Fibrinogen assays: a collaborative study of six different methods. C.I.S.M.E.L. Comitato Italiano per la Standardizzazione dei Metodi in Ematologia e Laboratorio

1991 ◽  
Vol 37 (5) ◽  
pp. 714-719 ◽  
Author(s):  
G Palareti ◽  
M Maccaferri ◽  
C Manotti ◽  
A Tripodi ◽  
V Chantarangkul ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Collaborative Study ◽  
Internal Standard ◽  
Calibration Procedure ◽  
Radial Immunodiffusion ◽  
Clotting Time ◽  
International Standard ◽  
Fibrinogen Content

Abstract This collaborative study, organized by the Hemostasis Subcommittee of C.I.S.M.E.L., evaluated the accuracy, precision, and comparability of the following six widely used fibrinogen assays: total clottable fibrinogen (Blombäck and Blombäck), clotting time (Von Clauss), turbidimetry (Ellis and Stransky), Chromotime System, prothrombin time (PT)-derived, and radial immunodiffusion (RID). The same frozen samples, with normal and high contents of fibrinogen, were examined in four laboratories. The methods were calibrated with an internal standard whose fibrinogen content was determined gravimetrically. Both the Von Clauss and the RID methods were reliable, accurate, and precise, if adequate calibration was used. The PT-derived method was highly reproducible, but had some problems with accuracy. We demonstrate that an adequate calibration procedure is indispensable for reliable fibrinogen measurements whatever method is used. Because neither the calibration procedures proposed by the manufacturers nor the use of lyophilized commercial plasmas is adequate for this purpose, we urge that an international standard for fibrinogen measurement be promptly established.

EFFECTS OF INTRAVENOUS INJECTIONS OF THE ANTICOAGULANT FRACTION OF INCUBATED FIBRINOGEN ON BLOOD COAGULATION

1960 ◽  
Vol 38 (1) ◽  
pp. 909-918 ◽  
Author(s):  
D. C. Triantaphyllopoulos
Keyword(s):  
Prothrombin Time ◽  
Bleeding Time ◽  
Jugular Vein ◽  
Coagulation Factors ◽  
External Jugular Vein ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Intravenous Injections ◽  
Fibrinogen Content ◽  
Ammonium Sulphate Saturation

AFIF (Anticoagulant Fraction of Incubated Fibrinogen precipitated between 25 and 50% ammonium sulphate saturation) prepared from Armour's incubated bovine fibrinogen was injected in a dosage of 46–106 mg (mean: 66.5 mg) tyrosine per kilogram body weight in the external jugular vein of 10 rabbits. Blood samples were withdrawn at [Formula: see text]- or 1-hour intervals until the aspirated blood began showing signs of coagulation. The anticoagulant effect was manifested immediately and lasted for [Formula: see text] to [Formula: see text] hours in the animal. Although the blood aspirated during this time remained unclotted even after 24 hours, the surgical wound in the neck of the animal did not bleed and autopsies revealed no sign of internal haemorrhage. The bleeding time, however, was found prolonged when tested by cutting the marginal vein of the ear. The level of the coagulation factors was determined both in oxalated specimens and in specimens with no anticoagulant added. Both kinds of sample showed: (1) clottable fibrinogen content normal, thus excluding fibrinolysis as the cause of the anticoagulant effect; (2) thrombin clotting time of infinity; (3) one-stage prothrombin time longer than 60 seconds; (4) no correction of the infinite thrombin clotting time of oxalated specimens following the addition of 0.25 mg protamine per ml. This makes unlikely any appreciable release of heparin by the animal. However, oxalated and non-oxalated specimens differed in the following respects: (1) prothrombin time determined after adsorption and mixing of the eluate with adsorbed plasma was found to be normal for the oxalated blood but variably increased for the non-oxalated specimen (10–34.5 seconds); (2) the plasma precursors of plasma thromboplastin were normal in oxalated but very low in native specimens. The serum precursors of plasma thromboplastin were normal.

