17. Preparation and titration of antisera for progesterone assays 147 17.1. Immunization of sheep, goats, horses or donkeys against progesterone 148 17.2. Titration of anti-progesterone serum and anti-rabbit gammaglobulin 149 Exercise 18. Radioiodination of sex steroids 154 18.1. Iodination procedure for progesterone 157 18.2. Iodination procedure for testosterone 160 Exercise 19. Radioimmunoassay of progesterone in serum and milk 160 19.1. Preparation of samples 160 19.2. Double-antibody separation with 12SI-progesterone 163 19.3. Radioimmunoassay procedure for extracted plasma or serum 166 19.4. Radioimmunoassay procedure for cow milk 169 19.5. Radioimmunoassay using 12SI-progesterone and antibody-coated tubes 169 19.6. Procedure using charcoal-dextran separation and [3H] progesterone 173 19.7. Titration of anti-progesterone serum using charcoal- dextran separation and [3H] progesterone 176 Exercise 20. Radioimmunoassay of testosterone in serum or plasma 178 20.1. Preparation of samples 179 20.2. Double-antibody separation with 125I-testosterone 180 20.3. Radioimmunoassay procedure for extracted plasma or serum 184 Exercise 21. Radioimmunoassay of luteinizing hormone in serum or plasma 185 21.1. Immunization of rabbits against ovine luteinizing hormone (LH) 185 21.2. Radioiodination of ovine LH with chloramine-T 186 21.3. Radioiodination of LH with lactoperoxidase 189 21.4. Separation of radioiodinated LH from unreacted iodide 190 21.5. Titration of anti-LH ovine serum and anti-rabbit gamma globulin and calculation of titres 193 21.6. Radioimmunoassay of ovine luteinizing hormone (LH).. 193 PART VI. APPENDICES 203

VI-1. How to put on and take off rubber gloves 203 VI—2. Radioactive waste control and disposal 204 VI—2.1. Waste collection 204 VI—2.2. Waste storage 204 VI—2.3. Effluent release to the environment 205 VI-2.3.1. General considerations VI-2.3.2. Effluent release to drains and sewers VI-2.3.3. Effluent release to the atmosphere VI—2.3.4. Burial of waste VI—2.3.5. Incineration of waste VI—3. Derived limits for controllable exposure 207

VI—4. Beta-particle range as a function of energy (Emax) 230 VI—5. Integration of equation (I—1 ). The radioactive decay law 231 VI-6. Derivation of equation (1-27) 232 VI—7. Characteristics of some common radionuclides used in biological research including radioimmunoassays 234 VI-8. Calculation of centrifuge speeds from g values 238 VI-9. Sources of some purified hormone preparations 239 VI-10. Basic materials and equipment for a radioimmunoassay laboratory 240 VI-11. Table of logit values 242

PART VII. GLOSSARY OF SOME BASIC TERMS AND CONCEPTS 245

SOME BASIC SYMBOLS AND UNITS FREQUENTLY USED IN THIS MANUAL

Symbol Description Dimensions and/or units

Z atomic number, i.e. proton number

A mass number

Ar relative atomic mass unified atomic mass units (u)

M, Mca, Mcaso4 gram-atomic or gram-molecular grams (g) mass

1 NA Avogadro's constant (number) NA = 6.022 X 10" mol"

T^ radioactive half-life time, e.g. years (a), days (d), hours (h), minutes (min), seconds (s)

X radioactive decay constant inverse time, i.e. s"'

t time in general time, i.e. s

T counting time (duration of) time, i.e. s

C accumulated counts in time T counts

R (= C/T) count-rate including background counts per second (counts/s) or blank

R(, count-rate of background or blank counts per second (counts/s)

Rs count-rate of sample counts per second (counts/s)

e counting efficiency = counting yield counts per 100 distintegrations

A* activity becquerels (Bq), curies (Ci) (becquerels s disintegrations per second)

A specific activity of a radioisotope activity per gram or mole (e.g. kBq/g, TBq/mol, mCi/g, etc.) or atoms % excess of stable isotope

S amount of tracee (substance mass (i.e. grams) or moles being traced)

b¡0i biological half-life (physiological time, e.g. s elimination)

T^ eff effective half-life (including effects time, e.g. s of physiological elimination and radioactive decay)

1 Symbol Description

LH luteinizing hormone FSH follicle-stimulating hormone PRL prolactin GnRH gonadotrophin-releasing hormones PMSG pregnant mare serum gonadotrophin TSH thyroid-stimulating hormone TRF thyroid-stimulating hormone release hormone CBG corticosteroid binding globulin SHBG sex hormone binding globulin

NSB non-specific binding or Bbl

MB maximum binding or B0

B0 maximum binding or MB PL placental lactogen

g relative centrifugal force EDTA ethylene diamine tetracetic acid, disodium salt PEG polyethylene glycol M molar solution, containing the gram molecular weight of the solute dissolved in 1 litre of solvent

2 PART I. GENERAL INTRODUCTION TO EXPERIMENTAL WORK WITH ISOTOPES AND RADIATION

This part of the Manual is intended to refresh the reader's memory of various aspects of atomic physics and radiation measurements — those aspects which are basic to carrying out the experimental studies which form the subject of the Manual. In addition, the reader is given information on safety measures to be considered when working with low-activity radioactive materials both in the laboratory and in the field. There then follows a discussion of certain specific techniques which, it is felt, will be of value to the reader in future practical studies.

1-1. PROPERTIES OF RADIONUCLIDES AND RADIATIONS

I—1.1. Atomic model. Radioactivity

An atom is composed of a positively charged nucleus surrounded by shells of negatively charged (orbital) electrons.1 The nucleus contains protons and neutrons as its major components of mass. A proton carries a positive (elementary) charge, and a neutron has no charge. The nucleus has a diameter of the order of 1CT12 cm and contains almost the entire mass of the atom. The atom, including the orbital electrons, has a diameter of the order of 1CT8 cm (or 1 Sngström). The number of protons, Z, in the nucleus, which is characteristic of a chemical element, is called the atomic number (proton number). The atomic nuclei of a particular element may, however, not all have the same neutron number, N. Atoms that have the same Z, but different numbers of neutrons, are called isotopes (of the chemical element corresponding to Z) because they occupy the same place in the periodic chart of the elements. As the neutrons and protons represent the major part of the mass of the atom and each has an atomic 'weight', i.e. an atomic mass2, close to unity, the

1 Many of the basic terms are included in a glossary for easy reference (Part VII). 2 The unified atomic mass unit (abbreviation u) is defined as exactly 1/12 the mass of the nuclide 12C: 1 u = 1.66053 X 10"27 kg approximately [ 1 ].

3 4 PART I. GENERAL INTRODUCTION

mass number, A, which is equal to the sum of protons and neutrons, is the

nearest whole number to the relative atomic mass, Ar. Thus:

Z + N = A - Ar in unified atomic mass units (see footnote 2)

Nuclides (any species of nuclei) are described symbolically by the designation:

2 El or A El or element-A (for example "Fe or 59Fe or iron-59)

where El represents the chemical symbol for the element. The nuclei of some nuclides are not stable. One by one they disintegrate spontaneously, each nuclide at a characteristic rate, and they are called 'radioactive'. In nature a number of unstable nuclides are known, and nowadays radioactive isotopes of nearly every element are produced artificially (e.g. in atomic reactors and by particle accelerators). The disintegration of radioactive nuclei is accompanied by the emission of various kinds of ionizing radiation. Radioactive nuclides are termed radionuclides, and other similar abbreviations are used (i.e. radioactive isotope -* radioisotope, etc.). Radioactive nuclei, upon disintegration, may emit alpha (a) or beta Q3) particles as well as gamma (7) rays. Alpha particles are fast-moving helium nuclei (^He), i.e. a combination of two protons and two neutrons. Beta particles are fast-moving electrons of either negative (ß~) or positive (ß+) charge. Gamma rays are electromagnetic energy packets (photons) of very short wavelength compared with that of visible light, but travelling at the characteristic speed of light. Natural isotopes of elements with low Z-numbers (except ordinary hydrogen) have approximately the same number of neutrons as protons in their nuclei (N - Z), and they are usually stable. As the atomic number of the element increases, the number of neutrons increasingly exceeds the number of protons with stability being maintained, but finally only unstable nuclei occur (above Z = 83, bismuth). Thus, the majority of radioisotopes in nature are found for elements of high Z-number with a neutron-to-proton ratio of the order of I5 : 1. The emission of alpha particles is characteristic of these heavy, unstable elements. The alpha particle is a very stable nuclear form which is ejected as a single particle from the nucleus of the heavy atom when it disintegrates. An example of alpha decay is given by the following nuclear reaction:

2&6Ra -» ¿He + 2gRh + Q(energy)

For Z less than 83 there appears to be a more or less well-defined optimum N : Z ratio for the stability of each element. When the number of neutrons in I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 5 the nucleus is excessive, the N-number tends to decrease by ejection of a negative beta particle (negatron) and a neutrino3 from the nucleus. This beta particle accompanies the transformation of a neutron into a proton:

n -*• p+ + ß~ + v (see footnote 3)

An example is:

nNa ->•_! e + nMg + v

An excess of protons in a nucleus may be counteracted by the ejection of a positron, i.e. a positive beta particle (regarding the definition of the energy unit MeV, see §1-1.5):

+ + 1.02 MeV + p -> n + |3 + v

An example is:

\lZn -»• ?e + f|Cu + v

Here, 1.02 MeV (1.63 X 10~13 J) is the minimum energy required for ^-emission and is equivalent to the rest mass of a positron plus an electron. An excess of protons in the nucleus may alternatively be reduced by the nucleus capturing one of its own orbital electrons, a process known as electron capture (EC) or K-capture since the electron is captured from the innermost or K-shell of orbital electrons:

+ p + e" -+• n + v

An example is:

IISr +-°e IvRb + v

EC is accompanied by the emission of a characteristic X-ray, most frequently representing the energy difference between an L and a K-shell electron in the element formed (a 'hole' in the K-shell being filled by an L-electron). After the ejection of an alpha or beta particle, or after EC, the energy level of the daughter nucleus may not be at its ground state. The excess energy of a nucleus thus excited is emitted in the form of one or more gamma photons.

3 A neutrino (v) possesses energy, but no charge and practically no mass, and will therefore not be detected by any of the instruments used in isotopic tracer techniques. 6 PART I. GENERAL INTRODUCTION

The excited nucleus may, however, interact with an orbital electron in the decaying atom, whereby the electron is expelled from the atom at a given velocity, and the expected gamma photon is not emitted. This process results in the combined emission of a fast electron and a characteristic X-ray, and is known as internal conversion (IC). The X-ray photon may in turn undergo IC, producing a so-called Auger electron. In some instances, two alternative modes of decay of the nucleus may occur. An example is seen for potassium-40 decay:

t°K _?e + 2oCa (ß~ decay, 89% of disintegrations)

?§K + _<}e-> $Ar (EC, 11% of disintegrations)

In the case of the EC mode a gamma photon is also emitted.

Another example is

+ 3oZn -*+®e + yCu (ß decay, 1.5% of disintegrations)

3oZn + .?e-+£>Cu (EC, 98.57* of disintegrations)

In this case, in 45.5% of disintegrations, EC is followed by the emission of a gamma photon. A large nucleus such as 235 U, either spontaneously or when it captures a neutron, will divide into two parts of approximately equal masses. This process is called fission and is accompanied by the release of neutrons. The primary fission products are unstable (excessive N), and each forms a series of radioactive daughter nuclides terminating with a naturally occurring stable nuclide. An example is:

^U + ¿n^93°Kr+'£Ba + 2¿n

Summarizing, radionuclides will emit particles and/or electromagnetic photons of the following nature: a-particle Doubly positively charged particle, containing two neutrons and two protons and originating at high speed from the nucleus /T-particle High-speed electron from the nucleus, negatively charged ß+-particle High-speed positron from the nucleus, positively charged I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 7

7-ray photon Electromagnetic energy packet coming from the nucleus at the speed of light X-ray photon Electromagnetic energy packet coming from an electron shell at the speed of light, following K-capture or internal conversion IC electron Electron emitted as a result of the interaction between a 7-ray and a valence electron Neutron Particle with no charge and a mass close to that of a proton.

1—1.2. The equation of Einstein

In almost every atomic or nuclear reaction a small quantity of mass is transformed to energy or vice .versa. An example of the energy, Q, involved in an alpha decay is given as follows:

»gRa-^He + 'gRn + Q

The Q of a nuclear reaction is related to the decrease in atomic mass in accordance with the general equation of Einstein:

E= mc2 where E = energy release (in the above case Q), m = mass decrease, c = speed of light.

Alternatively, Einstein's equation may be expressed either as:

E= 931m where E = energy release in mega-electronvolts (MeV), m = mass decrease in unified atomic mass units, or as

E = 5810m where E = energy release in attojoules (aJ = 10~18 J). 8 PART I. GENERAL INTRODUCTION

1—1.3. Radioactive decay law. Specific activity

The decay of radioactive atoms is comprised of individual random (unpredictable) events. However, if a sample contains a sufficiently large number of atoms of a radionuclide, their average statistical behaviour can be described by a precise law. The radioactive decay law is developed as follows: Let N be the number of radioactive atoms of a given radionuclide present at any time t. The change in N per unit time at any moment, dN/dt, is proportional to the number of atoms present at that moment, or:

dN = kN = — XN (1-1) dt where X is (numerically) the proportionality constant, termed the decay constant. The negative sign is used because the number N decreases witli time and X is chosen to be positive.

Rearranging Eq.(l-1 ) to solve for X:

1 dN X = (1-2) N dt

Thus, the decay constant is the fraction of radioactive atoms decaying per unit time at any moment. Equation (I—1) may be integrated4 to give:

Xt N = N0e" (1-3)

where N0 is the number present at any starting time (t = 0), and N is the number remaining after a period of time t. (e is the base of natural logarithms and equal to 2.71828.. .) It can be seen from Eq.(I—3) that the decay of radioactive atoms is

exponential with time. Also, the time for N0 to be reduced to half its initial

value is a constant, as shown below, independent of N0 and t.

Let N0 be reduced to \ N0 in a period of time (t = Tp termed the half-life. Then, from Eq.(I—3):

XT 5No = N0e- ^ (1-4)

4 The steps in this procedure are given in Part VI, Appendix VI-5. I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 9

Hence:

or exi> = 2 (1-5)

Thus, taking the natural logarithm of both sides:

XT^ =ln 2= 0.693 (1-6)

Therefore, since X is a constant characteristic of a given radionuclide, the same is true ofTj. The dimension of T^ is given in time units, whereas the decay constant is indicated in reciprocal time units. Since the rate of decay, -dN/dt, is termed the radioactivity or, simply, activity, A*, of the sample, then according to Eq.(l— 1 ):

A*=XN (Rutherford's equation) (1-7)

From Eq.(I—3), one therefore obtains:

A* = AS e"Xt (1-8)

Substituting X = (In 2)/Tj from Eq.(I-6) into Eq.(I-8), the following alternative equation is obtained:

A* = AJ (i)t/TV (I—8a)

The half-life of a radionuclide may be determined graphically by plotting the disintegration rate (or a constant fraction thereof, as determined by a suitable counting instrument) versus time on log-linear graph paper. Referring to Eq.(I-8a), if the common logarithm (logarithm to the base 10) is taken on both sides, the result is:

* * log 2 log A* = log A* - -2- (1—8b)

Therefore, a plot of A* (or count-rate) on the log co-ordinate versus time on the linear co-ordinate will be a down-grade straight line with a numerical slope of 0.301/TK This is graphically illustrated in Fig.I-1. Ti can be calculated, for example, as one third of the time it takes for Aj to fall to 5 Aj. The special unit of activity (radioactivity) has for many years been the curie (abbreviated Ci). This was originally defined as the radioactivity associated 10 PART I. GENERAL INTRODUCTION

A ON LOG SCALE

1 A* 2 0

1 A* A Ao

i A* t ON LINEAR SCALE fl Mo

FIG.I-1. Decay curve of a single radionuclide (log-linear plot).

with the quantity of radon in equilibrium with 1 g of radium (1910). The formal definition agreed in 1964, when the curie was accepted for use with the International System (SI), was:

1 Ci = 3.7 X 10'° disintegrations per second = 3.7 X 1010 s"1 (exactly)

Since 1976, a new unit of activity, the becquerel (Bq), has been defined as a derived unit of the SI:

1 Bq = 1 disintegration per second = 1 s"1

Hence

1 Ci = 3.7 X 1010 Bq = 37 GBq (exactly) 1 Bq = 27.027 pCi s 27.03 pCi

The old special unit, the curie, is to be phased out in the next few years. The following list will assist in obtaining a feel for the interrelationship. I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 11

106 Ci = 1 MCi = 37 PBq = 3.7 X 1016 Bq 103 Ci = 1 kCi = 37 TBq = 3.7 X 1013 Bq 10° Ci = 1 Ci = 37 GBq = 3.7 X 1010 Bq 10",-3 Ci = 1 mCi = 37 MBq = 3.7 X 107 Bq 10"i -6« Ci = lpCi = 37 kBq = 3.7 X 104 Bq ~ 2.7 X 10"" Ci = ~ 27 pCi = 1 Bq = 10° Bq 10~r'122 Ci = I pCi = 37 mBq = 3.7 X 10'2 Bq

In practice, a radionuclide will often be accompanied by variable quantities of one or more stable isotopes of that element. The stable form is called the carrier. Specific activity, A, is the term used to describe the ratio of radioactive atoms to carrier atoms. The specific activity is defined in general as the activity of a particular radionuclide per unit mass of its element or compound. Common units of specific activity are microcuries per gram of substance (i.e. kilo- becquerels per gram of substance).s

The absolute (carrier-free) specific activity, A0, of a (carrier-free) radionuclide, i.e. the special case in which all atoms of the element present are of the same radioisotope, can be obtained from Rutherford's equation (Eq.(I—7)) by 6 substituting Na, Avogadro's constant , for N, the number of atoms:

A0 = XNA (1-9)

-1 If X, the decay constant, is in reciprocal seconds (s ), A0 is obtained directly in becquerels (i.e. disintegrations per second) per mole, or in curies per mole after dividing by the curie-to-becquerel conversion factor:

i4 (Ci/mo,)= XNA 15 (I_1 a) A0 (Bq/mol) = XNA or ° iTxTÖ " °

The carrier-free specific activity per gram is obtained by dividing the appropriate form by the gram atomic mass of the radioisotope, M:

XNA A0 (Bq/g) = — or A0 (Ci/g) = (I—10b) M (3.7 X 10'°)M

5 Counts per second per gram are also frequently used in experimental work. 6 Avogadro's constant (or number), NA, is the number of atoms or molecules per mole 23 1 of substance: NA = 6.022 X 10 mol" . 12 PART I. GENERAL INTRODUCTION

It is worth bearing in mind that, from Eq.(I-6), X can be obtained from the half-life (expressed in seconds):

w In 2 0.693 Ms ')=—=— (1-11)

An example of the production of radioisotopes that are carrier-free will be given in § 1—1.6.4.

1—1.4. The energy of radiations

The energy unit most commonly used with regard to radiation is the electronvolt (eV). This is equivalent to the kinetic energy acquired by an electron (or any other singly charged particle) accelerated through a potential

32 P (I4d) ,37Cs (30 a) 1.7 MeV 1.17 MeV 92%\P 137 Bom (2 56 min)066 32« IT ((10% IC) 137

55 13N (10min) Fe (2.6 a) -1.20 MeV 0.22 MeV

100 °//o J-t <•0.1 8 100 °/o EC/ (75 °/o X-ray conversion 7 to Auger electrons) 13r A /- 0

60Co (5.26 a ) 2.81 MeV 2.84 MeV

FIG.1-2. Decay schemes showing characteristic radiations and energies of six radionuclides. IT = Isomeric transition. Different types of the same nucleus are called isomers. IC = Internal conversion of gamma photon. EC = Electron capture (K-capture). I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 13 difference of one volt in a vacuum. This unit is used with SI and has been determined experimentally to be:

1 eV = 1.602 19 X 10"19 J (approximately)

A commonly used multiple is mega-electronvolts (106 eV= 1 MeV).

1 MeV s 1.6 X 10~13 J = 1.6 X 10-6 erg

The kinetic energies of the particles and photons emitted by radionuclides have characteristic values. The energies of alpha particles, characteristic gamma and X-ray photons are constant or discrete. The energies of beta particles ejected by a given radionuclide vary, however, from zero up to a certain maximum energy (Emax) that is available to the beta particles. This is because a variable part of Emax is carried away by a neutrino in every beta particle decay. Neutrinos cannot be detected by ordinary methods as they have no charge and essentially no mass. As a consequence, the beta particles show a continuous spectrum of energies from zero to Emax. The beta energies given in a table or chart of nuclides are Emax-va!ues. The average beta- particle energy is usually about one-third of Emax. Internal conversion electrons, on the other hand, are monoenergetic. The characteristic radiations and energies for a given radionuclide are often shown in the form of a decay scheme. Examples of the decay schemes of six radionuclides are shown in Fig.I—2.

1—1.5. Interaction of radiation with matter

1—1.5.1. Alpha particles

The alpha particles ejected from any particular radionuclide are monoenergetic. Their initial kinetic energies are of the order of several MeV and, since ionization potentials and bond energies are in the range 1 — 12 eV, the alpha particles are capable of causing many ionizations as well as electronic excitations of the atoms or molecules along their path. Ionization is complete removal of the valence electron, and excitation is raising electrons to higher energy levels in their orbits. Since the valence electron participates in any chemical bond of the atom, ionization destroys the integrity of that bond. The term specific ionization is used to describe the intensity of ionization, i.e. the number of ionizations occurring per unit path-length of a particle. The specific ionization is directly proportional to the mass and charge of a particle and inversely proportional to its velocity. Since alpha particles are doubly charged and of comparatively heavy mass they have a high specific 14 PART I. GENERAL INTRODUCTION

FIG.1-3. Curve demonstrating the beta radiation as a function of absorber thickness. I = Intensity of transmitted beta radiation. B = Bremsstrahlung component (and gamma-ray component). R = Approximate maximum range of beta particles in absorber material.

ionization. Hence alpha particles lose energy in matter relatively rapidly by these processes. As the alpha particle dissipates its energy along its path, its velocity decreases, and at zero kinetic energy the particle acquires two electrons from its surroundings and becomes a helium atom. The range, i.e. the distance that an alpha particle can penetrate into any matter (absorber), depends on the initial energy of the particle and the density of the absorber. The range of an alpha particle is relatively small and amounts to several centimetres in air and several micrometres ( 1 fim = 10"3 mm) in tissue for energies of the order of 1 to lOMeV. Since all the energy of an alpha particle is lost in a relatively thin layer of matter, the LET (linear energy transfer) is high.

1—1.5.2. Beta particles

Beta particles lose energy in matter through ionization and excitation in the same way as alpha particles. The mass of the beta particle, however, is only 1/7300 of the mass of the alpha particle, and beta particles have only unit charge. They will, therefore, be scattered more, penetrate further into matter and produce a less dense track of ion pairs (i.e. electrons have a lower specific ionization and LET) than alpha particles. The range of beta particles in matter is also a function of the initial energy of the particle and of the density of the absorber, but this range is not well defined because of the tortuous path (due to scattering) of the electron. The range of beta particles of 1 MeV initial energy is approximately 3 m in air and 4 mm in tissue. (See also Part VI, Appendix VI-4.) Partly owing to the fact that beta particles have a continuous spectrum

of energies up to Emax, their absorption in matter is by chance approximately exponential with absorber thickness. Thus, when the beta radiation transmitted by an absorber is plotted on log-linear graph paper as a function of the I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 15

PHOTOELECTRIC ABSORPTION 1. GAMMA RAY COMPLETELY ABSORBED 2. ELECTRON EJECTED WITH GAMMA RAY'S ENERGY MINUS BINDING ENERGY

COMPTON EFFECT

1. GAMMA RAY OF LOWER ENERGY PROCEEDS IN NEW DIRECTION 2. ELECTRON IS EJECTED WITH THE ENERGY DIFFERENCE

COHERENT SCATTERING GAMMA RAY SCATTERED AFTER INTERACTION WITH ORBITAL ELECTRON WITHOUT CHANGING WAVELENGTH AND WHERE THE SCATTERED PARTICLES BEAR A PHASE RELATIONSHIP TO ONE ANOTHER (NEGLIGIBLE ENERGY CHANGE)

PAIR PRODUCTION ABSORPTION 1. GAMMA RAY ANNIHILATED 2. ELECTRON AND POSITRON CREATED AND SHARE GAMMA RAY'S ENERGY MINUS 1.02 MeV

FIG.I-4a. Gamma-ray interactions.

mass per unit area7 of the absorbing material, a fairly straight line is obtained over a portion of the curve (Fig.I—3). The total transmission curve becomes almost horizontal at R, which is the range of straight-pathed beta particles with an initial energy close to Emax. Although all the beta particles are stopped by this thickness of absorber, there is still some transmission of radiation, because the beta particles interact with the atoms of the absorber giving rise to non-characteristic X-rays, the bremsstrahlung. In addition, any gamma rays will contribute to this component. By subtracting this component (B) from the composite curve (I + B) the pure beta-transmission curve (I) is obtained. Positive beta particles or positrons lose their kinetic energy in matter in very much the same manner as negative beta particles. However, when the kinetic energy of the positron has been reduced to zero through

7 Mass per unit area is a product of the density of the absorber multiplied by its 'thickness'parallel to the incident radiation. Its units are: g/cm2 ; mg/cm2 ; kg/m2 (SI). Variously called thickness, area density, surface density, density thickness, etc. 16 PART I. GENERAL INTRODUCTION

(/> (- z UJ O

UJ O O Z o y- < D Z UJ I- K<

0.001

FIG.I-4b. Linear attenuation coefficients for gamma rays in water (the total Compton attenuation, oc = oa + aj,).

ionization and excitation, the positron undergoes annihilation with a nearby negative electron, giving rise to two characteristic annihilation photons of 0.51 MeV (8.17 X 10~14 J) each. (In accordance with Einstein's equation, 0.51 MeV (8.17 X 10~14 J) is the equivalent energy of the rest mass of an electron.) Absorption and scattering of beta particles are important in the measurement of the activity of beta samples. Absorption and scattering will occur in the sample cover, the detector window, the walls of the shield, the intervening air, and in the sample itself (self-absorption). These effects will all influence the count-rate, self-absorption being the most important. (This is illustrated in Part V, basic exercises 4 and 5.)

1—1.5.3. Gamma and X-ray photons

Electromagnetic radiation is considerably more penetrating than particulate radiation of the same energy. This is because the photon must first undergo a I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 17 special absorbing event, whereby either one or two 'secondary ionizing' electrons are produced, before any photon energy becomes dissipated. Gamma rays will be absorbed in matter as a function of the photon energy, as well as the Z and the density of the absorbing material (gamma rays, X-rays and annihilation rays differ only as to their origin, and they interact identically in matter). The following, illustrated in Fig.I-4a and described below, are the four types of attenuating event considered in the ICRU Report 19 [2]:8

(a) Photoelectric absorption (b) Compton effect (absorption and scattering) (c) Coherent scattering (d) Pair-production absorption.

(a) Photoelectric absorption is predominant for relatively low-energy gamma photons and for absorbing material of high Z. The gamma ray interacts with a K or L electron of the absorber atom and expels the electron from the atom with a kinetic energy equal to the initial gamma-photon energy minus the binding energy of that K or L electron. Thus, an electron is ejected witli kinetic energy, enabling it to produce ionizations and excitations along its path exactly in the manner of a beta particle. In Fig.I—4b, the coefficient for photoelectric absorption (T) is given for water as a function of gamma-photon energy, E^,. The absorption coefficient is a measure of the probability of absorption (average number of events per cm).

(b) Compton effect is the interaction of the gamma photon with an outer electron of the absorber atom or with a free electron. Part of the initial kinetic energy is absorbed (transferred to the electron), and the photon is scattered off in a new direction at a lesser energy. The photon will eventually after multiple scattering be absorbed through the photoelectric effect. As can be seen from Fig.I—4b, the coefficient for Compton absorption (0a) is at a maximum in water for gamma rays of about 0.5 MeV. The effect rises only slightly with increasing Z. The fast electron arising from a Compton event will produce ionizations and excitations again exactly in the manner of a beta particle.

8 This section has been revised to correspond with the concept of mass attenuation coefficient as defined in Radiation Quantities and Units, ICRU Report 19 [2], The attenuation coefficient is defined as that fraction of particles (including photons) that experiences interactions in traversing a given distance in a certain medium, where . . the term interactions refers to processes whereby the energy or direction of the indirectly ionizing particles is altered". 18 PART I. GENERAL INTRODUCTION

(c) Coherent scattering is a process in which photons are scattered after interaction with orbital electrons. The electrons return to their original state, there is no change in photon energy, and there is a relation in phase of the scattering from different electrons of the atom. Rayleigh scattering is another name for this process. Such scattering is important only for low-energy photons (<0.1 MeV) and high-Z materials.

(d) Pair-production absorption may occur when the gamma photon has an initial energy of at least 1.02 MeV (1.63 X 10"13 J). In this process, the gamma-ray photon interacts witli the positive field of the nucleus of the absorber atom and is completely used up in producing a positron-electron pair.9 Since it requires 1.02 MeV (1.63 X 10~13 J) for the formation of the two electron rest masses, this is the energy threshold for the pair-production event. Any gamma-photon energy above the required 1.02 MeV is imparted as kinetic energy to the positron-electron pair. Both the positron and the negative electron cause ionizations and excitations along their respective paths. The two 0.51 MeV photons produced upon annihilation of the positron are subsequently absorbed by a photoelectric event or a combination of Compton and photoelectric events. In Fig.I—4b, the absorption coefficient for pair production is labelled as K. Considering the above processes (single, random events), a beam of monoenergetic gamma rays is attenuated exponentially as a function of thickness, x, of the absorbing material. For a beam of intensity 1, the change in intensity per unit absorber thickness, dl/dx, is proportional to the intensity of the beam ut that point. Thus,

(1-12)

Equation (1—12) is identical with the well-known Lambert-Beer law for attenuation of monochromatic light. The proportionality constant (ß) is termed the total linear attenuation coefficient (Fig.I—4b). Exactly analogous to the radioactive decay constant, X, /J is the fraction of the original intensity removed from the beam per unit linear thickness of absorber. Equation (1—12) is mathematically identical with the radioactive decay law (Eq. (I-1 )) and may be integrated to give:

(1-13)

9 When the term electron is used in this text, it can be taken to refer to the negative electron, unless otherwise stated. I-l. PROPERTIES OF RADIONUCLIDES AND RADIATIONS 19

Here, n, the total linear attenuation coefficient for gamma and X-ray photons is given by:

M = r + ac+acoh + K (1-14) where r is the photoelectric attenuation coefficient;

ac is the total Compton attenuation coefficient (= aa + a^, see Fig.I—4b); °coh 's attenuation coefficient for coherent scattering; k is the pair-production attenuation coefficient.

The numerical value of n is dependent on the gamma-photon energy (E^) and the type of absorber material. Figure I—4b illustrates the attenuation probabilities for water as a function of E^. Again analogous to radioactive decay, the thickness at which I is reduced to one-half its initial value is termed the half-thickness or half-value layer(HVL), X^, and one finds:

In 2 0.693 X. = = (1-15) M P

an equation of a form similar to Eq.(I —11), see also Eq.(I—6). Equation (1—13) may alternatively be expressed as follows:

I=Io(f)x/x^ (I-13a) an equation of a form similar to Eq.(l—8a). An understanding of the interactions of high-energy electromagnetic radiation with matter is necessary in considerations of shielding, dose calculations, and measurement of gamma, X and annihilation photons.

1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY

The radiations which come from radioisotopes interact with all matter (gaseous, liquid or solid), causing chemical changes, ionizations and excitations as discussed previously. These effects are utilized in the various methods of detection and measurement of radioactivity. Of these reactions, the most commonly used are ionization in gases, orbital electron excitation in solids or liquids, and specific chemical reactions in sensitive emulsions. Commonly used detection methods employing these mechanisms are described below. 20 PART I. GENERAL INTRODUCTION

FIG.1-5. Block diagram illustrating an anti-coincidence unit used as an electronic shielding against cosmic radiation.

FIG.1-6. Block diagram illustrating a coincidence unit.

In radiography, for example, ionizing radiations are detected by their effect on photographic, X-ray or nuclear emulsion. In the ionization chamber, the gas-flow detector, the Geiger-Müller tube and the neutron detector, ions produced directly or indirectly by the radiation are collected on charged electrodes. In solid and liquid scintillation counting, emission photons (in the blue to ultraviolet region) form the basis of detection. Solid-state detectors, more properly called semiconductor radiation detectors, are crystals whose electrical conduction is altered by the absorbed radiation. Their operation depends on their semiconductor properties, and this class of detector has become of great importance in dosimetry. Besides a radiation detector, a monitoring or measuring set-up includes one or more of the following electronic units: Power unit. The primary source of power is either a battery or the mains supply. The detector potential requirements range from a few hundred to a few thousand volts, and good stabilization is generally necessary. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 21

Amplifier. The primary signal is often an electronic pulse or electric current that is too small for registering unless amplification is used. Furthermore, in proportional counting the amplification must be linear, i.e. the magnification factor must be independent of pulse size. Timing unit. This ranges from a stop-watch to an automatic unit which stops the detector at the end of a predetermined time interval or registers the time necessary for accumulation of a pre-set number of counts. Pulse input sensitivity. An electronic discriminator biased to reject all pulses below and/or above a certain size. This improves the signal-to-noise ratio. Anti-coincidence unit. This electronic unit rejects pulses that arrive 'in coincidence', i.e. both arriving within a very short time interval (e.g. 1 /us). An anti-coincidence unit is used for so-called electronic shielding against cosmic radiation (see Fig. 1—5) and for pulse-height analysis. Pulse-height analyser. This consists essentially of two variable discriminators (a lower and an upper one), together with an anti-coincidence unit. With this auxiliary equipment, only pulses within a set pulse-height interval are registered A fuller description is given in §1-2.3 on solid scintillation counting. " Coincidence unit. This unit rejects all single pulses but passes one pulse when two pulses arrive in coincidence (e.g. within 1 ps). A coincidence unit is generally used in conjunction with two scintillation detectors, in order practically to eliminate photomultiplier noise pulses (see Fig. 1-6). Registering unit. This may be a scaler, i.e. a set of decades displaying the sum count or a certain fraction thereof, a count-rate meter (visible or audible), a voltmeter reading out accumulated radiation dose, a sensitive electric-current meter displaying dose rate, or even a recording potentiometer. Historically, it might be noted that de Hevesy used a simple metal-leaf electrometer for his pioneer work, and since then a great deal of useful work in biological research has been done, and is still being done, with a Geiger- Müller counter, a stop-watch and a pocket dose meter or film badge. A number of detectors and some associated electronic equipment will now be described in more detail.

1-2.1. Autoradiography

This method is a photochemical process and the one used by Becquerel in 1896 in the discovery of radioactivity. In autoradiography, ionizing radiations interact with the silver halide in photographic emulsions. When radioactive material is placed near a photographic plate or film, a blackening will be produced on development of the emulsion. The blackened areas constitute a self-portrait of the activity in the material. The intensity of the blackening (as determined by eye or witli a densitometer)-at a given place will be a function of the exposure time and the amount of activity in the sample at that place. It also is a function of the LET (linear energy transfer) of the particular radiation. Gamma rays and 22 PART I. GENERAL INTRODUCTION

X-rays have a low LET and are of little use in autoradiography, because the photons from a given place in the sample material will penetrate throughout a large area of emulsion, producing an almost uniform fogging on development. Conversely, alpha particles and low-energy beta particles (3H, 14C, 3sS, 45Ca), which have a high LET, are very effective. High-energy beta particles produce diffuse radio- grams owing to the relatively long path-lengths of these particles in the emulsion. The properties of the emulsion should be a compromise between fine grain to increase the resolution and high sensitivity to reduce the exposure time. Usually, exposure times are long because, to obtain a good image, an absorption of about 107 soft beta particles is needed per square centimetre of emulsion. Thus, a thin histological section containing 1 to 10Bq/cm2 (1 to 10 dis-s_lcm~2 = 27 to 270 pCi/cm2) will require several weeks exposure to show optimal blackening. The method of autoradiography is particularly suitable when the distribution of a radioactive compound in biological material is to be studied. However, precautions should be taken that there is no chemical or pressure effect of the material on the emulsion as this may also produce an image. Various techniques have been worked out, each with specific advantages and disadvantages. Apart from the chemical effect on emulsions, complications with regard to the drying or pretreatment of samples, the transport of radioactive compounds under moist conditions and the self-absorption of low-energy particles in the biological material may occur; film development conditions will also affect the image. Hence the interpretation of autoradiograms of biological material is not straightforward. Autoradiography is frequently applied to the determination of the components of a paper chromatogram. Microautoradiography is useful when the distribution of radioactive compounds in a microscopic section is to be studied. Either the sections on the slides may be coated with melted emulsion, or a stripping film may be used to cover the sections on the slides. A more recent technique is the combined use of autoradiography and electron microscopy, for example in the study of sub-cellular organelles labelled with 3H.

1—2.2. Ionization detectors

A number of detectors are based on the principle that, in an electric field, negative particles will move to a positive electrode and positive particles to a negative electrode. Charged particles which arrive at an electrode will give rise to an electronic pulse, which can be amplified and registered. Alternatively, the pulses may be merged to form an electric current, which again can be amplified and measured. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 23

FIG.I- 7. Electroscope.

Alpha and beta particles arid IC electrons have a high specific ionization. Gamma and X-rays have a much lower primary specific ionization ; but at least one fast electron will be released by each photoelectric effect or Compton scattering (or pair production if the energy is very high), and these fast electrons will ionize just as do beta particles. Neutrons may also produce ions, directly (by collision) or indirectly (following nuclear absorption). Detection by ionization of these kinds of radiation is based on the fact that atoms of a gas (in the detector) will become ionized when they are hit by the radiation particles or photons. The number of ionizations in the gas is a direct measure of the quantity of ionizing particles or photons (a,/3,e,y, X or n) that reach the detector. When an electric field is created in the detector, the negative ions and/or electrons will start moving and, by hitting the positive electrode (anode), discharge. Likewise the positive ions will move toward the cathode. Five different types of ionization instrument will now be described.

1—2.2.1. Electroscope

In the electroscope or simple electrometer (see Fig. 1—7) the positive electrode is a rod with a wing or a metal string, and the negative electrode is the wall of the detector. When the electroscope is fully charged, the deflection of the wing or string will be maximum (A), the amount of deflection being a function of the charge accumulated. When a radioactive source is brought near the detector, the air in the detector will become ionized and electrons will move in the direction from wall to rod. As a consequence, the deflection will decrease (B). 24 PART I. GENERAL INTRODUCTION

This type of detector is commonly used as a 'pocket dose meter' and gives a measure of the accumulated dose of external radiation (gamma, X and hard beta radiation) to which a worker has been exposed during a certain period.

1—2.2.2. Gas-filled detectors with collector/cathode voltage bias

Not all the ions will discharge on the electrodes of an electroscope, since a certain number will have recombined before they reach the electrodes. If a bias voltage is applied between the cathode and collector (anode), the losses due to recombination in the volume of the detector decrease until, eventually, all the ions will be discharged on the electrodes. If the bias voltage is further increased up to a certain limit, the number of ion pairs that discharge will remain constant, i.e. each ionizing particle or photon that interacts will give rise to an electric pulse on the electrodes. Detectors operating in this mode are termed ionization chambers. Figure 1—8 shows a plot of pulse size against bias voltage. Region 1 is the ionization chamber region. Curves are drawn for an alpha and a beta particle traversing the sensitive volume of the detector. As the bias voltage is increased, the ions produced will move towards their respective electrodes with greater velocities, and at some voltage they will gain sufficient kinetic energy to cause further ionization in the gas, called secondary ionization. This process is known as gas amplification, and the flood of ions produced is termed the Townsend avalanche. As a result of gas amplification, each incident ionizing particle will lead to the formation of a relatively large electronic pulse. The pulse size produced increases rapidly with applied voltage. When the bias voltage results in gas amplification (region II), the size of the pulse produced by a particle (at a given voltage) is proportional to the number of primary ion pairs formed by the particle in the initial event. This is termed the proportional region. In region III, the bias voltage is so great that the charge collected on the anode attains a maximum size independent of the number of primary ions formed. In this region, at a given voltage, all pulses are of the same size, irrespective of the number of primary ions, and this is termed the Geiger-Müller (GM) region. In region IV (and to some extent in region 111) the discharge in a GM tube would continue indefinitely if it were not stopped or quenched. For this purpose certain quenching-gas molecules are added to GM tubes to stop the discharge, for example, gaseous halogens such as chlorine. When they collide with positive ions, quenching gas molecules dissociate rather than become ionized themselves and, in this fashion, the discharge is stopped. The halogen gas atoms subsequently recombine. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 25

HI I 12

200 400 V

FIG.I—8. Plot of logarithm of pulse size versus electrode voltage; I - ionization-chamber region; II - proportional region; III - Geiger-Müllerregion; IV - region of continuous discharge. (The voltage corresponding to a given region varies greatly from one make of instrument to another.)

1—2.2.3. Ionization chamber

When a detector is operating in region I (Fig. I—8) each ionizing particle or photon gives rise to an electric pulse on the electrodes. A constant stream of particles or photons gives rise to a continuous series of pulses, forming a weak electric current, which may be amplified and registered by an electronic circuit. Such detectors are termed ionization chambers, and they are often filled with air. A typical measuring circuit is shown in Fig. 1—9. The final scale reading will then be a measure of the energy dissipated in the ionization chamber per unit of time by the ionizing particles or photons. This kind of detection instrument is thus a dose-rate meter. (A well-known version of this type of meter is a radiation survey instrument known as the Cutie Pie.) When the walls of the ionization chamber are constructed of air-equivalent material, the instrument can be used to measure absorbed dose of gamma or X-rays in air. There are also chambers having biological tissue equivalence. A small, electrically-charged ionization chamber, held in place for instance by a finger ring, may be used to measure accumulated exposure dose. An electronic vacuum-tube voltmeter is often necessary to measure the charge reduction, which is proportional to dose. 26 PART I. GENERAL INTRODUCTION

IONIZATION CHAMBER

1 INSULATOR

ELECTROMETER T MEASURING •=• B CIRCUIT T

FIG.1-9. Schematic diagram of ionization-chamber circuit; B — voltage source (battery); R = resistor; C = capacitor; S = radioactive source.

1—2.2.4. Proportional counter

Proportional counters operate in region II (Fig. 1-8), when secondary ionization has become important. The electrons that have arisen from primary ionization will produce secondary ion pairs of the gas atoms in the counter tube as they are accelerated towards the anode. This process of secondary ionization becomes increasingly important as the voltage difference between the electrodes is further increased. The final pulse size will be proportional to the energy of the initial ionizing particle (as long as all tliis energy is dissipated in the detector), provided the applied voltage remains constant during the measurement. Usually the radioactive sample will be placed inside the detector, which will be transfused by a gas at atmospheric pressure (gas-flow counters). In this way particles of low energy, such as the ß~ from 14C, may be counted effectively ('windowless' counting), provided suitable amplification precedes the register.

1-2.2.5. Geiger-Müller (GM) counter

When the voltage difference between the electrodes of the detector is still further increased, secondary ionization becomes predominant and each primary ionizing event results in a discharge of a great number of electrons (avalanche). At this stage the large output pulse is independent of the energy of the initial particle or photon, and a further increase of the high voltage does not appreciably alter pulse size or count-rate. Geiger-Müller counter detectors (GM tubes) operate at this high-voltage plateau. The discharges of secondary electrons initiated by one ionizing particle or photon would continue if the detector were of an open design, as in the gas-flow counter (atmospheric pressure). GM tubes operate at a reduced gas pressure (about one-tenth atmosphere) and contain a certain amount oí quenching gas. Usually the closure of a GM tube is a very thin mica window (1-3 mg/cm2), and the filling gas is often a noble gas like argon with, 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 27

FIG.I-IO. Counter input circuit with a GM tube detector; C = capacitor; R=resistor.

for example, alcohol or halogen as the quenching gas. A certain number of molecules is dissociated during the quenching of a discharge with alcohol. Therefore, the quantity of quenching gas in the GM tube decreases steadily, and consequently the life of the tube is limited by this effect. This disadvantage does not exist when a halogen gas, e.g. chlorine, is used for quenching, because the atoms of the dissociated chlorine molecule recombine; the life of the tube is therefore determined by other effects, such as corrosion and leakage. Energetic beta particles, electrons and gamma or X-photons emitted by radioactive liquids may be counted with a thin glass wall 'dip-counter' GM tube, which is immersed in the liquid, or with a specially designed liquid detector that consists of a cylindrical glass container around the GM tube. The radioactive liquid thus surrounds the GM tube in both cases. Particles of low energy can obviously not be counted in this way because of absorption in the wall of the GM tube. Detectors operated in the Geiger-Müller region are very sensitive to beta particles, and very little additional amplification of the pulse is necessary to drive a counting circuit. In addition, they are almost insensitive to normal voltage fluctuations. Furthermore, they are relatively inexpensive. A simplified GM counter circuit is shown in Fig. I—10. The fact that some time is required to collect the flood of positive ions from each discharge, as well as for the quenching process to take place, implies that during this period the detector tube will be insensitive to other ionizing particles entering its sensitive volume. This period is 100 to 300 microseconds

for GM tubes and is termed the dead time, tdead. The time needed for a complete

recovery of pulse size after the dead time is termed the recovery time, trec. The time required before a subsequent pulse will again be recorded in the counting

system, termed the resolving time, r, lies between tdead and tdead+trec; as the

voltage amplification system is made more sensitive, so r approaches tdead. Since 28 PART I. GENERAL INTRODUCTION

Den crTOO PHOTOSENSITIVE LAYER CRYSTAL (NaKTU)

GAMMA RAY

LIGHT ELECTRONS ELECTRONS PHOTOMULTIPLIER TUBE

FIRST DYNODE

FIG.J-lla. Scintillation detector. Total light to tube is nearly proportional to gamma-ray energy. If 1 electron ejects 5 from a dynode, 11 dynodes result in 5" electrons, i.e. about 50 million electrons output.

PULSE-HEIGHT RECORDER PM AMP ANALYSER S N HV

FIG.I-llb. Block diagram of Nal(Tl) scintillation counter system. S = Gammá-ray source. DET = Nal(Tl) scintillation detector. PM = Photomultiplier tube. HV = High-voltage power supply. AMP = Linear amplifier.

UPPER DISCRIMINATOR BIAS

LOWER DISCRIMINATOR BIAS

FIG.I-12. Block diagram illustrating a single-channel pulse-height analyser. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 29 the dead time, and hence the resolving time, vary in a given tube, even from pulse to pulse, it is common practice to fix 7 at a value somewhat greater than tdead max electronically in the counting system. At high count-rates, counts will be lost owing to the inoperative period of the counting system, and hence a resolving- time correction must be made to obtain the true counts from the figure registered. Let R be the observed counts of a GM counter collected in one second and r (fraction of a second) the resolving time of the counter. During one second, the counter will have been ineffective for RT (fractions of a second). Therefore, R counts have really been registered (to a first approximation) in only (1 - RT) seconds. The true count-rate, RTRUE, is therefore (approximately):

Rtme = 7I^7 (1-16) where all count-rates are in counts/s. A method for determining the resolving time of a counter is given in Part V, Exercise 2. Correction is normally not necessary unless the count-rate exceeds about 50 counts/s. Equation (1—16) is only approximate, therefore it should not be used to make corrections that would be more than about 10% of R, otherwise it would be better to dilute the sample or to count it at a greater distance from the detector. GM counters are used most widely for the detection and measurement of beta particles. For gamma rays they are not very effective (1 —3% efficiency), because most of the photons will penetrate the gas without any interaction. For the detection of beta particles on glass-ware, benches or trays (i.e. contamination) monitors are used. A monitor consists of a GM tube connected to a power unit and a count-rate meter. Often a small loud-speaker is connected to the rate meter, so that a noise will warn the operator when the tube is in the vicinity of a contaminated spot. The necessary associated equipment for a GM counting system includes (besides the GM tube) a high-voltage supply, an amplifier, a recorder and a timer. This is illustrated in Fig. I—10.

1—2.3. Solid scintillation counting

A scintillator is a substance which emits a small flash of light when struck by a fast, charged particle. An example is (Ag)ZnS hit by an alpha ray. Solid scintillators (fluors) are particularly suited for the detection of gamma rays (besides X-rays and annihilation radiation) because of the high density and high-Z of certain solid crystals. The alkali halides, in particular Nal (activated with Tl), have been the most useful. A typical scintillation crystal detector is shown in Fig. 1-1 la, and a diagram of a counter system in Fig. 1-1 lb. 30 PART I. GENERAL INTRODUCTION

II il TOTAL ABSORPTION PEAK

UJ -1 ï~ LU ill 2Ï \ COMPTOCOMP N REG 8 -

PULSE HEIGHT (PROPORTIONAL TO Eabs)

FIGJ-13. Observed gamma-ray spectrum of a radionuclide emitting mono-energetic photons.

£"ab = Gamma-photon energy absorbed by crystal. Incident gamma-ray energy distribution.

When a gamma-ray photon is partially or totally absorbed in the scintillation crystal, at least one fast electron is liberated (it will be either a photoelectron, a Compton electron or pair-production electrons, depending upon the absorbing event). These fast electrons cause excitation and ionization along their paths in the crystal. When the atoms, thus excited, return to their ground-state they emit light photons with an intensity maximum in the violet or near-ultraviolet spectral region. The total number of light photons emitted will be proportional to the amount of the gamma-photon energy that is lost in, and absorbed by, the crystal. The photocathode of a photomultiplier tube is optically coupled to one face of the scintillation crystal, and the light photons produced in the crystal are internally reflected until they reach the photocathode. Here, by the photoelectric effect, they release photoeilectrons. The number of these photoelectrons again is proportional to the gamma-photon energy originally absorbed in the crystal. In the photomultiplier tube the photocathode is connected to a series of electrode stages or dynodes, each at a potential more positive than that of the preceding stage. Thus, photoelectrons released from the photocathode surface will be attracted to the first dynode and will gain sufficient kinetic energy to release two or more secondary electrons from the surface of this dynode. This multiplication process occurs at each stage and, at the end of ten or more stages in a typical photomultiplier tube, a large number of electrons will arrive at the anode. The size of this pulse of electrons will be proportional 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 31 to the original gamma-ray energy lost in the crystal. The pulse is then amplified linearly and directed to a scaler or to a pulse-height analyser.10 A pulse-height analyser consists essentially of two variable discriminators (a lower and an upper one), together with an anticoincidence circuit. With this auxiliary equipment, only pulses within a set pulse-height interval are registered (see Fig. 1-12). Such a unit, with only one set of discriminators, is called a single-channel pulse-height analyser. Instruments are also available allowing collection of counts in more than one channel simultaneously. In a multi- channel analyser the pulses are sorted according to their size by pulse height and stored in the appropriate portion of an electronic memory. After counts have been collected for a period of time, the read-out of the memory will be a gamma-ray spectrum of the radiation absorbed by the scintillation detector. The energy lost in the scintillation crystal by an incident gamma photon will range from zero to E^ depending upon the absorption event. For instance, the gamma ray can be absorbed by the photoelectric effect, or by a Compton interaction followed by photoelectric absorption of the scattered photon, or by any combination of processes that lead to total absorption of the gamma- photon energy within the crystal. If this occurs, then the output pulse will be stored in a location corresponding to the full E^-value. A typical monoenergetic gamma-ray spectrum is shown in Fig. 1-13 and the resulting peak is labelled as the total-absorption peak. If, however, the primary interaction in the crystal is of the Compton type and the scattered photon escapes from the detector, then the energy absorbed within the crystal will be less than E^. The range of possible Compton interactions results in a distribution of pulse sizes ('Compton smear'). This distribution is labelled as the Compton region in Fig. 1-13. The location of the total-absorption peak is characteristic of E^ and is useful in identifying the corresponding gamma-ray emitter in any sample. The area under the total-absorption peak is proportional to the activity of that radionuclide in the sample. The peak is actually broadened into a distribution due to (a) instrumental broadening and (b) statistical broadening as a result of the several conversion steps from gamma-photon absorption to final pulse. Pulse-height analysis is required when it is necessary to measure the activity of one gamma-ray emitter in the presence of one or more others. For most such experiments a single-channel or double-channel pulse-height analyser is sufficient. However, when one wishes to measure the amount of many tracers or absorption peaks, such as in multiple tracer experiments or neutron activation analysis, a multi-channel pulse-height analyser may be required.

10 The pulse height (voltage level) is used to determine the lower and upper levels of discrimination for pulse analysis. The term pulse size is sometimes used synonymously, and sometimes for the area under a pulse, which is obtained using a computer program linked to the pulse-height analyser output! Jn this manual, the common (commercial) term, pulse-height, is used wherever voltage-level discrimination would be used in the measurement. 32 PART I. GENERAL INTRODUCTION

Another distinct advantage of solid scintillation counting is the very short resolving time of such systems. This enables high count-rates to be determined (up to about 1000 counts/s) without the necessity of resolving-time corrections. (Additional details of gamma-ray spectral analysis are presented in Part V, Exercises 7 and 8.)

1—2.4. Liquid scintillation counting

Liquid scintillation counting techniques have promoted the application of radionuclides in the biological and agricultural sciences because they have allowed much wider use of low-energy beta-particle emitters such as 3H and 14C to be made. In this technique, the sample to be counted is placed in solution, together with an organic scintillator (the detector material), in anorganic solvent. Since each radioactive atom or molecule is closely surrounded by molecules of the scintillator, self-absorption preventing detection (see §1—2.7) is greatly reduced and the counting efficiency greatly increased. If the sample is insoluble in the organic solvent, it may often suffice to put it into uniform suspension. There are now many solute/solvent liquid scintillator systems in use. A very common one is PPO (2-5 diphenyloxazole) with toluene or dioxane as the solvent. The ionizing particles from the radioactive material cause excitation and ionization of the solvent molecules. These transfer their excitation energy to the PPO molecules which in turn fluoresce or scintillate, i.e. give rise to light photons on returning to their ground-state. The number of light photons emitted from the counting vial due to any one ionizing particle is proportional to the energy lost by that particle in the solution. The counting vial is optically coupled to a photomultiplier tube system to collect the emitted light. A block diagram of a simple liquid scintillation system is shown in Fig. I— 14. Normally, two photomultiplier tubes are used to collect the light emitted from the scintillation vial. This is done to increase the sample-to-background counting ratio as follows: After each single ionizing event, light photons will normally be registered at both photocathodes simultaneously. The coincidence circuit (Fig. 1 — 14) is designed to produce one output pulse if it receives an input pulse from each of the two photomultipliers simultaneously (within about 1 ¡is), i.e. in coincidence. Background or electronic noise pulses from either photomultiplier tube will seldom be in coincidence with those in the other, and will therefore be rejected. Thus, the ratio of true count-rate to background, and thereby the sensitivity, is increased. Since the size of the output pulse is proportional to the energy lost in the liquid scintillator, limited pulse- height analysis is possible. It is limited (a) because of the shape of beta spectra 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 33

REFRIGERATOR I

FIG.1-14. Block diagram of a typical liquid scintillation counter. S = Counting vial containing liquid scintillation solution and sample. PM = Photomultiplier tubes. Refrigerator = Refrigerating unit (optional!. Coincidence = Prompt coincidence and sum circuit. Pulse-height analyser = Upper- and lower-level discriminator.

and (b) because the pulse-height resolution is poor. However, it is generally possible to count, for instance, 3H and 14C simultaneously. The detector part of the system is often refrigerated to reduce thermally produced electron noise in the photomultiplier tubes.

For certain high-energy beta emitters (Emax > 0.26 MeV), it is often possible to employ so-called Cerenkov counting techniques using the liquid scintillation counter. In such cases, the radioactive sample need only be dissolved or suspended in water. An ionizing particle travelling through a medium (here water) at a velocity greater than the velocity of light in that medium produces a flash of Cerenkov light. The rate of production of the light flashes is proportional to the activity of the sample. Cerenkov counting techniques have proved useful with 42K, 24Na and other high-energy beta emitters. In particular, with this technique 32P can be counted in the presence of 33P without any interference from the latter (Emax <0.26 MeV). One of the main sources of error in liquid scintillation counting is the 'quenching' of light, often caused by compounds in the solution to be measured. This can be due to light absorption by coloured compounds or by certain chemicals. Since quenching commonly occurs, and its degree can be variable, it must always be considered. The three most important methods of correction are listed below: 34 PART I. GENERAL INTRODUCTION

(i) Removal of coloured material. The solution may be filtered through activated charcoal or treated by an ion-exchange technique to remove the quenching agent.

(ii) Channel-ratio method. In general, the net loss due to quenching is greatest for the most intense light flashes produced by the highest-energy particles. Therefore, when quenching occurs, the output pulse spectrum is shifted towards lower energy. If, by discriminator settings, the ratio of a lower energy part (channel) to a higher energy part (channel) of the spectrum can be obtained, then it is possible to observe the relation between counting efficiency, e, and channel ratio. This is done using a set of standards with a known constant amount of activity and measuring with increasing amounts of chemical quencher (i.e. increasing the channel ratio). A standard curve of e versus channel ratio can then be prepared and subsequently used to correct sample measurements for any decrease in count-rate due to quenching. However, this curve will not account for quenching due to coloured material.

(iii) External standard technique.11 In some instruments a standard source may be moved into position near the vial counting position. Thus, the relative decrease in the standard count for each sample counting vial will be proportional to the amount of quenching material and will provide the quench correction to be used for that vial count.

1—2.5. Semiconductor radiation detectors

The art of particle and gamma-ray spectroscopy has been significantly advanced by the relatively recent development of semiconductor radiation detectors. A semiconductor is a crystal with very high resistance to the flow of electric current at low temperatures due to the confinement of electrons in the valence bands of the crystal. At high temperatures, however, electrons may escape from the valence band to the conduction band where they are free to migrate and resistance is reduced, thus resulting in electronic 'noise'. In such crystals the energy difference between the valence and conduction bands must be quite small, e.g. < 1 eV, as compared with the 2-10 eV difference commonly seen in insulators even at high temperatures. Radiation detectors can be made from semiconductor crystals by adding impurities which provide either electron acceptor or electron donor properties to the crystal. Tetravalent germanium, for example, may be doped with trivalent

11 An internal standard technique may also be used; however, it makes it necessary to handle each sample twice. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 35 boron to produce a crystal with electron acceptor properties or 'holes' in the lattice which can be filled by nearby valence electrons, with only 0.01-0.1 eV being necessary to effect the transition. The new hole produced by this transition is filled in the same way, such that the 'holes' migrate in the valence band as electrons migrate in the conduction band. If germanium is doped with an electron donor impurity such as antimony, extra electrons are added to the lattice. In semiconductor radiation detectors a junction is formed between an electron-acceptor or p-type crystal and an electron-donor or n-type crystal. As a consequence, electrons from the n-type region flow across the junction and fill holes in the p-type region creating an electric field across the junction. The region near the junction, therefore, has a reduced number of both 'holes' and electrons and is called the 'depleted' region. By applying a voltage drop across this region the depleted region can be widened. If a charged particle or photon loses energy within this region, new electrons and 'holes' are formed and a pulse is generated. The depleted region, or sensitive volume, of the detector can be made even larger by a process called 'drifting', where lithium is deposited on the surface of the p-type crystal. Such a detector is called a Ge(Li) semiconductor detector. The size of the voltage pulse from a Ge(Li) detector is proportional to the charge collected by the electrodes,which is proportional to the number of electron-hole pairs produced, and the number of pairs is proportional to the energy deposited. Thus the pulse is directly proportional to the energy deposited. The pulse size is not affected by the type of radiation depositing the energy. The energy necessary per electron-hole pair is only about one-tenth of that necessary for ion-pair production in a gas or solid scintillation detector. Thus the number of pairs collected in a semiconductor is approximately ten times that in a conventional detector absorbing an equal amount of energy. Since the percentage relative standard deviation of the signal is inversely proportional to the square root of the number of ion pairs collected, it follows that the range of pulse size will be much smaller for a semiconductor detector than for a gas- filled or solid scintillation detector. Consequently the resolution of the semiconductor detector is superior. When spectral analysis is needed, such as in the evaluation of samples after neutron activation analysis, semiconductor detectors are extremely valuable. However, they have the disadvantage that they cannot be made as large as solid scintillation detectors and thus the counting yield is decreased. Also, in order to reduce electronic noise, they must be constantly maintained at very low temperatures requiring detector configurations in which good geometry (relationship of sample to detector) is difficult to achieve. 36 PART I. GENERAL INTRODUCTION

1—2.6. Inverse-square-law effect

A relationship met with in various branches of physics is that known as the inverse-square law. As applied to radiation, it states that the intensity of radiation emanating uniformly over the full solid angle (4 7r) from a point source in a vacuum decreases proportionally and monotonically with the square of the distance from the source. If I is the intensity of the radiation and d the distance from the point source, this can be expressed as:

IocT7 (1-17) d or

fl-18)

where k is the constant of proportionality.

