Distance Assisted Training Programme for Nuclear Medicine Technologists

Transcription

1 REGIONAL COOPERATIVE AGREEMENT INTERNATIONAL ATOMIC ENERG AGENC Distance Assisted Training Programme for Nuclear Medicine Technologists Edited by: Heather E. Patterson, Brian F. Hutton Radioimmunoassay Author: Vijay Kumar Liquid Scintillation Counter Author: Stefan Eberl Module 8 Units 14a & 14b The training material within this publication has been developed through Westmead Hospital, Sydney, under the auspices of the IAEA and sponsored by AusAID (Australian Agency for International Development). This material should be regarded as the property of the IAEA and should be reproduced or used only in accordance with the attached statement of ownership. (version 3.1)

2 Statement of ownership and disclaimer (2009) All materials which form part of the training program Distance assisted Training for Nuclear Medicine Technologists, including any translation of these materials, remain the property of the IAEA, Vienna. If the materials are to be reproduced or printed in any manner, the statement of ownership, as well as names of original authors and editors shall be included. The project materials are freely available to lecturers and students for use in Nuclear Medicine training, provided they are not used for commercial purposes. The materials will normally be made available only as part of national formal training programmes approved by the IAEA. This is encouraged to ensure that students undertaking the training have adequate supervision and guidance. Also formal recognition of students training will only be provided subject to formal student assessment via national training programmes. The IAEA, authors and editors make no guarantee regarding the accuracy of material presented and accept no responsibility for any action arising from use of the materials. our respect for the use of these materials will be very much appreciated. Please direct any queries regarding these materials or their use to: Nuclear Medicine Section International Atomic Energy Agency P.O. Box 100, A 1400 Vienna, Austria

3 Radioimmunoassay Flowchart Radioimmunoassay 14a CONTENTS Liquid Scintillation Counter Flowchart 1. Radioimmunoassays (RIA) Introduction 1.2 Definitions. 1.3 Types of assays 2. Materials & Methods Used in RIA Principles of RIA What is structurally specific immunoassay? 3.2 How to set up RIA assays 3.3 What is a standard curve? And How to measure ligand concentration? 4. Competitive Binding Assays (Equilibrium assays) Equilibrium assays 4.2 Displacement assays 4.3 Sequential assays 5. Non-Competitive assays (IRMA or Sandwich assays) Principles 5.2 Methods and Resulta 5.3 Graphs - calculations 6. Separation Methods Charcoal adsorption method 6.2 Non-specific precipitation of antigen-antibody complex 6.3 Immuno precipitation method 6.4 Solid phase antibody method. 7. Calculation Methods Linear plots 7.2 Semi-log plots 7.3 Logit-Log plots 8. Quality Control Procedures Statistics 8.2 Internal quality control 8.3 External quality control 9 Non-radioactive immuno assays Definitions Principles of the assays Advantages / Disadvantages 9.3 Enzyme Immuno Assays (EIA) 9.4 Fluoro Immuno Assays (FIA) 9.5 Chemiluminiscent Immuno Assays 10 Typical Commercial and research RIA kits. 33 Glossary 35 Page e f c

4 Liquid Scintillation Counter 14b CONTENTS page Outline 36 Theory of Liquid Scintillation counting 38 Instrumentation 40 - counting coincidence - multi-channel analyser - background and noise reduction Quantification, Quench and Sample preparation quench corrections - internal standardization - channel ratio - auto external standardization Glossary 52 d

5 Radioimmunoassay (RIA) Outline Definitions Types of Assays Materials & Methods of RIA Principles of RIA - antigens - setting up RIA assay - antibodies - measuring ligand concentration - separation methods - standard curve - buffers, standards & control Competitive Binding assay - equilibrium - displacement - sequential Non-competitive assays Separation Calculations Quality Control - charcoal - linear plots - internal - precipitation - semi-log plots - external - solid phase - logit-log plots Non-radioactive assays RIA kits - definitions - typical contents - enzyme immunoassay (EIA) - fluoro immunoassay (FIA) - chemiluminiscent Glossary e

6 Liquid Scintillation Counter Flowchart Outline Theory of Liquid Scintillation Counting Instrumentation Counting Multi-channel Background and Noise Coincidence Analyser Reduction Quench corrections - internal standardization - channel ratio - auto external standardization Quantification, Quench & Sample preparation f

7 Radioimmunoassays (RIA) Technical Writer: Vijay Kumar Production Editor: Heather Patterson Outline: In the mid-1950s Solomon Berson and Rosalyn alow, from the Veterans Administration Hospital in Bronx, New ork, were studying the role of insulin in diabetics when they noted that the treated diabetic patients carried antibodies to the peptide hormone. They discovered that these antibodies could bind radioactively labelled insulin, which paved the foundation for a new approach in science called radioimmunoassay (RIA). For her contribution of this important analytic technique to medical science, Rosalyn alow shared the 1977 Nobel Prize in Medicine and Physiology. It was not long before other investigators discovered that RIA could be used to measure other molecules besides peptide hormones. Assays for the thyroid hormones, drugs, and many more compounds were devised. Their effectiveness in readily measuring substances that previously could be measured only by long and laborious bioassays or indirect chemical methods led to revolutionise RIA within medical pathology. One of the important features of RIA is its ability to measure minute quantities of biological substances with the help of radioactively labelled molecules. Using RIA methods we are able to detect nano (10-9 )- or femtomoles (10 15 ) of molecules. These techniques have provided a large amount of information on the biochemical processes dealing with ligand - receptor systems and serum drug level estimation. After being accepted first by the medical practitioners, the field of RIA reached its peak in the early 1970s and continued to grow for two more decades. They are still widely used but because of the radioactive waste resulting from such assays, many molecules are now assayed with non-radioactive immunoassays such as those with enzyme labels, fluorescence labels and chemiluminescent labels. New assays are now added yearly but more importantly the technology is now fully automated. Although not all these methodologies are clinically useful, the core of clinically useful assays has grown significantly and spawned a new health care product industry to supply the needed reagents and supplies. This unit on radioimmunoassay aims to inform the nuclear medicine technologist on the different aspects of estimating small levels of ligand or antigens using the radioimmuno assay method. Many nuclear medicine departments do not perform RIA and in many countries the immunoassay procedures are carried out in Endocrinology laboratories. Whether you perform these investigations or not you should study this unit and understand the aims of the various procedures. 1

8 Objectives: On completion of this unit you will: 1. Understand the mechanism of antigen antibody reaction. 2. Understand various methods of Radioimmunoassays (RIA). 3. Know how to set-up a RIA run. 4. Understand the principles of Immunoradiometric assays (IRMA) 5. Understand four different separation techniques [to separate bound fraction from free fraction] 6. Be able to calculate using four different methods of plotting. 7. Understand internal and external quality control procedures 8. Have an introduction to non-radioactive immuno assays [EIA, FIA CIA etc]. Time Check: Allow 12 hours to study this unit and complete exercises in your Workbook. Note: As part of this subject and following this Unit 14a on Radioimmunoassay is the Unit 14b, Liquid Scintillation Counter describing the principles of operation. 2

9 1. Introduction to Radioimmunoassay Introduction: RIA is primarily based on the reaction between an antibody and an antigen whose concentration has to be quantified. Before describing assay systems in detail it is worth considering the most important terminologies used throughout the text in order to assist your understanding Time Check: Allow 2 hours to study sections 1 and 2 and complete exercises in your Workbook. 1.1 Definitions: Antibody: (AB) Antibody is a protein formed by the body s defence system as part of an immunologic response to a foreign substance. The antibody specifically combines with the foreign substance and to a variable extent with substances of similar structure. Antigen: (Ag) Antigen is a substance that is capable of inducing formation of antibodies which react specifically to the antibodies so produced. Antigens are also known as ligands. Ligands are usually the hormones, enzymes or other substances that are to be assayed. Hapten: A substance that is not immunogenic in itself, but becomes immunogenic when complexed to another compound. The antibody produced will bind the non-complexed hapten also. Examples: steroid hormones and drugs (e.g. Digoxin) usually complexed with Bovine Serum Albumin to form hapten. Sensitivity: Sensitivity is defined as the minimum quantity detectable using a particular RIA method. Sensitivity depends in part on assay precision. Specificity: Specificity is the ability to assay a single substance within a heterogenous mixture. For RIA, it is the capacity of the system to discriminate antigens of similar structures and identify the specific molecule of interest. 3

10 1.2 Types of assays Radioligand assay: When a radionuclide is used as the label for the ligand, the assay is called radioassay or a radioligand assay (eg. Vitamin B12 assay) Radioimmunoassay (RIA): When an antigen (unlabelled and/or radiolabelled) and an antibody (specific binder) are used, the assay is called RIA. Radioreceptor assay (RRA): When a receptor protein molecule (isolated from tissues) is used as the binder (instead of an antibody) it is called RRA. Competitive protein binding assays (CPBA): In a competitive protein binding assay, the unlabelled ligand competes with a labelled ligand for binding to a limited number of binding sites (ANTIBODIES). Immunoradiometric assays (IRMA): When the antibody is labelled with a radionuclide the assay is called an IRMA. It is also known as sandwich technique as the antigen is sandwiched between labelled and unlabelled antibodies. Enzymeimmunoassay (EIA): If an enzyme is used, instead of a radionuclide, then the technique is known as EIA. If the separation technique uses a solid phase antibody procedure, then it is generally known as enzyme linked immunoabsorbent assay (ELISA). Fluorescent immunoassay (FIA): If a fluorescent probe is used instead of a radionuclide, then the technology is known as FIA. Chemiluminiscent immunoassay (CIA): If a chemiluminiscent probe such as lanthanides is used instead of a radionuclide probe then it is known as CIA. The antibodies (binders) that specifically bind an antigen (ligand), can be classified into 3 major categories: protein antibodies, transport proteins naturally occurring receptors. The typical examples are summarised in the table below: PROTEIN ANTIBODIES: Table 1 LIGANDS Protein Hormones Steroid Hormones Drugs (Digoxin) Nucleotides (DNA, RNA etc) Viruses ANTIBODIES Specific antibody proteins Specific Steroid-Protein Conjugates Specific drug protein conjugate Specific Nucleotide-Protein Complex Antibody. Specific virus-antibody raised in animal or humans. 4

