Journal of Radioanalytical Chemistry, Vol. 46 (1978] 57-66

SOME PROBLEMS OF RADIOIMMUNOASSAY CONTROL M. Z/~VADA,* K. ~AFAR(~IK,* O. TOPOL~AN** *Department o f Nuclear Medicine, Regional Hospital, Ostrava (Czechoslovakia) **Medical University Clinic, Plzei~, (Czechoslovakia}

(Received April I0, 1978) Problems and aims of RIA control are discussed and the survey of the major characteristics applied to the control is presented. An arrangement and organization of RIA control system which enable the objective and rational control on different levels are proposed.

Introduction The recent development of radioimmunoassays (RIA) is characterized by a strong growth ~ of the range of assayed substances and by an increased number of appli -~ cations in routine clinical practice and the quantity of determinations. These methods are commonly used not only in research institutes but also in routineclinical laboratories provided with insufficient equipment and, very often, with an insufficiently trained staff. Large numbers of assay procedures, many commercial reagents and complete assay kits of very different qualities are used. On the other hand, there are increased requirements as to the reliability of observing minute changes of the substance concentrations 2 assayed by these methods. The main aim of any analytical method is to perform the assay with maximum accuracy and precision but with minimum expenses. 3 In the case of radioimmunological methods, the situation is rather complicated in consequence of a variety of circumstances. We wish to point out the main ones: (1) The basis of RIA is a considerably variable immunochemical reaction requiring the standard performance of assays under optimum conditions; (2) Heterogeneity of the most important components of the reaction mixture~ especially of antiserum and assayed samples, develops the danger of erroneous resuits due to cross reactions and non-specific processes and the problem of the specificity of immunoassay arises; (3) High demands on sensitivity connected with low concentrations of the assayed substances; (4) The high requirements for the level of experimental performance, and the correct calculation and estimation of the results. J. Radioanal. Chem. 46 (1978)

57

M. ZAVADA et al.: SOME PROBLEMSOF RADIOIMMUNOASSAY The large number of factors which may falsify the results of radioimmunoanalysis requires a realistic and critical attitude towards the results of assays and quality control of RIA. 4 The concept of quality control means a complex of experience, experimental results and parameters, on the basis of which conclusions may be drawn as to the accuracy, precision and reliability of the results obtained by the given analytical procedure, s'6 In regard of th6 pretentious and complicated control of radioimmunoassays, one of the actual problems in this field is working out an objective and rational control system which would permit: (1) The selection of the optimal procedures in view of accuracy and precision of the results, and the needs and possibilities of the users; (2) The selection of manufacturer's product for RIA in accordance with the demands presented under (1); (3) The effective control of the performance of routine assays including the reliability of the results; (4) The unification and standardization of RIA which may enable the specialization of laboratories and utilization of automation. RIA control has been discussed in a number of publications. 4'7-1~ The pioneers of these methods dealt, first of all, with the problems of specificity and sensitivity. Another extreme case is a method characterized by parameters of its precision only, without any respect to the accuracy of the obtained results. At present, great attention is paid to the problems of RIA control as demonstrated by the last International Symposium on RIA in Berlin. These problems, although in general terms, are solved even in some documents of international organizations. 11-12 One may agree with WALKER's demand 13 that quality control should be an integral part of each radioimmunoassay, which is not always carried out in practice.

Survey of parameters Many parameters, procedures and data, the use of which is recommended for the quality control of RIA have been described. The meaning and determination of each characteristic will not be discussed in this paper as has been done in former publications. 14, is The summary of the most important characteristics, the observation of which is, in our opinion, necessary for ensuring effective control is shown in Table 1. Classification into six groups will permit orientation in the large of characteristics although in some cases it may seem to be formal only. Table 1A presents the major characteristics used for the control of fundamental substances, viz. a specific antiserum, s'6-1a substances used as concentration standards 12'19'2~ and the labelled substance used as a radioactive tracer, s'21-23 Their 58