If distances d( and d2 correspond to intensities of I ! and I2, from Eq.(I-18):

dl , - = — or I,d? = I2dl (1-19) I2 d,

Hence it can be seen that if d2 = 10 d,, the intensity will have decreased 100 fold

at d2, i.e. a detector placed at 10 cm from a point source will see only 1% of the radiation seen by the detector placed at 1 cm. This indicates one method of reducing the count-rate from a source that may have too high an activity for the detection system. Since a point source is a theoretical concept, it is useful to note some practical requirements in making use of this relationship: (a) The radiation must not be focussed or collimated in any way. (b) A source can be considered as a point source if the detector is placed at a distance which is at least ten times larger than the largest dimension of the source, i.e. with a needle source of 1 mm dia and 1.0 cm long the detector would have to be placed at least 10 cm from the source (< 1% error). (c) Since radioactive decay is a random phenomenon, counting statistics have to be considered. Simply summarized, the counting time must be sufficiently long in relation to source activity to make random fluctuations in decay negligible (see §1-2.8). (d) Measurements are rarely made in a vacuum. The important consideration is that matter (gas, liquid or solid) that is between the source and the detector 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 37 should not attenuate the beam because of any kind of interaction (absorption, scattering, etc.) by more than {% in practical counting. This will vary with type of radiation and with the medium (see §1-1.6). For example, air at atmospheric pressure will not affect gamma counting at typical experimental distances.

1-2.7. Counting efficiency (counting yield)

Practically every tracer experiment involves a number of samples containing radioactivity, and the assay of the activity of these samples is an integral part of the complete experiment. When a radioactive atom decays, often more than one particle or photon is emitted. For example, a 60Co nucleus emits either one beta particle and two gamma photons or occasionally one of each (see Fig. 1-2). However, metastable states excepted, a disintegration including the emission of particle(s) and/or photon(s) requires only 10"10s or less, whereas the resolving times of even the fast counters are of the order of 10"7s. Thus, no practical counter will have a counting efficiency, e (counts per disintegration), of greater than one. The efficiency of a given counter in assaying a given sample is defined as follows: _ count-rate of sample _ R ~ Rb disintegration rate in sample A* (1-20)

where the efficiency, e, is in counts per second per curie or becquerel; the activity, A*, is in curies or becquerels, respectively; while R is the count- rate of sample plus background (counts/s) and Rb is the background count- rate (counts/s). In most counters the counting efficiency is considerably less than unity, that is to say, only a fraction of the total disintegrations in the sample are detected and registered by the counting system. With the exception of liquid scintillation counting, the reduction in e is caused by the following:

(a) Geometry factor. Events in the source are not 'seen' by the detector. This is a function of the geometry factor, i.e. the solid angle of the source/detector arrangement divided by 4 it. For a small source close to the detector window the solid angle is about 2 it and the geometry factor about 0.5. (b) Air and window absorption. Particles, particularly alpha and low- energy beta, and to a lesser degree photons, may be absorbed in the air or in the window or walls of the detector, never reaching the sensitive volume of the detector. (c) Self-absorption in the sample. Alpha and beta particles, and to a much lesser extent gamma photons, can be absorbed by the sample material in which the radionuclide is contained, and a significant fraction of the activity 38 PART I. GENERAL INTRODUCTION

SAMPLE THICKNESS (mg-crrf2)

FIG.1-15. Count-rate as a function of mass per unit area ("thickness") for samples of constant activity concentration.

radiation will not be counted. This is a very important consideration for low- energy beta particles. In consequence, the count-rate from a given sample will not increase in proportion to its thickness. For a sample of a given area, as the sample thickness of constant activity-concentration material increases, the count-rate will tend towards a maximum (Fig. 1-15). At thickness X (measured in units of mass per unit area, see footnote 7) the sample is considered to be of infinite thickness. A common method for assay of low-energy beta emitters using GM counting is to count all samples at infinite thickness. The count-rate,

Rx, is then proportional to the activity concentration in the sample. The value for infinite thickness of beta emitters is approximately equal to the range of beta particles in units of mass per unit area. (d) Scattering. Particles or photons may be scattered towards or away from the sensitive volume of the detector. This scattering occurs in the backing •material of the sample holder, the walls of the shield, and the air between the source and the window.

When it is necessary to know the value of e, it need seldom be determined by investigating each of the above effects individually. Instead, a calibrated standard, i.e. a source of known activity, prepared in the same way as the samples, is counted under the same geometry to determine e. Calibrated standards may be purchased from radionuclide suppliers. A local standard may be prepared from the radioactive material to be used in the experiment. In the latter case, the count-rate of all experimental samples can be compared, for instance, as a percentage of the experimental amount of tracer activity administered (% of dose). In purely comparative investigations it is sufficient if e can be kept constant from sample to sample, and its actual value need not be known. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 39

TABLE 1-1. NATURAL STANDARD DEVIATION OF ACCUMULATED COUNTS

Accumulated counts Natural standard Natural standard deviation . deviation3 (C)

100 10 10 1 000 31.6 3.2 10 000 100 1.0 100 000 316 0.3 1 000 000 1 000 0.1

In 1972, the ICRU published a report on the measurement of low-level radioactivity [3]. It is of interest for users of this manual, and particular reference is made to the definition and discussion of a figure of merit (Ref. [3 ], § 1-3) which should be studied in connection with this and the following section (§1-2.8).

1—2.8. Counting statistics (natural uncertainty)

If a single radioactive sample is counted several times under identical conditions using a perfect counter, and the count is corrected for radioactive decay (or the decay correction is negligible) then the individual number of counts will be observed to fall in the neighbourhood of a mean value. These deviations are due to the random nature of the radioactive decay (§ 1-1.3). This phenomenon may be termed natural uncertainty, as opposed to normal technical uncertainty due to the operator or the apparatus. The understanding of these statistical effects is necessary in the consideration of experimental design and in the interpretation of counting results. Disintegration statistics follow closely the Poisson probability distribution law. As a special consequence of the Poisson distribution, the natural standard deviation (onat c) of a registered number of counts (C), irrespective of the time it takes to accumulate them, is closely equal to the square root of that number, C, under the assumption that the duration of the counting is much less than the half-life of the radionuclide being counted. So, for C accumulated counts, to a close approximation:

«W^v'C (1-21) 40 PART I. GENERAL INTRODUCTION

Table I—1 gives the calculated natural standard deviation for some given

numbers of accumulated counts. As can be seen from the table, although anat ç increases as the square root of C, the natural uncertainty expressed as a percentage of the counts decreases as C increases. Referring to Eq. (1-21), if both sides are divided by the counting time, T, the result is the natural standard deviation of the count-rate, R, since R =C/T and T in this respect is constant. Thus:

qnat,C VC ^ °nat,R ~ j — ~T~~

However, since C = RT:

(1-23)

When C becomes large (> about 10), the Poisson distribution is closely approximated by the normal distribution. From the normal distribution, one standard deviation on either side of the mean value accounts for 68% or about 2/3 of the total area under the probability curve. A useful rule of counting is to try to accumulate so many counts that the percentage natural standard deviation is 2 to 3 times less than the percentage technical standard deviation. If 10000 counts, for instance, are accumulated, then from this single assay it can be stated that there is a 68% probability that the true mean C-value is within 10 000 ± 100, or in the range of 9 900 to 10 100. Two standard deviations (2a) account for approximately 95% of the area under a normal distribution curve, and in this case it can be stated that there is a 95% probability that the true mean C-value is within 10 000 ± 200. The accumulated counts (C) collected in any counting interval are due to

true counts of the sample (Cs), plus those from background (Cb). There is a significant radiation background in almost any location. This background comes from cosmic rays and cosmic-ray induced activity, such as l4C, and from naturally occurring radioactive materials in the earth's crust and elsewhere, e.g. 226Ra, 232Th and 40K. The latter all have associated gamma rays. The cosmic-ray contribution varies with altitude, and the composition of the earth's crust, etc., varies with location. All radiation detector/counter systems have an associated background from the above sources and from electronic noise. The background count-rate is commonly reduced by shielding or by special electronic circuitry. Obviously, every sample count is made in the presence of a background count-rate for that particular system. The background will be a function of the type of detector, as well as shielding, location, discriminator settings, etc. 1-2. RADIATION DETECTION AND ASSAY OF RADIOACTIVITY 41

The deviation of a background count is independent of that of a sample plus background count, so the appropriate uncertainty terms add as the sum of the squares. Therefore, the variance of the net sample count is, since

Cs = C-Cb:

= + (I 24) 4S °c °cb ~

where ÜQ = variance of net sample count;

Oq = variance of sample plus background count;

oí, = variance of background count.

The natural uncertainty of the net sample count then follows from Eqs (1-21) and (1-24):

CTnat,Cs = V^TcT (1-25)

where Onat.Q = natura' standard deviation of accumulated net sample counts; C = the total of accumulated counts due to sample plus background;

Cb = the part of accumulated counts due to background.

Similarly, it follows from Eq. (1-20) that the natural standard deviation the net coun = R R of the (°nat R ) t-™te (Rs ~ b) sample is given by

[R Kb On «-26>

where R = count-rate of sample plus background;

Rb = count-rate of background; T = time used for counting sample plus background;

Tb = time used for counting background.

In tracer experiments, the net count-rate of samples very commonly approaches, or is even less than, background count-rates. In order to divide

a given total period of counting time between T and Tb in such a way as to 42 PART I. GENERAL INTRODUCTION

statistically minimize anat Rs, the following formula may be used (by inserting 12 preliminary values for Rb and R, obtained during short periods of counting):

However, strict adherence to this criterion for optimum statistical partition of counting time is not critical in practice.

1-3. RADIATION PROTECTION

It is imperative that a knowledge of the safe use of radionuclides and radiation be gained before they are applied as tools in research. Ionizing radiation is hazardous to all biological systems, but with proper considerations for health protection measures, the hazard to personnel or experimental systems can be reduced to a tolerably low level. The health physics involved in the safe use of radionuclides and radiation is discussed in some detail under three headings:

§1-3.2. Protection of personnel §1—3.3. Control of contamination §1-3.4. Waste disposal

However, before considering these categories, an insight must be gained both into basic considerations involved in radiation protection and into units.

1—3.1. Basic considerations and units

The liberation of ion pairs by energetic photons is termed the exposure, X, and is defined as:

dQ dm

where dQ is the absolute value of the total charge of ions of one sign produced when all the electrons (e+ and e~) liberated by photons in a volume of air of mass dm are stopped in air. In SI units, the exposure is measured in coulombs per kilogram, while the present special unit of exposure, the roentgen (R), is defined as:

1 R= 2.58 X 10~4 C/kg

12 Derivation of Eq. (1-27) is given in Part VI, Appendix VI-6. 1-3. RADIATION PROTECTION 43

Using this definition the energy absorbed by one kilogram of air due to a total exposure of 1 R is equal to:

-4 2.58 X lo C/kg air ,0 , —— X 33 eV/ion pair X 1.6 X 10"'9 J/eV = 8.5 X 10"3 J/kg 1.6 X 10"19 C/electron

Using the factor 1 erg = 10 7 joules, it can be seen that the above value is identical to 85 erg/g of air, which was the accepted value using the old system of measurements.

To obtain some impression of the magnitude of such interaction, the following approximate figures are of assistance. The charge on an electron is about 1.6 X 10~19 C, and 1 cm3 of air.at STP has a mass of around 1.3 mg; hence, one roentgen will produce about 2.1 X 109 ion pairs per cubic centimetre of air and, since release of an ion pair requires some 33 eV, about 1.1 J of radiation energy are absorbed per cubic centimetre of air. Thus, one roentgen will dissipate 8.5 X 10"3 J/kg air (i.e. 85 erg/g). As discussed in §1—1.6, the energy of gamma rays absorbed per gram of various materials is a function of properties such as Z and density. Thus, the energy absorption per gram from exposure to 1 R will be slightly different for soft tissue, for water and for air, and it will be a function of photon energy. Further, for example, the energy absorption per gram of bone tissue from exposure to 1 R of X or gamma photons of energy below 0.01 MeV will be 2 to 5 times that of soft tissue exposed to 1 R of the same photons. In general, there is no simple relationship between the energy absorbed per gram and the exposure. The biological effect from a given type of irradiation is, however, proportional to the energy absorbed per gram, and a unit of absorbed dose was, therefore, introduced. The unit of absorbed dose in SI is the gray (Gy), representing the absorption of 1 joule per kilogram of the irradiated material. The special unit of absorbed dose at present is the rad, use of which is being phased out during the period to 1986. The interrelationships of interest are:

1 Gy = 1 J/kg = 100 rad (= 104 erg/g)

The dose absorbed by a material subject to a given exposure will vary with the nature of the material, depending to a large extent on the scattering power (electron density) of the constituent atoms. The units gray and rad may be used irrespective of the type of ionizing radiation being considered. 44 PART I. GENERAL INTRODUCTION

It is clear, however, that radiation dissipating 1 Gy (100 rad) with a high specific ionization, i.e. a high linear energy transfer (LET), will have a greater biological effect on an organism than a different quality of radiation dissipating 1 Gy with a low specific ionization. For example, alpha particles have a much higher LET than beta particles, and hence the biologically damaging effect of alpha particles will be greater than that of beta particles. This observation resulted in the use of a factor termed the Relative Biological Effectiveness (RBE), which was defined using the biological effect of irradiation with 200 keV X-rays as the basis for comparison:

Absorbed dose due to 200 keV X-rays causing a specific effect RBE = S— (1-28) Absorbed dose due to other radiation causing the same effect

Originally, the product of the absorbed dose in rads and the RBE were taken to give a value in rems (originally derived from the idea roentgen equivalent man) such that a figure of, say, 100 rems would represent the same biological effect of an irradiation, irrespective of the type of radiation used, i.e. the effect 100 rem beta rays = effect 100 rem gamma rays. This was to make it possible to sum such 'effective absorbed doses' or 'weighted absorbed doses' resulting from different exposures to different radiations at different times, a matter of practical importance. The use of the term rem for such values is no longer applicable, and the product absorbed dose and RBE is termed 'effective dose' or 'weighted dose', still measured in grays, J/kg or rads. For example:

Deff ,(rad) = D, XRBE,; Deff 2(rad) = D2 X RBE2

- Peff.tot (rad) = Deff), + Deffj 2 (1-29)

although the applicability of this idea must be proved for the biological system under investigation. The modern concept of dose equivalent, H, has been defined with regard to the human and his organs by the ICRP.and ICRU [4] as follows:

H = QND (1-30)

where D is the absorbed dose, Q is the quality factor of the radiation and N is the product of all other modifying factors. Q and N are dimensionless, and hence the dimensions of H are J/kg, as for absorbed dose. The use of dimensionally similar units for these two different concepts could have serious consequences in radiation protection, etc., and, hence, either sievert or rem should be used.13

13 The special name 'sievert', symbol Sv, was adopted in 1979 for the SI unit of dose equivalent, for use in radioprotection. Dimensionally: lSv=lJ/kg. 1-3. RADIATION PROTECTION 45

TABLE I -2. VALUES OF QUALITY FACTOR Qa USED IN DEFINING DOSE EQUIVALENT [7]

Radiation Q

X-rays, 7-rays, electrons and (3-rays 1.0 Fast neutrons and protons up to 10MeVb 10 a-particles from radioactive decay (for internal exposure) 10 Heavy recoil nuclei 20

a These values of Q are those chosen specifically for use in defining maximum permissible doses. k In the case of irradiation of the lens of the eye with particulate radiation of high LET, an additional modifying factor, N, must be used. N should be 3 when Q > 10.

Hence 1 Sv, i.e. 100 rem, of one kind of ionizing radiation is, for radiation protection purposes, defined as having the same biological effect in man as 1 Sv or 100 rem of another kind of ionizing radiation. In considering irradiation of the whole or part of the human body, the dose equivalent would be computed from the known or estimated absorbed dose, making use of the factors published by the ICRP [5] (see also Ref. [6]). Dose equivalents are additive for a given person or organ. Some data concerning quality factor, Q, are shown in Table 1-2. Apart from an exception noted in Table 1—2, N is assigned the value unity for all radiations from external sources [2,4,7],

1—3.2. Protection of personnel

The International Commission on Radiological Protection (ICRP) has recommended that the yearly dose to radiation workers must not exceed 5 rem per year. This is equivalent to an average rate of 0.1 rem per week; however, the ICRP recommendation does not stipulate any weekly rate [5, 6]. The yearly maximum of 5 rem applies to the dose (from both internal and external radiation) to the whole body, the gonads alone or the red bone marrow alone.

1—3.2.1. External exposure

Radiation dose to personnel must always be kept as low as possible. In the case of external exposure, this can be accomplished by an optimum combination of (i) shielding, (ii) increasing working distance from the source, and 46 PART I. GENERAL INTRODUCTION

TABLE 1-3. APPROXIMATE VALUE" OF THE HALF-THICKNESS OF LEAD AS A FUNCTION OF GAMMA-RAY ENERGY (>0.2 MeV)

£ Approximate half-thickness of lead shielding*1 (MeV) (cm)

0.25 0.25 0.5 0.5 1.0 1 1.5 1.5 2 to 4 2

a The value will depend on the geometrical relationship between source and absorber. With a point source very close to the absorber, the radiation meets the absorber at a wide range of angles of incidence (broad geometry). The half-thicknesses in such a case will be different to those if the absorber is sufficiently distant for the incident radiation to be nearly parallel (in effect a collimated beam; narrow geometry). The half-thickness values could vary by 20 to 30% down or up from the values given in the table above, which are for intermediate geometries more normally met with in measuring and experimental practice.

(iii) minimizing exposure time. Shielding of alpha emitters for external radiation is not required because the wall of the container or a few centimetres of air will absorb all particles. The same considerations generally apply to low-energy beta emitters such as 3H, 14C or 4SCa. High-energy beta emitters require only 1 to 2 cm of low-Z material, such as polymethyl methacrylate (Lucite, Perspex, etc.) for shields.14 In the case of gamma rays, a high-Z material, such as lead, provides the best shielding. Table 1-3 gives approximate values of half-thicknesses (half-value layers) of lead for shielding against gamma rays.

14 For strong beta-emitting sources the production of bremsstrahlung must be considered. Bremsstrahlung (braking radiation) is electromagnetic or photon radiation emitted by a high- speed electron as it is decelerated by the Coulombic field of atomic nuclei in high-Z absorbers (see Part VII, Glossary). 1-3. RADIATION PROTECTION 47

To obtain the approximate half-thicknesses of water, the corresponding half-thickness of lead may be multiplied by 10. (The density of water is about one tenth that of lead.) To obtain the approximate half-thickness of any other material, the necessary half-thickness of water is divided by the density of the other material. The photon-intensity attenuation factor, F. and the number, n, of half- thicknesses (X^, see Eq. (I—13a)) are related as follows:

• 'og.oF Fc = 2", i.e. n = —CI—31)

Work with radioactive sources must always be performed with sufficient shielding for personnel. The calculated dose rate after shielding must always be checked with a dose-rate meter, preferably an ionization chamber type. Sources not in use should always be stored behind shielding and access to the sources strictly controlled. Warning signs such as the following should be used:

Radioactive materials; external dose rates less than 2.5 mR'h"1 CAUTION RADIOACTIVE MATERIALS «6.5 X 10~7 C'kg"1'h"1)

External dose rates 2.5 to 100 mR h"1 CAUTION RADIATION AREA (6.5 X 10"7 to 2.5 X 10"5 C kg-' h"1)

External dose rates exceeding 100 mR h-1 CAUTION HIGH RADIATION AREA (>2.5 X lO-sc-kg-'-h"1)

It is important that before beginning any work with gamma-ray emitters the researcher should know how great the radiation dose from the source will be. The gamma-ray dose constant T (in various units for 1 m from a point source) is given in Table 1—4 for various radionuclides. For point sources of activity, gamma-ray intensity is inversely proportional to the square of the distance (see §1-2.6). Thus, once the exposure dose is known at any one distance, it may be calculated at any other distance by the inverse- square law. It was seen in §1-2.6 that distance is a very important factor in minimizing dose. Consider a point source from which the gamma-ray exposure dose was 1 mR'h"1 at 10 cm. Any manipulations with the source by means of long forceps or tweezers would produce a negligible finger or whole-body dose. However, if the source were handled without tweezers, for instance with rubber gloves as the only protection, the radiation exposure dose at 1 mm distance would be 10 000 mR'h"1 = 10 Rh-1 to the skin of the finger tips. 48 PART I. GENERAL INTRODUCTION

TABLE 1-4. GAMMA-RAY EXPOSURE DOSE LEVEL AT I m FROM A POINT SOURCE (D FOR SOME SELECTED RADIONUCLIDES

1" (at 1 metre) Predominant Radionuclide gamma-photon energy (R h"' per Ci) (R h-1 per Bq) (C kg-' h"1 per Bq) (MeV)

Na-22 1.2 3.2 X 10"" 8.4 X 10"'s 1.3 and 0.5a

Na-24 1.8 4.9 X 10"" 1.3 X 10"u 2.7 and 1.4 Mg-28 1.6 4.3 X 10"" 1.1 X 10"'4 1.8 and 1.4 (+ equíl. Al-28)

K-42 0.14 3.8 X 10'" 9.8 X 10"16 1.5

Cr-51 0.02 5.4 X 10"'3 1.4 X 10"'6 0.3 Mn-54 0.47 1.3X10'" 3.3 X 10"'s 0.8 Co-58 0.55 1.5 X 10"" 3.8 X I0"'s 0.8 and 0.5a

Fe-5 9 0.67 1.8 X 10"" 4.7 X 10"15 1.3 and 1.1

Co-60 1.3 3.5 X 10"" 9.1 X 10"'5 1.3 and 1.2 Cu-64 0.12 3.2 X 10"" 8.4 X I0"16 0.5a Zn-65 0.27 7.3 X 10"l! 1.9 X 10"'s 1.1 Se-15 0.20 5.4 X 10"" 1.4 X 10~'s 0.4, 0.3 and 0.1

Rb-86 0.05 1.4 X 10"" 3.5 X 10"" 1.1 Zr-95 0.41 1.1 X 10"" 2.9 X 10"'s 0.8 and 0.7 1-131 0.22 5.9 X 10'" 1.5 X 10"1S 0.4

Cs-137 0.31 8.4 X 10"" 2.2 X10"'5 0.7 (+ equil. Ba-137m)

Ta-182 0.68 1.8 X 10"" 4.7 X 10",s 1.2 and 0.2 Au-198 0.25 6.8 X 10"" 1.7 X 10"15 0.4 Ra-226 0.825 2.3 X 10'" 5.9 X10"'5 many different (+ equil. decay chain) with 0.5 mm Pt cover for calibration

a Annihilation photons following ß+.

Reduction of exposure time is also important in minimizing dose. Mani- pulations with sources should be performed rapidly but carefully. Monitoring of external dose can be accomplished by the use of personal dose meters [8]. These can be worn on the body, or attached to the hands or wrists if necessary. They provide an integrated dose reading, i.e. a dose value summed over the total working period. Pocket dose meters (electrometers), film badges or solid-state thermoluminescent dose meters [9] are the most common systems currently in use. 1-3. RADIATION PROTECTION 49

1—3.2.2. Internal exposure

The internal hazards of radionuclides involve some distinctly different considerations. Beta emitters and particularly alpha emitters become extremely hazardous on entry into the body. The protection against internal contamination largely involves prevention of accidental ingestion, of inhalation or of skin absorption of radionuclides. The International Commission on Radiological Protection (ICRP) has calculated (i) maximum permissible body burdens of all the radionuclides and (ii) the maximum permissible concentrations in water and air that would produce such body burdens if chronic exposure conditions existed [5, 6]. The factors that determine the maximum permissible body burden of any radionuclide are:

(a) Particle radiation energy, LET and radioactive half-life; (b) Absorption from the gastro-intestinal (Gl) tract, lung tissue or skin into body fluids; (c) Distribution into body organs, i.e. selective concentration (e.g. 1311 in the thyroid gland); (d) Biological half-life, i.e. the time required for a given body burden to decrease physiologically by one-half. The combined effect of radioactive decay and physiological excretion is given by the relation:

1 1 1 jT = Ti + - (I"32)

where T^ = radioactive half-life T^,eff = effective half-life T^.biol = biological half-life

Factors (b) and (c) also depend on the chemical and physical form of the radionuclide. Solubility in body fluids will largely determine the absorption and transport of the radionuclide. The relative radiotoxicities of all known radionuclides are given in Part VI, Appendix VI-3, while some typical examples are shown in Table 1—5.

1—3.3. Control of contamination

Contamination of laboratory, benches, glass-ware and operators by radionuclides must be avoided for two reasons:

(i) Laboratory contamination can result in internal exposure of the laboratory personnel, and it may even be spread to areas where other personnel may be exposed. (ii) Experimental results are likely to become uncertain. 50 PART I. GENERAL INTRODUCTION

TABLE 1-5. LIMITATION ON RADIOACTIVITIES IN VARIOUS TYPES OF WORKING PLACE OR LABORATORY" [7]

Radiotoxicily of Minimum Working place or laboratory required radionuclides significant quantity land examples of each) (pCil 1 k»q ) Type (' 1'ype B Type A

1. Very high 0.1 3.7 1*10 (i<"i or less JlO «Ci to 10 niCi JlO m('i or more (Sr-90.l'o-2l0.etc.) (0.37 MBq or less [0.37 MBq to 0.37 CBq [0.37 (;Bq or more

2. High 10 37 JlOOyCi or less flOO^fi to 100 m('i JlOO infï or more (Na.22.ra-45.C'o-60,Sr.X9.H3l.ete.) [3.7 MBq or less [3.7 MBq to 3.7 (,Bq [3.7 (.B<| or inore

3. Moderate 10 370 Jl mCi or less Jl mCi to 1 Ci Jl Ci or more KM4.P-32,S-35,K-42,Zn'65,Br-82,etc.) (37 MBq or less [37 MBq to 37 (¡Bq [37 (;Bq or more

4. Slight 100 3700 JlO mCi or less JlO mCi to 10 Ci Jl 0 Ci or more 111-3.Rb-K7.etc.) [0.37 (¿Bq or less [0.37 (iBq tu 0.37 TBq (0.37 TBq or more

3 Type C, Typo B and Type A have (he meanings normally used in the Classification of laboratories for handling radioactive materials. Type C is a good quality chemical laboratory. Type B is a specially designed radioisotope laboratory. Type A is a specialty designed laboratory for handling large activities of highly radioactive materials. In the case of a conventional modern chemical laboratory with adequate ventilation and fume hoods, as well as polished, easily cleaned, non-absorbing surfaces, etc.. it would be possible to increase the upper limits of activity for Type-C laboratories towards the limits for Typc-B laboratories for toxicity groups 3 and 4.

Control can best be achieved by regular maintenance of high standards. For this purpose, a number of staff should be appointed to the position of Radiation Safety Officer. The duties of this officer would include:

(a) Determining the previous radiation exposure of all workers in the isotope laboratory and arranging for blood counts if the exposure is considered serious; (b) Maintaining records of exposure of staff to ionizing radiation and arranging for blood counts at six-monthly intervals if considered necessary; (c) Arranging for the regular distribution and examination of film badges which should be worn at all times by workers in radioisotope laboratories; (d) Taking delivery of all shipments of radioisotopes and supervising their storage; maintaining detailed records of all shipments so that their use and final disposal can be traced; (e) Maintaining records of all closed radiation sources; (f) Regularly monitoring all working areas for radioactive spillage; (g) Ensuring that all workers, especially those inexperienced in handling radioisotopes, observe the laboratory rules.

A number of laboratory rules must, therefore, be strictly adhered to:

(a) Eating, drinking, smoking and application of cosmetics in the laboratory are strictly prohibited, as is combing of hair (because of the electrostatic charge induced). 1-3. RADIATION PROTECTION 51

(b) Each person should wear a laboratory coat. This coat should be worn in the laboratory space where the experiments with radionuclides are done, but not in separate counting rooms or outside the area of radioactivity. (c) When there is a significant risk that the hands may become contaminated, thin surgical gloves or disposable plastic gloves should be worn. The surgical gloves have to be put on and taken off in such a way that the inside never touches the outside in order to prevent direct contamination of the skin. A detailed description of the procedure for putting on or removing gloves is given in Part VI, Appendix VI-1. As soon as the risk for contamination of the hands is no longer present, the gloves should be removed, as they constitute a source of contamination of glass-ware, equipment, faucet handles, etc. (d) Pipetting or the performance of any similar mouth action is strictly prohibited. Syringes or propipettes must be used. (e) Protective eye glasses or shields are advantageous and should always be worn in high radiation areas of a radiochemistry laboratory. This will shield the lens of the eye'from beta particles and will minimize eye injury in the event of a chemical accident. (f) To prevent contamination of gloves, hands or equipment, paper tissues should be at hand and should always be used as a preliminary means of decontamination. After use, these tissues should be disposed of in foot- operated waste bins or large drums. (g) All operations involving volatile materials, heating or digestion must be done under a fume hood. The air velocity (suction) at the hood face should be approximately 1 m/s. (h) Any operation in which radioactive dust may arise should be carried out in a glove-box in which slightly negative pressure is maintained. In the exhaust system a dust filter must be present to collect radioactive particles. These precautions are imperative in the case of alpha activity. (i) All operations should be carried out over shallow trays. The bottoms of the trays should be covered with absorbent paper. (j) Storage bottles should be available for dumping of liquid waste (see Part VI, Appendix VI-2). These bottles should contain a small amount of ion- exchange resin to concentrate the activity. (k) Cross-contamination should be avoided by using glass-ware, tin openers, tweezers, etc., for one particular radionuclide only. (1) A thin-window GM survey meter should be available for contamination detection. In addition, it would be preferable to have an ionization-chamber survey meter for exposure dose measurements. (m) Frequent surveys of laboratory work areas, equipment and personnel should be performed with the GM survey meter to detect contamination. In the case of alpha emitters, 3H or other low-energy beta emitters, filter paper 52 PART I. GENERAL INTRODUCTION

should be used to swab the suspected areas. The swabs should be counted with an appropriate detector, (n) Before leaving the laboratory the hands, clothing and shoe soles should be checked with a suitable survey instrument or swabbed, the swabs being counted.

1—3.3.1. Decontamination

Decontamination of the skin should first be attempted with soft soap and water, possibly with a soft brush. Care should be taken to avoid damaging the skin by excessive washing. Often,washing with a carrier solution will aid in removal through exchange with the radioactive isotope. Obviously, the carrier solution must be non-toxic to the skin. Generally, the contamination of glass-ware, metal surfaces or painted surfaces which have been contaminated with radioactive material of high specific activity is greatly reduced by repeated washings with carrier solution. Stocks of carrier solution should therefore be present where contamination is likely to occur. A spreading agent may be very effective. Otherwise, materials may be decon- taminated as follows:

MATERIAL DECONTAMINATION SOLUTION

Glass Either 10% nitric acid, or 2% ammonium bifluoride, or chromic acid, or carrier in 10% hydrochloric acid.

Aluminium 10% nitric acid, sodium metasilicate or sodium metaphosphate.

Steel Phosphoric acid plus a spreading agent.

Lead 4N hydrochloric acid until a reaction starts, then a dilute alkaline solution, followed by water.

Linoleum Xylol or trichlorethylene to remove wax surface.

Painted surfaces Spreading agent and ammonium citrate or ammonium bifluoride.

Wood and concrete Difficult to decontaminate. Partial or complete removal of the contaminated material will usually be the only effective method.

1—3.3.2. Special laboratory design features

A laboratory in which work with radioactive materials is done should have facilities that: 1-3. RADIATION PROTECTION 53

(a) minimize the incidence and spread of contamination (b) make possible rapid decontamination. These facilities are further determined by the nature of the work that is to be carried out. Three types of laboratory may accordingly be described (see Table 1-5 and Ref. [7]). Usually, a Type A laboratory will be associated with reactor operations or waste processing plants. For biological research. Type B or C laboratories will generally be adequate. A Type C laboratory may be any ordinary laboratory that has a good ventilation system and an exhaust hood. Floors and benches should have a surface that can be cleaned easily. If larger quantities of radionuclides are to be used, for example for the dilution of stock solutions or the preparation of labelled compounds, then a Type B laboratory will be required.

The characteristics of a Type B laboratory may be listed as follows:

(a) The laboratory room should preferably be separate from the counting room(s). (b) Ventilation of the laboratory should be sufficient to exchange the total room volume 12 times hourly. The air flow should be from least active to most active areas. The fan for each hood should be at the top of the vent duct so there is negative pressure throughout the vent duct. Multiple hoods should automatically be vented simultaneously at the same air velocity. The ventilation to the room should be separate from that to other rooms, particularly counting rooms. There should be a particle filter in each exhaust duct. (c) Shielded, lockable, separate storage areas should be available for highly radioactive sources. (d) To facilitate decontamination, benches should be covered with melamine laminate and floors with vinyl or linoleum, preferably without seams. Under no circumstances should uncovered wooden or concrete floors and bench tops be allowed. Furniture should be of non-porous material. (e) The GM survey meter, the hand and foot monitoring station and laboratory coat hooks should be located just inside the entrance to the laboratory. (f) Water faucets should be of a foot or elbow-operated design to prevent contamination. (g) If possible, a shower for personnel decontamination should be located close to the laboratory. (h) Drains should be located in the floor. (i) There should be no ridges and corners in which dust may accumulate and which are difficult to clean. 54 PART I. GENERAL INTRODUCTION

1—3.4. Waste disposal Radioactive waste should be controlled and disposed of according to the recommendations of the 1CRP and IAEA (see Part VI, Appendix VI-2). Generally, liquid waste should be stored in polyethylene containers and not disposed of into the sanitary sewer system through sinks. High-volume, low-activity liquid waste may be treated by ion-exchangers to reduce the volume. Solid waste should be placed in foot-operated bins. All waste containers must have the appropriate label as well as a label stating the date and quantity of each radionuclide added. If possible, it is advisable to store all liquid and solid waste until the activities present have been reduced by radioactive decay such that it might be disposed of by usual methods. If this is not possible, as in the case of long-lived emitters, land burial may be necessary. In some countries a central organization is in charge of collection, storage and/or burial of radioactive materials. Waste disposal can be a serious problem, and if work with appreciable activity of long-lived radionuclides is expected, expert advice should be sought.

1—3.4.1. Disposal of radioactive animals

Radioactive animals should not be used for human food. Appropriate means of identification, such as ear tags, should be applied when the animal receives its first dose of radioactivity, and rules should be introduced to ensure that the animal is properly disposed of. It is taken for granted that the animal will be killed humanely - an intravenous overdose of barbiturate anaesthesia or saturated magnesium chloride will cause rapid death without the loss of radioactive blood. The disposal of the carcass should then proceed as with other wastes, especial care being taken that the carcass cannot be eaten by dogs or feral animals.

REFERENCES TO PART I

[1] BUREAU INTERNATIONAL DES POIDS ET MESURES, Le Système International d'Unités (SI), 2e ed., BIPM, Sèvres (1973) 14; English translations under the title The International System of Units (SI) have been prepared by the National Bureau of Standards, USA (NBS Special Publication 330), and by The National Physical Laboratory, UK. [2] INTERNATIONAL COMMISSION ON RADIATION UNITS AND MEASUREMENTS, Radiation Quantities and Units, ICRU Report 19, ICRU, Washington (1971). [3] INTERNATIONAL COMMISSION ON RADIATION UNITS AND MEASUREMENTS, Measurement of Low-Level Radioactivity, ICRU Report 22, ICRU, Washington (1972). [4] INTERNATIONAL COMMISSION ON RADIATION UNITS AND MEASUREMENTS, Dose Equivalent, Supplement to ICRU Report 19, ICRU, Washington (1973). BIBLIOGRAPHY TO PART I 55

[5] INTERNATIONAL COMMISSION ON RADIOLOGICAL PROTECTION, Report of Committee II on Permissible Dose for Internal Radiation, ICRP Publication 2, Pergamon Press (1959). [6] INTERNATIONAL ATOMIC ENERGY AGENCY, Basic Safety Standards for Radiation Protection, 1967 Edition, Safety Series No.9, IAEA, Vienna (1967). [7] INTERNATIONAL ATOMIC ENERGY AGENCY, Safe Handling of Radionuclides, 1973 Edition, Safety Series No.l, IAEA, Vienna (1973). [8] INTERNATIONAL ATOMIC ENERGY AGENCY, Personnel Dosimetry Systems for External Radiation Exposures, Technical Reports Series No. 109, IAEA, Vienna (1970). [9] BECKER, K., The future of personnel dosimetry, Health Phys. 23 (1972) 729.

BIBLIOGRAPHY TO PART I

RADIATION PHYSICS AND CHEMISTRY, UNITS AND DEFINITIONS

ARONOFF, S., Techniques of Radiobiochemistry, Iowa State College Press (1961). BIRKS, J.B., Theory and Practice of Scintillation Counting, Pergamon Press, Oxford (1964). BOYD, G.A., Autoradiography in Biology and Medicine, Academic Press, New York (1955). CHASE, G.D., RABINOWITZ, J.L., Principles of Radioisotope Methodology, 3rd ed., Burgess Publication Co., Minneapolis (1967). GLASSTONE, S., Source Book on Atomic Energy, Van Nostrand Publ. Co., New York (1958). LAPP, R.E., ANDREWS, H.L., Nuclear Radiation Physics, 3rd ed., Prentice-Hall, Englewood Cliffs ( 1963). PRICE, W.J., Nuclear Radiation Detection, 2nd ed., McGraw-Hill, New York (1964). SIEGBAHN, K. (Ed.), Alpha-, Beta- and Gamma-Ray Spectroscopy, Vol. 1, North-Holland Publishing Company, Amsterdam (1965). INTERNATIONAL COMMISSION ON RADIATION UNITS AND MEASUREMENTS, Washington, DC, ICRU Reports (selected as being of value in laboratory and field work with radioactive materials): ICRU Report 1 Ob, Physical Aspects of Irradiation ( 1964); ICRU Report 10c, Radioactivity (1963); ICRU Report 10e, Radiobiological Dosimetry (1963); ICRU Report 12, Certification of Standardized Radioactive Sources (1968); ICRU Report 14, Radiation Dosimetry: X Rays and Gamma Rays with Maximum Photon Energies between 0.6 and 50 MeV (1969); ICRU Report 16, Linear Energy Transfer (1970); ICRU Report 17, Radiation Dosimetry: X Rays Generated at Potentials of 5 to 150 kV (1970); ICRU Report 19, Radiation Quantities and Units (1971) ; Supplement to ICRU Report 19, Dose Equivalent (1973); ICRU Report 20, Radiation Protection Instrumentation and its Application (1971); ICRU Report 21, Radiation Dosimetry: Electrons with Initial Energies between 1 and 50 MeV (1972); ICRU Report 22, Measurement of Low-Level Radioactivity (1972); INTERNATIONAL ORGANIZATION FOR STANDARDIZATION: ISO 921, Nuclear Energy Glossary (1972); ISO 1000, SI Units and Recommendations for the Use of their Multiples and of certain other Units (1973); 56 PART I. GENERAL INTRODUCTION

ISO 31, Parts 0 to 12, Quantities, Units and Symbols (latest revisions). NATIONAL BUREAU OF STANDARDS, US Department of Commerce, Washington, DC, NBS Guidelines for Use of the Metric System, LC 1056 (latest Rev., Aug. 1975).

RADIATION PROTECTION

BOURSNELL, J.C., Safety Techniques for Radioactive Tracers, Cambridge Univ. Press (1958). MORGAN, K.Z., TURNER, J.E. (Eds), Principles of Radiation Protection, John Wiley, New York (1967). INTERNATIONAL ATOMIC ENERGY AGENCY, Vienna, IAEA Safety Series (selected as being of value in laboratory and field work with radioactive materials): No.l, Safe Handling of Radionuclides (1973 edition); No.8, The Use of Film Badges for Personnel Monitoring (1962); No.9, Basic Safety Standards for Radiation Protection (1967 edition); No. 12, The Management of Radioactive Wastes Produced by Radioisotope Users ( 1965); No.14, The Basic Requirements for Personnel Monitoring (1965); No. 19, The Management of Radioactive Wastes Produced by Radioisotope Users: Technical Addendum (1966); No.38, Radiation Protection Procedures (1973); No.40, Safe Use of Radioactive Tracers in Industrial Processes (1974). No.42, Radiological Safety Aspects of the Operation of Neutron Generators (1976). US DEPT. OF HEALTH, EDUCATION AND WELFARE, Radiological Health Handbook, Rev. ed., US Dept. of Health, Education and Welfare, Washington, DC ( 1970).

BIOLOGICAL EFFECTS OF RADIATION

CASARETT, A.P., Radiation Biology, Prentice-Hall, Englewood Cliffs ( 1968). BACQ, Z.M., ALEXANDER, P., Fundamentals of Radiobiology, 2nd ed., Pergamon Press, Oxford (1961). Peaceful Uses of Atomic Energy (Proc. 4th Int. Conf. Geneva, 1971) Vol. 13, Agenda Item 4.4., UN, New York, and IAEA, Vienna(1972) 361-502.

WORKING NOTES TO PART I WORKING NOTES TO PART I

NOTES (cont.) 58 PART I. GENERAL INTRODUCTION

NOTES (cont.) WORKING NOTES TO PART I

NOTES (cont.) 60 PART I. GENERAL INTRODUCTION

NOTES (cont.) PART II. MENTAL EXERCISES

(1) When the Z-number of all nuclides is plotted against the N-number, isotopes of a particular element will be found on a horizontal line. This kind of representation is usually given on nuclear charts. How can the decay products of a particular nuclide be found after the emission of one: (a) alpha particle? (b) ß -particle (electron)? (c) /T-particle (positron)? (d) gamma ray? (e) X-ray after electron capture? (0 electron after internal conversion? (g) neutron?

(2) With the aid of a nuclear chart, find the decay products of 14C, 22Na, 40K, 90Sr and 238 U. (3) Calculate the weight of 100 fJtCi (3.7 MBq) of carrier-free 14C and 100 ßCi (3.7 MBq) of carrier-free 22Na (Tt_ of ,4C and 22Na are 5730 and 2.62 years, respectively). (4) If a solution has a concentration of 100 juCi (3.7 MBq) of carrier-free 14C per ml, calculate its molarity (Ti. of 14C = 5730 years). (5) A sample of 60Co has an activity of 1 Ci (37 GBq); calculate its activity 2 years later (Tj_ of 60Co = 5.3 years). (6) A radionuclide has lost 15/16 of its original activity in 32 min; calculate the half-life of the nuclide. (7) 137Ba is formed from 137Cs. How many curies and becquerels of 137Bam will be formed from 100 mCi (3.7 GBq) of 137Cs in exactly 1, 2 and 20min? (Ti. of 137Bam = 2.55 min). '(baoösrOiouig'ööP1"* (bao ss'Od^ 6'\P '(haw I88) id1" 8'ei ^suy (8) Determine the daily decrement, in percentage of the activity, of any 32 P preparation (Ti. of 32P = 14.3 d). 2 (9) A 24Na sample (Ti. = 14.8 h) had a count-rate of 400 counts/s. One hundred 7 hours later it had a net count-rate of 4.40 counts/s. Roughly estimate the dead-time of the GM counter. (10) The activity of 14C in 8 g of natural carbon sample including background was found to be 10.2 counts/min. The background of the counter was 4.5 counts/min and the counting yield was 5%. Neglecting the statistical deviation, calculate the 14C abundance in atom per cent (Ti. = 5700 years). (11) The background count-rate of a GM counter system is 0.5 counts/s. A sample is counted giving a total of 450 counts in 100 seconds. The background is counted for 30 seconds. Calculate the net count-rate of the sample and the natural standard deviation as a percentage of the net count-rate.

61 62 PART II. MENTAL EXERCISES

(12) A 0.1 mg sample of pure 239Pu underwent a decay of 2.3 X 106 Bq (disintegrations per second). Calculate the half-life of this radioisotope. (13) Calculate the thickness of lead shielding necessary to reduce the exposure dose in air to 2.5 mR h"1 (0.645 juC kg-1 -h"1) at 1 m from a 100 mCi (3.7 GBq) 60Co source. Hint: Use Eq. (1-31) and assume the half-thickness of lead for 60Co gamma rays to be 1.3 cm. •UI3 ÇT, :j3MSuy (14) Indicate the increase or decrease in the number of neutrons (N) and protons (Z) and in the mass number (A) after the following nuclear reactions:

(n,p) (n,7) (n,a)

(15) Calculate the energy absorbed by a 70 kg man who has received a whole- body dose of 700 rad (7 Gy), an amount almost certain to be fatal. Assume the body to have the specific heat of water (1.0 cal g"' degC_1 or 4.18 kJ-kg"1 K"1) and calculate the resulting temperature rise (1 joule = 0.239 calories) using both the special and Sl-derived units. 'Do e-OI X LI 'S3[nofo6fr ^suy

WORKING NOTES TO PART II. WORKING NOTES TO PART II

NOTES (cont.) 64 PART II. MENTAL EXERCISES

NOTES (cont.) PART III. INTRODUCTION TO PRACTICAL WORK

There can be little question that almost all laboratories engaged in animal research will soon utilize radionuclide techniques just as they now use calorimetry, spectrophotometry, chromatography, microscopy and other such procedures. This is due to the advantages of their use and is possible because of the availability of many radionuclides and because adequate instrumentation is rather inexpensive. In addition, experimental techniques have become highly developed and a number of research personnel has become highly proficient in this area. Often the use of radionuclides can provide experimental data that would otherwise be unobtainable. However, the design of such experiments must be on a sound scientific basis and include considerations on statistics and radiological safety. Before attempting such experimentation the researcher must be acquainted with the principles of radionuclide methodology and obtain practice in special laboratory techniques and operation of counting instruments.

III-l. PRINCIPLES

The unique advantage of radionuclides is that their behaviour in a system is usually biologically identical with that of their stable counterpart, but they can be identified easily by their characteristic radiation. The criteria of an ideal tracer are that it is indistinguishable from the tracee in biological behaviour and that its introduction does not disturb the system. Radionuclides uniquely fit these criteria, since their chemical behaviour is identical with the stable counterpart and a great number of them can be produced in high specific activity. Since radiation can be detected with very high sensitivity, generally negligible mass as well as activity need be administered to the system under study.

Ill—2. BASIC CONSIDERATIONS

There are several basic considerations inherent in tracer methodology which must always be borne in mind. At a first glance, the listing of these may appear formidable. However, in practice, with proper experimental design and knowledge of the limitations, reliable experimental data may be obtained.

65 66 PART III. INTRODUCTION TO PRACTICAL WORK

III—2.1. Chemical effects

Most radionuclides are produced by methods which ensure a minimum of interfering chemicals. However, the investigator should be sure that the radionuclide preparation administered to the animal does not contain pyrogens, high concentrations of salts, high acidity or toxic metals. The amount of carrier or tracee in a radionuclide preparation may be an important consideration. A classical example'of the effect of mass on metabolic behaviour is the effect of stable iodide on the uptake of radioiodine by the thyroid. If kinetic response is to be measured, the tracer dose must be of high specific activity.

Ill—2.2. Radiochemical purity

It is very important in all tracer studies that the radionuclide solution does not contain radionuclides other than the one under study. Such extraneous radioactivity may arise from impurities in the target material or from the chemical separation processes used. Gamma-ray spectrometry, particularly using high- resolution detectors such as the semiconductor detectors, is an excellent method to check the primary solution for radioactive impurities. However, the half-life of the determined beta adsorption must be considered for pure beta emitters. An important problem of chemical state arises when carrier-free preparations are used, since the chemical behaviour at the extremely low concentrations that exist in carrier-free preparations may differ from that at ordinary concentrations. For example, the addition of a precipitating agent may not cause precipitation at carrier-free concentrations. Also, minute amounts of substances lost with trace impurities,and adsorption on vessel walls or on suspended particles of dust and silica, may be a sizeable fraction of the total at low concentrations, whereas at the usual macroconcentrations these losses still occur but constitute a negligible fraction. In some solutions carrier-free tracers behave more like colloids than do solutes. Such particles of colloidal dimension containing radioactive atoms are called radiocolloids and these may behave rather differently in biological systems than do true solutes. Another difficulty may arise from the decay of radioactive atoms to secondary radioactive atoms usually termed 'daughters'. Such parent/daughter relationships must always be considered when the radiation being measured originates from both the parent and the daughter. This is particularly true of the naturally occurring radioactive materials which commonly have many radioactive daughters resulting from a long-half-life parent (for example, see the decay scheme of 226Ra). ni-2. BASIC CONSIDERATIONS 67

III—2.3. Isotope effects

In most biological tracer work it is generally assumed that all isotopes of an element behave in an identical fashion. With the heavy elements this is generally a satisfactory assumption and any differences are certainly within experimental error. However, for the light elements, particularly hydrogen and carbon, it has been shown that biological fractionation of isotopes occurs, which is termed the 'isotope effect'. There are many data documenting this effect in plants but relatively few concerning animals. Nevertheless, the investigator should be aware of such possibilities, especially in processes that are dependent upon mass, such as diffusion or reaction rates.

Ill—2.4. Radiation effects

It is essential that the experimental results should not be affected by any radiation received by the biological material and therefore the minimum amount of radioactivity should be used. This procedure also minimizes possible personnel radiation exposure, contamination of the laboratory and problems with waste disposal. It is very important that the investigator carefully calculates the proper activity of radionuclide needed for each experiment. This will be based on factors such as the biological half-life of the radionuclide and the counting yield of the detection instrument. A factor of importance concerning biological effects of radiation is that the radionuclide may be incorporated into a critical biological molecule such as DNA where the radiation dose effect may be increased. Also, there may be chemical bond breaks by the recoil atom after disintegration or effects of transmutation. For example, consider a biological molecule that contains 32P tracer. At the time of disintegration, the phosphorus atom in the molecule will be transformed into a sulphur atom and this may have an effect on the integrity or the function of the molecule. In practice this is rarely a limiting factor, since the sensitivity of the method is so high that very small amounts of radioactivity are necessary. The significance of radiation effects can be determined by replicate experiments using graded levels of radioactivity. Another problem involving radiation effects is that of radiation decomposition. It is known that many 14C-labelled organic compounds will undergo self- decomposition because of the radiation from 14C. This means that labelled organic compounds, especially those that are purchased commercially and may have been in storage for some time, should always be examined chromatographically before use to make sure that there are no appreciable amounts of decomposition products present. Radiation decomposition can be reduced by several methods such as dissolution in a protecting solvent, adsorption on a solid matrix in a thin layer, conversion to a stable derivative, and storage under refrigeration and in a vacuum. 68 PART III. INTRODUCTION TO PRACTICAL WORK

III—2.5. Exchange reactions

Isotopic exchange is a special case of exchange in which atoms in a given element interchange between two or more chemical forms of the element. In most metabolic studies, radiotracers are used to provide information on the processes involved in the biological synthesis of important metabolic compounds. If the radionuclide becomes incorporated in the metabolic product merely by exchange, which requires no energy production, then these experiments are of no value in that there is no evidence for synthesis of the product. Thus it is necessary to carry out studies that can show whether or not exchange has occurred. This can be done by allowing the precursors and the product to react under conditions where the biological or energy-producing system is interfered with. For example, animal tissues are known to convert inorganic phosphate into phospholipid. This is probably not an exchange process because experimental observations showed that (a) inorganic phosphate does not exchange with the phosphate radical or phospholipid when sodium phosphate is shaken with a phospholipid solution, (b) homogenized liver failed to form radioactive phospholipid from inorganic 32P, and (c) respiratory inhibitors such as cyanide or carbon monoxide interfered with the formation of the tagged phospholipid. It is also important to ensure that there is no loss by exchange of the label from the molecule under study. For example, it would be of no avail to label the carboxyl hydrogen of an organic acid with tritium because, as soon as this labelled acid would be placed in the biological system, the tritium would readily exchange with the hydrogen of the body water. On the other hand, a tritium label in an aldehyde group would be stable until the aldehyde is oxidized.

Ill—2.6. Radionuclide preparation and delivery of dose

The investigator should have as much information as possible on the characteristics of the radionuclide preparation, particularly with regard to chemical or radiochemical impurities that may be present. It is necessary that the solution be suitable for administration to the animal regarding pH level and freedom from particulate material. The investigator should confirm the identity of the radionuclide and the radiochemical purity by determination of the half-life or energy spectrum. If dealing with a labelled organic material, it may be advisable to make a chromatographic evaluation to be sure that there are no decomposition products present. In most studies it is necessary to administer the radionuclide to the animal quantitatively. If necessary, extensive practical work should be undertaken with stable substances so that the investigator will be proficient before actually handling radioactive material. Standard methods can be used for oral, intravenous, sub- cutaneous, intramuscular and intraperitoneal injections. Inhalation is more m-2. BASIC CONSIDERATIONS 69 difficult and requires specialized procedures. It is usually necessary to prepare a standard solution. This should be done from the original preparation in such a way that the amount administered to the animal can be exactly correlated with the amount used to make up the standard solution. For example, this might involve using the same syringe or pipette for both processes.

Ill—2.7. Procedures with animals

It is always important that normal, healthy subjects are used and are so maintained during the study. Whenever possible, the animal should be precon- ditioned to the type of handling and management that it is to receive during the experimental period. Of course, the scientist has a moral obligation to practice the highest degree of humaneness with respect to experimental animals. Such endeavours automatically improve the reliability of the results obtained. After administration of radionuclides, the animal must be considered as a source of external radiation and of radioactive materials excreted and expired that can contaminate the surroundings. Provisions should be made for appropriate collection and disposition of radioactive excretions.'

Ill—2.8. Handling of samples

The proper collection of samples is just as important as the reliability of subsequent analysis. It is important that the sample should be truly typical of the material that it represents. In studies where the entire sample is not used, it is essential that proper mixing be done and that appropriate aliquots be taken. For example, care must be taken to mix faeces well before sampling because successive increments may differ widely in activity, especially shortly after administration of the radionuclide. With radionuclides, the conventional grinding of dry material causes considerable difficulties because of the dust produced. Therefore, grinding is not recommended unless precautions are taken against hazards. In radionuclide studies cross-contamination must be particularly guarded against because of the extremely high dilutions that are often measured. Any radioactivity found at the site of administration may well have arrived there mechanically rather than metabolically. For example, after intraperitoneal injection, radioactivity found in the liver or other abdominal organs may have been deposited there mechanically. If the radionuclide has been given orally and the animal killed shortly thereafter, it is important that tissue samples be not allowed to come in contact with intestinal contents.

1 See: GAY, W.I. (Ed.), Methods of Animal Experimentation, Academic Press (1965). 70 PART III. INTRODUCTION TO PRACTICAL WORK

When fresh weights are necessary for concentration calculations on this basis, the sample should be weighed before any appreciable moisture loss has occurred. With small samples such as adrenals, thyroids or pituitaries of laboratory animals, it becomes almost impossible to get accurate fresh weights and it is usually better to express the results on a dry-weight basis. Depending on the experiments, the samples should be dried as soon as possible after collection to minimize chemical and biological changes. The materials should be dried in a well-ventilated oven at 60—70°C, and if dry weights are required, the samples can be finished at 110°C. Depending upon the characteristics and amount of the radionuclide present, fluid samples such as blood, plasma, urine, bile and milk may be assayed directly by liquid counting. With gamma-ray emitters it is possible to count solid samples directly. Where simultaneous chemical analyses or chemical separations are required it is frequently necessary to oxidize completely the organic matter of the sample. Depending upon the degree of completeness of ashing necessary, the following methods can be used:

(a) Pseudo wet-ashing, which consists of dissolving small samples of tissue in concentrated nitric acid and making up to volume for liquid counting. (b) Conventional wet-ashing using Kjeldahl procedures or others that utilize oxidizing agents and various catalysts. (c) Conventional dry-ashing in a muffle furnace. With this procedure it is important to avoid losses due to volatilization, incorporation of the radionuclide into solid carbon particles, or adsorption onto the walls of the crucible. (d) Special oxidation procedures for 14C or 3H.

The form of the sample required for counting will be determined primarily by the energy of radiation emitted. In general, the samples must be geometrically and physically uniform, these requirements being more rigorous for the lower- energy beta emitters. The following procedures have been used for the preparation of solid samples from radioisotopes in solution or suspension:

(a) Direct evaporation. Most biological samples will, however, require a preliminary separation of unwanted soluble matter because of crystallization and creeping which results in non-uniform deposits. Direct evaporation is widely used with solutions such as calibration standards that have very little soluble material. (b) Many filtration devices have been developed for the purpose of quantitative collection of precipitates for radioassay. Techniques involving the use of filter paper suffer from the general disadvantages of variable texture, difficulty in reproducing constant weights, tendency of the paper and precipitate to buckle, and necessity for careful handling of the final sample to avoid damaging the precipitate. III-3. REPRODUCTIVE ENDOCRINOLOGY 71

(c) Settling and centrifuging methods have been used to overcome many of the difficulties encountered in the filtration technique. Several simple devices can be used for this purpose. (d) Electro-plating is an excellent method for the preparation of uniform thin films of many metals. Many types of electrolysis cells are available commercially for this purpose. (e) Dry powders may be pressed into shallow dishes with a spatula or may be formed into brickettes with a laboratory press and piston-cylinder apparatus.

The exercises of Part V describe specific procedures. These are not necessarily the only methods or the best ones, but they have been found adequate under given conditions. It is hoped that the user of this Manual will hereby have in his hands simple techniques to provide reliable data, and that he will be in a position to make such modifications as will suit any specialized needs.

Ill—3. REPRODUCTIVE ENDOCRINOLOGY IN MAMMALS

This presentation is relatively brief in its coverage of endocrinological events during the reproductive cycle. Readers interested in a more complete presentation of hormonal events involved in reproduction are referred to texts dealing specifically which the subject [1—4],

III—3.1. Hormones

By definition hormones are chemical substances synthesized and secreted by ductless endocrine glands directly into the bloodstream, by which they are carried to other parts of the body, where they regulate (decrease or increase) the rates of specific biochemical processes. The endocrine glands comprise the pituitary (the adenohypophysis and neurohypophysis), thyroid, parathyroid, adrenal cortex and medulla, islets of Langerhans of the pancreas, ovary, testis, placenta and the pineal gland. Hormones may be divided into three different groups of chemical structures: (i) peptide and protein hormones, e.g. follicle stimulating hormone (FSH), luteinizing hormone (LH) based on amino acids; (ii) steroid hormones, e.g. testosterone, progesterone having the common basic nucleus of perhydrocy- clopentanophenanthrene; (iii) unique fatty acids with hormone-like properties, e.g. prostaglandins. It is generally considered that after their synthesis the protein hormones are stored within the gland and released upon call. Steroids and prostaglandins, however, are not stored; they are released as produced. 72 PART III. INTRODUCTION TO PRACTICAL WORK

Steroid hormones are insoluble in water, and on release they are subsequently bound to binding proteins in the blood which are specific carrier proteins for the steroid hormones. Two such specific binding proteins are corticosteroid binding globulin (CBG) or transcortin and sex hormone binding globulin (SHBG). Steroid hormones are also bound to albumin, this binding being relatively weak (Kd 10~SM) and non-specific. Binding to CBG and SHBG is of much higher affinity (10"8-10'9M) and demonstrates steroid specificity. CBG binds both Cortisol and progesterone, whereas SHBG binds both androgens and oestrogens. 5-dihydrotestosterone is most avidly bound by SHBG (10"9M). The binding proteins assist in the circulation of the steroid hormones and also restrict diffusion through tissues as well as protecting against degradation and rapid elimination. A consequence of this binding is that the free fraction of a steroid hormone in peripheral blood is usually only a few per cent. Whether it is this small fraction of the hormone which is biologically active is not clear, although it is likely that protein-bound steroids are less available to cells. Support for such an assumption has been derived from studies of the metabolic clearance rate (MCR) of various steroids. The MCR, defined as the volume of blood that is irreversibly cleared of a substance per unit time, has been found to be inversely proportional to sex steroid binding affinity to SHBG. The free steroid hormone level is usually estimated by measuring the percentage of steroid that is free after the addition of a radioactive tracer hormone by using ultrafiltration or equilibrium dialysis and measuring the amount of material bound. Such a determination will reflect the minimum fraction of hormone in serum that is available to target cells. The albumin-bound steroids, which may account for 30—50% of the total, represent an intermediate between the free portion and the portion bound to specific binding protein. It is likely that the albumin-bound portion is more readily available to the cells in the target organ than is the portion bound to specific binding protein. The true physiological function of specific steroid-binding proteins remains to be elucidated. There is evidence suggesting that their function may, in fact, be to promote entry of the steroid into the target cell, e.g. CBG, which binds progesterone, may enter target cells in the uterus but not in the brain. Binding proteins are probably important to keep steroid hormones in the circulation and to avoid their 'leakage' to lipid-rich adipose or other non-target cells. Target tissues for hormones are characterized by having a specific mechanism responding to the action of the hormone. Thus, not all tissues in the body respond equally to hormone stimulation. Hormones accumulate in target tissue because they bind with high affinity to specific intracellular proteins called receptors. As pointed out above, it is not totally established whether the free steroid or the steroid-protein complex enters the target tissue. Steroid hormones are believed to enter the target cell by diffusion, while protein hormones are actively transported into the cell after binding to their receptors present on the target cell membrane. III-3. REPRODUCTIVE ENDOCRINOLOGY 73

For demonstration of specific tissue receptors for a hormone, the following criteria must be fulfilled:

(i) Saturation. The biological response upon exposure of a tissue to a hormone is a saturation phenomenon. If a hormone-receptor interaction is a prerequisite for biological response, the number of receptors must be limited and thus saturable. (ii) High affinity. The receptor must have a high affinity for the hormone. This is because the concentration of hormone in blood is usually very low (1CT10—10"8M). If a tissue is to produce a biological response upon hormone stimulation, the receptor must have an affinity for the hormone which parallels its concentration in blood. This does not exclude hormone-receptor interactions of weaker affinity provided the hormone concentration in blood is high. (iii) Tissue specificity. The receptor should have a high specificity for one hormone or a group of hormones (e.g. androgens). This specificity enables the target cell to have a specific response upon hormone stimulation without interference from other hormone 'signals'. (iv) Correlation with biological response. The number of oestrogen receptors that are bound to the cell nucleus has been shown to be proportional to uterine growth; at the same time, when specific receptors to a hormone are missing in a tissue, there is no hormone-induced effect. In pigs a correlation has been found between the number of free androgen receptors and ham growth. It is often difficult to correlate differences in blood levels of hormones and differences in biological response. Most hormone-secreting glands show diurnal or episodic variations in the release of hormones and consequently a single blood sample will seldom give a good estimate of the true secretion. Different sampling protocols are required which are dependent on the release pattern of the hormone studied. In the case of testosterone in bulls it has been demonstrated that a 3-fold increase in the repeatability estimates occurs after stimulation with gonadotrophin release hormone (GnRH). If the hormonal pattern shows a diurnal variation, it is advisable to obtain the samples at standardized times. When hormone concentrations show a pronounced variation between sampling occasions it is important to consider this in both the manner in which samples are taken, and the statistical treatment of the data.

