Multicenter Evaluation of a Chromogenic Substrate Method for Photometric Determination of Prothrombin Time

1987 ◽  
Vol 58 (03) ◽  
pp. 856-865 ◽  
Author(s):  
F Dati ◽  
M Barthels ◽  
J Conard ◽  
J Flückiger ◽  
A Girolami ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Anticoagulant Therapy ◽  
Photometric Determination ◽  
Coagulation Factors ◽  
Stable Phase ◽  
Photometric Method ◽  
Sensitivity Index ◽  
Chromogenic Substrate ◽  
Plasma Samples

SummaryA multicenter study of a chromogenic substrate method for photometric determination of prothrombin time was conducted in order to evaluate its clinical application. Seven laboratories pailicipaled in the study using a total of 742 plasma samples from 417 patients on oral anticoagulant therapy, 261 healthy subjects and 64 patients with different diseases especially of the liver as well as 30 patients with hereditary deficiency of coagulation factors II, V, VII, X. The chromogenic PT method was compared to a standardized coagulometric PT assay which uses the same sensitive human placenta thromboplastin calibrated against international reference preparations. A high correlation of the prothrombin ratio values of the chromogenic and the coagulometric assay was obtained in 402 plasma samples (r = 0.940; y = 1.02x − 0.1). The study showed that the chromogenic PT reagent is sensitive to deficiency of the coagulation factors of the extrinsic pathway but not affected by heparin up to 1 IU/ml because of the heparin antagonist added. The precision (coefficient of variation) of the photometric method ranged between 0.6 and 3% (intraassay CV) and between 1.4 and 5.8 (interassay CV). The International Sensitivity Index (ISI) obtained for the used lot was 1.09. The therapeutical range in percentage activity for patients in a stable phase of an anticoagulant therapy was found to be from 15 to 27 percent of normal. The results of the clinical evaluation proved the good comparability of the new chromogenic PT test with coagulometric methods, its high factor sensitivity, good reproducibility and easy performance.

APPLICATIONS OF INTERNATIONAL RECOMMENDATIONS ON THE STANDARDIZATION OF PROTHROMBIN TIME IN ORAL ANTICOAGULANT CONTROL

1987 ◽  
Author(s):  
F Dati ◽  
U Becker ◽  
N Heimburger
Keyword(s):  
Prothrombin Time ◽  
Oral Anticoagulant ◽  
Calibration Procedure ◽  
Sensitivity Index ◽  
Chromogenic Substrate ◽  
Clotting Factors ◽  
Reference Preparation ◽  
Broad Variation ◽  
The Mean

The determination of prothrombin time (PT) in oral anticoagulant control is affected by a broad variation. The responsible factors are: type of thromboplastin incorporated in the PT reagents, procedure for use, clotting factors or heparin inhibitors added to the reagent, method of expression of PT results. Recently, joint recommendations have been issued by International Committees (ICSH/ICTH) taking into account the system of International Thromboplastins and the statistical model for thromboplastin calibration established by WHO. The aim is a standardization of commercial thromboplastins for PT tests in order to allow the use of the international scale of oral anticoagulant intensity (INR: Intern. Normalized Ratio). Following such recommendations we have standardized two new PT tests, based on coagulometric and photometric methods which rely on the same sensitive human placental thromboplastin. The coagulometric PT test (Thromborel®S) is performed with conventional coagulometers. The photometric PT assay (Chromoquick®) uses a new chromogenic substrate specific for thrombin. This method is based on the measurement of the time necessary to reach a fixed increase of absorbance (0.1 A) using a special microprocessor-controlled photometer.The two PT reagents were calibrated either directly against a reference preparation (BCT) or via an intermediate standard thromboplastin in two multicentric studies. The calibration procedure by the WHO method allows to assign the corresponding ISI (Intern. Sensitivity Index) to the PT reagent used and the transformation of the obtained prothrombin ratio (PR) into INR by the equation INR = PRISI. The calculated ISI values were 1.08 for the coagulometric PT reagent (n = 330) and 1.07 for the photometric reagent (n = 365), respectively.The reproducibility of the ISI value for the new human placental thromboplastin for 64 different batches amounts to 3.6 %, the mean ISI value being 1.12.Comparison with the reference thromboplastins in PR values gave a good correlation.A) Coagul. PT assay (x): r = 0.964; y = 1.03x ™ 0.1;B) Photom. PT assay (x): r = 0.940; y = 1.02x ™ 0.1.

scholarly journals Monitoring "mini-intensity" anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F1+2 levels