11 TRANSPORT RECEPTOR ANTIBODIES: LIGANDS RECEPTORS (Transport Proteins) Thyroxine Vitamin B12 Testosterone Cortisol Folate Thyroxine Binding Globulin (TBG) Transcobalamin Testosterone-binding Globulin Cortisol binding Globulin (Transcortin) Folate Binding Globulin TARGET RECEPTOR ANTIBODIES: LIGANDS RECEPTORS (Target) Estrogen ACTH Insulin Vitamin-B12 Uterus Cytosol Receptor Adrenal Cortex Cell Membrane Receptors Liver, Placenta cell membrane receptor Intrinsic Factor Located in Stomach cell membrane as Receptor 2. Materials and Methods used in RIA. The following materials and methods are key to the understanding of RIA in the practical sense. All the materials are provided by the manufacturers and commercially available in the form of a kit. Often control solutions supplied with the kit are considered as the internal controls and it is advisable to run external quality controls to monitor the behaviour of the kit. 2.1 Radiolabelled antigens 2.2 Antibodies 2.3 Separation methods 2.4 Standards & Control solutions. 2.1 Radiolabelled antigens (ligands): Radiolabelled antigens are also known as ligands labelled with radionuclides. There are several radionuclides used in RIA such as 125 I, 57 Co, 3 H, 14 C etc. However the most commonly used isotope is 125 I due to several merits associated with it, such as high specific activity, practically suitable half-life (T 1/2 =60days) ease of labelling ligands with 125 I that are used in everyday life (for example: Thyroid hormones, Digoxin etc). Moreover, 125 I is predominantly a gamma emitter with very little beta emission, which is ideal to use in the laboratory and the design of the gamma counter can also be simplified. Finally iodination of several ligands can be performed with ease; the resultant product is stable and therefore they do not separate from iodine during assay procedures. 5

12 2.2 Antibodies: As discussed earlier, antibody is produced as a result of an immune response to a specific antigenic challenge in an experimental animal. If the antigen is peptide or protein the immune response is obtained directly and if the antigen is a non-protein substance such as a drug or steroid then the hapten molecule is required to initiate immune response. Hapten is the drug complexed with BSA (bovine serum albumin). It is important to know that the antibodies produced by this method are called polyclonal antibodies or a mixture of antibodies derived from different B-lymphocytes. On the other hand, Monoclonal antibodies (MoAB) are produced by hybrid cells, which are the sensitised single family of B-lymphocytes. (MoAB is highly specific to a given antigen and its cross reactivity with other antigens is very low.) Most of the RIA kits utilise MoAB to achieve high specific activity and high avidity in the Antigen-Antibody reaction. MoAB - are cloned from a single antibody, produced using the hybridoma technique, which is specific for only one antigenic determinant. 2.3 Separation method: One of the pre-requisite in RIA method is to separate the Bound fraction from the Free fraction without contaminating one another. Separation techniques will be discussed later under Separation method 2.4. Buffers, Standards and Controls: Buffers: Most of the RIA reactions are carried out in buffered solutions to prevent fluctuations in ph and ionic strength during the reaction. A buffer solution may be defined as a solution containing a weak acid and/or a weak base to give a set ph and ionic strength. Usually, buffer solution is supplied as part of the kit. Standards: Standards are reference preparations necessary to generate a doseresponse curve in order to compare the unknowns to be measured and are chemically identical to the ligands to be measured. The standards are supplied in pure form, given in known set amounts varying from 0 to maximum value, usually 5 to 6 vials. They are either freeze-dried or supplied in solution and supplied as part of the kit. The other important factor is that the standards are supplied in human serum (hormone-free) matrix so that it is exactly the same as the serum samples. Control analytes: Control serum is identical to standards in all respect, except it could be obtained or provided in 3 different known quantities so that they serve as controls at low, normal and high range of the ligands. They are treated exactly as unknown samples and the values are compared with the range so that performance of the kit is validated with the controls. This will be discussed in detail later. 6

13 3. Principles of Radioimmunoassay Time Check: Allow 2 hours to study this section and complete exercises in your Workbook. Important to Know 3.1 What is structurally specific immunoassay? RIA is a structurally specific immunochemical assay. The complementary shape of the antigen (ligand) and the antibody is responsible for specific recognition and binding of antigen-antibody complex and therefore called structurally specific immunochemical assay. There are two kinds of radioimmunoassay using Competitive Binding Assay and Non-competitive Binding Assay (Immunoradiometric Assay, IRMA) It is important to know that whether the antigen is unlabelled or labelled with a radionuclide, they both will bind to the antibody with equal specificity and affinity. Both the labelled and the unlabelled antigen will compete to bind to the antibody and form the Bound fraction. The unreacted labelled antigen is called the Free fraction. In order to estimate the antigen concentration, the bound and the free fractions have to be separated. There are several methods available to perform these tasks and these will be discussed in detail in the later chapters. The values are then plotted on a graph and the amount of antigen is estimated. [Ag + Ab] COMPLEX "BOUND" Fraction ANTIGEN (Ag) + ANTIBOD (Ab) (ligand) Figure 1. "FREE" Fraction Radioactive ligand Diagrammatic representation of a Radioimmunoassay The Antigen and the antibody have complementary shapes and coupled together produce a bound complex. Remember In summary, there are three important steps to be considered in understanding the basics of RIA: 7

14 STEP-1: Antigen-Ab Complex Formation resulting in a Bound and a Free fraction STEP-2: Separation of the Bound and the Free Fraction STEP-3: Calculation of ligand concentration. 3.2 How to set up RIA assay to measure the ligand estimation? The table 2 describes a typical set up for a RIA to analyse several patients serum for a particular ligand. The standards, controls and patients sera have two test-tubes each (duplicate). Certain other tubes of the set up are there to record various parameters of the Assay: Total Count tubes (Tc) These serve as a record of total activity added Non-specific Binding tubes (NSB)- These serve as a record of binding which is not due to the receptor Zero Binding or Zero Standard tubes (Bo) These serve as a record of the maximum binding possible in the system Buffers, antiserum, tracer solution and separating solutions are added to the tubes as indicated in the table. To the total activity tubes and NSB (non-specific binding) tubes only the indicated solutions are added. The exact procedure is described later. Description Tubes Buffer Std/Control /unknown Total Activity Antiserum Tracer solution T 1, T 2 No No No es No NSB 1,2 es Blank (0) No es es 0 Std (Bo) 3,4 es Blank (0) es es es Std Curve 5,6 7,8 9,10 11,12 13,14 15,16 Controls (QC) Patient s Sera (Unknown) 17,18 19,20 21,22 23,24 25,26 27,28 es Std A Std B Std C Std D Std E Std F Cont A Cont B Cont C Patient 1 Patient 2 Patient 3 Table 2 Separating solution External Controls 29,30 31,32 33,34 Ex Con 1 Ex con 2 Ex con 3 Reference: Radioimmunoassay -Principles and Practice (1998) Published by BARC, Bombay, India. 8

15 3.3 How to measure ligand concentration? What is a standard curve? The table 2 shows that tubes 5-16 have standards from A to F. Standard A contains zero (0) amount of ligand Standard F has the highest amount of the ligand. Add buffer, antiserum and labelled ligands to all the tubes. Incubate for the set amount of time and at the end of incubation add the separating solution. Centrifuge the tubes, decant the supernatant into a separate tube and then count both the pellet and the supernatant. Plot a standard graph using the proper calculation method. This graph as shown below represents Standard curve. The unknown values are estimated from the standard curve. Note! It is to be noted that the standard curves are generated every time an assay is made be it a single patient study or a group of patients. Step 1: Step 2: Step 4: Step 5: Add STD, Control or Pt serum into the tube Add Buffer, labelled antigen + antibody Add separating solution Separate the Sediment/supernatant STEP 3: Incubate Centrifuge Free BOUND FRACTION Figure 2. Typical experimental set up for a Radioimmunoassay The radioactivity associated with free and bound fractions are estimated using a gamma counter. The values are plotted in a graph (various methods will be described later) which is known as STANDARD CURVE. A typical graph is displayed as shown below in Figure 3: 9

16 CPM RADIOIMMUNOASSA Ligand (conc) Note: 1. The counts in the bound fraction is plotted against ligand concentration (conc). 2. The lowest concentration std 0 has the maximum counts 3. The highest concentration std 200 has the lowest counts 4. The inverse relationship between the bound fraction and ligand concentration is due to competition between labelled and the unlabelled ligands with the antibodies. 5. Unknowns, controls are read from this standard curve (Blue (darker) and Red arrows). Figure 3. Standard curve using cpm (vs) ligand concentration. Go To your Workbook, Radioimmunoassay section and answer questions 1-6 to document important points and show your understanding of the last three sections.. Key Points: The complementary shape of the antigen (ligand) and the antibody is responsible for specific recognition and binding of antigen-antibody complex and therefore called structurally specific immunochemical assay. What is an antibody? Antibody is a protein formed by the body s defence system as part of an immunologic response to a foreign substance. The antibody specifically combines with the foreign substance and to a variable extent with substances of similar structure. What is a Hapten? A substance that is not immunogenic in itself, but becomes immunogenic when complexed to another compound. The antibody produced will bind the non-complexed hapten also. Examples: steroid hormones and drugs (e.g. Digoxin) usually complexed with Bovine Serum Albumin to form hapten. What else can be used as an alternative to antibody? 1. Specific antibodies raised against an antigen 2. Naturally available transport RECEPTORS (eg TBG Thyroxin Binding Globulin) 3. Target receptors (eg. Uterus cytosol receptor for oestrogens) Note: see Table 1 for details 10