ÜI-3.2. Puberty

The onset of puberty in animals in which reproductive activity is related to seasonally changing factors), such as photoperiodicity, is usually influenced more by the time between birth and onset of breeding season than by age. Among the domestic species the horse, sheep and goat are seasonal breeders. In sheep there is considerable variation in age within breeds, and between breeds the onset of puberty can range between 6 and 17.5 months of age. The onset of puberty in 74 PART III. INTRODUCTION TO PRACTICAL WORK the mare usually occurs during the breeding season following birth at an age of between 12 and 17 months. In cattle the onset of puberty varies among breeds and occurs usually between 10 and 15 months of age. However, cyclic ovarian activity is usually present before the first oestrous period occurs. In gilts the first oestrus usually occurs at 4-9 months of age. Once breeding activity commences, it often continues for the life of the animal provided that the animal remains healthy. The counterpart of primate menopause is not observed per se in domestic animals.

Ill—3.3. Oestrous cycle

The major endocrine events that precede ovulation have been well documented in most domestic species. Some follicle growth occurs during the luteal phase in spite of the inhibitory nature of progesterone, the major hormone secreted from the corpus luteum. While follicles do not ovulate during the luteal phase in most species, this can occur with the mare. With regression of the corpus luteum (or corpora lutea) follicles grow rapidly due to gonadotrophin stimulation prior to ovulation. During development the follicles secrete increasing amounts of oestrogen, which is important for initiating the onset of sexual receptivity, as well as for the initiation of the surge release of gonadotrophins that is essential for the ovulatory process. In most species the preovulatory surge of gonadotrophins begins approximately 24 hours before ovulation and is usually of short duration, for example, 8-10 hours in the cow and sheep. The mare is an exception in that large amounts of LH are released during an 8 to 9-day period with ovulation occurring on the third day. Following ovulation, a corpus luteum is formed under the influence of pituitary gonadotrophins. In most species both LH and prolactin are considered to be luteotrophins, although the role of prolactin is less certain. If pregnancy does not ensue, the corpus luteum again regresses, permitting follicular growth, and oestrogen secretion from the growing follicle initiates the surge release of gonadotrophins, resulting in ovulation. This well-timed sequence occurs repetitively at set intervals if not interrupted by pregnancy. But it is as well to remember that in nature an animal comes into oestrus, is mated, ovulates and becomes pregnant, hence repeated oestrous cycles are abnormal. Seasonal breeders such as the mare, ewe and doe undergo cyclic ovarian activity only during the breeding season, while the cow and sow are affected little, if any, by photoperiod and can have cyclic ovarian activity the entire year. Oestrous cycle length is approximately 3 weeks in the cow, mare and sow and 2.5 weeks in the ewe and doe. m-3.4. Control of the corpus luteum

The regression of the corpus luteum (luteolysis) is the key event responsible for the well-timed oestrous cyclicity seen in most domestic species. The importance III-3. REPRODUCTIVE ENDOCRINOLOGY 75 of the uterus in the control of corpus luteum lifespan has been documented through hysterectomy in the cow, ewe, sow and mare. Removal of the uterus from these species during the luteal phase results in a pronounced prolongation of the luteal activity. It is now well established that the uterus in these species synthesizes and releases prostaglandin F2 (PGF2alpha)> which causes the corpus luteum to regress. The temporal release patterns of PGF2alpha> usually in spurts lasting a few hours, have been described in most domestic species. Some of the problems involved in determining PGF2aipha (short half-life, formation by platelets at collection) can be avoided if the main blood plasma metabolite 15-keto-13, 14-dihydro-PGF2aipha is determined. Regression of corpora lutea is usually accomplished within 48 hours following the onset of the prostaglandin release. Factors that control the initiation of PGF2aipha synthesis and release are not yet completely understood. ffl-3.5. Early pregnancy

Suppression of prostaglandin release is essential for the establishment of pregnancy in those species (cow, ewe, mare, doe and sow) having this compound as the luteolysin. The rapid elongation of foetal membranes, which precedes the critical time of the initiation of luteal regression by about 3 days in the non-pregnant animal, appears to be important for the prevention of prostaglandin release. In the sow it has been suggested that oestrogens synthesized by the early preimplantation embryo may be the messenger for maternal recognition of pregnancy. It has been suggested that the establishment of pregnancy involves redirection of PGF2aipha secretion from the blood vascular system (endocrine) to the uterine lumen (exocrine). Significant increases in intraluminal PGF2aipha have been observed in pigs and cattle (but not in horses) beginning about two weeks post-conception. Possible uterine antiluteolytic factors include PGEj and PGE2, particularly as increased production of PGE2 by endometrial tissue has been demonstrated in ewes during early pregnancy. The net result is that luteal function is maintained in the cow, ewe, mare and sow during early pregnancy.

Ill—3.6. Pregnancy and parturition

In the cow the presence of a corpus luteum is necessary for the maintenance of pregnancy in the vast majority of animals. In the ewe the presence of corpora lutea is required for the first 50—60 days of gestation. After this time period the foetoplacental unit secretes significant amounts of progesterone. It has been suggested that this progesterone synthesis is caused by a luteotrophin of placental origin called ovine placental lactogen. The pig and doe, like the cow, require luteal support throughout gestation. 76 PART III. INTRODUCTION TO PRACTICAL WORK

The necessity of the secondary corpora lutea formed in the mare between days 40 and 60 of gestation has been discussed over the years. The secondary corpora lutea may be a result of pregnant mare serum gonadotrophin (PMSG) secretion by the endometrial cups which are formed from a circular band of cells of placental origin (chorionic girdle cells) that invade the endometrium and form isolated endocrine organs of temporary function. The secondary corpora lutea are formed either through the luteinization or rupture (ovulation) of follicles. Recently, it has been demonstrated that PMSG has a close immunological relationship with equine LH; further, a luteotrophic effect of PMSG has been shown by incubation studies with corpora lutea. The clinical importance of this resides in the fact that if foetal loss occurs after the formation of endometrial cups, the continuing PMSG production will support luteal activity, even to the point of making lysis of these corpora lutea difficult in conjunction with pharmacological administration of PGF2aipha- Progesterone support of pregnancy in the mare begins to be taken over by the placenta as early as Day 50 of gestation but is not complete for all mares until approximately 140 days, a time that coincides with regression of both the primary and secondary corpora lutea. One of the first important endocrine changes that occurs prior to parturition in the cow, ewe, doe and sow involves an increase in the synthesis and release of oestrogens by the foetoplacental unit. This increased oestrogen synthesis is reflected in elevated plasma oestrone concentrations in the pregnant cow beginning between 20 and 30 days pre-partum. In the ewe the increase in oestrogen occurs abruptly about 2 days before parturition. The pig shows two distinct peaks of oestrogen during gestation, one at around 25—30 days and the other beginning 1 week before delivery. A different oestrogen pattern is observed in the mare in that significant oestrogen production begins early in gestation (around 90 days), with high values obtained from Day 150 to parturition. The mare produces two oestrogens during pregnancy that are unique to equids, equilin and equilenin, both of which have unsaturated B rings. In the cow parturition is preceded by an abrupt fall in the peripheral blood plasma concentrations of progesterone between 48 and 24 hours prior to delivery. In the ewe, mare and sow partial withdrawal of progesterone occurs prior to delivery. In the cow, ewe and doe it has been demonstrated that the release of prostaglandin initiates the regression of the corpus luteum and thus is responsible for the decline in progesterone. High oestrogen and prostalgandin combined with low progesterone concentrations increase the contractile state of the uterus. Prostaglandins may also initiate cellular changes within the cervix which result in softening and dilation. Cervical stimulation, a result of the initial entry of the foetus into the pelvic canal, results in the reflex release of oxytocin from the posterior pituitary and possibly the ovaries. This increases the intensity of the uterine contractions and thus aids the final steps of the delivery process. III-3. REPRODUCTIVE ENDOCRINOLOGY 77

IH—3.7. Applications of radioimmunoassay in assessing reproductive efficiency

The development of radioimmunoassay techniques for hormone determinations in domestic species has created laboratory procedures that are relatively simple to perform, inexpensive, specific and sensitive and that have potential usefulness as diagnostic aids in reproductive studies. For some reproductive hormones relatively well-defined indications for their use are known at present and more are likely to be identified in the future. For hormone analyses to be useful in assessing reproductive efficiency certain criteria have to be fulfilled. The concentration of the hormone at the site of sampling (usually a peripheral vein) should provide an indication of the release of the hormone from the endocrine gland. Many hormones are released in a pulsatile manner and consequently single blood samples will convey little information of value. To use single samples for diagnosis, it is necessary to examine only those hormones that are released at a steady level to obtain valid information on the secretory status of the endocrine gland. Several reproductive hormones do not fulfil these criteria, and thus their determination is not as useful from a routine diagnostic view. For example, the short duration of the LH peak observed in conjunction with ovulation in most domestic species except the horse may be cited. In the cow the preovulatory LH peak has a duration of 8-10 hours, requiring samples to be obtained every 30 min in order to detect the peak. In the mare the duration of the peak is considerably longer, 8—9 days, which allows a less frequent sampling interval. However, the long duration of LH peak in the mare prevents the determination from being useful in predicting ovulation. Furthermore, LH is secreted in a pulsatile manner during the oestrous cycle in most species, with the amplitude and frequency of the pulses being indicative of the state of the oestrous cycle. Recent studies have suggested that the frequency of sampling requires! to be in the order of 10 minutes to ensure detection of all the pulses. In the male the pulsatile behaviour of LH has been found to be followed by a pulse of testosterone in rams and bulls. Seasonal breeding in sheep may result in different profiles at different times of the year. Levels of LH remain at about 1 ng/ml, with pulsatile spikes occurring at levels of up to 9 ng/ml, the number of spikes increasing in the breeding season. These spikes are followed by increased blood concentrations of testosterone from basal levels of 1 ng/ml to over 20 ng/ml. Rams maintained in the long day length typical of the non- breeding season in areas at high latitudes and treated with GnRH so that spikes of LH are obtained similar in amplitude and frequency to those occurring during the breeding season show a corresponding pattern of testosterone secretion. Further, if the treatment is maintained for several weeks, a characteristic rise in the tonic level of plasma FSH occurs as well as typical breeding season behaviour. Studies of these 3 hormones in the male in areas where well-marked breeding seasons not affected by day length occur could well supply valuable information. 78 PART III. INTRODUCTION TO PRACTICAL WORK

Another hormone much affected by photoperiod in both the male and female of many species is prolactin. In sheep plasma levels rise with day length and reach peak levels at about 14 hours day length and remain high until close to the onset of the next breeding season. It was suggested earlier that elevation of PRL may cause suppression of the oestrous cycle and thus bring about the anoestrous period but recent studies on sheep with widely differing breeding seasons indicate that PRL levels rise and fall at the same time in the different breeds. There therefore seems to be no obvious relationship between PRL levels and breeding season. The influence that PRL has on reproduction in sheep and cattle still has to be established, but measurement of plasma levels in areas with small photoperiod changes may provide useful information. One reproductive hormone, progesterone, has been found to be of significant clinical value in most domestic species. Other hormones with established or potential clinical use will be discussed further for each species. In most cases the determination of hormones has been utilized for the detection of pregnancy or sexual activity.

Ill—3.8. Hormones during pregnancy in cattle

III—3.8.1. Progesterone

The cyclic behaviour of progesterone levels during the oestrous cycle and the relatively high levels during pregnancy has resulted in this hormone being widely used as a pregnancy test. As well as plasma progesterone, progesterone in whole milk, milk fat and fat-free milk has been examined and a high correlation found between the values [5]. The basis of the test rests on the difference that exists in both blood plasma and milk progesterone concentrations 19—24 days after conception as compared with a non-fertile insemination. The plasma progesterone concentration in pregnant cows at 21 days after insemination is almost always greater than 2 ng/ml (6.4 nmol/litre) and usually 6—8 ng/ml (19.1—25.5 nmol/litre), as compared with 0.5 ng/ml (1.6 nmol/litre) or less in the non-pregnant animal at the same time. The finding of elevated progesterone concentrations, however, is not necessarily indicative of the presence of a foetus in utero. If a cow has a prolonged oestrous cycle after breeding (e.g. a 27-day cycle instead of a 21-day cycle), this animal could have a corpus luteum on Day 21, and thus the progesterone level would be high and the animal would be falsely considered pregnant. Consequently, the accuracy of the forecast for pregnancy (positive forecast) can be relatively low, and in most cases will range between 75 and 90%. The negative forecast, however, will be more accurate since cows having low progesterone levels in milk or blood 21 days post-breeding will not be pregnant. The accuracy of the forecast will be dependent to some extent on the intermediate values used for III-3. REPRODUCTIVE ENDOCRINOLOGY 79 separating progesterone concentrations in pregnant cows from those in non- pregnant animals. The accuracy of the positive forecast can be increased if progesterone analyses are also carried out on samples obtained at the time of insemination. Cows inseminated during the luteal phase will be in the luteal phase 21 days later, and if a sample is assayed at this time, the animal will be considered pregnant even though it is non-pregnant. The examination of cows at 40 days after insemination to confirm the presence of a foetus in animals previously designated pregnant will minimize the problem of false positive diagnoses. Sequential progesterone determinations in cattle assist in establishing the secretory status of the corpus luteum. Thus, if conducted at approximately 5-day intervals during an oestrous cycle, the ovarian cyclicity can be established in cases in which oestrus is poorly expressed or missed. Progesterone determinations have also been used for confirming the absence of an active progesterone- secreting corpus luteum at the time of insemination in cattle. If high progesterone levels are recorded at this time, this indicates that the insemination has been performed in the presence of a corpus luteum, i.e. at the wrong time. Since inadequate detection of oestrus is the most common cause of low fertility in herds utilizing artificial insemination, the determination of progesterone levels at the time of insemination is a useful tool when herds with fertility problems are encountered. The use of milk progesterone in these cases allows the farmer to take the sample. In one study improper timing of insemination, as judged from milk progesterone determinations, was performed in 15% of the cases in a controlled field test, while under practical field conditions the figure rose to 26%. The use of progesterone determinations in milk obtained at the time of insemination in cows with questionable heat signs and inconclusive genital tract findings can serve as a valuable tool of educating the staff responsible for this work. Another area in which progesterone determinations are useful is the confirmation of a clinical diagnosis, e.g. anoestrus, failure to manifest oestrus, cystic ovaries, pyometra. They are also useful for following the effect of treatment. For example, ovarian cysts in cattle usually require treatment with human chorionic gonadotropin (HCG) or Gn-RH; however, when the cysts are partly luteinized, a better effect can be obtained using PGF2alpha- Diagnosis of the type of cyst present by rectal examination sometimes produces inconclusive results, but the determination of progesterone in such cows will aid in the establishment of a correct clinical diagnosis. Progesterone analysis is also useful for assessing the effect of a particular treatment. If milk progesterone analyses are used, the farmer can collect samples following the treatment and the clinical response of the animal can be evaluated from subsequent progesterone analysis in milk samples. In cases of follicular cysts the original progesterone level should be low and, following successful treatment (HCG or Gn-RH), a progressive luteinization of the cysts should occur, resulting in increased progesterone 80 PART III. INTRODUCTION TO PRACTICAL WORK concentrations in subsequent milk samples. In the case of a luteinized cyst the original progesterone value will be higher than for the follicular cyst and after treatment (prostaglandin) the progesterone level should decrease, reflecting the lysis of the luteal cyst. The same principle can also be applied in cases of pyometra and mummification of the foetus wherein a persistent luteal activity is a common finding. Progesterone determinations in blood have been used to assess the success of superovulation in cattle in conjunction with ova transfer. In some, but not all studies a positive correlation has been found between .progesterone concentrations and the number of corpora lutea formed following superovulation. \

III—3.8.2. Other hormones

Prolonged gestation in both Holstein-Fresian and Guernsey cows has been described in animals with foetuses having malfunctioning pituitaries and adrenal glands. It has been shown that cows carrying such foetuses do not exhibit increased peripheral blood plasma concentrations of oestrone around the expected due day. The determination of blood plasma concentrations of oestrone could aid in the diagnosis of the condition. There is substantial evidence that oestrone sulphate is synthesized by the normal conceptus, and milk oestrone sulphate determinations by radioimmunoassay at 15 weeks after mating have recently been shown to provide useful added evidence for the diagnosis of pregnancy. Placental lactogen, although present in foetal fluids, cannot be detected in the maternal blood of cattle and hence cannot be used as an indicator of pregnancy.

HI—3.9. Hormones during pregnancy in sheep

III—3.9.1. Progesterone

The same basic principle of using progesterone determination as an early test for pregnancy has also been used in sheep. In the ewe progesterone determinations have to be carried out on blood samples since most breeds of sheep are not lactating at the time of breeding. Maximal luteal phase progesterone levels in the ewe are approximately 2-4 ng/ml (6—13 nmol/litre), while the concentration at oestrus is from 0.15 to 0.8 ng/ml (0.5-2.5 nmol/litre). A marked increase in the progesterone concentration from 2-4 ng/ml (6—13 nmol/litre) to 12-20 ng/ml (38-63 nmol/litre) also occurs between days 60 and 125 of pregnancy. This rise is due to an increased progesterone production from the placenta/conceptus. The contribution of the corpus luteum to the progesterone level at this time is relatively low. III-3. REPRODUCTIVE ENDOCRINOLOGY 81

The accuracy of the forecast and the pitfalls associated with using progesterone determinations as a pregnancy test in the ewe are similar to those previously outlined for cattle. However, in the ewe the accuracy of the forecast will be increased if samples are obtained relatively late in the gestation period since the progesterone concentration at this stage is considerably higher than during the luteal phase, allowing differentiation between non-pregnant and pregnant ewes. Furthermore, because of the limited reproductive season in some breeds, ewes not bred at this time become anoestrous and, as a result, have low progesterone concentration. In a trial that determined plasma progesterone concentrations in 46 ewes on the 18th and 70th days after mating, correct pregnancy diagnoses were made for 80 and 94% of ewes, respectively. Since the elevated progesterone concentration seen in blood during the later part of pregnancy in the ewe is due to an increased production by the placenta/conceptus, several attempts have been made to use progesterone determinations to differentiate between single and twin pregnancies. However, the use of progesterone determinations to predict litter size has generally been poor. Thus workers in the Netherlands correctly predicted litter size in about 80% of cases in 34 Texel ewes using the plasma progesterone concentration at 70-110 days after mating, but when 194 ewes of different breeds were used, correct classifications were reduced to 65% of cases. In general, the larger the number of foetuses, the more accurate is the prediction. However, the effect of litter size on plasma progesterone concentration recorded on Day 100 of gestation also depends on the level of nutrition. When ewes were fed according to litter size, plasma progesterone appeared to be little affected by litter size, but low feed intake caused a marked increase in plasma progesterone concentrations with increase in litter size.

Ill—3.9.2. Other hormones

Ovine placental lactogen (oPL) has been demonstrated in the peripheral blood of pregnant ewes. The increase in maternal oPL concentrations parallels the rise in progesterone that occurs during days 60-125 of pregnancy and therefore the determination of oPL concentrations can be used as a basis for a specific pregnancy test in the ewe. In the ewe an abrupt increase of unconjugated oestrogens (oestrone and oestradiol-17beta) occurs about 2 days prior to parturition, and a conjugated oestrogen (i.e. oestrone sulphate) can be detected at around 100 days of gestation; this steroid is probably secreted from the conceptus or placenta. Whether determinations of oestrone sulphate can be useful as a pregnancy test in the ewe remains to be elucidated. 82 PART III. INTRODUCTION TO PRACTICAL WORK

III—3.10. Hormones during pregnancy in pigs

III—3.10.1. Progesterone

Luteal phase progesterone concentrations in the pig are considerably higher than in cattle and sheep, namely 20-50 ng/ml (65—160 nmol/litre), while concentrations at oestrus are similar to those observed in cattle and sheep, i.e. approximately 0.5 ng/ml (1.6 nmol/litre). The difference in progesterone concentration between non-pregnant and pregnant animals 19—24 days after service has been used as an early pregnancy test. In the pig progesterone determination has to be performed on blood. Due to the sensitivity of the assay systems, the analyses can be performed on a small volume of blood (about ten drops) allowing the sample to be obtained through a small incision in an ear vein. The limitations associated with using progesterone determinations as a pregnancy test in the pig are similar to those previously discussed for cattle. Progesterone analyses have also been used to determine ovarian activity in clinically anoestrous gilts as well as to establish the stage of the oestrous cycle in gilts. Correct dating of the oestrous cycle is possible in most cases, from two progesterone determinations 1 week apart.

Ill—3.10.2. O estrone sulphate

The blood concentration of oestrone sulphate rises markedly in pigs around Day 30 of pregnancy and it is possible that this reflects foetal synthesis. Determina- tion of oestrone sulphate in early pregnancy in the pig is thus a specific pregnancy test, the index of discrimination between oestrone sulphate concentrations of pregnant versus non-pregnant pigs around 30 days after breeding being very high. During the latter part of pregnancy the maternal blood concentrations of oestrone sulphate, oestrone and oestradiol-17beta are very high and can thus be used to confirm pregnancy at this stage of the gestation period.

Ill—3.11. Hormones during pregnancy in mares

III—3.11.1. Pregnant mare serum gonadotrophin (PMSG)

While pregnancy has been diagnosed in mares through the measurement of PMSG, there are several drawbacks to its use. First, PMSG cannot be detected until about Day 40 of gestation and thus is not useful for early pregnancy diagnosis. Secondly, the presence of PMSG does not guarantee the presence of a foetus. It has been shown that endometrial cups have some autonomy and can continue to secrete PMSG for a period of time in spite of loss of the foetus. III-3. REPRODUCTIVE ENDOCRINOLOGY 83

III—3.11.2. Progesterone

Plasma progesterone has been assayed to support the diagnosis of a persistent corpus luteum. The progesterone levels in mares with persistent luteal activity (2-4 ng/ml; 6-13 nmol/litre) suggest the clinical syndrome especially as luteal activity is often reduced by 14 days after ovulation due to PGF2aipha release, which is insufficient to cause complete luteolysis. Progesterone analysis can also be useful in mares that fail to manifest sexual receptivity, yet have cyclic ovarian activity. Progesterone analysis at 5-day intervals over 20 days can verify the presence or absence of cyclic ovarian activity and predict the time of ovulation within a 2 to 3-day interval, which can be helpful to the veterinary practitioner anticipating the next time of ovulation. Artificial insemination may have to be used in these circumstances.

Ill—3.12. General comments

Appreciable variability in assay results occurs between laboratories and it is important that clinical endocrinology laboratories know the results to expect from their assay systems in relation to particular clinical syndromes. For example, the actual concentration of plasma progesterone during the follicular phase of the oestrous cycle of a domestic animal species is approximately 100 pg/ml (318 pmol/litre). Some laboratories, however, report basal values of 1-2 ng/ml (3.2-6.4 nmol/litre) for progesterone. Workers often use 1 ng/ml (3.2 nmol/litre) as the lowest concentration of progesterone compatible with an actively secreting corpus luteum (especially for the cow, ewe, mare and sow) and organize their assays to achieve maximum precision at these values. Assays may prove to give differing levels of basal progesterone; however, the mark that divides active and inactive corpus luteum function must be precisely known. The reference values developed by laboratories are dependent upon the type of assay used as well as the specificity of the binding protein, purification steps, and purity of reagents. There is a general tendency for values obtained from competitive protein-binding assays to be somewhat higher than those obtained by radioimmunoassay. It is possible to have a wide range of values for a particular hormone and still have normal physiological conditions. For example, luteal-phase plasma progesterone concentrations in the cow can range between 2 and 12 ng/ml (6—40 nmol/litre) with no adverse effect. Hormone values can also depend upon the type of material used in the assay. In fat-free milk luteal-phase progesterone values are approximately 4 ng/ml (12 nmol/litre); in whole milk the corresponding value varies between 5 and 35 ng/ml (16-110 nmol/litre) and if milk fat is used the progesterone value is approximately 250 ng/ml (795 nmol/litre). Another factor influencing hormone values in blood is the time interval elapsed from bleeding to separation of serum or plasma. Storage of unseparated blood samples 84 PART III. INTRODUCTION TO PRACTICAL WORK from cattle can result in a significant lowering of the assayable progesterone concentration. Consequently, sample handling procedures from blood collection to the freezing of serum or plasma should be standardized for each species and hormone determined.

REFERENCES TO PART III

[1 ] AUSTIN, C.R., SHORT, R.V., Reproduction in Mammals. (3) Hormones in Reproduction, Cambridge University Press, Cambridge (1972). [2] COLE, H.H., CUPPS, P.T., Eds, Reproduction in Domestic Animals, 3rd Ed., Academic Press, New York (1972). [3] HUNTER, R.H.F., Physiology and Technology of Reproduction in Female Domestic Animals, Ist Ed., Academic Press, London (1980). [4] SCARAMUZZI, R.J., LINCOLN, D.W., WEIR, B.J., Eds, Reproductive endocrinology of domestic ruminants, J. Reprod. Fert. Suppl. 30 (1980). [5] HEAP, R.B., HOLDSWORTH, R.J., Modern diagnostic methods in practice. Hormone assays in reproduction and fertility, Br. Vet. J. 137 ( 1981) 561 -71. PART IV. GENERAL INTRODUCTION TO RADIOIMMUNOASSAY

IV-1. PRINCIPLES OF RADIOLIGAND ASSAYS

Radioligand assays allow one to monitor hormone concentrations in body fluids and tissues, e.g. during the various reproductive phases. These techniques consequently have become powerful tools in studying the endocrine control of reproductive processes in domesticated species of animals and in elucidating factors affecting their reproductive efficiency. The methods at present in use derive from those reported by Berson and Yalow [1] and Ekins [2], for which Yalow eventually received the Nobel prize in 1977. Before radioligand assays were available, the smallest amounts of hormones that could be effectively measured by biological assay or chemical methods ranged from milligram (mg, 10~3g) to microgram levels (/ug, 10"6g). As a result, hormones could not be quantified in small volumes of blood. However, with radioligand assays hormones in nanogram (ng, l(T9g), picogram (pg, lCT12g) and even femtogram (fg, 10"15g) levels can be measured in a millilitre or less of serum or plasma. A wide range of compounds can now be measured by radioligand assays, in addition to protein, steroid and other hormones. These include drugs, antibiotics, cyclic nucleotides, neurotransmitters, amino-acid derivatives, and vitamins. In many cases the presence of microbial or parasitic pathogens also can be detected in blood samples. More specifically, the radioligand assay can be divided into the following types:

(i) Competitive protein binding assay: where the binding protein is a naturally occurring protein, e.g. a serum protein. Steroid hormones are poorly soluble in water and they are transported in blood bound to specific carrier proteins. Two such proteins, corticosteroid binding globulin (CBG) and sex hormone binding globulin (SHBG), have been used for the assay of steroid hormones. These proteins do not bind steroids with a high degree of specificity, e.g. CBG binds progesterone, 17alpha-hydroxyprogesterone and corticosteroids, whereas SHBG binds oestrogens and androgens. Mainly because of their lack of specificity and sensitivity, competitive protein binding assays have in many cases been replaced by more specific radioimmunoassays. (ii) Radioreceptor assay: since several hormones, e.g. FSH, LH and prolaction (PRL), are proteins, they are transported in a 'free' state in blood, but when they reach their target organ they are bound to relatively specific cell membrane receptors. Such receptors have been used for the determination of protein hormones by applying the general

85 86 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

principle of radioligand assay. In some cases problems of specificity can arise and in general sensitivity is poor. Radioimmunoassays are generally used for the determination of protein hormones. (iii) Radioimmunoassay (RIA): largely because of specificity and sensitivity, RIA is the most widely used technique for the determination of hormone concentrations. These assays utilize antibodies specific for the hormone as the binding protein. Antibodies against many antigens are available commercially and others can be produced by immunizing rabbits, sheep, guinea-pigs or horses. Consequently, in the following presentation only RIA techniques are described — in particular those that are suitable for use in the study of reproductive functions in domestic animals. Radioligand assays are based on the ability of non-radiolabelled antigen Ag (e.g. hormone) in a specific volume of standard solution or in an unknown sample to compete with a fixed amount of a radiolabelled antigen Ag* for a limited number of binding sites on a specific binding antibody protein Ab. This reaction between antibody and antigen can be expressed as follows:

k Ag + Ab ^ AgAb unlabelled antigen-antibody complex ki and

k2 Ag* + Ab ^ Ag*Ab labelled antigen-antibody complex k2 where Ag and Ag* are the unlabelled and labelled antigen respectively; Ab is the antibody; klj2 are the association rates; and k'lj2 are the dissociation rates. Since the quantities of binding protein Ab and radiolabelled Ag* are held constant in a radioimmunoassay, inhibition of binding of radiolabelled antigen to the binding protein is related to the concentration of the antigen Ag in the standard solutions and samples (Fig. IV—1). This process may be viewed as a simple

Radiolabelled Sample or antigen standard 6®-v r-60 V

3®,30 Binding O protein J—* Unknowns Incubate s Separate bound Count & ® from free calculate ft 3

FIG.IV-l. Principles of radioligand assays. IV-1. RADIOLIGAND ASSAYS 87

competition in which unlabelled antigen, Ag, reduces the amount of free antibody, Ab, and thereby decreases the availability of the antibody to labelled antigen Ag*. Thus, as the concentration of antigen in standards or samples increases, the percentage of Ag* that binds to Ab decreases. The concentration of Ag in unknown samples can then be calculated from an inhibition curve generated by standard solutions. The sensitivity of the assay, i.e. the smallest mass of the substance that can be distinguished from zero, can be adjusted by varying the concentrations of the antiserum and the radiolabeled antigen. Decreasing these concentrations will increase the sensitivity. The antibody-antigen reaction is a reversible one, and obeys the Law of Mass Action which states that, at equilibrium, the ratio of the products of the concentration on the two sides of the equation will be constant. Therefore, at equilibrium we get in molar concentrations:

kf[AgAb]_ ki [Ag] [Ab]

and

K*>[Aë*Ab] (IV-2) kí [Ag*] [Ab]

where K and K* are the affinity or equilibrium constants, usually expressed in litres/mol.1 Non-radiolabelled antigen in unknown samples and in standard solutions competes with radiolabeled antigen for a limited number of binding sites on specific binding proteins, e.g. corticosteroid binding globulin (CBG), sex hormone binding globulin (SHBG), or antibody. After appropriate incubation, bound and unbound (free) radiolabeled antigen are separated. As the quantity of non-radiolabelled antigen in samples and standards increases, the quantity of radiolabeled antigen that binds to the protein decreases. The concentration of antigen in samples can then be determined from a standard curve. Assuming that the antigen is unchanged by labelling, K = K*. The theoretical basis for this can be explained as follows. If the bound/free ratio is represented by R, then

[Ag*Ab] R- * (IV—3) [Ag*]

1 Affinity refers to the properties of the antigen, whereas avidity refers to those of the antibody. Avidity is defined as the energy of the antibody for its antigen and is numerically equal to the energy of binding of the antigen for its antibody. 88 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

FIG.IV-2. Diagram of a Scatchard plot. The bound/free ratio is plotted against the total molar concentration of bound antigen, the slope is —K, which has dimensions in litre/mole. The intercept on the abscissa where the bound/free ratio is 0, gives the total number of binding sites in mole/litre.

and the total concentration of bound antigen is B where

B = [AgAb] + [Ag*Ab] (1V-4) and

q = [Ab] + [Ag*Ab] + [AgAb] (IV-5) where q is the total concentration of antibody. By combining Eqs (IV-1) - (IV-5), the affinity constant K may be expressed as:

q-B K = or R = (q-B)K"1 (IV-6) R

Hence by plotting the bound/free ratio R against the bound fraction B, it is possible to obtain an estimation of K. This curve (Fig. IV—2) is known as the Scatchard plot [3]. The slope of the curve is -K and shifts in the plot in terms of slope and position determine thé accuracy and precision of the assay. IV-2. RADIOIMMUNOASSAY 89

In the case of multivalent antibodies the Scatchard plot will not yield a straight line, but a quadratic curve representing the combination of lines from each specific antibody population. When this occurs, it can act as a warning that one of the assumptions made for the mathematical treatment, i.e. that both antigen and antibody are univalent, is no longer valid. A high K-value would imply that at equilibrium the concentration of the Ag - Ab complex will be much greater than that of free Ag and free Ab. A fuller treatment of the mathematical theory and statistical aspects of RIA can be found elsewhere [4, 5],

IV—2. RADIOIMMUNOASSAY

IV—2.1. Production of antibodies

Radioimmunoassay systems utilize, as the binding protein, antibodies to the hormone to be measured. Steroid hormones and prostaglandins have low molecular weights and are thus not immunogenic per se. However, these structures can be rendered immunogenic if covalently linked to large carrier molecules such as bovine serum albumin, and specific antibodies can be elicited in this fashion. In order for such a hormone-protein conjugate to be immunogenic, approximately 10—20 hormone molecules (e.g. steroid) should be present per molecule of protein. In the case of bovine serum albumin about 30% of the sites available for conjugation should be occupied. Most naturally occurring steroid hormones or prostaglandins contain hydroxyl or ketone groups, which are used to prepare derivatives containing active groups such as carboxyl or amino groups. These groups are then activated so that they react with amino or carboxyl groups of the protein molecule. The specificity of the antisera obtained by immunization with a steroid-protein conjugate is dependent on the site used for conjugating the steroid to the protein and the nature of the covalent bridge. More specific antisera are obtained if the hapten (steroid) is attached to the protein at a site remote from the characteristic functional groups of the hormone. During immunization the developing antibody titre is monitored, and bleedings are performed when a suitable titre has been achieved. Antisera seem to be quite stable when stored at —20°C, although the usual preferred temperature is —70°C. A few millilitres of a high-titre antiserum are usually sufficient for millions of radioimmunoassay determinations. The immunization procedures used for production of antibodies against progesterone in sheep are given in Exercise 17. Hormones such as LH and FSH, which are glycoproteins with molecular weights of around 30 000, are themselves antigenic because of their size and chemical composition. 90 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

At present there are only a few polypeptide reproductive hormones available with a purity suitable for both immunization and preparation of the radioactive tracer. If an assay for bovine LH utilizes an antibody to bovine LH, radiolabeled bovine LH as tracer, and bovine LH as the standard, the assay system is completely species specific and is said to be of the homologous type. Such a system represents the ideal radioimmunoassay system for measuring a polypeptide hormone. However, because of the limited availability and sometimes lack of sufficiently pure polypeptide hormone preparations, heterologous assay systems have been developed. In such cases an antiserum to a polypeptide hormone of one species is used for the determination of the same polypeptide hormone in another species, and only relative and not absolute values are obtained; these values may, nevertheless, provide meaningful biological information. It should be noted in this context that the majority of commercial ready- made kits for the assay of protein hormones are designed to be used in humans. Such kits cannot be applied for measurements in domestic animals unless the assay is validated for the species in question, and even if satisfactorily validated, such systems can be used to determine only relative changes of hormone concentrations rather than the absolute concentrations. There are polypeptide hormone antisera available which show a high degree of cross-reactivity. For example, there is an LH antiserum available raised against ovine LH which reacts specifically with LH from other species and has been used for the determination of LH in approximately 45 species including the cow, sheep, pig, goat and horse [6]. The immunization procedure used for production of rabbit anti-ovine LH serum is given in Exercise 21.1.

IV—2.2. Production of labelled antigens

Most radioimmunoassay systems for steroid hormones and prostaglandins utilize commercially available tritiated forms of these molecules. Because of the long half-life and soft beta radiations, such labelled preparations are very stable, can be stored with little radiation damage, and represent a minimal external radiation hazard to personnel. On the other hand,3H is counted with an efficiency of only 15% and hence high concentrations of labelled antigen are needed. Furthermore, counting of3 H-labelled compounds is expensive because of the need for scintillation fluids, and disposal of relatively large quantities of flammable and toxic radioactive waste is frequently a major problem. Such disadvantages have led recently to the development of steroid and prostaglandin assays that utilize readioiodinated tracers. With such tracers it is easily possible to design assay protocols so that one fraction (preferably the antibody- bound tracer) remains in the assay tube and may be counted directly, leading to greater convenience, precision and to decreased costs. Against this advantage must be set two factors: first, the short half-life of 1251, which necessitates a fresh labelled hormone every 2—3 months; and secondly, the decreased sensitivity often observed. The latter effect arises because of the necessity for a chemical bridging structure between the steroid and the carrier protein (often bovine serum albumin) IV-2. RADIOIMMUNOASSAY 91

H-0

GLUCURONIDE BRIDGE

125,I == 11a-progesterone glucuronide-tyramine

HEMISUCCINATE BRIDGE

BSA-NH

11a-progesterone-hemisuccinate-bovine serum albumin

FIG.IV-3. Examples of bridge structures attached at the i 7a|pha position of progesterone to give labelled antigen and an immunogen.

in the immunogen, and between the steroid and a substance (such as histamine or tyramine) capable of being iodinated in the tracer. Antibodies raised against such steroid-protein conjugates will recognize the bridge as well as the steroid and, if the same bridge is present in the tracer, it will usually bind to antibody with much greater affinity than the unlabelled steroid, resulting in a considerable loss in assay sensitivity. Successful 125I-based steroid assays have relied on the use of selected antisera that do not suffer excessively from bridge-binding; on particular bridge structures (notably 3- (O-carboxymethyl oxime) that are not well recognized by antisera; or on heterologous systems where different positions on the steroid structure are used for attachment of the bridge in 92 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY the immunogen and tracer, respectively. In many cases these systems result in rather shallow standard curves compared with assays using tritiated labelled steroids, with a consequent loss in precision. More recently methods of synthesizing bridges have been devised that retain the slope of the standard curve and it can be confidently predicted that as these are extended to cover the whole range of steroid assays, nsI-labelled tracers will replace 3H-labelled material. At present the preparation of 125I-labelled steroids is largely an 'in-house' procedure, often using materials prepared in the worker's own laboratory. However, this situation is changing rapidly as commercial suppliers exploit recent research and radioiodinated ligands for steroid assays are now becoming increasingly available, at least for the hormones of most physiological importance, e.g. Cortisol, progesterone and testosterone. In Part V several exercises are described that exploit this approach — indeed, both the 125I-labelled progesterone and testosterone employed in Exercises 18.1 and 18.2 are available from commercial suppliers. Figure IV-3 gives examples of bridge structures attached at the 11 alpha position of progesterone to give labelled antigen and an immunogen. In radioimmunoassay techniques for polypeptide hormones the homologous highly purified antigen (hormone) is most commonly used for preparing the radioactive tracer. Usually, radioactive iodine, 125I, is used for the iodination of the antigen. All radioiodination procedures involve the oxidation of iodide (I") to iodine (I2), which in turn reacts with tyrosine or histidine residues on proteins. The most widely used procedure for incorporating radioiodine into a protein involves the use of chloramine-T, a mild oxidizing agent [7]. The reaction is timed precisely and terminated by the addition of excess sodium metabisulphite, a reducing agent. The time of reaction and the amount of chloramine-T in the reaction mixture must be carefully controlled to minimize structural damage and to maintain the antigenicity of the protein. The chloramine-T procedure is technically demanding but simple and rapid to perform, but oxidation damage to the hormone may occur. A detailed description of the iodination of steroid and protein hormones using the chloramine-T procedure is given in Exercises 18.1, 18.2 and 21.2. Another common procedure for radioiodinating proteins is the use of lactoperoxidase, an oxidizing enzyme. In the presence of hydrogen peroxide

H202 the enzyme will iodinate proteins via tyrosyl residues. H202 may be added directly or be generated during iodination by the reaction of glucose oxidase with D-glucose. Generally, the quantity of H202 added can be used to control the degree of iodination. The reaction is stopped by adding 10 to 20 volumes of cold buffer. The major advantage of the lactoperoxidase procedure is that less oxidative damage to the protein occurs than with the chloramine-T procedure. However, too much H202 can cause excessive iodination and loss of antigenicity, and lactoperoxidase itself can be iodinated in the process, introducing an iodinated contaminant. Other disadvantages include variation in commercial sources of lactoperoxidase, changes in lactoperoxidase activity with age, and protease contamination which may destroy the protein. A detailed IV-2. RADIOIMMUNOASSAY 93

protocol for radioiodination using the lactoperoxidase procedure is given in Exercise 21.3. If the protein to be radioiodinated is particularly susceptible to oxidative damage by iodination reagents, the iodine may first be reacted with a suitable carrier molecule and then the iodinated carrier can be conjugated to the protein (Bolton-Hunter reaction). A useful reagent for this purpose is N-succinimidyl 3-(4-hydroxyphenyl) propionate. This is iodinated by the chloramine-T reaction and the labelled material extracted with organic solvent and dried. It is then reacted with the protein in aqueous solution to form peptide bonds with the amino groups of the protein. In this procedure the protein is not exposed to oxidizing and reducing agents and, therefore, chemical damage is minimal. Its disadvantages are the introduction of a large organic molecule onto the protein which may affect its antigenicity; the procedure's technical complexity; the need for frequent manipulation of radioactive materials; the lower iodination yields; and the expense of reagents. A similar technique for the steroid testosterone using histamine as the reagent to be iodinated is described in Exercise 18.2 Irrespective of the technique used, it is necessary to separate the labelled hormone preparation from unreacted iodide. This separation may be carried out by gel-filtration chromatography, ion-exchange chromatography, or by electrophoresis. The procedure using thin-layer chromatography is described in Exercise 18.1, and that using gel-filtration chromatography in Exercise 21.4.

IV—2.3. Separation of antibody-bound from free hormone [4, 5, 7]

An essential part of any radioimmunoassay system is an efficient procedure for separating that portion of labelled antigen that has bound to antibody (bound fraction) from that which has not (free fraction). This process is known as phase separation. There are a number of methods of achieving this separation but they may be grouped together under four broad headings: chemical precipitation of antibody-bound hormone (e.g. with ammonium sulphate, ethanol or polyethylene glycol (PEG)); adsorption of free hormone (e.g. with coated charcoal or protein A); immunological (i.e. using a second antibody prepared against the first antibody); and solid phase separation using antibody-coated tubes or matrix.

IV—2.3.1. Chemical precipitation

In chemical precipitation, it is the antibody-bound label that is precipitated. The method can be readily used with large numbers of assay tubes, the precipitation is fairly rapid and the process does not disturb the final equilibrium between the bound and free phases. It does suffer, however, from a number of serious disadvantages, e.g. it has a tendency to trap some of the free phase in the precipitate; it reduces the precision of the assay; it cannot be used in the assay of large protein molecules, e.g. the protein and polypeptide hormones, as it results in the co-precipitation of the labelled antigen, and the presence of ammonium sulphate can cause problems in the liquid scintillation counting of tritium-labelled antigens. 94 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

Ethanol has also been used to precipitate the bound phase but the method is very temperature dependent (it must be carried out at 4°C precisely) and any increase in temperature above that point will result in resolubilization of the precipitate. Polyethylene glycol (PEG) has had a rather chequered history as a phase separation medium and is now mainly used as an accelerator of the double antibody precipitation method.

IV—2.3.2. Adsorption with coated charcoal

By far the most important adsorption system in separating antibody-bound from free steroid hormone is coated charcoal [5]. In this procedure an activated charcoal (Norit A) is coated with dextran, albumin, gelatin or serum, which in turn acts as a molecular sieve. When coated charcoal is added to the assay tube at the end of incubation, the activated charcoal core adsorbs the free labelled antigen, providing it is small enough to penetrate the coating. Due to its much larger molecular size, the antibody-bound label will be excluded and remain in solution, thus permitting a complete separation of the bound and free phases. The final separation is achieved by centrifugation, followed by either décantation or aspiration of the supernatant. The main advantages of this method are that it is a simple procedure, in terms of preparation of the reagent and its dispensing (see Exercise 19.7); additionally, phase separation takes place immediately after adding the coated charcoal to the assay tube and does not require a further period of incubation as with the second antibody procedure. On the other hand, the method is only suitable for small molecules, such as steroids, drugs and peptides up to a molecular weight of 10 000 daltons, because above this molecular weight range the coating begins to exclude both the free and bound forms of the label; this problem may be partially overcome by increasing the molecular weight of the dextran used as a coating. Moreover, since serum proteins may be used to coat the charcoal particles, the method can be very sensitive to variations in the protein content of the incubate. However, the most serious disadvantage is that the charcoal may disturb the equilibrium between the free and bound forms of the antigen by stripping off the antigen bound to antibody. This is particularly important when low avidity antibodies are used since in this situation the affinity of the coated charcoal for the antigen is greater than the avidity of the antibody. The net result is that there is a transfer of the bound antigen into the free pool, which is then adsorbed by the charcoal. There have been several instances where the appearance of antibodies in an immunized animal has been completely missed because coated charcoal was used as the phase separation medium. In this situation another means of phase separation, e.g. ammonium sulphate or second antibody, has to be used. Finally, this separation method may be also temperature and time dependent since the Law of Mass Action applies both to the antigen-antibody binding reaction and also to the adsorption of the free antigen by the charcoal. Since low temperatures reduce the rate of dissociation of the antigen-antibody complex, it is recommended IV-2. RADIOIMMUNOASSAY 95 that the charcoal suspension and the tubes are kept at 4°C and that the centrifugation also is carried out at 4°C (refrigerated centrifuge). By the same reasoning the charcoal must not be left for a long time in contact with the incubate.

IV—2.3.3. Adsorption with protein A

Protein A is a component of the Staphylococcus aureus bacteria which specifically binds the Fc fragment of immunoglobulins. It is very useful as a separating agent in steroid hormone assays but cannot be recommended for separating free and bound phases of protein hormones. After incubation of antibody and tracer, a suspension of killed, dried S. aureus is added to the assay tubes. The tubes are incubated and then centrifuged and the antibody-bound hormone precipitated as a complex with the protein A preparation. The major advantage of using protein A is that the binding reaction (precipitation) is rapid and irreversible. For steroid hormones in plasma an extraction step is necessary, and preferably an 125I-labelled tracer should be used.

IV—2.3.4. Second antibody separation

This technique is the basic immunological phase separation method, of which there are now many variations [4]. The aim is to create a sufficiently large complex, or micelle, incorporating the first antibody-specific hormone complex, so that it is possible to separate the bound antigen phase by ordinary centrifugation. The molecular weight of the first antibody-specific hormone complex (150 000 - 210 000) is too small itself to be precipitated by the centrifugal force generated by most ordinary centrifuges. To increase the overall weight of the complex, the idea was conceived of making the first antibody become the antigen of a second antigen-antibody reaction. This can be achieved by raising an antibody to the first antibody in another species to that in which the first antibody was raised. For example, if the first antibody is raised in guinea-pigs then gamma globulins from normal guinea-pigs are injected into a rabbit to produce a rabbit anti-guinea-pig gamma globulin antiserum. Such an antiserum will react with any guinea-pig gamma globulins whether specific for the first antigen or not. This second antibody is normally added at the end of the incubation period of the first antibody with its specific antigen and the system allowed to incubate for a further period, which may be between 8-24 hours. The assay tubes are then centrifuged and the resulting supernatant fluid removed, leaving the precipitated first-second antibody complex at the bottom of the tube. It should be stressed that not all anti-gamma globulin antisera are suitable for use as precipitating second antibodies in a radioimmunoassay. It is necessary to screen them first for their ability to do this. Phase separation using second antibody has several advantages over other methods. First, the use of such antibodies does not depend on the molecular weight of the antigen, since the second antibody primarily reacts with the Fc fragment of the first antibody; thus, second antibodies are now applied to all 96 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY types of assay systems, even for the small molecular weight drugs and steroid hormones. Secondly, the system is more or less independent of time once precipitation is complete but leaving assays over the weekend when they should be spun down on the Friday beforehand is not advisable since changes do occur in the end-point, particularly in sequential-type assays. Thirdly, since the separation is based essentially on an immunological reaction between the Fc fragment of the first antibody and the Fab fragment of the second antibody, the equilibrium between the first antibody and its specific antigen is not disturbed; the method is thus admirably suited to low avidity antisera. And finally, the method is much less sensitive to variation in protein content of the incubate than is coated charcoal. Nevertheless, such an apparently perfect method is not without several serious disadvantages. It involves a second, somewhat lengthy, period of incubation of up to 24 hours. Also, since the amount of precipitate produced by the reaction of the second antibody with the first specific antibody is normally very small, there may be significant losses during the décantation or aspiration stage, resulting in loss of precision in the assay. This may be overcome by the addition to the assay tubes of 'carrier' serum (i.e. normal serum from the same species as that in which the first antibody was raised) to increase the bulk of the final precipitate, or by using the first antibody at a final dilution of less than 1:3000. Obviously, the use of carrier serum will increase the amount of second antibody that has to be present to ensure complete precipitation of the first antibody-antigen complex. Consequently, each assay system must be optimized with regard to the amount of second antibody and carrier serum that must be added. Such a titration of second antibody must be performed simultaneously with the titration of carrier serum. When one reagent is exhausted, the whole system must be re-optimized. The formation of the insoluble micelle is also susceptible to interference from complement, which may be inactivated by adding the chelating agent, ethylene diamine tetra-acetic acid, di-sodium salt (EDTA). However, the chelator should only be added with the second antibody, because if present during the first antigen-antibody reaction, it may interfere with that reaction and reduce the percentage binding of the labelled antigen to antibody. This may be acceptable in some assays, particularly in the presence of sera and plasma proteins, as precipitation of the first antigen-antibody complex may be potentiated by the presence of serum or plasma. It is therefore necessary to check that the second antibody being used does not possess a serum/ plasma effect. Anticoagulants, e.g. heparin, may also influence precipitation and therefore plasma samples should not be assayed using standards diluted in serum, nor vice versa. Most second antibodies are raised in donkeys, on account of the volume of reagent required. Such antibodies may exhibit a 'prozone' — i.e. the insoluble micelle of the first-second antibody complex may resolubilize in the presence of excess second antibody, causing an apparent loss in binding. Again, the second antibody must be checked for the absence of this effect. Com- IV-2. RADIOIMMUNOASSAY 97 pared with coated charcoal and ammonium sulphate, the second antibodies increase the cost of assays because of their cost of production. In addition, the antisera produced have to be checked for their ability to precipitate the first antigen-antibody complex and their freedom from prozone and protein effects, and, since each antiserum is unique, there is considerable variation between different batches of second antibody, even those produced by the same animal; it is therefore essential to check each batch before use.

IV-2.3.5. Solid phase separation

In the solid phase group of separation media, the common aim is to immobilize the specific antibody before the assay is performed so as to avoid the later addition of other reagents to secure this separation. Two approaches have been used. The first involves coating the walls of the assay tube with the specific antibody being used. The assay is performed in the coated tube after first washing off the excess antibody. At the end of the incubation period the contents of the assay tube, containing the free fraction, are poured off and the tube counted for the residual radioactivity. Although simple in operation, it does, like the other methods, suffer from a number of disadvantages. The first require- ment for this type of assay system is a plastic assay tube capable of binding the antibody with sufficient affinity that it does not leach off into the assay medium during incubation. Plastic tubes, however, vary enormously in their ability to do this even within the same batch and this results in considerable loss of precision. Coating of tubes for RIA is technically complicated. Several manufacturers sell pre-coated assay tubes which have proved to be precise and accurate. A coated tube technique for the quantification of progesterone is described in Exercise 19.5. An alternative approach is to covalently link purified antibodies to very fine insoluble dextran particles. The particles are first activated with cyanogen bromide and the antibody molecules then linked in a condensation reaction with the activated solid phase to form the immunosorbent. Aliquots of this slurry are then transferred to each assay tube and after a period of incubation, the tubes are spun and the supernatant decanted. Since the immunosorbent particles trap some of the free phase, the particles must be washed several times. Whilst this repeated washing of the bound phase reduces the level of non-specific binding to almost zero (with concomitant increase in precision and sensitivity), the number of times the precipitated fraction must be washed is a disadvantage. Also the fact that incubation has to be carried out in a solid phase means that the assay tubes require to be shaken during the incubation period to keep the reaction mixture in suspension. It is, however, possible to separate the free antigen from the bound complex by sedimentation, thus avoiding the necessity for a large refrigerated centrifuge.

IV—2.4. Optimizing the conditions for an assay

Once the titre of an antiserum has been established, using a specified quantity of labelled antigen, it is possible to produce a standard curve. This, of 98 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY course, requires a source of unlabelled antigen to prepare standards. The optimum dilutions for all the reagents, however, require careful experimentation and adjustment. The first condition to optimize is the length of the incubation period for the hormone and the antiserum before adding the phase separation medium. Generally, the larger the molecular weight of the antigen being measured, the longer the incubation time required to reach equilibrium; with small molecular weight compounds, equilibrium is attained in anything from 30 minutes to a few hours, while the larger pituitary hormones may take anything up to five days or more to do so. Another factor which greatly influences the speed with which equilibrium is attained is the avidity of the antiserum being used; the more avid the antiserum, the quicker will it reach equilibrium. The equilibration or association time for the assay system to reach maximum binding may be determined by setting up a series of 15—20 sets of maximum binding (MB) and non-specific binding (NSB) tubes, put up in duplicate. The MB tubes contain no standard hormone but radiolabelled hormone and antibody and represent maximum binding of the labelled hormone to the antibody and are usually named 'zero standards'. The NSB tubes contain no antibody but labelled hormones and provide information on the efficiency of the separation system. A set of MB and NSB tubes is removed at regular time intervals, depending on the molecular size of the antigen, and the phases separated as quickly as possible. In the case of a RIA of a low molecular weight compound it may suffice to remove sets at 5-min intervals for the first half hour, then at 10-min intervals for the next half hour and finally at 15-min intervals for the remaining 3 hours or so. In the case of the large molecular weight antigens, the intervals between each observation will be much longer being as much as every 8 hours in the final stages. Equilibrium is reached when the amount of label bound by the antibody attains a maximum and plateaus out. The time taken to do this is the association time. As the incubation is continued beyond the association time, the amount of label bound to antibody may appear to decrease, probably because of increased levels of NSB. Thus, an assay that is due to be taken off on a Friday afternoon should not be left incubating over the weekend in the mistaken belief that the level of binding will not alter and may even increase. When developing an RIA, the objectives must be clearly defined in terms of the levels of the substance under investigation. Obviously, the assay must be sensitive enough to measure values over the range anticipated. In some cases this may not be known until a number of samples have been assayed. If, on the other hand, it is known that very small levels of the compound are likely to be encountered, then one should aim to make the assay as sensitive as possible over this range of the standard curve, by using as small a quantity of label and as large a sample volume as possible. Alternatively, if one is studying a hyper- secreting endocrinopathy, an assay with a working range capable of measuring a higher range of values is required and this can be achieved by using a relatively large amount of label and a small sample volume. It is most important to ensure that the assay is most sensitive over that part of the standard curve where it is most required. IV-2. RADIOIMMUNOASSAY 99

One method of making an assay more sensitive is to preincubate the unlabelled antigen with the antiserum and to add the label later. This type of assay is called a disequilibrium, or sequential assay, and results in a more rapid fall in binding. The one factor that decides whether or not a sequential type of assay will improve sensitivity is the avidity of the antiserum being used. A very avid antiserum is required because the antigen-antibody reaction is reversible and obeys the Law of Mass Action. When equilibrium is reached, the antigen-antibody complex dissociates at the same rate at which it is formed. With a strongly avid antibody there is very little dissociation of the complex and, at equilibrium, the reaction is shifted almost exclusively to the right. Thus, if the unlabelled antigen is first allowed to react with the antibody and the labelled antigen introduced later, the labelled antigen has to compete with the remaining unbound antigen molecules for the binding sites that are still left unfilled. There is no displacement by the labelled antigen molecules of unlabelled antigen molecules already bound to antibody, since there is no dissociation of the complex. On the other hand, if the antiserum is only weakly avid, the reaction is not shifted so far to the right at equilibrium and there is an appreciable amount of dissociation. By introducing the labelled antigen into a system of unlabelled antigen and antibody that has come to equilibrium, the labelled molecules in time will be substituted for the unlabelled molecules previously bound to antibody due to the relatively high dissociation rate. Eventually there will be the same number of labelled molecules bound to antibody as there would have been if all three components had been added to the tube simultaneously in an equilibrium type assay. Thus, with a weakly avid antibody there is no point in attempting to increase the sensitivity of the assay using disequilibrium techniques. The degree of dissociation may be determined in one of two ways by incubating sets of MB and NSB tubes until they have reached equilibrium. If the antigen has a small molecular weight, dextran-coated charcoal may be added, left in contact with the incubate for various lengths of time, and contact with the charcoal terminated by centrifugation. If the antiserum is only weakly avid, then the degree of dissociation of the antigen-antibody complex will be quite high and labelled antigen molecules will be adsorbed by the activated charcoal, which has a higher affinity for them than the antibody binding sites. Thus the charcoal effectively strips the labelled antigen off the antibody and the level of bound antigen falls precipitously. In the case of a strongly avid antiserum there is little or no dissociation of the antigen-antibody complex and the presence of charcoal in the tube has no effect on the number of labelled molecules bound to antibody. Instead of using dextran-coated charcoal, an excess of unlabelled antigen can be added to the MB and NSB tubes after the labelled antigen and antiserum have come to equilibrium. If the antibody is very avid, there will be no change in the level of labelled antigen bound to antibody. On the other hand, if the antiserum is only weakly avid, unlabelled antigen will eventually displace most of the labelled antigen bound to antibody and the percentage binding of the label will 100 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY fall. This alternative method of determining the dissociation rate is more suitable for the larger molecular weight protein hormones with which dextran-coated charcoal cannot be used.

IV-2.5. Validation of RIA

Once a specific antibody, a highly purified hormone standard and a radiolabelled hormone are obtained, the radioimmunoassay must be validated to determine its reliability before being used to quantify the hormone. Four criteria for validation of the assay must be examined whenever a radioimmunoassay is used to measure a hormone in a different species, medium or tissue extract and whenever a previously validated assay in one laboratory is used in another laboratory, i.e. specificity, accuracy, precision and sensitivity.

