Simplex Optimization of the Blue Tetrazolium Assay Procedure for α-Ketol Steroids

Abstract This new assay procedure for diaphorase eliminates problems of high blank rates and nonlinear kinetics associated with other methods. The dye thiazolyl blue tetrazolium bromide is reduced in the presence of NADH and diaphorase to yield a colored formazan, which as maximum absorbance at 560 nm.

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Quantitative nitro blue tetrazolium test in febrile patients. Correlation with diagnosis and bacterial activity of leukocytes

Archives of Internal Medicine ◽

10.1001/archinte.135.12.1569 ◽

1975 ◽

Vol 135 (12) ◽

pp. 1569-1574

Author(s):

J. Silva

Keyword(s):

Bacterial Activity ◽

Nitro Blue Tetrazolium ◽

Blue Tetrazolium ◽

Tetrazolium Test

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Reliability of Laboratory Tests for the Control of Oral Anticoagulation

Thrombosis and Haemostasis ◽

10.1055/s-0038-1647716 ◽

1974 ◽

Vol 32 (02/03) ◽

pp. 483-491

Author(s):

E. A Loeliger ◽

M. J Boekhout-Mussert ◽

L. P van Halem-Visser ◽

J. D. E Habbema ◽

H de Jonge

Keyword(s):

Human Brain ◽

Laboratory Tests ◽

Test Procedure ◽

Rabbit Brain ◽

Normal Range ◽

Test Procedures ◽

Discrimination Power ◽

Power Of The Test ◽

Assay Procedure ◽

Low Sensitivity

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.

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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.

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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.

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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.

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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.

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Human Prothrombin Activation: Determination of Plasma and Serum Levels of Prothrombin Fragment 3 by Radioimmunoassay

10.1055/s-0038-1665668 ◽

1979 ◽

Author(s):

Daniel Walz ◽

Thomas Brown

Keyword(s):

Blood Clotting ◽

Factor Xa ◽

Heart Association ◽

Serum Levels ◽

Cross Reactivity ◽

Assay Procedure ◽

A Chain ◽

Prothrombin Activation ◽

Theoretical Amount

Human prothrombin activation is unique in that, in addition to the release of fragment 1.2 (FI.2) from the NH-terminus of prothrombin by factor Xa during the generation of thrombin, an additional 13 residue polypeptide, fragment 3 (F3), is autocatalytically removed from the amino-terminus of the thrombin A chain. We have developed a rapid radioimmunoassay for human F3 which incorporates short incubation times and the use of a preprecipitated second antibody; the assay can be performed in three hours. Specificity studies in buffer systems show prothrombin and prethrombin 1 cross-reacting at a level of 0.001; purified thrombin does not cross-react. In the presence of 5% BSA, prothrombin displays considerably less cross-reactivity. No immunoreactive material to F3 antibodies could be detected in 400 μL of plasma. Serum, obtained from whole blood clotting, contained measurable quantities of F3 (40-100 ng/mL). This amount in serum represents only 5-10% of the theoretical amount available should all of the fragment be hydrolytically cleaved during the conversion of prothrombin to thrombin. This assay procedure is currently being utilized to monitor the activation of purified human prothrombin in the absence and presence of selected plasma inhibitors. (Supported in part by NIH 05384-17 and the Michigan Heart Association).

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Determination of Factor XIII Activity and of Factor XIII Inhibitors Using an Ammonium-Sensitive Electrode

Thrombosis and Haemostasis ◽

10.1055/s-0038-1665256 ◽

1983 ◽

Vol 50 (02) ◽

pp. 563-566 ◽

Cited By ~ 3

Author(s):

P Hellstern ◽

K Schilz ◽

G von Blohn ◽

E Wenzel

Keyword(s):

Factor Xiii ◽

Normal Subjects ◽

The Other ◽

Activity Measurement ◽

Factor Xiii Deficiency ◽

Assay Procedure ◽

Stabilization Factor ◽

Release Method ◽

Sensitive Electrode

SummaryAn assay for rapid factor XIII activity measurement has been developed based on the determination of the ammonium released during fibrin stabilization. Factor XIII was activated by thrombin and calcium. Ammonium was measured by an ammonium-sensitive electrode. It was demonstrated that the assay procedure yields accurate and precise results and that factor XIII-catalyzed fibrin stabilization can be measured kinetically. The amount of ammonium released during the first 90 min of fibrin stabilization was found to be 7.8 ± 0.5 moles per mole fibrinogen, which is in agreement with the findings of other authors. In 15 normal subjects and in 15 patients suffering from diseases with suspected factor XIII deficiency there was a satisfactory correlation between the results obtained by the “ammonium-release-method”, Bohn’s method, and the immunological assay (r1 = 0.65; r2= 0.70; p<0.01). In 3 of 5 patients with paraproteinemias the values of factor XIII activity determined by the ammonium-release method were markedly lower than those estimated by the other methods. It could be shown that inhibitor mechanisms were responsible for these discrepancies.

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