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WORLD HEALTH ORGANIZATION ORGANISATION MONDIALE DE LA SANTE
vrn:o/Bs/so.12s1 ENGLISH ONLY
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EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15-22 April 1980 INTERNATIONAL COLLABORATIVE STUDY OF FOUR CANDIDATE REFERENCE PREPARATIONS FOR TF.E ANTIGENIC AND ENZYMATIC MEASUREMENT OF HUMAN SERUM COMPLEMENT COMPONENTS by 1 . 2 3 4 5 L. van Es, S. J.Smith, P.H. Schur, G.Hauptman, W. Leskovar, 8 0 P. Spath, 6 G. Fust, 7 P . Lachmann, U. Rother, 9 R. A.Thompson,1 T. B. L. Kirkwoodll ABSTRACT Scientists in seven countries evaluated antigenic and haemolytic human serum complement in four freeze-dried pools of human sera which were prepared in three The nations as candidate international or national reference preparations. complement components measured for antigenic activity were Clq, C3, C4, CS, and Total functional complement was also measured. One collaborator factor B. Candidate preparations measured the functional activity for C4, CS, and factor B. were evaluated by using single radial immunodiffusion, nephelometry, and haemolytic assay methods. Criteria for accepting each candidate as a possible reference preparation included linearity of response-relative concentration curves, and validity with slope-ratio or parallel-line assay techniques when evaluated versus the other candidates. Appropriate transformation of the analytical response and relative concentration variables gave linear curves that were analytically suitable for all four candidate preparations except that one preparation yielded considerably lower activity for total functional complement than the other preparations. 1 2 3 4 5 6
Central Laboratory for Blood Transfusion, Amsterdam, The Netherlands, Statistical Activities, Center for Disease Control, Atlanta, Georgia 30333. P.H. Schur, Robert B. Brigham Hospital, Boston, M.A. 02120, United States of America. G. Hauptman, Centre de Transfusion Sanguine de Strasbourg, Strasbourg, France. W. Leskovar, Kli nisches Laboratorium, ·Graz A8036 , Austria. P, Spath, Med. Abteilung des Landeskrankenhauses, Graz, Austria.
G. Fust, International Institute of Haematology and Blood Transfusion, Budapest, Hungary. Lachmann, Laboratory of Molecular Biology, The Medical School, Cambridge CB2 2QH, United Kingdom. 9 U. Rother, Institut fur Immunologie der Universitat, 69 Heidelberg, Federal Republic of Germany. 10 R. A. Thompson, Regional Immunology Laboratory, East Birmingham Hospital, Birmingham BY55ST, United Kingdom. ll T. B. L. Kirkwood, National Institute for Biological Standards and Control, London NW3 GRB, United Kingdom. The issue of this document does not constitute formal publication. It should not be reviewed, abstracted or quoted without the agreement of the World Health Organization. Authors alone are responsible for views expressed in signed articles. Ce document ne constitue pas une publication. II ne doit faire l'objet d'aucun compte rendu ou resume ni d'aucune citation sans l'autorisation de !'Organisation Mondiale de la Sante. Les opinions exprimees dans les articles signes n'engagent que Jeurs auteurs.
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wHo/Bs/so.12s1 page 2 The candidate international reference preparation (preparation 4) was shown to be suitable as a standard, since its thermal stability and analytic suitability (comparability with other preparations) have been proved satisfactory. Relative potencies for the individual complement components in the other three preparations were obtained in terms of preparation 4, and these may serve to calibrate them for use as national reference preparations. Variability among collaborators was found to account for the greater part (657.) of the total variability in relative potency estimation. The coefficient of variation of the mean relative potency averages approximately 10% for individual complement components. On the basis of these findings, preparation 4 is to be submitted to the World Health Organization (WHO) Expert CDllmittee on Biological Standardization for consideration as the WHO Reference Preparation for Human Serum Complement. If it is agreed that this is a suitable preparation it would be appropriate to assign a unitage of 100 International Units per ampoule for each of the analytes studied and in which there had not been an assigned unitage previously.