IV—2.5.1. Specificity

Assay specificity or freedom from interference by substances other than the one to be measured can be attained in one of three ways. First, the hormone antigen used for immunization must be very pure and stimulate production of a specific antibody. Secondly, the hormone that is radiolabeled must be extremely pure. Thirdly, the hormone to be quantified can be separated from other interfering materials in the sample, e.g. through chromatography. Demonstration of the specificity of a radioimmunoassay for large protein hormones such as LH and FSH is difficult and relies more or less upon indirect criteria. Since it is not possible to synthesize these hormones, they have to be isolated and purified from biological material. The purity of such preparations is variable, and the most common cause of non-specificity is impurity of the immunogen. Furthermore, antibodies against LH frequently cross-react with thyroid-stimulating hormone (TSH) and vice versa. This is probably due not so much to the presence of impurities, but to the fact that part of the molecules of these proteins are similar. Both LH and TSH are composed of two polypeptide sub- units. The alpha subunit is identical for the two hormones, whereas the beta subunit is unique for each. The antiserum may contain binding sites that will react only with the alpha subunit, in which case the dose-response curve of LH and TSH utilizing such an antiserum will be parallel, and the assay system will not be hormone specific and thus will be invalid for the measurement of LH. Further purification of a hormone interfering in an assay will usually reveal the reason for its interference. If an antibody is specific for a particular antigen, it is implied that the antibody will only combine with that antigen and not with any related compounds. In actual practice this is rarely the case, since most antisera do cross-react to a greater or lesser extent with analogues, metabolites, fragments and molecules IV-2. RADIOIMMUNOASSAY 101

GH, ACTH, 100

10 100 1000 10000 ng/ml

FIG.IV-4. Specificity of an LH assay as assessed by cross-reactivity curves.

possessing a similar amino acid sequence. It is necessary to determine whether or not an antiserum is completely specific for the antigen in question, and if it is not, to what extent it cross-reacts with associated structures. The specificity of an antiserum is assessed by deriving cross-reactivity curves for each compound whose cross-reactivity with the antiserum is being checked. In this procedure a series of standard curves are set up, one consisting of increasing concentrations of the specific antigen (e.g. LH) and the others of increasing concentrations of compounds that might possibly cross-react (e.g. TSH, FSH, etc.). The same amounts of labelled specific antigen and antiserum are added to each tube. After incubation and phase separation, the amount of label that has bound to LH antibody in the presence of each of these compounds is determined. The results are plotted in the form of a series of standard curves (Fig. IV—4 ). The degree of cross-reaction is conventionally defined as the ratio of the weight of specific antigen required to reduce binding of the label at zero by 50% to the weight of the cross-reacting compound that reduces the binding by the same amount, multiplied by 100. An antiserum that cross-reacts 100% with another compound would measure that compound equally well as the specific antigen. In this example, the %TSH cross-reactivity is 10%, that of FSH only 1% and that of most other hormones less than 0.001%. Cross-reaction data for steroids are frequently presented in the form of tables, of which Table IV-1 is an example. A cross-reactivity of 1% implies that the particular compound tested marginally combines with the antibody over the range studied and would not be likely to interfere in the. assay for the specific hormone. This way of determining specificity is open 102 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

TABLE IV-1. PERCENTAGE OF CROSS-REACTION FOR DIFFERENT STEROIDS IN A PROGESTERONE RADIOIMMUNOASSAY

Cross-reaction (%)

Progesterone 100.0 11 alpha-hydroxyprogesterone 51.1 Corticosterone 0.8 Pregnenolone 0.5 Deoxycorticosterone 0.5 17alpha-hydroxyprogesterone 0.5 Testosterone 0.1 Cortisone 0.1 Oestradiol-17beta 0.1 Cholesterol 0.1 Cortisol 0.1

to the criticism that the amount of binding of the cross-reacting substance is determined in the complete absence of unlabelled specific antigen, a situation that is not normally likely to occur. However, this is the maximum degree of cross-reactivity that the antibody is likely to exhibit with this particular compound since it has been shown that the level of cross-reactivity is reduced as the amount of the specific antigen present is increased and the degree of cross-reactivity may be reduced by as much as half in the presence of the highest concentration of the specific antigen standard. This criticism may therefore be overcome by determining the degree of cross-reactivity for a particular substance in the presence of low, medium and high concentrations of the specific antigen. Specificity should not be assessed solely on the basis of a cross-reaction study and a further method which is widely used is the test for parallelism. If a radioimmunoassay is immunologically specific, serial dilutions of the assay material (serum, plasma, urine, tissue extract, etc.) should inhibit binding of the radiolabeled antigen to the antibody in a manner parallel to the inhibition curve for the standard solutions. In Fig. IV—5 a standard curve (inhibition curve) is plotted with the log of the dose on the abscissa and the per cent binding on the ordinate. Also plotted is the per cent binding for each of two samples of serum serially diluted from full strength to 1: 16 in assay buffer. Parallel inhibition or IV-2. RADIOIMMUNOASSAY 103

Dilution

FIG.IV-5. Example of an RIA inhibition curve, with two serially diluted serum samples superimposable on the standard curve.

superimposable curves suggest that the same material in the sample is inhibiting binding as is in the standard solutions. However, if the curves are not superimposable or not parallel, then the use of that particular antiserum will be prone to interference from other, possibly unidentifiable, compounds. .Specificity may be evaluated by examination of the physical properties of the antigen being measured. For example, in Fig. IV-5 the chromatographic properties of a particular hormone after elution through a gel-filtration column are illustrated. A small quantity of 12sI-labelled hormone is passed through the column and the counts per minute(counts/min) in each fraction determined. After chromatography of an extract of tissue that secretes the hormone the concentration of the hormone in each fraction is measured. As can be seen, the assay detected the hormone in the fraction that eluted from the column at the same rate as those that contain highly purified radiolabeled hormone. Coincident elution of the assayable material and the highly purified material suggests that the assay is measuring the same hormone as was radiolabeled. Therefore, the assay is probably specific. All the above procedures can be used to demonstrate immunological specificity of a radioimmunoassay. However, before an assay can be used to quantify a hormone, biological specificity should also be shown. The radioimmunoassay should measure changes in concentrations of the hormone that correspond to expected changes after stimulation, suppression, or removal of the source of the hormone. The 104 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

c 6

4 8 12 16 20 24 Hours

FIG.IV-6. Test of biological specificity. The effect of GnRH on plasma LH levels, followed by hypophysectomy (removal).

concentration of LH in serum is shown in Fig. IV-6. At 4 hours the source of the hormone, e.g. pituitary gland, was stimulated with GnRH, and an increase in LH was measured. In addition, removal of the source of the antigen (e.g. by hypophysectomy) caused a decrease in the antigen to undetectable levels. This assay shows biological specificity because it measures changes in concentration of the hormone that are expected from previous studies. The same proof of specificity has to be undertaken for the radioimmunoassay of steroid hormones. This is relatively simple, because small molecular weight hormones can easily be purified and, in most cases, produced synthetically. In most steroid hormone radioimmunoassays, the hormone is extracted by an organic solvent from a blood plasma or serum sample. Many radioimmunoassay procedures designed to measure progesterone utilize antisera developed against a progesterone 11 -protein conjugate. Most such antisera described show a minor cross-reaction with corticosteroids. By using a non-polar solvent such as petroleum ether for the extraction of progesterone from serum or plasma samples, about 80-90% of progesterone is extracted, leaving the more polar corticostéroids in the plasma. The use of such a selective extraction system increases the overall assay specificity. Certain radioimmunoassay systems require a further purification of the plasma extract in order to achieve an acceptable specificity. Such purification steps might include thin-layer chromatography, column and affinity chromatography, etc. Some idea as to the specificity of an antiserum to a steroid hormone can be gained from the position of the steroid molecule that is used for conjugation to the protein. If an antiserum to oestradiol-17beta is produced through the use of an antigen conjugated via the hydroxyl group at carbon 17 of the steroid, the IV-2. RADIOIMMUNOASSAY 105 resulting antiserum will react almost equally well with oestrone and thus would have a relatively poor specificity. This is because the only structural difference between the oestradiol-17beta and oestrone steroid skeletons is the configuration at position 17. When this position is used for conjugation, the resulting antiserum will recognize the remaining steroid structure which is identical for both oestrone and oestradiol-17beta. In general, steroid antibodies are more specific for the portion of the steroid molecule that protrudes from the carrier protein and less specific for the portion of the steroid used for linkage to the protein. Thus, in the case of oestradiol-17beta, highly specific antibodies have been developed after immunization with conjugates when carbon 6 of the beta ring has been used as the site of attachment to the protein. However, recent advances in the development of affinity chromatography have resulted in major improvements in the assay of oestradiol-17beta and testosterone [8]. Linkage of a specific antibody to a solid base, such as agarose, has made it possible to pass serum or plasma directly through columns containing the solid phase, thus separating the steroid from interfering substances, such as other steroids and plasma proteins. It is then possible to elute the steroid from the column by means of an organic solvent, and assay it by a normal technique. The same fundamental principle concerning the selection of appropriate sites for conjugation has allowed production of specific antibodies against progesterone conjugated to the carrier protein through carbon 11 and against testosterone through carbon 1 and 3. Nevertheless, it should be noted that even if a highly specific antibody is used for steroid RIA, chromatography of the material to be analysed must be undertaken initially to check the specificity of the assay. Demonstrating specificity is usually a minor problem for prostaglandin radioimmunoassay systems. The main problem in radioimmunoassay of primary prostaglandins is that they are formed in large amounts from various sources, e.g. platelets, when blood samples are collected. The concentrations of PGF2aipha reported in blood serum or plasma appear, in most cases, to be 100— 1000 times higher than the actual values. The primary prostaglandins have a very short half- life in the circulation and are rapidly converted to their corresponding 15-keto-13, 14-dihydro derivatives. The latter have a considerably longer half-life and occur in higher concentrations than'the parent compounds. Radioimmunoassay systems utilizing antibodies to 9alpha, 1 l-dihydroxy-15-ketoprost-5-enoic acid and 5 alpha, 7 alpha-dihydroxy-11-ketotetranorprosta-l, 16-dioic acid have been developed [9]. Most problems involved in the determination of the primary prostaglandins are avoided if their main metabolites, the 15-keto-13, 14-dihydro compounds, are measured. 106 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

IV-2.5.2. Sensitivity

The sensitivity of an assay is defined as the smallest dose of standard hormone that is significantly different from the zero standard dose. This dose can be derived from the variation of the counts in the zero standard dose (containing no un- labeled hormone). Of more importance is the sensitivity of the assay in the measurement of a hormone level in a biological fluid. This kind of sensitivity is dependent upon the sensitivity of the standard curve, the presence of assay blanks, e.g. solvent residues and, if applied, the recovery of the hormone after its extraction or purification. The presence of a solvent blank can easily be detected by comparing the counts in the 'zero' tubes with the counts present in tubes containing solvent residues (after evaporation of the appropriate amount). A useful way to establish a practical detection limit of an assay is to analyse a 'hormone-free' sample. Such a sample is, however, very difficult to establish. In the case of progesterone determinations in cattle the surgical removal of both ovaries will result in low progesterone levels in peripheral blood. Performing such an operation on an animal with a functioning corpus luteum will also enable the biological specificity criteria to be established. Blood from an oophorectomized cow will not be totally free of progesterone since this steroid is also secreted in small quantities from the adrenal glands. However, the mean ±2 S.D. of the progesterone concentration in such blood will give valuable information on the lowest level of progesterone of ovarian origin which can be measured.

IV—2.5.3. Accuracy

The determination of accuracy for an assay is made by adding known quantities of an antigen to samples of serum which are then tested in the radioimmunoassay being validated to measure the concentration of the hormone in the samples. Quantitative recovery of the added antigen is indicated by the slope of the curve in Fig. IV—7 which suggests that other components of serum, such as cross- reacting antigens, do not interfere with the estimation of the antigen in question and that the assay is accurate. Accuracy may also be demonstrated by comparing the results obtained by radioimmunoassay with those obtained by some other method (bioassay, gas chromatography, mass spectrometry, etc.).

IV—2.5.4. Precision

The quality of a radioimmunoassay is also judged by its precision, which is assessed by measuring the within-assay and between-assay variation. The within-assay (intra-assay) variability is usually obtained by calculating the coefficient of variation of replicate measurements performed in a single assay. These may be IV-2. RADIOIMMUNOASSAY 107

FIG.IV-7. Quantitative recovery of known amounts of added hormone to serum.

several measurements of a representative plasma sample(s) (plasma or serum pool), or duplicate measurements of all unknown plasma samples. In the latter case the information obtained from the duplicates of individual samples is pooled to obtain an average estimate of the within-assay variation. These calculations have an additional benefit in that duplicates exhibiting an excessively high variation can easily be discovered and rejected. The between-assay (inter-assay) variation, usually expressed as coefficient of variation, is a measure of the reproducibility of the results on the same samples yielded by different assays. It is obtained by assaying a plasma pool (or several pools) in every assay. The between-assay variation is a very important quality control criterion which should be systematically checked.

IV-2.6. Quality control in RIA

In no other branch of clinical biochemistry is quality control so important as in RIA. This is because one is dealing with biological reagents of very variable specificities, since each antiserum produced against a given antigen is a unique reagent with its own particular profile of cross-reactions. If the results obtained by RIA are to be useful, it is important that the same sample assayed both in the same laboratory on different occasions and in different laboratories should give as closely as possible the same value. In view of this inherent variability, it is absolutely essential that stringent quality control procedures are practised in every RIA laboratory. 108 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

Quality control (Q.C.) schemes can be divided into two main categories — internal and external. An internal scheme is one that is run entirely within a particular larboratory, whilst an external one involves the collaboration of at least two laboratories, preferably more. Internal schemes can be subdivided still further into 'within-assay' schemes and 'between-assay' schemes. The data emanating from the within, or intra-assay scheme enable a decision to be made about a particular assay without reference to any of the previous assays. The between, or inter-assay schemes provide data whose significance is purely relative to the previously performed assays.

IV—2.6.1. Internal quality control schemes

(a) Within-assay. The principal objectives are to provide a check on the immuno- reactivity of the reagents being used (particularly the label and antiserum) and the errors caused by the manipulation of the reagents used. This will include statistical errors in the reproducibility of dispensing equipment used, errors due to the amount of unbound labelled antigen present in the final antibody-antigen complex and the amount of antibody-antigen complex lost in the separation procedure. The information is obtained by running tubes in the assay to measure the non- specific-binding (NSB) and maximum binding (MB) of the label, usually with its binding to antibody in the absence of unlabelled antigen, i.e. zero standard tubes.

These 'zero' tubes, often referred to as B0, also provide a check on assay drift if placed at regular intervals throughout the assay, say after every 20 tubes. This can be appreciated by considering the distribution of radioactivity in the assay tube. Some of the radioactivity added will bind non-specifically to the walls of the assay tube and to the protein in the diluent buffer. The level of binding of the label in the absence of antibody should not exceed 5% of the total added; in most steroid and drug assays it is less than 2%, but may be somewhat more in peptide hormone assays. Yet another portion of the radioactivity added to the tube is not immunoreactive, and therefore not available for binding. This unreactive component comprises the free radionuclide that has become detached from the labelled antigen, together with any breakdown products of both the nuclide and labelled antigen. It can be measured by incubating an aliquot of the label with an excess of the antibody (as much as 10 times that present in the other assay tubes) and determining the amount of radioactivity that is bound specifically to the antibody. It will be appreciated that the standard curve can only be constructed between the limits delineated by the NSB and MB values. As the shelf-life of the label increases, there is a tendency for the MB to decline and the NSB to increase slightly. The point at which the labelled preparation deteriorates rapidly is characterized by a rapid fall in maximum or 'zero' standard binding, and sometimes by a sudden increase in the NSB. A rapid fall in MB indicates that the label is no longer of any use and that another should be prepared. It may be IV-2. RADIOIMMUNOASSAY 109

necessary to discard a label after the first time it is used if the control parameters do not come up to expectation, as may happen on checking a new preparation of the label immediately after iodination. It is helpful to plot a graph of these parameters against the shelf-life of the label. Critical evaluation of this graph would not only indicate when it was necessary to prepare a fresh batch of label, but an increase in the rate of deterioration of successive batches of label would prompt an investigation into the storage conditions employed. Another within-assay quality control parameter is the '50% intercept', which is defined as the concentration of unlabelled antigen that depresses the MB by 50%, after first of all subtracting the NSB. It is an indirect measurement of the slope of the standard curve, which, if computer facilities exist, can be calculated directly. (b) Between-assay. The object of these schemes is primarily to determine whether a particular assay is 'in control'. In other words, they indicate when results for unknown samples should be rejected through the assay being 'out of control'. In addition, they provide information on the precision of a particular assay method; if the precision is poor, then the quality control results should provide the necessary stimulus to improve it by modifying the assay protocol to reduce the imprecision. This assessment is obtained by repeated analysis of the same sample, or better still, samples, in every assay and comparing the imprecision of the results with the pool means. The procedure for setting up an internal, between-assay, quality control scheme as follows. First, accumulate three pools of material containing low, medium and high levels of the hormone, which span the analytical range of the assay to be controlled. The concentration of the antigen in the low pool should be just greater than the detection limit of the system. In the medium pool the level of the antigen should ideally fall about the midpoint of the curve and the high at the point just before the curve begins to flatten out. If all the unknowns are expected to fall within a relatively narrow concentration range, the concentrations of the control chosen may be related to the range over which the majority of samples are likely to be encountered, or at the points which separate the normal from the pathological. Several criteria are used for the selection of material for the quality control pools. First, they must be the same fluid; if, for example, the unknown samples are sera, then sera must be used for the pools, and not plasma. Plasma would contain fibrinogen which can influence the binding of antigen to antibody in some peptide hormone assays. Secondly, the material must come from the same species, since, in the case of peptide hormones, the hormone from another species may have a different amino acid composition or sequence. Thirdly, the material for the quality control pools must be in the same form as that being measured in the samples. This is most important. The precursors and fragments of the peptide hormone must also be present as must the metabolites of steroids and drugs, since 110 PART IV. INTRODUCTION TO RADIOIMMUNOASSAY

14 • • • • • 12

_10 E g> 8 OO O O 0 O 0 O OOOO 6

I I I I I I I I I I I J—I—L_L 1 3 5 7 9 11 13 15 Assay Number

FIG.I V-8. Quality control chart: the levels of high, medium and low controls in 15 sequential assays.

they may cross-react with the antibody being used and affect the final result. For this reason, do NOT use blank material 'spiked' with a pure preparation of the antigen since it underestimates the imprecision of the assay. This is because the coefficient of variation of 'spiked' samples can be less than that of samples containing the endogenously secreted or metabolized hormone as there are no cross-reacting substances present. In addition, most chemical treatments for producing blank material for RIA purposes, in particular treatment with charcoal, leave the product very different in composition to the starting material. This is because charcoal removes other material besides the hormone to be measured. When the necessary pools have been accumulated, they should be dispensed into aliquots sufficient for analysis in one assay. These should be stored either deep frozen at —24°C or freeze dried. The significance of this temperature for storing deep frozen material is that it is the eutectic point of saline, above which serum and plasma samples are not completely frozen. An aliquot should be assayed from each pool in 10 separate assays and the mean and S.D. for each pool should be calculated. A quality control chart can then be constructed embodying these data. This might take the form of a simple graph showing the mean and limits of 1 and 2 S.D. from this value; or it may be a more refined cusum chart. Finally, something which is frequently forgotten until it is too late is that the procedure must be repeated on a replacement pool before the existing pool is finished so that one can start using the new pool with confidence. Normally, one accumulates sufficient material for a pool to last at least six months, so that there are as few changes in the control values as possible. When the pools are in use in an internal quality control scheme, one aliquot from each pool is IV-2. RADIOIMMUNOASSAY 111 assayed in every assay and the apparent hormone contents determined. The values are then plotted on a quality control chart, since nothing creates such a striking impact as a visual display (Fig. IV—8). Having plotted the results, one is then faced with the problem of what to do with them, i.e. how to interpret them and decide whether or not an assay is in control. As yet there is no universal convention but almost as many different ones as there are operators. One rule commonly applied is to reject an assay if the results for two or more pools deviate more than 2 S.D.s away from the mean. If one pool has a greater variability than the others, it may be because it is not as stable as the others. Under these circumstances, it would be advisable to set up a replacement pool as soon as possible.

REFERENCES TO PART IV

[1] BERSON, S.A., YALOW, R.S., ill The Hormones (PINCUS, G., THIMANN, K.V., ASTWOOD, E.V., Eds) IV, Academic Press, New York (1964). [2] EKINS, R.P., The estimation of thyroxine in human plasma by an electrophoretic technique, Clin. Chim. Acta S (1960) 453. [3] SCATCHARD, G., The attraction of proteins for small molecules and ions, Ann. N.Y. Acad. Sei. 51 (1949) 660-72. [4] ODELL, W. DAUGHADAY, W., Eds, Principles of Competitive Protein-Binding Assays, Lippincott, Philadelphia (1971). [5] DICZFALUSY, E., Ed., Steroid Assay by Protein Binding, 2nd Karolinska Symp. Research Methods in Reproductive Endocrinology, Acta Endocrinol. Suppl. 147, Stockholm (1970). [6] NISWENDER, G.D., REICHERT, L.E., Jr., MIDGLEY, A.R., Jr., NALBANDOV, A.V., Radioimmunoassay for ovine and bovine luteinizing hormone, Endocrinol. 84 (1969) 1166. [7] HUNTER, W.M., in Handbook of Experimental Immunology I, 2nd ed. (WEIR, D.M., Ed.), Blackwell Scientific Publ., London (1973). [8] GLENCROSS, R.G., ABEYWARDENE, S.A., CORLEY, S.J., MORRIS, H.S., The use of oestradiol-17beta antiserum covalently coupled to sepharose to extract oestradiol-17beta from biological fluids, J. Chromat., Biomed. Appl. 223 (1981) 193. [9] BERGSTROM, S., GREEN, K., SAMUELSSON, B., Eds, WHO Research and Training Centre on Human Reproduction, Karolinska Inst., Stockholm (1972).

PART V. LABORATORY EXERCISES

SECTION A. BASIC EXERCISES

EXERCISE 1. THE PLATEAU AND OPERATING VOLTAGE OF A GM COUNTER

Geiger-Müller counter assemblies in normal operation often show an appreciable variation in performance from one time of measurement to another. It is thus useful to have a reference source by which day-to-day counting may be standardized. The half-life of such a standard should be so long that no correction for decay need be made. A suitable reference source may be made from black uranium oxide (U308). This combines the required chemical stability and long half-life (4.5 X 109 a). The oxide should not have been treated chemically for at least a year, during which time any significant daughter products removed by previous treatments will have again come to radioactive equilibrium. The disintegration scheme of the mixture of isotopes which forms natural uranium is complex, and it is advisable to filter out all particles except the beta particles of 2.3 and 1.5 MeV from 234Pam. (The 2.3-MeV beta particle is given off in more than 99% of the disintegrations.) This may be done by covering the source with aluminium foil of approximately 35 mg/cm2 thickness. If this is done,

the U308 source may be used as an absolute standard. With this or a similar standard beta reference source the following properties of a GM tube may be determined: (a) The starting potential and threshold voltage. (b) The characteristic curve of count-rate versus tube voltage and the counting plateau. (c) The optimum operating voltage. (d) The counting efficiency e (i.e. counts per 100 dis).

MATERIALS AND EQUIPMENT

(1) Beta-emitting source giving about 100 counts/s (about 0.1 pCi, i.e. 3.7 kBq) (2) Tweezers (3) GM counter and timer

113 114 PART V. LABORATORY EXERCISES

ELECTRODE BIAS VOLTAGE (V)

FIG. V-l. Characteristics of a GM tube showing the relationship between count-rate and voltage.

PROCEDURE

(1) Obtain a source counting about 100 counts/s (5000 counts/min). (2) Put the source (on a planchet) into the planchet holder in the shield. Be sure that the high-voltage switch is turned off and at its minimum position. Turn on the master power switch and the instrument to 'count' mode. Now turn on the high-voltage switch. (3) Increase the voltage slowly until the first counts are obtained. This voltage is called the starting potential. (4) Determine the count-rate with increasing voltage. A total of 2000 counts for each voltage step is adequate. Increase the voltage in steps of 25 or 50 volts. (5) When the count-rate does not change appreciably as the voltage is increased, the GM tube is operating in the plateau region. No further high-voltage steps should be attempted once it is noticed that the count-rate is again beginning to increase. This is termed continuous discharge, and above this voltage the counter will start to race and damage to the GM tube is likely to occur. (6) Calculate the slope as percentage increase in count-rate per 100 volts and

the plateau length (see Fig.V-1), V2~V,. The slope is:

(R2-R,)/Rw — X 100 (% per 100 V) (Vj - V, )/100

in which Rw is the count-rate at the working voltage (see below). (7) As the counter ages, the threshold voltage (VT) tends to increase and the

racing voltage VR to decrease. To allow for this, choose the working

voltage at VT + 75 volts or¿(VT + VR) if the plateau length (V2 - V,) is less than 150 volts. Occasional checks on the plateau characteristics of a tube are necessary with age (number of counts). APPLIED EXERCISES 115

(8) At the operating voltage, if the disintegration rate of the standard source is known, calculate the counting efficiency e at each shelf position in the shield. R (counts/s) X 100 e" A* (Bq)

EXERCISE 2. THE RESOLVING TIME OF A GM COUNTER

The resolving time, i.e. the time after each pulse that the GM tube (and counting system) does not register pulses, can be determined in various ways. The method by which a series of samples of increasing strength is counted is straightforward. From the difference between the expected count-rate as extrapolated from low count-rates and the observed count-rate, the dead time can be estimated.

Let the true count-rate be Rtrue counts/s and the observed count-rate R counts/s. If the resolving time is rseconds, the counter has been inoperative during Rr seconds per second. R counts have therefore been registered in 1—Rr effective seconds. The corrected count-rate RtmP is thus as follows:

R (V-l)

If the R of a radioisotope of known half-life is plotted against time on log-linear graph paper, R^g for the highest count-rates can be extrapolated from the R of the lowest count-rates, and r can then be estimated approximately with the aid of Eq. (V—1). (At very high count-rates, it may turn out that r is no longer constant but equal to some function of R.) Another approximation of the dead time r may be obtained by the method of twin samples, i.e. from a comparison of the count-rate of two samples counted together,with the sum of the count-rates of each sample counted separately. Let Rtrue,1 ' Rtnie,2> ^tiue,l + 2 ancl ^true.b l'le true count-rates (background included) of sample 1, sample 2, samples 1 plus 2, and a blank sample, respectively.

Also let R,, R2, RI + J and Rb be the corresponding observed count-rates. Then by definition:

Rtiue.l + Rtrue,2 = ^true.l + 2 + ^true.b and thus from Eq. (V—1):

R2 R1+2 Rb + = + —-— (V-2) 1-R,r 1-R2T 1-R1+2T L-RbT 116 PART V. LABORATORY EXERCISES

2 Since (Rít) 1, and if Rbr < Rjf, the following approximations can be made:

Ri „ R Ri1 + Rf1 T and ^ Rb (V-3) 1 - R¿r 1 - RbT

Therefore, after substituting, one obtains:

R, + R2-R1+2-Rb T — . (V—4) R]+2 -Rî-Rl or

R, + Rj R1+2 - Rb ^ (V-5) since

2 R] + 2 -(R, + R2)

MATERIALS AND EQUIPMENT

( 1 ) Laboratory neutron source, for example 5 Ci (185 GBq) Pu-Be source or a similar one (2) Coin of appreciable silver content (3) Pure silver foil (4) Stop watch (5) GM counter

(6) S03-H-type cation-exchange resin (e.g. Amberlite IR-120 or Dowex-50) (7) About 200 juCi (7.4 MBq) of 131Cs in solution (8) A conventional burette (9) EDTA solution (approximately 0.3% adjusted to pH 11-12 with NaOH) (10) Plastic counting container (11) Two beta-emitting sources giving about 200 counts/s ( 12 000 counts/min) (e.g. 402Tb) (12) Two blank (i.e. empty) planchets (13) Tweezers APPLIED EXERCISES 117

PROCEDURE

(1 ) Using the laboratory neutron source, activate a sample containing silver to a count-rate of approximately 400 counts/s (25 000 counts/min) (l02Ag + ¿n -*• ,08Ag). Within 3 minutes of activation begin counting for periods of 30 seconds, separated by intervals of 30 seconds, and continue this process for 10-12 minutes. If more convenient, obtain 137Bam using the procedure described in Exercise 9. Place about 5 ml of the 137Bam solution (i.e. about 400 counts/s = 25 000 counts/min) in a counting dish and cover with a plastic cap. Immediately start counting as above. (2) After half an hour determine the residual count-rate (background), then plot the count-rates corrected for background versus time on log-linear graph paper. (3) A straight line with its slope corresponding to the tenth-life1 is drawn through the last three or four points. From the small deviations of the first few points from this straight line, estimate the resolving time of the GM counter, using Eq.(V-l). Note: Verify that T, X3.33=T, . i re For the method of'twin-samples' the procedure is as follows:

(1) Count the first sample in a sample holder with two holes. In the second hole insert an empty counting planchet. (2) After counting the first sample, remove the blank cup without touching the active sample. Put the second sample in the holder and count both samples together. (3) Remove the first sample and replace by a blank planchet without touching sample 2. Count sample 2. (4) Determine the background by counting with the two blank planchets in counting position. (5) Calculaté r with the aid of Eqs (V-4 or 5). (6) Repeat the complete r-determination three times and calculate the relative standard deviation of the average value, r, according to the usual formula:

/So--?)2 loo °r =A — ' — % T \J n(n-l) r

where n = number of replicate determinations. (7) » Use an end-window GM tube survey meter to survey for personnel or equipment contamination.

1 The time required for the radioactivity to decrease to one tenth of its initial value. 118 PART V. LABORATORY EXERCISES

EXERCISE 3. COUNTING AND SAMPLING STATISTICS

In scientific experimentation, the standard deviation (calculated from replicates) should always be given together with the results to permit assessment of the uncertainty. When the standard deviation a is calculated from replicates, it automatically includes all sources of uncertainty. When a series of identical counts is made on a sample which is not moved between individual counts, assuming the counter functions correctly, the standard 1/2 deviation of the sum-count will be found to be anat = C , where C is the sum- count. This is a measure of the natural uncertainty inherent in radioactive decay. Note that this type of uncertainty can be calculated after a single counting. However, when the sample is moved between countings or a number of identical samples are counted in succession, a larger figure is likely to be obtained than can be explained by natural uncertainty alone. This is because of random irregularities in geometry and sample preparation. This form of added deviations (including erratic counter performance) we will call technical uncertainty. An experimental evaluation of these two types of uncertainty will be made.

MATERIALS AND EQUIPMENT

(1) 32P solution of approximately 1 juCi/ml (37 kBq/ml) (2) 0.1 ml pipette and pro-pipette (e.g. rubber bulb or syringe) (3) 25 counting planchets (4) Infra-red drying lamp (5) Tweezers (6) Geiger counter and timer (7) GM survey meter

PROCEDURE

(1) Using the pro-pipette, pipette 25 samples containing 0.1 ml each. Dry each sample under an infra-red lamp. Do not allow the samples to boil or spatter. (2) Place one of the dry samples in the planchet holder and make 25 countings of 2 min each, without moving the sample. Record the counts and make the following calculations: (a) The mean or average (C), i.e. the sum of all the counts divided by the number of observations (n) (b) The deviation of each observation from the mean (A), i.e. (Q - C) (c) The sum of the deviations (2A). (This should be zero and should be determined as a check on the arithmetic.) (d) The mean deviation from the average (A) APPLIED EXERCISES 119

(e) The square of each deviation (A2) (f) The sum of the square of deviations (SA2) (g) The corrected mean-square deviation (corrected for small numbers of observations), i.e. 2A2/(n - 1) (h) The total standard deviation (a) which is the square root of the corrected 2 mean-square deviation, i.e. atot =\/2A /(n - 1) (i) The probable error P (P = 0.6745 a at the 50% confidence level) (j) The probable percentage error, i.e. 100 P/C% Show that the observations follow a Poisson distribution by making the following statistical tests: (a) Calculate the square root of the mean value (\/C) and show that it is approximately equal to the standard deviation (a) (b) Show that the ratio of the mean deviation to the standard deviation, i.e. ¿/a, is approximately 4/5, i.e. 0.797 (c) Count the number of times that the deviation, regardless of sign, is greater than the standard deviation and show that this occurs in approximately a third of the observations (d) Similarly, show that the deviation is greater than twice the standard deviation in about one observation in twenty (4.6%)

Now count all 25 samples for 2 minutes each. Calculate the standard deviation and compare it with the value obtained above.

EXERCISE 4. EXTERNAL ABSORPTION OF BETA PARTICLES

The absorption of beta particles in matter is very nearly independent of the atomic number of the absorbing material, provided the thickness is expressed in mg/cm2 (thickness X density). Beta particles have a spectrum of energies ranging from zero to a maximum value for each particular radionuclide. Phosphorus-32 emits only beta particles with a maximum energy of 1.7 MeV and an average energy of 0.7 MeV. The thickness of matter which can absorb all incident beta particles is called the 'range' and this is determined by the maximum energy particles. However, only a small fraction of the beta particles from any source have this maximum energy and the range is therefore not sharply defined. For 32P the range is approximately 800 mg/cm2 (8 kg/m2). A transmission curve of 32P beta particles through aluminium is to be obtained in the present experiment. A simplified method to determine the beta- particle range and energy is also demonstrated. 120 PART V. LABORATORY EXERCISES

MATERIALS AND EQUIPMENT

( 1 ) Source containing about 0.2 /uCi (7.4 kBq) 32P (about 200 counts/s) (2) Source containing about 8 pCi (296 kBq) 32P (3) Blank source container (4) Tweezers (5) Set of aluminium absorbers (6) Holder to accommodate absorber between source and detector (about 5 cm apart) (7) GM counter and timer (8) GM survey meter

PROCEDURE

(1) Prepare a 32P sample containing approximately 0.1 ßCi/m\ (3.7 kBq/ml) by pipetting 100 /d onto a planchet and drying under an infra-red lamp. (2) Prepare a second sample by pipetting 100 ¡il of a 32P solution containing about 50 fxCi/ml (1.85 MBq/ml) onto a planchet and dry as above. (3) Count the low-activity source in the GM counter. Obtain at least 170 counts/s (10 000 counts/min). Place an aluminium filter of about 20 mg/cm2 between the counter window and the source and count again. (4) Continue counting at increasing absorber thickness until a count-rate of about 3.5 counts/s (200 counts/min) is obtained. (5) Repeat steps (3) and (4) above, using the high-activity source, and calculate the average ratio between low- and high-activity source count-rates. (Note: resolving-time corrections must be made for the high-activity source counts and the background must be subtracted in the case of the low-activity source counts.) This ratio can be used to transform the low-activity source count- rates into the high-activity source count-rates. The high-activity source cannot be counted at zero or low absorber thicknesses. (6) Continue counting the high-activity source with increasing absorber thickness until an almost constant count-rate is obtained. The count-rate now is due to bremsstrahlung or continuous X-rays produced by interaction of the beta particles with the aluminium nuclei. (7) Plot the observed and calculated net count-rates of the high-activity source on the log co-ordinate versus absorber thickness on the linear co-ordinate. (To the absorber thickness add the window thickness of the GM tube and the air thickness, in mg/cm2, from GM tube window to source.) (8) Extrapolate the bremsstrahlung component to zero absorber thickness and subtract this contribution from the net count-rate. Plot the corrected curve. (9) Determine by inspection the point at which the uncorrected transmission curve appears to intersect the bremsstrahlung curve. This point corresponds to the range of 32P beta particles and should be approximately 800 mg/cm2. APPLIED EXERCISES 121

A simplified method of determining the beta-particle range is based on the determination of the fifth half-thickness of absorber. The half-thickness is the thickness of absorber required to reduce the count-rate by a factor of two. The fifth half-thickness is the amount required to reduce the count-rate by a factor of 32. The fifth half-thickness has been found empirically, in most cases, to be approximately equal to half the range of the maximum-energy beta particle.

( 1 ) From the corrected curve, determine the thickness of absorber that was required to reduce the count-rate at zero absorber by a factor of 32. Multiply this value by two and determine how well it estimates the range of 32P particles. (2) How well does the initial portion of corrected curve approximate a straight line? (3) Use the end-window GM survey meter to survey for personnel or equipment contamination.

EXERCISE 5. SELF-ABSORPTION AND SELF-SCATTERING OF BETA PARTICLES

It is often necessary to measure the radioactivity of sources which contain appreciable amounts of solid material. This introduces errors from self-absorption and from source scattering. Self-absorption decreases the expected count-rate and is most important with low-energy beta emitters whose maximum energy is less than 0.5 MeV. Scattering tends to increase the count-rate and is most noticeable with high-energy beta emitters. (The effect of self-absorption and self-scattering also exists with gamma-ray emitters, but is usually negligible since gamma radiation has a greater penetrating power.) A third source of error when voluminous samples of varying thickness are involved may be called self-geometry, i.e. the top of the sample is relatively closer to the counter as the sample thickness increases. The combined effects of self-absorption, self-scattering and self-geometry normally result in a lower count-rate than expected.

The principal method used to correct for self-absorption is based on the assumption that the absorption of beta particles is an exponential function of absorber thickness. (To verify this, observe that in the previous experiment the absorption was closely approximated by an exponential function over the first decade or two.) Therefore, assume that the decrease in count-rate per unit sample thickness is proportional to the sample thickness or 122 PART V. LABORATORY EXERCISES

SAMPLE THICKNESS (mg/cm2)

FIG. V-2. Count-rate as a function of thickness for samples of constant specific activity for a soft beta emitter.

da* - — =Ma (V—6) dx where a* = transmitted activity (per cm3 sample) from depth x below the surface of the sample x = sample layer thickness (cm)

ß = linear absorption coefficient (characteristic of Eßmax and density of sample material).

Equation (V—6) can be integrated to

a* = ÜQC'V* (V—7)

where aj = number of beta particles per cubic centimetre of the sample.

The (total) transmitted activity A* from a sample of cross-sectional area ß and thickness X is then obtained by integration from top to bottom of the sample as follows:

aJ8(l - e'"x) Ap(l - e~^x) A* = ajBe-^dx = (V—8) ß MX

Finally, defining a self-absorption factor f as the ratio between observed

(i.e. self-transmitted) activity and theoretical zero-absorption activity A0, one lias

», » (1 -e-',x) A /Aq = f = • (V—9) tiX APPLIED EXERCISES 123

MASS OF SAMPLE

FIG. V-3. Curve plotted to determine an estimate of the 'true' count-rate per unit mass by extrapolating the experimental line to zero mass of sample.

1.0 r O ÏJ 0.8 • Z -J O < 0.6 - P> O< 3 0.4 - acc no LL H

0I MASS FIG. V-4. Calibration curve for self-absorption.

where ß may be taken as the mass absorption coefficient (characteristic of E/?max alone) and X as mass per unit area (mg/cm2). The factor f may be used to correct mathematically observed count-rates for self-absorption. If it is impossible to prepare infinitely thick samples, the following adaptation may be used. If the data for Fig.V-2 are replotted as count-rate per unit sample mass versus sample mass, a curve as in Fig. V—3 will result. The curve is extrapolated to zero mass to obtain the estimate of the 'true' count-rate per milligram. This is assigned the value 1, and the value at each sample mass is calculated as a fraction of the 'true' value. These fractions are plotted against sample mass to give a calibration curve for self-absorption in samples of intermediate thickness (see Fig.V—4). The total activity of a sample of unknown concentration and intermediate thickness may be determined by: (a) Weighing and counting the sample; (b) Determining its specific activity; 124 PART V. LABORATORY EXERCISES

(c) Determining from a graph, such as Fig.V-4, the fraction of the sample activity that will be counted; (d) Dividing the apparent specific activity by the above fraction to obtain the specific activity; (e) Total activity = mass of sample X specific activity.

The following experiment will demonstrate these methods.

MATERIALS AND EQUIPMENT

(1) Eight 1-in planchets (2) Filter apparatus (3) Eight 1-in glass filter papers (4) 50, 100, 150, 250, 400 and 500-fd pipettes, 1 and 2 ml pipettes l4 (5) Solution of Na2 C03 (approximately 3 mg/ml and 0.05 /nCi/ml (1.85 kBq/ml))

(6) BaCl2 solution (7) Tweezers (8) GM counter and timer (9) GM survey meter

INFRA-RED LAMP

GLASS CYLINDER SPRING OF RUBBER BAND FILTER PAPER SINTERED GLASS FILTER STICK

SUCTION

FIG. V-5. Exploded view of filtering assembly. APPLIED EXERCISES 125

PROCEDURE

( 1 ) Place the following measured aliquots of a known stock solution of radioactive sodium carbonate in six centrifuge tubes: 50, 100, 150, 400 and 500 jd, and 1, 2 and 5 ml.

(2) Precipitate the radioactive carbonate by the addition of excess BaCl2 solution. (3) Filter the samples on pre-weighed 1 -in filters and wash three times with 5 ml of distilled water (Fig.V—5). Air-dry. Fix the precipitate with 1 ml of collodian solution to prevent spread of contamination. Be sure that the samples are thoroughly dry. (4) Weigh the samples. Place in planchets and count. (5) Count the background with an empty planchet in the counter. (6) Prepare curves from background-corrected counting data as described in Part I, i.e. count-rate versus sample mass; count-rate/mg versus sample mass; and fraction of true count-rate versus sample mass. (7) Calculate the value of p, the mass absorption coefficient. For 14C it should, be approximately 0.28 cm2/mg. p can be determined from any point at infinite thickness, since at infinite thickness X becomes large, therefore e~'jX -* 0, 1 e"^x->-l. (8) Use the end-window GM survey meter to survey for personnel or equipment contamination.

EXERCISE 6. SOLID INTEGRAL SCINTILLATION COUNTING

The following solid scintillation crystals are used to detect radiation: Alpha particles: ZnS (activated by silver) spread as a thin layer, 10-20 mg/cm2 Beta particles: anthracene, trans-stilbene Gamma rays: Nal (activated by thallium at about 0.1% concentration)

By far the most important application of solid scintillation crystals is in the detection and measurement of gamma rays. Scintillation detectors have three distinct advantages over GM tubes for the measurement of gamma rays:

(a) Higher detection efficiencies (20 to 40 times); (b) No significant resolving-time corrections up to about 1700 counts/s (10s counts/min); (c) The output pulse-height is proportional to the gamma-ray energy absorbed in the crystal; therefore, gamma-ray spectrometry is possible.

Nal is hygroscopic and is encased in an air-tight metal can. The detectors are single crystals and should be protected against mechanical shock or temperature changes. The crystal and photomultiplier tube combination is generally sealed 126 PART V. LABORATORY EXERCISES in an air-tight metal case and shielded by lead in the counting position. In spite of shielding, the background of a scintillation counter will generally be considerably higher than that of a GM counter. This is due partly to electronic noise, but mostly to the high efficiency of Nal(Tl) for background gamma rays. A lower-level pulse- height discriminator is used to reject noise pulses, since they are generally of smaller pulse-height than pulses due to the photons being measured.

MATERIALS AND EQUIPMENT

(1) Nal(Tl) scintillation crystal - photomultiplier detector assembly (2) Single-channel pulse-height analyser and scaler (3) High-voltage supply (4) Gamma-ray sources of 137Cs, 60Co and l33Ba, approximately 0.1 fiCi (3.7 kBq) each.

PROCEDURE

( 1 ) Read the instruction manual of your counter and modify the directions below to meet the requirements of the particular counter. (2) Set the lower-level discriminator at 5% of maximum. Set the differential- integral switch at integral or disengage the upper-level discriminator. The scaler will now count all pulses exceeding the lower-level setting. This is termed integral counting. (3) Set the amplifier gain controls to their minimum value. (4) Insert one of the above sources in the counting chamber. (5) Increase the high voltage to approximately 600 V and turn the scaler to the count mode. (6) Increase the amplifier gain until the scaler begins counting. (7) Keep the gain constant and begin recording the count-rate with increasing high voltage. Increase the high voltage in steps of 50 V to a maximum of 1500 V (serious damage to the photomultiplier tube will otherwise result). (8) Repeat counting the background only. (9) Repeat with the other two sources. (10) Plot the above data as counts/s versus high voltage on four- or five-cycle log-linear graph paper. (11) Using one of the three sources, increase the gain of the amplifier by a factor of two and repeat steps (7 — 10) above. What shift do you note? What change in photomultiplier tube voltage is equivalent to a gain of two in amplified gain? ( 12) Leave the gain the same as in ( 11 ) but increase the lower-level discriminator by a factor of two. You should obtain the original curve for your source. Why? APPLIED EXERCISES 127

(13) If the activities of the three sources are known, plot the counting yield (counts/gamma ray) versus energy in the highest-counting Hat portion of your curve. The geometry should be essentially the same for all sources. Explain the variation in counting yield versus source gamma-ray energy. (Be sure to consider the number of gamma rays per disintegration from the decay schemes as well as the half-life and age of the source.) (14) Set the high voltage at the value corresponding to the start of the plateau for the 60Co curve. Use the 60Co source and, with the lower-level discriminator set at 2 V, measure counts with increasing discriminator setting until the count-rate reaches the background. Plot the integral curve. Explain the inflections of the slope of this curve. (15) What settings would you use to measure 60Co in the presence of 137Cs?

EXERCISE 7. SOLID DIFFERENTIAL SCINTILLATION COUNTING

This procedure is used when radioisotopes may be present which will produce counts not related to the isotope being studied. Differential counting is particularly important when it is desired to determine accurately the amount of two or more isotopes present in a sample or if it is necessary to reduce the background to a minimum.

MATERIALS AND EQUIPMENT

As for Exercise 6.

PROCEDURE

(1) Read the instruction manual of your counter and modify these directions to suit the capability of the machine you are using. (2) Place 137Cs in the well of the crystal. Set the gain or attenuator control to its mid-point. Set the high voltage as low as possible and place the control switch so that both the lower and upper discriminators are operative. (3) Since 137Cs has a 0.662 MeV gamma emitter, we shall want to set the mid- point of the window at 662 so that, when calibrated, the full-scale calibration of the ten-turn potentiometer is 1.0 MeV or 1000 keV. For this, set the lower discriminator at 657 (consider that the ten-turn potentiometer has 1000 divisions full scale) and the upper discriminator at 667. The window has now ten divisions (10 keV). This is a 1% window (10/1000-100). (4) Counting with a long-time setting on the timer, turn the high-voltage control up, gradually increasing the voltage. When the first counts appear on the scaler, proceed more slowly. By turning the high-voltage control knob back and forth, try to estimate the point where a peak count is found. 128 PART V. LABORATORY EXERCISES

(5) When an approximate peak is found, start counting at short time intervals (0.1, 0.2 min, etc.) and find the high-voltage setting that gives the peak count- rate. When this is found, the machine is calibrated so that the discriminator potentiometers read from 0 to 1 MeV, with one division equal to 1 keV. Lock the high-voltage control and do not disturb it during this period of operation. (6) To reset for counting a particular gamma energy of less than 1 MeV, only the lower and upper discriminators need to be adjusted. For example, to count a 0.51 MeV gamma ray with a 10% window (10% of 1000 = 100 units or 100 keV), the mid-point of the window must be 510. Since the window is to be 100 units wide, this means 510-100/2 and 510 + 100/2, or 460 to 560, as the settings on the lower and upper discriminator, respectively. This would translate into a setting that accepts any gamma energy from 0.46 to 0.56 MeV. The window width is a variable determined by the operator. (7) If energies greater than 1 MeV are desired, the gain control can be manipulated. For example, by reducing the gain to one-half its original setting the calibration of the discriminators is increased by a factor of two. In the above case the full-scale calibration would be 1000 units = 2.0 MeV (1 unit = 2 keV). The settings of 460 to 560 on the lower and upper discriminator, respectively, would now accept gamma energies of 0.92 to 1.12 MeV. Note that the high- voltage setting must be unchanged! Place an appropriate gamma emitter in the well of your counter and verify the function of the gain control. (8) If it is wished to expand the scale, the gain control can also be used. If the gain is doubled from the setting originally used, the calibration is now changed from 0-1 MeV to 0-0.5 MeV. The settings of 460 and 560 would allow counting of gamma energies from 0.23 to 0.28 MeV. (9) Separating the counts of two or more gamma emitters is a simple extension of the above system. When possible, one channel is set to count the gamma radiation of the highest-energy emitter without interference from the lower- energy emitter. The lower channel is set to count the characteristic peak energy of the second isotope. However, some counts collected in the lower channel will be those of the higher-energy isotope. There is a constant ratio that can be determined for particular settings of the counts of the higher- energy isotope in its own channel to its counts in the channel set for the lower-energy isotope. By multiplying the counts of the higher-energy isotope in its channel by this factor the counts for this isotope in the lower-energy channel can be determined and subtracted from the total count in the lower channel to give the counts for the lower-energy isotope. If both channels contain counts from both isotopes it will be necessary to determine the amount of 'cross-over' of each isotope into the other channel and to use simultaneous equations for solution. APPLIED EXERCISES 129

EXERCISE 8. ESTIMATING THE EFFICIENCY OF A GAMMA COUNTER

It is frequently desirable to convert counts per minute to millicuries or becquerels or to some proportion of the dose of nuclide given. By definition, 1 mCi is equal to 37 MBq (= 37 X 106 dis/s = 2.22 X 109 dis/min), and the conversion of count-rates to millicuries or becquerels requires information on counting yield, i.e. the relationship between disintegration rate and the registered count-rate. As the yield will vary with such factors as self-absorption, scattering and quenching, it is important to measure the counting yield under the same conditions that apply to experimental measurements. The efficiency of the counter can be measured by counting standard preparations of known activity (prepared by the manu- facturer of the counter) containing radionuclides of long half-life. This is a valuable check on the counter itself, but for many purposes it does not duplicate the counting conditions of the experiment, and other counting standards must be prepared. For practical purposes it is generally satisfactory to assume that the amount of radionuclide recorded on the container when dispatched from the reactor is accurate. The activity present at a later date can be calculated from knowledge of the decay rate of the radionuclide. With this information, counting standards of known activity can be prepared. However, the high specific activity of 12SI used in RIAs means that it is extremely difficult to carry out a dilution that is sufficiently precise to obtain more than an approximate estimate of efficiency. Many manufacturers will supply a known gamma source and it is advisable to make use of this when possible.

MATERIALS AND EQUIPMENT

( 1 ) 1251 of approximately 3.7 GBq/ml ( 100 ¿tCi/ml) (2) Micropipettes (Eppendorff type) (3) 100 ml volumetric flasks (4) Polystyrene or glass assay tubes

PROCEDURE

(1 ) Calculate the decay factor for the date of test from the information sheet supplied by the supplier of the 125I. 130 PART V. LABORATORY EXERCISES

(2) Accurately measure 1 jul of 125I (approximately 100 ¿¿Ci) and dilute to 100 ml with the relevant general assay diluent to give approximately 1 fzCi/ml. (3) Dispense 100 jul of this solution into each of 3 counting vials and count for 100 seconds. (4) Deduct the background reading obtained by counting 100 p\ general assay diluent. (5) Calculate the activity that was pipetted in the 100 ßl aliquots in (3) above (e.g. 0.1 pCi X decay factor). (6) Convert above quantity of 125I into Bq (1 kBq = 27.03 X 10"3 juCi). (7) Calculate the counting yield as:

counts/s per lOOul1251 E = ttt— kBq per 100 fil1251

(1 kBq = 1000 disintegrations/s)

EXERCISE 9. RAPID RADIOACTIVE DECAY

The primary purpose of this exercise is to investigate the general law of radioactive decay in one short laboratory period. For this a short-lived radioisotope is used, and a secondary effect (of less importance) may be considered, since the determination of the activity of a sample containing an isotope of short half-life becomes complex when the time which is required to obtain sufficient counts to give a desired accuracy is long compared with the half-life of the isotope. If the disintegration rate at a certain time t is called A*, the count-rate, being proportional to the disintegration rate (i.e. activity) may be expressed as

Rt = cAf (V-10) where e is the counting yield. The disintegration rate, however, changes witli time according to:

A*=A2e~Xt (cf. Eq.(I-8)) (V-ll) APPLIED EXERCISES 131

ACTIVITY

TIME t + T •V T FIG. V-6. Curve showing the exponential decay of the activity of a sample as a function of time.

where AQ - the disintegration rate at zero time, X = disintegration constant = 0.693/T^, t = time elapsed since zero time. If the substance is decaying rapidly during the measurements, i.e. if the duration of counting time T is similar in magnitude to T^, then the disintegration rate Af+y at the end of the counting period will be significantly lower than at the beginning:

A * _ = A * p-\(t + T) At + T o (V—12)

The decrease in activity of a sample (expressed, e.g., as number of radioactive atoms N) during the counting time T is shown in Fig.V-6. The observed count-rate R is the average over the counting period T.

R and the sample activity at the beginning of the counting period Rt are related as follows:2

e e -XTM R = eA* = — (Nt -Nt+T) = — [Nt( 1 -e" )] (V—13) since, from Eqs(IV-14 and 15):

XT Nt+T = Nte- (cf. Eq.(I-3)) (V—14)

2 If the counting time T is not greater than , the relation Rt — Rt + LT(i.e. the average activity is about equal to the activity in the middle of the counting period) is correct within 1%. This approximation can be useful. 132 PART V. LABORATORY EXERCISES

Hence, as A* = XNt:

Thus, using Eq. (V—10):

_ R.(l-e-XT) R = — (V—16) XT where

0.693 X = — î and Ti and T are in seconds. 2

With the aid of Eq. (V-16) the ratio of Rt to R may be calculated for various durations of counting. If the duration of each count T is the same throughout a series of consecutive counts, then the ratio Rt : R is constant. In the following experiment, Eq. (V-16) will be used to correct the count- rate of 137Bam. Barium-137m is the metastable isomer of 137Ba, and it emits 0.66 MeV gamma photons.

REAGENTS AND MATERIALS

(1) SO3H type cation exchange resin (e.g. Amberlite 1R-120 or Dowex 50) (2) About 200 AiCi (7.4 MBq) of 137Cs (3) A conventional burette (4) EDTA solution (about 0.3% adjusted to pH 11-12 with NaOH) (5) Plastic counting container (6) Stop-watch

PROCEDURE

(1) Saturate 50 g of resin with Na+ by leaving it overnight in 10% NaCl solution. Put a glass-wool plug at the bottom of the burette, and fill it half way up with resin. Run 1 litre of distilled water upwards through the resin to remove excess NaCl and air bubbles. Allow the resin to settle and wash with EDTA solution. Never allow the surface of the EDTA solution to come below the top of the resin column. APPLIED EXERCISES 133

(2) Lower the surface of the EDTA solution to the top of the resin and apply the 137Cs. Elute the column repeatedly with EDTA solution at 10 min intervals until the amount of 137Ban1 coming through each time no longer increases. At each elution the 137Bam concentration in the effluent will be maximum after about a half or a third of the resin-column volume of EDTA solution has run through (but the peak is not sharp). (3) Take about 0.5 ml of the effluent rich in 137Bam in a plastic counting container; start counting immediately, using a scintillation (well-type) detector. (4) The counting should be carried out at 1 min intervals for a duration of 1 min counting time and a total running time of 30 min, without removing the sample container. (5) At the end of 30 minutes from the start of counting, the 137Bam remaining in the liquid will be much less than 1% of the original quantity, and most of the count-rate observed above empty-container background results from some l37Cs leached by the EDTA effluent. (6) Repeat counting for 1 min at 5 min intervals until the count-rate no longer decreases, and subtract the final count-rate from the observed count-rate and plot this net count-rate, from 137Bum, against time on log-linear graph paper. Deduce the half-life of 137Bam from this plot. (7) Use Eq. (V-16) to obtain the net count-rates at the beginning of each counting period, and plot these corrected values of net count-rate against time on log-linear graph paper.

EXERCISE 10. INVERSE-SQUARE LAW AND ATTENUATION OF GAMMA RAYS

The intensity of the rays emitted from a source of radiation can be reduced in the following two ways, (i) by increasing the distance from the source of radiation, and (ii) by increasing the attenuation in the path of the rays.

A. INVERSE-SQUARE LAW

For a point source, the radiation intensity is inversely proportional to the square of the distance if no intervening matter is present between the source and the target. This is usually referred to as the inverse-square-law effect and applies to all electromagnetic radiation. The intensity at a distance d from a point source (or other source whose dimensions are small in comparison with d) is thus given as

Id = k/d2 where Id = intensity at d cm distance, k = proportionality constant (see Part I, §1-2.6). 134 PART V. LABORATORY EXERCISES

MATERIALS AND EQUIPMENT

(1) 60Co source, approximately 5-10 ßC.i (0.19-0.37 MBq) (2) Nal(Tl) scintillation counter system

PROCEDURE

(1) Apply the operating voltage previously set in Exercise 7 for the scintillation counter. (2) Determine the background count-rate. (3) Determine the count-rate as a function of distance from the source. Increase the distance until the background count is obtained. (4) Plot the net count-rate versus the distance on log-log graph paper, and draw the best straight line through the points. (5) Determine the slope of the line and explain the reason for discrepancies, if any, between the result and the inverse-square law. (6) Calculate the exposure dose-rate in mR/h at 1 metre from the source.

B. ATTENUATION

The attenuation by matter of a collimated beam of monoenergetic gamma photons is exponential. The attenuation of gamma rays from 60Co which emits gamma photons of two characteristic energies (1.17 and 1.33 MeV) will be investigated.

MATERIALS AND EQUIPMENT

(1) 60Co source, approximately 5-10 ¿¿Ci (0.19-0.37 MBq) (2) Nal(Tl) scintillation counter system (3) Lead shielding (4) Absorber set, preferably lead (5) Micrometer for measuring absorber thickness

PROCEDURE

(1) The previous experimental set-up is used except that the distance between the sample and counter is now kept fixed. The source should be collimated by lead shielding. APPLIED EXERCISES 135

(2) Determine the count-rate of the sample without adding any absorber between source and counter. (3) Determine the count-rate after placing one layer of lead absorber between source and detector, and keep on repeating with increasing the thickness of the absorber by adding more layers. (4) Remove the source completely and determine the background (with all layers of absorber in place). (5) The net count-rate from the sample is taken as a measure of the radiation intensity, and this is plotted against linear absorber thickness on log-linear graph paper. (6) Determine the half-thickness of lead for 60Co gamma rays and compare with the table value (Part I, §1-3.2.1, Table 1-3). (7) Explain any discrepancy between the results and the simple exponential law (i.e. not a straight line when plotted on log-linear graph paper).

EXERCISE 11. LIQUID SCINTILLATION COUNTING: DETERMINATION OF OPTIMUM COUNTER SETTINGS

Liquid scintillation counting has the advantage that the sample is dissolved in the liquid organic fluor and there is no self-absorption of the beta (or alpha) particle. For this reason, liquid scintillation counting is the method of choice for counting low-energy beta emitters. Both 3H and 14C, the two most important radiotracers in biological research, emit only beta particles of very low energy. Organic fluors also exhibit a very short decay time of the fluorescence produced, and resolving-time corrections are not necessary. In addition, liquid scintillation counters can be adapted to sample changers which allow automatic counting of large numbers of samples. The general description of a liquid scintillation counter is found in Part I, §1-2.4. The following experiment is designed to illustrate its use, including methods to correct for 'quenching'. An optional exercise demonstrating Cerenkov counting is also presented.

MATERIALS AND EQUIPMENT

( 1 ) Liquid scintillation counter, refrigerated or at ambient temperature (similar to Packard Instrument Co. Model 314 EX series) (2) Liquid fluor system: Toluene 0.5 litre PPO (2-5 diphenyloxazole) 4 g/1 POPOP ( 1,4-bis-2-5 phenyloxazoloyl) 0.05 g/1 136 PART V. LABORATORY EXERCISES

PPO is the primary solute. POPOP is termed a secondary solute and is used to shift the fluorescence spectrum of PPO to longer wavelengths for better matching to the spectral response of photomultiplier tubes. This procedure increases the counting yield and is generally used in measurement of 3H. (3) Benzoic acid-7-14C dissolved in toluene: approx. 0.02 pCi/ml (0.8 kBq/ml). Benzoic acid-3H dissolved in toluene: approx. 0.1 pCi/ml (3.7 kBq/ml). Other available compounds of l4C and 3H soluble in toluene can also be used. (4) Chloroform (5) Sample counting vials

PROCEDURE

(1) Take sufficient time to read the instrument instruction manual and become thoroughly familiar with operating controls. (2) Fill the sample counting vials with equal measured volumes of the liquid scintillation solution (15 ml). (Pipette accuracy is not necessary.) (3) Pipette 100 pi of the 14C solution into one vial. Pipette 100 jul of the 3H solution into one vial. Use one vial containing only the liquid scintillation solution as a background vial. (4) For a dual-channel instrument: Set the lower discriminator of channel A at 10% of scale. Set the upper discriminator of channel A at 50% of scale. Set the lower discriminator of channel B at 50% of scale. Set the upper discriminator of channel B at Channel A will now record all pulses with pulse heights between 10 and 50% of scale, and channel B will record all pulses with pulse heights between 50% of scale and

(5) Determine the background count-rate Rb in both channels as a function of increasing high voltage. Begin at about 600 V. (6) Determine the count-rate of each sample in both channels as a function of increasing high voltage. At one particular voltage, the count-rate in channel A will be approximately equal to the count-rate in channel B. This voltage will very closely correspond to what is known as balance-point operation. At this point, a decrease in counts from channel A due to quenching will be offset by additional counts from channel B due to quenching. Balance-point operation provides a condition of high counting yield and at the same time a minimum sensitivity to quenching. The balance-point settings will not be the same for the two radionuclides. (7) Determine the apparent counting yield (e) at the balance-point settings for each radionuclide. At this point the extent of quenching (if any) in the benzoic acid standards is not known. APPLIEÍ) EXERCISES 137

EXERCISE 12. PREPARATION OF SAMPLES FOR LIQUID SCINTILLATION COUNTING

Since water and toluene are not appreciably miscible, the aim is to form an emulsion to bring the beta particles into intimate contact with the primary and secondary scintillators.

REAGENTS AND MATERIALS

(1) Scintillator mixture: Toluene (S-free) containing 4 g/1 diphenyl oxazole and 0.1 g/1 dimethyl; POPOP mixed with Triton X-100 in proportions of 2 to 1 (2) Same scintillator mixture as under (1), but without addition of Triton X-100 (3) 20 ml glass vials + caps (4) Pipettes (5) l4C sodium acetate solution (0.1 /iCi/ml or 3.7 kBq/ml) (6) Plasma (7) Three test tubes

PROCEDURE

(1) Pipette 1 ml 14C sodium acetate into two test tubes. (2) Add 9 ml water to one test tube (solution A) and 9 ml plasma to the other (solution B); mix well. (3) Pipette into three counting vials 1 ml each of solution A, solution B and water (background). (4) To each vial add 4 ml water and 15 ml scintillator mixture ( 1 ). Cap vials and shake well. (5) Repeat steps (3) and (4), this time adding 15 ml of scintillator mixture (2). (6) Set up the counter as in Exercise 11 and count the samples for 1 minute each. (7) Subtract appropriate background counts from the counts of solutions A and B. (8) On the basis of 1 ¡id (37 kBq) = 37 X 103 dis/s (2.22 X 106 dis/min), estimate the counting yield for all solutions, as in Exercise 11. (9) Repeat this exercise with tritiated water. Use a solution containing 0.5 juCi/ml (2 kBq/ml) 3H and alter the settings on the scintillation counter appropriately.

