An international standard for low molecular weight heparin.

An international collaborative study has been carried out with the aim of establishing an international standard for low molecular weight (LMW) heparin. Three preparations of LMW heparin were assayed against the International Standard for unfractionated heparin (UFH) by 25 laboratories in 13 countries, using nine different assay methods. The results confirmed previous findings of non-parallel assays, wide interlaboratory variability and differences between methods when LMW heparins are assayed against a UFH standard. Use of one of the LMW heparins as a standard for the other two gave parallel assays and much closer agreement between laboratories. The preparation in ampoules coded 85/600 was selected as likely to give the best agreement with the largest number of LMW heparins; potencies were assigned by taking the mean of all the anti-Xa assays, and the mean of the thrombin and APTT assays, to represent the two major groups of activities. Preparation 85/600 has been established by WHO as the 1st International Standard for LMW heparin, with potencies of 1,680 iu/ampoule by anti-Xa assays and 665 iu/ampoule by thrombin inhibition and APTT assays.

A collaborative study to establish the 2nd international standard for tissue plasminogen activator (t-PA).

An international collaborative study involving ten laboratories located in eight different countries was undertaken in order to replace the current International Standard (I.S.) for tissue plasminogen activator (t-PA). Two lyophilised candidate preparations of high purity were assessed in comparison with the current I.S. for t-PA using only a clot lysis assay. One preparation (coded 86/670) was purified from a cultured melanoma cell supernatant and was about 98% single chain t-PA while the other preparation (coded 86/624) was derived from Chinese hamster ovary (CHO) cells following DNA recombinant procedures and was 75% single chain t-PA. Both candidate preparations of t-PA compared in quite a satisfactory manner with the current I.S. from the viewpoint of the biometrics of parallel line bioassays and both preparations were quite stable for long periods at low temperatures and stable from up to 1 month at temperatures of 20 degrees and 38 degrees C. Both fulfil the criteria to serve as a satisfactory 2nd International Standard for t-PA. The Fibrinolysis Subcommittee of the International Committee for Thrombosis and Haemostasis recommended the melanoma source t-PA (86/670) as the next I.S. in order to maintain continuity with the 1st I.S. which was also a melanoma-type preparation. The data from the ten laboratories indicated that each ampoule of the new proposed standard contains 850 international units of t-PA activity by the clot lysis assay. It is planned to present the results of this study to the Expert Committee on Biological Standardization of the World Health Organization at its next meeting and to request that the preparation to t-PA, coded 86/670, be established as the 2nd International Standard for t-PA.

Bioavailability in rats of human recombinant tissue plasminogen activator after subcutaneous and intramuscular injection.

The bioavailability of human recombinant tissue plasminogen activator (rt-PA) in rats was measured after subcutaneous (s.c.) and intramuscular (i.m.) injection. Rt-PA was absorbed after both i.m. and s.c. injection, giving peak plasma concentrations within 30 min and 1 h, respectively, with detectable concentrations up to 6 h. These peak values of bioavailable t-PA were obtained in a functional fibrin plate assay of euglobulin precipitates and expressed as +88% and +243% (for s.c. and i.m. routes respectively) above basal rat fibrinolytic activity. Prior injection of rt-PA, s.c. or i.m., significantly reduced the weights of thrombi induced in the inferior vena cava after injection.

British standard for human serum vitamin B12.

Seven laboratories took part in a collaborative study of an ampouled preparation of normal serum labelled 81/563. Each laboratory calibrated the preparation in terms of pure cyanocobalamin by use of Euglena assay. The inclusion of pernicious anaemia serum in the study and additional tests for safety and stability indicated that 81/563 would be a suitable standard for diagnostic testing. In 1985 the National Biological Standards Board established the preparation as the British Standard for Human Serum Vitamin B12 and, with the agreement of participants in the study, assigned it a potency of 320 picograms per ampoule.

Low molecular weight heparin: a better drug?