INTRODUCTION The availability of stable international reference preparations for human serum proteins, the imm.unoglobulins and a number of hormones and antibodies has increased markedly over the past several years (Reimer, Smith et al., 1978). Although collaborative results for one complement component (C3) have been published (Reimer, Smith et al., 1978), a study specifically for the measurement of several complement components in freeze-dried candidate reference sera has not been reported. In order to determine if suitable reference preparations for complement components can be made available, the International Union of Immunological Societies (IUIS) Complement Standardization Subcommittee and the Biometrics Subcommittee have organized a multinational study of four processed, freeze-dried human serum pools. The general objective of the study was to determine by several immunological methods the analytic suitability of each candidate preparation for the measurement of human serum complement components and to compare the results obtained by several collaborators. Experimental data are presented that relate to (1) the effect of dilution of each candidate preparation upon the analytical response variable for specific complement components with particular attention to linearity of response-relative concentration curves, assay validity for slope-ratio and parallel-line assay, and response variance over the relative concentration (RC) or dilution range of interest; (2) the estimation of the antigenic and haemolytic (functional) relative potency of those proteins of interest in the candidate preparations that are analytically suitable; (3) an examination of the antigenic and functional thermal stability of the four candidate preparations; (4) a recommendation of preparation 4 to the World Health Organization (WHO) Expert Committee on Biological Standardization as the proposed WHO reference preparation for calibrating the concentration of the human complement components analysed. An International Unit(I.UJwould then be assigned to this standard for each complement component evaluated that had not already been assigned an international unitage; (5) assigningl.U.to complement proteins in the other preparations that are analytically suitable. Subgoal (2) was restricted to antigenic activity and whole complement activity, because too few data were obtained for fun~tional activity. The results of a pilot study on functional activity are given in Appendix 1. A form was circulated to the invited participants to elicit information on the methods that they routinely used to assay complement components. On the basis of the replies, a detailed protocol was developed and sent to each participant in advance of the samples. Results were received from eight participants in seven countries. These participants are listed as authors and they are identified only by code letters which bear no relation to the order in which they are listed.
WHO/Bs/80.1281 page 3 MATERIALS Candidate preparations Preparation No. 1 (Netherlands standard for serum proteins), Serum was obtained from the blood of 30 unpaid donors between the ages of 18 and 60 years. Each donor supplied 500 ml . To prevent the collection of activated plasmin, 10 ml of blood was taken initially. The 500 ml glass bottles were stored for four hours at 37°C. Serum and clotted cells were then separated by centrifugation. The serum was cooled to 4°C and subsequently mi xed with cold trichlorotrifluorethane (Freon 113) so that the 500 ml glass bottles were nearly filled. The mixtures were intensively stirred for 30 minutes at 4°C, and the serum and Freon were separated by centrifugation. The delipidated serum was finally centrifuged for 30 minutes at SO 000 g to remove a small amount of gelatinous material. The 30 samples were stored for 36 hours at 4°C awaiting the results of the tests for HBs antigen. No antigen was found and the sera were pooled to yield a lot of about 6000 ml. Glass vials were filled with 1 ml of Residual serum and ~reeze-dried as one lot. The vials were sealed by glass fusion. moisture (Karl Fisher method) found in three sealed vials was 0,1%. The mean dry weight of the contents of 10 vials was 93.9 mg and the range was 92.6-95.4 mg.
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Preparation No. 2 UUIS Lot 3 - (United States) Center for Disease Control (CDC) Reference Preparation for Human Serum Proteins (CDC 120575C)). Preparation of this material is described in detail as No. 3 in Reimer, Smith et al. (1978, p. 135). Approximately 6000 borosilicate vaccine vials (1.5 ml/vial) were prepared and freeze-dried as one lot at the (United States) CDC and then capped with natural rubber stoppers while still under vacuum. This preparation was found to be slightly more potent than the proposed WHO International Reference Preparation for six human serum proteins studied in Reimer, Smith et al. (1978). It was found to be less stable, however, than candidate preparations sealed in all-glass ampoules when measured after exposure to elevated temperatures. Pre aration No. 3 (National Institute for Biological Standards and Control (NIBSC) preparation 74 522). The source material for this preparation is described as preparation No. 1 in Reimer, Smith et al. (1978, p. 135). This description applies down to the twelfth line of the paragraph, ending in "were sent at 4°C to NIBSC". Two litres of material were mixed with 500 ml of Richardson's additive sterilized by millipore filtration. The bulk was frozen on liquid nitrogen and then placed at 4°C. Thawing was completed at 20°C under water. The whole bulk was distributed into ampoules in 0.5 ml amounts. During the filling procedure, 66 ampoules were test weighed: the mean weight of liquid contents was 0.575 gm (coefficient of variation= 0.17%). The complete lot of filled vials was freeze-dried and secondarily dried to constant weight in a single operation. The ampoules were filled with pure dry nitrogen, sealed by fusion of glass, and stored at -20°C in the dark. The mean dry weight of the contents of six vials was 69 . 1 mg and the range was 68.9-69.2 mg. Preparation No. 4 (IUIS Lot 5). Preparation of this material is described in detail as No. 5 in Reimer, Smith .et al. (1978, p. 136). All glass ampoules (1.3 ml/ampoule) were filled with serum, freeze-dried, and sealed at the Central Laboratory of the Blood Transfusion Service (CLB) in Amsterdam. This preparation was found to be approximately equipotent to the WHO International Preparation for six human serum proteins studied in Reimer, Smith et al. (1978). Thermal stability was not found to differ from the other sealed-glass ampoule preparations included in that study. Handling and reconstitution of candidate preparations All preparations were kept frozen at -30"C at the CLB and mailed in containers with dry ice. Collaborators were asked to store materials at -20"C or below until use and to reconstitute each ampoule with 1 ml of distilled water. Five ampoules of each candidate preparation were mailed in a single package and each ampoule was marked preparation 1, 2, 3, or 4, according to the above description. No attempt was made to conceal the identity of the candidate preparations since the glass ampoules from three countries were of different shapes and sizes, recognizable to some collaborators.
