H.H. Dale and the London Centre for WHO standards--some milestones of the early years.

Henry Dale, a Nobel laureate and statesman of science, helped to organize the meetings in 1923 and 1925 to set up international standards for insulin and other biologicals. He made the National Institute for Medical Research one of the two world centres for standards. Some milestones in the work of the London centre are described: (i) the first standards; (ii) vitamins and hormones; (iii) WHO standards for many antibiotics; (iv) the provision of an international working standard for ACTH; (v) the old and new methods of ampouling; (vi) the impact of research and immunoassay on the need for standards; and (vii) the special ECBS sessions for endocrinology and haematology. Dale tells how he had to preserve the first batch of insulin for use as the standard instead of for treatment.

Basic features of biomedical assays.

All biomedical assays have certain aspects in common, the most striking of which is that they generally involve protein-binding. Ligand-ligator interactions and the resulting response form the basis of most assays. This paper reviews the advantages and limitations of different types of assays ranging from long-term bioassays to in vitro assays using cell-free components. Consideration of these aspects helps in the understanding of the different assays, selection of appropriate assays for different applications, and the better interpretation of assay results. Clinical chemists, hematologists, endocrinologists, immunologists, and scientists from other disciplines have each developed their own measurement methods and units. This paper seeks to identify those aspects and principles common to assays in all such disciplines, concentrating on protein-binding systems. Understanding what and how different assays measure may clarify why different assay methods often give different results.

Assays for human growth hormones.

Human growth hormone is, in effect, defined by its activity in an in vivo bioassay and the standard used with it, growth being measured as the increase in body weight in hypophysectomised immature rats. The assay reflects the hormone's survival and metabolism in vivo, its cell-cell interactions, the activation and effects of its secondary hormones, such as GF1 and GF2, and various feedback mechanisms. Although it is insensitive, imprecise, easily influenced by contaminants TSH and vasopressin, it is the only practical assay that reflects all the in vivo properties of "hGH". The in vivo tibial epiphysis bioassay is more sensitive and precise, but the response reflects only the elongation of bone. Both these bioassays are well established. By contrast, in vitro receptor assays do not reflect in vivo properties; there may be different natural forms of receptor molecules, they may be altered during their extraction, and the measured response (like those of immunoassays) is not relevant to the biological action of the hormone. The validity of a bioassay depends on the use of a suitable standard. The collaborative study of the International Standard for human growth hormone (in 1984) revealed marked disparities between results with different assay methods. When a growth hormone protein (such as somatotropin, 191 amino acids) is produced in quantity, reproducibly, and with physicochemical properties consistently related to in vivo bioassay results, it may then be reasonable to use physico-chemical tests for control purposes. Many such tests require international reference materials for comparison purposes.

World Health Organization International Standards for highly purified human, porcine and bovine insulins.

The 4th International Standard (IS) for Insulin, established in 1958, consists of a mixture of relatively impure bovine and porcine insulins and is not suitable as a standard for the assay of highly purified single-species insulins presently used in the treatment of diabetes. Preparations of human, bovine and porcine crystalline insulins, representative of current highly purified therapeutic insulins, have now been studied in an international collaborative study carried out by twenty-three laboratories in fifteen countries. In the collaborative study described here, each of the three preparations was found to be suitable for use as a standard for insulin for bioassay and each was established by WHO in 1986 as an international standard. The 4th IS of Insulin bovine/porcine (code numbered 58/6) has been discontinued. Insulin preparations should now be calibrated in terms of International Units defined by the standard for the appropriate species: the International Standard for Insulin, Human, the International Standard for Insulin, Bovine, or the International Standard for Insulin, Porcine.

The International Standard for Human Growth Hormone for Bioassay: calibration and characterization by international collaborative study.

