Photometric assay of antithrombin III with a chromogenic substrate.

A simple assay procedure for antithrombin III is described. The synthetic product benzoyl-Phe-Val-Arg-p-nitroanilide (S-2160) is used as substrate. By thrombin p-nitroaniline is split from the tripeptide molecule. The yellowish color of this split product can be measured in a photometer. Inactivation of thrombin by antithrombin III results in inhibition of this reaction. The percentage of thrombin inhibited is in direct relation to the activity of antithrombin III. Various modifications were tested to optimate the method. Comparable results were obtained between the coagulation method of HENSEN and LOELIGER and the photometric method.     

Comparison of antithrombin III assays using biological and chromogenic substrates

S ummary . Antithrombin III (At III) levels in plasma samples were determined by incubation of diluted plasma with thrombin, either with or without heparin, followed by measurement of residual thrombin using clotting and amidolytic methods. All assays were of multidose bioassay design suitable for parallel line analysis. Assays without heparin showed only a small difference between amidolytic and clotting methods, which was not significant at the 95% level. Assays with heparin showed a much larger difference between clotting and amidolytic methods which is shown to be attributable to the heat defibrination step used in the clotting assay. Heat defibrination and ancrod defibrination methods are compared using the amidolytic heparin cofactor assay and it is shown that heat defibrination can cause the loss of nearly 50% of the functional At III in a reconstituted freeze-dried plasma which was used as a standard. No loss occurs when ancrod is used for defibrination. It is an advantage of the amidolytic heparin cofactor assay that defibrination is not required.     

Determination of rivaroxaban by different factor Xa specific chromogenic substrate assays: reduction of interassay variability

Rivaroxaban and other oral direct factor Xa inhibitors (ODiXa) are currently developed for prophylaxis and treatment of thromboembolic diseases using fixed doses. Although routine monitoring is not required, assessing the intensity of anticoagulation may be useful under certain clinical conditions. ODiXa prolong coagulation times of several clotting assays and, thus, their concentration may be determined in factor Xa specific chromogenic substrate assays. So far, no standardized and validated assay is commercially available. Here, five methods (A through E) are studied and optimized to reduce interassay variability. Human pooled plasma was spiked by a serial dilution of rivaroxaban (25–900 ng/ml). The release of para-nitroaniline from the chromogenic substrates was measured by the optical density (OD) at 405 nm. Method B was identified to yield the lowest sum of deviations from the mean value of the OD concentration curve calculated from all assays. Spline functions were developed for OD versus concentration curves for all methods. The calculated OD versus concentration curves overlapped for all methods. The coefficient of variation for all assays and concentrations of rivaroxaban decreased from 25.3 ± 11.4% using the original data to 3.8 ± 2.2% using the calculated data (P < 0.0001). The robustness of the chromogenic assay (method B) remains to be corroborated in interlaboratory comparisons.     

Coagulation and chromogenic assays of factor VIII activity: general aspects,standardization, and recommendations

Factor VIII (FVIII) is assayed by one-stage and two-stage clotting methods and by chromogenic methods, although the chromogenic method has largely replaced the two-stage clotting assay. Clinical plasma samples are assayed mostly by one-stage assays, but most manufacturers of concentrates use the chromogenic method, which is more precise and is the reference method of the European Pharmacopoeia and the International Society on Thrombosis and Haemostasis (ISTH). For most plasma-derived concentrates, assays against the World Health Organization (WHO) concentrate standard give similar results with the one-stage and chromogenic methods, but for products produced by the "method M" monoclonal antibody process, the one-stage potency is 25 to 30% higher than the chromogenic potency. For full-length recombinant products assayed against a plasma-derived concentrate standard, one-stage potencies are about 10% lower than chromogenic potencies, but for the B-domain deleted recombinant product ReFacto, the discrepancy is larger-from 20 to 50%. These discrepancies emphasize the need for an international methodology for labeling of concentrates. In ex vivo assays of hemophilic plasmas after infusion of concentrates, large discrepancies are found among laboratories and with different assay methods when a plasma standard is used. In most studies, the chromogenic potencies are higher than the one-stage potencies, and the discrepancy is highest for recombinant products. This discrepancy can be largely eliminated by the use of concentrate standards, diluted in FVIII-deficient plasma, to assay postinfusion plasma samples.     

Photometric assay of platelet factor 4 with a chromogenic substrate

A photometric assay procedure for platelet factor 4 is described. The synthetic oligopeptide benzoyl-Ile-Glu-Gly-Arg-p-nitroanilide (S-2222) is used as a substrate. By the action of factor Xa, p-nitroaniline (pNA) is split form the peptide bond. The amount of pNA liberated from S-2222 per minute is in direct relation to the activity of factor Xa. This reaction permits a photometric assay. Addition of heparin to an activation system consisting of plasma, thromboplastin and calcium chloride inhibits development of Xa activity. Since platelet factor 4 neutralizes heparin, its activity can be measured in such a system when all other components are kept at a constant level. Experimental details of the reactions involved and clinical results of the assay in comparison to a clotting method are described.     