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l WHO/Bs/so.12s1 page 4 METHODS Assay design Each participant was asked to analyse all four complement preparations concurrently by reconstituting a single vial of each and preparing two aliquots from which separate serial dilutions were to be made. A minimum of three, and preferably five, dilutions were to be made from each aliquot and each of these was to be tested in duplicate for each complement component. The testing of all four preparations in this manner by one assay method for one complement component was considered to be a single assay. Participants were asked to complete one run per day consisting of a single assay of each complement component by one assay method, Each participant was asked to complete three such runs for each component measured. In practice, most participants were able to complete a run as defined above in a single day, but a few required a second day. They used their own antisera. Participants were free to estimate the linear portion of the response curves using excess material and to choose their own dilution ranges within the linear response region. Dilution ranges were not suggested to the participants in advance. The dilution ranges that • were used varied from fourfold to tenfold for the antigenic assays and from approximately 1.7-fold for CHSO (functional haemolytic total complement) to a maximum of eightfotd for the haemolytic assays of individual components. All participants used at least three dilutions and most used four or five. Assay methods Most participants performed both antigenic (single radial imnunodiffusion (SRID); nephelometryl) assays and functional (whole haemolytic) assays. The methods used by each participant are shown in Table 1. Seven collaborators performed antigenic assays, four analysed CH50. Collaborator F measured the individual haemolytic activity for factor B, C4, and CS. The response variables for haemolytic, SRID, and nephelometric assays are the fraction of total cells haemolysed, the reaction diameter and the peak height, respectively. Two collaborators reported their source of antiserum as sheep; the remainder reported a rabbit serum source, Collaborator F reported mouse and guinea-pig . serum sources. Statistical methods All data were sent to the (United States) Center for Disease Control for statistical analysis. Antigenic SRID data were analysed as slope-ratio assays, whereas all other assays were analysed as parallel-line assays (Finney, 1978). In all cases, preparation 4 was regarded as the standard, and potency ratios for preparations 1, 2 and 3 were calculated with respect to this standard. Appropriate mathematical transformations of the basic assay response-relative concentration variables were used to achieve linearity. These response transformations are shown in Table 2, For assays to be accepted as statistically valid, it was necessary that the slope-ratio assays conform with the two basic requirements: (i) linearity of response and relative concentration curves and (ii) equal intercepts for zero complement concentration; for parallel-line assay, however, the corresponding requirements were: (i) linearity and (ii) parallelism. To assess linearity, the data were inspected graphically, but constancy of slope or intercept between preparations for each participant were assessed by analysis of variance between assays for parallel-line or slope-rati~ assays, respectively. No attempt was made to assess the validity of individual assays of aliquots by analysis of variance since it was thought that no reliance could be placed on the estimates of residual error obtained from duplicate responses. Estimation of relative potency from valid assays was made in the usual way, except that weighted rather than unweighted regression was used for the SRID assays because there was clear evidence of response-variable heteroscedasticity (heterogeneity of response precision over the relative concentration range of interest): i.e., dependence of response variance on
WHO/Bs/80.1281 page 5 response magnitude. For these assays, an iterative weighting procedure (Williams, 1959; Rodbard, 1976) was used whereby: (i) an initial unweighted regression was fitted, (ii) the inverse variances of the estimated responses were used to weight the observed responses, and (iii) this procedure was iterated to convergence. The weights were the inverses of the squared reaction diameters multiplied by a constant. The potency ratio estimates for the individual assays from each participant were combined for each component of each preparation by taking the unweighted geometric mean. Likewise, overall mean potency ratio estimates for each component of each preparation were combined by taking an overall unweighted geometric mean. In each case, appropriate confidence limits to these geometric means were calculated. RESULTS Assay validity Graphical inspection of the data confirmed that satisfactory linearity was achieved by use of the transformations listed in Table 2, with the exception of the nephelometric assays for C3 by participant H which were rejected as invalid. The mean coefficient of determination is .983 for .456 standard curves from slope-ratio assays and .968 for 119 standard curves from parallel-line assays. No activity was detected for preparation 3 in the haemolytic assays for total complement activity (participants A, B, E, G) or in two cases for antigenic assays (participant G, component CA; participant H, component CS); potency ratios were not estimated in these cases. Collaborator D measured weak whole functional activity in preparation 3. Analysis of variance to test equality of intercepts among the four preparations for the slope ratio assays showed nine instances of significant differences at the 1% probability level for the 25 cases tested. However, these differences were small and it was decided to accept the assays as valid for this study. Similar tests for equal slopes for