J. Radioanal. Chem. 46 (1978}

M. Zti.VADA et al.: SOME PROBLEMS OF RADIOIMMUNOASSAY

Table 1 Summary of parameters, procedures and experiments used for RIA control on different levels of the control system. The symbol + means necessity of the systematic observation on the given level of control; - means only periodical observation or control after the change of the procedure or in using a new substance; without symbol - no control. The symbol in brackets means observation according to the possibilities Control level Local

Central

Routine

Between laboratories

(-)

(+)

No. Selectire

Guarantee

Introductory

(A) Control o f basic substances

(+) (+)

1 Antiserum - equilibrium constant 2.Antiserum - binding capacity 3 Antiserum - specificity (cross-reactivity data) 4 Antiserum - dilution curve, working titer 5 Standard - calibration with national standard

(+)

6 Standard - calibration with international standard

+

7 Labelled - specific radioactivity substance

+

8 Labelled - specific radioactivity by selfdisplacement

+

(--)

(+)

+

9 Labelled - contents of free radionuclide 10 Labelled - radiochemical purity

+

+

11 Labelled - immunoreactivity

+

+

(B) Control o f other substances 12 Quality and dilution o f second antibody

+

13 Quality of sorbents

+

14 Quality o f biological substances

+

15 Buffers

+

(C) Control o f RIA - common parameters +

16 Total activity o f sample

+

17 Mass of labelled substance in sample

+

18 Binding ability - percent ratio of counts for zero standard to total counts

+

+

19 Non-specific binding - percent ratio

+

+

J. Radioanal. Chem. 46 (1978)

59

M. ZAVADA et al.: SOME PROBLEMS OF RADIOIMMUNOASSAY Table 1 (cont.) Control level Local

Central

No.

Selectire

Guaran-

Intro-

tee

ductory

R outine

Between

laboratories

21 50% Intercept

+ q-

+

22 Control samples (serum pool samples)

+

+

23 Between-assay variability

+

24 Sensitivity - calculated

+

20 Shape of dose-response curve

(D) Control o f RIA - special procedures 25 Sensitivity - experimental

+

26 Experimental precision index of assay

+

27 Within-assay variability 28 Precision and confidence limits for different concentrations

+

29 Recovery experiments 30 Parallelism of dose-response curves

+

31 Specificity of RIA 32 Normal values of healthy individuals 33 Values of pathological individuals

+ + + +

34 Reliability

(E] Comparison o f results 35 Pathophysiologieal state of patients 36 References of scientific literature 37 Other methods of assay 38 Between laboratories

(F) Control o f assay techniques 39 Pipetting accuracy 40 Pipetting reproducibility 41 Absolute efficiency of radioactivity measurement 42 Reproducibility of radioactivity measurement 43 Stability of radioactivity measurement