EXERCISE 13. QUENCH CORRECTION

It is incorrect to compare the count-rates of two biological samples unless it is certain that they are counting at the same efficiency. As quenching is quite variable, it is unlikely that the same counting efficiency will be observed even 138 PART V. LABORATORY EXERCISES between samples present in the same medium. The measurement of quenching entails estimating counting efficiencies and the use of the relationship:

counts/s becquerels = disintegrations per second = X 100 1 v efficiency (%)

There are at least three methods of estimating counting efficiency, i.e. (1) the internal standard method, (2) the channels ratio method, and (3) the external standard ratio method. These methods are illustrated by the following exercises.

A. THE INTERNAL STANDARD METHOD

In this method, each sample is counted; then a known amount (0.1 ml) of radioactivity is added to each and the samples are recounted. The counting efficiency is calculated as:

difference in counts/s Counting efficiency = X 100 Bq per 0.1 ml added

The activity of the sample (in Bq) is then calculated. These calculations can also be made using curies as the measure of activity. This method is most accurate but laborious and renders the sample useless for later counting.

REAGENTS

( 1 ) Haemolysed plasma or dark-coloured urine (2) l4C sodium acetate solution (0.1 //Ci/ml or 3.7 kBq/ml) (3) Tritiated water (0.5 ¿iCi/ml or 18.5 kBq/ml) (4) ,4C n-hexadecane (0.2 ¿iCi/ml or 7.4 kBq/ml) (5) 3H n-hexadecane (0.2 ¿tCi/ml or 7.4 kBq/ml)

PREPARATION OF SAMPLES

(1) Pipette 1, 2, 3 and 4 ml plasma or urine into four test tubes and dilute to 9 ml. Pipette 9 ml water into a fifth test tube. Prepare a duplicate series of test tubes in the same way. (2) To each test tube of the first series add 1 ml 14C sodium acetate; to each test tube of the second series add 1 ml tritiated water. ,(3) Mix the contents of each tube thoroughly. These solutions will be used in this and the following two exercises. APPLIEÍ) EXERCISES 139

PROCEDURE

(1 ) Pipette into each of five counting vials 1 ml from each of the l4C-series test tubes. (2) Repeat the procedure of step (1) with the 3H-series test tubes. (3) Into two counting vials pipette 0.1 ml l4C n-hexadecane and 3H n-hexadecane. (4) Into one counting vial pipette 1 ml water (background). (5) Add to each vial 4 ml water and 15 ml scintillator mixture (2:1 POPOP:Triton X-100 as in Exercise 12). (6) Count each sample for 1 minute and subtract background counts. (7) To the five counting vials of the 14C series, accurately pipette 0.1 ml 14C n-hexadecane. To the vials of the 3H series, pipette 0.1 ml 3H n-hexadecane. (8) Recount the samples and again subtract background counts. (9) Estimate the counting efficiency as indicated above.

B. THE CHANNELS RA TIO (CR) METHOD

The spectrum of a beta-emitting isotope will shift to lower energy values (downscale) on quenching. Hence, if the spectrum is divided into two parts (dual-channel instrument), the ratio of the lower part to the upper part will increase with quenching. By counting a selected series of quenched samples to cover the range of efficiencies in the unknown samples a graph relating the channels ratio to the counting efficiency can be produced. When an unknown sample is counted and the channels ratio calculated, the counting efficiency may be read from the graph. (Note: this technique has limited value when the samples have low count-rates, as the channels ratios may be subject to large errors.)

PROCEDURE

(1 ) Prepare two sets of samples, one containing 14C and the other 3H, as outlined in Exercise A. (2) With the 14C machine standard, set the two channels so that one gives approximately half the counts of the other; for instance, with l4C, the channels might be set to read between 50—1000 and 350—1000. (3) Count the samples and subtract the background counts for each channel. (4) Calculate the channels ratio for each sample. (5) From the known amount of activity present in each sample, calculate the efficiency of counting in the 50—1000 channel. (6) Prepare a graph relating counting efficiency to channels ratio. (7) Reset the counter with a 3H machine standard and repeat steps (3) to (6) with 3H samples. 140 PART V. LABORATORY EXERCISES

C. THE EXTERNA L STANDARD RA TIO (ESR) METHOD

This method depends on the observation that the gamma irradiation of a sample in a counting vial and scintillator blank solution with an external gamma source produces Compton electrons; these have properties similar to beta particles emitted by the 14C or 3H in the counting vial and are quenched to approximately the same extent. A series of samples covering a wide range of quenching is prepared. After each sample is counted, a gamma source is automatically brought alongside the counting vial and a one-minute count recorded. The counter automatically calculates a ratio of two parts of the spectrum of the Compton electrons, and this ratio can be related to the counting efficiency of the series. This method has the advantages of speed and of being applicable to mixtures of two isotopes. However, the Compton electrons do not behave exactly as the weaker beta particles in a sample. The ESR method is also affected by the volume of the sample being counted, and it is difficult to maintain a constant geometry between the gamma source and the sample.

PROCEDURE

(1) Use the same sets of samples as prepared in Exercise A, together with another sample containing 20 ml scintillator mixture. (2) Count all samples. After each count, bring the machine gamma source into position and record the counts and ESR. (3) Assuming that the scintillator blank solution is counted with 100% efficiency, calculate the efficiency of counting the plasma or^urine samples. Prepare a graph relating ESR to counting efficiency for'l4C and for 3H.

EXERCISE 14. REDUCING THE^FFECTS OF QUENCHING

Quenching of samples is generally associated with colour or with the presence of organic matter. Reduction of the effects of quenching can be achieved in many ways. These include: (1) removal of colour by oxidation, (2) removal of interfering organic material by 'wet oxidation', and (3) extraction of the molecules under study by conversion to a volatile gas during combustion in an atmosphere of oxygen. These methods are illustrated in the following exercises.

A. COLOUR REMOVAL

As colour is generally the main factor inducing quenching, the removal of colour also reduces quenching. The simplest such procedure involves oxidation with peroxides. APPLIEÍ) EXERCISES 141

MATERIALS AND EQUIPMENT

(1 ) Plasma showing some haemolysis or containing an indicator such as phenol red (2) l4C sodium acetate (0.1 ¿iCi/ml (3.7 kBq/ml)) (3) 3H water (0.5 ptCi/ml (18.5 kBq/ml)) (4) Scintillator solution (2:1 POPOP:Triton X-100) (5) Counting vials (6) Pipettes (7) Benzoyl or hydrogen peroxide (8) Test tubes

PROCEDURE

(1) Pipette 9 ml plasma into each of two test tubes. Ad 1 ml l4C to one tube (solution A) and 1 ml 3H solution to the other tube (solution B) and mix well. (2) Set up the following counting vials: 5 ml water (background) 1 ml solution A + 4 ml water 1 ml solution A + 1 ml benzoyl peroxide + 3 ml water 1 ml solution B + 4 ml water 1 ml solution B + 1 ml benzoyl peroxide + 3 ml water. (3) To each vial add 15 ml scintillator solution and shake well to mix. (4) Count all samples and estimate the counting efficiency by the channels ratio or external standard ratio methods (Exercise 13, B or C).

B. WET OXIDATION

Interfering substances sometimes must be removed by more severe oxidizing conditions than those provided by hydrogen peroxide. Such conditions, provided by a mixture of nitric and perchloric acids, are particularly suitable for estimating isotopes such as phosphorus-32. This exercise involves not only wet oxidation but also Cerenkov counting. This method of counting takes advantage of the ability of high-energy beta emitters such as 32P to produce in aqueous solutions 'Cerenkov light', which can be measured in scintillation counters and which makes the use of scintillation fluids unnecessary.

MATERIALS AND EQUIPMENT

(1) 3ÎP solution (1 pCi/ml (37 kBq/ml)) (2) Sheep faeces, ground with pestle and mortar (3) Two 10 ml measuring cylinders 142 PART V. LABORATORY EXERCISES

(4) Kjeldahl flask and digestion rack (5) 100 ml digestion mixture: 10 parts concentrated nitric acid:4 parts perchloric acid

PROCEDURE

(1) Weigh 1 g faeces into the first measuring cylinder and the Kjeldahl flask. (2) Pipette 1 ml 32P solution into each. (3) Fill up the volume in the measuring cylinder to 10 ml with water. (4) To the Kjeldahl flask add 20 ml digestion mixture. Boil on the Kjeldahl digestion rack for approximately 2 hours until only a few millilitres remain. (5) Cool the contents of the flask. Transfer it quantitatively to the second 10 ml cylinder and fill up to volume. (6) Transfer the contents of both cylinders to counting vials, using 5 ml additional water to rinse the cylinders. (7) Prepare a standard by pipetting 1 ml standard solution plus 14 ml water into a counting vial. (8) Prepare a water blank in a fourth vial. (9) Count for 10 minutes in a liquid scintillation counter using Cerenkov radiation (threshold 0.3 MeV). (10) Subtract the background readings from the other three counts, and determine the effectiveness of wet oxidation in reducing quenching.

C. OX Y GEN FLASK COMB USTION

In this method, the isotope, usually 14C, 3H or 3SS, is oxidized by combustion in an atmosphere of oxygen. The resulting gas is dissolved in a scintillator solution, transferred to a counting vial and counted.

MATERIALS AND EQUIPMENT

(1) One 2 litre Büchner flask with neoprene stopper and 5 cm neoprene tubing on the side-arm (2) Hoffman screw clamp for the side-arm (3) Platinum gauze basket for the sample, attached to sealed glass tubing inserted through neoprene stopper (4) Oxygen (5) 14C sodium acetate solution (33 kBq/ml) (6) Plasma (7) Scintillator solution: Toluene containing 0.6% PPO and 0.1% bis-MSB (p-bis-ortho-methyl-styryl benzene):ethanolamine:methanol (10:1:9) APPLIEÍ) EXERCISES 143

PROCEDURE

(1) Prepare two counting solutions by adding 1 ml 14C sodium acetate each to 9 ml water and to 9 ml plasma. (2) Pipette 0.1 ml of each solution into counting vials and add 20 ml scintillator solution. (3) Pipette 0.1 ml of each solution onto paper tissue and dry in an oven at 50°C for 10 minutes. (4) Attach a filter paper wick to the dried sample and place it in the platinum gauze basket. (5) Close the side-arm of the Büchner flask and flush the flask with oxygen for 30 seconds. (6) Working in a fume cupboard, light the wick and plunge the basket into the flask, pushing the stopper in firmly. Wait 5 minutes after complete ignition for the flask to cool. (7) Introduce 15 ml of scintillator solution quickly through the side-arm and swirl the flask until the gases are absorbed. (8) Transfer the contents of the flask to a counting vial with 5 ml scintillator solution. (9) Count all samples and compare.

EXERCISE 15. CERENKOV COUNTING IN A LIQUID SCINTILLATION COUNTER

Cerenkov counting can be a very useful counting procedure for many beta- emitting isotopes whose beta emissions exceed 0.26 MeV. Its major advantages are that sample preparation is greatly simplified, the cost of the counting solution is minimal, and chemical quench has no effect on the system. While it is not as efficient in detecting the emitted betas as liquid scintillation counting using a 'cocktail' containing fluors, the efficiency usually exceeds several-fold that which can be obtained with a GM counter and, in addition,the procedure is not disturbed by factors such as self-absorption and scatter. The purpose of this exercise is to introduce a Cerenkov counting procedure and to demonstrate quench correction. Since the number of light photons produced by a beta emission resembles that of tritium in a liquid scintillation cocktail containing fluors, the best procedures for counting will be those which produce the best results for tritium. 144 PART V. LABORATORY EXERCISES

MATERIALS AND EQUIPMENT

(1) Standard radioactive solution of 42 K or 32 P, or any other relatively energetic beta emitter. The solution should contain a known amount of activity per millilitre; this can approximate 0.01 pCi/ml (0.4 kBq/ml) and can be in an aqueous solution (2) Liquid scintillation counter with two independent channels (A and B) (3) A yellow dye. This can be an indicator, food dyes, a solution of a chromatic salt, etc. (4) Concentrated acid or base

PROCEDURE

A. Determination of counting efficiency

Fill a counting vial with 20 ml of water and add 100 pi of the standard radioactive solution to it. In another vial place only 20 ml of H20 to serve as the background. Set the lower discriminator of one channel as low as possible and the upper discriminator as high as possible. Using the gain control (or the attenuator, depending on the machine), determine the balance point, i.e. the setting of the gain control (or attenuator) that gives the maximum number of counts within the window. Count the sample and the blank. From the count-rate obtained and the known number of pCi (kBq) in the sample determine the counting efficiency. How does this compare with GM counting? How does it compare with liquid scintillation counting using a fluor?

B. Preparation of a quench correction chart

(1 ) Place 20 ml of water in nine counting vials. One of these is to be capped and labelled (on the cap) as the blank. To each of the remaining eight vials add 100 pi of the standard radioactive solution. Label these from 1 through 8. Vial No.l is capped and is considered the 'unquenched' sample. To vials Nos.2 through and including 7, add the yellow dye. Do this in a stepwise fashion — the least dye into vial No.2 and increasing amounts into the other vials. It is not desirable to have a very intense colouration in this series. Vial No.2 should be only slightly different in its colour from vial No.l. Vial No.8 is to be saved for additions of base or acid. (2) Using standard No.l, place both channels of the liquid scintillation counter at balance point. Determine the count in each channel. Then adjust the upper discriminator of channel A so that the count is reduced to 30% of the original count. For channel B adjust the lower discriminator so that the count is reduced to 70% of its original count. APPLIEÍ) EXERCISES 145

Now place the colour quench series in the counter and count all of the samples, including the background. Using the count-rate in channel A and the channels ratio (A/B), prepare a quench correction curve. For the efficiency evaluation, use the unquenched member of the series (No.l) as 100% and express the others in relation to this sample. How can you use this chart to correct for colour present in a sample? (3) Effect of chemicals on quenching: Count sample No.8 at the settings determined for the quench correction curve. To this sample add one drop of concentrated base or acid and count again. Add two drops and count again. Each time determine the count-rate and the channels ratio. What is the effect of chemicals on quenching? Discuss the possible uses of Cerenkov counting in your research programme.

SECTION B. APPLIED EXERCISES

EXERCISE 16. SAMPLING AND HANDLING OF ASSAY DATA

General

This exercise describes techniques that are applicable to all RIA methods and should be used in conjunction with the relevant assay exercise.

Exercise 16.1. Collection and storage of samples for radioimmunoassay

Blood (1 ) Collect blood from sheep, goats, cattle and other large animals via a jugular vein (jugular venepuncture). Cattle may also be bled from the tail vein; pigs can be bled from an ear vein and birds from a vein in the wing. (2) Avoid stress to animals during restraining and bleeding; stress can cause aberrant increases in hormone levels. (3) Many hormones are released episodically. Therefore, a single blood sample may not reflect accurately the overall concentration of hormone in blood. If samples are being taken daily, collect at the same time of each day to avoid effects of circadian rhythms, feeding, etc. Under circumstances of frequent sequential bleeding, an indwelling catheter is often advisable. (4) Immediately after collecting a blood sample, place the tubes in an ice-water bath to prevent deterioration of the hormone. 146 PART V. LABORATORY EXERCISES

4 8 12 16 10 100 Arithmetic Dose Log Dose

LOG IT

10 100 Log Dose

FIG. V- 7. Different forms of RIA standard curves: (a) tracer bound plotted against linear standard concentrations; (b) as a, but with logarithmic standard concentrations; (c) the logit value of percentage of zero tube plotted against logarithmic standard concentrations.

(5) Plasma from anti-coagulated blood or serum from clotted blood may be used for measuring most hormones. (6) To prepare blood collection tubes, place a rubber stopper in the tube and evacuate with a needle connected to a vacuum pump or syringe, which has been inserted through the stopper. (7) For collecting plasma, use the following anti-coagulation preparations: (a) 0.2 ml of a 15% solution of EDTA, potassium or lithium salts, per 20 ml of blood; (b) 300 USP units of sodium heparin per 20 ml of blood.

Milk

Milk samples may be collected from any normal quarter of the mammary gland. For preservation, store in a refrigerator for up to 1 week, a freezer for up to 6 months, or add a sodium azide preservation tablet. Samples should be well mixed and representative of the whole milking (i.e. not Strippings). The comments made in (3) above are also relevant.

Exercise 16.2. Construction of standard curves

In constructing the standard curve it is usual to plot some function of the bound counts against standard (or ligand) concentration. This may be bound counts/min, % bound, bound over free, free over bound, etc. The graph shown in Fig.V-7(a) is a standard curve where the percentage bound tracer against the APPLIEÍ) EXERCISES 147

standard concentration is on a linear scale. A semi-logarithmic plot of percentage tracer bound against the log concentration of standard is shown in Fig.V-7(b). It is common to express percentage bound as a percentage of the activity in the zero standard or MB tube of the standard curve and a frequently used plot is the so-called logit-log transformation (Fig.V-7(c)). This type of plot will in most cases yield a straight line rather than a curve and is used in most computer programs handling RIA data. The logit value is derived in the following manner: If B is the count of tracer bound to antibody in standards and unknowns,

B0 is the count of tracer bound in the presence of no unlabelled antigen, B! is the count of tracer bound in the presence of no antibody (NSB), then B - B) Y = X 100 B0-B then

where log n is the Naperian log and Y is %. For use in the calculation of RIA results, logit values can be conveniently read from logit-log graph paper, which is commercially available, or from the Tables in Appendix VI-11. Many computer programs are available to handle the raw data from radioimmunoassays, and many modern counting instruments contain microprocessors programmed for this purpose. Programs for processing of RIA data on programmable calculators are available.

BIBLIOGRAPHY

Programs for data processing in radioimmunoassay using the HP-41C programmable calculator. IAEA-TECDOC-252, IAEA, Vienna (1981); Suppl. Analysis of results from quality control specimens, IAEA, Vienna (1982).

FELDMAN, H., RODBARD, D., in Principles of Competitive Protein Building, Chaps.VII and VIII, Lipincott, Philadelphia (1971).

EXERCISE 17. PREPARATION AND TITRATION OF ANTISERA FOR PROGESTERONE ASSAYS

The types of bridge structures for both immunogen and tracer to be used for steroid assays are discussed in section IV-2.2. In the case of progesterone, 148 PART V. LABORATORY EXERCISES the labelling technique for the conjugate with an 1 lalpha-glucuronide-tyramine bridge is given (Exercise 18.1). To obtain an antiserum that will give the maximum assay sensitivity, it is necessary to use the 11 alpha-hemisuccinate-bovine serum albumin conjugate (see Fig.IV-3). With different methods of iodination, different immunogen conjugates may be required. These specialized immunogens are slowly becoming available from commercial suppliers, but in some cases it will be necessary to request these from the workers who originated the technique. The exercises which follow describe a standard method of immunization, of titrating antiprogesterone serum using an iodinated tracer and a second antibody used for separation (anti-rabbit gamma globulin). In Exercise 19.7 a titration procedure is described for a progesterone antiserum employing a 3H-labelled tracer and dextran-coated charcoal for separation.

Exercise 17.1. Immunization of sheep, goats, horses or donkeys against progesterone

MATERIALS AND EQUIPMENT

(1) Progesterone — bovine serum albumin conjugate (BSA) (2) Sodium chloride (3) Freund's Complete Adjuvant (4) 5 ml glass syringe (5) Disposable needles, short 21 G (6) Animal hair clippers or curved scissors (7) High-speed, small-volume mixer or syringe for emulsification (8) Distilled water

PREPARATION OF MATERIALS

(1) Physiological saline solution: dissolve 9 g of NaCl in 1000 ml of distilled water. Mix well. (2) Progesterone emulsion for immunization: mix 2 mg of progesterone-BSA with 1 ml of physiological saline solution and 1 ml Freund's Complete Adjuvant (for one animal). Mix well until material is white and very viscous. This is best done using a high-speed mixer. The hormone may also be emulsified by repeatedly aspirating through a syringe. It is very important to have a good emulsion. To determine if emulsion is complete, place a small drop of emulsion onto the surface of water. If well emulsified, the material will not disperse; if further emulsification is needed, the material will disperse over the surface of the water. APPLIEÍ) EXERCISES 149

IMMUNIZATION PROCEDURE

(1) Clip wool or hair from left side of midline on back of animal. (2) Using the syringe and needle, inject 2 ml (2 mg) of the emulsion intradermally. Divide the dose into about 10 sites, each of 200 pi. (3) Repeat (1) and (2) at weekly intervals for a total of six weekly injections alternating on the left then on the right side. (4) Then continue injections at monthly intervals. (5) After 3 months collect blood by jugular venepuncture. Allow blood to clot for a minimum of 3 h in a refrigerator at 4°C. Break clot loose from the tube and store for an additional 18 to 24 h at 4°C. (6) Centrifuge clotted blood and draw off antiserum. (7) Collect antiserum as above regularly 5 to 7 days after each booster injection. (8) Titrate antiserum by the procedure given below to monitor antibody titre. A usable titre is generally obtained after 6 to 9 months.

(Note: Rabbits may be immunized by the method described in Exercise 21.)

BIBLIOGRAPHY

VAITUKAITIS, J.L., ROBBINS, J.B., NIESCHLAG, E., ROS, C.T., A method for producing specific antisera with small doses of immunogen, J. Clin. Endocrinol. Metab. 33 (1971) 988—91.

Exercise 17.2. Titration of anti-progesterone serum and anti-rabbit gamma globulin

MATERIALS AND EQUIPMENT

(1 ) Sodium phosphate dibasic, anhydrous (Na2HP04)

(2) Sodium phosphate monobasic (NaH2P04H20) (3) Disodium ethylenediamine tetraacetate (EDTA) (4) Sodium chloride (NaCl) (5) Non-immune, normal rabbit serum (NRS) (6) Anti-progesterone serum prepared as in Exercise 17.1 (7) Sheep, goat or donkey anti-rabbit gamma globulin (ARGG) serum (8) Refrigerator and/or cold room set at 4°C (9) Refrigerated centrifuge (10) Gamma counter (11) Microlitre pipettes in 100 pi to 500 pi range (12) Disposable glass culture tubes (12 X 75 mm) (13) Test tube racks 150 PART V. LABORATORY EXERCISES

(14) Vortex mixer (15) Radioiodinated progesterone solution (" 1 x tracer") diluted to approximately 25 000 to 35 000 counts/min per 100 Ml in phosphate buffered saline, pH 7.0 containing 0.1% gelatin (16) Felt-tip pen (17) Sodium azide (18) Gelatin

PREPARATION OF MATERIALS

(1) Buffers (a) 0.01 M phosphate-buffered saline (PBS), pH 7.0: dissolve 1.42 g of

sodium phosphate dibasic, anhydrous (Na2HP04) in 1 litre of distilled water. Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04H20) in 1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04 solution to give a final pH of 7.0. Add 9 g of sodium chloride (NaCl) to 1 litre of PBS. Add 1 g/1000 ml of sodium azide as preservative. (b) Phosphate-buffered saline-gelatin (PBSG); dissolve 1 g of gelatin in 1 litre of PBS. Heat gently to dissolve gelatin. (c) Phosphate-buffered saline -0.05M EDTA (PBS-EDTA), pH 7.0: dissolve 18.6 g of disodium ethylenediamine tetraacetate in 1 litre of PBS. Adjust pH with 5N NaOH. (2) Prepare 1 : 400 dilution of NRS by adding 100 /il of NRS to 40 ml of PBS-EDTA (lc). (3) Since in general it is not possible to obtain as high titres for anti-progesterone as for anti-LH (cf. Exercise 21.5), it may be necessary to carry out more dilutions at low concentrations (i.e. 1 : 800 — 1 : 6400) and less at higher. These dilutions may be made as follows: To make 1 : 400, add 100 pi of antiserum to 40 ml of PBS-EDTA. Store in 500 pi aliquots at -20°C. To make 1 : 800, add 500 pi of 1 : 400 antiserum to 500 pi of 1 : 400 NRS. To make 1: 1600, add 500 pi of 1 : 400 antiserum to 1.5 ml of 1: 400 NRS. To make 1: 3200, add 250 pi of 1: 400 antiserum to 1.75 ml of 1: 400 NRS. To make 1: 6400, add 100 pi of 1: 400 antiserum to 1.5 ml of 1: 400 NRS. To make 1:10 000, add 100 pi of 1: 400 antiserum to 2.4 ml of 1: 400 NRS. To make 1: 20 000, add 50 jul of 1 : 400 antiserum to 2.45 ml of 1: 400 NRS. To make 1: 40 000, add 40 pi of 1: 400 antiserum to 3.96 ml of 1: 400 NRS. To make 1: 80 000, add 20 pi of 1: 400 antiserum to 3.98 ml of 1: 400 NRS. To make 1: 160 000, add 10 pi of 1: 400 antiserum to 3.99 ml of 1: 400 NRS. APPLIEÍ) EXERCISES 151

(4) Prepare dilutions of ARGG as follows: To make 1: 5, add 1 ml of ARGG to 4 ml of PBS-EDTA. To make 1:10, add 500 jul of ARGG to 4.5 ml of PBS-EDTA. To make 1: 20, add 250 p\ of ARGG to 4.75 ml of PBS-EDTA. To make 1: 25, add 200 pi of ARGG to 4.8 ml of PBS-EDTA.

(Note: These dilutions will depend on the avidity and affinity of the antibody/antigen systems.)

TITRATION PROCEDURE

(1 ) Write a protocol as shown in Table V-l. (2) Number assay tubes (glass) with felt-tipped pen. (3) Add 300 Ml of PBSG to all tubes except numbers 1 and 2. (4) Add 200 fil of 1: 400 NRS to tubes 3, 4, 19, 20, 35, 36, 51, 52, 67 and 68 (5) Add 200 p\ of each anti-progesterone serum dilution to tubes as indicated on the protocol. (6) Add 100 jul of lx tracer solution to all tubes. (7) Mix tubes well on a vortex mixer and incubate for 3 h at room temperature. (8) Add 200 pi of each ARGG dilution to tubes as indicated on the protocol. (9) Mix well on a vortex mixer and incubate for 16—24 h at 4°C (overnight). (10) Add 1 ml cold PBS to all tubes except numbers 1 and 2. (11) Centrifuge all tubes (except numbers 1 and 2) at 1000 g for 30 min at 4°C. Determine the correct centrifuge speed by applying the equation given in Appendix VI-8. (12) Carefully decant the supernatant from each tube by up-turning the tube and collecting the supernatant in a radioactive liquid waste container. Allow the tube to drain on to absorbent paper. (13) Count each tube in a suitable gamma counter and record counts (Table V-l).

CALCULATION OF ANTI-PROGESTERONE AND ARGG TITRES

(1) Determine mean counts per minute for each pair of duplicate tubes (Table V-l). (2) Calculate corrected counts/min for each antiserum dilution by subtracting the respective mean non-specific binding counts/min (NSB). (3) Determine corrected binding for each dilution by dividing corrected counts/min for each dilution by mean counts/min in total tubes. That is,

Corrected counts/min for each dilution X 100 = % binding Mean counts/min for total tubes 152 PART V. LABORATORY EXERCISES

TABLE V-1. PROTOCOL AND RESULTS FOR TITRATIONS OF ANTI-PROGESTERONE AND ARGG

Tube Identification PBSG Antiserum lx ARGG Mean %of No. (300 Ml) (200 Ml) (100 MD (200 /A) counts/ total min

1 Total counts - - + - 27 525 -

2 - - + - 3 NSB + 1 400a + 1 5 316 1.1 4 + 1 400a + 1 5 5 Antiserum + 1 400 + 1 5 2 400 7.6 6 + 1 400 + 1 5 7 + 1 1000 + 1 5 2 168 6.7 8 + 1 1000 + 1 5 9 + 1 10 000 + 1 5 2 283 7.1

10 + 1 10 000 + 1 5 11 + 1 20 000 + 1 5 1 982 6.0 12 + 1 20 000 + 1 5

13 + 1 40 000 + 1 5 1 458 4.1 14 + 1 40 000 + 1 5 15 + 1 80 000 + 1 5 1 319 3.6 16 + 1 80 000 + 1 5 17 + 1 160 000 + 1 5 1 101 2.8 18 + 1 160 000 + 1 5

a 19 NSB + 1 400 + 1 10 322 - 20 + 1 400a + 1 10 21 Antiserum + 1 400 + 1 10 4 954 16.8 22 + 1 400 + 1 10 23 + 1 1000 + 1 10 5 780 19.8 24 + 1 1000 + 1 10 25 + 1 10 000 + 1 10 5 230 17.8 26 + 1 10 000 + 1 10 27 + 1 20 000 + 1 10 3 578 11.8 28 + 1 20 000 + 1 10 29 + 1 40 000 + 1 10 2 477 7.8 30 + 1 40 000 + 1 10 31 + 1 80 000 + 1 10 2 064 6.3 32 + 1 80 000 + 1 10 33 + 1 160 000 + 1 10 1 116 2.9 34 + 1 160 000 + 1 10

a 35 NSB + 1 400 + 1 15 416 - APPLIED EXERCISES 153

TABLE V-l (cont.)

Tube Identification PBSG Antiserum lx ARGG Mean % of No. (300 (il) (200 MD (100 Mi) (200 MD counts/ total min

a 36 NSB + 1 400 + 1 15 37 Antiserum + 1 400 + 1 15 13 763 48.5 38 + 1 400 + 1 15 39 + 1 1000 + 1 15 15 139 53.5 40 + 1 1000 + 1 15

41 Antiserum + 1 10 000 + 1 15 13 487 47.5 42 + 1 10 000 + 1 15 43 + 1 20 000 + 1 15 10 597 37.0 44 + 1 20 000 + 1 15 45 + 1 40 000 + 1 15 7 432 25.5 46 + 1 40 000 + 1 15

47 + 1 80 000 + 1 15 4 955 16.5 48 + 1 80 000 + 1 15 49 + 1 160 000 + 1 15 1 927 5.5 50 + 1 160 000 + 1 15 51 NSB + 1 400a + 1 20 428 1.6 52 + 1 400a + 1 20 53 Antiserum + 1 400 + 1 20 13 683 48.2 54 + 1 400 + 1 20 55 + 1 1000 + 1 20 14 982 52.9 56 + 1 1000 + 1 20 57 + 1 10 000 + 1 20 12 523 43.9 58 + 1 10 000 + 1 20 59 + 1 20 000 + 1 20 11 683 40.9 60 + 1 20 000 + 1 20 61 + 1 40 000 + 1 20 7 430 25.4 62 + 1 40 000 + 1 20 63 + 1 80 000 + 1 20 4 565 15.0 64 + 1 80 000 + 1 20 65 + 1 160 000 + 1 20 1 827 5.1 66 + 1 160 000 + 1 20

a 67 NSB + 1 400 + 1 25 310 - 68 + 1 400a + 1 25

69 Antiserum + 1 400 + 1 25 10 460 36.9 70 + 1 400 + 1 25 71 + 1 1000 + 1 25 11 423 40.4 154 PART V. LABORATORY EXERCISES

TABLE V-9. (cont.)

Tube Identification PBSG Antiserum 1 x ARGG Mean % of No. (300 ill) (200 Ml) (100 Ml) (200 Ml) counts/ total min

72 Antiserum + 1 1000 + 1 : 25 73 + 1 10 000 + 1 : 25 9 909 34.9 74 + 1 10 000 + 1 : 25 75 + 1 20 000 + 1 : 25 7 707 26.9 76 + 1 20 000 + 1 : 25 77 + 1 40 000 + 1 : 25 4 954 16.9 78 + 1 40 000 + 1 : 25 79 + 1 80 000 + 1 : 25 2 753 8.9 80 + 1 80 000 + 1 : 25 81 + 1 160 000 + 1 : 25 1 376 3.9 82 + 1 160 000 + 1 : 25 a 1 : 400 NRS only, no antiserum.

(4) Select the greatest dilutions of anti-progesterone serum and ARGG that bind 20-50% of the lx tracer. For example, in Table V-l, select 1 : 40 000 anti- progesterone and 1 : 20 ARGG.

EXERCISE 18. RADIOIODINATION OF SEX STEROIDS

The comments made in Exercise 17 with respect to steroid conjugates also apply to this exercise.

MATERIALS AND EQUIPMENT

(1) 1251 solution for radioimmunoassay: sodium salt, highly specific, carrier free

(2) Sodium phosphate dibasic, anhydrous (Na2HP04) (3) Sodium phosphate monobasic (NaH2P04H20) (4) Gelatin (5) Distilled water (6) Sodium chloride (NaCl) (7) 1 la-progesterone glucuronide-tyramine conjugate (8) Testosterone-3(0-carboxymethyl) oxime (9) Histamine APPLIEÍ) EXERCISES 155

(10) Chloramine-T (11) Sodium metabisulphite (12) Cysteine hydrochloride (13) Dioxane. Before use, the dioxane should be passed through a chromatography column containing aluminium oxide (chromatography grade) with 0.5% of water added (14) Tri-n-butylamine (15) Iso-butylchloroformate (16) 0.1 M NaOH (17) 0.1MHC1 (18) Ethyl acetate (19) Anhydrous sodium sulphate (20) Thin layer chromatography (TLC) plates, silica gel on plastic backing (21) TLC elution mixture, chloroform: methanol: acetic acid, 90:10: 1 (22) TLC glass tank (23) 15 ml glass-stoppered centrifuge tubes (24) Micropipettes in the range 10—400 /d with disposable tips (25) Pasteur pipettes (26) Small glass vials, about 1 ml capacity (27) Glass scintillation vials (28) Sheets of filter paper, about 20 X 20 cm (29) Waterbath containing ice (30) Vortex mixer (31 ) 5-ml glass-stoppered tubes (32) Radiation monitor with a sodium iodide crystal probe sensitive to gamma radiation. The probe should be fitted with a standard detachable cap of a heavy metal (stainless steel) having a slit about 1 cm long and 0.5 mm wide. (33) Cylinder of nitrogen.

PREPARATION OF REAGENTS

(1) Buffers

(a) 0.5M sodium phosphate buffer, pH 7.5: dissolve 7.1 g of sodium phos-

phate dibasic, anhydrous (Na2HP04) in 100 ml of distilled water.

Dissolve 6.9 g of sodium phosphate monobasic (NaH2P04H20) in

100 ml of distilled water. Mix approximately 80 ml of Na2HP04

solution with 20 ml of NaH2P04H20 solution to give a final pH of 7.5. Store frozen in small aliquots. This will tend to crystallize out at low temperatures, and will require warming and re-dissolving before use. 156 PART V. LABORATORY EXERCISES

_ 20 X 20 cm TLC Plate

Start line r~ 2 cm L_

FIG. V-8. The marking of the TLC plate.

(b) 0.05M sodium phosphate buffer, pH 7.5: dilute 0.5M sodium phosphate buffer, pH 7.5, 1 : 10 with distilled water. (c) 0.25M sodium phosphate buffer, pH 7.5: dilute 0.5M sodium phosphate buffer, pH 7.5, 1: 2 with distilled water.

(2) Solutions for progesterone iodination

(a) Progesterone solution: dissolve the 1 la-progesterone glucuronide- tyramine conjugate in ethanol to give a concentration of approximately 10/ig/ml. Aliquot several vials containing 100//I and store at —20°C. Do not use automatic de-frost freezer for storing hormones. (b) Chloramine-T solution (5 mg/ml in buffer): dissolve 25 mg of chloramine-T in 5 ml of 0.25M sodium phosphate buffer, pH 7.5. Make fresh on day of use. Store small portions of dry chloramine-T in vials at 0°C in a container containing dessicant. (c) Cysteine hydrochloride solution (10 mg/ml): dissolve 50 mg of cysteine hydrochloride in 5 ml 0.05M sodium phosphate buffer, pH 7.5. Make fresh on day of use.

(3) Solutions for testosterone iodination

(a) Chloramine-T solution (5 mg/ml in water): dissolve 0.5 g chloramine-T in 100 ml of distilled water. Prepare fresh on the day of use. APPLIEÍ) EXERCISES 157

(b) Sodium metabisulphite solution (30 mg/ml): dissolve 3 g sodium metabisulphite in 100 ml of distilled water. Prepare fresh on the day of use. (c) Histamine reagent: dissolve 1.1 mg of histamine in 5 ml 0.5M phosphate

buffer, pH 8.0 (adjust pH 7.5 buffer with Na2HP04 solution to give pH 8.0, using a pH meter). (d) Tri-n-butylamine solution: add 1.0 ml of tri-n-butylamine to 5 ml dioxane. (e) Iso-butylchloroformate solution: add 0.5 ml iso-butylchloroformate to 5 ml dioxane.

(4) Thin layer chromatography

Before commencing iodination prepare the chromatography tank by lining the walls with discs of filter paper and allowing them to equilibrate with approximately 100 ml TLC elution mixture placed in the bottom of the tank. The lid should be sealed firmly to the top of the tank. The TLC plate should be marked as shown in Fig.V-8.

Exercise 18.1. Iodination procedure for progesterone

(1 ) All procedures must be carried out in a well-ventilated fume cupboard or hood at normal room temperature. (2) Pipette 40 pi of the progesterone conjugate solution into a 15 ml glass- stoppered centrifuge tube (preferably set into a lead castle) mounted in a fume cupboard. Dry down in a stream of nitrogen and dissolve in 10 pi of the 0.25M sodium phosphate buffer. (3) Add 37 MBq (1 mCi) 125I (approximately 10 pi); mix by vortexing. (4) Add 10 pi of the chloramine-T solution in buffer and vortex vigorously for 30 seconds. (5) Immediately add 10 pi of cysteine hydrochloride solution and again mix briefly. (6) Add 200 pi of 0.05M phosphate buffer, pH 7.5 and mix again. (7) The centrifuge tube containing the reaction mixture should now be checked in a gamma counter for counts/10 s. (8) Add 400 pi of ethyl acetate and vortex for 2 min, and allow to stand until the phases cleanly separate. Remove the top solvent layer with a pipette, a little at a time, until approximately 300 pi has been removed, and transfer to a small glass vial. Take care not to remove any aqueous layer. Check the counts of both solvent and aqueous layers and see that 30-50% of the count is in the ethyl acetate layer. 158 PART V. LABORATORY EXERCISES

(9) The ethyl acetate layer is now applied to the TLC plate along the start line as a streak. (Care, this is a difficult procedure.) Allow the ethyl acetate layer to rise up in a capillary tube and apply it to the start line, moving it smoothly along the line until empty. Repeat the process until all the ethyl acetate layer has been applied as equally as possible along the length of the start line. The TLC plate is then allowed to dry by normal air currents or a small fan, and is then transferred to the equilibrated tank. The separation is allowed to continue for approx. h until the liquid level reaches 1—2 cm from the top. The plate is then removed and dried for 30 min, using a small fan. ( 10) The plate is placed in a suitably sized plastic bag and either scanned with a TLC scanner, or an autoradiograph may be prepared, using X-ray film. It is also possible to use a radiation monitor, with the probe fitted with a slit made of heavy metal, such as stainless steel. The probe may be passed manually over the plate. The bands may be marked sufficiently accurately by observing the meter. If "Screen" X-ray film only is available, cut in a dark-room to a suitable size to fit the TLC plate. Carefully place the film on the TLC plate, and mark the ordinates and start-line in pencil, using a suitable dark-light for illumina- tion. Leave in the dark-room for a 48 h exposure time before removing the X-ray film and developing, then drying and replacing in position on the chromatogram. Two bands are obtained on the X-ray film, monitor or scanner, about midway from the origin and solvent front and the leading band should be marked on the plastic bag enclosing the chromatogram with the back of a pair of scissors. Without removing the plate from the bag, the active section sljould be carefully cut out and removed from the remaining part of the plastic bag. (Care — this is highly radioactive! Carry out under a fume hood. ) (11) In a fume cupboard the strip of thin layer plate is carefully scraped, a section at a time, into a glass bottle (a scintillation vial is suitable). As each section is cleaned of silica gel, the backing material is cut away, until the whole area of gel has been transferred to the bottle. To the silica gel powder scraped from the plate add 3 ml ethanol, mix and stand overnight. Aspirate off the ethanol to a centrifuge tube and centrifuge to remove any solid matter. Wash the powdered gel with another 3 ml of ethanol, centrifuge and amalgamate the supernatants. This is the concentrated label and can be stored at 4°C. For working strength, it can be diluted 1: 200 by dispensing a suitable aliquot and drying it down in a stream of nitrogen. The residue may then be taken up in the assay diluent to give the correct dilution, with vigorous shaking. APPLIEÍ) EXERCISES 159

This should be checked in a gamma counter by dispensing 100 p\ into an assay tube and counting. It should give a count in the order of 30 000 counts/min. The dilution may be adjusted to achieve this count, but if it is widely different from this, the labelling procedure should be re-investigated, and the label treated with caution. If all is in order, this is the working strength and may be used in assays. The concentrated label should be diluted only when required and may be stored at 4°C for about 2 months before the activity becomes too low.

Exercise 18.2. Iodination procedure for testosterone

(1) All procedures must be carried out in a well-ventilated fume cupboard or hood at normal room temperature. (2) In a glass-stoppered tube, large enough to hold 5 ml, add 10 yul of histamine reagent. (3) Add 37 MBq (1 mCi) 125I (approximately 10 /xl); mix by vortexing. (4) Add 10 /d of chloramine-T solution in water, mix thoroughly and stand for 60 s at room temperature. (5) Add 10 pi of sodium metabisulphite solution to stop the reaction. Cool to 1°C. (6) Dissolve 1.26 mg testosterone-3(0-carboxymethyl) oxime in 100 pi dioxane in a 5-ml glass-stoppered tube and cool to 10°C. (7) Add 10 jul tri-n-butylamine solution cooled to 10°C. (8) Add 10 pi iso-butylchloroformate solution cooled to 10°C. (9) Mix intermittently for 30—45 min in a waterbath containing ice. (10) Add 1.5 ml of dioxane (at the same temperature), mix and transfer 50 pi of this diluted activated oxime to the iodination reaction mixture. Add 10 pi of 0.1M NaOH and stand in the ice/water bath (5—10°C) with occasional mixing. Care must be taken with the temperature as the mixture freezes at 0°C. It is advisable to place the tube in a beaker containing dioxane, and place that in the ice/water bath to prevent freezing.

Extraction

(11) Acidify the reaction mixture with 1 ml 0.1 M HCl and then add 1 ml ethyl acetate and vortex for 1 second to extract the activators. Discard ethyl acetate layer by aspiration. (12) Add 1 ml 0.1M NaOH, followed by 1 ml 0.5M sodium phosphate buffer,

pH 7.5 (after adjusting pH to 7.0 with NaH2P04 solution using a pH meter). (13) Extract the reaction mixture twice with 0.5 ml portions of ethyl acetate by briefly vortexing the tube (do not use more than 1.5 ml in total) and combine and dry the acetate extracts over a small amount of anhydrous sodium 160 PART V. LABORATORY EXERCISES

sulphate in a stoppered glass tube. The amount of sulphate should be such that it remains solid, but care should be taken to prevent too large an excess. The extract can be left for 2—3 days before the next step.

Thin layer chromatography

(14) Reduce the volume of the ethyl acetate extract to about 300 jul, using air from a pasteur pipette connected to a blowball or a pump. (15) Follow out the procedure given for the iodination of progesterone in Exercise 18.1 from step (9). Only one band of active material is present on the chromatogram. (16) As with progesterone, the concentrated label may be stored at 4°C for 2 months. (17) A suitable aliquot of the concentrated label should be dried down in a stream of nitrogen and diluted with assay buffer so that 100 jul gives a count of about 15 000 counts/min. Only sufficient for one assay should be diluted at any one time.

EXERCISE 19. RADIOIMMUNOASSAY OF PROGESTERONE IN SERUM AND MILK

Several different techniques are available for assaying progesterone involving different radiolabelled hormones (3H, 125I), separation procedures (double-antibody, dextran-charcoal, protein A, solid phase), extraction with organic solvent, and counting techniques (gamma counting, liquid scintillation counting). The exercises that follow describe a technique for extracting progesterone from blood and monitoring procedural losses, together with a number of different assay procedures.

Exercise 19.1. Preparation of samples

Progesterone is bound to binding proteins in blood. To estimate accurately concentrations of progesterone in serum or plasma, it is necessary to dissociate progesterone from its binding protein. This is simply done by extraction with petroleum ether. During the process of recovery certain losses of endogenous progesterone occur because extraction may be incomplete, some progesterone may stick to the glassware and some solvent may be spilled in the process. To monitor procedural losses of progesterone, 3H progesterone is added to the samples before extraction is begun. After extraction is complete the 3H progesterone is quantified in a liquid scintillation counter and recovery is determined. The concentration of progesterone determined by radioimmunoassay is then corrected for the procedural loss. APPLIEÍ) EXERCISES 161

If 3H progesterone is used as the tracer for the assay, 3H progesterone should not be used for monitoring procedural losses in the samples to be assayed. It is suggested that losses be monitored in a second set of samples handled simultaneously and in exactly the same manner.

MATERIALS AND EQUIPMENT

(1) Petroleum spirit, 40-60°BP (60-80°C in hot climates), free from aromatic hydrocarbons, preferably re-distilled in an all-glass still. Each batch must be tested for recovery from water before widespread use (2) 18 X 150 mm glass tubes. If used more than once, these tubes should be washed in distilled water and preferably baked in an oven at 250°C before re-use. If handshaking only is possible, these tubes must have ground-glass stoppers (3) 12 X 75 mm glass tubes. If used more than once, prepare as in (2) (4) Vortex mixer (5) Methanol, analytical reagent (6) Dry ice (solid carbon dioxide) + technical methanol or technical acetone bath of a size suitable to hold a rack containing the extraction tubes (7) 3H progesterone: prepare a dilution of 3H progesterone, which may be obtained from a supplier of radiochemicals, by drying down a suitable aliquot in a stream of nitrogen and dissolving in pure methanol, so that 50 fü in a suitable scintillation cocktail gives a count in a beta scintillation counter of approximately 2000 counts/min (8) A liquid scintillation counter, set to measure 3H (9) A scintillation cocktail suitable for RIA. These are available from many suppliers, and are usually based on toluene or xylene with 2,5-diphenyloxazole (PPO) and 1,4-bis-2—5 phenyloxazoloyl (POPOP) (see Exercise 11 )

PROCEDURE

The methods for extraction and for monitoring procedural losses are as follows:

(1) Dispense 500 p 1 of each serum or plasma sample into an 18 X 150 mm glass tube. (2) Dispense 500 jd of distilled water into a second set of 18 X150 mm glass tubes. (3) Add 50 fil 3H progesterone (reagent (7)) to all samples in the first set of tubes containing serum or plasma (1). Simultaneously add 50 jul 3H progesterone to two scintillation vials labelled "total counts". Set "total counts" and two "background" empty vials aside for later counting. 162 PART V. LABORATORY EXERCISES

(4) Allow the 3H progesterone to equilibrate in the samples for a minimum of 2 h at room temperature or 12—18 h at 4°C. This is to ensure binding to proteins (e.g. CBG) of the label simulating the endogenous situation in the samples. (5) Add 5 ml of petroleum spirit to each tube containing plasma sample. (6) Mix content of the tubes well on a vortex mixer for 1 min or shake by hand for 1 min. In this case the tubes require ground-glass stoppers, which may be sealed by dipping the stopper in distilled water. (7) Place tubes in a dry ice-acetone or -methanol bath to freeze the bottom aqueous layers (serum or water). If placed in a freezer, one must be absolutely sure that the freezer is explosive-proof and at sufficiently low temperature to freeze the aqueous layer. (8) Pour solvent layer into the second set of 18 X 150 mm tubes containing water as in (2) above. (9) Mix the second set of tubes for 30 s and freeze the aqueous layer as in (7) above. (10) Pour solvent (containing progesterone) into corresponding 12 X 75 mm glass tubes and dry with a gentle stream of nitrogen or air. If available, a gently heated block heater accelerates this process (not above 40°C). (11) After the serum has thawed extract again by repeating steps (5)—( 10) above. Combine the second extract with the first and dry. (12) Add 1 ml of assay diluent to the dried extract. Mix well and place in refrigerator at 4°C for 12-18 h. (13) Transfer 100 /il of each reconstituted extract to scintillation vials and 100 jul of assay diluent to the "total counts" and "background" vials. (14) Add 10 ml scintillation fluid to all vials, mix, and quantify radioactivity in a liquid scintillation counter. (15) Determine extraction recovery by the following equation:

counts/min in sample tubes —mean counts/min in background tubes Recovery % = X 100 mean counts/min in total tubes

(16) Adjust concentrations of progesterone determined by radioimmunoassay according to the recovery for each sample as follows:

uncorrected concentration Corrected concentration = recovery

(17) The remaining reconstituted extracts are used for the radioimmunoassays described below. APPLIEÍ) EXERCISES 163

(Note: Provided that the precision is acceptable for the extraction recovery step (c.v. 5%), a mean extraction recovery figure can be used substituting the need for monitoring individual recoveries. It is preferable that such a mean extraction figure is established for each batch of organic solvent cited. Testing has to be done for a large number of samples.)

Exercise 19.2. Double-antibody separation with 125I-progesterone

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04) (2) Sodium phosphate monobasic (NaH2P04H20) (3) Disodium ethylenediamine tetraacetate (EDTA) (4) Gelatin (5) Non-immune, normal rabbit serum (NRS) (6) Rabbit anti-progesterone 11 alpha -bovine serum albumin serum (see Exercise 17) (7) Sheep, goat or donkey anti-rabbit gamma globulin (ARGG) (8) Progesterone for standard solutions (9) Refrigerator or cold room set at 4°C (10) Refrigerated centrifuge (11) Gamma counter (12) Microlitre pipettes in 100—500 /il range (13) Disposable glass culture tubes (12 X 75 mm) (14) Test tube racks (15) Vortex mixer (16) Radioiodinated progesterone diluted to approximately 25 000 to 35 000 counts/min per 100 jul in phosphate-buffered saline, pH 7.0 containing 0.1 % gelatin (see Exercise 18.1) (17) Felt-tip pen (18) Distilled water (19) Sodium azide (20) Methanol (21) Sodium chloride (22) Magnetic and heated stirrer (optional) 164 PART V. LABORATORY EXERCISES

PREPARATION OF MATERIALS

(1) Buffers

(a) 0.01M phosphate-buffered saline (PBS), pH 7.0: dissolve 1.42 g of sodium

phosphate dibasic, anhydrous (Na2HP04) in 1 litre of distilled water. Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04H20)in 1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04H20 solution to give a final pH of 7.0. Add 9 g of sodium chloride (NaCl) to 1 litre to give PBS. Add 1 g of sodium azide per litre as preservative. (b) Phosphate-buffered saline-gelatin (PBSG): dissolve 1 g of gelatin in 1 litre of PBS. Heat gently to dissolve gelatin, using a magnetic, heated stirrer (if available). (c) Phosphate-buffered saline 0.05M EDTA (PBS-EDTA),pH 7.0: dissolve 18.6 g of disodium ethylene diamine tetraacetate in 1000 ml of PBS. Adjust pH with 5N NaOH.

(2) 1: 400 NRS: add 1 ml of normal rabbit serum (or species in which the first antiserum is raised) to 400 ml of PBS-EDTA. Mix well. (3) Diluted anti-progesterone serum. Prepare appropriate dilution of the anti- progesterone serum as described in Exercise 17.2, using 1:400 NRS. Note that the correct 'bridge' must be used to obtain adequate binding and sensitivity. (4) Diluted ARGG: prepare appropriate dilution of the ARGG as described in Exercise 17.2 using PBS-EDTA. (5) Standard progesterone solutions: A pure progesterone standard preparation (MW 315 daltons) should be obtained from a reputable supplier, and a primary standard solution must be prepared in pure ethanol. A convenient dilution for this is 1 mg/ml. This may be prepared by weighing the progesterone on an accurate balance, say, 100 mg. No attempt should be made to weigh this quantity exactly, but the amount weighed should make up to the relevant volume with ethanol, e.g. if the accurately weighed amount of progesterone is 92.5 mg, then make up to 92.5 ml. This solution remains stable for long periods if stored at 4°C. An intermediate standard can be prepared by diluting the primary standard 1/1000 with ethanol, i.e. 100 pi primary standard transferred accurately to a volumetric flask and made up to 100 ml with ethanol. This has a concentration of 1 pg/ml progesterone, and may be used for dilution to working standards, as given below for milk or plasma. Methanol may be used as an alternative to ethanol as diluent. APPLIEÍ) EXERCISES 165

TABLE V—2. PROTOCOL FOR THE PROGESTERONE RADIOIMMUNOASSAY (Extracted plasma serum)

Tube Identification PBSG Standard Sample Antiserum lx ARGG No. (Ml) (Ml) (Ml) (Ml) (Ml) (Ml)

1 Total counts 0 0 0 0 100 0 2 0 0 0 0 100 0 3 0 0 0 0 100 0 4 NSB 500 0 0 200a 100 200 5 500 0 0 200a 100 200 6 500 0 0 200a 100 200 7 B„ control 500 0 0 200 100 200 8 500 0 0 200 100 200 9 500 0 0 200 100 200 10 Stnd-O.S ng/ml 300 200 0 200 100 200 11 Stnd-1.0 ng/ml 300 200 0 200 100 200 12 Stnd-2.0 ng/ml 300 200 0 200 100 200 13 Stnd-4.0 ng/ml 300 200 0 200 100 200 14 Stnd-8.0 ng/ml 300 200 0 200 100 200 IS Stnd-16.0 ng/ml 300 200 0 200 100 200 16 Stnd-32.0 ng/ml 300 200 0 200 100 200 17 Sample #1 300 0 200 200 100 200 18 300 0 200 200 100 200 19 Sample #2 300 0 200 200 100 200 20 300 0 200 200 100 200 21 Sample #3 300 0 200 200 100 200 22 300 0 200 200 100 200

(continue samples as needed in duplicate)

B0 control 500 0 0 200 100 200 500 0 0 200 100 200 500 0 0 200 100 200 Stnd-0.5 ng/ml 300 200 0 200 100 200 Stnd-1.0 ng/ml 300 200 0 200 100 200 Stnd-2.0 ng/ml 300 200 0 200 100 200 Stnd-4.0 ng/ml 300 200 0 200 100 200 Stnd-8.0 ng/ml 300 200 0 200 100 200 Stnd-16.0 ng/ml 300 200 0 200 100 200 Stnd-32.0 ng/ml 300 200 0 200 100 , 200 a 1:400 NRS only, no antiserum. 166 PART V. LABORATORY EXERCISES

(6) After first drying down the equivalent working standard in a stream of nitrogen or air, standard solutions of 64 ng/ml, 32 ng/ml, 16 ng/ml, 8 ng/ml, 4 ng/ml, 2 ng/ml, 1 ng/ml and 0.5 ng/ml are prepared as follows:

For the 64 ng/ml standard, add 10 ml of PBSG to 640 pi of dried stock (1 Mg/ml); For the 32 ng/ml standard, add 10 ml of PBSG to 320 pi of dried stock; For the 16 ng/ml standard, add 10 ml of PBSG to 160 pi of dried stock; For the 8 ng/ml standard, add 10 ml of PBSG to 80 jul of dried stock; For the 4 ng/ml standard, add 10 ml of PBSG to 40 pi of dried stock; For the 2 ng/ml standard, add 10 ml of PBSG to 20 pi of dried stock; For the 1 ng/ml standard, add 20 ml of PBSG to 20 pi of dried stock; For the 0.5 ng/ml standard, add 40 ml of PBSG to 20 pi of dried stock.

Mix all solutions well. Freeze 1 ml aliquots in well-sealed tubes for future use. (7) Radioiodinated progesterone solution (called "lx tracer"); for labelling procedure see Exercise 18.1.

BIBLIOGRAPHY

CORRIE, J.E.T., HUNTER, W.M., MacPHERSON, J.S., A strategy for radioimmunoassay of plasma progesterone with the use of a homologous-site 12SI-labelled radioligand, Clin. Chem. (Winston-Salem, N.C.) 27 (1981) 594-99.

Exercise 19.3. Radioimmunoassay procedure for extracted plasma or serum

(1 ) Write a protocol as shown in Table V-2. (2) Number tubes with a felt-tip pen. (3) Add PBSG to the various tubes as indicated on protocol (4) Add sample (extracted plasma or serum) and standard solution in the volumes indicated on protocol shown in Table V—2. (5) Add 200 Ml of 1:400 NRS to tubes 4, 5 and 6 and 200 pi of diluted antiserum to the remaining tubes (except 1, 2 and 3). (6) Add 100 /xl of the lx tracer solution to all tubes. (7) Mix tubes well on a vortex mixer and incubate at room temperature for 3 h. (8) Add 200 pi of diluted ARGG to all tubes except numbers 1, 2 and 3. (9) Mix tubes well on a vortex mixer and incubate at 4°C for 16-24 h (overnight). (10) Add 1 ml of cold PBS to all tubes except numbers 1, 2 and 3. Do not mix. APPLIEÍ) EXERCISES 167

(11) Centrifuge all tubes except numbers 1, 2 and 3 at 1000 g for 30 min at 4°C. Determine correct centrifuge speed for 1000 g by using the equation in Appendix VI-8. (12) Carefully decant the supernatant of each tube into a radioactive liquid waste container. Keep the tube inverted and allow to drain on absorbent paper. (13) Quantify radioactivity in the precipitates remaining in the tubes with a gamma counter. Record counts.

CALCULATION OF RESULTS

(1) Determine uncorrected binding in the B0 control tubes: This is done by

calculating the mean counts per minute in the B0 tubes and dividing by the mean counts/min in tubes 1, 2, and 3. That is,

Mean counts/min in B0 tubes Uncorrected B0 binding (%) = X 100 Mean counts/min in total tubes

(2) Determine non-specific binding (NSB): This is done by dividing the mean counts/min in tubes 4, 5, and 6 by mean counts/min in tubes 1, 2, and 3. This number should be less than 5%.

Mean counts/min in NSB tubes Non-specific binding % = X 100 Mean counts/min in total tubes

(3) Determine corrected B0 binding:

Corrected B0 binding = Uncorrected B0 binding - NSB

When expressed as per cent of mean total counts this number should range from 20 to 40%.

(4) Since corrected B0 binding is the maximum possible in a particular assay, consider B„ binding as 100%.

(5) Determine corrected B0 binding for each standard solution: i.e.

Mean counts/min in standard - mean NSB counts/min % of B0 binding = X 100 Corrected B0 binding counts/min 168 PART V. LABORATORY EXERCISES

TABLE V—3. PROTOCOL FOR THE PROGESTERONE RADIOIMMUNOASSAY IN MILK

Tube Identification PBSG Standard Sample Antiserum 1 x ARGG No. (Ml) (Ml) (Ml) (Ml) (Ml) (Ml)

1 Total counts 0 0 0 0 100 0 2 0 0 0 0 100 0 3 0 0 0 0 100 0 4 NSB 350 0 0 200a 100 200 5 350 0 0 200a 100 200 6 350 0 0 200a 100 200

7 B0 control 350 0 0 200 100 200 8 350 0 0 200 100 200 9 350 0 0 200 100 200 10 Stnd-1.0 ng/ml 300 50 0 200 100 200 11 Stnd-2.0 ng/ml 300 50 0 200 100 200 12 Stnd-4.0 ng/ml 300 50 0 200 100 200 13 Stnd-8.0 ng/ml 300 50 0 200 100 200 14 Stnd-16.0 ng/ml 300 50 0 200 100 200 15 Stnd-32.0 ng/ml 300 50 0 200 100 200 16 Stnd-64.0 ng/ml 300 50 0 200 100 200 17 Sample # 1 300 0 50 200 100 200 18 300 0 50 200 100 200 19 Sample # 2 300 0 50 200 100 200 20 300 0 50 200 100 200 21 Sample # 3 300 0 50 200 100 200 22 300 0 50 200 100 200

(continue samples as needed in duplicate)

B„ control 350 0 0 200 100 200 350 0 0 200 100 200 350 0 0 200 100 200 Stnd-1.0 ng/ml 300 50 0 200 100 200 Stnd-2.0 ng/ml 300 50 0 200 100 200 Stnd-4.0 ng/ml 300 50 0 200 100 200 Stnd-8.0 ng/ml 300 50 0 200 100 200 Stnd-16.0 ng/ml 300 50 0 200 100 200 Stnd-32.0 ng/ml 300 50 0 200 100 200 Stnd-64.0 ng/ml 300 50 0 200 100 200

a 1:400 NRS only, no antiserum. APPLIEÍ) EXERCISES 169

(6) Plot a standard curve on semi-log graph paper using the known concentration of hormone in standard solutions and binding for each concentration. See Exercise 16.2 for an example. (7) Determine binding for each unknown sample by subtracting mean counts/min in NSB tubes from mean counts/min for each unknown sample and dividing by

corrected B0 binding.

Mean counts/min in samples — mean NSB counts/min % of B0 binding = — X 100 Corrected B0 binding çounts/min

(8) From the standard curve, determine the concentration of hormone which has the same binding. This concentration (ng/ml) is the concentration in the unknown sample, which should be corrected for recovery (Exercise 19.1).