parallelline assays showed no significant differences at the 1% probability level for 10 cases . The mean coefficient of variation (CV) among slopes estimated per run was 4% for the CHSO assays and 14% for the individual complement component assays done by collaborator F. The mean between-duplicate response CV is given in Table 3 for all participants and These CVs are highly collaborator-dependent, but they do not appear complement components. to differ substantially among the complement components analysed. The mean between-duplicate response CVs are 4% and 7% for antigenic (slope-ratio) and functional (parallel line) assays, respectively. Relative potency The geometric mean relative potency (RP) and the 95% upper and lower confidence limits are given in Table 4. Preparation No. 1 is about 70-97% as antigenically potent as preparation No. 4 for the components measured according to the observed results. The antigenic potency of preparation No. 2 is about 90-110% of preparation No. 4 for these components, whereas the antigenic potency of preparation No. 3 is about one-third that of preparation No. 4. Table 4 indicates that preparation Nos. 1 and 2 are about 74-95 7- as potent as preparation No. 4 for total functional complement, respectively. Collaborator F did functional assays for CA, CS, and factor B, individually, and the results are reported in Table 4. The overall mean fraction of within-collaborator log RP variability to the total log RP variability is 35% for all components including CHSO measured by two or more collaborators. This result indicates that most of the log RP variability was among collaborators, rather than within collaborator variability. The mean arithmetic CV of the within collaborator RP is 12% for all collaborators and it is largest for Clq and smallest for factor B. The arithmetic CV of the geometric mean RP averages 11% for CHSO and the components measured by
WHo/Bs/80.1281 page 6 two or more collaborators. Its mean value is smallest for C4 (8%) and largest for Clq (11%). Its mean value for C3 is 9%, which may be compared to 5% obtained by Reimer, Smith et al, (1978) in a study with nine collaborators, five candidate standards, with an average of about 18 determinations per candidate standard. The overall mean among collaborators CV of the RP is 18%. Th.is CV varies ~rom Oto 40% and it does not appear to be related to the preparation number or complement component. TI-.e individual collaborator geometric mean RPs for preparations 1, 2, and 3 are given in Tables 5, 6, and 7, respectively, for all complement components analysed. These tables indicate the degree of among-collaborators RP variability. Certain collaborators tended to obtain relatively larger or smaller RPs for all complement components that they measured, compared to the other results. The range of the geometric mean RPs is the largest for preparation No. 1 and smallest for preparation No. 3. Thermal stability The stability of the antigenic activity of preparation No. 4 has been previously evaluated in a thermal degradation study for human serum proteins including C3 (preparation No. 5 in Reimer & Smith, 1978, p. 149). It was reported that this preparation was stable when subjected to moderate thermal treatment and little thermal degradation was observed when more drastic thermal treatment was applied. Using SRID, Collaborator B measured the antigenic activity of the four candidate preparations in samples stored for three months and 12 months at 2o•c and 37°C. No loss of potency was detected for Clq, C3, C4, CS and factor Bon the basis of measurements from these thermally elevated samples. A three-collaborators study on the functional and antigenic stability of about one dozen individual complement components was also done. These results are summarized in Appendix 1. DISCUSSION For practical reasons only a limited number of the 15 components of the complement system are routinely quantitated in the average laboratory, This limitation is due to the low concentration of some components and lack of suitable antisera. Levels of C3, C4, CS, Clq, factor Band whole functional activity are valuable tools for the clinician, in particular when follow-up studies are done. Though elevated levels are more frequent than reduced levels, the former observation is not specifically related to a pathologic situation. In contrast, reduced levels are often characteristic for a disease (Whicher, J, T. (1978) and Editorial 1976, Complement activation and disease, Brit. med. J., 21 February 416). For a proper estimation of abnormal patterns, a reliable benchmark for normal values is required. As complement is a system of very specifically collaborating proteins with an enzymatic function, quantitation of functional and of antigenic activity is possible. For reasons of simplicity, antigenic quantitation is used more frequently than functional activity quantitation. Only a few laboratories have the skill for testing functional potency of individual components. The IUIS Subcommittee's principal task was the preparation of a standard sertnn for the antigenic quantitation of those components which are most useful for diagnosis and whole complement functional activity. The Subcommittee agreed to study the possibilities for functional standardization as well. Two important factors had to be considered before preparing standard materials. First, spontaneous activation during sampling and further treatment must be prevented, and second, a pool of sera must be obtained from the blood of many donors because of wide limits of individual component concentration among normal donors. The ranges of normal serum concentration values for Clq, C3, C4, CS and whole complement activity were found as 69-128%, 66-124%, 37-161%, 73-135% and 68-133% respectively from a standard pool of 250 healthy donors collected at the Central Laboratory of the Blood Transfusion Service in Amsterdam where the pool concentration was defined as 100%.