+

44 Processing and calculation o f results

60

J. Radioanal. Chem. 46 (1978)

M. Z.~VADA et al.: SOME PROBLEMSOF RADIOIMMUNOASSAY qualities influence the choice of assay conditions, its specificity and sensitivity as well as the accuracy of the results. The proper specification of manufacturer's products should be given by their producers. 12 Many characteristics of these substances depend on the RIA design 12 and the procedure used. Radioimmunoanalysis is a delicate method and it is necessary to pay attention to quality control of other substances used 12,14 as is shown in Table lB. The purity of additional biological substances (e.g. albumin, antigen-free serum, animal sera, etc.) is also important, especially from the point of view of their contamination by substances causing cross reactions and non-specific effects. In this connection we wish to point out the necessity of maintaining optimal and standard conditions when collecting, preparing and storing samples for analyses as well as antigenfree and control sera. 11'14'16 The following two parts of Table 1 present parameters characterizing the given radioimmunological system. The parameters presented in Table 1C may be obtained from the results of usual routine assay without any special procedures and, first of all, they are criteria for the optimal conditions of the assay under which it was carried out. The binding ability (No. 18) and non-specific binding (No. 19) 4,8,24 inform us about the immunochemical reaction of the given assay. The next two parameters (Nos 20 and 21) are connected with the shape of the dose-response curve and, along with sensitivity, determine the range of concentrations for which the given assay is applicable. 8-~~ The control by means of suitable control samples (No. 22) of a known concentration o f the assayed substance (for example serum pool samples) 7'11'26 is the basis for estimating the accuracy of the results of a given assay and for the calculation of between-assay variability of this method. 4,7,26 The parameters shown in Table 1D characterize, first of all, the accuracy and reliability of the method and it is necessary to use special experimental procedures and estimation methods for their assignments. Parameters Nos 26-28 characterize the precision and within-assay variability; they are significant for eliminating erroneous results 4,7'1~ and depend on the technical performance of the analysis. The next three characteristics are of fundamental consequence for estimating specificity of the assay and accuracy of its results. 1~176 Normal and pathological values26 should be determined in the individual laboratory under the given conditions. Observing the reliability of routine assays by means of the suitable parameters 4's'7'26 is also very important. Fig. 1 illustrates the results of three months' observation of RIA thyroxine as an example of the observation of radioimmunoassay reliability. On the left side of Fig. 1, the observed parameters are shown; viz. percent ratio of binding ability (Bo/T) and non-specific binding (Z/T), calculated sensitivity, 50% intercept (Is o) J. Radioanal. Chem. 46 (1978)

61

M. ZAVADA et al.: SOME PROBLEMS OF RADIOIMMUNOASSAY

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I t I ~ I 26 28 30 NO. ofexperiment

Fig. 1. The results of three months observing RIA thyroxine

as a characteristic of the shape of dose-response curve, residual scatter (R.R.)'as a parameter of the assay precision and thyroxine level for three control pool sera collected from patients with hyperfunctional (SH), eufonctional (SE) and hypofunctional (SN) state of thyroid. The solid lines in the middle show the mean values of the observed parameters, broken lines show the 95% confidence limits. The results of experiment No. 21 were out of control. For the complex estimation of the given method the results should be compared (as shown in Table 1E) first of all with the pathological state of the patients and with similar data reported in the literature. By comparing the results with those of other methods, both radioimmunological l~ and others, t information of great 62

J. Radioanal. Chem. 46 (1978)

M. ZAVADA et al.: SOME PROBLEMS OF RADIOIMMUNOASSAY significance may be obtained. Comparison of results between laboratories 11'26'29 is also very valuable. The precision and reproducibility of the results depend on the level of assay performance, especially on the experimental technique and the experience of the staff. The major problems of RIA are the exact pipetting of a large number of small solution volumes s,l~ and the measurement of the radioactivity of the final samples 7'11 (Table 1F). The choice of the suitable processing method and calculation of the results 3~ is also very significant. The conclusion about the correctness of the results obtained by the use of control methodics may be inferred only from the complex assessment of the characteristics presented in Table 1'. 12,14'1s In conclusion of this survey, we wish to point out the significance of the individual parameters for RIA control, which is in some way, dependent on the characteristics of the assayed substances and sampies as well as on the applied radioimmunological procedure. The problems of RIA control are rather complicated and laborious. The extent and level of RIA control depend on a great number of circumstances; they will be different in the case of introducing a new method from those of usual and routine assay control and from those performed with complete manufacturer's kits or by methods worked out in the individual laboratory. They are also dependent on the approach of each experimenter and on the level of each laboratory. Obviously, it is neither possible nor useful to carry out RIA control to the full extent in a routine laboratory. It would be favourable, therefore, to assume such a system of RIA control which would solve the problems as a whole, including the recommendations for the optimal parameters and the extent of control in accordance with the real needs on the given level. 14

System o f control ~ s

In our opinion, two fundamental types of control should exist: Central Control which, being very laborious, should be performed by top laboratories, having wider, for instance national influence, and Local Control which, being much simpler, should be performed in the individual laboratories in a routine way. Each of them should exist in two forms assigned to verify quality as well as to chose newly introduced methods (selective and introductory), and for the current and lohg-run control as well as observing reliability (guarantee and routine). Further, it would be useful to carry out comparative measurements between laboratories regarding the possibilities of wider assessment of the remits of a certain assay for the purpose of unification of the procedures and results. J. Radioanal. Chem. 46 H978)