Exercise 19.4. Radioimmunoassay procedure for cow milk

This method is suitable for pregnancy testing, and does not require an extraction step.

(1) Milk sampling: A well-mixed sample of milk should be taken. It is important to standardize the time of milking for accurate studies. The milk may be preserved with sodium azide tablets which are available commercially for this purpose. (2) Carry out the procedure given in Table V-3.

(Note: The effect of milk proteins on the assay should be checked by including samples of known progesterone-free milk. If appreciable interference is obtained, casein buffer should be added as described in Exercise 19.5.)

Exercise 19.5. Radioimmunoassay using 12sI-progesterone and antibody-coated tubes

This method is based on polypropylene-coated tubes and 125I-progesterone supplied by a commercial firm. Consequently, no description of the methods required to prepare antibody and antigen are given.

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04)

(2) Sodium phosphate monobasic (NaH2P04H20) (3) Bovine serum albumin, Fraction V (BSA) (Note: gelatin may not be suitable) 170 PART V. LABORATORY EXERCISES

TABLE V-4. PROTOCOL FOR THE PROGESTERONE COATED-TUBE METHOD FOR MILK

Tube Identification PBS-BSA Standard Sample lx Casein buffer No. (/il) Oil) (Ml) (Ml) (MO

1 Total counts 0 0 0 100 0 2 0 0 0 100 0 3 0 0 0 100 0 4 NSB 650 0 50 100 50

5 650 0 50 100 50

6 650 0 50 100 50

7 B0 control 700 0 0 100 50 8 700 0 0 100 50

9 700 0 0 100 50

10 Stnd-1.0 ng/ml 650 50 0 100 50

U Stnd-2.0 ng/ml 650 50 0 100 50

12 Stnd-4.0 ng/ml 650 50 0 100 50

13 Stnd-8.0 ng/ml 650 50 0 100 50 14 Stnd-16.0 ng/ml 650 50 0 100 50

15 Stnd-32.0 ng/ml 650 50 0 100 50

16 Stnd-64.0 ng/ml 650 50 0 100 50

17 Sample #1 700 0 50 100 0

18 700 0 50 100 0

19 Sample #2 700 0 50 100 0

20 700 0 50 100 0 21 Sample #3 700 0 50 100 0 22 700 0 50 100 0

(continue samples as needed in duplicate)

Bo control 700 0 0 100 50 700 0 0 100 50 700 0 0 100 50 APPLIED EXERCISES 171

TABLE V-l (cont.)

Tube Identification PBS-BSA Standard Sample lx Casein buffer No. (/Ü) (/il) 0*1) (Ml) (MO

Stnd-1.0 ng/ml 650 50 0 100 50 Stnd-2.0 ng/ml 650 50 0 100 50 Stnd-4.0 ng/ml 650 50 0 100 50

Stnd-8.0 ng/ml 650 50 0 100 50

Stnd-16.0 ng/ml 650 50 0 100 50 Stnd-32.0 ng/ml 650 50 0 100 50

Stnd-64.0 ng/ml 650 50 0 100 50

Note: High progesterone sample used for NSB. Take care: Danger of cross-contamination.

(4) Polypropylene assay tubes coated with rabbit anti-progesterone serum raised against the 1 la-succinyl-BSA conjugate (5) Progesterone for standard solution (6) Incubator set at 37°C (7) Sodium azide (8) Gamma counter (9) Microlitre pipettes in 50-1000 p\ range (10) Vortex mixer (11) Radioiodinated progesterone: progesterone-1 l-125I-tyrosine methyl ester in organic solvent (12) Felt-tip pen (13) Distilled water (14) Sodium azide (15) Sodium chloride ( 16) Büchner vacuum flask attached to vacuum line with tubing and pasteur pipette ( 17) Casein (vitamin-free) 172 PART V. LABORATORY EXERCISES

PREPARATION OF MATERIALS

(1) Buffers (a) 0.01M phosphate buffer, pH 7.0: dissolve 1.42 g of sodium phosphate

dibasic, anhydrous (Na2HP04) in 1 litre of distilled water. Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04H20) in 1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04 solution to give a final pH of 7.0. (b) Phosphate-buffered saline (PBS): add 9 g of sodium chloride (NaCl) to 1 litre of O.OlM phosphate buffer. Add 1 g of sodium azide per 1 litre for preservative. (c) Phosphate-buffered saline BSA (PBS-BSA): dissolve 5.0 g of BSA in 1 litre of PBS. (2) Dissolve 1.25 g of casein in 100 ml phosphate buffer (la). Heat to 37°C and then filter solution through glass wool. (3) Standard progesterone solutions: Prepare a stock solution and standard solutions as described in Exercise 19.2 using PBS-BSA as diluent. (4) Radioiodinated progesterone solution (called "lx tracer"): The working solution is prepared by dispensing a suitable aliquot of concentrate 125I-progesterone, drying in a stream of nitrogen and making up in PBS-BSA so that 100 pi contains 15 000 to 20 000 .counts/min. Make up only sufficient for the assay. This may be provided ready diluted by the manufacturers.

ASSAY PROCEDURE

(1) Write a protocol as indicated by Table V-4 for milk; for extracted plasma or serum note changes in standard solutions (3). (2) Number tubes with a felt-tip pen. (3) (a) Milk: Add reagents in the volumes as indicated in the protocol followed by the casein buffer solution where indicated. (b) Serum or plasma: Add extracted samples as given in Exercise 19.1, followed by PBS instead of casein buffer, where indicated in the protocol. (4) Add 50 pi of intermediate standard to NSB tubes. Take special care to avoid cross-contamination. (5) Add PBS-BSA as indicated on protocol. (6) Add 100 pi of 12SI progesterone to all tubes. (7) Mix tubes well and incubate for 3 h at 37°C or at 22°C overnight. (8) Aspirate contents of all tubes except numbers 1, 2 and 3. (9) Rinse tubes (except 1, 2 and 3) twice with 0.5 ml distilled water. The wash must be discarded within 5 minutes. (10) Quantify radioactivity in each tube with a gamma counter. Record counts per minute. APPLIEÍ) EXERCISES 173

CALCULATION OF RESULTS

Proceed as described in Exercises 19.3 and 16.2.

BIBLIOGRAPHY

BOER, G. de, ETCHES, R.J., WALTON, J.S., A solid-phase radioimmunoassay for progesterone in bovine plasma, Can. J. Anim. Sei. 60(1980) 783-86.

GOWAN, E.W., ETCHES, R.J., A solid-phase radioimmunoassay for progesterone and its application to pregnancy diagnosis in the cow, Theriogenology 12 (1979) 327—44.

Exercise 19.6. Procedure using charcoal-dextran separation and [3H] progesterone

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04)

(2) Sodium phosphate monobasic (NaH2P04H20) (3) Sodium chloride (NaCl) (4) Gelatin (5) Progesterone for standard solutions (6) Refrigerator set at 4°C (7) Refrigerator centrifuge (8) Liquid scintillation counter (9) Microlitre pipettes in 100-500 jul range (10) Disposable glass culture tubes (12 X 75 mm) (11) Test tube racks ( 12) Vortex mixer (13) [3H] progesterone (specific activity 90-100 Ci/mM or better) in phosphate buffered saline, pH 7.0 containing 0.1% gelatin (14) Felt-tip pen (15) Distilled water ( 16) Sodium azide (17) Scintillation fluid (see Exercise 19.1) ( 18) Norit A charcoal (19) Dextran T-70 (20) Anti-progesterone serum (21) Scintillation vials 174 PART V. LABORATORY EXERCISES

TABLE V-5. PROTOCOL FOR THE PROGESTERONE RADIOIMMUNOASSAY (extracted plasma or serum)

Tube Identification PBSG Standard Sample Antiserum lx Dextran- No. charcoal (Ml) (Ml) (Ml) (Ml) (Ml) (ml)

1 Total counts 1600 0 0 0 100 0 2 1600 0 0 0 100 0 3 1600 0 0 0 100 0 4 NSB 600 0 0 0 100 1.0 5 600 0 0 0 100 1.0 6 600 0 0 0 100 1.0

7 B0 control 500 0 0 100 100 1.0 8 500 0 0 100 100 1.0 9 500 0 0 100 100 1.0 10 Stnd-0.5 ng/ml 300 200 0 100 100 1.0 11 Stnd-1.0 ng/ml 300' 200 0 100 100 1.0 12 Stnd-2.0 ng/ml 300 200 0 100 100 1.0 13 Stnd-4.0 ng/ml 300 200 0 100 100 1.0 14 Stnd-8.0 ng/ml 300 200 0 100 100 1.0 15 Stnd-16.0 ng/ml 300 200 0 100 100 1.0 16 Stnd-32.0 ng/ml 300 200 0 100 100 1.0 17 Sample #1 300 0 200 100 100 1.0 18 300 0 200 100 100 1.0 19 Sample #2 300 0 200 100 100 1.0 20 300 0 200 100 100 1.0 21 Sample #3 300 0 200 100 100 1.0 22 300 0 200 100 100 1.0 (continue samples as needed in duplicate'

B0 control 500 0 0 100 100 1.0 500 0 , 0 100 100 1.0 500 0 0 100 100 1.0 Stnd-0.5 ng/ml 300 200 0 100 100 1.0 Stnd-1.0 ng/ml 300 200 0 100 100 1.0 APPLIED EXERCISES 175

TABLE V-l (cont.)

Tube Identification PBSG Standard Sample Antiserum lx Dextran- No. charcoal (Ml) (Ml) (MO (Ml) (Ml) (ml)

Stnd-2.0 ng/ml 300 200 0 100 100 1.0 Stnd-4.0 ng/ml 300 200 0 100 100 1.0 Stnd-8.0 ng/ml 300 200 0 100 100 1.0 Stnd-16.0 ng/ml 300 200 0 100 100 1.0 Stnd-32.0 ng/ml 300 200 0 100 100 1.0

PREPARATION OF MATERIALS

(1) Buffers (a) 0.01M phosphate-buffered saline (PBS), pH 7.0: dissolve 1.42 g of sodium

phosphate dibasic, anhydrous (Na2HP04) in 1 litre of distilled water. Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04H20) in

1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04 solution to give a final pH of 7.0. Add 9 g of sodium chloride (NaCl) to 1 litre of PBS. Add 1 g of sodium azide per litre for preservative. (b) Phosphate-buffered saline-gelatin (PBSG): dissolve 1 g of gelatin in 1000 ml of PBS. Heat gently to dissolve gelatin. (2) Diluted anti-progesterone serum: prepare appropriate dilution of the anti- progesterone in PBSG so that 100 binds about 50% of the total counts/min of [3H] progesterone added. See Exercise 19.7 below. (3) Standard progesterone solutions: prepare a stock dilution and standard solution as described in Exercise 19.2. (4) [3H] progesterone solution called "lx tracer": dilute [3H] progesterone to approximately 10 000 counts/min per 100 pi in PBSG. (5) Norit A charcoal-dextran suspension: prepare a stock solution by adding 12.5 g of Norit A and 1.25 g of dextran T-70 to 500 ml of PBS. Mix for 1 h. Store in refrigerator at 4°C. Immediately before use, mix stock suspension for 30 min and dilute 1:10 in PBSG. 176 PART V. LABORATORY EXERCISES

ASSAY PROCEDURE

(1) Write a protocol as indicated in Table V-5. (2) Number tubes with a felt-tip pen. (3) Add PBSG to the various tubes as indicated on protocol. (4) Add sample and standard solutions in the volume indicated on the protocol. Extraction procedure for progesterone is given in Exercise 19.1. (5) Add 100 u¡ of diluted antiserum to the appropriate tubes. (6) Add 100 pi of [3H] progesterone containing approximately 10 000 counts/min to all tubes. (7) Mix tubes well on a vortex mixer and incubate at 4°C for 24 h. (8) Add 1 ml of diluted charcoal suspension to all tubes except total counts. This should be added as quickly as possible. (9) Mix all tubes and incubate for 15 min at 4°C. Timing is very important. ( 10) Centrifuge all tubes except total counts at 1000 g for 10 min. (11) Decant each supernatant fraction into a scintillation vial containing scintillation fluid suitable for aqueous materials. Shake the vial vigorously and count in a liquid scintillation counter for 5 to 10 minutes.

CALCULATION OF RESULTS

The same procedure can be used as for Exercise 19.3.

Exercise 19.7. Titration of anti-progesterone serum using charcoal-dextran separation and [3H] progesterone

MATERIALS AND EQUIPMENT

See Exercise 19.6.

PREPARATION OF MATERIALS

(1) Buffers: prepare the same buffers as in Exercise 19.6, la and b. (2) Dilute anti-progesterone serum for determination of titre as follows: (a) Pipette 100 /ul of anti-progesterone serum into a stoppered test tube containing 9.9 ml of PBSG. Mix gently by inverting 10 times (1/100 dilution). (b) From (a) pipette 1 ml into a stoppered test tube containing 9.0 ml of PBSG. Mix gently by inverting 10 times (1/1000). APPLIEÍ) EXERCISES 177

TABLE V-6. TITRATION OF ANTI-PROGESTERONE SERUM (PROTOCOL FOR PROGESTERONE RADIOIMMUNOASSAY)

Tube Identification PBSG Antiserum Tracer Dextran- No. (Ml) (100 ML) (MD charcoal (Ml)

1 Total counts 1600 - 100 0

2 1600 - 100 0

3 NSB 600 - 100 1.0

4 600 - 100 1.0 5 Antiserum 500 1/100 100 1.0 6 500 1/100 100 1.0 7 500 1/1000 100 1.0 8 500 1/1000 100 1.0 9 500 1/5000 100 1.0 10 500 1/5000 100 1.0 11 500 1/10 000 100 1.0 12 500 1/10 000 100 1.0 13 500 1/20 000 100 1.0 14 500 1/20 000 100 1.0 15 500 1/40 000 100 1.0 16 500 1/40 000 100 1.0 17 500 1/80 000 100 1.0 18 500 1/80 000 100 1.0

(c) From (a) pipette 200 m1 into a stoppered test tube containing 9.8 ml of PBSG. Mix gently by inverting 10 times (1/5000). (d) From (a) pipette 100 yl into a stoppered test tube containing 9.9 ml of PBSG. Mix gently by inverting 10 times (1/10 000). (e) From (b) pipette 500 m1 into a stoppered test tube containing 9.5 ml of PBSG. Mix gently by inverting 10 times (1/20 000). (f) From (b) pipette 250 m1 into a stoppered test tube containing 9.75 ml of PBSG. Mix gently by inverting 10 times (1/40 000). Prepare the 9.75 ml of PBSG by pipetting out 10 ml and removing 250 ¡Á. (g) From (b) pipette 125 m1 into a stoppered test tube containing 9.875 ml of PBSG. Mix gently by inverting 10 times (1/80 000). 178 PART V. LABORATORY EXERCISES

(3) Dilute [3H] progesterone to approximately 10 000 counts/min per 100 pi in PBSG. (4) Prepare Norit A charcoal-dextran suspension as outlined in Exercise 19.6 (5).

ASSAY PROCEDURE

( 1 ) Write protocol as indicated in Table V-6. (2) Number tubes (12 X 75 culture tubes) with a felt-tip pen. (3) Add PBSG to the various tubes as indicated on protocol. (4) Add 100 nl of [3H] progesterone containing approximately 10 000 counts/min to all tubes. (5) Add 100 /il of diluted antiserum (various dilutions) to the appropriate tubes. (6) Mix the contents of tubes gently but well on a vortex mixer and incubate at 4°C for 4 to 24 h. (7) Add 1 ml of diluted dextran-charcoal to all tubes except total counts (as quickly as possible). (8) Mix contents of all tubes on a vortex mixer and incubate for 10 min at 4°C. (9) Centrifuge all tubes except total counts at 1000 g for 10 min. ( 10) Decant supernatant fraction into a scintillation vial containing scintillation fluid suitable for aqueous materials. Shake the vial vigorously and count in a liquid scintillation counter for 5 to 10 min.

CALCULATION OF RESULTS

counts/min at each antibody dilution — NSB % binding for each dilution = X 100 total counts

Generally the antibody titre that corresponds to 50% binding is selected for RIA of the standards and unknowns.

EXERCISE 20. RADIOIMMUNOASSAY OF TESTOSTERONE IN SERUM OR PLASMA

As with progesterone, it is possible to use several different techniques for assaying testosterone. The following exercise describes a double-antibody method based on iodinated testosterone. APPLIEÍ) EXERCISES 179

Exercise 20.1. Preparation of samples

Testosterone requires to be extracted by organic solvents, to remove it from interfering materials in the serum or plasma. As some interfering matter is extracted with the solvent, it is advisable that standards and blanks should contain equivalent amounts of testosterone-free plasma or serum.

MATERIALS AND EQUIPMENT

( 1 ) Diethyl ether, analytical reagent (2) 18 X 150 mm extraction tubes (see Exercise 19.1) (3) 12 X 75 mm glass tubes (4) Vortex mixer (5) Methanol, analytical reagent (6) Test tube racks (7) Dry ice (solid carbon dioxide) + technical methanol or technical acetone bath of a size suitable to hold a rack containing the extraction tubes (8) 3H testosterone: prepare a dilution of 3H testosterone, which may be obtained from a supplier of radiochemicals, by drying down a suitable aliquot in a stream of nitrogen and dissolving in pure methanol, so that 50 jul in a suitable scintillation cocktail gives a count in a beta scintillation counter of approximately 2000 counts/min (9) A liquid scintillation counter, set to measure 3 H ( 10) A scintillation cocktail suitable for RIA. These are available from many suppliers, and are usually based on toluene or xylene with 2,5-diphenyloxazole (PPO) and 1,4-bis-2-5-phenyloxazoyl (POPOP)

PROCEDURE

The methods for extraction and monitoring procedural losses are as follows:

(1) Dispense 100 /ul of each serum or plasma sample into an 18 X 150 mm glass tube (including 3 standards diluted in testosterone-free plasma). (2) Dispense 100 pi of distilled water into an 18 X 50 mm glass tube. (3) Add 50 f/13H testosterone (reagent 8) to all samples in the set of tubes containing serum or plasma (1). Simultaneously add 50 pi 3H testosterone to two scintillation vials labelled "total counts". Set "total counts" and two "background" empty vials aside for later counting. (4) Allow the 3H testosterone to equilibrate in the samples for a minimum of 2 h at room temperature or 12-18 h at 4°C. This is to ensure binding to proteins (e.g. SHBG) simulating the endogenous situation in the samples. 180 PART V. LABORATORY EXERCISES

(5) Add 4 ml of diethyl ether to each tube containing sample. (6) Mix content of the tubes well on a vortex mixer for 1 min or shake by hand for 1 min. (7) Place tubes in a dry ice-acetone or -methanol bath to freeze the bottom aqueous layer (serum or water). If placed in a freezer, one must be absolutely sure that the freezer is explosion-proof and at sufficiently low temperature to freeze the aqueous layer. (8) Pour solvent (containing testosterone) into corresponding 12 X 75 mm glass tubes and dry with a gentle stream of nitrogen. If available, a gently heated block heater accelerates this process. (9) Add 1 ml of assay diluent to the dried extract. Mix well and place in refrigerator at 4°C for 12-18 h. (10) Transfer 100 /il of each reconstituted extract to scintillation vials and 100 ¡A of assay diluent to the "total counts" and "background" vials. (11) Add 10 ml scintillation fluid to all vials, mix, and quantify radioactivity in a liquid scintillation counter. (12) Determine extraction recovery by the following equation:

counts/min in sample tubes — mean counts/min in background tubes Recovery % = X 100 mean counts/min in total tubes

(13) As standards are treated in the same manner as unknown plasma or serum samples, no correction for recovery is necessary. However, at least 85% recovery should be achieved, and there should be good agreement between different samples (c.v. < 5%). (14) The remaining reconstituted extracts are used for the radioimmunoassay described below.

Exercise 20.2. Double-antibody separation with 125I-testosterone

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04)

(2) Sodium phosphate monobasic (NaH2P04H20) (3) Disodium ethylenediamine tetraacetate (EDTA) (4) Gelatin (5) Non-immune, normal rabbit serum (NRS) (6) Rabbit (or sheep) anti-testosterone-3-0(carboxymethyl-oxime)-BSA serum (see Exercise 17 — a similar procedure may be used for testosterone) (7) Sheep, goat or donkey anti-rabbit gamma globulin (ARGG) (8) Testosterone for standard solutions APPLIEÍ) EXERCISES 181

(9) Refrigerator or cold room set at 4°C ( 10) Refrigerated centrifuge (11) Gamma counter (12) Microlitre pipettes in 100—500 /il range (13) Disposable glass culture tubes (12 X 75 mm) ( 14) Test tube racks (15) Vortex mixer (16) I25I-histamine-testosterone (see labelling procedure, Exercise 18.2) (17) Felt-tip pen (18) Distilled water (19) Sodium azide (20) Methanol (21) Sodium chloride (22) Magnetic and heated stirrer (optional)

PREPARATION OF MATERIALS

(1) Buffers (a) 0.01 M phosphate-buffered saline (PBS), pH 7.0: dissolve 1.42 g of

sodium phosphate dibasic, anhydrous (Na2HP04) in 1 litre of distilled water. Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04H20) in 1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04 solution to give a final pH of 7.0. Add 9 g of sodium chloride (NaCl) to 1 litre to give PBS. Add 1 g of sodium azide per litre as preservative. (b) Phosphate-buffered saline-gelatin (PBSG): dissolve 1 g of gelatin in 1 litre of PBS. Heat gently to dissolve gelatin, using a magnetic, heated stirrer (if available). (c) Phosphate-buffered saline-0.05M EDTA (PBS-EDTA), pH 7.0: dissolve 18.6 g of disodium ethylenetetraacetate in 1000 ml of PBS. Adjust pH with 5N NaOH. (2) 1:400 NRS: add 1 ml of normal rabbit serum (or species in which the first antiserum is raised) to 400 ml of PBS-EDTA. Mix well. (3) Diluted anti-testosterone serum: prepare appropriate dilution as given below, of the anti-testosterone serum, using PBS-EDTA (no NRS). Note that the correct 'bridge' must be used to obtain adequate binding and sensitivity. (4) Diluted ARGG: prepare appropriate dilution of the ARGG as given below using 1:400 NRS (2) as diluent. This solution should be freshly prepared. (5) (a) General. A pure testosterone preparation (MW 288 daltons) should be obtained from a reputable supplier, and a primary standard solution must be prepared in pure ethanol (or methanol). A convenient dilution would 182 PART V. LABORATORY EXERCISES

TABLE V-7. PROTOCOL FOR THE TESTOSTERONE RADIOIMMUNOASSAY (extracted plasma or serum)

Tube Identification PBSG Standard Sample Antiserum lx ARGG No. (Ml) (pi) (jul) (Ml) (Ml) (Ml)

1 Total counts 0 0 0 0 100 0 2 0 0 0 0 100 0 3 0 0 0 0 100 0 4 NSB 700 0 0 0 100 200 5 700 0 0 0 100 200 6 700 0 0 0 100 200 7 Bo control 500 0 0 200 100 200 8 500 0 0 200 100 200 9 500 0 0 200 100 200 10 Stnd-0.125 ng/ml 300 200 0 200 100 200 11 Stnd-0.25 ng/ml 300 200 0 200 100 200 12 Stnd-0.50 ng/ml 300 200 0 200 100 200 13 Stnd-1.00 ng/ml 300 200 0 200 100 200 14 Stnd-2.00 ng/ml 300 200 0 200 100 200 15 Stnd-4.00 ng/ml 300 200 0 200 100 200 16 Sample #1 300 0 200 200 100 200 17 300 0 200 200 100 200 18 Sample #2 300 0 200 200 100 200 19 300 0 200 200 100 200 20 Sample #3 300 0 200 200 100 200 21 300 0 200 200 100 200

(continue samples as needed in duplicate]

B0 control 500 0 0 200 100 200 500 0 0 200 100 200 500 0 0 200 100 200 Stnd-0.125 ng/ml 300 200 0 200 100 200 Stnd-0.25 ng/ml 300 200 0 200 100 200 APPLIED EXERCISES 183

TABLE V-l (cont.)

Tube Identification PBSG Standard Sample . Antiserum lx ARGG No. (Ail) (pi) (pi) (pi) (pi) (pi)

Stnd-0.50 ng/ml 300 200 0 200 100 200 Stnd-1.00 ng/ml 300 200 0 200 100 200 Stnd-2.00 ng/ml 300 200 0 200 100 200 Stnd-4.00 ng/ml 300 200 0 200 100 200

be 50 mg of testosterone, accurately weighed, made up to 50 ml in a volumetric flask with ethanol. An intermediate standard can be prepared by diluting the primary standard 1:1000 with ethanol, i.e. 100 pi primary standard transferred accurately to a volumetric flask and made up to 100 ml with ethanol. This has a concentration of 1 pg/ml and may be used for dilution to a working standard of 100 ng/ml (i.e. 10 ml diluted to 100 ml with ethanol). Methanol may be used as an alternative to ethanol. (b) Standard testosterone solutions are prepared by dispensing into 10 ml volumetric flasks the following aliquots of working standard: 400, 200, 100, 50, 25 and 12.5 pi. Evaporate to dryness in a stream of nitrogen with gentle heat and make up to the 10 ml mark with a specimen of serum of plasma that is free from testosterone or with extremely low levels, preferably from the species under test. 400 pi aliquots of each standard can be stored at -20°C, along with 200 pi aliquots of the diluting plasma or serum for future assays. This will provide standards of 4000, 2000, 1000, 500, 250 and 125 pg/ml. (6) nsI-histamine-testosterone in methanol. The working solution is prepared by taking an appropriate amount of the stock solution and evaporating off the methanol in an airstream in a conical flask, then adding an amount of assay diluent and allowing it to stand for several minutes with occasional shaking. This solution should be prepared freshly for each assay and should contain about 15 000 counts/min per 100 pi when the tracer is fresh. Since the half-life of 12SI is 60 days, longer counting times will be required as the tracer ages. 184 PART V. LABORATORY EXERCISES

Titration of anti-testosterone serum and anti-i rabbit) gamma globulin and calculation of titres

See instructions given for the titration of anti-progesterone serum and anti- rabbit gamma globulin given in Exercise 17.2. The protocol (Table V-l ) may be used in a similar fashion for anti-testosterone serum. Calculate the anti-testosterone and anti-ARGG titres as described and use the greatest dilutions that bind 40-60% of the lx tracer.

Exercise 20.3. Radioimmunoassay procedure for extracted plasma or serum

(1) Write a protocol as shown in Table V-7. (2) Number tubes with a felt-tip pen. (3) Add PBSG to the various tubes as indicated on protocol. (4) Add sample (extracted plasma or serum) and standard solution in the volumes indicated on protocol shown in Table V-7. (5) Add 200 /il of diluted antiserum to all tubes (except 1, 2 and 3). (6) Add 100 /il of the 1 x tracer solution to all tubes. (7) Mix tubes well on a vortex mixer and incubate at room temperature for 1 -2 h. (8) Add 200 /il of diluted ARGG containing NRS to all tubes except numbers 1, 2 and 3. (9) Mix tubes well on a vortex mixer and incubate at 4°C for 16-24 h (overnight). (10) Add 1 ml of cold PBS to all tubes except numbers 1, 2 and 3. Do not mix. (11) Centrifuge all tubes except numbers 1, 2 and 3 at 1000 g for 30 min at 4°C. Determine correct centrifuge speed for 1000 g by using the equation in Appendix VI-8. ( 12) Carefully decant the supernatant of each tube into a radioactive liquid waste container. Keep the tube inverted and allow to drain on absorbent paper and remove any remaining drops of liquid from the rim by a pasteur pipette attached to a water pump via a Büchner flask. (13) Quantify radioactivity in the precipitates remaining in the tubes with a gamma counter. Record counts.

CALCULATION OF RESULTS

Follow instructions described in Exercise 19.3. APPLIEÍ) EXERCISES 185

EXERCISE 21. RADIOIMMUNOASSAY OF LUTEINIZING HORMONE IN SERUM OR PLASMA

The iodination procedures and assay that will now be described for LH • may be regarded as examples that may be followed for all other peptide hormones and applied for the assay of FSH, insulin, prolactin and growth hormone. Both prolactin and FSH are best iodinated by the lactoperaxidase method and prolactin should be separated on a Sephadex G-l 00 column. In this case, 3 peaks are obtained; the first peak is a dimerized inactive form of the hormone and consequently only the second peak should be used. Only a limited number of homologous FSH antisera have been obtained and as a result many measurements of blood levels in farm animals have been obtained using heterologous assays (i.e. anti-human FSH sera). Highly purified FSH preparations are not easily obtained and attempts at immunization are not advised at present. However, purified FSH for labelling may on occasion be obtained from a few specialist research workers.

Exercise 21.1. Immunization of rabbits against ovine luteinizing hormone ( LH)

MATERIALS AND EQUIPMENT

(1) Purified ovine LH (2) Sodium chloride (NaCl) (3) Freund's Complete Adjuvant (4) 5 ml glass syringe (5) Short, 25 G disposable needles (6) Animal hair clippers or curved scissors (7) High-speed, small volume mixer or 5 ml syringe for emulsifying hormone solution (8) Büchner flask, 250 ml (9) Vacuum water aspirator or motor pump (10) Rubber tubing (11) Distilled water (12) Centrifuge (13) Scalpel

PREPARATION OF MATERIALS

(1) Physiological saline solution: dissolve 9 g of NaCl in 1000 ml of distilled water. Mix well but avoid frothing. 186 PART V. LABORATORY EXERCISES

(2) Purified LH solution: dissolve 500 pg of LH in 2.5 ml of physiological saline solution. (3) LH emulsion for immunization: to 2.5 ml of purified LH solution, add 2.5 ml of Freund's Complete Adjuvant. Mix very well but avoid frothing until material is white and very viscous. This emulsifying is best done using a high-speed mixer. The mixture may be emulsified by repeatedly forcing through a syringe. To determine if emulsification is complete, place a small drop of the emulsion on the surface of water. If well-emulsified, the material will not disperse; if further emulsification is needed, material will disperse over surface of water. It is very important to have a good emulsion. This volume of emulsion is sufficient for immunizing 4 to 5 rabbits.

IMMUNIZATION PROCEDURE

(1) Restrain rabbit by placing rabbit on a bench and grasping firmly around neck and rump. (2) Clip hair over back. An area about 15 cm X 10 cm is sufficient. (3) Fill the 5 ml glass syringe with the emulsified LH preparation. Attach a short 25 G needle. (4) With the bevel of the needle up, inject 1 ml (100 ßg of LH) of the emulsion intradermally. Divide the dose into 30 to 40 sites over the back, each of 25 to 30 pi. (5) Four weeks after the initial immunization, inject 100 pg of purified LH solution (in 1 ml of saline without adjuvant) intravenously into the marginal ear vein. (6) One week later collect blood from the marginal ear vein into a 50 ml centrifuge tube by making a small ( 1 mm) cut in the vein with the point of a scalpel. (7) Allow blood to clot for a minimum of 3 hours in a refrigerator at 4°C. Break clot loose from the centrifuge tube and store at 4°C for 18 to 24 hours. (8) Centrifuge clotted blood and draw off antiserum. (9) Freeze rapidly in small aliquots and store at -20°C. (10) Collect antiserum about every 10 days. Titrate antiserum as described in Exercise 17.2 to monitor antibody titre.

Exercise 21.2. Radioiodination of ovine LH with chloramine-T

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04) (2) Sodium phosphate monobasic (NaH2P04H20) (3) Distilled water APPLIEÍ) EXERCISES 187

(4) Sodium chloride (NaCl) (5) 5N NaOH (6) Highly purified, iodination-grade ovine LH (7) Chloramine-T (8) Sodium metabisulphite (9) 1 ml serum vials with stoppers ( 10) Hamilton glass syringes (10, 100 jul) (11) Disposible 1 ml plastic syringes (12) Long 25 G disposable needles (13) Stopwatch (14) Analytical balance (15) High specific activity, carrier-free Na125I (37 MBq or 1 mCi) (16) Sodium azide

PREPARATION OF MATERIALS

(1) Buffers (a) 0.5M sodium phosphate buffer, pH 7.5: dissolve 7.1 g of sodium

phosphate dibasic, anhydrous (Na2HP04) in 100 ml of distilled water. Dissolve 6.9 g of sodium phosphate monobasic (NaH2P04H20) in 100 ml of distilled water. Mix approximately 80 ml of Na2HP04 solution with 20 ml of NaH2P04H20 solution to give a final pH of 7.5. Store frozen in small aliquots. Note that this solution will crystallize out at low temperatures, and may require warming and re-dissolving before use. (b) 0.05M sodium phosphate buffer, pH 7.5: dilute 1 ml of 0.5M sodium phosphate buffer, pH 7.5, with 9 ml distilled water. (c) 0.01 M phosphate-buffered saline (PBS), pH 7.0: dissolve 1.42 g of

use. Store small portions of dry chloramine-T in vials at -20°C in a container containing dessicant. (c) Sodium metabisulphite solution (2 mg/ml): dissolve 10 mg of sodium metabisulphite in 5 ml of 0.05M sodium phosphate buffer, pH 7.5. Make fresh on day of use.

IODINATION PROCEDURE

(1 ) All procedures may be carried out at room temperature, under an efficient fume hood. (2) To an unstoppered 1 ml serum vial containing 5 pg of hormone to be iodinated, add 50 pi of 0.5M sodium phosphate buffer. Mix gently by tapping the vial. (3) Add 1 mCi (37 MBq) of 12SI using a 10-jul Hamilton syringe. (Use this syringe only for this purpose. Store under hood and label with radioactive tape.) (4) Close vial with the rubber stopper. Mix contents by tapping gently. (5) With the 100 pi Hamilton syringe attached to the needle, add 15 pi of the chloramine-T solution to the vial by injecting through the rubber stopper. When injecting into the vial, position the tip of the needle just above the top of the liquid in the vial. Mix vial by gentle tapping for 2 min, timing with a stopwatch. (6) After 2 min, immediately inject 30 pi of sodium metabisulphite solution into the vial using a 1 ml disposable syringe and needle. Position needle just above top of mixture. Mix for 30 s. (7) Separate radioiodinated hormone from unreacted iodide as described in Exercise 21.4.

PRECAUTIONS

(1) Once the 125I has been added to the serum vial, close the vial with the rubber stopper and keep closed. Inject chloramine-T, metabisulphite, transfer, and rinse solutions through the rubber stopper. (2) When adding chloramine-T and sodium metabisulphite to the iodination vial, position the tip of the needle just above the level of the reaction mixture in the vial. Then add solution without the needle touching the mixture.

BIBLIOGRAPHY

HUNTER, W.M., GREENWOOD, F.C., Preparation of iodine-131 labelled human growth hormone of high specific activity, Nature (London) 194 (1962) 495-96. APPLIEÍ) EXERCISES 189

Exercise 21.3. Radioiodination of LH with lactoperoxidase

MATERIALS AND EQUIPMENT

(1) Highly purified iodination-grade ovine LH

(2) Sodium phosphate dibasic, anhydrous (Na2HP04)

(3) Sodium phosphate monobasic (NaH2P04H20) (4) Distilled water (5) Sodium chloride (NaCl) (6) Sucrose

(7) 30% hydrogen peroxide solution (H202) (8) Lactoperoxidase (9) 1 ml serum vials with rubber stopper (10) Hamilton glass syringes (10 pi, 100 pi) (11) Long 25G disposable needles

PREPARATION OF MATERIALS

(1) Buffers (a) 0.5M sodium phosphate buffer, pH 7.5: dissolve 7.1 g of sodium

phosphate, anhydrous (Na2HP04) in 100 ml of distilled water. Dissolve 6.9 g of sodium phosphate monobasic (NaH2P04H20) in 100 ml of distilled water. Mix approximately 80 ml of Na2HP04 solution with 20 ml of NaH2P04 solution to give a final pH of 7.5. Store frozen in small aliquots. (b) 0.05M sodium phosphate buffer, pH 7.5: dilute 0.5M sodium phosphate buffer 1 to 10 with distilled water. (2) Solutions (a) LH solution: dissolve purified, iodination-grade hormone in 0.05M phosphate buffer to a concentration of 1 mg/ml. Add 5 pi (5 pg) to a 1 ml serum vial. You may aliquot several vials and freeze at -20°C for future use.

(b) H202 working solution: add 250 pi of 30% H202 to 100 ml of distilled water. Prepare immediately before you are ready to iodinate. Discard unused portion. (c) Lactoperoxidase stock solution: add 10 mg of lactoperoxidase to 10 ml of 0.05M sodium phosphate buffer, pH 7.5. Make fresh on day of use. 190 PART V. LABORATORY EXERCISES

IODINATION PROCEDURE

( 1 ) Maintain all reagents and reaction solutions at 4°C. (2) Add 50 pi of 0.5M sodium phosphate buffer to serum vial containing 5 pg of LH. Mix gently by tapping vial. (3) Add 1 mCi (37 MBq) of 12SI solution using a 10 pi Hamilton syringe. Mix. (4) Close vial with the rubber stopper. Mix contents by gently tapping.

(5) With the 100 pi Hamilton syringe attached to a needle, add 10 pi of H202 working solution to the vial by injecting through the rubber stopper. (6) Add 5 pi (5 pg) of lactoperoxidase solution to vial. (7) Mix contents for 5 min. (8) Add 500 pi of cold sodium phosphate buffer. (9) Separate radioiodinated LH and unreacted 125I as described below.

Exercise 21.4. Separation of radioiodinated LH from unreacted iodide

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04)

(2) Sodium phosphate monobasic (NaH2P04H20) (3) Gelatin (4) Distilled water (5) Sodium chloride (6) Sucrose (7) Sephadex G-25-150 beads for gel filtration (8) Disposable serological pipettes (10 ml) (9) Disposable 1 ml plastic syringes ( 10) Long 25 G disposable needles (11) 12X75 mm glass culture tubes ( 12) Test-tube rack ( 13) Sodium azide (14) Gamma counter ( 15) Glass wool (16) Microlitre pipettes (10-100 pi) (17) Sodium azide

PREPARATION OF MATERIALS

(1) 0.01M phosphate-buffered saline-gelatin (PBSG): dissolve 1.42 g of sodium

phosphate dibasic, anhydrous (Na2HP04) in 100 ml of distilled water. APPLIEÍ) EXERCISES 191

Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04H20) in 1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04 solution to give a final pH of 7.0. Add 9 g of sodium chloride (NaCl) to 1 litre of 0.01M sodium phosphate buffer. Add 1 g/litre of sodium azide as preservative. Dissolve 1 g of gelatin in 1 litre of phosphate-buffered saline. Heat gently to dissolve gelatin. (2) Transfer solution (16% sucrose): dissolve 16 g of sucrose in 100 ml of PBSG. Store frozen in small aliquots. (3) Rinse solution (8% sucrose): dissolve 8 g of sucrose in 100 ml of PBSG. Store frozen in small aliquots. (4) Sephadex G-25-150: add approximately 50 g of Sephadex G-25-150 to 400 ml of PBSG. Swirl until there is a homogeneous suspension. Let Sephadex swell at least 12 h at 4°C or 1 h at room temperature. Store in a flask at 4°C until used. (5) Disposable Sephadex G-25-150 columns: a 10 ml disposable serological pipette works very well for a column. Pack a small piece of glass wool into the top of the pipette using a smaller diameter pipette. Pack firmly but avoid overpacking as this will greatly reduce the flow rate. Attach a short (3—4 cm) piece of rubber tubing to the tip of the pipette and attach a small clamp to close off the column. Add PBSG to the column until approximately half full. Fill the remainder of the column with Sephadex slurry and allow the Sephadex to settle until 1 to 2 cm has accumulated at the tip of the pipette. Open the clamp and continue adding Sephadex slurry until the column bed is 5 cm from the top of the column. Close clamp and store columns to 4°C until used.

SEPARATION PROCEDURE

(1) Inject 100 ¡Û of transfer solution into the 1 ml vial containing reaction mixture using a 1 ml disposable syringe and needle. Leave syringe and needle positioned in vial. Mix gently. (2) Draw the reaction mixture into the syringe and apply to the top of the Sephadex without disturbing the bed. (Before applying radioiodinated hormone to the top of the Sephadex bed in the column, allow excess PBSG to drain from the column until the buffer is just above the top of the bed. Allow the radioiodinated hormone and transfer solution to enter the bed before adding rinse solution.) (3) Inject 70 jul of rinse solution into the vial with a 1 ml syringe and needle. Remove syringe and use the syringe in (1) above to withdraw the mixture from the vial. Apply rinse solution to column. 192 PART V. LABORATORY EXERCISES

TABLE V-8. COUNTS PER MINUTE ELUTED FROM SEPHADEX G-25 COLUMN

Fraction number Counts/min per 10 p\ (thousands)

1 1 2 1 3 1 4 36 5 Radioiodinated 412 6 hormone; 'peak 972 7 tubes' 377 8 152 9 1163 10 Unreacted 125I 1486 11 220 12 191 13 143 14 81 15 56 16 42 17 21 18 18 19 10 20 5

(4) Collect 1 ml fractions of eluate from the columns into 122 X 75 mm disposable culture tubes using PBSG as eluting buffer: After 20 fractions are collected, aliquot 10 to 20 jul of each fraction into another set of tubes. Determine radioactivity in the second set of tubes using a gamma counter. Two peaks of radioactivity should be noted. The first peak is radioiodinated hormone and is usually in fractions 3 to 6. The free, non-reacted iodide peak occurs in the 9th, 10th, and 11th fractions. See Table V-8. (5) Select a 'peak tube' containing radioiodinated hormone. To 100 ml of PBSG, add a few drops of the radioiodinated hormone solution until 100 /d contains 25 000 to 35 000 counts/min. Label this mixture 'lx tracer, counts/min APPLIEÍ) EXERCISES 193

per 100 ßV. Store in refrigerator until added to assays. Label 'peak tubes' with hormone name and fraction number. Store frozen.

Note: For a rapid and simple method of detection of the 'peak' tubes, it is possible to measure the radioactivity of the first set of tubes by placing them individually in a rack situated a fixed distance from the head of a sensitive radiation monitor, and reading the 'counts/s' value from the meter.

Exercise 21.5. Titration of anti-LH ovine serum and anti-rabbit gamma globulin and calculation of titres

These are as described in Exercise 17.2 except that anti-ovine LH serum (Exercise 21.1) and radioiodinated ovine LH (Exercises 21.2 and 21.3) are required. Write a protocol as described in Table V-9. Select the greatest dilution of anti-LH serum and ARGG that binds 20-40% of the 1 x tracer.

Exercise 21.6. Radioimmunoassay of ovine luteinizing hormone (LH)

MATERIALS AND EQUIPMENT

(1) Sodium phosphate dibasic, anhydrous (Na2HP04) (2) Sodium phosphate monobasic (NaH2P04H20) (3) Disodium ethylenediamine tetraacetate (EDTA) (4) Gelatin (5) Non-immune, normal rabbit serum (NRS) (6) Rabbit anti-ovine LH serum (7) Sheep, goat or donkey anti-rabbit gamma globulin (ARGG) (8) Purified ovine LH for standard solutions (9) Refrigerator and/or cold room set at 4°C (10) Refrigerated centrifuge (11) Gamma counter (12) Microlitre pipettes in 100—500 pi range (13) Disposable glass culture tubes (12 X 75 mm) (14) Test-tube racks (15) Vortex mixer (16) Radioiodinated LH diluted to approximately 25 000 to 35 000 counts/min per 100 /il in phosphate-buffered saline, pH 7.0, containing 0.1% gelatin (17) Felt-tip pen (18) Distilled water (19) Sodium azide 194 PART V. LABORATORY EXERCISES

TABLE V-9. PROTOCOL AND RESULTS FOR TITRATIONS OF ANTI-LH SERUM AND ARGG

Tube Identification PBSG Antiserum lx ARGG Mean %of No. (300 i/1) (200 pi) (100 fil) (200 f/1) counts/ min total

1 Total counts - - + - 27 525 -

2 - - + - 3 NSB + 1:400a + 1:5 316 1.1 4 + 1:400a + 1:5 S Antiserum + 1:400 + 1:5 2 400 7.6 6 + 1:400 + 1:5 7 + 1:1000 + 1:5 2 168 6.7 8 + 1:1000 + 1:5 9 + 1:10 000 + 1:5 2 283 7.1 10 + 1:10 000 + 1:5 11 + 1:20 000 + 1:5 1 982 6.0 12 + 1:20 000 + 1:5 13 + 1:40 000 + 1:5 1 458 4.1 14 + 1:40 000 + 1:5 15 + 1:80 000 + 1:5 1 319 3.6 16 + 1:80 000 + 1:5 17 + 1:160 000 + 1:5 1 101 2.8 18 + 1:160 000 + 1:5

a 19 NSB + 1:400 + 1:10 322 - 20 + 1:400a + 1:10 21 Antiserum + 1:400 + 1:10 4 954 16.8 22 + 1:400 + 1:10 23 + 1:1000 + 1:10 5 780 19.8 24 + 1:1000 + 1:10 25 + 1:10 000 + 1:10 5 230 17.8 26 + 1:10 000 + 1:10 27 + 1:20 000 + 1:10 3 578 11.8 28 + 1:20 000 + 1:10 29 + 1:40 000 + 1:10 2 477 7.8 30 + 1:40 000 + 1:10 31 + 1:80 000 + 1:10 2 064 6.3 APPLIED EXERCISES 195

TABLE V-9. (cont. )

32 + 1:80 000 + 1:10 33 + 1:160 000 + 1:10 1 116 2.9 34 + 1:160 000 + 1:10

a 35 NSB + 1:400 + 1:15 416 - 36 + 1:400a + 1:15 37 Antiserum + 1:400 + 1:15 13 763 48.5 38 + 1:400 + 1:15 39 + 1:1000 + 1:15 15 139 53.5 40 + 1:1000 + 1:15 41 + 1:10 000 + 1:15 13 487 47.5 42 + 1:10000 + 1:15 43 + 1:20 000 + 1:15 10 597 37.0 44 + 1:20 000 + 1:15 45 + 1:40 000 + 1:15 7 432 25.5 46 + 1:40 000 + 1:15 47 + 1:80 000 + 1:15 4 955 16.5 48 + 1:80 000 + 1:15 49 + 1:160 000 + 1:15 1 927 5.5 50 + 1:160 000 + 1:15 51 NSB + 1:400a + 1:20 428 1.6 52 + 1:400a + 1:20 53 Antiserum + 1:400 + 1:20 13 683 48.2 54 + 1:400 + 1:20 55 + 1:1000 + 1:20 14 982 52.9 56 + 1:1000 + 1:20 57 + 1:10 000 + 1:20 12 523 43.9 58 + 1:10 000 + 1:20 59 + 1:20 000 + 1:20 11 683 40.9 60 + 1:20 000 + 1:20 61 + 1:40 000 + 1:20 7 430 25.4 62 + 1:40 000 + 1:20

63 + 1:80 000 + 1:20 4 565 15.0 64 + 1:80 000 + 1:20 65 + 1:160 000 + 1:20 1 827 5.1 196 PART V. LABORATORY EXERCISES

TABLE V-9. (cont.)

Tube Identification PBSG Antiserum lx ARGG Mean %of No. (300 pi) (200 Ml) (100/Ltl) (200 Ml) counts/min total

66 Antiserum + 1:160 000 + 1:20

a 67 NSB + 1:400 + 1:25 310 - 68 + 1:400a + 1:25 69 Antiserum + 1:400 + 1:25 10460 36.9 70 + 1:400 + 1:25 71 + 1:1000 + 1:25 11 423 40.4 72 + 1:1000 + 1:25 73 + 1:10000 + 1:25 9 909 34.9 74 + 1:10000 + 1:25 75 + 1:20 000 + 1:25 7 707 26.9

76 + 1:20 000 + 1:25 77 + 1:40 000 + 1:25 4 954 16.9 78 + 1:40 000 + 1:25 79 + 1:80 000 + 1:25 2 753 8.9 80 + 1:80 000 + 1:25 81 + 1:160 000 + 1:25 1 376 3.9 82 + 1:160 000 + 1:25

8 NRS only, no antiserum.

PREPARATION OF MATERIALS

(1) Buffers (a) 0.01 M phosphate-buffered saline (PBS), pH 7.0: dissolve 1.42 g of

sodium phosphate dibasic, anhydrous (Na2HP04) in 1 litre of distilled water. Dissolve 1.38 g of sodium phosphate monobasic (NaH2P04 • H20) in 1 litre of distilled water. Mix approximately 600 ml of Na2HP04 solution with 400 ml of NaH2P04 solution to give a final pH of 7.0. Add 9 g of sodium chloride (NaCl) to 1 litre of PBS. Add 1 g of sodium azide per 1 litre for preservative. (b) Phosphate-buffered saline-gelatin (PBSG): dissolve 1 g of gelatin in 1 litre of PBS. Heat gently to dissolve gelatin. APPLIEÍ) EXERCISES 197

(c) Phosphate-buffered saline-0.05M EDTA (PBS-EDTA), pH 7.0: dissolve 18.6 g of disodium ethylenediamine tetraacetate in 1 litre of PBS. Adjust pH with 5N NaOH. (2) 1:400 NRS: add 1000 ¿d of NRS to 400 ml of PBS-EDTA. Mix well. (3) Anti-LH serum: adjusted to the optimum dilution as described in Experiment 22.1. (4) Anti-rabbit serum (ARGG): adjusted to the optimum dilution as described in Experiment 21.5. (5) Standard LH solutions: prepare a stock solution of ovine LH in a concentration of 1 pg/ml by dissolving 10 jug of purified LH in 10 ml of PBSG. Standard solutions of 200 ng/ml, 100 ng/ml, 50 ng/ml, 25 ng/ml, 12.5 ng/ml, 6.25 ng/ml, 3.13 ng/ml, 1.56 ng/ml and 0.78 ng/ml are prepared as follows:

For the 200 ng/ml standard, add 2 ml of stock to 8 ml of PBSG; For the 100 ng/ml standard, add 1 ml of stock to 9 ml of PBSG; For the 50 ng/ml standard, add 0.5 ml of stock to 9.5 ml of PBSG; For the 25 ng/ml standard, add 0.25 ml of stock to 9.75 ml of PBSG; For the 12.5 ng/ml standard, add 0.125 ml of stock to 9.875 ml of PBSG; For the 6.25 ng/ml standard, add 0.0625 ml of stock to 9.938 ml of PBSG; For the 3.13 ng/ml standard, add 0.0313 ml of stock to 9.969 ml of PBSG; For the 1.56 ng/ml standard, add 0.0156 ml of stock to 9.984 ml of PBSG; and For the 0.78 ng/ml standard, add 0.0078 ml of stock to 9.992 ml of PBSG.

Mix all solutions well. Freeze 1 ml aliquots in well-sealed test tubes for future use. (6) Radioiodinated LH solution (called 'lx tracer'): to 100 ml of PBSG, add a few drops of radioiodinated LH from the 'peak tube' until 100 M1 contains 25 000 to 35 000 counts/min.

ASSAY PROCEDURE

(1) Write a protocol as shown in Table V-l0 for setting up the assay. (2) Number tubes with a felt-tip pen. (3) Add PBSG to the various tubes as indicated on protocol. (4) Add sample and standard solutions in the volumes indicated on the protocol. (5) Add 200 Ml of 1:400 NRS to tubes 4, 5 and 6 and 200 m1 of diluted antiserum to the remaining tubes (except 1,2 and 3). 198 PART V. LABORATORY EXERCISES

TABLE V-10. PROTOCOL FOR THE LH RADIOIMMUNOASSAY

Tube Identification PBSG Standard Sample Antiserum lx ARGG No. (MO (MO (MD Oil) (M» (Ml)

1 Total counts 0 0 0 0 100 0 2 0 0 0 0 100 0 3 0 0 0 0 100 0 4 NSB 500 0 0 200a 100 200 5 500 0 0 200a 100 200 6 500 0 0 200a 100 200

7 B0 control 500 0 0 200 100 200 8 500 0 0 200 100 200 9 500 0 0 200 100 200 10 Stnd-0.78 ng/ml 300 200 0 200 100 200 11 Stnd-1.56 ng/ml 300 200 0 200 100 200 12 Stnd-3.13 ng/ml 300 200 0 200 100 200 13 Stnd-6.25 ng/ml 300 200 0 200 100 200 14 Stnd-12.5 ng/ml 300 200 0 200 100 200 15 Stnd-25.0 ng/ml 300 200 0 200 100 200 16 Stnd-50.0 ng/ml 300 200 0 200 100 200 17 Stnd-100.0 ng/ml 300 200 0 200 100 200 18 Stnd-200.0 ng/ml 300 200 0 200 100 200 19 Sample #1 300 0 200 200 100 200 20 300 0 200 200 100 200 21. Sample #2 300 0 200 200 100 200 22 300 0 200 200 100 200 23 Sample #3 300 0 200 200 100 200 24 300 0 200 200 100 200

(continue samples as needed in duplicate)

B0 control 500 0 0 200 100 200 500 0 0 200 100 200 500 0 0 200 100 200 APPLIED EXERCISES 199

TABLE V-l 1. (cont.)

Tube Identification PBSG Standard Sample Antiserum lx ARGG No. (Ml) (Ml) (Ml) (Ml) (MO (MO

Stnd-0.78 ng/ml 300 200 0 200 100 200 Stnd-1.56 ng/ml 300 200 0 200 100 200 Stnd-3.13 ng/ml 300 200 0 200 100 200 Stnd-6.25 ng/ml 300 200 0 200 100 200 Stnd-12.5 ng/ml 300 200 0 200 100 200 Stnd-25.0 ng/ml 300 200 0 200 100 200 Stnd-50.0 ng/ml 300 200 0 200 100 200 Stnd-100.0 ng/ml 300 200 0 200 100 200 Stnd-200.0 ng/ml 300 200 0 200 100 200

1:400 NRS only, no antiserum.

(6) Mix tubes well on a vortex mixer and incubate at 4°C for 24 h. (7) Add 100 pi of the 1 x tracer solution to all tubes. (8) Mix tubes well on a vortex mixer and incubate at 4°C for 24 h. (9) Add 200 fx 1 of diluted ARGG to all tubes except numbers 1, 2 and 3. (10) Mix tubes well on a vortex mixer and incubate at 4°C for 72 h. (11) Add 3 ml of cold PBS to all tubes except numbers 1, 2 and 3. Do not mix. (12) Centrifuge all tubes except numbers 1, 2 and 3 at 1000 g for 30 min at 4°C. Determine correct centrifuge speed for 1000 g by applying the equation given in Appendix VI-8. (13) Carefully pour the supernatant of each tube into a radioactive liquid waste container. Blot rim of the tubes with absorbent paper. (14) Quantify radioactivity in the precipitates remaining in the tubes with a gamma counter. Record counts (Table V-l 1).

CALCULATION OF RESULTS (TABLE V-l 1)

(1) Determine uncorrected binding in the B0 control tubes by calculating the mean counts per minute in the B„ tubes and dividing by the mean counts/min in tubes 1, 2 and 3.

Mean counts/min in Bo tube Uncorrected B0 binding = X 100 Mean counts/min in total tubes 200 PART V. LABORATORY EXERCISES

TABLE V-l 1. CALCULATION OF RESULTS FOR THE LH RADIO- IMMUNOASSAY

Tube Counts/min % of Bo LH concentration No. binding (ng/ml)

1 31 965 2 32 017 3 31899 4 255 5 245 6 260 7 10 643 100.0 8 10 748 9 10 800 10 10 140 92.2 11 9 652 87.8 12 8 814 79.8 13 7 470 67.4 14 5 900 52.7 15 3 972 34.7 16 2 361 19.7 17 1 499 11.7 18 740 4.5 19 ' 7 556 68.3 20 7 601 21 1 416 12.5 22 1 596 23. 4 980 .44.2 24 5 001

10 640 APPLIED EXERCISES 201

TABLE V-l 1. (cont.)

10 629 10 710 10 141 9 662 8 806 7 488 5 904 3 968 2 360 1 505 720

1.0 10.0 100.0 Hormone (ng/ml)

FIG. V—9. Standard curve for LH radioimmunoassay. 202 PART V. LABORATORY EXERCISES

(2) Determine non-specific binding by dividing the mean counts/min in tubes 4, 5 and 6 by mean counts/min in tubes 1, 2 and 3. This number should be less than 5%.

Mean counts/min in NSB tubes Non-specific binding % = X 100 Mean counts/min in total tubes

(3) Subtract the NSB from uncorrected B0 binding. This is corrected B0 binding.

Uncorrected B0 binding — NSB = corrected Bo binding

When divided by the mean total counts, this number should range from 20 to 40%. If less than 20%, the radioiodinated hormone was severely damaged during or after iodination, the antiserum is too dilute, or the ARGG is diluted incorrectly. If greater than 40%, the antiserum should be diluted further.

(4) Since the B0 binding is the maximum possible in a particular assay, consider

B0 binding as 100%. (5) Determine corrected binding for each standard solution by subtracting mean counts/min in NSB tubes from mean counts/min for each standard

concentration and dividing by corrected B0 binding.

Mean counts/min in standards — mean NSB counts/min % of B0 binding = X 100 Corrected B0 binding counts/min

(6) Plot a standard curve on semi-log graph paper using the known concentration of hormone in standard solutions and binding for each concentration as shown in Fig. V-9. (7) Determine binding for each unknown sample by subtracting mean counts/min in NSB tubes from mean counts/min for each unknown sample and dividing

by corrected B0 binding.

Mean counts/min in samples — mean NSB counts/min % of B0 binding = X 100 Corrected Bo binding counts/min

(8) From the standard curve, determine the concentration of hormone which has the same binding. This concentration (ng/ml) is the concentration in the unknown sample.

(Note: See also Exercises 16.2. and 19.3.) PART VI. APPENDICES

Appendix VI —1

HOW TO PUT ON AND TAKE OFF RUBBER GLOVES1

The technique employed in this procedure is such that the inside of a glove is not touched by its outer side or the outside of the other glove, nor is any part of the outside of a glove allowed to come into contact with the bare skin. The procedure is as follows:

Putting on the gloves ( 1 ) The gloves should be dusted internally with talcum powder. (2) The cuff of each glove should be folded over, outwards, for about 5 cm. (3) Put one glove on by grasping only the internal folded-back part' with the other hand. (4) Put the second glove on by holding it with the fingers of the gloved hand tucked in the fold and only touching the outside of the glove. (5) Unfold the gloves (cuffs) by manipulating the fingers inside the fold. Taking off the gloves (6) In taking off the gloves, seize the fingers of one glove by the other gloved hand and pull free. (7) Take off the other glove by manipulating the fingers of the free hand under tlie cuff of the glove and fold it back so that an internal part is exposed which may be seized, and the remaining hand freed.

It is a great advantage if the inside and the outside of the gloves are distinctly different, for example in colour or texture.

1 Adapted from BOURSNELL, J.C., Safety Techniques for Radioactive Tracers, Cambridge University Press, London (1958).

203 204 PART VI. APPENDICES

Appendix VI—2

RADIOACTIVE WASTE CONTROL AND DISPOSAL2

VI-2.1. WASTE COLLECTION

Suitable receptacles should be available in all working places where radioactive waste may originate.

Solid waste should be deposited in refuse bins with foot-operated lids. The bins should be lined with removable plastic or paper bags to facilitate removal of the waste without contamination.

If no other facilities for liquid-waste disposal exist, liquid waste should be collected in bottles kept in pails or trays designed to retain all their contents in the event of a breakage. Containers are available for liquid waste, which are provided with a suitable absorbent so that the waste is held in a solid form for subsequent storage or disposal.

All receptacles for radioactive waste should be clearly identified. In general, it will be desirable to classify radioactive waste according to methods of storage or disposal, and to provide separate containers for the various classes of waste. Depending upon the needs of the installation, one or more of the following bases for classifying waste may be found desirable:

(a) Gamma radiation levels (high, low); (b) Total activity (high, medium, low); (c) Half-life (long, short); (d) Combustible, non-combustible. For convenient and positive identification, it may be desirable to use both colour Coding and wording.

Shielded containers should be used when necessary (e.g. for gamma emitters).

It is generally desirable to maintain an approximate record of the quantities of radioactive waste released to drainage systems or to sewers, or for burial. This may be particularly important in the case of long-lived radioisotopes. For this purpose it may be desirable or necessary to maintain a record of estimated quantities of radioactivity deposited in various receptacles, particularly for high levels of activity or long-lived radionuclides. Depending upon the system of control used by the installation, it may be desirable to provide for the receptacle to be marked or tagged with a statement of its contents.

Radioactive waste should be removed from working places by designated personnel under competent supervision.

VI —2.2. WASTE STORAGE

All waste which cannot be immediately disposed of in conformity with the requirements of the competent authority must be placed in suitable storage.

Storage may be temporary or indefinite. Temporary storage is used to allow for decrease of activity, to permit regulation of the rate of release of activity, to permit monitoring of materials of unknown degree of hazard or to await the availability of suitable transport. Indefinite centralized storage in special places must be provided by the competent authority for the more hazardous waste for which no ultimate disposal method is available to the particular user.

