A chromogenic assay for activated protein C resistance

Summary. Resistance to activated protein C (APC) diagnosed on the basis of prolongation of clotting time in an activated partial thromboplastin time (aPTT) assay is now considered a major cause of inherited thrombophilia. The majority of patients with APC resistance carry a factor V molecule with a point mutation at one APC cleavage site (Arg506Gln) which prevents the optimal inactivation of activated factor V by APC. To overcome the limitations of aPTT-based assays in the diagnosis of APC resistance, we have developed a chromogenic assay which is based on the capacity of APC to limit the generation of factor Xa by inactivating factor Villa in plasma. The ratio of the factor Xa amidolytic activity in a sample without APC to its factor Xa activity with the addition of APC reflects the response of the plasma coagulation system to APC. The normal range in 44 healthy individuals was 1.62-2.06. APC response ratios as measured by the chromogenic assay correlated with ratios measured by the aPTT assay and were below the normal range in 23.24 individuals with Arg506Gln mutant factor V from three different families with familial thrombosis and from 11 unrelated asymptomatic individuals. In reconstitu-tion experiments, purified factor V corrected the decreased APC response in plasma samples from patients with the Arg506Gln mutation as well as with factor V deficiency, and increased the APC response in normal plasma, whereas the addition of activated factor V had no enhancing effect.     

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.     

Determination of antithrombin-dependent factor Xa inhibitors by prothrombin-induced clotting time

Prothrombinase-induced clotting time (PiCT) determines the anticoagulant effects of heparins, low molecular weight heparins (LMWHs), and direct thrombin inhibitors. At present, this is the only method that measures the effects of all of these inhibitors, in contrast to the prothrombin time, activated partial thromboplastin time (aPTT), Heptest, ecarin clotting time, and the chromogenic assays. The antithrombin-dependent direct factor (F) Xa inhibitors fondaparinux and idraparinux were compared with the LMWH dalteparin on PiCT, aPTT, Heptest, and chromogenic anti-FXa assays in pooled human normal plasma samples. Fondaparinux and idraparinux prolonged the coagulation times in the PiCT, Heptest, and chromogenic FXa assays in a dose-dependent manner, in contrast to the aPTT. We conclude that PiCT is a suitable assay to determine the anticoagulant effects of these two new FXa inhibitors in patients receiving treatment with these compounds.     

Drotrecogin alfa activated (recombinant human activated protein C) in combination with heparin or melagatran: effects on prothrombin time and activated partial thromboplastin time.

Recombinant human activated protein C (rhAPC) has recently been demonstrated to be a promising candidate to improve the outcome for patients with severe sepsis. Plasma-derived activated protein C and unfractionated heparin (UH) exert anticoagulant synergy due to mechanisms that simultaneously decrease thrombin generation. Melagatran, a new direct thrombin inhibitor, does not bind to plasma proteins or requires antithrombin as a cofactor. The latter is often consumed in patients with severe sepsis. We investigated the anticoagulant efficiency in combined administration of rhAPC and UH or melagatran in terms of prolongation of the standard clotting assays activated partial thromboplastin time (aPTT) and prothrombin time (PT) in pooled plasma samples in vitro. RhAPC dose-dependently prolonged the aPTT but not the PT. The ability of UH and melagatran to prolong the aPTT was significantly enhanced in combination with rhAPC. The combined administration of rhAPC and melagatran, but not UH, resulted in additive prolongation of the PT. In control measurements the capability of rhAPC to suppress prothrombin fragment 1.2 generation dose-dependently increased in combination with heparin and melagatran. Our study demonstrates the respective effects of rhAPC, UH, melagatran and further different additive effects in combined administration of rhAPC and UH or melagatran on the prolongation of the aPTT and PT clotting assays usually used to monitor anticoagulant treatment.     