17 Three most important steps to be considered in understanding the basics of RIA: 1. Antigen-Antibody Complex Formation. The reaction gives two products: a high percentage of Bound fraction and a small percentage of Free fraction. 2: Separation of Bound and Free Fraction. It is very important that there is no cross over or contamination of one fraction into the other. It will cause error. 3: Calculation of ligand concentration. 11

18 Introduction: 4. Competitive Binding Assays: RIA is classified under three different categories: Equilibrium Assays which is also known as competitive binding assays: In this method all the reagents (labelled and unlabelled antigens and antibodies) are added at the same time Displacement Assays: It is literally the displacement of tracer-ligand from binding protein by unlabelled ligand Sequential Assays: A sequence of three different steps involved in this method. i. First Incubation where the Standards, Controls, Patient s serum and antibody are incubated for certain time period (usually between 30 min and 2 hrs) ii. Second Incubation: Add tracer to the above mixture and incubate. iii. Separate bound and free ligands by appropriate technique. Time Check: Allow 1 hour to study this section and complete exercises in your Workbook Principles of Equilibrium Assay or Competitive Binding Assays: There are several ways to quantify the antigen concentration but the most frequently used method is competitive binding assay. In this assay a known quantity of radioactively labelled antigen is mixed with a series of "cold" antigen. Since unlabelled and labelled antigens compete with each other for the same antibody binding sites, a high concentration of antigen will result in little radioactive antigen in the bound fraction and vice versa. After a fixed time, a second antibody directed against the first antibody, which leads to the formation of large complexes, which will sediment upon centrifugation. This is called the bound fraction, which is counted in a radioactive counter. (For example: Wallac: LKB-Automatic Gamma Counter 1480 with 3 NaI crystal or equivalent) This bound fraction contains two components: i. the "cold" or the unlabelled antigen and ii. the radioactive antigen (bound to the specific antibody), while the supernatant contains the unbound antigen Results The serially diluted standards yield points on a curve relating radioactive counts to the concentration of standard antigen: a so-called standard or reference curve. Using this reference curve, an unknown quantity of antigen in a serum or solution can be quantified by preforming the same reactions with first specific, then unspecific antibody and a fixed amount of radioactive antigen. Identification of the radioactive counts in the 12

19 centrifugate and use of the reference curve yields the unknown antigen concentration. The competitive binding assay is schematically represented as follows: labelled antigen + antibody unlabelled antigen bound + free "BOUND"fraction separation "FREE" fraction Figure Graphs: Flow chart of various steps involved in RIA analysis. The percentage uptake of free fraction and the bound fraction can be calculated using any one of the Methods described under Methods section [Refer sections 3.2 and 3.3]. The unknown sample value can be read from the standard curve. 4.2 Displacement Assays Principles of displacement assay are the same as described for competitive binding assays as described above. The fundamental principle is that the amount of antibody and labelled antigen are constant in the assay system. The only variable is the quantity of antigen in each assay tube. Under those conditions it can be seen in the diagram below, Figure 5, that when the antigen is 0 there is maximum binding of labelled antigen ( ) with the antibody. When the quantity of ligand increases, as in the case of tubes 2, 3, 4 and 5, then the binding of labelled antigen becomes lower. Note! The highest standard has the lowest binding. This is called competitive binding. Note: Displacement of labelled antigen by unlabelled antigen from the Antibody. 13

20 Std Figure 5. Diagrammatic representation of Displacement binding assays. 4.3 Sequential Assays: It has a sequence of two incubation reactions: In the first incubation the ligand (antigen) and antibody interact and form the Antigen-Antibody complex. In the second incubation the labelled antigen competes for binding with unlabelled antigen. SEQUENTIAL ASSA: STD / CONTROL / PATIENT SERUM INCUBATE + ANTIBOD (FIRST) ANTIGEN - ANTIBOD COMPLEX ADD TRACER ANTIGEN INCUBATE (SECOND) COMPETITIVE BINDING OCCURS BOUND UNREACTED + ANTIGEN-ANTIBOD LABELLED ANTIGEN SEPARATE Figure 6. COUNT THE BOUND FRACTION Flow chart of a typical sequential assay 14

21 5. Non-Competitive Binding Assays (or) Immunoradiometric Assay (IRMA) 5.1 Principles: IRMA is the quantitative estimation of an antigen (ligand) using the immuno reaction principle. Labelled antibody is added in excess unlike RIA where labelled antigen is added in excess. IRMA is characterised by the antibody being labelled rather than the antigen. Two or more antibodies are used with different specificities. Often one or both the antibodies are monoclonal. Specificity and sensitivity at low Ag concentrations is greatly enhanced. It is also known as a Sandwich assay, as characteristically an antigen is sandwiched between a solid phase antibody (immobilised onto a solid phase such as tube or a bead) and a labelled antibody (usually in liquid form), as shown in the diagram below: Time Check: Allow 1 hour to study this section and complete exercises in your Workbook. 5.2 Methods and Results IRMA - Immuno Radio Metric Assay is performed in the sequence of steps described in the section below. The reaction reaches equilibrium after the set period of incubation [varies from 30min to 2hrs, depending on the reaction]. The free and bound fractions are separated as described in Chapter 6. Calculations are performed as indicated in the methods section [Refer sections 3.2 and 3.3]. The unknown sample value can be read from the standard curve RADIOIMMUNOASSA CPM Ligand (conc) Figure 7a. Standard curve using cpm (vs) ligand concentration. 15

22 IRMA - Immuno Radio Metric Assay AB - 1 ANTIGEN AB - 2 AB1-Ag-AB2 (Unlabelled) (Ligand) ( Labelled) (Complex) AB = ANTIBOD Figure 7. Diagrammatic representation of IRMA - Immuno Radio Metric Assay Step 1: Step 2: Step 4: Add Std, Control or Pt serum into the tube Add Buffer + labelled antibody Step 3 Incubate Discard the supernatant containing free fraction Free - supernatant Antibody coated tubes BOUND FRACTION Figure 8. A diagrammatic representation of IRMA assay procedure: 16

23 5.3 Graphs [Calculation] The radioactivity associated with free and bound fractions are estimated using a gamma counter. The values are plotted in a graph which is known as STANDARD CURVE. The percentage uptake of free fraction and the bound fraction can be calculated using any one of the Methods described under Methods section [Refer sections 3.2 and 3.3]. The unknown sample value can be read from the standard curve. A typical graph is displayed as shown below (linear curve): IRMA Method Counts cpm Ligand Conc Figure 9. A typical IRMA standard curve Note: The bound activity and ligand concentration are directly proportional to each other. Go To your Workbook, Radioimmunoassay section and answer questions 6-7 to document important points and show your understanding of this last section. Key Points: What is an Equilibrium Assay? Equilibrium assay is also known as competitive binding assay: In this method all the reagents (labelled and unlabelled antigens and antibodies) are added at the same time. They reach equilibrium within the incubation period and form a bound fraction depending on the concentration of antigen and antibody 17

24 Displacement Assays: It is literally the displacement of tracer-ligand from antibody by unlabelled ligand, typical in antibody coated tubes. Here the displacement is proportionate to the amount of unlabelled ligands. Sequential Assays: A sequence of three different steps involved in this method. 1. First Incubation where the Standards, Controls, Patient s serum and antibody are incubated for certain time period (usually between 30 min and 2 hrs) 2. Second Incubation: Add tracer to the above mixture and incubate. 3. Separate bound and free ligands by appropriate technique. What is an IRMA? (immuno radiometric assay) It is also known as a Sandwich assay, as characteristically an antigen is sandwiched between a solid phase antibody (immobilised onto a solid phase such as tube or a bead) and a labelled antibody (usually in liquid form). The percentage of radioactive binding is proportionate to the concentration of ligand. 18

25 Introduction: 6. Separation Methods There are different ways to separate the bound fraction from free fraction. But most commonly four methods are used in practice: 1. Charcoal method 2. Polyethylene Glycol method 3. Immuno-precipitation method using second antibody 4. Solid-phase method including magnetic separation method. In the following section we will discuss each of these methods, their advantages and limitations. Time Check: Allow 1 hr to read and understand the following section on separation methods. 6.1 Charcoal method (Non-specific adsorption of Antigen): Charcoal adsorption of bound antigen from the antibody is a typical example of non-specific adsorption of antigen. Charcoal is coated with dextran, albumin or gelatin. Charcoal is also used in its natural acid treated form without any coating. Tube containing Ant-Ab complex and free fraction Pour / add activated charcoal Centrifuge, Decant supernatant. Count the pellet / bound fraction Mix, Incubate 5 mins Figure 10. Charcoal separation method BOUND FRACTION 19