Clinical utility and impact of the use of the chromogenic vs one-stage factor activity assays in haemophilia A and B

Treatment of haemophilia A/B patients comprises factor VIII (FVIII) or factor IX (FIX) concentrate replacement therapy, respectively. FVIII and FIX activity levels can be measured in clinical laboratories using one-stage activated partial thromboplastin time (aPTT)-based clotting or two-stage chromogenic factor activity assays. We discuss strengths and limitations of these assays, providing examples of clinical scenarios to highlight some of the challenges associated with their current use for diagnostic and monitoring purposes. Substantial inter-laboratory variability has been reported for one-stage assays when measuring the activity of factor replacement products due to the wide range of currently available aPTT reagents, calibration standards, factor-deficient plasmas, assay conditions and instruments. Chromogenic activity assays may avoid some limitations associated with one-stage assays, but their regulatory status, perceived higher cost, and lack of laboratory expertise may influence their use. Haemophilia management guidelines recommend the differential application of one or both assays for initial diagnosis and disease severity characterisation, post-infusion monitoring and replacement factor potency labelling. Efficient communication between clinical and laboratory staff is crucial to ensure application of the most appropriate assay to each clinical situation, correct interpretation of assay results and, ultimately, accurate diagnosis and optimal and safe treatment of haemophilia A or B patients.     

Maintenance control of oral anticoagulant therapy by an automated chromogenic substrate assay of factor X

Summary An amidolytic assay of factor X based on the new chromogenic peptide substrate S 2337 (Kabi Diagnostica) was adapted for use with the Kem-o-Mat (Coulter Electronics) automated substrate analyser. Factor X was assayed in 25 healthy controls and in 375 patients on Warfarin therapy. The results in the control group correlated well with a one stage coagulation factor X assay. A good correlation was also found when the S 2337 factor X assay was compared with Thrombotest (Nyegaard & Co.) results in the patients. From the regression line of the S 2337 factor X assay on the Thrombotest results, the comparable range for factor X amidolytic activity in well controlled anticoagulated patients was found to be 22.5–37.5% with this method. Concordant classification of patients by both tests according to proposed therapeutic ranges demonstrated fully concordant information in 76% and fully discordant information in none. This study demonstrates that chromogenic substrate assays for anticoagulant control can be readily automated. Amidolytic assays of factor X based on the S 2337 substrate, therefore, warrant further clinical investigation as a potential method for controlling maintenance oral anticoagulant therapy.     

Maintenance control of oral anticoagulant therapy by an automated chromogenic substrate assay of factor X

Summary An amidolytic assay of factor X based on the new chromogenic peptide substrate S 2337 (Kabi Diagnostica) was adapted for use with the Kem-o-Mat (Coulter Electronics) automated substrate analyser. Factor X was assayed in 25 healthy controls and in 375 patients on Warfarin therapy. The results in the control group correlated well with a one stage coagulation factor X assay. A good correlation was also found when the S 2337 factor X assay was compared with Thrombotest (Nyegaard & Co.) results in the patients. From the regression line of the S 2337 factor X assay on the Thrombotest results, the comparable range for factor X amidolytic activity in well controlled anticoagulated patients was found to be 22.5-37.5% with this method. Concordant classification of patients by both tests according to proposed therapeutic ranges demonstrated fully concordant information in 76% and fully discordant information in none. This study demonstrates that chromogenic substrate assays for anticoagulant control can be readily automated. Amidolytic assays of factor X based on the S 2337 substrate, therefore, warrant further clinical investigation as a potential method for controlling maintenance oral anticoagulant therapy.     

Recombinant factor IX: discrepancies between one‐stage clotting and chromogenic assays

New and modified recombinant factor IX (rFIX) products are in development and accurate potency estimation is important to ensure the consistency of production and efficacy of these therapeutics. Collaborative study data obtained during the replacement of the 3rd International Standard (IS) for FIX concentrate suggested that there was a discrepancy between potency estimates for rFIX using clotting and chromogenic methods, when the rFIX candidate was measured against the plasma‐derived FIX (pdFIX) IS. This study explores potential chromogenic and one‐stage clotting method discrepancies in more detail. Five batches each of rFIX and pdFIX were assayed against the 4th IS FIX concentrate (a pdFIX) by activated partial thromboplastin time (APTT) one‐stage clotting assay and specific functional chromogenic assay. The potency of rFIX by chromogenic assay was consistently around 70% of the one‐stage clotting potency (average 78 and 108 IU mL^?1 respectively). These differences were not observed with pdFIX, which had similar potencies (average 96 IU mL^?1) by each assay method. In addition, different APTT reagents yielded different potency estimates for rFIX when assayed against the pdFIX IS, with a variation of up to 23%. In all cases, the differences were largely resolved when a rFIX reference was used as the standard. This study highlights some of the challenges associated with assay of rFIX products in the laboratory and that careful consideration needs to be given to the choice of reference material used. This is especially important with the imminent arrival of new and modified rFIX products.     

Control of anticoagulant therapy with a chromogenic substrate.