Two preparations of human growth hormone (hGH) were prepared as candidates for the International Standard for Human Growth Hormone for Bioassay and were studied by 22 laboratories in 10 countries in an international collaborative study. The 2 candidate preparations, freeze-dried in ampoules coded 80/505 and 80/521, were assayed against the International Standard for Growth Hormone, bovine, for Bioassay (ISbGH), by in vivo assays; against the International Reference Preparation of Growth Hormone, human, for Immunoassay (IRP hGH), by receptor-, immunoassays and other in vitro methods; and against each other by various methods. Both preparations contained the 2 recognized main growth hormone components (22 kDa and 20 kDa forms) and other components, but that in ampoules coded 80/505 had less deamidated hGH and contaminant hormones and pyrogen than that in ampoules coded 80/521. The estimates by in vivo bioassays, using immature hypophysectomized rats, of the potency of 80/505 in terms of the IS bGH were heterogeneous (1-6 IU/ampoule), probably because of the dissimilarity of the preparations of bovine and human GH. Estimates with receptor- and immunoassays of 80/505 against the IRP hGH were also heterogeneous (3-9 IU/ampoule). Nevertheless, the majority of estimates from all assays tended to be between 3 and 6 IU/ampoule. Although 80/521 and 80/505 differed in relatively minor respects, assays of one directly against the other gave relative potency estimates with in vivo assays which were significantly different from estimates with in vitro methods. With the agreement of the participants in the study, the WHO Expert Committee on Biological Standardization established the preparation in ampoules coded 80/505 as the First International Standard for Human Growth Hormone for Bioassay, with the defined potency of 4.4 IU/ampoule. This corresponds to an approximate 2.5 IU/mg of hGH extract and maintains reasonable continuity of the unit.

Interim report of results from an international collaborative study of the Proposed International Reference Preparation of porcine pancreatic kininogenase.

A collaborative study of the ampouled preparation of porcine pancreatic kininogenase, the Proposed International Reference Preparation (PIRP), was carried out, on behalf of the World Health Organization, by 19 laboratories in seven countries. The PIRP was studied using in vivo bioassays, immunoassays and in vitro enzyme assays involving natural protein substrate and small molecular ester and peptide substrates. An analysis of results obtained from a majority of the participating laboratories is presented. Comparison of the PIRP with the widely used Bayer house standard of porcine pancreatic kininogenase showed general agreement for estimates of relative activity, although differences were detected in some assay systems. The PIRP was shown to be stable and appears to be suitable to serve as an international reference preparation. Comparison of the PIRP with a preparation of highly purified human urinary kininogenase showed a wide diversity of estimates so that no general comparison of activities for these preparations is possible.

Use of a common standard for comparison of insulin C-peptide measurements by different laboratories.

A synthetic human C-peptide analogue has been used as a common standard for the comparison of insulin C-peptide measurements in seven assay systems in six laboratories. Even in terms of this common standard there was statistically significant numerical heterogeneity between laboratories for estimates of the C-peptide content of the same plasma samples. However, the consistency in ranking order of estimates of C-peptide in the plasma samples between laboratories suggests that laboratories are in most cases measuring at least similar immunoreactive constituents and that a reference plasma might prove useful in comparing results between laboratories. Until a more suitable reference material is available, the synthetic analogue, 64 formyllysine C-peptide, in ampoules coded 76/561, will be made available for research purposes.

International collaborative study of assay of anti-D (anti-Rh) immunoglobulin.

An international collaborative study of anti-D assays has been carried out by 21 laboratories in 11 countries. Samples of anti-D immunoglobulin assayed in this study included two dilutions of a preparation used in clinical trials to determine a dose-protection relation, a national standard, commercial clinical preparations and the proposed international reference preparation in coded ampoules. Manual, automated haemagglutination and isotope labelling methods all gave similar relative potencies. Several of these estimates were significantly (P=0.95) heterogeneous and some modifications to improve assay design and procedure are suggested. The coded preparation was shown to be stable and suitable for comparative assays. It was estimated to contain 60 microgram of of anti-D IgG immunoglobulin per ampoule, and 150 i.u./ampoule when assayed against the International Standard for Incomplete Anti-D Blood Typing Serum. Thus for this preparation 1 microgram of IgG anti-D immunoglobulin identical to 2.5 i.u. anti-D antibody. At its 28th meeting the Expert Committee on Biological Standardization of WHO established the preparation 68/419 as the International Reference Preparation of Anti-D Immunoglobulin and assigned to it a potency of 150 i.u. per ampoule. The Preparation has been widely used (with a nominal content of 60 microgram of IgG anti-D immunoglobulin) for control of clinical preparations.

The first international standard for human urinary FSH and for human urinary LH (ICSH), for bioassay.