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2034-2038 ◽  
Author(s):  
MM Millenson ◽  
KA Bauer ◽  
JP Kistler ◽  
S Barzegar ◽  
L Tulin ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Factor Xa ◽  
High Sensitivity ◽  
International Normalized Ratio ◽  
Target Range ◽  
Lower Sensitivity ◽  
Sensitivity Index ◽  
Plasma Samples ◽  
Prothrombin Activation

Treatment with warfarin using a target International Normalized Ratio (INR) range of 1.7 to 2.5 is efficacious for many clinical indications, but the minimal intensity of anticoagulation required for antithrombotic protection has yet to be determined. To evaluate whether patients could be reliably monitored with a less intense regimen, we anticoagulated patients with warfarin for several months using a target INR range of 1.3 to 1.6 as determined by prothrombin time (PT) using a sensitive thromboplastin (Dade IS, International Sensitivity Index [ISI] = 1.3). Plasma measurements of F1+2, a marker of factor Xa action on prothrombin in vivo, were also obtained to determine the suppressive effect of warfarin on hemostatic system activity. Overall, 20 of 21 patients with a history of cerebrovascular events (mean age, 61 years) could be reliably regulated with warfarin in the target INR range. F1+2 levels were significantly suppressed from baseline in all patients, with a mean reduction of 49% (range, 28% to 78%). We found a significant relationship between the extent of suppression of prothrombin activation levels and the baseline measurements. A mean reduction of 65% was observed for those patients with baseline F1+2 greater than or equal to 1.5 nmol/L, but only 38% for baseline F1+2 less than or equal to 0.5 nmol/L. Overall, 68% of plasma samples obtained during stable anticoagulation were within the target INR range. PTs were also determined on all plasma samples with two thromboplastins of lower sensitivity (C+, ISI = 2.09; and automated simplastin, ISI = 2.10). Only 47% and 35% of PT determinations, respectively, were within the target range with these reagents. We conclude that prothrombin activation can be significantly suppressed in vivo with use of warfarin in an INR range of 1.3 to 1.6. This level of anticoagulation can be reliably achieved by monitoring PTs with a thromboplastin of high sensitivity.

Automatic Flame Photometric Determination of Potassium in Fertilizers. A. Elimination of Anion Exchange Cleanup. B. Using Direct Available P2O5 Extract

1965 ◽  
Vol 48 (6) ◽  
pp. 1095-1100
Author(s):  
James P Ussary ◽  
Charles W Gehrke
Keyword(s):  
Anion Exchange ◽  
Primary Standard ◽  
Photometric Determination ◽  
Photometric Method ◽  
Potassium Salts ◽  
Commercial Fertilizer ◽  
Aoac Official ◽  
Primary Standards ◽  
Standard Grade

Abstract Three primary standard grade potassium salts, eight Magruder check samples, and 18 commercial fertilizer samples were analyzed by three methods. Primary standards gave an average recovery of 100.0% and an average range of 0.21% K20. Magruder check samples averaged 0.09% K20 higher by the modified flame photometric method than the grand averages of the STPB results on the respective Magruder reports. The modified flame photometric method averaged 0.02% K20 lower than the official flame photometric method and 0.11% K20 higher than the official STPB method on 18 commercial fertilizer samples. The automatic flame photometric method, without anion exchange cleanup, is rapid enough for routine analysis and is as accurate and precise as the AOAC official methods. The method was also applied to the direct available P205 extract. Results on three primary grade potassium salts, seven Magruder check samples, and 13 commercial fertilizer samples were as accurate and precise as the official STPB method.

scholarly journals Monitoring "mini-intensity" anticoagulation with warfarin: comparison of the prothrombin time using a sensitive thromboplastin with prothrombin fragment F1+2 levels

Blood ◽  
1992 ◽  
Vol 79 (8) ◽  
pp. 2034-2038 ◽  
Author(s):  
MM Millenson ◽  
KA Bauer ◽  
JP Kistler ◽  
S Barzegar ◽  
L Tulin ◽  
...  
Keyword(s):  
Prothrombin Time ◽  
Factor Xa ◽  
High Sensitivity ◽  
International Normalized Ratio ◽  
Target Range ◽  
Lower Sensitivity ◽  
Sensitivity Index ◽  
Plasma Samples ◽  
Prothrombin Activation