2 Based on §8 of Safe Handling of Radionuclides, 1973 Edition, Safety Series No.l, IAEA, Vienna (1973). See also: The Management of Radioactive Wastes Produced by Radioisotope Users, Safety Series No. 12, IAEA, Vienna (1965); The Management of Radioactive Wastes Produced by Radioisotope Users: Technical Addendum, Safety Series No.19, IAEA, Vienna (1966). APPENDIX VI-2. RADIOACTIVE WASTE CONTROL AND DISPOSAL 205

Storage conditions should meet the safety requirements for storage of sources, as set forth in the IAEA's Safety Series No. 1, §4. 3

The storage site should not be accessible to unauthorized personnel. (Control of animals should not be overlooked.)

The method of storage should prevent accidental release to the surroundings.

Appropriate records should be kept of the storage.

VI-2.3. EFFLUENT RELEASE TO THE ENVIRONMENT

VI-2.3.1. Genera] considerations

Radioactive effluent releases to the environment should be carried out in accordance with conditions established by the radiological health and safety officer and by the competent authority.

The ways in which radioactive materials may affect the environment should be carefully examined in relation to any proposed method of effluent release.

The capacity of any route of disposal to accept radioactive effluent safely depends on the evaluation of a number of factors, many of which depend on the particular local situation. By assuming unfavourable conditions with respect to all factors, it is possible to set a permissible level for effluent release which will be safe under all circumstances. This usually provides a very considerable safely factor. The real capacity of a particular route of effluent release can only be found from a lengthy study by experts.

The small user should first try to work within restrictive limits which are accepted as being safe and which will usually provide a workable solution to the problem of effluent release. Such a restrictive safe limit may be arrived at by identifying: (a) The critical radionuclide; (b) The critical pathway to man; (c) The critical group of the population concerned; (d) The critical organ. Thereafter it will be possible to calculate the amount and rate of radioactivity that may safely be released at a given point in the environment, taking into account any alternative requirements the competent authority may impose if local studies by experts provide reasonable justification for other levels. More detailed guidelines for this exercise can be found in the appropriate Safety Series publications of the International Atomic Energy Agency. In practice, however, the small user seldom requires to go into such detailed considerations since the type of work and the amount and nature of the radionuclide involved would not in all probability pose a significant environmental problem.

VI-2.3.2. Effluent release to drains and sewers

The release of radioactive effluent into drains does not usually need to be considered as a direct release into the environment.. Hence, a restrictive safe limit will usually be provided if the concentration of radioactive effluent, based on the total available flow of water in the system averaged over a moderate period (a day or a month), does not lead to exposure of individual members of the public to doses in excess of the dose limits prescribed in the Agency's Basic Safety Standards for Radiation Protection". In arriving at the dose rates and amounts of discharge the factors summarized in the last paragraph of the previous section may need to be considered in some cases. Finally, before release of radioactive

3 INTERNATIONAL ATOMIC ENERGY AGENCY, Safe Handling of Radionuclides, 1973 Edition, Safety Series No.l, IAEA, Vienna (1973). 4 INTERNATIONAL ATOMIC ENERGY AGENCY, Basic Safety Standards for Radiation Protection, 1976 Edition, Safety Series No.9, IAEA, Vienna (1967). 206 PART VI. APPENDICES effluent to public drains, sewers or rivers, the competent authority should be consulted to ascertain that no other radioactive effluent is being released in such a way that the accumulated releases will create a hazardous situation.

Radioactive release to drains should be readily soluble or dispersible in water. Account should be taken of possible changes of pH-value due to dilution or other physico-chemical factors, which might lead to precipitation or vaporization of diluted materials.

In general, the excreta of persons being treated by radioisotopes do not call for special consideration.

Wastes should be flushed down the pipe by a copious stream of water.

The dilution of radioactive effluent by the addition of stable isotopes of the radioactive elements present in the effluent may be considered.

Maintenance work on active drains within an establishment should only be carried out with the knowledge of the radiological health and safety officer and under competent supervision. Special attention should be given to the possibility that small sources may have been dropped into sinks and retained in traps or catchment basins.

The release of radioactive effluent to sewers should be done in such a manner as not to require protective measures during maintenance work on sewers outside the establishment, unless other agreement has been reached with the authority in charge of those sewers. This authority should be informed of the release of radioactive effluent into the sewer system; mutual discussion of the technical aspects of the waste disposal problem is desirable to provide protection, and to avoid unnecessary anxiety.

VI-2.3.3. Effluent release to the atmosphere

Any release of radioactive effluent in the form of gases or aerosols into the atmosphere should con- form with the requirements of the competent authority.

If protection is based on an elevated release point from a stack, levels of release should only be set after examination of local conditions by an expert.

The need for filtration of gases or aerosols before release as waste should be assessed.

Used filters should be handled as solid waste.

VI-2.3.4. Burial of waste

Burial of waste in soil sometimes provides a measure of protection not obtained if the waste is released directly to the environment. The possibilities of safe burial of waste should always be appraised by an expert.

Burial under a suitable depth of soil (about one metre) provides economical protection from the external radiation of the accumulated deposit.

A burial site should be under the control of the user, and adequate steps to exclude the public from it should be taken.

A record should be kept of disposals into the ground.

VI—2.3.5. Incineration of waste

If solid waste is incinerated to reduce the bulk to manageable proportions, adequate precautions should be taken.

The incineration of active waste should only be carried out in equipment embodying such features for filtration and scrubbing as may be necessary for the levels of activity to be disposed of.

Residual ashes should be prevented from becoming a dust hazard, for example by damping them with water, and should be properly dealt with as active waste. APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 207

Appendix VI-3

DERIVED LIMITS FOR CONTROLLABLE EXPOSURE5

The limits of doses for controllable exposure are given in detail in the Agency's Basic Safety Standards for Radiation Protection (1967 Edition)6. To ensure, in the case of internal radiation exposure, that the maximum permissible doses and dose limits are not exceeded, derived limits are available for practical application. The more appropriate derived limit for this purpose would be the body or critical organ content of the radionuclides giving the maximum permissible dose rate. However, in practice, the use of this derived limit is not easy and therefore another derived limit which may be used is the maximum permissible annual intake in air or water, subject to the qualifications indicated in the Basic Safety Standards®. The intakes are given in Tables IIA and IIB in the Basic Safety Standards. However, the estimation of intakes is not always easy and, for practical control of internal exposure, a third derived standard is used, i.e. the derived concentration limit (DCL), formerly known as maximum permissible concentration. The derived concentration limit is not given in the Basic Safety Standards, though a method of computing is indicated. For the convenience of the user, DCL values for occupational exposure are given in the following Table. Derived air concentration (DAC) and derived water concentration (DWC) are also given for practical convenience. For control purposes, keeping within the DCL will ensure that the dose equivalent to the worker does not exceed the maximum permissible dose for occupational exposure, provided there is no external exposure. It should be noted that the DCL values in this Table apply to workers only. Furthermore, in practice, the DWC values should normally be of technical interest only, since workers should not, at their working place, consume water containing radioactivity at higher concentrations than in public water supplies.

Table of derived concentration limits commences overleaf.

5 INTERNATIONAL ATOMIC ENERGY AGENCY, Safe Handling of Radionuclides, 1973 Edition, Safety Series No.l, IAEA, Vienna (1973), Table Al-IIa. 6 INTERNATIONAL ATOMIC ENERGY AGENCY, Basic Safety Standards for Radiation Protection, 1967 Edition, Safety Series No.9, IAEA, Vienna (1967). 208 PART VI. APPENDICES

DERIVED CONCENTRATION LIMITS OF RADIONUCLIDES IN AIR AND WATER FOR OCCUPATIONAL EXPOSURE (40 h/week)

These tables give curies as units of radioactivity; the data for (id (or ¿/Ci/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration^ Radionuclide Critical organ a permissible concentration (ingestion) dose rate (fiCi/cms) (/jCi/cmS) (MCi)

J H (HTO or SH,0) (soluble) Body tissue 5 x 10"s 0.1 Total body 1.2 x 101

(SH,) (submersion) Skin 2 x 10"!

J Be (soluble) Gl (LU) 0. 05 Total body 5.6 x 10! 6 x 10"®

(insoluble) Lung 52 10"' Gl (LLI) 0. 05

"c (CO,) (soluble) Fat 1. 6 x 10! 4 x 10"s 0. 02

(submersion) Total body 5 x 10"*

"9F (soluble) GI (SI) 5 x 10"« 0.02 (insoluble) GI(ULI) 3 x 10"« 0. 01

„Na (soluble) Total body 12 2 x 10"' 10"1

(insoluble) Lung 1 9 x 10"9 Gl (LLI) 9 x 10"4

8 s nNa (soluble) GI (SI) 10" 6 x 10"

(insoluble) GI (LLI) 10-' 8 x 10"4

Jlo, 14 (soluble) Gl (S) 6 x 10"« 0. 03 (insoluble) Gl (ULI) 10'« 6 x 10"s

32 D 4 15 (soluble) Bone 3.1 1 X 10-« 5 x 10" (insoluble) Lung 1.2 8 x 10"1 Gl (LLI) 7 x 10"4

s "16s (soluble) Testis 0.2 3 x 10-' 2 x 10" (insoluble) Lung 15 3 x 10"' GI (LLI) 8 x 10"1

ÎS" (soluble) Total body 15 4 x 10-' 2 X 10"' (insoluble) Lung 3.2 2 x 10'' GI (LLI) 2 x 10"'

"CI (soluble) Gl (S) 3 x 10"« 0. 01 (insoluble) Gl (S) 2 x 10"« 0.01

> (submersion) Skin 6 x 10"3

{¡Ar (submersion) Total body 2 x 10"®

3 The abbreviations GI, S, SI, ULI, and LLI refer to gastro-intestinal tract, stomach, small intestine, upper large intestine, and lower large intestine, respectively, k The derived water concentration values should not be interpreted to mean that the worker is liable, at his working place, to drink water containing radioactivity at higher concentrations than in public water supplies. APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 209

These tables give curies as units of radioactivity; the data for/¿Ci (or/iCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (fiCi/cm3) OiCi/cm') OiCi)

42 K (soluble) Gl (S) 2 x 10"« 9 x 10"'

(insoluble) GI (LLI) 10" 6 x 10"'

ilP* (soluble) Bone 26 3 x 10"« 3 x 10"« (insoluble) Lung 9.7 10-' Gl (LLI) 5 x 10-'

î'.c» (soluble) Bone 4.2 2 x 10"' 10"' (insoluble) GI (LLI) 2 x 10"' 10"' Lung 1 2 x 10-'

il (soluble) Gl (LLI) 2 x 10"' 10"' Liver 2.2 2 x 10"'

(insoluble) Lung 1. 3 2 x 10"' GI (LLI) 10"'

Il. (soluble) Gl (LLI) 6 x 10"' 3 x 10"' (insoluble) Gl (LLI) 5 x 10"' 3 x 10"'

41 Sr ¡1 (soluble) GI (LLI) 2 x 10"' 8 x 10"' (insoluble) GI (LLI) 10" 8 x 10"4

4 «ÎSV (soluble Gl (LLI) 2 x 10"' 9 x 10" (insoluble) Lung 0. 93 6 x 10"' GI (LLI) 8 x 10"4

5 ÄCr (soluble) GI (LLI) 10" 0. 05 Total body 780 10"5

(insoluble) Lung 60 2 x 10"6 Gl (LLI) 0.05

SM» (soluble) GI (LLI) 2 x 10"' 10"' (insoluble) Lung 0. 87 10"' GI (LLI) 9 x 10"4

»Mn (soluble) GI (LLI) 4 x 10"' Liver 6. 2 4 x 10"'

(insoluble) Lung 3. 6 4 x 10"' GI (LLI) 3 x 10"'

*Mn (soluble) Gl (LLI) 8 x 10"' 4 x 10"'

(insoluble) GI (LLI) 5 X 10"' 3 x 10"'

(soluble) Spleen 19 9 x 10"' 0. 02

(insoluble) Lung 130 10"« GI (LLI) 0. 07 210 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate 0iCi/cm') OjCi/cms) Oí Ci)

g Fe (soluble) Gl (LU) 2 x 10"' Spleen 0. 31 10-'

(insoluble) Lung 2 5 x 10-« Gl (LLI) 2 x 10"!

fJCo (soluble) Gl (LLI) 3 x 10"® 0.02

(insoluble) Lung 16 2 x 10"' Gl (LLI) 0.01

m |'Co (soluble) Gl (LLI) 2 x 10"s 0.08

(insoluble) Lung 4.2 9 x 10"« Gl (LLI) 0.06

„Co (soluble) Gl (LLI) 8 x 10"' 4 x 10"1 Total body 32

(insoluble) Lung 3 5 x 10"' Gl (LLI) 3 x 10"'

SJCo (soluble) Gl (LLI) 3 x 10"' 10-J Total body 13

(insoluble) Lung 1.2 9 x 10"' Gl (LLI) 10"»

UNÍ (soluble) Bone 1400 5 x 10"' 6 x 10"»

(insoluble) Lung 110 8 x 10"' Gl (LLI) 0.06

4 "NÎ8 i (soluble) Bone 100 6 x 10"' 8 x 10" (insoluble) Lung 40 3 x 10"' Gl (LLI) 0. 02

S5 NÍ (soluble) Gl (ULI) 9 x 10"' 4 x 10"»

(insoluble) Gl (ULI) 5 x 10"' 3 x 10"'

„Cu (soluble) Gl (LLI) 2 x 10"« 0.01

(insoluble) Gl (LLI) 10"« 6 x 10"'

(soluble) Total body 61 10"' 3 x 10"J Prostate 0.1 10" Uver 9.5 10"'

(insoluble) Lung 5.6 6 x X0-« Gl (LLI) 5 x 10"'

(soluble) Gl (LLI) 4 X 10"' 2 x 10"J Prostate 0.013 4 x 10"'

(insoluble) Gl (LLI) 3 x 10"' 2 x 10"' APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 211

These tables give curies as units of radioactivity; the data for /¿Ci (or /¿Ci/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Critical organ permissible concentration Radionuclide (ingestion) dose rate 0i Ci/cm3) OiCi/cmS) diCi)

Gl (S) 0. 05 30 (soluble) Prostate 0.015 1 x 10"«

(insoluble) Gl (S) 9 x 10"« 0. 05

(soluble) Gl (LU) 2 x 10"' 10"3

(insoluble) Gl (LLI) 2 x 10"' 10"3

lice (soluble) Gl (LLI) 10~s 0. 05

(insoluble) Lung 84 6 x 10"6 GI (LLI) 0. 05

(soluble) GI (LLI) 0. 01 Total body 320 2 x 10"®

(insoluble) Lung 20 4 x 10"' GI (LLI) 0. 01

(soluble) GI (LLI) 3 x 10"' 2 x 10"3

(insoluble) Lung 2. 2 10"' GI (LLI) 2 x 10"3

ÏA» (soluble) GI (LLI) 10"' 6 x 10-' (insoluble) Gl (LLI) 10" ' 6 x 10"4

»As (soluble) GI (LLI) 5 x 10"' 2 x 10"1

(insoluble) GI (LLI) 4 x 10"' 2 x 10"3

'¿Se (soluble) Kidney 3.5 10"® 9 x 10"3 Total body 98 10"6

(insoluble) Lung 8. 9 10"' GI (LLI) 8 x 10"'

> (soluble) Total body 11 10"® 8 x 10"3 GI (SI) 8 x 10"!

(insoluble) GI (LLI) 2 x 10"' 10"3

'¿Kr"1 (submersion) Total body 6 x 10"«

y Kr (submersion) Total body 10"5

»Kr (submersion) Total body 10"«

S«b (soluble) Total body 28 3 x 10"' 2 x 10"3 Pancreas 0. 09 3 x 10"' 2 x 10"3 Liver 2.2

(insoluble) Lung 1.3 1 x 10"* Gl (LLI) 7 x 10"« 212 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ(o r jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate OiCi/cm3) (M Ci/cm') (MCI)

7 SRb (soluble) Pancreas 0. 065 5 x 10" 3 x 10"' Total body 220 Liver 16

(insoluble) Lung 9.3 7 x 10"* GI (LLI) 5 x 10"'

!„sSrm (soluble) GI (SI) 4 x 10" 5 0.2

(insoluble) GI (SI) 3 x 10"5 0.2

> (soluble) Total body 59 2 x 10"' 3 x 10"' (insoluble) Lung 5.2 10"' GI (LLI) 5 x 10"3

> (soluble) Bone 3.9 3 x 10"' 3 x 10"4 (insoluble) Lung 1. 5 '4 x 10"' Gl (LU) 8 x 10"4

'„°Sr (soluble) Bone 2 1 x 10"® 1 x 10"s

(insoluble) Lung 0.76 5 x 10"' GI (LLI) 10" 5

5¡Sr (soluble) GI (LLI) 4 x 10"' 2 x 10"1

(insoluble) GI (LLI) 3 x 10"' 10"'

> (soluble) Gl (ULI) 4 x 10"' 2 x 10"! (insoluble) Gl (ULI) 3 x 10"' 2 x 10"'

90r » v (soluble) Gl (LLI) 10" 6 x 10"4

(insoluble) Gl (LLI) 10-' 6 x 10" 4

«lyni 5 59* (soluble) Gl (SI) 2 x 10" 0.1 (insoluble) Gl (SI) 2 x 10"s 0.1

4 JSY (soluble) Gl (LLI) 3 x 10" Bone 3. 8 4 x 10"'

(insoluble) Lung 1.4 3 x 10"' Gl (LLI) 8 x 10"4

ÏV (soluble) Gl (ULI) 4 x 10"' 2 x 10"' (insoluble) Gl (ULI) 3 x 10"' 2 x 10"'

(soluble) Gl (LLI) 2 x 10"' 8 X 10"4 2* (insoluble) Gl (LLI) 10"' 8 x 10"4 APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 213

These tables give curies as units of radioactivity; the data for /iCi (or/iCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (pCi/cm') 0iCi/cm') OiCi)

95Zr (soluble) Gl (LLI) 0. 02 Bone 100 10"

(insoluble) Lung 43 3 x 10"' GI (LLI) 0. 02

UZr (soluble) GI (LLI) 2 x 10"' Total body 18 10"'

(insoluble) Lung 1.6 3 x 10"' Gl (LLI) 2 x 10->

> (soluble) GI (LLI) 10"' 5 x 10"* (insoluble) Gl (LLI) 9 x 10"» 5 X 10"«

(soluble) GI (LLI) 0.01 Bone 91 10-'

(insoluble) Lung 22 2 x 10"' Gl (ai) 0.01

"Nb (soluble) Gl (LLI) 3 x 10"' Total body 38 5 x 10"'

(insoluble) Lung 3.2 10"' GI (LLI) 3 x 10"'

ÎÎNb (soluble) Gl (ULI) 6 x 10"s 0.03

(insoluble) Gl (ULI) 5 x 10"5 0.03

J|MO (soluble) Kidney 0. 56 7 x 10"' 5 x 10"' Gl (LLI)

(insoluble) Gl (LLI) 2 x 10"' 10"'

Tcm (soluble) Gl (LLI) 8 x 10"s 0.4

(insoluble) Lung 1. 3 3 x 10"' Gl (LLI) 0.3

«Te (soluble) Gl (LLI) 6 x 10"' 3 x 10"'

(insoluble) Gl (LLI) 2 x 10"' 10"'

î',Tcm (soluble) Gl (LLI) 2 x 10"s 0.01

(insoluble) Lung 9.3 2 x 10"' GI (LLI) 5 x 10*'

r>Tc (soluble) GI (LLI) 10"' 0. 05 Kidney 13 10"'

(insoluble) Lung 42 3 x 10"' GI (LLI) 0. 02 214 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ(o r jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (jj Ci/cm3) ((jCi/cm3) ÜiCi)

99-TV m 5 (soluble) Gl (ULI) 4 x 10" 0.2

(insoluble) Gl (ULI) 10-5 0. 08

"Tc (soluble) Gl (LLI) 2 x 10"' 0. 01

(insoluble) Lung 8.9 6 x 10"' Gl (LLI) 5 x 10"3

> (soluble) Gl (LLI) 2 x 10"« 0.01 (insoluble) Gl (LLI) 2 x 10"« 0. 01 Lung 2 x 10"«

(soluble) Gl (LLI) 5 x 10"' 2 x 10"3

(insoluble) Lung 3. 1 8 x 10"' Gl (LLI) 2 x 10"3

(soluble) Gl (ULI) 7 x 10"' 3 x 10"'

(insoluble) Gl (ULI) 5 x 10"' 3 x 10"3

'«Ru (soluble) Gl (LU) 8 x 10"' 4 x 10"'

(insoluble) Lung 0.6 6 x 10"' Gl (LLI) 3 x 10"'

(soluble) Gl (S) 8 x 10"s 0.4

(insoluble) Gl (S) 6 x 10"« 0.3

(soluble) Gl (LLI) 8 x 10"' 4 x 10"3

(insoluble) Gl (LLI) 5 x 10"' 3 x 10"3

(soluble) Gl (LLI) 0.01 Kidney 4. 10 10"«

(insoluble) Lung 13 7 x 10"' Gl (LLI) 8 x 10"3

-Pd (soluble) Gl (LLI) 6 x 10"' 3 x 10"3

(insoluble) Gl (LLI) 4 x 10"' 2 x 10"3

(soluble) Gl (LLI) 6 x 10"' 3 x 10"3

(insoluble) Lung 2.9 8 x 10"' Gl (LLI) 3 x 10"'

'ÎÎAg™ (soluble) Gl (LLI) 2 x 10"' 9 x 10"'

(insoluble) Lung 1 10-" Gl (LLI) 9 x 10"' APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 215

These tables give curies as units of radioactivity; the data for /iCi (or /uCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (jiCi/cm3) OiCi/cm3) üiCi)

3 'U AA 10" 41 g (soluble) Gl (LLI) 3 x 10"' (insoluble) Gl (LLI) 2 x 10"' io->

'S Cd (soluble) Gl (LLI) 5 x 10"' Liver 14 5 x 10"' Kidney 2.6

(insoluble) Lung 8.4 7 x 10"' Gl (LLI) 5 x 10"s

mcdm (soluble) Gl (LLI) 7 X 10"< Liver 2.3 4 x lo"' Kidney 4 x 10"«

(insoluble) Lung 1.4 4 x 10"' Gl (LLI) 7 X 10"4

'»Cd (soluble) Gl (LLI) 2 x 10"' 10"'

(insoluble) Gl (LLI) 2 x 10"' 10"»

us. m „In (soluble) Gl (ULI) 8 x 10"« 0.04

(insoluble) Gl (ULI) 7 x 10"® 0. 04

lu. m 4 49 (soluble) Gl (LLI) 10" 5 X 10" Kidney 0. 27 lo"' Spleen 0.14 10"'

(insoluble) Gl (LLI) 5 x 10"4 Lung 0. 89 2 x 10"»

lis, ra 49111 (soluble) Gl (ULI) 2 x 10"® 0.01 (insoluble) Gl (ULI) 2 x 10"® 0. 01

5 s«Sn (soluble) Gl (LLI) 2 x 10" Bone 16 4 x 10"'

(insoluble) Lung 3.6 5 x 10"' Gl (LLI) 2 x 10"5

> (soluble) Gl (LLI) 10"' 5 X 10"4 (insoluble) Lung 0. 87 8 x 10"' Gl (LLI) 5 x 10*4

lIïsb (soluble) Gl (LLI) 2 x 10"' 8 x 10"4

(insoluble) Gl (LLI) 10-' 8 x 10" 4

'lîSb (soluble) Gl (LU) 2 x 10"' 7 x 10"4 Total body 2 x 10"'

(insoluble) Lung 0.91 2 x 10"« Gl (LU) 7 x 10"4 216 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (fiCi/cm1) (pCi) (M Ci/cm')

'"si, • 51 Sb (soluble) Gl (LLI) 3 x 10"' Lung 3. 3 5 x 10"' Total body 56 Bone 18

(insoluble) Lung 3. 3 3 x 10"' GI (LLI) 3 x 10"'

125 .p m 52 Te (soluble) Kidney 1. 8 4 x 10"' 5 x 10"' Gl (LLI) 5 x 10"' Testis 0.1

(insoluble) Lung 6 10"' Gl (LLI) 3 x 10"'

1!7 m S2 le (soluble) Kidney 0.79 10"' 2 x 10"' Testis 0. 063 10"' 2 x 10"' GI (LLI) 2 x 10"'

(insoluble) Lung 2.6 4 x 10"' Gl (LLI) 2 x 10"'

'gTe (soluble) Gl (LLI) 2 x 10"« 8 x 10"!

(insoluble) Gl (LLI) 9 x 10"' 5 x 10"'

(soluble) GI (LLI) 10"' Kidney 0. 32 8 x 10"» 10"' Testis 0. 016 10"'

(insoluble) Lung 1 3 x 10"' GI (LLI) 6 x 10"'

129 ^ 6 S2 (soluble) Gl (S) 5 x 10" 0. 02 (insoluble) Gl (ULI) 4 x 10"s 0. 02

131 Tam 52 ie (soluble) Gl (LLI) 4 x 10"' 2 x 10"' (insoluble) Gl (LLI) 2 x 10"' 10"'

«Te (soluble) Gl (LLI) 2 x 10"' 9 x 10"'

(insoluble) Gl (LLI) 10"' 6 x 10"'

126 i s 53 ' (soluble) Thyroid 0.21 8 x 10"' 5 x 10"

(insoluble) Lung 4.7 3 x 10"' Gl (LLI) 3 x 10"'

m, 53 (soluble) Thyroid 0.49 2 x 10"' 10"' (insoluble) Lung 10 7 x 10"' Gl (LLI) 6 x 10"' APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 217

These tables give curies as units of radioactivity; the data for /aCi (or ¿/Ci/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (MCi/cm3) (fjCi/cm3) («Ci)

131, 5 531 (soluble) Thyroid 0.15 9 x 10"® 6 x 10" (insoluble) GI (LU) 3 x 10"' 2 x 10"3 Lung 2. 8 3 x 10"'

13 i, S31 (soluble) Thyroid 0. 052 2 x 10"' 2 x 10"3

(insoluble) Gl (ULI) 9 x 10"' 5 x 10"3

1331 4 53 1 (soluble) Thyroid 0. 062 3 X 10"' 2 x 10" (insoluble) GI (LLI) 2 x 10-7 10"'

! 531 (soluble) Thyroid 0.041 5 x 10"' 4 x 10" (insoluble) Gl (S) 3 x 10"« 0. 02

1351 4 53* (soluble) Thyroid 0.065 10-1 7 x 1G"

(insoluble) Gl (LLI) 4 x 10"1 2 x 10-s

m s 's';xe (submersion) Total body 2 x 10"

's»Xe (submersion) Total body 10-s

'»Xe (submersion) Total body 4 x 10"'

s 'lies (soluble) Total body 680 10" 0. 07 Liver 60 10" s

(insoluble) Lung 35 3 x 10"« GI (LLI) 0. 03

'gCsm (soluble) Gl (S) 4 x 10"s 0. 2

(insoluble) Gl (ULI) 6 x 10"« 0. 03

,s4 SSC s (soluble) Total body 18 4 x 10"' 3 x 10"4

(insoluble) Lung 1.5 10"» GI (LLI) 10" 3

13s 3 ss Cs (soluble) Liver 22 5 x 10"' 3 x 10" Spleen 1.9 5 x 10"' Total body 300

(insoluble) GI (LLI) 7 x 10"3 Lung 13 9 x 10"'

136 Cs (soluble) Total body 30 4 x 10"' 2 x 10"3

(insoluble) Lung 2.4 2 x 10"' Gl (LLI) 2 x 10"3 218 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (MCi/cm3 ) (liCi/cm1) (JJCÍ)

'Sc. (soluble) Total body 33 6 x 10"« 4 x 10"' Liver 3.5 Spleen 0.34 Muscle 14

(insoluble) Lung 2 10"« GI (LLI) 10"s

3 'S«» (soluble) CI (LLI) 10"« 5 x 10" (insoluble) Lung 4.4 4 x 10"' Gl (LLI) 5 x 10"3

(soluble) Gl (LLI) 8 x 10"« Bone 2. 6 10"'

(insoluble) Lung 0. 6 4 x 10"! GI (LLI) 7 x 10"*

""Lsi"a (soluble) Gl (LLI) 2 x 10"' 7 x 10"' (insoluble) GI (LLI) 10"' 7 x 10"«

"'Ce (soluble) Gl (LLI) 3 x 10"' Liver 7.9 4 x 10"' Bone 14

(insoluble) Lung 4.7 2 x 10"' GI (LLI) 3 x 10"3

143 3 SB Ce (soluble) GI (LLI) 3 x 10-' 10" (insoluble) GI (LLI) 2 x 10"' . 10"3

'"Ce (soluble) Gl (LLI) 3 x 10"« Bone 1.7 10"« Liver 10"»

(insoluble) Lung 0.64 6 x 10"' GI (LLI) 3 x 10"«

,c 59P r (soluble) GI (LLI) 2 x 10"' 9 x 10"« (insoluble) GI (LLI) 2 x 10"' 9 x 10"«

(soluble) GI (LLI) 3 x 10"' 10"3

(insoluble) Lung 2 x 10"' Gl (LU) 10"3

(soluble) Gl (LU) 4 x 10"' 2 x 10"3 Uver 4.5 4 X 10"'

(insoluble) Lung 2.8 2 x 10"' Gl (LLI) 2 x 10"3 APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 219

3 These tables give curies as units of radioactivity; the data for/¿Ci (or fiCi/cm ) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate OiCi/cm3) OiCi/cm3) üiCi)

'SNd (soluble) GI (LLI) 2 x 10"' 8 x 10"'

(insoluble) Gl (ULI) 10"« 8 x 10"'

(soluble) Gl (LLI) 6 x 10"' Bone 31 6 x 10"'

(insoluble) Lung 12 10"' GI (LLI) 6 x 10"'

(soluble) GI (LLI) 3 x 10"' 10"'

(insoluble) Gl (LLI) 2 x 10"' 10"'

! 3 62 Sm (soluble) Bone 9. 5x 10" 7 x 10"" 2 x 10" GI (LLI) 2 x 10"3

(insoluble) Lung 0. 036 3 x 10"'° Gl (LLI) 2 x 10"'

151 c_ 62 Sm (soluble) GI (LLI) 0. 01 Bone 84 6 x 10"'

(insoluble) Lung 20 10"' GI (LLI) 0. 01

'SSn> (soluble) GI (LLI) 5 x 10"' 2 x 10"' (insoluble) GI (LLI) 4 x 10"' 2 x 10"'

•SH» (soluble) GI (LLI) 4 x 10"' 2 x 10"' (9. 2 h) (insoluble) GI (LLI) 3 x 10"' 2 x 10"'

(soluble) GI (LLI) 2 x 10"' (13 yr) Kidney 1 10""

(insoluble) Lung 2. 5 2 x 10"' GI (LLI) 2 x 10"'

'S*« (soluble) GI (LLI) 6x10"* Kidney 0. 33 4 x 10"® Bone 4.1 4 x 10"'

(insoluble) Lung 0. 97 7 x 10"' GI (LLI) 6 x 10"4

•gf (soluble) GI (LLI) 6 x 10"' Kidney 3 9 x 10"« Bone 39

(insoluble) Lung 8. 8 7 x 10"' GI (LLI) 6 x 10"' 220 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ(o r jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (Ii Ci/cmJ) 0i Ci/cm') OiCi)

'„Gd (soluble) GI (LLI) 6 x 10"' Bone 47 2 x 10"

(insoluble) Lung 8.5 9 x 10-' GI (LLI) 6 x 10"

',¡Gd (soluble) GI (LLI) 5 x 10"' 2 x 10"

(insoluble) GI (LLI) 4 x 10" 2 x 10"3

'IsTb (soluble) GI (LLI) 10"' Bone 10 10-' Kidney 10" Total body 10"

(insoluble) Lung 1.7 3 x 10"' GI (LLI) lo"

'^Dy (soluble) Gl (ULI) 3 x 10"« 0. 01

(insoluble) Gl (ULI) 2 x 10"' 0. 01

'^Dy (soluble) Gl (LLI) 2 x 10"' 10"'

(insoluble) Gl (LLI) 2 x 10"' 10"'

'"Ho (soluble) Gl (LLI) 2 x 10" 9 x 10"4

(insoluble) Gl (LLI) 2 x 10"' 9 x 10"4

'^Er (soluble) Gl (LLI) 6 x 10"' 3 x 10"'

(insoluble) Lung 3.8 4 x 10" Gl (LLI) 3 x 10*'

'¡¡Er (soluble) Gl (ULI) 7 x 10"' 3 x 10"'

(insoluble) Gl (ULI) 6 x 10-1 3 x 10"'

'™Tm (soluble) Gl (LLI) 10" Bone 6. 5 4 x 10"'

(insoluble) Lung 2.5 3 x 10"* Gl (LLI) 10"

"¡Tm (soluble) Gl (LLI) 0. 01 Bone 73 10"

(insoluble) Lung 28 2 x 10" Gl (LLI) 0. 01

"jYb (soluble) Gl (LLI) 7 x 10" 3 x 10"'

(insoluble) Gl (LLI) 6 x 10"' 3 x 10"' APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 221

These tables give curies as units of radioactivity; the data for /¿Ci (or /LtCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (jiCi/cm3) (pCi/cm3) (pCi)

'¡1 Lu (soluble) GI (LLI) 6 x 10-' 3 x 10"3

(insoluble) GI (LLI) 5 x 10"' 3 x 10"3 Lung 5. 2

Hf (soluble) GI (LLI) 2 x 10"' Spleen 0. 50 4 x 10"'

(insoluble) Lung 2.9 7 x 10"' GI (LLI) 2 x 10"'

'{J Ta (soluble) GI (LLI) 10-s Liver 2. 6 4 x 10"'

(insoluble) Lung 1. 5 2 x 10"' GI (LLI) 10"3

•;jW (soluble) GI (LLI) 2 x 10"s 0.01

(insoluble) Lung 9.6 10" GI (LLI) 0. 01

(soluble) GI (LLI) 8 x 10"' 4 x 10'!

(insoluble) Lung 6 10" GI (LLI) 3 x 10"3

(soluble) GI (LLI) 4 x 10"' 2 x 10"3

(insoluble) GI (LLI) 3 x 10"' 2 x 10"3

'"Re (soluble) GI (LLI) 0. 02 Total body 82 3 x 10"« 0. 02

(insoluble) Lung 8.4 2 x 10"' GI (LLI) 8 x 10"3

•;|Re (soluble) GI (LLI) 6 x 10"' 3 x 10"3

(insoluble) GI (LLI) 2 x 10"' io-3

•;¡Re (soluble) GI (LLI) 0. 07 Skin 280 9 x 10"6

(insoluble) Lung 70 5 x 10"' GI (LLI) 0. 04

'Js'Re (soluble) Gl (LLI) 4 x 10"' 2 x 10"3

(insoluble) GI (LLI) 2 x 10"' 9 x 10"4

'"Os (soluble) GI (LLI) 5 x 10"' 2 x 10"3

(insoluble) Lung 2.9 5 x 10"' GI (LLI) 2 x 10"3 222 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡jLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (fjCi/cms) (p Ci/cm3) OiCi)

191 m s 76 Os (soluble) Gl (LLI) 2 x 10" 0. 07 (insoluble) Lung 6.4 9 x 10"® GI (LLI) 0. 07

'So. (soluble) GI (LLI) 10"® 5 x 10"3 (insoluble) Lung 7 4 x 10"' Gl (LLI) 5 x 10"3

(soluble) GI (LLI) 4 x 10"' 2 x 10"3

(insoluble) Gl (LLI) 3 x 10"' 2 x 10"3

-ir (soluble) GI (LLI) 10"® 6 x 10"3

(insoluble) Lung 5. 2 4 x 10"' GI (LLI) S x 10"3

> (soluble) Gl (LLI) 10"3 Kidney 0. 5 10"' Spleen 10"'

(insoluble) Lung 1.4 3 x 10"' Gl (LLI) 10"3

194, 3 7711 (soluble) GI (LLI) 2 x 10"' ÎO" (insoluble) GI (LLI) 2 x 10"' 9 x 10"'

191 pt 71 rl (soluble) Gl (LLI) 8 x 10"' 4 x 10"' (insoluble) Gl (LLI) 6 x 10"' 3 x 10"3

193ptm 78 (soluble) GI (LU) 7 x 10"' 0. 03 (insoluble) Gl (LLI) 5 x 10"® 0. 03 Lung 26

I9Spt 78 (soluble) Kidney 18 10"® 0. 03 (insoluble) Lung 44 3 x 10"' Gl (LLI) 0. 05

197 Df m 78 rl (soluble) Gl (ULI) 6 x 10"' 0. 03 (insoluble) Gl (ULI) 5 x 10"® 0. 03

197pt 3 78 (soluble) Gl (LLI) 8 x 10"' 4 x 10" (insoluble) Gl (LLI) 6 x 10"' 3 x 10"3

6 3 '»A79 u (soluble) Gl (LLI) 10" 5 x 10" (insoluble) Lung 4 6 x 10"' Gl (LLI) 4 x 10"' APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 223

These tables give curies as units of radioactivity; the data for juCi (or£/Ci/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate QiC i/cm3) (pCi/cm1) (MCi)

>u (soluble) Gl (LU) 3 x 10"' 2 x 10"'

(insoluble) Gl (LLI) 2 x 10-' 10"»

>u (soluble) Gl (LLI) 10"' 5 x 10"'

(insoluble) Gl (LLI) 8 x 10"' 4 x 10"'

'SHgm (soluble) Kidney 1.4 7 x 10-' 6 x 10"»

(insoluble) Gl (LLI) 8 x 10"' 5 x 10"»

J T.Hg (soluble) Kidney 5.9 10"« 9 x 10" (insoluble) GI (LLI) 3 x 10"6 0. 01

'I'M (soluble) Kidney 1.7 7 x 10"' 5 x 10"* (insoluble) Lung 4.9 10"' Gl (LLI) 3 x 10"5

6 il 11 (soluble) Gl (LLI) 3 x 10" 0. 01 (insoluble) Gl (LLI) 10"« 7 x 10"'

201 ri 3 81 11 (soluble) GI (LLI) 2 x 10"® 9 x 10" (insoluble) GI (LLI) 9 x 10"' 5 x 10"3

202 Ti SI 1 (soluble) Gl (LLI) 8 x 10"' 4 x 10"' (insoluble) Lung 3. 1 2 x 10"' GI (LLI) 2 x 10"'

204 T1 11 (soluble) GI (LLI) 3 x 10"' 81 Kidney 1 6 x 10"'

(insoluble) Lung 3.4 3 x 10"' GI (LLI) 2 x 10"'

"|Pb (soluble) GI (LLI) 3 x 10"' 0. 01

(insoluble) GI (LLI) 2 x 10"' 0. 01

«»Pb (soluble) Kidney 0. 025 10-" 4 X 10"6 Total body 4 x 10"«

(insoluble) Lung 0. 034 2 x 10"" GI (LLI) 5 x 10"'

(soluble) 0. 0031 4 'S» Kidney 2 x 10"' 6 x 10" Gl (LLI) 6 x 10"4

(insoluble) Lung 0. 010 2 x 10"! GI (LLI) 5 x 10"4

>¡$Bi (soluble) GI (LLI) 2 x 10"' 10"3 Kidney 0.43 2 x 10"'

(insoluble) Lung 1 IQ"' GI (LLI) 10"' 224 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate OiCi/cm') (jjCi/cm3) diCi)

T,Bi (soluble) GI (LLI) 2 x 10"' Kidney 0.76 2 x 10"'

(insoluble) Lung 1.9 10"' GI (LLI) 2 x 10"'

'S" (soluble) Gl (LLI) 10"' Kidney 0. 013 6 x 10"9

(insoluble) Lung 0.032 6 x 10-9 GI (LLI) 10" 3

83 (soluble) GI(S) 0. 01 Kidney 0. 0030 10-'

(insoluble) Lung 0. 010 2 x 10"' Gl (S) 0.01

!1°Po (soluble) Spleen 0.002 5x10"10 2 x 10"s Kidney 0. 0045 5 x 10"10 2 x 10"s

(insoluble) Lung 0. 015 2 x 10"10 GI (LLI) 8 x 10"4

!llAt SS (soluble) Thyroid 0. 00047 7 x 10"' 5 x 10-« Ovary 0. 000031 7 x 10"' 5 x 10"s

(insoluble) Lung 0. 11 3 x 10"! Gl (ULI) 2 x 10"3

Lung 3 x 10"'a

! a > Lung 3 x 10"sa

(soluble) Bone 0. 039 2 x 10"9 2 x 10"s

(insoluble) Lung 3 x 10° 2 x 10"10 Gl (LLI) 10"4

(soluble) Bone 0.039 5 x 10"' 7 x 10"s

(insoluble) Lung 0. 0029 7 x 10"'° GI (LLI) 2 x 10"4

(soluble) Bone 0.1 3 x 10"" 4 x 10"'

(insoluble) GI (LLI) 2 x 10"' 9 x 10"«

(soluble) Bone 0. 058 7 x 10"" 8 x 10"'

(insoluble) Lung 0.0052 4 x 10"11 GI (LLI) 7 x 10"4 a The daughter elements of and miRJI are assumed to be present to the extent that they occur in unfiltered air. For all other radionuclides, the daughter elements are not considered as part of the intake; if daughter elements are present, they must be considered on the basis of the rules for mixtures. APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 225

These tables give curies as units of radioactivity; the data for ¡id (or juCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (fjCi/cm3 ) (fiCi/cm3) (pCi)

(soluble) Bone 0.011 2 x 10",! 6 x 10"s

(insoluble) Lung 0. 0036 3 x 10'» GI (LLI) 9 x 10'3

(soluble) Gl (ULI) 3 x 10"' Bone 0. 011 Liver 0. 026 8 x 10"'

(insoluble) Lung 0. 0052 2 x 10-« Gl (ULI) 3 x 10"3

TK 90 Th (soluble) Gl (LU) 0. 03 5 x 10"« Bone 0. 011 3 x 10"'°

(insoluble) Lung 0. 0036 2 x 10"'° Gl (LLI) 5 x 10"4

221 ! 4 90 Th (soluble) Bone 0. 011 9 x 10"' 2 x 10"

(insoluble) Lung 3. 5 x 10"3 6 x 10"'! GI (LLI) 4 x 10"4

s 90 Th (soluble) Bone 0. 046 2 x 10"" 5 x 10"

(insoluble) Lung 0. 017 10-" GI (LLI) 9 x 10'4

231 tu 3 90 Th (soluble) GI (LLI) 10" 7 x 10"

(insoluble) GI (LLI) 10"« 7 x 10"3

a s 90 1,1 (soluble) Bone 0.041 2 x 10"" 5 x 10" (insoluble) Lung 0. 018 10-" GI (LLI) lo"3

234 « 4 th GI (LLI) »0 (soluble) 5 x 10" Bone 2.4 6 x 10"«

(insoluble) Lung 0. 93 3 x 10"' Gl (LU) 5 x 10-4

,/h (natural)2 (soluble) Bone 2 x 10"1!a 3 x 10_s

(insoluble) Lung 4 x IQ"" GI (LLI) 3 x 10"4

Provisional values for löTh and natural thorium. Although calculations and animal experiments suggest that natural thorium, if injected intravenously, is perhaps as hazardous as plutonium and therefore the above-listed values are indicated, experience to date has suggested that in industry the hazard of natural thorium is not much greater than 11 3 that of natural uranium. Therefore, pending further investigation, the value of (DAC)a = 3 x 10' pCi/cm for occupational exposure (40 h/week) is recommended as a provisional level, permissible for exposure to inhaled natural thorium or 2ÄTh. However, the above values are listed to indicate the possibility that further evidence may require lower values and to urge especially that exposure levels for these radionuclides be kept as low as is opera- tionally readily achievable. It may be possible to show that similar considerations apply to other inhaled long- lived thorium isotopes under conditions in which the physical characteristics of the airborne particulates are much the same as in the case of natural thorium and where there is a large amount of airborne material serving as an effective carrier for the thorium. 226 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (pCi/cm *) ftiCi/cm3) CCI)

"°P91 a (soluble) GI (LLI) 7 x 10"' Bone 0. 034 2 x 10"®

(insoluble) Lung 0. 014 8 x 10"'° GI (LLI) 7 x 10'3

1! s 91 Pa" (soluble) Bone 0.015 10" 3 x 10"

(insoluble) Lung 0.016 10-" GI (LLI) 8 X 10"*

! 91 (soluble) GI (LLI) 4 x 10" Kidney 1.7 6 x 10*'

(insoluble) Lung 4.7 2 x 10"' GI (LLI) 3 x 10"J

»a., a s 9Î (soluble) Kidney 0. 00072 3 x 10"'° 7 x 10" 1 (insoluble) Lung 0. 0024 lo" « GI (LLI) 10"4

0 'S"' (soluble) Bone 0. 0091 lo"' 2 x 10"5 (insoluble) Lung 0. 004 3 x 10"" Gl (LLI) 8 x 10"4

(soluble) Bone 0.044 5 x 10_1° 10"4

(insoluble) Lung 0.017 10-10 GI (LLI) 9 x 10"4

(soluble) Bone 0. 046 6 x 10"10 lO"1

(insoluble) Lung 0. 017 10-10 Gl (LLI) 9 x 10"4 a > (soluble) Kidney 1. 9 x 10° 5 x 10"'0 10"4 Bone 0.048 10"4

(insoluble) Lung 0. 018 10-10 GI (LLI) 8 x 10"'

(soluble) Bone 0. 047 6 x 10"'° 10" 4

(Insoluble) Lung 0. 018 10-10 GI (LLI) 10" 3

4 'Sua (soluble) Kidney 3. 1 x 10" 7 x 10"" 2 x 10"s (insoluble) Lung 0. 02 10-10 GI (LLI) 10"3

a Because of the chemical toxicity of natural uranium, !"U, "®U, and !35U in soluble form, inhalation of uranium of any isotoplc composition should not exceed 2. 5 mg of soluble uranium per day; ingestion of soluble uranium should not exceed 150 mg per day. APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 227

These tables give curies as units of radioactivity; the data for ßC'\ (or/¿Ci/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (fjCi/cm3) ((iCi/cm') (MCI)

„U (natural) a (soluble) Kidney 7 x 10"" 2 x 10"s

(insoluble) Lung 6 x 10'" GI (LLI) 5 x 10"4

(soluble) GI (LLI) 2 x 10"' 10"'

(insoluble) GI (LLI) 2 x 10"' 10"'

KI« ,! s 93 NP (soluble) Bone 0. 044 4 x 10" 9 x 10" (insoluble) Lung 0. 017 10-10 GI (LLI) 9 x 10"4

tJ).. N (soluble) GI (LLI) 8 x 10'' 4 x 10° » P (insoluble) GI (LLI) 7 x 10-7 4 x 10"'

'»Pu (soluble) Bone 0. 039 2 x 10'" 10"4

(insoluble) Lung 0. 015 3 x 10'" GI (LLI) 8 x 10'4

»Pu (soluble) Bone 0. 041 2 x 10'" 10'4

(insoluble) Lung 0. 016 4 x 10-1' Gl (ai) 8 x 10"4

'«Pu (soluble) Bone 0. 041 2 x 10'" 10"4

(insoluble) Lung 0. 016 4 x 10"" GI (LLI) 8 x 10'4

»¡Pu (soluble) Bone 0.78 9 x 10"11 7 x 10'3

(insoluble) Lung 16 4 x 10'« GI (LLI) 0. 04

•s Pu (soluble) Bone 0.044 2 x 10"1! 10'4

(insoluble) Lung 0. 016 4 x 10'" Gl (LLI) 9 x 10"4

(soluble) Gl (ULI) 2 x 10"« 0.01

(insoluble) Gl (ULI) 2 x 10"« 0. 01

(soluble) Bone 0.045 2 x 10'" 10'4

(insoluble) Lung 0.017 3 x 10'" Gl (LLI) 3 x 10'4

a Because of the chemical toxicity of natural uranium, mU,I36U, and2îSU in soluble form, inhalation of uranium of any isotopic composition should not exceed 2. 5 mg of soluble uranium per day; ingestion of soluble uranium should not exceed 150 mg per day. 228 PART VI. APPENDICES

These tables give curies as units of radioactivity; the data for ¡JLCÍ (or jjCi/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (MCi/cm3) ()j Ci/cm') (pCi)

(soluble) Kidney 0.0044 6 x 10-" lo"4 Bone 0. 039 6 x 10-" 10"

(insoluble) Lung 0. 015 10-" GI (LLI) 8 x 10"4

m Am (soluble) Bone 0. 036 6 x 10'" 10"4 Kidney 6 x 10"" lo""

(insoluble) Lung 0. 037 3 x lO"10 GI (LLI) 9 x 10"4

-Am (soluble) GI (LLI) 0. 098 4 x 10"' Liver 0. 023 4 x 10"«

(insoluble) Lung 0. 037 5 x 10"" GI (LLI) 4 x 10"'

>m (soluble) GI (SI) 0.1 Bone 0.044 4 x 10"s Kidney 0. 044 4 x 10"«

(insoluble) Lung 0. 52 2 x 10"s Gl (SI) 2 x 10"s 0. 1

'S Cm (soluble) GI (LLI) 7 x 10"4 Liver 0.018 io">°

(insoluble) Lung 0. 013 2 x 10"10 GI (LLI) 7 x 10"4

'S Cm (soluble) Bone 0. 037 6 x lo"" 10"4

(insoluble) Lung 0. 014 10-10 GI (LLI) 7 x 10"4

144 rm (soluble) Bone 0. 037 9 x 10"" 2 x 10"4

(insoluble) Lung 0. 014 10-10 GI (LLI) 8 x 10"4

«Cm (soluble) Bone 0. 039 5 x 10"" 10"4

(insoluble) Lung 0.015 10-" Gl (LLI) 8 x 10*4

!J|Cm (soluble) Bone 0. 039 5 x 10"" 10" 4

(insoluble) Lung 0. 015 10"'° GI (LLI) 8 x 10"4 247 96 Cm (soluble) Bone 0. 041 5 x 10"" lO"4

(insoluble) Lung , 0.015 10-1» Gl (LU) 6 x 10"4

'«Cm (soluble) Bone 0. 0048 6 x 10"" 4 x 10"s

(insoluble) Lung 0. 0018 10"" GI (LLI) 4 X 10"s APPENDIX VI-3. DERIVED LIMITS FOR CONTROLLABLE EXPOSURE 229

These tables give curies as units of radioactivity; the data for pCi (or /¿Ci/cm3) should be multiplied by 37 to obtain data in kBq (or kBq/cm3).

Organ content Derived water giving maximum Derived air concentration Radionuclide Critical organ permissible concentration (ingestion) dose rate (pCi/cm3) (fiCi/cm3) (MCI)

'¡¡Cm (soluble) Gl (S) 10"» 0. 06 Bone 0.41 10-»

(insoluble) Gl (S) 10"s 0. 06

'«Bk (soluble) Gl (LU) 0. 02 Bone 0. 55 9 X 10"'°

(insoluble) Lung 12 10-' Gl (LLI) 0. 02

!^Bk (soluble) Gl (ULI) 6 x 10"3 Bone 0. 038 lo"'

(insoluble) Gl (ULI) 10"® 6 X 10"3

!«Cf (soluble) Bone 0. 037 2 x 10"'! 10"«

(insoluble) Lung 0.014 10"1» Gl (LLI) 7 x 10"4

™Cf (soluble) Bone 0. 035 5 x 10",! 4 x 10"4

(insoluble) Lung 0. 014 io-'° Gl (LLI) 7 x 10"4

'JJCf (soluble) Bone 0. 038 2 x 10"'! 10"4

(insoluble) Lung 0.014 lO"10 GI (LLI) 8 x 10"4

"!,Cf (soluble) GI (LLI) 2 x 10"4 Bone 0. 01 6 x 10-"

Spontaneous fission Lung 0. 004 3 x 10'" (insoluble) GI (LLI) 2 x 10"4 230 PART VI. APPENDICES

Appendix VI—4

BETA-PARTICLE RANGE AS A FUNCTION OF ENERGY (Emax)

10 20 30 ¿0 50 60 80 100 200 300 400 600 8001000 2000 3000 5000 MAXIMUM RANGE IN ALUMINIUM, p -X (mg cm2) ' Al APPENDIX VI S. INTEGRATION OF EQ.(I-l) 231

Appendix VI—5

INTEGRATION OF EQUATION (1-1). THE RADIOACTIVE DECAY LAW

Given Eq.(l-I) in l'art I:

dN .... „ . — = -AN (VI-5.1 ) dt

Separating variables for the indefinite integration:

dN — = -X / dt + constant (VI-5.2) N '

Now integrating

In N = -M + (' (VI — 5.3>

where C is the constant of integration. C is evaluated at any initial conditions which are:

N = N0 at t = t„

That is, N0 is the number of atoms present at any initial time, t0. Therefore, at t = t0:

C = In N0 + Xt0 (VI-5.4)

Substituting Eq.( VI -5.4) into (VI-5.3):

In N-In N0 =-Xt + Xt0 (VI-5.5)

(VI-5.5')

Now taking the antilogarithm:

,.-Mt - i0) (VI-5.61

X(, ,o) N = N„ c- ~ (VI-5.7)

In the special case of t0 =0, Eq.( VI—5.7) becomes:

Xt N = N0e"

giving the desired equation (1-3). 232 PART VI. APPENDICES

Appendix VI-6

DERIVATION OF EQUATION (1-27)

From Eq. (1-26) in Part I:

ÍR RhM"

where anai,Rs is the natural standard deviation of the net sample count-ratc. By squaring and taking the time derivative of both sides

X.I.R, •<«»«,t.k, = * £ dT - Tí

To obtain the mininiuin value, set donai.Rs = 0; and since the given total period counting time, T1(,t. has to he apportioned between T and Tj,:

, = T + Th (VI-6.3)

(VI 6.4)

dT = - dTh (VI -6.5)

Therefore, rearranging Eq.( VI-6.2) for danal r = 0:

(VI-6.6)

T2 R dT

and introducing Eq.(VI-6.5):

' ^r'^t

which is the desired equation (1-27).

The partition of T|0t (between T and Th) in conformity with Eq.(VI- 6.7) corresponds mathe-

matically to the smallest, i.e. best-partition, onui.Rs value which is obtainable under the given conditions. If we call the fractional natural standard deviation of the net count-rate of the sample f, then, from formula (1-26): '-T-iihgt APPENDIX VI-6. DERIVATION OF EQ. (1-27) 233

and using Tb + T = T|0t and Eq.(VI-6.7) to substitute in Eq.(VI-ft.S) for Tt, and T. one obtains for the best partition value, fbpv:

fbpv = -r—'1 r «V1-6-9)

For a given sample activity in a proportional counter (e.g. gas-flow or scintillation), the values of R and Rb may be altered independently by variation of the high voltage and/or the input-bias voltage. For a certain setting of these two variables, the so-called optimum setting, (y'R-v/Rb) W'H attain its

maximum value, and the natural uncertainty (for the best partition of the given Tlol) will, according to Eq.(VI-6.7), attain its minimum value, fhpvjiiin- f'or another safjjple containing a different activity

the optimum setting corresponding to fbpV,min will in general be different. Theoretically, the choice of operating conditions (high voltage and input-bias voltage) on the basis of minimum natural uncertainty is thus a complex problem. However, natural counting uncertainty, at least in biological experimentation, is usually not critical in comparison with technical uncertainty, except when low activity samples (R — Rb) are to be measured. But when R is not much greater than Rb. the difference R^ - R^ may be approximated7 by |(R - R|>)/2Rj], so that the optimum setting (corresponding to fbpv.min ) may be approximated by that for which [(R - Rb)/2R£] or (R - Rh)2/Rb attains its maximum value. Since this approximate optimum criterion for low activities is equivalent to e3/Rh attaining its maximum value, it in independent of sample activity (e is the overall counting yield). In tracer work (non-GM counter) operating conditions arc usually chosen as optimum on the basis of minimum natural uncertainty for very low-activity samples; i.e. the maximum of (R - Rb)2/Rb or eJ /Rb is taken as the criterion. However, for expediency, a medium or high-activity source is normally used in finding the operating conditions that give the maximum value of (R - Rb)2/Rb. which is permissible becausc this maximum, as mentioned above, is independent of sample activity.

so, for R - Rh, using tlie binomial expansion:

f& - R- * R¡¡ 11 + i (R - Rh)/R„ - 11 = RV ( R - R„)/2Rb or

RV-RTa(R-Rh)/:RV 234 PART VI. APPENDICES

Appendix VI—7

CHARACTERISTICS OF SOME COMMON RADIONUCLIDES USED IN BIOLOGICAL RESEARCH INCLUDING RADIOIMMUNOASSAYS

Radionuclide Radiation emitted

Symbol _ . ,, ,, Additional radiation Z and Half-life Type Energies m MeV from (Per cent disintegration) mass No. daughter nuclide .

3 1 H 12.35 a r max. 0.0186 (100)

,4 6 C 5730 a r max. 0.156 (100)

l8 9 F 109.7 min r max. 0.635 (97) e (3) y

11 »Na 15.0 h r max. 1.389 (100) y 2.754 (100); 1.369 (100) 12 28Mg 21.1 h fT max. 0.46(100) ya 1.34 (69) ; 0.94 (28) ; 0.40 (31 ) ; 28Al radiations 0.031 (95) 13 28 Al 2.24 min r max. 2.86 (100) y 1.780(100)

15 32p 14.3 d r max. 1.710(100) 35 16 S 88.0 d ß- max. 0.167 (100) 17 38 CI 37.3 min ß- max. 4.91 (56); 2.74(11); y 1.10(33) 2.17(44); 1.64 (33) *> 19 K 1.28 X 10'a ß- max. 1.314(89); avg. 0.490 e (11) y 1.460(11)

19 42 K 12.36 h ß- max. 3.52 (82); 2.00 (18); others y 1.524 (18); 0.31 (0.2); others 20 45 Ca 164 d ß' max. 0.26(100) 20 47Ca 4.53 d ß- max. 1.98 (18); 0.69 (82); others y 1.30 (75); 0.815 (7); 0.49 (7); 47Sc radiations others 21 47Sc 3.40 d ß- max. 0.600(30); 0.44 (70) y 0.160(70)

23 18 y 16.0 d r max. 0.696 (49) e (51) y 2.241(3); 1.312(97); 0.983 (100); 0.945 (10)

24 51 Cr 27.7 d e 0.0050(22) y' 0.320 (10)

a Gamma-ray emission is significantly affected by internal conversion (ejection of an atomic orbital electron occurring sometimes instead of the emission of a gamma photon). APPENDIX VI-7. CHARACTERISTICS OF RADIONUCLIDES 235

Appendix VI—7 (cont.)

Radionuclide Radiation emitted

Symbol . . ,, ,, Additional radiation Energies in MeV , Z and Half-life Type • , from (Per cent disintegration) mass No. daughter nuclide

25 "Mn 5.7 d r max. 0.575 (28); avg. 0.24 e 0.0055 (17) y 1.434 (100); 0.935 (94); 0.744 (85); others

25 MMn 313 d e 0.0055 (24)

7a 0.835 (100) 26 ssFe 2.7 a e 0.0060(26)

26 "Fe 44.6 d ß' max. 0.475 (53); 0.27 (46);others 7 1.292(44); 1.095 (56); 0.192 (2.8); 0.143 (0.8)

27 "Co 270 d e 0.0065 (54) y 0.692(0.14); 0.136(11); 0.122 (86); 0.014 (9)

58 27 Co 71.3 d r max. 0.474 (15) e 0.0065 (26) y 1.67 (0.6); 0.865 ( 1.4); 0.810 (99) 27 "Co 5.26 a r max. 0.32 (100); others y 1.332 (100); 1.173 (100)

M 29 Cu 12.8 h ß- max. 0.573 (38); avg. 0.175 ß* max. 0.65 (18) e 0.0076 (14) y' 1.34(0.5) 67 29 Cu 59 h ß~ max. 0.57(100) 7a 0.184 (40); 0.092 (23) 6s 30 Zn 244 d r max. 0.327 (1.7) e 0.0081 (35) 7a 1.115 (51) 33 «As 17.9 d ß- max. 1.36 (32) r max. 1.54 (29) e (39) y 0.635 (14); 0.596 (61)

° Gamma-ray emission is significantly affected by internal conversion (ejection of an atomic orbital electron occurring sometimes instead of the emission of a gamma photon). 236 PART VI. APPENDICES

Appendix VI—7 (cont.)