63

M. ZAVADAet al.: SOME PROBLEMS OF RADIOIMMUNOASSAY At each of the 44 parameters and procedures in Table 1, there are shown the distinctions defining their applications on the given control level. The task of Central Selective Control is an all-round verification of the methodics, kits or substances, which are supposed to be in large-scale use. The Central Selective Control along with manufacturer's information and economic points of view is the basis for the optimal decision with respect to their establishment into practice. This control, at least of several deliveries or lots, should be performed in parallel by several laboratories and the results should be assessed by mean of a general discussion by various laboratory workers. Central Guarantee Control should ensure the current and long-ran control of the individual control of deliveries and lots. It should be performed at least by one laboratory of high reputation, with respect to the quality claim of the producer and with warning to all users. The task of Local Introductory Control is to verify the quality of newly introduced methods in individual laboratories through the major parameters of control, gathering normal and pathological values in the given laboratories and comparing them with similar data presented in the literature. The aim of this control is to judge the convenience of the assayed methodics under the given conditions in the individual laboratory. Local Routine Control is assigned to observing long-run reliability of routine assay results in the individual laboratory by the use of 14 current parameters and control samples. It is very simple and does not require any special procedures and in substance it is identical with observing long-run RIA reliability. Furthermore, the shape of the dose-response curve (No. 20) is examined, the results are compared with the clinical state of patients as well as with the data presented in the literature, and the control of the stability of radioactivity measurements as well as processing and calculation are carried out. If central control for a certain RIA does not exist, local control must be extended in the corresponding way. The proposed system is favourable not only for the control of manufacturer's kits but also for other modifications of radioimmunoanalyses performed on the basis of proper or manufacturer's substances. The control and specification of manufacturer's substances and kits performed by their producers is defined in great detail in a WHO exports' report 12 and it is desirable for it to be applied in practice. The documentation of the precision and accuracy of RIA used in the publications is also very important. It should include, in our opinion, data on sensitivity, within-assay and between-assay variability, recovery, parallelism of dose-response curves, specificity, normal and pathological values and their comparison.

64

J. Radioanal. Chem. 46 (1978)

M. ZAVADA et al.: SOME PROBLEMS OF RADIOIMMUNOASSAY Application in

practice

The application o f the proposed RIA control in practice will not be easy in this country. The choice o f RIA methodics and substances is strongly dependent on the arbitrariness o f the individual laboratory staff. In Czechoslovakia there is no Central National Laboratory, which would deal with RIA control and no National Assay Service for Hormones, as is recommended by WHO. 12 The central control and coordination o f the activities related to RIA of a certain substance or a group of substances should be committed to a laboratory provided with the suitable equipment and an experienced staff. This coordination should include both RIA control problems (preparing national standards, preparing control samples, local control instructions, between-laboratory comparing measurements) and RIA optimization and unification, establishing a non-kits method, service for other laboratories, specialization of laboratories, etc. Obviously, the load o f these selected laboratories would increase but the reciprocity of the services would provide a certain compensation for them. At present, the complete control must be performed by each laboratory separately, which is neither realistic nor economical. The establishment of this RIA control system would contribute to ensuring, correct results for clinical practice, RIA development as well as a much more economical and rational utilization o f expenses for the purpose.