EFFECTS OF INTRAVENOUS INJECTIONS OF THE ANTICOAGULANT FRACTION OF INCUBATED FIBRINOGEN ON BLOOD COAGULATION

1960 ◽  
Vol 38 (8) ◽  
pp. 909-918 ◽  
Author(s):  
D. C. Triantaphyllopoulos
Keyword(s):  
Prothrombin Time ◽  
Bleeding Time ◽  
Jugular Vein ◽  
Coagulation Factors ◽  
External Jugular Vein ◽  
Anticoagulant Effect ◽  
Clotting Time ◽  
Intravenous Injections ◽  
Fibrinogen Content ◽  
Ammonium Sulphate Saturation

AFIF (Anticoagulant Fraction of Incubated Fibrinogen precipitated between 25 and 50% ammonium sulphate saturation) prepared from Armour's incubated bovine fibrinogen was injected in a dosage of 46–106 mg (mean: 66.5 mg) tyrosine per kilogram body weight in the external jugular vein of 10 rabbits. Blood samples were withdrawn at [Formula: see text]- or 1-hour intervals until the aspirated blood began showing signs of coagulation. The anticoagulant effect was manifested immediately and lasted for [Formula: see text] to [Formula: see text] hours in the animal. Although the blood aspirated during this time remained unclotted even after 24 hours, the surgical wound in the neck of the animal did not bleed and autopsies revealed no sign of internal haemorrhage. The bleeding time, however, was found prolonged when tested by cutting the marginal vein of the ear. The level of the coagulation factors was determined both in oxalated specimens and in specimens with no anticoagulant added. Both kinds of sample showed: (1) clottable fibrinogen content normal, thus excluding fibrinolysis as the cause of the anticoagulant effect; (2) thrombin clotting time of infinity; (3) one-stage prothrombin time longer than 60 seconds; (4) no correction of the infinite thrombin clotting time of oxalated specimens following the addition of 0.25 mg protamine per ml. This makes unlikely any appreciable release of heparin by the animal. However, oxalated and non-oxalated specimens differed in the following respects: (1) prothrombin time determined after adsorption and mixing of the eluate with adsorbed plasma was found to be normal for the oxalated blood but variably increased for the non-oxalated specimen (10–34.5 seconds); (2) the plasma precursors of plasma thromboplastin were normal in oxalated but very low in native specimens. The serum precursors of plasma thromboplastin were normal.

Results of Calibration by the Dutch National Reference Laboratory of the Thromboplastins Included in the ICTH/ ICSH Collaborative Study of Prothrombin Time Standardization

1979 ◽  
Vol 42 (04) ◽  
pp. 1128-1131 ◽  
Author(s):  
E A Loeliger ◽  
L P van Halem-Visser
Keyword(s):  
Prothrombin Time ◽  
Collaborative Study ◽  
Calibration Procedure ◽  
Reference Laboratory ◽  
National Reference Laboratory ◽  
Calibration Line ◽  
Calibration Constant ◽  
National Reference ◽  
Pass Through ◽  
Simplified Procedure

SummaryResults obtained with the original calibration procedure of Biggs and Denson obtained by one expert laboratory compare well with those obtained from the ICTH/ICSH collaborative study.The simplified calibration procedure described in 1975 should only be used for the assessment of inter-batch variability of a given brand of thromboplastin; for the calibration of unlike thromboplastins, the simplified procedure should be revised by using more than two abnormal plasmas, e.g. different plasmas representing seven levels of anticoagulation between international calibrated ratios (ICRs) from 1.5 to 4.5.The formula for the calculation for the proposed ICRs based on the calibration constant should be modified to allow for instances where the calibration line for dissimilar thromboplastins, fitted in the therapeutic range, does not pass through the origin of ratio 1,1.

A Collaborative Study to Establish the 2nd International Standard for Tissue Plasminogen Activator (t-PA)

1987 ◽  
Vol 58 (04) ◽  
pp. 1085-1087 ◽  
Author(s):  
P J Gaffney ◽  
A D Curtis
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Collaborative Study ◽  
Chinese Hamster ◽  
World Health ◽  
Clot Lysis ◽  
Expert Committee ◽  
Single Chain ◽  
International Standard ◽  
Tissue Plasminogen

SummaryAn 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 861670) was purified from a cultured melanoma cell supernatant and was about 98% single chain t-PA while the other preparation (coded 861624) 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° and 38° C. Both fultil the criteria to serve as a satisfactory Znd International Standard for t-PA. The Fibrinolysis Subcommittee of the International Committee for Thrombosis and Haemostasis recommended the melanoma source t-PA (861670) as the next I.S. in order to maintain continuity with the 1st I.S. which was also a melanomatype 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 of t-PA, coded 861670, be established as the 2ndlnternational Standard for t-PA.