Preliminary clinical trials suggest that at least some low molecular weight (LMW) heparins are as effective as heparin in preventing post-operative deep vein thrombosis, and need only be injected once a day. However, a firm basis does not exist for assigning potency estimates to different LMW heparins. Caution is therefore necessary in determining appropriate dosages for various clinical indications. Until the problems of standardization and dosage are resolved, LMW heparins are unlikely to achieve their potential usefulness, or prove safer than standard heparin.

Standardization of low molecular weight heparins: a collaborative study.

A collaborative study was carried out, in which eight laboratories each assayed eight low molecular weight (LMW) heparins against the International Standard (IS) for heparin. APTT assays and three types of anti-Xa method were used. The results of this study showed that: LMW heparins cannot be validly assayed against the IS by APTT or anti-Xa methods. Potencies of LMW heparins vs. the IS differed considerably between the four types of assay method used and also between different laboratories using the same type of method. Adoption of a single LMW heparin standard would improve validity, improve inter-laboratory variation, and largely abolish the differences between the three types of anti-Xa method. However, since calibration of a LMW heparin standard against the IS would give potencies that differ widely by the different assay methods, a single assay method such as the anti-Xa amidolytic, plasma, would need to be chosen for this calibration.

Standardization of prekallikrein activator (PKA): the 1st International Standard for PKA.

A international collaborative study was carried out to establish an international standard for prekallikrein activator (PKA). The candidate material, coded 82/530, was assayed against the Office of Biologics (FDA) PKA reference preparation No. 2 (OoB 2) and the 1st British Reference Preparation for PKA. There was good agreement between participant laboratories on the relative PKA activity of the three preparations. Preparation 82/530 has been established by the World Health Organization as the 1st I.S. for PKA, with an assigned potency of 85 International Units per ampoule.

A collaborative study of a proposed international standard for tissue plasminogen activator (t-PA).

An international collaborative study involving seven laboratories was undertaken to assess which of three lyophilised preparations might serve as an International Standard (I.S.) for tissue plasminogen activator (t-PA). Two of the preparations were isolates from human melanoma cell cultures while one was of pig heart origin. A clot lysis assay was used by all participants in the study. The data suggested that both preparations of human cell origin were comparable, in that their log dose-response lines were parallel, while that of the porcine preparation was not. Accelerated degradation studies indicated that one melanoma extract (denoted 83/517) was more stable than the other and it was decided to recommend preparation 83/517 as the standard for t-PA. The International Committee for Thrombosis and Haemostasis (Stockholm 1983) has recommended the use of this material as a standard and it has been established by the Expert Committee on Biological Standardization of the World Health Organization as the International Standard for tissue plasminogen activator, with an assigned potency of 1000 International Units per ampoule.

A comparison of heparin potency estimates obtained by activated partial thromboplastin time and british pharmacopoeial assays.

Heparin samples from five manufacturers were assayed by the revised British Pharmacopoeia (BP) heparin assay and the results compared with those obtained using the activated partial thromboplastin time (APTT) assay. The United States Pharmacopoeia (USP) reference heparin preparation and the 4th International Standard (IS) for heparin were also assayed by the two methods relative to the 3rd IS. The results obtained by the revised BP assay were in close agreement with those obtained by the APTT assay for all the heparins that were tested. The assays revealed that there is at least a 10% discrepancy between the International Unit for heparin and the USP unit.

Standardization of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4): a collaborative study to establish international standards for beta-TG and PF4.

An international collaborative study was carried out to determine the suitability of freeze-dried preparations of beta-TG and PF4 to serve as international standards, and to compare these materials with other purified preparations and with plasma samples. Although problems remain with the accurate measurement of these proteins, it has been demonstrated that common standards improve the precision of measurement by RIA and provide an essential foundation for future work into the effects of assay system differences. The World Health Organization established in 1984 the purified preparation of beta-TG (83/501) and the purified preparation of PF4 (83/505) as International Standards, with assigned potencies of 500 International Units per ampoule and 400 International Units per ampoule, respectively.

A collaborative study designed to establish the 4th International Standard for Heparin.