References 1.J. J, PRATT, M. G. WOLDRING, J. Radioanal. Chem., 35 (1977) 45. 2.J. WOO, D. C. CANNON, Am. J. Clin. Pathol., 65 (1976) 854. 3.R.P. EKINS, Proc. Symp. Radioimmunoassay and Related Procedures in Medicine, Istanbul, 1973, Vol. I, p. 91. 4.D. RODBARD, P. L. RAYFORD, J. A. COOPER, J. Clin. Endocrin. Met., 28 (1968) 1412. 5. L. P. WHITBY, F. S. MITCHELL, D. W. MOOS, Adyant. Clin. Chem., 10 (1967) 65. 6. M. ZAVADA, XIV. Dny nukle~rnl mediclny, Ostrava, 1974. 7.D. RODBARD, in Principles of Competitive Protein-Binding Assays, W.D. ODELL, W. H. DAUGHADAY (Eds), Philadelphia, 1972, p. 204. 8.S.A. BERSON, R. S. YALOW, Clin. Chim. Acta, 22 (1963) 51. 9.R. ~. EKINS, G.B. NEWMAN,Acta Endocrin. (KBH) Suppl., 142 (1970) I1. 10.A. ~R. MIDGLEY, G. N. NISWENDER, R.W: REBAR, Acta Endocrin. Suppl., 142 (1969) 163. ll.Repo(t of an IAEA Panel on Standardization of Radioimmunoassay Procedures, Intern. J. Appl. Radiation Isotopes, 25 (1974) 145. 12.WHO Expert Comittee on Biological Standardization, 26th Report, Geneva, 1975. 13.W.H.C. WALKER, Clin. Chem., 23 (1977) 384. 14.M. ZAVADA, O. TOPOL~AN, V. KARLI~EK, Metody kontroly radioimunologichk~,ch stanoveni, Souhrnn~ zpr~va 6. 04-SZ-01, UISJP, 1977. J. RadioanaL Chem. 46 (1978) 5

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M. ZAVADA et al.: SOME PROBLEMS OF RADIOIMMUNOASSAY 15.M. Z/tVADA, K. SAFARCIK, O. TOPOL~AN, Metody kontroly radioimunologickych stanovenf, Souhrnn~ zpr~va & 04-SZ-02, UISPJ, 1978. 16.S.J. GOLDSMITH, Sem. in Nucl. Med., 5 (1975) 125. 17.W.D. ODELL, G. A. ABRAHAM, W. R. SKOWSKY, in Principles of Competitive ProteinBinding Assays, W. D. ODELL, W. H. DAUGHADAY(Eds), Philadelphia, 1972, p. 57. 18.J.J. PRATT, W. KOOPS, M. G. WOLDRING,Eur. J. Nucl. Med., 1 (1976) 37. 19.D.R. BANGHAM, P. M. COTES, in Radioimmunoassay Methods, K. E. KIRKHAM, W. M. HUNTER (Eds), Edinburgh, 1971, p. 163. 20.D.R. BANGHAM,Acta Endocrin., 71 (1972) 625. 21. R. S. YALOW, S. A. BERSON, in Principles of Competitive Protein-Binding Assays, W. D. ODELL, W. H. DAUGHADAY(Eds), Philadelphia, 1972, p. 1. 22. R. A. CARO, V. A. CISCATO, S. M. V. DE GIACOMINI, Intern. J. Appl. Radiation Isotopes, 26 (1975) 527. 23.L. RAO CHERVU, D. R. K. MURTY, Sem. in Nucl. Med., 5 (1975) 157. 24.J; D. M. ALBANO, R. P. EKINS, G. MARITZ, Acta Endocrin., 70 (1972) 487. 25.H. FELDMAN, D~ RODBARD, in Principles of Competitive Protein-Binding Assays, W. D. ODELL, W. H. DAUGHADAY(Eds), Philadelphia, 1972, p. 158. 26.P. M; WEIGAND,J. Nucl. Med. Technol., 3 (1975) 154. 27.E. ROLLERI, R. MALVANO, in Recent Progress in Reproductive Endocrinology, P. G. CROSIGNANI, V. H. T. JAMES (Eds), London, 1974, p. 113. 28.R. MALVANO, G. G. ZUCCHELLI,T. QUESADA, J. Nucl. BioL Med., 18 (1974) 80. 29.A. CONSTANTINI,O. LOSTIA, R. MALVANO, J. NucL BioL Med., 19 (!975) 164. 30.V. ULLMAN,M. ZAVADA, ~as. L6k. ~es., 114 (1975) 1392.

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