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WHO/Bs/80.1281 page 7 Most of the assays done for this study have been SRID assays because nephelometric assay is only suitable for the relatively highly concentrated proteins C3, C4, and factor B. Measurements obtained by the eight collaborators indicate that the four preparations analysed are acceptable for comparative analytical dilution assays of the antigenic complement components studied. Preparations 1, 2, and 4 are suitable for assaying total functional complement. The geometric mean RPs of Table 4 are the best available comparisons of the potency of the four candidate preparations as obtained by several independent collaborators using two assay methods. We believe that the observed among-collaborators RP differences are chiefly due to the use of different antisera and to differences in methodological procedures. The results for C4 and factor B from a single collaborator (G) using nephelometry appear to be consistent with the results from those using SRID. Although the among-collaborators RP variability exceeds the within-collaborator variability obtained in this study, Table 4 indicates that the RP means are determined with an overall imprecision (CV) of about 10%. We propose that candidate preparation 4 be recommended as the international standard for the components studied herein, except for C3 that has been calibrated in a preceding study (Reimer, Smith et al. 1978). The concentration of preparation 4 should be defined as 100 I.U./ampoule. Upon acceptance of preparation 4 by the WHO Expert Committee on Biological Standardization, l.U. valuesshould be assigned to preparations 1, 2, and 3 according to the Table 4 relative potencies. We also recommend the assignment of I.U. for whole functional complement activity (CH50). In pilot studies among five collaborators, it was demonstrated that more precise agreement was obtained when functional activity was related to the common preparation included (Van Es (1978)). We encourage preparation of specialized local reference materials. Normal ranges, however, of individual components may differ markedly among local populations so that local normal ranges must be determined. We advise the use of antisera from several sources for the calibration of local standards because complement component reaction products made with antisera from different producers may differ substantially. We also recommend that interassay variability be carefully considered and that a quality control procedure be used. Reimer, Smith et al. (1978) reviewed the problems encountered when individual variation is related to variation in a population. They concluded that meaningful variations in an individual are sometimes so small that the usual immunodiagnostic methods are not sufficiently precise. We believe that data for the diagnosis of complement-related problems will be improved by the use of a stable reference material. Major reagent suppliers should have direct access to the proposed international complement standard (preparation 4) according to the agreement between WHO and the IUIS Standardization Committee. Requests for secondary _standards should be addressed to CDC (Atlanta), NIBSC (London), or CLB (Amsterdam). None of the preparations should be used as a reference in routine assays but in setting up and checking in-house or laboratory standards at appropriate intervals. We believe that the supply of preparation 4 is sufficient for at least 10 years, given this policy. We hope that suppliers of immunodiagnostics will increase and improve the standardization of their reagents by using several standards and reference preparations already available for many serum proteins. We do not believe the components in this study should be quantitated in mg per ml. In a study on C3 in commercial kits wherein the standard serum was calibrated with purified C3 protein, wide variations in four kits were found (Vladutlu, A. D. & Winiarsky, B. M., 1976). If quantitation is required, we reconunend that the investigator calibrate his preparation himself and use one specific antiserum. BRIEF SUMMARY The antigenic activity of five serum complement components and whole functional activity was measured in four freeze-dried pools of human sera as candidates for an international reference preparation in a multinational study. Collaborators used single-radial inununodiffusion, nephelometry, and haemolytic assay methods. Candidate preparations were evaluated
WHO/Bs/80.1281 page 8 for linearity of response-relative concentration curves, validity with slope-ratio on parallelline assay techniques, and potency. The selected candidate international preparation is to be submitted to the World Health Organization (WHO) Expert Counnittee on Biological Standardization for consideration as the WHO Reference Preparation for Human Serum Complement. Potency ratios for the complement components studied in each candidate preparation were obtained relative to the ' selected candidate international preparation. The three remaining candidate preparations will serve as national complement standards. ACKNOWLEDGEMENTS The dedicated assistance of many colleagues and friends helped in the realization of this project. We particularly thank the following: R. E. Ritts, jr, Chairman and Irene Batty, Secretary, International Union of Immunological Societies Standardization Committee; Anna-Britta Laurell, Bacteriological Institute,University of Lund; N. R. Cooper, Scripps Clinic and Research Foundation; R. M. Zarco, Cordis Laboratories, W. Opterkuch, Institute Medical Microbiology,Bochum; K. W. Poundman and A. Hannema, Central Laboratory of the Netherlands Red Cross Blood Transfusion Service. The financial support of the Netherlands Ministry of Health and Environmental Hygiene (grant R497a) and the facilities of the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service were indispensable for the fulfilment of the subcommittee's task. The collaboration of Waltraut Leskovar and P. Spath .wasmade possible by the support of the Austrian Fund for Advancement of Scientific Studies Project nr30fl. Drafts and final manuscript were typed by the Word Processing Facility of the CDC. REFERENCES Finney, D. J. (1978) Statistical Methods in Biological Assay, 3rd ed., London; Griffin The
Katamura, H. et al. (1977) The cold activation of the classical complement pathway. cause of the difference between plasma and serum complement in liver cirrhosis, Clin. Exp. Immunol., ~ 34 Kondo, M., Hosokana, K. & Masuda, M. (1976) Cold activation of complement. coagulation related activator, J. Immunol., 117, 486 1.