An international collaborative study, in which 22 laboratories participated, was carried out to establish a replacement for the International Standard for Heparin. A total of 248 assays were analyzed, including APTT, thrombin inhibition and anti-Xa assays, as well as pharmacopoeial assays. Overall, there was less than 5% difference in the mean potency estimates of the candidate preparations, by all assay methods. The freeze-dried preparation 82/502 demonstrated the closest parallelism by bioassay to the existing standard and was established by WHO as the 4th International Standard for Heparin, with an assigned unitage of 1780 i.u. per ampoule.

The establishment of a standard for plasminogen (glu-type).

Following an international collaborative study, a preparation of glutamic acid-(glu-)human plasminogen was established as a British Standard. This study compared the activity of the proposed standard following full activation to plasmin with the 2nd International Reference Preparation (IRP) for plasmin using both fibrinolytic and chromogenic assays. There was good agreement between the results of the two assay methods. The activity of the standard was 10 units per ampoule, these units being equivalent to those defined by the 2nd IRP for plasmin.

Standardization of Factor VIII-IV. Establishment of the 3rd International Standard for Factor VIII: C concentrate.

An international collaborative study was carried out to establish a replacement for the current (2nd) international standard for Factor VIII:C, concentrate. Twenty-six laboratories took part, of which 17 performed one-stage assays, three performed two-stage assays and six used both methods. The proposed new standard, an intermediate purity concentrate, was assayed against the current standard, against a high-purity concentrate and against an International Reference Plasma, coded 80/511, previously calibrated against fresh normal plasma. Assays of the proposed new standard against the current standard gave a mean potency of 3.89 iu/ampoule, with good agreement between laboratories and between one-stage and two-stage assays. There was also no difference between assay methods in the comparison of high-purity and intermediate purity concentrates. In the comparison of the proposed standard with the plasma reference preparation, the overall mean potency was 4.03 iu/ampoule, but there were substantial differences between laboratories, and the two-stage method gave significantly higher results than the one stage method. Of the technical variables in the one-stage method, only the activation time with one reagent appeared to have any influence on the results of this comparison of concentrate against plasma. Accelerated degradation studies showed that the proposed standard is very stable. With the agreement of the participants, the material, in ampoules coded 80/556, has been established by the World Health Organization as the 3rd International Standard for Factor VIII:C, Concentrate, with an assigned potency of 3.9 iu/ampoule.

The stability of tissue plasminogen activator (t-PA).

Since tissue-type plasminogen activator (t-PA) derived from tissue culture and recombinant DNA procedures has been proposed for use in thrombolytic therapy in man, it is essential that the t-PA molecule should display reasonable stability in a lyophilised state to facilitate its usefulness. In this study, four laboratories compared the potencies of three preparations of t-PA following storage at 4 degrees, 20 degrees, 37 degrees and 45 degrees C, using each t-PA stored at -20 degrees C as a reference (100% activity) in each case. A pig heart extract of t-PA was the most stable, losing no activity when stored for 30 days at 37 degrees C, while two melanoma cell tissue culture extracts varied in their storage behaviour. One was quite stable at 37 degrees C (losing about 3% of its activity) while the other lost about 16%. Thus both the pig heart t-PA and one t-PA from melanoma cell culture proved suitable for the further development of reference standards for t-PA activity.

Standardization of beta-thromboglobulin and platelet factor 4: a collaborative study to investigate the sources and extent of variation in the measurement of platelet specific proteins.

A collaborative study involving seven laboratories has been carried out to investigate the sources and extent of variation in the measurement by radioimmunoassay of beta-thromboglobulin (beta-TG) and platelet factor 4 (PF4). Three plasma samples, one purified beta-TG sample and two purified PF4 samples were assayed against house standards for the respective antigens by each laboratory. The main source of variation of both beta-TG and PF4 measurements was inter-assay (particularly for PF4). It was found that a significant component of this variation was due to the participants' use of different preparations as house standards for beta-TG and for PF4. This indicates that the first step in the standardization of beta-TG and PF4 measurement should be the introduction of a reference for each.