Prothrombin is determined with the aid of a recently developed assay, based on the amidolysis of a chromogenic substrate. The assay proved to be reliable when it was compared with more conventional coagulation assays in the control of oral anticoagulant therapy, both in the therapeutic range and in a case of overdosage. As is the case in coagulation tests, heparin therapy remains a disturbing circumstance. The prothrombin concentration was measured (a) in the plasma of 50 long-term anticoagulated patients, and the results were compared with those obtained with a one-stage coagulation assay and with those obtained with Thrombotest determinations, and (b) during vitamin K administration in the plasma of a patient with a severe intoxication of a vitamin K antagonist.     

Monitoring therapy with LMW heparin: a comparison of three chromogenic substrate assays and the Heptest clotting assay

Three LMW heparins (LMWH), one unfractionated heparin (UH), and international standards of LMWH and UH were compared in three chromogenic substrate (CS) assays and the 'Heptest' clotting assay. With a two-stage CS assay, linear standard curves were obtained in the 0.1-1.0 U/ml range, nearly coinciding for all preparations. With the one-stage CS assays, standard curves were curvilinear and similar for UH and the LMWH groups. In the Heptest assay, standard curves were linear for UH but not for LMWH. Mean recovery of LMWH, added to patients' plasma samples was 70-98% for the four assays. Variation between individual recoveries was much greater with Heptest (coefficient of variation (CV) 35-44%) than with one-stage CS assays (CV 14-21%) or two-stage CS assays (CV 7-8%). For monitoring LMW heparin therapy, CS assays seem preferable to Heptest. The two-stage CS assay had superior accuracy, but the one-stage CS assays were easier to perform.     

改良发色底物法测定胃肠道肿瘤患者外周血单核细胞中组织因子活性

目的：建立一种简便易行的测定组织因子（TF）活性的方法,研究胃肠道肿瘤患者外周血单核细胞中TF活性与肿瘤的关系。方法：采用已商品化的冻干人凝血酶原复合物（PPSB）作为因子Ⅶ,Ⅹ的来源,活化的Xa水解底物S2222释放对硝基苯胺,405nm处的吸光度与标准液中的组织凝血活酶含量成良好的双对数关系（r=0.998),采用此法测定32例胃癌,45例结直肠癌患者和30例正常人外周血单核细胞TF的促凝活性。结果：该方法简单,灵敏,能定量,性范围大,胃肠道肿瘤患者外周血单核细胞的活性明显高于对照组（P＜0．05）,且与肿瘤恶性程度成正比（P＜0．05）。结论：检测TF在外周血单核细胞中的活性水平将有助于评估肿瘤的恶性程度。,     

Automated assays for von Willebrand factor activity

von Willebrand factor (VWF) ristocetin cofactor activity (VWF:RCo) by platelet aggregometry has been considered the gold standard for evaluating the ability of VWF to bind platelets for over 40 years. Many automated systems no longer require platelets and rather rely on agglutination of latex particles. Automated methods of measuring VWF activity have improved performance characteristics and are performed on the same coagulation instruments used for routine testing via immunoturbidimetric methodology. Alternatively, a newer chemiluminescence assay system for measuring VWF activity demonstrates excellent performance characteristics. As these methods are becoming widely used, it is important to assess their performance in diagnosing and monitoring different types of von Willebrand disease. We review the automated methodologies and the published performance of these VWF assays. Advantages and limitations of these automated methods are discussed.     

Assessment of plasma fibrinolytic system with use of chromogenic substrate.

A block of tests employing a chromogenic substrate H-D-Val-Leu-Lys-pNA (S-2251, Kabi AB, Stockholm) is proposed for a quantitative assessment of the plasma fibrinolytic system. It allows measurement of spontaneous plasmin activity, plasminogen content, activator activity (ability of plasma to activate plasminogen), immediate and progressive antiplasmin levels and alpha2-macroglobulin. All these assays could be performed within 45 min using a total of 0.4 ml of plasmin.     

Role of chromogenic assays in haemophilia A and B diagnosis

The laboratory diagnosis and monitoring of factors VIII and IX have been primarily by one‐stage clotting assay (OSA) for many years. Chromogenic assays (CSA) have been available only in specialist laboratories and not for routine use. Significant differences, of more than 1.5‐fold in results between the 2 assay methods, have been described in Europe and Australia in approximately one‐third of patients with mild haemophilia A. In certain discrepant groups with restricted F8 gene mutations, the OSA results are more than 1.5‐fold higher than CSA and risk a missed or misleading diagnostic result. More recently, an assay discrepancy in haemophilia B has been reported. With the introduction of extended half‐life (EHL) FVIII and FIX products, it is likely most coagulation laboratories will need to evaluate at least one CSA and gain experience with this technique. The validation of CSA involves a careful appraisal of calibration curve linearity, limit of detection, precision, reference range, quality control material, sample analysis, method comparison and cost. This review will discuss the current status of FVIII and FIX CSA for the diagnosis of haemophilia A and B and describe approaches to implement CSA into the laboratory repertoire.