Presence of
Reimer, C. B. et al, (1978) Progress towards international reference standards for human serum proteins, Journal of Biological Standardization, §., 133-158 Rodbard, D. et al. (1976) Statistical characterization of the random errors in the radioimmunoassay dose-response variable, Clinical Chemistry,~ 350-358 Van Es, L. (1978) Critical evaluation of complement titration in relation to standardization. In: Opterkuch, W., Rother, K. & Schultz, D. R., eds, International Symposium, Clinical Aspects of the Complement System, Thieme Stuttgart Vladutlu, A. D. & Winiarsky, B. M. (1976) Complement C3 in serum and plasma as measured by radial immunodiffusion with four counnercial kits, Clinical Chemistry, 1£, 267-269 Whicher, J. T. (1978) The value of complement assays in clinical chemistry, Clinical Chemistry, 24, 7-22 Williams, E. J. (1959) Regression Analysis, New York, Wiley
WHO/Bs/ao .12a 1 page 9 TABLE 1. SUMMARY OF ANTIGENIC AND FUNCTIONAL ASSAYS BY COLLABORATOR
Complement component Collaborator Clq A B C3 Factor B C4
cs S(3,2) S (3, 2)
CHS~ H(3, 2) H(2, 2) H(3, 1) H(3, 2)
S(3,2)E. . S (3, 2) S(3,2) S(3,2) S (3, 2) S(3,2) S (3, 1) S (3, 2) S (3, 2) H(3, 2).£ N(2,1) S (3, 2) N(2,l) S (3, 2) S(2,2) S(3,1)
S(3,2) S(3,2) S (3, 1) S (3, 2) S (3, 2) H(3, 2).£ N(2, 1) S(3,2)
c D E F G
H(3, 2)-£ H(2, 1) S(3,2)
H
~ Functional assay.
£ Assay type Ls°=Single Radial Immunodiffusion; N=Nephelometry; H=Haemolyti£7 (number of vials, number of aliquots) . .£ Functional haemolytic assay for individual complement component.
TABLE 2.
RESPONSE TRANSFORMATIONS
Assay type SRID* Nephelometry Haemolytic (CHSO) Haemolytic (other) D= R= Y= Z
Response transformations Dz R logefj/ (1-YV
z
Reaction diameter. Peak height. Fraction of total cells haemolysed. Diameter of ring of lysis.
*
SRID = Single radial immunodiffusion.
WHO/Bs/ao.12s1 page 10
TABLE 3.
RESPONSE IMPRECISION
Collaborator
Mean coefficient of variation of replicate response Clq
Total degrees of freedom
A
6i.
B D
F H
3 4 11 4 C3
69 112 95 72
99
A B
c D
E G
5 2 8 2 6 3 Factor B
72 116 24 96 96 34
B
c G
F H
2 6 3 3 3 C4
79 36 36 96 108
A B
c D
E F G H
7 2 5 3 7 3 5 4
72
118 36 64 95 64 30 108
cs A B
F H
15 2 5 4 CHSO
72
120 96 81
A B
E F G
4 5 27 13 12
55 37 41 54 29
'"".:"'
GEOMETRIC MF.AN AND IMPRECISION OF RELATIVE POTENCY TABLE 4. VERSUS PREPARATION 4 FOR ANTIGENIC AND FUNCTIONAL ASSAYS Arithmetic CV of the geometric mean RP 17i. 9 8 6 8 12 7 11 14 9 6 9 14 8 8 12 9
' Among-collaborator arithmetic CV of the RP 40% 11 15 14 16 29 13
Complement component
Preparation No.
Total No. of estimates
No. of collaborators
Geometric mean relative potency (RP)
Lower 95i. confidence limit~
Upper 95% confidence limit!!.