This paper describes the preparation and nature of the First International Standard for Human Urinary Follicle Stimulating Hormone and for Human Urinary Luteinizing Hormone, for Bioassay, and of two batches of working standard which were prepared from the same material. A collaborative study of these materials was carried out by six laboratories in six different countries. The FSH and LH activities of the Standard were assayed in terms of those of the Second International Reference Preparation of Human Menopausal Gonadotrophins, Urinary, for Bioassay, which it replaces. The results from 20 valid FSH assays and 30 valid LH assays (using four different methods) obtained in this way gave a weighted combined potency estimate for FSH of 53.7 IU, with 95 % fiducial limits of 47.2-61.1 IU, and for LH of 46.2 IU, with 95 % fiducial limits of 43.3-49.3 IU. Accelerated degradation studies of the Standard stored at elevated temperatures suggested that the stability of both FSH and LH activities under normal storage conditions would be satisfactory. The FSH and LH activities of the two batches of working standard (WS-A and WS-B) were compared with those of the Standard and were not found to differ significantly, except for the LH activity of WS-B which appeared to be slightly higher than that of the Standard. Accelerated degradation studies did not show any significant differences in stability between the Standard and batches of working standards. On the basis of these results the Standard has been established by WHO and allocated a potency for FSH of 54 IU per ampoule and for LH 46 IU per ampoule. The International Units for FSH, Human Urinary, for Bioassay and for LH (ICSH), Human Urinary, for Bioassay are thus defined as the activities contained in 0.1138 mg and 0.13369 mg of the International Standard, respectively.

Four heparin preparations: anti-Xa potentiating effect of heparin after subcutaneous injection.

Four different heparin preparations--sodium and calcium salts of the same batch of heparin (mean molecular weight 15,000), low molecular weight sodium heparin (mean m.w. 9,000) and high molecular weight sodium heparin (mean m.w. 22,000) were injected subcutaneously on different days each into 6 healthy young volunteers in a randomized trial. Plasma heparin levels were measured using the anti-Xa assay at 1 hour, 3-4 hours and 6-7 hours after the injection. The highest anti-Xa potentiating effect was obtained after the injection of the low molecular weight sodium heparin (mean 0.381 i.u./ml) at 3-4 hours after the injection. With sodium heparin (m.w. 15,000) the highest values (0.135 i.u./ml) were found at 1 hour. Significantly lower anti-Xa potentiating effect was obtained 1 hour after the injection of calcium heparin and in particular after the injection of high molecular weight heparin (mean values 0.072 i. u./ml and 0.043 i. u./ml respectively). Both these preparations showed an increase from 1 hour after injection to 3-4 hours after injection (mean values 0.082 i. u./ml and 0.057 i. u./ml at 3-4 hours after injection). These results indicate that the salt and the molecular weight of the preparation may strongly influence the degree of anticoagulation achieved after subcutaneous injection.

Stability of the 4th International Standard for Insulin.

The stability of the (W.H.O.) 4th International Standard for Insulin, has been assessed by accelerated thermal degradation studies. This is a crystalline preparation of insulin, freed from proteolytic enzymes, sealed in ampoules containing air and with a moisture content of 5--6%. Of the original biological activity 95.8 (92.8--98.9;P = 0.95)% was retained after storage for 12 years in the dark at 20 degrees C and 65.7 (63.4--68.1;P = 0.95)% after 14 years at 37 degrees C. Degradation rate constants were calculated from these data for the Standard at 20 degrees C and 37 degrees C, assuming first order kinetics. The degradation constant at 37 degrees C did not differ significantly from those found in earlier degradation studies at 37 degrees C over shorter periods, thereby supporting the assumption that the degradation of crystalline insulin, at least at 37 degrees C, is a first order reaction. Extrapolation of these data suggest that the Standard stored at -20 degrees C for 20 years would have retained at least 99.93% (P = 0.95) of its original activity and so for practical purposes can be considered to be stable.

Standard for human thrombin.

Ten laboratories took part in an international study of the proposed standard for human thrombin. A freeze-dried preparation (coded 70/157) was shown, using clotting time tests, to be suitable to serve as a standard for the assay of human and bovine thrombin preparations. The study shows that assay variation within laboratories was much less than between laboratories. The preparation was stable, having a negligible estimated loss of potency after ten years under the recommended storage conditions (--20 degrees C in the dark). Each ampoule of the standard was assigned a potency of 100 units of thrombin activity, and one unit was defined as the activity contained in 0.0853 mg of the freeze-dried preparation. It was agreed by the participants in the study and by the International Committee on Thrombosis and Haemostasis (Vienna, 1973) that the thrombin preparation coded 70/157 is suitable to serve as an international standard for human thrombin.