26 6.2 Non-specific Precipitation of Antigen-Antibody Complexes: PEG (Polyethylene glycol) binds water and makes the bound complex insoluble. It uses the property of molecular sieving and captures the macromolecules (antigen-antibody complex). Definition: Molecular sieve: PEG acts as a molecular sieve. The structure of PEG is described as a cage with specific pore size. When PEG is added to the end-product of the antigen-antibody reaction, the macro molecules (big size ant-ab complex) are trapped inside the cage like structure and the free (unreacted) component is squeezed out of the cage. On centrifugation, the PEG and the bound fraction is sedimented as a pellet and the free fraction is decanted. 6.3 Immunoprecipitation of Antigen-Antibody Complex: A polyclonal antibody raised against the second antibody (or the antigenantibody complex) is used to achieve complete precipitation of the end product of the Immune-reaction. This process is further enhanced by PEG addition. The separation of bound from the free antigens is achieved very satisfactorily using this method. What is a second antibody? The first antibody is raised for a specific antigen in an animal (eg. Rabbit). The serum containing IgG from the rabbit is injected into another large animal (eg. Sheep, donkey etc), which will produce anti-rabbit IgG. This is called the second antibody. A big animal is selected, as a large volume of second antibody is required. The first antibody, which is in the form of bound complex with the antigen is selectively and quantitatively precipitated by the second antibody and therefore it enhances the separation process to completion. What is IgG: When a protein molecule is injected into an animal it elicits an immune reaction and produces immunoglobulin. IgG is a specific fraction or a family of immunoglobulins responsible for antibody response. As shown in the figure 11 the second antibody has several binding sites for the Ag -Ab complex and can produce a very large complex. When PEG is supplemented with the second antibody the resultant complex can be sedimented very easily by centrifugation and quantitatively precipitate Ag -Ab complex. The diagram below illustrates that the second antibody is coated on a glass bead and binds the antigen-antibody complex. They expose several binding sites per glass bead. 20

27 ANTIGEN-Ab COMPLEX SECOND ANTIBOD Figure 11. Immuno precipitation method of separation using a second antibody. Note! In the case of the magnetic separation method, the central core of the glass bead is filled with ferrous iron particles. When it is subjected to a tray containing a magnetic field, it sediments and thus the Ag-Ab complex is separated from the supernatant, which contains the free fraction. 6.4 Solid-phase Adsorption of Antigen: The reaction tube is coated with antibody to bind the antigen (labelled or unlabelled). Solid phase could use beads or magnetic particles to achieve solid-phase adsorption. Figure 12. Solid phase separation method using antibody coated tubes. 21

28 Go To your Workbook, Radioimmunoassay section and answer questions 8-10 to document important points and show your understanding of this last section. Key Points: Separation techniques. There are different ways to separate the bound fraction from free fraction. But most commonly four methods are used in practice: see 1. Charcoal method is a non-specific adsorption method. 2. Polyethylene Glycol (PEG) method. The underlying principle of PEG separation is by molecular-sieve mechanism. Therefore the separation of bound from free is distinct or complete. 3. Immuno-precipitation method using second antibody. Two antibodies are used and the resulting complex is a macromolecule, which is separated with PEG method to further assist complete separation of bound and free fractions. 4. Solid-phase method including magnetic separation method. In this method an antibody coated tube is used as a solid phase. This is the most commonly used method. Magnetic separation is also used extensively. Advantages of IRMA over RIA 1. Avoids difficulties of radio-iodination of some antigens such as peptide hormones. 2. IRMA is useful when purification of the antigen is difficult or where radio-labelling will alter the immunological property of the antigen. 3. Labelled Ab more stable during incubation. 4. Generally IRMA is more sensitive than conventional RIA. 5. There is a linear relationship between amount of tracer bound and Ag concentration. 6. Some antigens such as Australian Ag is highly contagious and it is much safer to work with the non-contagious labelled Ab. 22

29 Introduction: 7. Calculation Methods: Analysing the raw data to get a meaningful result is very important in RIA. The results can be calculated in several ways. However, certain assays are better calculated using a particular method, so that there is nothing called a universal or general method which can be recommended. We will describe various methods of calculation and discuss their merits and limitations. From the experimental design, it is apparent all the assays are done in duplicate. Therefore an average of the duplicate samples is used in the calculation. [All the tubes are counted for the set time, for example: 1 min (60sec)]. The NSB [non-specific binding] values are subtracted from the individual reading so that the true binding values are used in the calculation: Average cpm- NSB = True cpm [corrected mean cpm] Time Check: Allow 3 hours to study sections 7 and 8 and complete exercises in your Workbook. 7.1 Linear Plots: Cpm vs ligand concentration: In this method of calculation, the true counts per minute (cpm) are plotted on the -axis and the concentration of standards is plotted on the X-axis. See figure 13a The typical standard curve is as shown in the graph. If the ligand concentration is reasonably high as in the case of digoxin assay then it is suitable to calculate using this method. The sensitivity is good at the low end to the middle part of the curve. It is very insensitive to measure at the higher ligand levels. As you can see a small difference in the cpm at the y-axis can make a big difference in the ligand estimate at the higher end of the curve on the x-axis. Therefore this method is not suitable to measure high levels of ligands. Especially, if the curve is extrapolated, then the insensitivity becomes worse. 23 CPM RADIOIMMUNOASSA LIGAND (CONC) Figure 13a: Linear plot using cpm (vs) ligand conc

30 The cpm values can be expressed as % B/F (%Bound/Free) or as % B /Total. Note: The shape of the curve remains the same. Therefore the low-end sensitivity and high end insensitivity is comparable to the [cpm vs ligand] conc method discussed earlier. See Figures 13b & 13c. %B/Free %B/Total %B/Free %B/Total ligand conc ligand conc. Figure 13b. Linear plot using Figure 13c. Linear plot using %B/free (vs) ligand conc %B/total (vs) ligand conc 7.2 Semi-log Plot %B/Bo Semi - Log Plot Log [Ligand] Semi-Log Plot: The calculation methods discussed above have severe limitation of highend insensitivity. The B/Bo values are used in the semilog plot as shown in the graph. It is apparent that the shape of the plot is not a typical curve nor is it a straight line. Therefore, the high-end sensitivity is better than the curve plot but cannot be used to extrapolate and read higher values of the unknown samples. Figure 14a Semi-log plot using %B/Bo (vs) log of ligand conc 24

31 7.3 Logit vs Log Plot: Logit - Log Plot Logit B/Bo vs log of ligand conc: The graph is usually generated by computer using the logit [B/Bo] and log [ligand] parameters. Here, ln [B/B0] Logit B/B0 = B/Bo where ln=log to the base e (Natural or Naperian Base) (Refer: Pillai & Bhandarkar, Radioimmunoassay, Principles and Practice. (BARC, BOMBA for more details) Logit [B/Bo] Key Points: Log [Ligand] Figure 14b. Semi-log plot using %B/Bo (vs) log of ligand conc Being a straight line the values can be read across the spectrum of the ligand concentrations, with equal confidence. Also, a straight line graph can be easily extrapolated to read even higher values so that the potential is very much higher than other methods. There are 3 different ways the results can be calculated: 1. Linear Plot: The values on the x-axis are always the Ligand concentration. The values an the y-axis is expressed in three different ways a. cpm (radioactive counts of the bound fraction b. %B/Free fraction as a ratio c. %B/Total counts as a ratio. 2. Semi-Log Plot: In this plot the x-axis is expressed as the logarithm of ligand concentration. The y-axis has the B/Bo values plotted in a linear plot. Note: Bo is the activity (cpm) associated with the Bound fraction at zero concentration of the standard. B = is the activity (cpm) associated with the Bound fraction except the zero concentration of the standard or the unknown sample. 3. Log-Logit Plot: In this plot x-axis has the Log of the ligands concentration -axis has the logit of %B/Bo values. 25

32 Introduction: 8. Quality Control Procedures The quality control procedure is an important aspect in RIA assay. It is mandatory to include QC samples every time you do RIA assay, as it is the only way you can monitor the performance of the RIA-kit. For instance, the reagents in the kit have expired due to bad quality of the reagents or due to storage conditions affecting the antibodies or other reagents, then it will reflect on the performance of the QC. QC components are usually provided to cover low, normal and high range of the standard curve for each assay. The QC values should fall within the range prescribed for each item. If the QC value is higher or lower than the range, then the assay should be repeated. Therefore, do QC every time with a RIA assay. It is important and advisable not to validate a RIA assay, unless the QC values are satisfactory. 8.1 Statistics Fundamentals terminology especially terms like systematic error and random error are commonly used in Statistics. An in depth knowledge is not required but the basic concepts, which are described below, are essential to know. The normal probability distribution is very commonly used in statistics because of a rule called the Central Limit Theorem, which states that, under certain conditions, as the number of random factors affecting some phenomenon increases, the distribution of the sum (or average) of these factors approaches the normal distribution. The Figure 15a shows several normal distributions with different means and standard deviations. The mean of the distribution is a measure of its location. Since the spread is symmetrical with one peak in the centre [the mean μ] is also the mode and the median of the probability distribution; thus μ is the point where the function is the highest and the point that splits the area under the curve in half. The area to each side of μ is equal to 0.5. The standard deviation of the distribution is a measure of the spread, or variability, of the distribution. When the standard deviation is large, the functional spread is wide; when the standard deviation is small, the curve is narrow and high. We will denote the distribution, mean and variance of a normal random variable by a simple notional statement : X = N(μ,σ) This means that random variable X has a normal distribution (N) with mean μ and standard deviation σ. [For further information on statistical analysis, you may refer to the text book Statistics Concepts and Applications by Amir D. Aczel. Ed. Richard D Irwin (1995). ISBN: X]. 26