Within-collaborator arithmetic CV of the RP 13% 18 11 7 9 8 6 7 8
Clq Clq Clq c3.S cJ.S c3.S Factor n Factor n Factor B C4 C4 C4
1 2 3 1 2 3 1 2 3 l 2 3 1 2 3 1 2d 31 2 3 1 2 3 1 2 3
26 27 27 28 28 28 12 12 12 JO 30 28 15 15 12 15 18
5 5 5 6
.82 1.11 .37 .96 1.10 .33 .94 1.14 .38 . 97 1.06 .33 . 72
. 71 1.02 .34 .91 1.03 .29 .86 1.00 .32 .89 1.01 .30 .62 .84 . 28 .66 .87
6 6 4 4 4 7 7 6 3 3 2 5 s
-
-
.95 1. 21 .39 1.02 1.18 .36 1.02 1. 29 .44 1.05 1.12 .36 .83 1.00 .33 .83 1.03
21 27 20 9 11 21
12 12 17 20 7 14 16 6
cs cs cs CHSO CHSO CHSO C4~ C4~ ~
.92 .30 .72 .95
17 0 15 18
4 4 4 6 6 6 6 6 6
1 1 1 1 1 1 1 1 1
.75 .95 .68 .63 1.03 . 37 .67 1.04 . 34
.62 . 71 .56 .58 .83 . 30 .58 .96 .29
.89 1. 27 .81 .69 1. 28 .46 .78 1.11 .40
-
-
-
-
8 13
-
11 19 11
8 s 13 13
cs~ cs~ cs~ e Factor nFactor~ Factor~
-
-
8 21 21 14 7 15
9 4 10
'O Pl ll" fl)
;,: .....____
:£! 0 t,l
~ Lower 95% confidence limit for true geometric mean RP.
,.... C/l
t
,.... .....____ ex,
Upper 95% confidence limit for true geometric mean RP.
£ Preparation 4 contains 116 r.u./ml of CJ relative to WHO International Standard for CJ.
i No detectable functional activity for CHSO. ~ Enzymatic assay done by collaborator F only.
,.... ex, '" ,....
0
WH0/Bs/80.12s1 page 12
TABLE 5.
INDIVIDUAL COLLABORATOR GEOMETRIC MEAN RELATIVE POTENCY PREPARATION NUMBER 1 VERSUS PREPARATION NUMBER 4
Complement Component Collaborator Clq A B
C3 1.12 .85 .87 .90 .94 1.29
Factor B
C4 1.16 .90 .87 1.00 .93 1.47 .66 .97
cs .64 .90
CHSO .60 .73
1.17 .98
.86 .87
c D E
1.03 .48
1.16 .99
-
.83 .93 .81
£ H
.58 .82
.59
.96
.74
Overall
GMRP£.
.94
• 72
~ Nephelometry was used for the antigenic assays by this collaborator.
~ Geometric Mean Relative Potency.
TABLE 6.
INDIVIDUAL COLLAEORATOR GEOMETRIC MEAN RELATIVE POTENCY PREPARATION NUMBER 2 VERSUS PREPARATION NUMBER 4
Complement Component Collaborator Clq A B
C3 1.18 1.19 .89 1.32 .92 .90
Factor B
C4 1.12 1.03 .99 1.13 1.04 .82 1.26
cs .83 l.09
CHSO .73 1.06
.92 1.11
LOS .99
c D
1.25 1.10
1.02 1.56
-
1.09 1.02 1.08
E
G~ H Overall GMRPQ.
1.34
.81
-
.95
1.11
1.10
1.14
1.06
.92
~ Nephelometry was used for the antigenic assays by this collaborator.
~ Geometric Mean Relative Potency.
WHO/Bs/Bo.1281 page 13 TABLE 7.
INDIVIDUAL COLLABORATOR GEOMETRIC MEAN RELATIVE POTENCY PREPARATION NUMBER 3 VERSUS PREPARATION NUMBER 4
Complement Component Collaborator Clq A
C3 .44 .23 .3 7 .40 .26 .35
Factor B
C4 .32 .29 .35 .39 .37
cs .31 .30
.41 .32
.32 ,3 6
B
c D
.38 .42
-
E
.32 .56
.30
~ H
.28
.2 7
-
Overall GMRP~
.3 7
.33
.38
.33
~ Nephelometry was used for the antigenic assay by this collaborator.