A Collaborative Study to Establish the Second International Standard for Streptokinase

1990 ◽  
Vol 64 (02) ◽  
pp. 267-269 ◽  
Author(s):  
A B Heath ◽  
P J Gaffney
Keyword(s):  
High Purity ◽  
Collaborative Study ◽  
World Health Organisation ◽  
World Health ◽  
Clot Lysis ◽  
Current Standard ◽  
International Standard ◽  
Accelerated Degradation ◽  
The World ◽  
International Collaborative Study

SummaryAn International Standard for Streptokinase - Streptodomase (62/7) has been used to calibrate high purity clinical batches of SK since 1965. An international collaborative study, involving six laboratories, was undertaken to replace this standard with a high purity standard for SK. Two candidate preparations (88/826 and 88/824) were compared by a clot lysis assay with the current standard (62/7). Potencies of 671 i.u. and 461 i.u. were established for preparations A (88/826) and B (88/824), respectively.Either preparation appeared suitable to serve as a standard for SK. However, each ampoule of preparation A (88/826) contains a more appropriate amount of SK activity for potency testing, and is therefore preferred. Accelerated degradation tests indicate that preparation A (88/826) is very stable.The high purity streptokinase preparation, coded 88/826, has been established by the World Health Organisation as the 2nd International Standard for Streptokinase, with an assigned potency of 700 i.u. per ampoule.

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

1992 ◽  
Vol 67 (04) ◽  
pp. 424-427 ◽  
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.

A Collaborative Study of a Proposed International Standard for Tissue Plasminogen Activator (t-PA)

1985 ◽  
Vol 53 (01) ◽  
pp. 134-136 ◽  
Author(s):  
P J Gaffney ◽  
A D Curtis
Keyword(s):  
Plasminogen Activator ◽  
Tissue Plasminogen Activator ◽  
Collaborative Study ◽  
International Committee ◽  
World Health ◽  
Clot Lysis ◽  
Expert Committee ◽  
International Standard ◽  
Tissue Plasminogen ◽  
Health Organization

SummaryAn 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 Collaborative Study Designed to Establish the 4th International Standard for Heparin

1984 ◽  
Vol 52 (02) ◽  
pp. 148-153 ◽  
Author(s):  
D P Thomas ◽  
A D Curtis ◽  
T W Barrowcliffe
Keyword(s):  
Collaborative Study ◽  
International Standard ◽  
Thrombin Inhibition ◽  
Freeze Dried ◽  
Assay Methods ◽  
The Mean ◽  
International Collaborative ◽  
International Collaborative Study

SummaryAn 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.

Standardization of Factor VIII – IV. Establishment of the 3rd International Standard for Factor VIII: C Concentrate

1983 ◽  
Vol 50 (03) ◽  
pp. 697-702 ◽  
Author(s):  
T W Barrowcliffe ◽  
A D Curtis ◽  
D P Thomas
Keyword(s):  
Factor Viii ◽  
High Purity ◽  
Collaborative Study ◽  
World Health ◽  
Current Standard ◽  
Two Stage ◽  
International Standard ◽  
One Stage ◽  
The One ◽  
Health Organization

SummaryAn 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.

Flame ionization detector response to coeluting hydrocarbons and carbon disulphide and its application to the determination of the sum of C6-C11 alkanes and cycloalkanes in air

1983 ◽  
Vol 48 (3) ◽  
pp. 722-734
Author(s):  
Martin Koval
Keyword(s):  
Carbon Disulphide ◽  
Internal Standard ◽  
Calibration Procedure ◽  
Detector Response ◽  
Column Temperature ◽  
Flame Ionization ◽  
Other Substances ◽  
Flame Ionisation Detector ◽  
Ionisation Detector

The flame ionisation detector response to C6-C11 aliphatic hydrocarbon solutions in carbon disulphide in the concentration range between 1.3-9.5 mg ml-1 retained lineary despite the excess of solvent entering the detector simultaneously with the analyte. Pure carbon disulphide exhibited a small positive detector response which did not interfere in calibration procedure and which, under certain GC conditions, inverted to negative values. This response was not proportional to the injected volume and was strongly influenced by the column temperature and/or bleed. On the basis of these findings, a method compatible with the widely used charcoal tube carbon disulphide desorption procedure was developed and evaluated. It consists of static desorption of the sum of aliphatic alkanes and cycloalkanes from the activated charcoal after which an internal standard is added to the supernatant eluate. The resulting carbon disulphide solution is analysed on a highly polar stationary phase 1,2,3-tris(2-cyanoethoxy)propane where the solvent and the analyte coelute in a single peak, the height of which is practically proportional to the sum of alkanes and cycloalkanes present. This also makes determinations of other substances present in the sample more simple. The field test of the proposed method yielded values comparable in precision and accuracy with a control infrared spectrophotometric method.

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