33 σ σ Figure 15a. μ The Standard Normal Distribution. 8.2 Internal quality control: Monitoring Internal Quality Control: When setting up a RIA assay, it is mandatory to run a set of controls each time. Three controls are usually included per run comprising, low, normal and high levels The control range with the upper limit and the lower limit is set as shown in the graph, which is supplied by the manufacturer which is standardised and determined already. The value for each control is plotted in a graph every time. If the control values of all 3 controls fell within the range, then the RIA assay is validated. If the control values of all 3 fell outside the range, then the RIA-assay is not acceptable. If one control value is constantly on the lower side of the range and eventually falls outside the lower limit, then it may suggest that the assay kit has exceeded the expiry date. Usually it indicates the antibody titre is getting lower and predict the failure of the kit. When a trend is established, then it is time to decide and change the kit before the trend goes off the limits. Figure 15. Internal quality control (QC) monitoring chart for low, normal and high controls. 27

34 8.3 External Quality Control: If identical samples of digoxin is assayed using several RIA kits supplied by different manufacturers, then the values obtained can be pooled and analysed. Two parameters can be calculated: 1. Mean value: the mean value is the average of the pooled values. 2. Standard deviation: standard deviation is a measure of deviation of each sample value from the mean value. Remember The important point is lesser the deviation from the mean value, better the performance of the kit. If the individual values are plotted in a graph, then the bell-shaped curve as shown below will be obtained. If a control value using a single particular RIA kit fell within the bell shape closer to the mean, then it is the best kit. Up to 2 Standard deviation from the mean value is acceptable; but if the value fell more than +2S.D then it is positive bias and <2S.D then it is negative bias. RIA kits giving values outside the 2 S.D is not acceptable. No. of Measurements Frequency Figure Standard Deviations Frequency distribution of repetitive measurements. External quality control is an essential part of RIA assays, in terms of assessing the quality of the RIA kit used for a particular assay method. For instance, there are 10 different manufacturers supply kits for measuring digoxin levels. Some manufacturers might use good quality antibodies and others might compromise the quality for cost effectiveness. The message is useful to select the ideal RIA kit to use for a particular assay. 28

35 Precision, Accuracy and Random: It is important to know three more terminologies precision, accuracy and random. If one single sample is analysed by several centres using different kits, then the individual values are pooled and the mean true value of the sample is calculated. If the values were higher, then they have positive bias and if the values were lower than the mean then they have negative bias. The following representation will explain three parameters: accuracy, precision and random distribution. ACCURAC PRECISION RANDOM Figure 17a 17b 17c External QC to assess Accuracy, Precision and Random performance. Each value is represented by the coloured dots. Accuracy: The values are consistent and close to the true value of the estimate, they will be precise and accurate. [in a dart board it represents the bull s eye]. The values are then accurate. Precision: The values are consistent and reproducible but not close to the true value. It is higher than or lower than the true value. It is precise but not accurate due to positive or negative bias of the results. Random: It is neither accurate nor precise. The mean value of the random values may fall close to being accurate. But the values are distributed at random. Conclusion: If the RIA kit is accurate and precise then it is most desirable. If the RIA kit is precise, then it is useable but the bias factor has to be taken into consideration. If the RIA kit has random performance, then the values are unacceptable. Go To your Workbook, Radioimmunoassay section and answer questions to document important points and show your understanding. Key Points: The terminologies are explained in the text. Internal quality control External quality control Mean value Accuracy Standard deviation and confidence limits Precision Quality control performance chart. Random 29

36 Introduction: 9. Non-radioactive Immuno Assays: RIA is still widely used but because of the radioactive waste resulting from such assays, many ligands are now assayed with non-radioactive immunoassays with enzyme labels, fluorescence labels and chemiluminescent labels etc. New assays are now added yearly but more importantly the technology is now fully automated. Time Check: Allow 2 hours to study sections 9 and 10 and complete exercises in your Workbook. 9.1 Enzyme Immuno Assay Enzymes can act as labels because their catalytic properties allow the detection of small quantities of labelled immune antigens. The following is the sequence of events which are characteristic of Enzyme Immunoassay. I. antigen + antibody Ag-Ab (Ag) (Ab-excess) (complex) Complex is formed as per principles discussed under RIA. II. antigen + antibody + enzyme Ag-Ab-Enzyme (excess) (complex) Note: III. Enzyme concentration is proportionate to anti-ab complex. Enzyme reaction: antigen - antibody - enzyme H 2 O 2 colour from Horse Radish Peroxidase Chromogenic substrate Horse-radish Peroxidase is used as the enzyme. One molecule of Peroxidase can oxidise several million molecules of substrates per minute. Reaction Kinetics is dependent on the amount of Antigen- Antibody Complex. Excess amount of Hydrogen Peroxidase is used as the substrate. Advantages: Enzymes have long shelf-life. Usually up to 12 months. No radiation hazard. The kits which are commercially available could be used until the expiry date. It becomes economical for infrequently requested tests or low volume esoteric tests. RIA is limited to the half-life of the isotope used. Typical half-life of 125 I is 6-8 weeks, used most commonly. 30

37 Disadvantages: EIA laboratory procedure is cumbersome. It involves too many steps; more than RIA. Contamination and carry-over of enhancing solution or fluorescence solution are high. Influenced by factors affecting enzyme reactions, thus might create artefacts. For example: Metal ion inhibition of enzyme reaction. Drug therapy interaction with enzyme reaction. 9.2 Fluorescence Immuno Assay (FIA). In fluorescence or photoluminiscence light of the appropriate energy (wavelength) excites the molecule from its ground state to a higher energy state. The return to the ground state is accompanied by the release of energy in the form of light or longer wavelength. Definition: FIA is a competitive binding assay that employs a fluorophore or fluorogen as the label, instead of radioisotope-probe as in RIA. The assay is based on a label that is a fluorogenic enzyme substrate (β Galactosyl-umbelliferone). When the label is hydrolysed by a specific enzyme it yields a fluorescent product. Binding of the substrate-labelled ligand by specific antibody prevents the enzyme from hydrolysing the substrate label. Since fluorescence would not be produced by antibody bound label, the bound label can be distinguished from unbound. β Galactosyl-umbelliferase β Galactosyl-umbelliferone (substrate) Umbelliferone (fluorescent product) Fluorescent emission time of most fluorescent compounds, such as Fluorescein, is in the range of nano seconds and this is not suitable to measure for laboratory use. So time resolved fluorescence is used. The metal chelate such as diketone - Europium have very long fluorescence time ( μ sec) when the interference from light scattering is minimum. Advantages: Fluorescent products can be detected at times lower quantities than colorimetric procedures of EIA. Incubation times are shorter. Sensitivity is greater. Limitations: FIA requires specialised instrumentation. An additional separation step is involved in the assay, because chelates cannot be measured in body fluids. 31

38 9.4 Chemiluminiscence Immuno Assays (CIA): Definition: A Chemiluminiscent reaction is any chemical reaction in which one of the products of the reaction is light. A typical chemiluminicscent reaction is represented as follows: 2 H 2 O 2 + LUMINOL + enhancer light + H 2 O + hν peroxidase (chemiluminiscence) The enzyme peroxidase can react with molecules such as luminol to yield light as part of the reaction product. Photon emission is in the range of nm. Low photon yield of the reaction has limited sensitivity and its application. Adding enhancer molecules such as luciferin the photon output is increased by several thousand times. The reaction time can be followed up to 30 min or longer. Advantages: Very sensitive; one molecule of peroxidase can turn over several million molecules of substrate per minute. Newer chemiluminiscent probes such as acridinium esters are insensitive to serum catalysts (serum haem proteins etc) The newer labels have improved signal to noise ratio. Signals are prolonged for longer times, hence makes the measurement easier. Limitations: Separation step is required because H 2 O 2 and luminol must be in a system free from common biological matrices such as serum. Reaction is performed in a heterogenous system in which Peroxidase is attached to a surface. 32

39 10. Typical Commercial and Research RIA kits A typical RIA kit is supplied with the following ingredients: Each kits is sufficient for 100 RIA tubes (14 standards and 86 unknowns) and contains the following components: RIA buffer Primary antiserum titered for 100 RIA tubes Reference standards and internal controls 125 I- Labelled tracer packaged with carrier IgG Precipitating second antibody Instruction booklet and graph paper Each kit should be stored at 4 o C until used. Then follow the specific storage instructions for each of the individual components. Shipping and special packaging conditions are stipulated when the kits are shipped overnight in insulated containers with cold packs. Since each RIA kit contains Iodine-125, their receipt, acquisition, possession, use and transfer are subject to the regulations as laid down by the appropriate regulatory authority. Federal regulations require suppliers of 125 I-labeled materials to verify prior to shipment that the purchaser is authorised to receive the type, form and quantity of 125 I being ordered. Usually a photocopy or facsimile of the purchasers' current specific or general license is required, before any 125 I-labeled materials are shipped. For Research RIA KITS- Not for Use in Diagnostic Procedures Atrial Natriuretic Peptide RIA Kit Calcitonin (Salmon) RIA Kit Calcitonin Gene Related Peptide RIA Kit Dynorphin A[1-13] RIA Kit b-endorphin (Human) RIA Kit Endothelin-1 RIA Kit Epidermal Growth Factor (Human) RIA Kit Insulin-like Growth Factor-1 RIA Kit Insulin-like Growth Factor-2 (Human) RIA Kit Protein Kinase Ca RIA Kit PTH Related Peptide [1-35] RIA Kit Vasoactive Intestinal Peptide RIA Kit Go To your Workbook, Radioimmunoassay section and answer questions to document important points and show your understanding of the last two sections. 33

40 Key Points: 1. RIA is based on antigen antibody binding mechanism. 2. It is important to know that whether the antigen is unlabelled or labelled with a radionuclide, they both will bind the antibody with equal specificity and affinity. 3. Both the labelled and the unlabelled antigen will compete to bind the antibody and form the Bound fraction. 4. The unreacted labelled antigen is called Free fraction. 5. IRMA (Immuno radiometric assay) is based on the same principles of RIA except, the second antibody is labelled (not the antigen as with RIA). 6. In order to estimate the antigen concentration, the bound and the free fractions are to be separated. 7. There are 4 different methods available to perform these tasks. 8. The values are plotted into a suitable calculation method and the amount of antigen is estimated. 9. Quality control (QC). Internal QC is a guide to monitor the performance of one kit over a period of time External QC is a guide to monitor the performance of several manufacturer s kits for a single ligand. 34