Radionuclide Radiation emitted

Symbol ..,,,, Additional radiation Energies m MeV Z and Half-life Type . . from (Per cent disintegration) mass No. daughter nuclide

34 15 Se 120.4 d e 0.011 (54) 0.401 (12); 0.280 (25); 0.265 (84); 0.13 (72); 0.121 (17); 0.097 (3.3); 0.066(1) 35 8îBr 35.4 h r max. 0.444 (98); others 7 1.475 (17); 1.317 (28); 1.044 (29); 0.828 (24); 0.777 (83); 0.698 (27); 0.619 (43); 0.554 (72)

38 85Sr 64.5 d e 0.014(60) ya 0.514(99) 42 "Mo 66.2 h ß- max. 1.23 (80); 0.45 (19); others 7 0.780 (5); 0.740 (14); 0.372(1); "Tc1" radiations 0.18 (7); 0.041 (2)

m 43 "Tc 6.02 h 7' 0.140(90) 44 106 Ru 368 d ß- max. 0.039(100) 48 l09Cd 453 d e (100) 7a 0.088 i.3 m 49 ]n 1.66 h 7a 0.393 (64) 50 "3Sn 115 d e 0.025 (97) 7 0.26 (2) "3Inm radiations I2S, 5,3 60.2 d € 0.027 (86) 7' 0.035 (7) 53 8.06 d ß' max. 0.806 (100); 0.61 (90); 0.33 (7); others e 0.030 (5) 7B 0.723 ( 1.6); 0.637 (6.8); 0.364 (82); 0.284 (6); 0.08 (2.6)

l34 55 Cs 2.07 a ß- max. 0.662 (71); 0.09 (27); others 7 1.365 (34); 1.168(1.9); 1.038(1); 0.796 (99); 0.605 (98); 0.57 (23)

' Gamma-ray emission is significantly affected by internal conversion (ejection of an atomic orbital electron occurring sometimes instead of the emission of a gamma photon). APPENDIX VI-7. CHARACTERISTICS OF RADIONUCLIDES 237

Appendix VI—7 (cont.)

Radionuclide Radiation emitted

Symbol Additional radiation Energies in MeV Z and Half-life Type from (Per cent disintegration) mass No. daughter nuclide

55 l37Cs 30.0 a r max. 1.17 (6); 0.51 (94)

7 a 0.662(85) 56 l37Bam 2.554 min y' 0.662 (85)

198 79 Au 2.697 d r max. 0.962 (99); others y1 1.088 (0.2); 0.676(1); 0.412(96)

80 '"Hg 64.1 h e 0.070(72) y' 0.268 (0.15); 0.191 (2); 0.077(20)

80 203 Hg 46.6 d ß' max. 0.214(100) e 0.08 (13)

7a 0.279(82)

' Gamma-ray emission is significantly affected by internal conversion (ejection of an atomic orbital electron occurring sometimes instead of the emission of a gamma photon).

BIBLIOGRAPHY

LEDERER, C.M., et al., Table of Isotopes, 6th ed., Wiley, New York ( 1967). MARTIN, M.J., BLICHERT-TOFT, P.H., Nuclear Data Tables, Sect. A, Vol.8, Nos. 1 -2, Academic Press, New York (1970). 238 PART VI. APPENDICES

Appendix VI-8

CALCULATION OF CENTRIFUGE SPEEDS FROM g VALUES

The relative centrifugal force may be calculated from the following equation:

g= RX N2X 118 X 10"8 where R is the radius in cm from the centrifuge spindle to the point along the tube containing the solution, and N is the speed of the centrifuge in revolutions per minute (rev/min).

Hence, the speed of a centrifuge at 1000 g would be: APPENDIX VI-9. SOURCES OF HORMONE PREPARATIONS 239

Appendix VI-9

SOURCES OF SOME PURIFIED HORMONE PREPARATIONS

VI-9.1. WHO INTERNATIONAL AND REFERENCE STANDARDS

Although most of the preparations are of human origin, some purified animal hormones are available from: National Institute of Biological Standards and Control Holly Hill Hampstead London NW3 6RB United Kingdom.

VI-9.2. PURIFIED PITUITARY HORMONE PREPARATIONS FROM SPECIES INCLUDING RAT, SHEEP, PIG AND COW

Distributed for research purposes only by the National Institute of Arthritis, Metabolism and Digestive Diseases, USA. Availability of these materials is published periodically in Endocrinology and in the Journal of Clinical Endocrinology and Metabolism, and recent copies of these journals should be examined for materials available. Supplies of these materials may be obtained by submitting a one-page letter IN DUPLICATE to: Dr. Salvatore Raiti, Director, National Pituitary Agency, Suite 503-9, 210 West Fayette Street, Baltimore, Maryland, 21201, USA. Applications (one-page letter) must include (a) a progress report and/or recent publication list of results from materials previously awarded, (b) a description of the proposed use of materials, brief, but sufficient to justify the kinds and amounts requested, (c) evidence of experience with radioimmunoassay with all new requests for reagents for radioimmunoassay, and (d) your correct mailing address including country (if from outside the USA); (e) letters from trainees must also be signed by a senior staff member. Only one vial, or one kit, of the reagents, animal or human for radioimmunoassay, is awarded at a time. Requests for large amounts of materials may be granted, subject to review by the Committee on Hormone Distribution.

VI-9.3. PURIFIED ANIMAL HORMONE PREPARATIONS

These are occasionally available to bona fide research workers from a number of independent laboratories and individuals in Europe and the United States of America, and some manufacturers supply commercially preparations of animal hormones of varying levels of purity. Many of these preparations are of a very high standard, but should be checked against acknowledged reference standards where possible. 240 PART VI. APPENDICES

Appendix VI-10

BASIC MATERIALS AND EQUIPMENT FOR A RADIOIMMUNOASSAY LABORATORY

VI-10.1. THE RADIOCHEMICAL LABORATORY

This should be equipped to Type B standards (see 1-3.3.2), with an efficient fume hood or cupboard. It need only be a small room and will be required for iodination only. The basic equipment should be:

(a) A sensitive radiation monitor (b) A vortex mixer (c) Pipettes with disposable tips 10-500 ¡j\ (d) Radioactive waste bins (e) Disposable pipettes and general laboratory glassware (f) Scissors, spatulas, can openers, etc. (g) Ball-point pen (not to be removed) (h) Lead bricks (i) Trays lined with absorbent paper.

VI-10.2. THE ASSAY LABORATORY

This may be equipped to Type C standards (see 1-3.3.2). Benches should be covered with plastic-backed absorbent paper for easy disposal of contamination, and the laboratory should contain a fume hood. The basic equipment should be:

(a) Refrigerated centrifuge with multiple-tube rotor head and balance for rotor heads (b) Culture tubes (c) Polystyrene tubes (d) Tube racks (e) General laboratory glassware, including assorted sizes of volumetric flasks, beakers, test tubes, graduated cylinders, pasteur pipettes, serological pipettes, Erlenmeyer flasks, Büchner flasks (f) Multiple vortex mixer (g) Eppendorff-type pipettes, assorted sizes with disposable tips (h) Radioactive disposal bins (i) Trays of various sizes lined with absorbent paper (j) Water pump (k) A refrigerator at 4°C.

VI-10.3. THE COUNTING ROOM

If possible separate from the assay laboratory, and containing:

(a) Large table or bench for samples (b) A ß and/or 7-counter (c) Radioactive disposal bins. APPENDIX VI-10 LABORATORY MATERIALS AND EQUIPMENT 241

VI-10.4. ANCILLARY

An accurate analytical balance or micro-balance should be available, and large volumes of preferably double-distilled water. A cold room at 4°C, or a large-medium refrigerator is required for incubating assays and for storing buffer solutions; a deep freeze is required for storing samples. A multi-tube solvent evaporator is useful equipment for extracted steroid assays. Chemicals are given under the relevant applied exercise. 242 PART VI. APPENDICES

Appendix VI-11

TABLE OF LOGIT VALUES

%' 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

0 _ 6.91 6.21 5.81 5.52 5.29 5.11 4.95 4.82 4.70 1 4.60 4.50 4.41 4.33 4.25 4.18 4.12 4.06 4.00 3.94 2 3.89 3.84 3.79 3.75 3.71 3.66 3.62 3.58 3.55 3.51 3 3.48 3.44 3.41 3.38 3.35 3.32 3.29 3.26 3.23 3.20 4 3.18 3.15 3.13 3.10 3.08 3.06 3.03 3.01 2.99 2.97 5 2.94 2.92 2.90 2.88 2.86 2.84 2.82 2.81 2.79 2.77 6 2.75 2.73 2.72 2.70 2.68 2.87 2.65 2.63 2.62 2.60 7 2.59 2.57 2.56 2.54 2.53 2.51 2.50 2.48 2.47 2.46 8 2.44 2.43 2,42 2.40 2.39 2.38 2.36 2.35 2.34 2.33 9 2.31 2.30 2,29 2.28 2.27 2.25 2.24 2.23 2.22 2.21

10 2.20 2.19 2,18 2.16 2.15 2.14 2.13 2.12 2.11 2.10 11 2.09 2.08 2.07 2.06 2.05 2.04 2.03 2.02 2.01 2.00 12 1.99 1.98 1.97 1.96 1.96 1.95 1.94 1.93 1.92 1.91 13 1.90 1.89 1.88 1.87 1.87 1.86 1.85 1.84 1.83 1.82 14 1.82 1.81 1.80 1.79 1.78 1.77 1.77 1.76 1.75 1.74 15 1.73 1.73 1.72 1.71 1.70 1.70 1.69 1.68 1.67 1.67 16 1.66 1.65 1,64 1.64 1.63 1.62 1.61 1.61 1.60 1.59 17 1.59 1.58 1,57 1.56 1.56 1.55 1.54 1.54 1.53 1.52 18 1.52 1.51 1.50 1.50 1.49 1.48 1.48 1.47 1.46 1.46 19 1.45 1.44 1,44 1.43 1.42 1.42 1.41 1.41 1.40 1.39

20 1.39 1.38 1,38 1.37 1.36 1.36 1.35 1.34 1.34 1.33 21 1.32 1.32 1.31 1.31 1.30 1.30 1.29 1.28 1.28 1.27 22 1.27 1.26 1.25 1.25 1.24 1.24 1.23 1.23 1.22 1.21 23 1.21 1.20 1.20 1.19 1.19 1.18 1.17 1.17 1.16 1.16 24 1.15 1.15 1.14 1.14 1.13 1.13 1.12 1.11 1.11 1.10 25 1.10 1.09 1.09 1.08 1.08 1.07 1.07 1.06 1.06 1.05 26 1.05 1.04 1.04 1.03 1.03 1.02 1.02 1.01 1.00 1.00 27 0.99 0.99 0,98 0.98 0.97 0.97 0.96 0.96 0.95 0.95 28 0.94 0.94 0,93 0.93 0.92 0.92 0.91 0.91 0.91 0.90 29 0.90 0.89 0.89 0.88 0.88 0.87 0.87 0.86 0.86 0.85

30 0.85 0.84 0.84 0.83 0.83 0.82 0.82 0.81 0.81 0.80 31 0.80 0.80 0,79 0.79 0.78 0.78 0.77 0.77 0.76 0.76 32 0.75 0.75 0.74 0.74 0.74 0.73 0.73 0.72 0.72 0.71 33 0.71 0.70 0.70 0.69 0.69 0.69 0.68 0.68 0.67 0.67 34 0.66 0.66 0.65 0.65 0.65 0.64 0.64 0.63 0.63 0.62 35 0.62 0.62 0.61 0.61 0.60 0.60 0.59 0.59 0.58 0.58 36 0.57 0.57 0.57 0.56 0.56 0.55 0.55 0.55 0.54 0.54 37 0.53 0.53 0,52 0.52 0.52 0.51 0.5 1 0.50 0.50 0.49 38 0.49 0.49 0,48 0.48 0.47 0.47 0.46 0.46 0.46 0.45 39 0.45 0.44 0,44 0.43 0.43 0.43 0.42 0.42 0.41 0.41

40 0.41 0.40 0.40 0.39 0.39 0.38 0.38 0.38 0.37 0.37 41 0.36 0.36 0.36 0.35 0.35 0.34 0.34 0.34 0.33 0.33 42 0.32 0.32 0.31 0.31 0.31 0.30 0.30 0.29 0.29 0.29 43 0.28 0.28 0.27 0.27 0.27 0.26 0.26 0.25 0.25 0.25 44 0.24 0.24 0.23 0.23 0.22 0.22 0.22 0.21 0.21 0.20 45 0.20 0.20 0,19 0.19 0.18 0.18 0.18 0.17 0.17 0.16 46 0.16 0.16 0.15 0.15 0.14 0.14 0.14 0.13 0.13 0.12 47 0.12 0.12 0.11 0.11 0.10 0.10 0.10 0.09 0.09 0.08 48 0.08 0.08 0.07 0.07 0.06 0.06 0.06 0.05 0.05 0.04 49 0.04 0.04 0.03 0.03 0.02 0.02 0.02 0.01 0.01 0.00

a In the percentage range 0.0-49.9 logit values are negative. APPENDIX VU 1. LOGIT TABLE 243

Appendix VI-11 (cont.)

%" 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

50 0.00 0.00 0.01 0.01 0.02 0.02 0.02 0.03 0.03 0.04 51 0.04 0.04 0.05 0.05 0.06 0.06 0.06 0.07 0.07 0.08 52 0.08 0.08 0.09 0.09 0.10 0.10 0.10 0.11 0.11 0.12 53 0.12 0.12 0.13 0.13 0.14 0.14 0.14 0.15 0.15 0.16 54 0.16 0.16 0.17 0.17 0.18 0.18 0.18 0.19 0.19 0.20 55 0.20 0.20 0.21 0.21 0.22 0.22 0.22 0.23 0.23 0.24 56 0.24 0.25 0.25 0.26 0.26 0.26 0.27 0.27 0.27 0.28 57 0.28 0.29 0.29 0.29 0.30 0.30 0.31 0.31 0.31 0.32 58 0.32 0.33 0.33 0.34 0.34 0.34 0.35 0.35 0.36 0.36 59 0.36 0.37 0.37 0.38 0.38 0.38 0.39 0.39 0.40 0.40

60 0.41 0.41 0.41 0.42 0.42 0.43 0.43 0.43 0.44 0.44 61 0.45 0.45 0.46 0.46 0.46 0.47 0.47 0.48 0.48 0.49 62 0.49 0.49 0.50 0.50 0.51 0.51 0.52 0.52 0.52 0.53 63 0.53 0.54 0.54 0.55 0.55 0.55 0.56 0.56 0.57 0.57 64 0.58 0.58 0.58 0.59 0.59 0.60 0.60 0.61 0.61 0.61 65 0.62 0.62 0.63 0.63 0.64 0.64 0.65 0.65 0.65 0.66 66 0.66 0.67 0.67 0.68 0.68 0.69 0.69 0.69 0.70 0.70 67 0.71 0.71 0.72 0.72 0.73 0.73 0.74 0.74 0.74 0.75 68 0.75 0.76 0.76 0.77 0.77 0.78 0.78 0.79 0.79 0.80 69 0.80 0.80 0.81 0.81 0.82 0.82 0.83 0.83 0.84 0.84

70 0.85 0.85 0.86 0.86 0.87 0.88 0.88 0.88 0.89 0.89 71 0.90 0.90 0.91 0.91 0.91 0.92 0.92 0.93 0.93 0.94 72 0.94 0.95 0.95 0.96 0.96 0.97 0.97 0.98 0.98 0.99 73 0.99 1.00 1.00 1.01 1.02 1.02 1.03 1.03 1.04 1.04 74 1.05 1.05 1.06 1.06 1.07 1.07 1.08 1.08 1.09 1.09 75 1.10 1.10 1.11 1.11 1.12 1.13 1.13 1.14 1.14 1.15 76 1.15 1.16 1.16 1.17 1.17 1.18 1.19 1.19 1.20 1.20 77 1.21 1.21 1.22 1.22 1.23 1.24 1.24 1.25 1.25 1.26 78 1.27 1.27 1.28 1.28 1.29 1.30 1.30 1.31 1.31 1.32 79 1.32 1.33 1.34 1.34 1.35 1.36 1.36 1.37 1.37 1.38

80 1.39 1.40 1.40 1.41 1.41 1.42 1.42 1.43 1.44 1.44 81 1.45 1.46 1.46 1.47 1,48 1.48 1.49 1.50 1.50 1.51 82 1.52 1.52 1.53 1.54 1.54 1.55 1.56 1.56 1.57 1.58 83 1.59 1.59 1.60 1.61 1.61 1.62 1.63 1.64 1.64 1.65 84 1.66 1.67 1.67 1.68 1.69 1.70 1.71 1.71 1.72 1.73 85 1.73 1.74 1.75 1.76 1.77 1.77 1.78 1.79 1.80 1.81 86 1.82 1.82 1.83 1.84 1.85 1.86 1.87 1.87 1.88 1.89 87 1.90 1.91 1.92 1.93 1.94 1.95 1.96 1.96 1.97 1.98 88 1.99 2.00 2.01 2.02 2.03 2.04 2.05 2.06 2.07 2.08 89 2.09 2,10 2.11 2.12 2.13 2.14 2.15 2.16 2.18 2.19

90 2.20 2.21 2.22 2.23 2.24 2.25 2.27 2.28 2.29 2.30 91 2.31 2.32 2.34 2.35 2.36 2.38 2.39 2.40 2.42 2.43 92 2.44 2.46 2.47 2.48 2.50 2.51 2.53 2.54 2.56 2.57 93 2.59 2.60 2.62 2.63 2.65 2.67 2.68 2.70 2.72 2.73 94 2.75 2.77 2.79 2.81 2.82 2.84 2.86 2.88 2.90 2.92 95 2.94 2.97 2.99 3.00 3.03 3.06 3.08 3.10 3.13 3.15 96 3.18 3.20 3.23 3.26 3.29 3.32 3.35 3.38 3.41 3.44 97 3.48 3.51 3.55 3.58 3.62 3.66 3.71 3.75 3.79 3.84 98 3.89 3.94 4.00 4.06 4.12 4.18 4.25 4.33 4.41 4.50 99 4.60 4.70 4.82 4.95 5.1 1 5.29 5.52 5.81 6.21 6.91

b In the percentage range 50.0-99.9 logit values are positive.

PART VU. GLOSSARY OF SOME BASIC TERMS AND CONCEPTS

Bold words in an explanation refer the reader to another Glossary entry.

Certain definitions, mainly of quantities and units, are based on or taken from standards published by international bodies such as BIPM, ISO and ICRU. The references are listed below:

[Gl] BUREAU INTERNATIONAL DES POIDS ET MESURES. Le Système International d'Unités (SI), 3e Edition, OFFILIB, 48 rue Gay-Lussac, F-75005 Paris (1977): (An English translation entitled the International System of Units (SI) is available as National- Bureau of Standards Special Publication 330, 1972 Edition, from the US Government Printing Office, Washington, DC, 20402, or from HMSO, London: the corresponding editions appear later than the French original.) [G2] INTERNATIONAL ORGANIZATION FOR STANDARDIZATION, International Standard ISO 1000(1973), and International Standard ISO 31/0—XIII (the latter standard is published in 13 parts with a general introduction ISO 31/0: the various parts have various publishing dates). These two standards concern themselves with quantities, units and symbols. [G3] INTERNATIONAL COMMISSION ON RADIATION UNITS AND MEASUREMENTS, Radiation Quantities and Units, ICRU Report 19, ICRU, Washington, DC ( 1971 ), and Supplement to ICRU Report 19(1973). [G4] INTERNATIONAL ORGANIZATION FOR STANDARDIZATION, Nuclear Energy Glossary, International Standard ISO 921 (1972).

245 246 GLOSSARY absorbance, A, or optical density. The logarithm to the base 10 of the reciprocal of the transmittance, T, of an optically absorbing medium in a densitometric or spectrophotometric measurement:

A = !og10- r

where T = I/I0, I0 being the incident luminous flux of the light and I the transmitted luminous flux [G2/VI], For example, A = 1.0 represents 10% incident light transmitted, A = 2.0 represents 1% transmitted, etc. When this quantity is measured at a given wavelength of light, X, it is called spectral absorbance, A^, or spectral optical density.

absorbed dose, D. The mean energy, de, imparted by ionizing radiation to matter in a suitably small volume element divided by the mass, dm, of matter in that volume element [G3]:

D = —- dm

The SI derived unit of absorbed dose is the gray (Gy) [Gl ]; the traditional special unit is the rad, use of which is being phased out.

absorbed dose rate, D. The increment in absorbed dose, dD, divided by the time interval, dt, in which it is accumulated [G3]:

. dD D = — dt

In SI the unit of measurement is the gray per second (Gy/s), or multiples or submultiples thereof [Gl ]. The traditional special unit was the rad per second (rad/s), or any multiples or submultiples thereof; use of these is being phased out.

absorber. Any matter placed in the path of a radiation beam. Such matter causes a reduction in the radiation flux and, often, a change in radiation quality in the beam behind the absorber, the magnitude of the change varying with the type and spectrum of the radiation and the density and atomic constituency of the material. GLOSSARY 247 absorber, semi-infinite. An absorber whose thickness is greater than the practical range of penetration of the radiation and whose lateral dimensions are considerably greater than those of the radiation field.

absorbing event. An interaction, such as elastic or inelastic scattering, photoelectric absorption, the Compton effect or pair-production, by which radiation energy is transferred to matter.

absorption coefficient, see energy absorption coefficient. activity, A. The number, N, of spontaneous nuclear transformations occurring in a given quantity of radioactive nuclide during an incremental interval of time, dt, divided by that interval of time [G3]:

The SI derived unit of activity is the becquerel (Bq) [Gl ]; the traditional special unit is the curie (Ci), use of which is being phased out. amber Perspex dose meter, see PMMA dose meters.

annihilation. Commonly used for the event when a positron and electron annihilate on interaction; their rest energy is converted into two photons, each of 0.51 MeV energy (see annihilation radiation).

annihilation radiation. Electromagnetic radiation of 0.51 MeV energy resulting from an annihilation interaction between a positron and an electron.

area density, see mass per unit area.

atomic fluorescence. The emission of characteristic X-rays as a result of the photoelectric effect: the photoelectron is ejected from an atom, being replaced by an electron from an outer atomic orbit, accompanied by the emission of the K-shell, L-shell, etc. X-rays (the fluorescence yield increasing with the atomic number). It is in competition with the Auger effect. 248 GLOSSARY attenuation coefficient (linear attenuation coefficient, p, or mass attenuation coefficient, pip). Of a substance i, the quantity (/i)¡ is defined for a

parallel beam of specified radiation, where the expression (pA\)i represents the fraction of indirectly ionizing particles that is removed from the incident beam by interactions in passing through a thin layer of thickness (Ax), of that substance. It varies with the spectral energy of the radiation. According as (Ax)¡ is expressed in terms of length or mass per unit area, the attenuation

coefficient is termed linear, (p)it or mass, (plp)v where p represents the density of the substance [G4].

The ICRU definition is also given for completeness [G3j: The mass attenuation coefficient, pjp, of a material for indirectly ionizing particles of specified energy is the quotient of dN/N by pdx, where dN/N is the fraction of particles that experience interactions in traversing a distance dx in a medium of density p:

p 1 . dN p pN dx

NOTES: (a) The term interactions refers to processes whereby the energy or direction of indirectly ionizing particles is altered, (b) For X or 7-ray photons:

p_ J_ ! °c | °coh K P P P P P

where 7"/p is the photoelectric mass attenuation coefficient, ac/p is the total Compton

mass attenuation coefficient, acoh/p is the mass attenuation coefficient for coherent scattering, and x/p is the pair production mass attenuation coefficient. (This equation applies if the nuclear interactions are not important. An extra term for such interactions may be required for X or 7-ray energies in excess of a few million electronvolts.)

Auger effect. The non-radiative transition occurring in an atom, especially one of low atomic number, as a result of the photoelectric effect, in which orbital electrons (Auger electrons) other than photoelectrons are ejected from the atom as a result of electron orbital readjustments from excited states to lower energy states. It is in competition with atomic fluorescence.

Auger electron. Fast electron ejected as a result of interaction between an X-ray photon and an orbital (valence) electron, resulting in a non-radiative transition of an atom to a lower excited electronic energy state (see Auger effect, isomeric transition).

background. Signals (e.g. radiation) that are recorded by a measuring device (e.g. dose meter) and which do not emanate from the radiation source of interest. Background radiation may be from a source external to the dose meter or arise from radioactive contamination of the dose meter or holder. Because of the high levels of radiation used in food irradiation, this problem will not normally be of GLOSSARY 249

importance in dosc-meter calibration procedures. (Another 'background' effect is the alteration of the dose meter reading by allowing light, heat or aging to affect the sensitive volume of the dose meter.) See also noise. becquerel (Bq). The SI derived unit of activity, being one radioactive disintegration per second of time. It has dimensions of s~', and its relationship to the traditional special unit, the curie (Ci), is:

1 Bq = 2.7Ó27 X 10"" Ci

The term disintegration refers to a nuclear transformation, i.e. either a change of nuclide or an isomeric transition.

beta-particle, ß-particle. A /T-particle is a high-speed (negatively charged) electron ejected from a nucleus during radioactive decay. A j3+-particle is a high-speed positron (positively charged electrón) ejected from a nucleus during radioactive decay.

bilateral irradiation. Irradiation of a product from two opposite sides simultaneously (see irradiator). binding energy. (1) For a particle in a system, the net energy required to move it from the system to infinity. (2) For a system, the net energy required to decompose it into its constituent particles. biological indicator. Biological system that undergoes a measurable and reproducible change on being irradiated.

BIPM, see SI.

bremsstrahlung. Photon radiation emitted by fast-moving charged particles that arc decelerated or deflected by an electric or magnetic field (the originally German word means, literally, braking radiation). It is usually high-energy penetrating photon (electromagnetic) radiation produced by deceleration of fast electrons in high-atomic-number absorbers. It is physically identical to X-rays and similar to gamma rays and shows a broad spectral energy distribution having a maximum energy corresponding to the maximum energy of the incident electrons.

broad beam. (1) The nature of a stream of incident radiation where the diameter is relatively large with respect to the size of the absorber or target (as con- trasted with narrow beams). (2) In beam attenuation measurements, a beam in which the unscattered and some of the scattered radiation reach the detector. 250 GLOSSARY build-up. In the passage of radiation through a medium, the increase with depth in energy deposition due to the forward-moving secondary radiation produced. It leads to a maximum in the depth-dose curve. For example, with 60Co 7-radiation, this maximum is at a depth of 0.5 cm in water. bulk density. Weight per unit volume of the product 'en mass', as it would be irradiated (e.g. with potatoes, the air space between the potatoes is also considered in determining the bulk density). calibration curve. The response curve (radiation effect as a function of absorbed dose) established under controlled conditions in which the doses are determined by comparison with a standard reference dose meter. calorimeter. A device for determining energy deposition by means of the resulting temperature change in the calorimeter body or of some other thermal effect.

Compton effect. Interaction (elastic, incoherent scattering) between a (high- energy) photon and a free or loosely bound electron, whereby the photon suffers a change in direction and a loss in energy, and the electron gains an amount of kinetic energy equivalent to that lost by the photon. confidence level, see confidence probability. confidence limits. The upper and lower values in a distribution of data within which a mean value from a succeeding measurement falls with the confidence probability. Both values of the confidence limits are calculated from the parameters of the distribution (e.g. design uniformity ratio, U, and appropriate standard deviation, sy) by the use of the t-value.

confidence probability or confidence level. The probability with which a result will fall within specified limits.

conversion efficiency (X-rays) or conversion ratio (X-rays). In the production of bremsstralilung by the slowing down of electrons or other charged particles, the ratio of energy flux density of resulting photons to the energy flux density of incident electrons or particles.

curie (Ci). The special unit of activity, which is being superseded by the becquerel (Bq). The curie is defined as: 1 Ci = 3.7 X 1010 disintegrations per second = 3.7 X 10,oBq

The term disintegration refers to a nuclear transformation, i.e. either a change of nuclide or an isomeric transition. GLOSSARY 251 decay law, radioactive. The fractional rate of decrease of the number of radioactive atoms of a specific radionuclide is constant, is independent of its age and surroundings, and is characteristic of that radionuclide. __ J_ dN N dt where N is the number of radioactive atoms of a specific radionuclide at time t and X is the decay constant for the radionuclide. This equation can be integrated to give:

Xt N = N0e"

where N0 is the number of radioactive atoms present at t = 0. degraded spectrum. A radiation spectrum that has been shifted to lower energies owing to interaction with an absorber. density thickness, see mass per unit area. deptli dose. The variation of absorbed dose with depth of penetration of a primary radiation beam incident perpendicularly on a planar medium, as determined along the central axis of the beam. direction, one- or two-, see irradiator. directly ionizing particles are charged particles (electrons, protons, a-particles, etc.) having sufficient kinetic energy to produce ionization by collision [G3]. dose, see absorbed dose dose distribution. The spatial variation in absorbed dose throughout the product,

the dose having the extreme values Dmax and Dmin. dose meter. A device, instrument or system having a reproducible and measurable response to radiation that can be used to measure or evaluate the quantity termed absorbed dose, exposure or similar radiation quantity. [The word dosimeter has been replaced by dose meter [G4] as standard terminology (dose meter, but dosimetry, dosimetric).] dose meter probe. A dose meter that is inserted into material to be irradiated. dose rate, see absorbed dose rate. dose uniformity, see uniformity ratio. dosimeter, see dose meter. dosimetry. The measurement of radiation quantities, specifically absorbed dose and absorbed dose rate, etc. elastic scattering, see scattering. 252 GLOSSARY electron. A small particle having a rest mass of 9.107 X 10"28 g, an atomic mass 1837 a hydrogen atom, a diameter of 10~12 cm, and carrying one elementary unit of positive or negative charge (1.602 X 10~I9C). The positively charged electron is called the positron, while the negatively charged electron is usually just termed electron (the term negatron is rarely used). See also beta particle. electron accelerator. A device for imparting large amounts of kinetic energy to electrons. electron beam. An essentially monodirectional stream of (negative) electrons which have usually been accelerated electrically or electromagnetically to high energy.

electron capture (EC). Mode of radioactive decay of an atom in which its nucleus captures one of its orbital electrons, whereby a proton in the nucleus is transformed to a neutron and a neutrino is emitted.

electron equilibrium, also called charged-particle equilibrium [G4]. The condition existing at a point, P, within a medium uniformly exposed to gamma or X-radiation whereby the sum of the kinetic energies of the electrons (liberated by the primary photons) entering an incremental volume containing P equals the sum of the kinetic energies of electrons leaving that volume. This condition is approximately satisfied if the point P is surrounded on all sides by homogeneous material having a thickness equal to the maximum range of scattered electrons. Briefly stated — electron equilibrium exists if the energy fluence of the electrons throughout the immediate vicinity of the point P is constant.

electronvolt (e V). A unit of energy. One electronvolt is the kinetic energy acquired by an electron in passing through a potential difference of one volt in a vacuum. It is defined as [Gl ]:

1 eV = 1.602 19 X 10"19 J, approximately

It is a unit used with the SI whose value is obtained experimentally [Gl ].

energy absorption coefficient (linear energy absorption coefficient, pen, or mass energy absorption coefficient, Men/p)- Of a substance i, the quantity (Men)i is defined for a parallel beam of specified radiation, where the

expression (¿ien Ax)¡ represents the fraction of the energy of indirectly ionizing particles that is absorbed from the incident beam as it passes through a thin layer of thickness (Ax)¡ of that substance. It varies with the spectral energy of the radiation. According as (Ax)¡ is expressed GLOSSARY 253

in terms of length or mass per unit area, the absorption coefficient is termed

linear, (/Jen)i. mass, (Men/p)i> where p represents the density of the substance. The absorption coefficient is that part of the attenuation coefficient resulting from energy absorption only [G4]. The 1CRU definition is also given for completeness [G3|: The mass energy absorption coefficient, iifn/p, of a material for indirectly ionizing particles of specified

energy is the product of the mass energy transfer coefficient, ptr/p, for that energy and (1-g), where g is the fraction of energy of secondary charged particles that is lost to bremsstrahlung in the material.

KM. = M tr('-g) P P While the two coefficients can differ considerably when the kinetic energies of the secondary particles are comparable with or larger than their rest energies, particularly for interactions in high-Z materials, in most applications considered in this Manual g is small and, hence, the two coefficients are, for practical purposes, identical. energy fluence, At a given point in space or in an absorbing medium undergoing irradiation, the sum of the energies, exclusive of rest energies, of all particles incident during a given time interval on a suitably small sphere centred at that point divided by the cross-sectional area of that sphere. It is the same as the time integral of energy flux density [G4], energy flux density, \p, or energy fluence rate. At a given point in space or in an absorbing medium undergoing irradiation, the sum of the energies, exclusive of rest energies, of all particles incident per unit time on a suitably small sphere centred at that point divided by the cross-sectional area of that sphere. It is identical with the product of the particle flux density and the average energy of the particles [G4].

energy transfer coefficient (linear energy transfer coefficient, ¿itr, or mass energy

transfer coefficient, ptr/p). Of a substance i, the quantity 0itr)¡ is defined

for a parallel beam of specified radiation, where the expression (/^trAx)¡ represents the fraction of the energy of indirectly ionizing particles transferred in passing through a thin layer of thickness (Ax)¡ of that substance. It varies with the spectral energy of the radiation. According as (Ax)¡ is expressed in terms of length or mass per unit area, the transfer coefficient is termed linear,

(U^i, or mass, (/utr/p)¡, where p represents the density of the substance.

The ICRU definition is also given for completeness [G3J: The mass energy transfer

coefficient, Hulp, of a material for indirectly ionizing particles of specified energy is the

quotient of dEtr/E by pdx when dEtr/E is the fraction of incident particle energy (excluding rest energies) that is transferred to kinetic energy of charged particles by interactions in traversing a distance dx in a medium of density p. Pjr _ J_ dE p pE dx

(From Ref. [G3], which should be consulted for more detailed information.) 254 GLOSSARY entrance dose, surface dose. The absorbed dose in a product at the entrance surface (or extrapolated to this surface), i.e. where the radiation beam enters the product. error probability. The probability with which a result will fall outside the specified limits. ethanol-chlorobenzene dose meter. A close meter in which radiation dissociates chlorobenzene with the formation of hydrochloric acid and other radiolytic products. Useful close range: 40 krad to 20 Mrad (0.4 to 200 kGy). exit dose. The absorbed dose in a product at the exit surface (or extrapolated to this surface), i.e. where the radiation beam leaves the product. exposure, X. For gamma or X-rays in air, the sum of the electrical charges of all the ions of one sign produced in air when all electrons (negatrons and positrons) liberated by photons in a suitably small volume element of air are completely stopped in air divided by the mass of air in the volume element [G3]. The special unit of exposure is the roentgen, R:

1 R = 2.58 X 10-4 C/kg

In SI, the unit is coulombs per kilogram: there is no derived unit for this quantity. (See kerma.)

exposure rate, X. The increment of exposure during a suitably small interval of time divided by the time interval. The unit of exposure rate is of the form roentgens per time, e.g. R/s, mR/h, etc., or, in SI units, C kg"1 • s~l, etc. external standard. (1) Any suitable radioactive source that is accurately defined and can be used as a standard to calibrate a measuring system. (2) Any standard or master system that is used to calibrate a measuring system (e.g. 'master' ferric ion solution for calibrating the Fricke dose meter).

FAO. Food and Agricultural Organization of the United Nations, Rome (Italy).

ferrous sulphate/cupric sulphate dose meter. A dosimetry system similar to the ferrous sulphate (Fricke) dose meter system, but whose sensitivity is

decreased by the addition of CuS04. The absorbed dose range covered is -200-800 krad (2-8 kGy), although it can be used down to ~60 krad (0.6 kGy) with some loss of accuracy.

ferrous sulphate dose meter, see Fricke dose meter.

ferrous sulphate dosimetry. Dosimetry making use of the oxidation of ferrous ions to ferric by ionizing radiation. GLOSSARY 255 film badge. Small radiographic film in light-tight envelope worn by personnel working in radiation areas to register exposure to ionizing radiation. fluence, energy, see energy fluence. fluence, particle, see particle fluence. flux density, energy see energy flux density. flux density, particle see particle flux density. frequency distribution. An arrangement of statistical data that exhibits the frequency of occurrence of the values of a variable. Fricke dose meter. Dose meter using the change in ultraviolet absorption caused by oxidation by ionizing radiation of ferrous ions to ferric. It is accepted as a standard system for calibrating other dose meters. The absorbed dose range covered is ~ 4 X 103 to ~ 4 X 104 rads (40-400 Gy). With oxygen saturation of the dose meter solution (this system is called the "super" Fricke dose meter) it can be used up to ~ 2 X 10s rads (2 kGy). gamma rays, 7-rays. Penetrating electromagnetic radiation (photons) emitted from a specific radionuclide in the process of nuclear transition or from any material as a result of particle annihilation. With food irradiation, gamma rays are generally high-energy penetrating photons as emitted from 60Co or 137Cs radionuclide sources.

Gaussian distribution, see normal distribution. geometry. A term used loosely to designate the arrangement in space of the various components of an irradiation or measuring system. This designation includes positions of source, detector and any intervening absorber. The solid angle around the source that is irradiated or measured is also sometimes indicated, e.g. '2rr geometry'. (See irradiation geometry.) glass dose meters. The absorption of ionizing radiation in silver-activated phosphate glass gives rise to fluorescing or colour centres. The darkening can be measured spectrophotometrically, or use can be made of the radiophotoluminescence. gray (Gy). The SI derived unit of absorbed dose of ionizing radiation, being equal to one joule of energy absorbed per kilogram of matter undergoing irradiation. It has dimensions of J/kg, and its relationship to the traditional special unit, the rad, is:

1 Gy = 100 rad (= 1 J/kg)

G-value. The radiation yield of chemical changes in an irradiated substance, in terms of the number of specified chemical changes produced per 100 eV or 256 GLOSSARY

per joule of energy absorbed from ionizing radiation. Examples of such chemical changes are production of particular molecules, free radicals, ions, etc. In the case of molecules affected, it is sometimes called molecular yield.

At present, most data are given as number of chemical changes per 100 eV. To convert to SI units, transform to per-eV value and divide by 1.602 X 10~19 to obtain data in per-joule value, i.e.

15.6 per 100eV- 15.6 X 10~2eV'-»(, J'1 -> 9.74 X lO'T1 \1.602 A 10 J

half-life, radioactive, T1/2- For a single radioactive decay process, the time required for the (radio) activity to decrease to half its value by that process. heat capacity. The quantity of heat (i.e. energy) required to raise the temperature of a given mass of substance by one degree of temperature difference. (In the SI system, the unit would be J/K, and the specific heat capacity, or heat capacity per unit mass, would be J-kg"' K"1.)

IAEA. International Atomic Energy Agency, Vienna (Austria).

ICRP. International Commission on Radiological Protection, Sutton (Surrey, UK).

ICRU. International Commission on Radiation Units and Measurements, Washington, DC (USA). indirectly ionizing particles are uncharged particles (neutrons, photons, etc.) which can liberate directly ionizing particles or can initiate nuclear transformations.

inelastic scattering, see scattering.

integrated dose. The total absorbed dose received by a material, determined by summing up all individual dose contributions over a period of time or after completion of an irradiation process.

internal conversion (IC). A process whereby an atomic nucleus, that would otherwise emit a 7-ray photon, de-excites by interacting with one of its own orbital electrons (usually in the K, L or M shell), the electron being ejected at high velocity. The ejected electron (termed the conversion electron) has a kinetic energy which is the difference between the transition (de-excitation) energy and the binding energy. inverse-square law. A law stating that the intensity of radiation emanating uniformly over the full solid angle (4ÎT) from a source in vacuum decreases proportionally and monotonically with the square of the distance from the source, i.e. is inversely proportional to the square of the distance.

ionization. Production of ion pairs, one of which may be an electron. GLOSSARY 257 ionization chamber. A device used in dosimetry, in particular for the measurement of absorbed dose and exposure. Its operation is based on measuring the number of ions produced by the radiation in a gas-filled vessel (by measuring the charge collected). ionizing radiation. Any radiation, consisting of directly or indirectly ionizing particles, or a mixture of both.

ISO. International Organization for Standardization, Geneva (Switzerland). isomeric transition (IT). Decay with a measurable half-life of an isomer (in a metastable state) to an isomer of lower energy. De-excitation of a nucleus may occur by emission of a gamma photon or by internal conversion (IC), with emissions of X-rays and/or Auger electrons. isotopes. Nuclides having the same atomic number (i.e. the same chemical element) but having different mass number (i.e. same Z, different A). kerma, K. (This quantity may replace exposure in the near future.) The kerma

is the quotient of dE^ by dm, where dEfr is the sum of the initial kinetic energies of all the charged particles (electrons) liberated by indirectly ionizing particles (photons) in a volume element of the specified material, and dm is the mass of the matter in that volume element:

The SI derived unit of kerma is the gray (Gy); the traditional special unit is the rad, use of which is being phased out. Kerma may be a useful quantity in photon dosimetry when electron equilibrium exists in the material at the point of interest, and bremsstrahlung losses are negligible. It is then equal to the absorbed dose at that point [G3]. k-value. In statistical treatment of measured data, the k-value is a function of the fraction of measurements considered, the confidence probability required and the number of measurement values available. labyrinth. A passage linking two areas that is designed to follow a tortuous path such that no radiation originating in one area can pass into the other area without undergoing at least a single reflection or absorption at a passage- wall interface.

LET, see linear energy transfer. linear absorption coefficient, ¿uen, see energy absorption coefficient. 258 GLOSSARY linear attenuation coefficient, p, see attenuation coefficient. linear energy transfer (LET), also called restricted linear collision stopping power, L^. The average energy locally imparted to a medium by a charged particle of specified energy along a suitably small clement of its path, divided by that element [G4].

The ICRU definition is also given for the sake of completeness [G3|: The linear energy transfer, L¿, of charged particles in a medium is the quotient of dE by dx, where dx is the distance traversed by the particle and dE is the energy loss due to collisions with energy transfers less than some specified value A.

(a) Although this definition specifies an energy cut-off and not a range cut-off, the energy losses are sometimes called "energy locally imparted". (b) In order to simplify notation and to ensure uniformity it is recommended that

A be expressed in electronvolts. Thus, L100 is the LET for an energy cut-off of 100 eV.

(c) L00=SC,)|: see stopping power. linear stopping power, S, see stopping power. limiting dose uniformity ratio, see uniformity ratio. luminescence. The property of a material which causes it to emit light as a result of some excitation or 'stimulation' (the term does not include incandescence). The two types of luminescence are (i) fluorescence, present only as long as the excitation is applied, and (ii) phosphorescence, which persists after the excitation has ceased. AH such materials are termed phosphors, and may show either or both of the two effects. Luminescence is classified by the method of excitation, e.g. photoluminescence — luminescence caused by excitation with light, usually u.V., thermoluminescence — excitation with heat, etc. If the luminescence arises owing to prior irradiation, the complex term is prefixed by radio-, e.g. radiophotoluminescence, radiothermoluminescence, effects that can be used in dosimetry. Radio- fluorescence, i.e. luminescence in a phosphor due to prompt release of energy absorbed from ionizing radiation, is termed scintillation. lyoluminescent dose meters. Certain organic and inorganic materials which emit light when dissolved in water or other solvent after irradiation (lyoluminescence). A measurement of the integrated light output gives a measure of absorbed dose. The absorbed dose range covered is ~ 103— 106 rads (0.01-10 kGy). GLOSSARY 259 mass absorption coefficient, Men/P. see energy absorption coefficient. mass attenuation coefficient, pip, see attenuation coefficient. mass energy absorption coefficient, see energy absorption coefficient. mass energy transfer coefficient, see energy transfer coefficient. mass per unit area. A parameter used for specifying thickness of absorber that, for a given radiation, is independent of the absorbing material itself over a wide range. It is obtained by multiplying the absorber thickness, i.e. pathlength through the absorbing medium, by the density of the medium. There is no agreement on a name for this, it being called variously surface density, density thickness, area density, mass thickness and, simply, thickness. The dimensionally descriptive title is used in this Manual. mass stopping power, S/p, see stopping power. master ferric ion solution. A standard ferric ion solution prepared to enable a spectrophotometer to be calibrated before determining absorbed dose as derived from the yield of ferric ions due to irradiation of the ferrous sulphate (Fricke) or the ferrous sulphate/cupric sulphate dose meter, molar extinction coefficient, e¡ or molar absorption coefficient. A constant relating the absorbance of an optically absorbing medium at a given wavelength (spectral absorbance) per unit pathlength to the molar concentration of that medium in its host substance:

d [i]

where A\ is the spectral absorbance at a given wavelength X,d is the optical pathlength, and [i] is the concentration of the absorbing medium. Units of measurement are:

traditional units SI units

d cm m 3 [i] mol/1 or M mol/m l-moP'-cm 1 or M~'cm 1 mJ raol"1

Traditional data for e¡ are multiplied by 10 1 to obtain SI values. molecular yield, see G-value. monitoring. General routine measurements of parameters related to the radiation treatment. Monitoring may involve either simply a qualitative indicator measurement or a precise measurement of radiation quantities. 260 GLOSSARY

monoenergetic radiation. Radiation comprising photons or particles that all have the same spectral energy.

narrow beam. In beam attenuation measurements, a beam in which only the unscattered and small-angle forward scattered radiations reach the detector. (See also broad beam.)

negatron, see electron.

noise. Background signals and those originating in the measuring equipment that interfere with the measurement and, in measuring small signals, determine the limiting sensitivity of the measuring system.

normal distribution, or Gaussian distribution. Symmetrical arrangement of replicate values that deviate randomly on either side of a mean value. This bell-shaped distribution is described mathematically by the Gaussian equation. It is completely determined by two parameters, the mean value and the standard deviation.

nuclear transformation. This term designates a change of nuclide or an isomeric transition.

nuclide. Any given atomic species characterized by (1) the number of protons, Z, in the nucleus, (2) the number of neutrons, N, in the nucleus, and (3) the energy state of the nucleus (in the case of an isomer).

optical density, see absorbance.

pair production. The simultaneous formation of a positive and negative electron (positron and negatron) as a result of the interaction of a photon of sufficient energy (>1.02 MeV) with the field of an atomic nucleus or other particle. (The reverse process produces annihilation radiation.)

particle fluence, 4>. At a given point in space or in an absorbing medium undergoing irradiation, the number of particles incident during a given time interval on a suitably small sphere centred at that point divided by the cross-sectional area of that sphere. It is the same as the time integral of particle flux density [G4],

particle flux density, <¡>, or particle fluence rate. At a given point in space or in an absorbing medium undergoing irradiation, the number of particles incident per unit time on a suitably small sphere centred at that point divided by the cross-sectional area of that sphere. It is the same as the product of the particle density and the average speed of the particles [G4],

peak dose. Dmax in a depth-dose curve. GLOSSARY 261

per cent transmission. The transmittance represented as a percentage, i.e. multiplied by 100 (see absorbance).

photoelectric absorption, or photoelectric effect. The complete absorption of a photon by an atom. The energy of the photon is transferred to an (inner) orbital electron that is then ejected from the atom. Atomic fluorescence (a characteristic X-ray) is emitted as this orbital electron is replaced by an electron from an outer atomic orbit. photon. A quantum or unit of electromagnetic radiation, the photon being considered as an elementary particle. A photon of frequency v has an energy of hi>, where h is Planck's constant (6.6256 X 10"34 J ' s). plaque source. An arrangement of radionuclide sources in planar configuration. plastic film dose meters. Dose meters consisting of plastic films which undergo an irradiation effect that can be used as a measure of absorbed dose. In most cases, the measured effect is a change of absorbance (optical density), which at specified optical wavelengths can be correlated with the dose by means of a calibration curve.

PMMA dose meters. This plastic (polymethyl methacrylate) undergoes changes in absorbance (optical density) at certain wavelengths that are correlated with absorbed dose of ionizing radiation. Depending on the thickness, the dose ranges measurable are 100 krad to 10 Mrad (1 to 100 kGy). Red and amber varieties are used as well as the colourless, i.e. "clear". Trade-names for PMMA are Perspex, Lucite, Plexiglas, etc.

pocket.dose meter. A robust electrometer-type instrument designed to be worn in the pocket to register the integrated dose of penetrating (photon) radiation to which, for example, an operator in an irradiation facility might have become exposed.

polymethyl methacrylate dose meters, see PMMA dose meters.

polyvinyl chloride dose meter, see PVC dose meter.

position, single-, 2-, 3-,. . . or multi-, see irradiator.

positron, see electron.

primary radiation. Incident radiation before interaction with an absorber.

PVC dose meter. A plastic film (polyvinyl chloride) which undergoes changes in absorbance that are correlated with absorbed dose. It is not recommended for accurate dosimetry, but can be used for monitoring functions. 262 GLOSSARY rad (rad). The special unit of absorbed dose [G3], which is being superseded by the gray (Gy). The rad is defined as:

1 rad = 0.01 Gy = 0.01 J/kg (= 100 erg/g) radiation. To be understood as referring to ionizing radiation in this Manual. radiation energy. The spectral energy of the particles in the radiation beam. The beam may be monoenergetic, comprise particles of a number of discrete energies or comprise a mixture of energies giving rise to a continuous energy spectrum (see spectral energy). radiation quality. A loose qualitative term indicating the spectral characteristics of a radiation field. For example, softness and hardness as radiation qualities represent, respectively, relatively low and high spectral energies in the given radiation field.

radiation quantity. A quantity characteristic of a particular radiation and capable of being measured. Thus particle flux density is a radiation quantity, while hardness (see above) is not [G4]. radiation source. An apparatus or radioactive substance in a suitable support that constitutes the origin of the ionizing radiation (e.g. cobalt-60 source rods in a frame, or an electron accelerator).

radiation spectrum. The distribution of spectral energy of a given radiation.

radioactive decay law, see decay law, radioactive.

radioactivity, see activity.

radiochromic dye dosimetry system. This system is based on the intense, perma- nent coloration produced by ionizing radiation in triphenylmethane dye cyanide, dye methoxide, or other leuco dye solutions. Both the solutions themselves and thin plastic films impregnated with the solutions can be used for dosimetry. Solutions have a useful dose range of 1 — 1000 krad ( 10— 104 Gy); the film dose ranges extend from 1 krad to 30 Mrad (10 Gy to 300 kGy), depending on the film type.

radiochromic dye system. A precursor of a dye that is transformed to dye by the absorption of radiation energy; used in dosimetry.

radionuclide. A radioactive nuclide [G4].

radiophotoluminescent material. A substance that, after irradiation, emits luminescence radiation when stimulated by optical radiation (e.g. ultraviolet or visible radiation). GLOSSARY 263 range. The distance that a charged particle will penetrate a given substance before its kinetic energy is reduced to such a level that it will no longer cause ionization. As used of alpha and beta particles of given (maximum) energy, the maximum distance they will penetrate a given substance in a specified direction (since ß~ particle tracks, in particular, are tortuous). red Perspex dose meter, see PMMA dose meters. reference dose meter. A standard dosimetric system with which absorbed dose can be determined in terms of measurements of radiation-induced changes of physical quantities, ionization current, electric charge or temperature, or in terms of measurements of radiation chemical yields from standard solutions that are readily available from stock and can be accurately reproduced at any time in any laboratory. These reference systems do not need calibration against some other standard dose meter, relative biological effectiveness (RBE). A factor which allows for the fact that radiations with different specific ionizations, i.e. with different linearenergy transfers, will produce different effects in an organism for the same absorbed dose. rem (rem). The unit of dose equivalent used for radiation protection purposes only (see ICRU Report 19 and its Supplement [G3]). Its dimensions are joules per kilogram (i.e. energy per mass), response, f. The measured radiation effect per unit absorbed dose.

rest energy, electron, or rest-mass energy of an electron. The energy equivalent of the mass of an electron at rest, namely 0.51 MeV. roentgen (R). The special unit of exposure (q.v.), defined as: 1 R= 2.58 X 10~4 C/kg There is no SI derived unit for this quantity (see kerma). routine dose meter. A dose monitor or dose meter having a reproducible response, which must, however, be calibrated against a reference dose meter in order to make an accurate dose interpretation. scattering. Change in direction of a particle or photon due to a collision or interaction with another particle, an atom or a system, (i) If the total kinetic energy is conserved, the process is termed elastic scattering, (ii) If some of the total kinetic energy raises a target atom or nucleus to a higher energy state, the process is termed inelastic scattering, (iii) If the scattering centres act such that the scattered particles bear a phase relationship to one another, coherent scattering results; for example, a crystal lattice will scatter particles in a way that results in a diffraction pattern, (iv) If no phase relationship exists, the scattering is incoherent; for example, Compton scattering (see Comp ton effect), (v) If the scattering angle is less than 90°, the term forward scattering is used; is it greater than 90°, the term back-scattering is used, (vi) A photon or particle may undergo single or multiple scattering. 264 GLOSSARY secondary radiation. Radiation resulting from the interaction of primary radiation with an absorbing medium.

SI. The abbreviation for the International System of Units. This coherent system is administered by the International Bureau of Weights and Measures (B1PM) in Sèvres, France. The Resolutions and Recommendations are taken by the General Conference on Weights and Measures (CGPM). Most countries are now committed to changing over to SI, which will eventually be a single, practical, worldwide system of units. The CGPM, which started its work in 1889, introduced the new radiation units, the becquerel and the gray, in 1975, in co-operation with ICRU.

To facilitate conversion of units that may be met with into SI equivalents, a conversion table has been appended at the end of this Glossary to aid the reader. This is followed by a list of most of the SI units. source strength. Of a gamma-ray radiation source, the strength defines the activity of the radioactive nuclide source material; it is expressed in becquerels (or curies). specific activity. The number of spontaneous nuclear disintegrations per unit mass of a given material per unit time interval. Expressed in becquerels or curies per mole or per gram. (See activity.)

specific heat capacity. The quantity of heat (i.e. energy) required to raise the temperature of unit mass of a substance by one degree of temperature difference. In the SI system, the unit of specific heat is the joule per kilogram per degree of temperature difference in Kelvin. The relation between the SI and the metric system unit, the thermochemical calorie, is:

1 Jkg"1 • K"1 = 0.2390 cal-kg"1 • K"1 = 2.390 X 10~4 cal - g"1- K"1

spectral energy. (1) For a particle, the energy (quantum energy) carried by the particle, usually given in units of electronvolts (eV); it is proportional to frequency, i.e. is inversely proportional to wavelength (see photon). (2) For continuous radiation spectra, the energy contained in a given interval of quantum or particle energy.

spectrophotometer, transmission. An instrument for measuring either transmittance of light or the absorbance (optical density) of a material as a function of wavelength. In dosimetry with films or solutions, it is usually used to compare the absorbance (optical density), at a specified wavelength for the dosimetry system being used, either before and after irradiation or after irradiation against that of a standard.

spectrum, radiation, see radiation spectrum. GLOSSARY 265 stopping power, for charged particles (linear stopping power, S, or mass stopping power, S/p). A measure of the energy lost (dE) by a charged particle of specified incident energy while traversing an incremental distance (dx)¡ in a substance i. It varies with the spectral energy of the particles. It is defined as Sj = (dE/dx)¡. According as dx is expressed in terms of length or mass per unit area, the stopping power is termed linear, S¡, or mass, (S/p)¿, where p represents the density of the substance.

The ICRU definition is also given for completeness [G3]: The total mass stopping power, 6/p, of a material for charged particles is the qygtient of dE by pdx, where dE is the energy lost by a charged particle of specified energy in traversing a distance dx, and p is the density of the medium.

S = TdE p p dx

For energies at which nuclear interactions can be neglected, the total mass stopping power is:

where Scu| is the linear collision stopping power (see linear energy transfer) and Sraj is the linear radiative stopping power. surface dose, see entrance dose and exit dose. technical uncertainty. Statistical deviations in replicate observations as a result of instrumental and operational variations and/or errors. thermoluminescent dose meter (TLD). A dose meter making use of the phenomenon of thermoluminescence, comprising a holder and some thermoluminescent material. thermoluminescent material. Material that luminesces when excited (stimulated) by heat (see luminescence). tolerance limits. The upper and lower values in a distribution of data within which a certain fraction of succeeding individual measurements falls with a certain confidence probability. Both limit values are calculated from

the parameters of the distribution (e.g. the average of the Dmin or Dmax measurements and the appropriate standard deviations, SBD) by the use of k-values.

total attenuation coefficient, see attenuation coefficient.

transmittance, see absorbance. 266 GLOSSARY t-value, or Student's t-value. The value used in statistical analysis to determine the range and reliability of measurements and results. It is a function of the confidence probability required and the number of measurement values available.

uniformity ratio, U, or dose uniformity ratio. The ratio of maximum to minimum

absorbed dose in the product, i.e. U = Dmax/Dmin-

units. To facilitate conversion of units that may be met with into SI equivalents, a table has been appended at the end of this Glossary to aid the reader.

utilization efficiency. The fraction of radiation energy emitted that is absorbed by the total product after the completion of an irradiation cycle. water equivalence. The quality of a material being irradiated such that its radiation absorption characteristics correspond closely to those of water.

WHO. World Health Organization, Geneva (Switzerland). X-rays. Penetrating electromagnetic radiation (photons) usually produced by high-energy electrons impinging upon a metal target. (See atomic fluorescence, Auger effect, photoelectric absorption, Comp ton effect, brenisstrahlung).

Z. Atomic number, i.e. proton number of a nucleus. High-Z or low-Z refers to high- or low-atomic-number substances, respectively. GLOSSARY 267

SI base units are the metre (m), kilogram (kg), second (s), ampere (A), kelvin (K), candela (cd) and mole (mol).

Multiply by to obtain

Radiation units becquerel 1 Bq (= 2.7027 X 10"u Ci) disintegrations per second (1 dis/s) = 1.00 X 10° Bq 10 curie (= 3.7 X 10'°dis/s) 1 Ci = 3.70 X 10 Bq -4 roentgen 1 R = 2.58 X 10 c/kg gray 1 Gy (= 1.00 X 10° J/kg) 2 rad 1 rad = 1.00 X 10" Gy (= 1.00 X 10~2 J/kg) rem (in radiation protection only) dimensions of J/kg

Mass unified atomic mass unit 12 27 (75 of the mass of C) 1 u = 1.661 X io- kg pound mass (avoirdupois) 1 lbm = 4.536 X lo-' kg ounce mass (avoirdupois) 1 ozm = 2.835 X 10' g 3 ton (long) (= 2240 Ibm) 1 ton = 1.016 X 10 kg 2 ton (short) (= 2000 lbm) 1 short ton = 9.072 X 10 kg 3 tonne (=metric ton) 1 t = 1.00 X 10 kg

Length statute mile 1 mile = 1.609 X 10° km yard 1 yd = 9.144 X 10"' m foot 1 ft = 3.048 X 10-' m 2 inch 1 in = 2.54 X 10" m -3 2 mil (= 10 in) 1 mil = 2.54 X 10" mm

Area hectare 1 ha = 1.00 X 104 m2 2 2 2 (statute mile) 1 mile = 2.590 X 10° km 3 2 acre 1 acre = 4.047 X 10 m 2 2 2 yard 1 yd = 8.361 X 10-' m 2 2 foot 1 ft = 9.290 X 10'2 m2 2 inch 1 in2 = 6.452 X 102 mm2 barn 1 b 1.00 X 10'28 m2 268 GLOSSARY

Multiply by to obtain

Volume

3 yard 1 yd3 = 7.646 X 10-' m3 foot3 1 ft3 = 2.832 X 10"2 m3 inch3 1 in3 = 1.639 X 104 mm gallon (Brit, or Imp.) 1 gal (Brit) = 4.546 X 10"3 m3 gallon (US liquid) 1 gal (US) = 3.785 X 10"3 m3 litre 1 1 = 1.00 X 10~3 m3

Force dyne 1 dyn 1.00 X 10"5 N kilogram force 1 kgf 9.807 X 10° N poundal 1 pdl 1.383 X 10"' N pound force (avoirdupois) 1 Ibf 4.448 X 10° N

Density pound mass/inch3 1 lbm/in3 = 2.768 X 104 kg/m3 pound mass/foot3 1 lbm/ft3 = 1.602 X 10' kg/m3

Energy

British thermal unit 1 Btu = 1.054 X 103 calorie 1 cal = 4.184 X 10° electronvolt 1 eV s 1.602 X 10"'9 erg 1 erg = 1.00 X 10"7 foot-pound force 1 ft-lbf = 1.356 X 10° kilowatt-hour 1 kWh = 3.60 X 106

Temperature, energy I area' time

Fahrenheit, degrees-32 3F—32 5 °C Rankine 3R 9 K 2 1 Btu/ft s 1.135 X 104 W/m2 2- 1 cal/cm min 6.973 X 102 W/m2 GLOSSARY 269

INTERNATIONAL SYSTEM OF UNITS (SI)

> approved for use with SI for the time being

A ampere 1, L litre (use L t> Â angström throughout if the ell and the one t> a are on the typeface are identical or t> atm atmosphere similar, e.g. not 1 1 but 1 L) > bar bar lm lumen C> b barn lx lux Bq becquerel m metre cd candela »» minute (of angle) C coulomb > min minute (time) Ci curie (1 Ci = 37 GBq) mol mole > d day N newton degree (of angle) n ohm °C degree Celsius Pa pascal (= N/m2) eV elect ronvolt rad rad (100 rad = 1 Gy) F farad rad radian g gram t> R röntgen (1 R = 2S8 ¿iCi/kg) Gy gray (1 Gy= 1 J/kg) ' second (of angle) > ha hectare s second (time) H henry S siemens (= ohm"1) t> h hour sr steradian J joule T tesla K Kelvin t tonne (1000 kg) Hz hertz (= cycles per second) u unified atomic mass unit kg kilogram V volt W watt Wb weber

Prefixes

d (deci) 10"' da (deka) 101 2 c (centi) 10~ h (hecto) 102 3 m (milli) 10~ k (kilo) 103 M (micro) 10"6 M (mega) 106 9 n (nano) 10~ G (giga) 109 12 p (pico) 10~ T (tera) 1012 f (femto) 10"15 P (peta) 1015 18 a (atto) 1CT E (exa) 1018 HOW TO ORDER IAEA PUBLICATIONS

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