~ Geometric Mean Relative Potency
WHO/Bs/so.12s1 page 14 APPENDIX 1 A pilot study of complement functional activity and thermal stability of individual complement components was ~erformed. A 500 ml blood pool from 100 donors was collected at NBTS and allowed . to clot at 20 °C overnight. Clotting was allowed at 20°C because at 0°C a strong reduction of CHSO haemolytic activity was found. This reduction is possibly due to cold activation (Kondo, Hosokana & Masada (1976); Katamura, Nazaki, et al (1977)). The processed serum pool was aliquoted (1 ml per vial) lyophilized, and stored at -70°C. Samples were mailed in dry ice and measured at -70°C, -20°C and 20°C over a period of zero, three, and six months. The percentage activity of thermally elevated samples versus the samples stored at -70°C was determined. The individual collaborator results are shown in Tables 1-2. A statistical evaluation of the data was not done due . to the fact that only one estimate per time-temperature combination per laboratory was obtained. Thus, we were not able to evaluate within-laboratory precision. The Table 1 data indicate the relative stability of the components In general, the observed perCl-C9 at -20°c and 20°C as obtained by collaborators A and B. centage functional activity is lower at 20°C than at -20°c. Also, the observed percentage functional activity declines during the observed time period. Considerable reduction of functional activity for C3, C4 and CS, is demonstrated after 6 months. The results of collaborator C indicate a reduction in functional activity for C4 and CS, but the collaborator B results do not agree for C4. Whole complement activity is reduced upon storage at 20°C, but does not appear to be reduced at -20°c. Collaborator Bused SRID to evaluate the thermal stability of preparations 1-4 stored for 3 and 12 months at 20°C and 37°C. Complement components Clq, C3, C4, CS and Factor B were assayed. The relative potencies of these samples were evaluated versus samples stored at -20°C by slope-ratio assay and they are listed in Table 3. These samples maintained about two-thirds or more of their original poten~y after exposure at 37°C for 12 months. TABLE 1. PERCENTAGE ACTIVITY OF TIIERMALLY ELEVATED SAMPLES RELATIVE TO A SAMPLE STORED AT -70°C
Collaborator a AA A A A
Complement Component Cl C2 C3 C4 cs C6 C7 CB
Time of Storage
Storage Temperature
O months -2o·c 91.1% 95.0 85.7 99.5 87 .2 76.7 83. 7 75.8 98.6 79.5 83.0 135.0 62 .o 106.0
3 months -20°c 20°0 98.1% 94.8 76.7 86.4 83 .6 84.3 79.2 60.l 60.2 80.0 86.0 123 .o 62 .o 94.0 79 .2% 94.2 76.3 78.7 66.4 80.2 67.6 59.9 52 .4 75.0 89.0 116.0 62 .o 75.0
6 months -20°c 20°c 76.2% 80.0 75.7 67.3 91. 7 75.7 90.3 82 .8 83.9 81.6 93.0 140.0 58.0 86.0 68.8% 63.5 35 . 1 39.5 44.2 80.0 91.2 78 .9 90.3 57.9 84.0 95.0 52.0 62 .o
A A
A A A
C9 CHSO Clll c2b. c4b. CHSO
B B B B
~ Collaborator A measured the complement components after 10 days for the colunm labelled "O months".
£.
CH63 units.
WHO/Bs/so.12s1 page 15 Appendix 1 TABLE 2. PERCENTAGE ACTIVITY OF THERMALLY ELEVATED SAMPLES RELATIVE TO A SAMPLE STORED AT -70°C
Collaborator
Complement Component Cl.a CZ C3 C4 cs CB C9 CHSO
Time of Storage
Storage Temperature
3 months -zo•c 4700 h.u. 100% 100 91 89 95 78 94 zo·c
c c c c c c c c
.a.
34 000 h .u. .a. 73% 100 60 54 73 62 56
.a. -70°C data lost. Note: h.u.
= hemagglutination
units.
TABLE 3.
ESTIMATED RELATIVE POTENCY VERSUS SAMPLE STORED AT -20°c
Preparation Months Stored Storage Temperature 3
1 12 3
z. 12
3 3 3
4 12
l
-
-
-
-
--
-
-1.08 1.11
-
----1.01 .98
-Clq -
-- - -- - -- -- - -- -1.00 .91 .85 .66 1.06 1.03
zo·c 37°c zo·c 37°c
- - - - - - - - - - - - zo•c 37°c
-
-
--
.93 .96 - - C3
------
-
----- -1.00 .97 .95 .90
.97 .87
.97 .86
-
- - -
-- -
1.14 1.22
-
1.03 .98
- - -
-.90 .90 .90
.98 .87 - - C4 1.01 .94 - cs
-- ---- --1.06 1.21
.98 .65
.94 .85
-
-
- - - -
-
- -- - -- 1.00 1.03
.94 .94
---
.87
----.81 .74
- - -
---
- -
zo•c 37°c
- zo•c 37°c
-
-
-
---
- - -
-.99 1.04
.88 .93 .88 .90 - Factor B - -
---- -----
1.07 1.10
1.03 1.01
.BO - .79 .85
-
-
.95 .79
-
-
Not measured