Abstract Treatment with warfarin using a target International Normalized Ratio (INR) range of 1.7 to 2.5 is efficacious for many clinical indications, but the minimal intensity of anticoagulation required for antithrombotic protection has yet to be determined. To evaluate whether patients could be reliably monitored with a less intense regimen, we anticoagulated patients with warfarin for several months using a target INR range of 1.3 to 1.6 as determined by prothrombin time (PT) using a sensitive thromboplastin (Dade IS, International Sensitivity Index [ISI] = 1.3). Plasma measurements of F1+2, a marker of factor Xa action on prothrombin in vivo, were also obtained to determine the suppressive effect of warfarin on hemostatic system activity. Overall, 20 of 21 patients with a history of cerebrovascular events (mean age, 61 years) could be reliably regulated with warfarin in the target INR range. F1+2 levels were significantly suppressed from baseline in all patients, with a mean reduction of 49% (range, 28% to 78%). We found a significant relationship between the extent of suppression of prothrombin activation levels and the baseline measurements. A mean reduction of 65% was observed for those patients with baseline F1+2 greater than or equal to 1.5 nmol/L, but only 38% for baseline F1+2 less than or equal to 0.5 nmol/L. Overall, 68% of plasma samples obtained during stable anticoagulation were within the target INR range. PTs were also determined on all plasma samples with two thromboplastins of lower sensitivity (C+, ISI = 2.09; and automated simplastin, ISI = 2.10). Only 47% and 35% of PT determinations, respectively, were within the target range with these reagents. We conclude that prothrombin activation can be significantly suppressed in vivo with use of warfarin in an INR range of 1.3 to 1.6. This level of anticoagulation can be reliably achieved by monitoring PTs with a thromboplastin of high sensitivity.

Elimination of Aliquoting in Automated Flame Photometric Determination of K2O in Fertilizers

1984 ◽  
Vol 67 (4) ◽  
pp. 847-850
Author(s):  
Layna D Steele ◽  
Kelly J Ramsey ◽  
Peter F Kane
Keyword(s):  
Full Range ◽  
Flow Analysis ◽  
Photometric Determination ◽  
Photometric Method ◽  
Official Method ◽  
Significant Saving ◽  
Large Numbers ◽  
Continuous Flow Analysis ◽  
Automated Instrument

Abstract Current automated instrument systems used in conjunction with the official AOAC flame photometric method for K2O in fertilizer, 2.D06, all require a dilution step to bring the fertilizer extract to the appropriate concentration range. Two CFA (Continuous Flow Analysis) automated instrument systems are described which together eliminate aliquoting over the full range of fertilizer potassium, a significant saving of analyst effort if large numbers of samples are analyzed. The systems have performance characteristics well within the limits in the official method, and produce results comparable to current methods on routine samples.

Comparison of a New Automated Amidolytic Assay of Thrombin Generation with “ Prothrombin Time” and “Thrombotest”.

1979 ◽  
Author(s):  
J.J.C. Jonker ◽  
L.H.M. van Riel ◽  
M.M.P. Paulssen
Keyword(s):  
Prothrombin Time ◽  
Whole Blood ◽  
Thrombin Generation ◽  
Chromogenic Substrate ◽  
Automated Method

An automated method was developed for the determination of thrombin generation in citrated whole blood during activation by thromboplastin. The thrombin generated was allowed to split the chromogenic substrate Tos-Gly-Pro-Arg-pNA yielding p-Nitroaniline. The comparison with this method and “Prothrombin time” in 50 samples was significant (r = 0,88 P < 0,001) and also in 503 samples with “Thrombotest” (r = 0,73 P < 0,001). This assay may serve as a specific, precise and fast and cheaper alternative for “prothrombin time” and “Thrombotest” in large Thrombosis Services.

Correction of instrument- and reagent-based differences in determination of the International Normalized Ratio (INR) for monitoring anticoagulant therapy.

1989 ◽  
Vol 35 (5) ◽  
pp. 840-843 ◽  
Author(s):  
J L van Rijn ◽  
N A Schmidt ◽  
W P Rutten
Keyword(s):  
World Health Organization ◽  
Prothrombin Time ◽  
Anticoagulant Therapy ◽  
International Normalized Ratio ◽  
World Health ◽  
Individual Values ◽  
The World ◽  
Health Organization ◽  
Reference Samples

Abstract As recommended by the World Health Organization, standardization of prothrombin time assays involves conversion of prothrombin times into International Normalized Ratios (INR). We investigated the effect of two different methods (Nycomed's Thrombotest, and Instrumentation Laboratory's PT-fibrinogen) and three coagulation instruments (Schnitger & Gross, KC-10, and ACL) on calculations of INR. The INR plots showed considerable scatter of individual values around the regression lines when the two different methods were compared. Systematic differences in the outcome of INR calculation were related to the use of the different coagulation instruments. Prothrombin times obtained with the different instruments were linearly correlated. We used the bias of these lines to correct results for both the patients' samples and the reference samples. This correction yielded INR values from the different instruments that agreed well.