41 Afinity Avidity Bovine Chelate Cross reactivity Esters Esoteric Fluorescence Fluorophore Fluorogen Hapten Immune Lanthanides Monoclonal Glossary: A measure of force of attraction of Ag to Ab. Measure of strength of the bound Ag-Ab, the greater the strength the greater the Ag-Ab complex formed. Ox or cow Bind The inverse of specificity. Interaction with other substances that are closely related or structurally similar to the substance to be assayed. Delete not important Specialised Type of light with specific wavelength A substance that can emit fluorescence Is the same as fluorophore Substances that cannot stimulate an antibody response but can be made antigenic when coupled to a high molecular weight substance such as a protein. Response by immunologic cells Group of metals emit fluorescence for longer time Antibodies derived from a single B cell lymphocyte population (hybrid cells) that are specific for only one antigenic determinant Photoluminiscence Light emitted at range of wavelength (eg nm) Sensitvity Specificity Substrate Titered Closely related to affinity, the greater the sensitivity of the Ab the more rapid the attainment of equilibrium. The degree to which the antiserum or Ab will react with the substance assayed and only that substance. Basic reaction substance (eg. Enzyme acts on substrate to produce product) Measure of antibody 35

42 Liquid Scintillation Counter Technical Writer: Stefan Eberl Production Editor: Heather Patterson - Outline: Prerequisites: Resources: The liquid scintillation counter is perhaps less commonly encountered in Nuclear Medicine laboratories than other radiation detectors and counting instrumentation, as it is particularly well suited for measuring activity of low energy beta emitting isotopes, alpha emitting isotopes and very low energy gamma rays. Thus it tends to be used more for counting samples from the life sciences such as 3 H and 14 C labelled samples, in environmental monitoring, and for carbon dating of samples by measuring 14 C in the sample. Liquid scintillation counting is also used for quantifying wipe test samples of areas where low energy beta or alpha emitters are used. While the use of liquid scintillation counters is perhaps limited in nuclear medicine, a basic understanding of the principle and instrumentation of liquid scintillation counting can be expected from technical staff working in nuclear medicine. The range of low energy beta particles and alpha particles in any material is very small (much less than a mm), so they are very difficult to detect and quantify with the commonly used radiation detection equipment in nuclear medicine, as even the protective housing of the radiation detector will cause substantial, if not total absorption of the emitted radiation. To overcome this problem, the sample is mixed with a liquid scintillator, which emits light photons when the emitted particle interacts with it, hence the name liquid scintillation counter. In this module, the basic principles of liquid scintillation counting and instrumentation will be introduced. It will be assumed that you are familiar with scintillation counting and associated equipment and have successfully completed the module Instrumentation Part 1, Scintillation Counter. If your institution has a liquid scintillation counter, much useful information can be found in its manual. The Perkin Elmer web page ( has good application notes on theory and practice of liquid scintillation counting. Select Application Notes or Technical Information from the main web page and then search on liquid scintillation to bring up a list of available Application Notes or Technical Info documents. Most of these documents are in PDF format and can thus be downloaded and saved. 36

43 The recommended reference text book is Physics in Nuclear Medicine, Simon R Cherry, James A Sorenson and Michael E Phelps eds, 3 rd edition, pages , Saunders, Philadelphia, However, it is not required that you have access to this reference book to complete the module. Objectives: On completion of this unit you should have a basic understanding of 1. Basic principle of liquid scintillation counting 2. Liquid scintillation counting instrumentation 3. Corrections and quantification in liquid scintillation counting 4. Sample preparation for liquid scintillation counting As access to liquid scintillation counters is potentially limited, exercises in this module do not require access to a liquid scintillation counter. However, if you have access to a liquid scintillation counter, you are encouraged to explore some of the exercises and topics raised on your counter. Time Check: Allow a total of 4 hrs for completion of this unit and Workbook exercises. 37

44 Part 1 Theory of Liquid Scintillation Counting Time Check: This section should take you no longer than about 1 hr to complete. Introduction: Low energy beta particles and alpha particles have a very short range (much less than a mm) in most materials. Thus it is difficult to accurately and efficiently detect the particulate emissions with conventional radiation detectors. However, some of these beta emitting isotopes are invaluable in the life sciences and in hospital laboratories such as in immunology departments. The main characteristics of beta emitters of particular interest in the life sciences are listed in Table 1. Table 1: Beta emitting radionuclides used in the life sciences Radionuclide Half-Life Maximum β Energy 3 H 14 C 35 S 32 P 12.3 yr 5730 yr 87.0 yr 14.3 d 18 kev 156 kev 167 kev 1710 kev Because of the short range of these particles, the scintillator, which converts the energy from the particle into light photons has to be very close to the particle emission. This is achieved by a having a liquid scintillator which can be mixed with the radioactive material. Liquid Scintillator: The basic principle of the liquid scintillation process is shown in Figure 1. Radioactive Atom Solvent Molecule Primary Scintillator Secondary Scintillator β - Light λ 1 Light λ 2 Figure 1: Basic principle of liquid scintillation The radioactive atom emits a beta particle (β - ), which imparts its energy to the solvent molecule. The solvent molecule than imparts the energy to the primary scintillator (some times also referred to as the fluor molecule), which emits the energy as light. However, the wavelength of the emitted light λ 1 is usually not well matched to the spectral efficiency curve of light detectors, such as photomultiplier tubes, and hence a secondary scintillator (or wave shifter) is used, which absorbs the light from the primary scintillator and emits it at a different wavelength λ 2 which is better suited for the light detectors used. The light from the secondary scintillator is then detected externally by usually a photomultiplier tube. 38

45 Common solvent molecules include toluene and xylene. While these are very well suited for the purpose, they are quite toxic and are more commonly replaced with newer solvents such as DIN (diisopropylnaphthalene) and PXE (phenylxylethane) which are more environmentally friendly due to their lower toxicity. For specific applications, and depending on the sample, a mixture of other solvents may also be included to ensure that the radioactive sample is properly dissolved in the liquid scintillation cocktail. It is obviously also important that the solution is transparent to the light emitted by the secondary scintillator. A common primary scintillator is PPO (2,5-diphenyloxazole) and is used frequently with the secondary scintillator POPOP (1,4-di-[2-5- phenyloxazole] benzene). Go To your Workbook, Liquid Scintillation section and answer question 1-3 to assess your understanding of this section. Summary: The energy of low energy beta and alpha emitters is insufficient to be efficiently and quantitatively measured with conventional radiation detectors equipment, as even the protective cover over the detector may substantially or completely absorb the emitted radiation. Instead, the radioactive material is dissolved in a liquid scintillator cocktail, which allows the energy from the emitted particle to be efficiently transferred to the scintillator/wave shifter combination to produce light which is then externally detected by photo multiplier tubes. Key Points: Most α & β emissions are of insufficient energy ranges to be detected by conventional radiation detection equipment and require a scintillating liquid mixed with the radioactive material, which converts the energy from the radioactive emissions into light which can be detected by a photomultiplier tube. In liquid scintillation counting β emitting radioactive samples are dissolved in a liquid that scintillates, called a cocktail. 3 main ingredients of the cocktail are: - an organic solvent - a primary scintillator or fluor molecule - a secondary scintillator or wave shifter The primary fluor scintillates when energy is transferred to it from the solvent but the wavelength of the emitted light is not well suited for efficient detection by a photomultiplier tube. The secondary fluor / wave shifter changes the wavelength of the emitted light for efficient detection by a photomultiplier tube 39

46 Part 2 Liquid Scintillation Counting Instrumentation Introduction: As detailed in Part 1, the scintillator which converts the ionising radiation to light is part of the sample. Thus no additional external scintillator such as a NaI(Tl) crystal is required as part of the radiation detection system. In fact, the liquid scintillator replaces the function of say a NaI(Tl) scintillation crystal of the more familiar radiation detection systems in nuclear medicine. However, the light emitted by the liquid scintillator still needs to be detected and converted to electrical signals for counting and measuring of the activity in the sample. As in other radiation probes and in the gamma camera, the light is detected by photomultiplier tubes (PMTs), which are described in detail in the module: Module 2, Unit 4a, Instrumentation Part 1, Scintillation Counter. Because of the low energy of the particulate emissions, the number of photons of light emitted and hence the number of photo electrons emitted from the photomultiplier tube photocathode is very small (up to about 25 photoelectrons for the maximum energy of a 3 H beta particle). In addition, particularly with the long half-life of the isotopes used (see Table 1) with liquid scintillation counters, the number of particles emitted per unit time is quite small. Thus noise in the system has to be kept to an absolute minimum to allow samples to be counted in a reasonable length of time. The main potential sources of noise are: Electronic noise in the PMT and processing electronics such as spontaneous thermal emissions of electrons from the photocathode of the PMT. Background from external radiation Natural phosphorescence (emission of light) from the sample after being exposed to light. As detailed below in Figure 2, a number of steps in the instrument design and preparation of samples are taken to ensure low background and high sensitivity counting. Time Check: This section should take you no longer than 1hr to complete. 40

