Estimation of Limiting Nutrient by Algal Assay Procedure with Indigenous Phytoplankton Assemblage.

SummaryThe present study concerned the reproducibility of the so-called prothrombin time as assessed with a series of more commonly used modifications of the Quick’s onestage assay procedure, i.e. the British comparative reagent, homemade human brain thromboplastin, Simplastin, Simplastin A, and Thrombotest. All five procedures were tested manually on pooled lyophilized normal and patients’ plasmas. In addition, Simplastin A and Thrombotest were investigated semiautomatically on individual freshly prepared patients’ plasmas. From the results obtained, the following conclusions may be drawn :The reproducibility of results obtained with manual reading on lyophilized plasmas is satisfactory for all five test procedures. For Simplastin, the reproducibility of values in the range of insufficient anticoagulation is relatively low due to the low discrimination power of the test procedure in the near-normal range (so-called low sensitivity of rabbit brain thromboplastins). The reproducibility of Thrombotest excels as a consequence of its particularly easily discerned coagulation endpoint.The reproducibility of Thrombotest, when tested on freshly prepared plasmas using Schnitger’s semiautomatic coagulometer (a fibrinometer-liJce apparatus), is no longer superior to that of Simplastin A.The constant of proportionality between the coagulation times formed with Simplastin A and Thrombotest was estimated at 0.64.Reconstituted Thrombotest is stable for 24 hours when stored at 4° C, whereas reconstituted Simplastin A is not.The Simplastin A method and Thrombotest seem to be equally sensitive to “activation” of blood coagulation upon storage.

Download Full-text

A in Vivo Assay for Standardizing Heparin Preparations

Thrombosis and Haemostasis ◽

10.1055/s-0038-1646810 ◽

1979 ◽

Vol 41 (03) ◽

pp. 576-582

Author(s):

A R Pomeroy

Keyword(s):

In Vitro Assays ◽

British Pharmacopoeia ◽

In Vitro Method ◽

Standard Preparation ◽

Reliable Estimation ◽

Assay Procedure ◽

Accuracy And Precision ◽

Heparin Preparation

SummaryThe limitations of currently used in vitro assays of heparin have demonstrated the need for an in vivo method suitable for routine use.The in vivo method which is described in this paper uses, for each heparin preparation, four groups of five mice which are injected intravenously with heparin according to a “2 and 2 dose assay” procedure. The method is relatively rapid, requiring 3 to 4 hours to test five heparin preparations against a standard preparation of heparin. Levels of accuracy and precision acceptable for the requirements of the British Pharmacopoeia are obtained by combining the results of 3 to 4 assays of a heparin preparation.The similarity of results obtained the in vivo method and the in vitro method of the British Pharmacopoeia for heparin preparations of lung and mucosal origin validates this in vivo method and, conversely, demonstrates that the in vitro method of the British Pharmacopoeia gives a reliable estimation of the in vivo activity of heparin.

Download Full-text

A Collaborative Study to Establish an International Standard for Alpha-Thrombin

Thrombosis and Haemostasis ◽

10.1055/s-0038-1648464 ◽

1992 ◽

Vol 67 (04) ◽

pp. 424-427 ◽

Cited By ~ 3

Author(s):

P J Gaffney ◽

A B Heath ◽

J W Fenton II

Keyword(s):

Elevated Temperatures ◽

Collaborative Study ◽

World Health Organisation ◽

Significant Loss ◽

Thrombin Inhibitors ◽

World Health ◽

Expert Committee ◽

International Standard ◽

Assay Procedure ◽

And Control

SummarySince 1975 an International Standard for Thrombin of low purity has been used. While this standard was stable and of value for calibrating thrombins of unknown potency the need for a pure a-thrombin standard arose both for accurate calibration and for precise measurement of thrombin inhibitors, notably hirudin. An international collaborative study was undertaken to establish the potency and stability of an ampouled pure a-thrombin preparation. A potency of 97.5 international units (95% confidence limits 86.5-98.5) was established for the new a-thrombin standard (89/ 588) using a clotting-assay procedure. Stability data at various elevated temperatures indicated that the standard could be transported and stored with no significant loss of potency.Ampoules of lyophilised a-thrombin (coded 89/588) have been recommended as an International Standard for a-thrombin with an assigned potency of 100 international units per ampoule by the International Society for Thrombosis and Haemostasis (Thrombin and its Inhibitors Sub-Committee) in Barcelona, Spain in July 1990 while the Expert Committee on Biological Standardisation and Control of the World Health Organisation will consider its status at its next meeting in Geneva in 1991.

Download Full-text

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

Thrombosis and Haemostasis ◽

10.1055/s-0038-1651628 ◽

1993 ◽

Vol 69 (05) ◽

pp. 430-435 ◽

Cited By ~ 16

Author(s):

Colin Longstaff ◽

Man-Yu Wong ◽

Patrick J Gaffney

Keyword(s):

Specific Activity ◽

Collaborative Study ◽

Residual Activity ◽

Quality Standard ◽

Upper Limit ◽

Assay Procedure ◽

Specific Activities ◽

International Collaborative ◽

Range Of Values ◽

International Collaborative Study

SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

Download Full-text

Two-Stage Procedure for the Quantitative Determination of Autoprothrombin III Concentration and Some Applications

Thrombosis and Haemostasis ◽

10.1055/s-0038-1655029 ◽

1967 ◽

Vol 18 (01/02) ◽

pp. 198-210 ◽

Cited By ~ 5

Author(s):

Ronald S Reno ◽

Walter H Seegers

Keyword(s):

Prothrombin Time ◽

Factor Vii ◽

Two Stage ◽

Pronounced Increase ◽

One Stage ◽

Assay Procedure ◽

Bovine Plasma ◽

The One ◽

Autoprothrombin C

SummaryA two-stage assay procedure was developed for the determination of the autoprothrombin C titre which can be developed from prothrombin or autoprothrombin III containing solutions. The proenzyme is activated by Russell’s viper venom and the autoprothrombin C activity that appears is measured by its ability to shorten the partial thromboplastin time of bovine plasma.Using the assay, the autoprothrombin C titre was determined in the plasma of several species, as well as the percentage of it remaining in the serum from blood clotted in glass test tubes. Much autoprothrombin III remains in human serum. With sufficient thromboplastin it was completely utilized. Plasma from selected patients with coagulation disorders was assayed and only Stuart plasma was abnormal. In so-called factor VII, IX, and P.T.A. deficiency the autoprothrombin C titre and thrombin titre that could be developed was normal. In one case (prethrombin irregularity) practically no thrombin titre developed but the amount of autoprothrombin C which generated was in the normal range.Dogs were treated with Dicumarol and the autoprothrombin C titre that could be developed from their plasmas decreased until only traces could be detected. This coincided with a lowering of the thrombin titre that could be developed and a prolongation of the one-stage prothrombin time. While the Dicumarol was acting, the dogs were given an infusion of purified bovine prothrombin and the levels of autoprothrombin C, thrombin and one-stage prothrombin time were followed for several hours. The tests became normal immediately after the infusion and then went back to preinfusion levels over a period of 24 hrs.In other dogs the effect of Dicumarol was reversed by giving vitamin K1 intravenously. The effect of the vitamin was noticed as early as 20 min after administration.In response to vitamin K the most pronounced increase was with that portion of the prothrombin molecule which yields thrombin. The proportion of that protein with respect to the precursor of autoprothrombin C increased during the first hour and then started to go down and after 3 hrs was equal to the proportion normally found in plasma.

Download Full-text