Levels of Platelet Factor 3 in Citrate Plasma and in Whole Blood as Determined by A Chromogenic Peptide Substrate Assay

1979 ◽  
Author(s):  
H. Sandberg ◽  
A.-K. Gellerbring ◽  
L.-O. Andersson
Keyword(s):  
Whole Blood ◽  
Platelet Rich Plasma ◽  
Blood Sampling ◽  
Chromogenic Substrate ◽  
Plasma Samples ◽  
Peptide Substrate ◽  
Platelet Factor ◽  
No Release ◽  
Sensitive Method

A newly developed sensitive method for determination of platelet factor 3 (PF 3) using a chromogenic substrate (S 2238) (Sandberg and Andersson, Thromb. Res., in press) was used for comparison of the PF 3 levels in platelet-rich plasma (PRP) in citrate with the levels in PRP in the anticoagulant EDTA/citrate/PCE 1/theophylline. It was shown that in citrate PRP, release of PF 3 was started after about 20 minutes from blood sampling. Release of β-thromboglobulin (β-tg) was found to occur simultaneously with the release of PF 3. No release of PF 3 or β-tg could be detected within 3 hours from blood sampling in PRP in the EDTA/citrate/PCE 1/theophylline anticoagulant.The PF 3 level in whole blood was measured by a method which was a modification of the method used for plasma samples. It was found that, using the same donor, the PF 3 level of plasma and blood in the EDTA/citrate/PCE 1/theophylline anticoagulant was essentially the same. The levels of PF 3 found in blood upon standing in citrate or in EDTA/citrate/PCE 1/theophy11ine anticoagulant were in accordance with the levels found in plasma. Experiments with whole blood without any anticoagulant showed that release of PF 3 begin to occur simultaneously with release of β-tg, about 5 minutes before clotting. Thus the data show that PF 3 and βtg are released in parallel.

Determination of the Biological Activity of Heparin by Use of a Chromogenic Substrate

1979 ◽  
Vol 42 (05) ◽  
pp. 1446-1451 ◽  
Author(s):  
Knut Bartl ◽  
Elfriede Dorsch ◽  
Helmut Lill ◽  
Joachim Ziegenhorn
Keyword(s):  
Biological Activity ◽  
Therapeutic Range ◽  
Antithrombin Iii ◽  
Photometric Determination ◽  
Chromogenic Substrate ◽  
Standard Samples ◽  
Spectrum Line ◽  
At Iii ◽  
Specific Determination

SummaryWe describe a new method for determining the biological activity of heparin in plasma with use of thrombin and the substrate Tos-Gly-Pro-Arg-pNA. The procedure is based on the photometric determination of the inactivation of thrombin after incubation with plasma in the presence of endogenous antithrombin III (At III). The method allows the specific determination of heparin concentrations from 0.02 USP to 0.8 USP/ml of plasma in the presence of normal At III levels. It has been carried out manually by use of an Eppendorf spectrum line photometer or automatically by use of a Vitatron Akes analyzer. For evaluation, the results were compared with two standard samples which contained heparin in the low and high therapeutic range, respectively.

Issues with the Assay of Factor VIII Activity in Plasma and Factor VIII Concentrates

2000 ◽  
Vol 84 (12) ◽  
pp. 942-948 ◽  
Author(s):  
Henry Kingdon ◽  
Kenneth Mann ◽  
Gilbert White ◽  
Roger Lundblad
Keyword(s):  
Factor Viii ◽  
Hemophilia A ◽  
Coagulation Factors ◽  
Chromogenic Substrate ◽  
In Vitro Measurements ◽  
Factor Viii Concentrates ◽  
The One

SummaryA review of the literature suggests that assays accurate for the determination of factor VIII in plasma samples may not necessarily retain this accuracy when used for the determination of factor VIII in high-purity factor VIII concentrates such as Hemofil ® M. Review of assay data suggests that it is imperative to obtain maximal activation of the factor VIII in the sample with thrombin when using an assay system of isolated coagulation factors such as the two-stage assay or the various chromogenic substrate assays. Based on a combination of ease and reproducibility of performance and correlation of in vivo and in vitro measurements, it is recommended that the one-stage activated partial thromboplastin time performed with plasma from an individual with severe hemophilia A be used for the measurement of factor VIII potency. Chromogenic substrate assays can be used if care is taken to assure optimal activation of factor VIII by thrombin in the assay and the presence of sufficient factor IXa, phospholipid and calcium ions to stabilize factor Villa during the assay process.

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