47 Instrument Overview: A block diagram of a liquid scintillation counting system and its main components is shown in figure 2. Light tight chamber with Light guide reflective coating Sample Vial Pre Amp Pre Amp PMT PMT Coincidence Circuit Summation Circuit and Amplifier Coincidence Trigger Multi Channel Analyser MCA Computer Figure 2: Block diagram of liquid scintillation counter. The basic components of the liquid scintillation counter include, as shown in figure 2 the following components. A light tight chamber into which the sample is placed. The light tight chamber keeps external light, not emitted from the sample, from reaching the photo multiplier tubes. The sample chamber also includes reflective coating and light guides to guide as much of light emitted in the sample as possible towards the photomultiplier tube. Two photomultiplier tubes which convert the emitted light to electrical signal. Initial amplification of the electrical signal from the PMTs is performed by the pre amplifiers (Pre Amps) A coincidence circuit which will be described in more detail below The summation circuit sums (adds) the signals from the two PMTs. As in the gamma camera, the sum of the PMT outputs is proportional to the total amount of light detected by the PMTs and hence related to the energy of the emitted particle. An amplifier amplifies the summed signal further to allow processing by the subsequent stages. The multi channel analyser produces an energy spectrum of the detected counts and will be discussed in more detail below. 41

48 Coincidence Counting: As the name suggests, in coincidence counting, a count is only accepted when there is an event detected from each of two detectors within a predefined time window, i.e. the two events from the two detectors have to be in coincidence or have to coincide. This is illustrated in Figure 3 below: Counts PMT 1 Counts PMT 2 Coincidence Trigger Coincidence? N N N N N Figure 3: Illustration of coincidence counting. As shown in figure 3, a coincidence trigger output is only produced when there is a pulse within the timing window from both PMTs as indicated by the coincidence circuit output ( Coincidence Trigger ) and by the in the Coincidence? row. When there is a pulse from either PMT not matched by a pulse from the other PMT, then there is no output from the coincidence circuit, indicated by N in the Coincidence? row. A finite time has to be defined for the coincidence time window to make sure that all true coincidences are detected. This finite time window accounts for imperfect time resolution of the electronics, as well as the finite time taken for the light to travel from the point of emission to the photomultiplier tube. Thus, if pulses from the two photomultiplier tubes are detected within this time window, then they are considered to be in coincidence, even though their arrival time is slightly different, but still within the specified time range. Typical coincidence time windows used in liquid scintillation counters are about Why Count in Coincidence? τ = 15 ns (15 x 10-9 s). Coincidence counting is one of the most effective ways for reducing noise and background in liquid scintillation counting. Because light is emitted in all directions when the beta or alpha particle interacts with the liquid scintillator, light will be detected by both photomultiplier tubes simultaneously and hence produce a coincidence event. In contrast, pulses from the PMT due to electronic noise will occur randomly in each PMT and hence there is only a very low likelihood that electronic noise will produce a coincidence event. Hence it is a very effective way of eliminating the background due to electronic noise. 42

49 There is, however, a finite probability that two noise pulses produce a coincidence event. The probability is a function of the coincidence time window τ and the rate of the noise pulses (R n ) and is given by R r = (2τ)R n 2 Where R r is coincidence count rate produced by the noise, usually referred to as random or accidental coincidence rate. As an example, if Then Noise count rate R n = 1000 cps Time window τ = 15 ns (15 x 10-9 s) Random coincidence rate R r = 0.03 cps Reducing the background noise count rate due to electronic noise to 0.003% of that which would have been observed without coincidence counting. This clearly illustrates the vital part coincidence counting plays in background noise reduction in liquid scintillation counting. A noise count rate of 1000 cps from each PMT might at first seem excessive. However, due to the small number of photoelectric emissions from the cathode of the PMT from the low energy of the beta particles, the signal is very small, requiring the threshold to be set quite low, which gives rise to the relatively high counting rate from spontaneous thermal emissions and other electronic noise sources. Thus, without the use of coincidence detection, it would be very difficult to count these low activity, low energy tracers. Multi Channel Analyser: Particulate emission, such as beta particles, give rise to a continuous spectrum of energies up to the maximum energy for the radioisotope being measured. Hence a simple pulse height analyser as used in scintillation probes detailed in the Module 2, Unit 4a Instrumentation Part 1, Scintillation Counter is insufficient for the energy discrimination requirements in liquid scintillation counting. In addition, as discussed in Part 3 of this unit, the energy spectrum changes due to changes in efficiency of energy transfer to the liquid scintillator and light detection caused by the sample composition, which is referred to as quenching. Hence it is important to collect the full energy spectrum of the sample being counted. The multi channel analyser (MCA) is used to generate the energy spectrum. It is basically an analogue to digital converter with memory which analyses the height of the incoming summed signal from the PMTs and increments the appropriate memory location based on the height of the incoming signal. For example: As an example consider an incoming summed signal which can vary from 0V to 10V. The MCA analogue to digital converter can produce output values from 0 to 9 i.e. an incoming voltage between 0 and 1V would produce an output of 0, between 1 and 2V an output of 1 and so 43

50 on. The memory required to store this coarse spectrum would require 10 bins (bins 0-9). Let us look at a sequence of incoming pulses, the corresponding digital output signal and the bin number incremented and the total number of counts in each bin as follows: Summed Signal Size MCA Digital Output Value Memory Bin Incremented Total Counts in the Bin being Incremented 5.2V V V V V V V V V V Thus at the end of collecting 10 counts, bin 5 contains 3 counts, bin 3 contains 2 counts, bins 0, 4, 6, 7 and 8 contain one count each and the remaining bins (bins 1, 2 and 9) contain zero counts each. If the number of counts in each bin are plotted versus bin number a simple energy spectrum shown in Figure 4 would be produced. Number of Counts in Bin Number Figure 4: Simple spectrum produced by example detailed in text In practice, MCAs have typically several hundreds to thousands of bins and many more than 10 counts are collected, providing a smooth spectrum similar to that shown in Figure 5. To only collect coincidence counts, the conversion and storing of counts in the bins is triggered by the output from the coincidence circuit as shown in Figure 2. 44

51 Relative Number of counts 3H 14 C Figure 5: Typical energy spectra produced by MCA for 14 C and 3 H. Background and Noise Reduction Techniques: As mentioned above, coincidence counting is the main technique used to reduce background. An additional approach used to reduce electronic noise and keep the output from the PMTs stable is to refrigerate the detectors to keep them at constant temperature of typically -10 o C. Keeping the PMTs at a low temperature reduces particularly spontaneous thermal emissions from the photo cathode. There are also other important techniques used to minimise noise and background. Energy Window: As shown in figure 5, the maximum energy for 3 H is much less than for 14 C, so different energy windows should be used to only encompass the energies expected for the particular radioisotope being counted. Thus for 3 H, the upper energy window cut-off would typically be set much lower than for 14 C, as any counts detected above the maximum 3 H energy would be noise. Counting Vial: Selection of counting vial also requires careful consideration. Normal glass contains potassium (K) including naturally occurring radioactive potassium ( 40 K) which can increase the background count by as much as cpm when counting 3 H or 14 C. Thus, either low potassium content glass vials have to be used or if the liquid scintillation fluid permits, polyethylene vials. Polyethylene vials are well suited for dioxane based solvents, but should not be used for solutions containing toluene, since toluene interacts with polyethylene and can cause it to swell and distort, which can jam the sample changer. Other suitable materials for vials include quartz, but these are less frequently used. Suitable liquid scintillation vials for the range of available liquid scintillation cocktails are commercially available from a number of suppliers, and their specifications and details can be consulted to select the most appropriate vials for particular applications. 45

52 Phosphorescence Effect: Exposure of the vial with the liquid scintillator solution to bright light, including bright sunlight, will cause energy to be absorbed which produces background light emissions called phosphorescence, which may take hours to decay. Thus samples are often stored in a dark, refrigerated enclosure before counting to allow the phosphorescence to decay following sample preparation. This is referred to as dark adaptation of the samples. Go To your Workbook, Liquid Scintillation section and answer question 4-7 to assess your understanding of this section. Summary: Specific instrumentation is required for liquid scintillation counting and special techniques are used to reduce background counting. Using 2 photomultiplier tubes for the light detection allows coincidence counting which is fundamental to reducing background counts from noise from the electronics and photomultiplier tubes. Liquid scintillation counters also generally incorporate a multi channel analyser, which allows the detected energy spectrum to be stored and analysed for optimum counting. Minimising exposure of samples to light and keeping them refrigerated as well as the use of suitable counting vials also helps to minimise the background. Key Points: Basic components of the liquid scintillation counter. - light tight chamber for the radioactive sample - Two photomultiplier tubes convert light to electrical signal - A coincidence circuit determines if pulses occur at the same time in both PMTs and selects them. - selecting the coincidence counts within a window of limits reduces background counts due to noise signals. - A multi-channel analyser produces an energy spectrum of detected counts. Noise should be kept to a minimum to allow samples to be counted within a reasonable length of time. The type of vials needs consideration e.g. low potassium glass or polyethylene - depending on the cocktail ingredients. Samples should be stored in the dark and refrigerated before counting because of light induced phosphorescence, increasing background light emissions and thermic emissions. 46

53 Part 3 Quantification, Quench Correction and Sample Preparation Time Check: Allow about 1hr to complete the following section Introduction: It is often necessary to quantitatively know the amount of radioactivity in the sample. In liquid scintillation counting the activity in the sample is usually expressed in terms of disintegrations per minute (DPM) due to the low concentrations of activities. This compares with disintegrations per second, which is the definition of the Bq activity unit ie. 1 Bq is one disintegration per second. For light to be detected by the photomultiplier tubes, a number of processes have to take place as illustrated in Figure 6. Radioactive Atom Solvent Molecule Primary Scintillator Secondary Scintillator Light λ Light λ 1 2 Chemical Quench Colour Quench Figure 6: Process Production of Light and Quenching in Energy Transfer The energy from the particle has to be transferred to the scintillators via the solvent molecule and the light has to then travel through the sample to be detected. The efficiency with which these energy transfers occur depends on the sample composition. The loss of efficiency is referred to as quenching. Chemical quenching refers to loss of efficiency in transferring the energy from the emitted particle to the primary scintillator, Colour quenching refers to the absorption of some of the emitted light in the sample before it reaches the PMT. The net effects of quenching result in a decreased efficiency of counting as well as a shift in energy spectrum of the sample as shown in Figure 7. To obtain accurate quantitative results, the amount of quench occurring in a sample has to be estimated and corrected. 47

54 Counts Unquenched Quenched Energy Figure 7: Quench Corrections Effect of quenching on Energy Spectrum There are a number of different techniques which can be used to estimate the amount of quench occurring in a sample and hence estimate correction factors to provide quantitative results. These include: 1. Internal standardization method 2. Channel ratio method 3. Automatic external standardisation method Internal Standardisation Method: With this method, the sample is first counted. After the initial count has been completed, a known amount of activity from a calibrated standard solution is added to the sample and the sample is counted again. The counting efficiency is then determined from the counts with and without the standard present as follows: Efficiency = (cps(standard+sample)-cps(sample))/(standard Activity (Bq)) Where cps(standard+sample) is the count rate obtained after adding the standard to the sample, cps(sample) is the count rate obtained from the sample alone before adding the standard and (Standard Activity (Bq) is the amount of standard activity added. The sample activity is then given by Sample(Bq) = cps(sample)/efficiency. The main disadvantages of this method are that the sample has to be counted twice (with and without the standard), an accurate standard solution has to be available and addition of the standard can change the sample composition and hence the quenching. The latter can cause loss of accuracy in the correction. 48

55 Channel Ratio Method: This method takes advantage of the fact that the energy spectrum changes (shifts) with increasing amount of quenching as shown in Figure 8. Counts Channel 1 Channel 2 Unquenched Quenched Energy Figure 8: Illustration of the channel ratio method Two energy channels are used and counts are obtained in each channel. Channel 1 encompasses the whole expected energy range for an unquenched sample, while channel 2 covers a lower section of the energy spectrum range (Figure 8). As quenching shifts the energy spectrum, the ratio of counts in the two channels will change. i.e. the ratio of Channel 2 to Channel 1 counts will increase as more and more quenching shifts the energy spectrum lower and lower. A quench correction curve is established by adding increasing amounts of quenching agent to a sample with known activity and the channel ratio is plotted as a function of efficiency for each amount of quenching present. For the unknown sample, the channel ratio is determined and from the channel ratio, the amount of quenching and counting efficiency is read off the previously established calibration curve and the counts from the sample are corrected by the efficiency determined from the quench calibration curve. The main disadvantage of this technique is that at very low counting rates, the counting statistical errors can cause quite large errors and uncertainty in the estimated channel ratio and hence the correction factor. In addition for very low energy beta emitters, such as 3 H, the shift in energy spectrum as a function of amount of quench can be quite subtle, again leading to large uncertainties in estimating the appropriate correction factors from the quench calibration curve. Also, it can not be used when two radioisotopes (eg. 3 H and 14 C) are counted simultaneously in the same sample. 49

56 Automatic External Standardization Method: This method incorporates features of both internal standardization method and channel ratio method by employing an external gamma emitting radioactive source. The sample is first counted with the external source shielded. It is then recounted (usually for only a short time e.g. 1 min) with the sample irradiated by the external gamma emitting source such as 137 Cs or 133 Ba. The gamma rays undergo Compton interactions within the sample, which produce recoil electrons, which then interact with the scintillator similar to the beta particles emitted by the sample being measured. Again, a channel ratio calibration method is established which relates the channel ratio of the counts from the external source to the amount of quenching present in the sample and hence the counting efficiency. The main advantage of the external standard method is that it provides high counting statistics for the channel ratio method, as it does not rely on the radioactivity contained in the sample. It also overcomes the limitations of the channel ratio method for very low energy beta emitters such as 3 H. While it corrects for both chemical and colour quenching, it does not correct for losses due to beta particle self-absorption or losses due to sample distribution effects. Correction Techniques Commercially Available: Commercially available liquid scintillation counters incorporate the above described quench correction techniques and come factory equipped with external standardisation source as well as pre calibrated quench curves for radioisotopes commonly counted on liquid scintillation counters. The exact implementations of the quench corrections are substantially more sophisticated than detailed above to ensure maximum accuracy and reliability. More details can be found in the technical product descriptions on the web sites of liquid scintillation counter manufacturers (eg. which is particularly informative). Sample Preparation Proper sample preparation is an essential aspect of liquid scintillation counting. Care has to be taken in the selection of the scintillation cocktail: 1. To ensure that the radioactive sample is thoroughly dissolved and dispersed in the liquid scintillation fluid. 2. The amount of quenching introduced by the sample should also be minimised which may for example require bleaching of the sample to reduce colour quenching effects. Due to the wide variety of samples used and preparation techniques, these techniques are not discussed here in detail. 3. However, it should be emphasised that if intending to perform liquid scintillation counting of a sample, appropriate references should be looked up to determine the most appropriate liquid scintillation cocktails and sample preparations. Again application notes found on company web sites provide a good starting point for applicable information. 50

57 The substances used in some liquid scintillation cocktails, such as toluene, are toxic and care needs to be taken that skin is not contaminated by these chemicals, their fumes are not inhaled and that they are not ingested. Appropriate protective clothing has thus to be worn and adequate ventilation must be provided, such as dispensing the solution in a fume cupboard. Disposal of liquid scintillation vial after counting must also be carefully considered. In general, the amount of radioactivity contained in the sample is negligible and hence the main concern is the toxic nature of some of the solutions used. Thus the chemicals must be disposed in accordance with toxic liquid chemical waste disposal procedures in place for your institution. Go To your Workbook, Liquid Scintillation section and answer question 8-11 to assess your understanding of this section. Summary: Quantitative measurements are frequently required for liquid scintillation counting. For quantitative measurement, the efficiency loss in counting, called quenching, needs to be determined for each sample and then a correction factor needs to be applied to the counts to correct for the loss of counts due to quenching. Quenching can be measured in several ways, the most commonly employed being the automatic external standardisation method, which uses analysis of the energy spectrum when the sample is irradiated by an external gamma emitting source to determine the amount of quenching. Key Points: Quenching refers to the loss of efficiency during the number of processes in the energy transfers - chemical quench: efficiency loss from the emitted particle to the primary scintillator - colour quench: efficiency loss during the transfer of light emission to the PMT For quantification of results a correction factor is required to compensate for quenching. Proper sample preparation is required 1. dissolved and dispersed 2. quenching minimized 3. check references for appropriate procedures for cocktails and sample preparations. Due to the toxic nature of some of the solutions, the chemicals must be disposed in accordance with toxic liquid chemical waste disposal procedures in place for your institution. Generally, the amount of radioactivity contained in the sample is negligible. 51

58 Glossary Cocktail Coincidence Counting Compton Interaction Fluor Molecule Phosphorescence Photocathode Photoelectric Emissions Quench Scintillation Self-absorption A mixture of different liquid substances. In liquid scintillation counting the cocktail mixture consists of the solvent to dissolve the radioactive substance, the scintillator or fluor molecule, which converts the particle energy to light, and the secondary scintillator or wave shifter, which changes the wavelength of the light suitable for detection by the photomultiplier tubes. Coincidence counting refers to the counting technique where a valid count is only registered if it is detected by 2 photomultiplier tubes within a set time period or time window. In Compton interaction, a gamma ray looses part of its energy by ejecting an orbital electron from its shell. The gamma ray will then have reduced energy and travel in a different direction. For liquid scintillation counting, Compton interactions produce electrons which behave similar in the cocktail to the beta particles emitted by the radioactive substance to be counted. Also sometimes referred to as primary scintillator, the fluor molecule converts the energy from the radioactive emission into light photons. Some materials, when stimulated by light or heat, will continue to loose the imparted energy by emitting light. This is referred to as phosphorescence. The photocathode is part of the photomultiplier tube. When hit by light photons, the photocathode with emit electrons, which are then amplified by the other stages in the photomultiplier tube to produce an electrical signal. The emission of electrons from the Photocathode in response to being hit by light photons is called photoelectric emission. Term used to describe the losses in efficiency in the liquid scintillation cocktail of converting the energy from the radioactive emissions to light photons which can be detected by the photomultiplier tubes. Scintillation is the emission of light by a substance in response to having absorbed some energy from eg a beta particle or gamma ray. Some of the radioactive emissions will be absorbed by the material they are in, without transferring their energy to the solvent molecule and hence without producing a scintillation. This is referred to as self-absorption. 52

59 Solvent Molecule Thermal Emissions Time Window Toluene Wave Shifter Xylene The molecule which is used to dissolve the radioactive substance and which transfers the energy from the radioactive emission to the primary scintillator Emissions of electrons from the photocathode of the photomultiplier tube due to thermal (temperature/heat) energy rather than light photons. Can also refer to light emissions from the sample due to its temperature which is referred to as phosphorescence. A small set period of time, used for coincidence counting. If counts are detected by two PMTs in the set time window, then they are said to be in coincidence. A toxic chemical substance/solvent which is well suited as the solvent molecule for liquid scintillation counting. A chemical substance which can change the wavelength of light. In liquid scintillation counting it is used to change the wavelength of the light emitted by the primary scintillator to a wavelength better suited for detection by photomultiplier tubes. A toxic chemical substance/solvent which is well suited as the solvent molecule for liquid scintillation counting. 53