Impact of telavancin on prothrombin time and activated partial thromboplastin time as determined using point-of-care coagulometers

Telavancin is approved in the United States, Canada, and Europe (At the time of submission, the telavancin European marketing authorization for nosocomial pneumonia was suspended until Theravance provides evidence of a new European Medicines Agency approved supplier) as an antibiotic to treat certain Gram-positive bacterial skin infections. Telavancin has been shown to prolong plasmatic prothrombin (PT) and activated partial thromboplastin (aPTT) clotting times in clinical diagnostic lab-based assays. In this study, we evaluated the potential for telavancin to prolong whole blood PT/International Normalized Ratio (INR) and aPTT tests on point-of-care (POC) instruments. Whole blood collected from 8 healthy subjects was supplemented with telavancin to final concentrations of 0, 10, 20, and 100 μg/ml. Final concentrations were selected to match trough, twice trough, and peak plasma levels following the approved 10 mg/kg dose. Four widely employed POC coagulation instruments were chosen to be representative of the POC platforms currently in use.. These systems were the Roche Coaguchek XS, the Abbott iSTAT, the ITC Hemochron SIG+, and the Alere INRatio2 POC devices. The PT/INR measured by the Coaguchek XS showed the greatest sensitivity to the presence of telavancin. The PT/INR measured by the Hemochron SIG+ and iSTAT were sensitive to telavancin but to a lesser extent. The INRatio2 was the least sensitive to the presence of telavancin when testing the whole blood PT/INR. Only the Hemochron SIG+ device was capable of measuring aPTT and showed a concentration-dependent increase in aPTT. This study supports the current recommendation that PT and aPTT monitoring be conducted immediately to the next dose of telavancin when coagulation parameters are tested using POC instrumentation.     

Assaying the circulating factor VIII activity in hemophilia A patients treated with recombinant factor VIII products

Large discrepancies between factor VIII assay methods have been reported from pharmacokinetic studies of recombinant factor VIII concentrates. In the assay of postinfusion patient plasma samples, traditional activated partial thromboplastin time (aPTT)-based one-stage clotting methods usually give results that are 20 to 50% lower than those obtained by chromogenic substrate assays. Investigations into the cause of these discrepancies have shown that the choice of phospholipid in the one-stage assay is crucial. The use of platelets or liposomes resembling platelet factor 3 instead of traditional aPTT reagents results in an increase in the apparent one-stage activity and a fairly good correlation with the chromogenic results. These and other functional test results, antigen measurements as well as clinical data, support the view that the chromogenic assay most accurately reflects the therapeutic effect. In addition to the differences among assay methods, there is also a discrepancy between the World Health Organization (WHO) standards for concentrates and plasma. The use of product-specific standards, prepared by diluting the factor VIII concentrate into hemophilic plasma, when assaying postinfusion plasma samples seems to be a feasible approach to overcome the problems encountered in pharmacokinetic studies.     

Monitoring the new antithrombotic drugs

Today there is a diverse group of anticoagulant and antithrombotic drugs available that includes warfarin derivatives, heparin, low-molecular-weight heparins, thrombin inhibitors, factor Xa inhibitors, and various antiplatelet agents. Many of these new drugs do not alter measurable blood coagulation parameters, yet they are effective antithrombotic agents through their actions on vascular endothelial cells and proteins. Thus, these new agents do not affect the traditional clot-based prothrombin time/International Normalized Ratio (PT/INR) and activated partial thromboplastin time (aPTT) tests, and monitoring and standardization require the development of new methods. In addition to clot-based assays, chromogenic assays, enzyme-linked immunosorbent assay (ELISA), high-performance liquid chromatography (HPLC), flow cytometry, and other techniques have been used to monitor these new drugs. On the other hand, some of the new antithrombotic drugs do affect the PT, aPTT, and activated clotting time (ACT); however, they behave differently from the warfarin derivatives and heparin. The traditionally used relationship of target time to clot values and INR to clinical effect cannot necessarily be transferred to the new drugs. Unfortunately, monitoring is not as simple as it was for warfarin and heparin. Although the new antithrombotic drugs have been approved for clinical use, assay systems for monitoring most of them are still in development or have not been clinically validated. This applies to each of the clinical settings targeted for prophylaxis, treatment, or interventional procedures (i.e., high- and low-dosing regimens typically require different monitoring methods). In addition to basic monitoring, other issues such as sensitivity of the drug to different laboratory monitoring reagents and instrumentation, drug combination monitoring, and patient-related factors that contribute to the variability of the results still need to be addressed.     

Effects of a heparin-binding protein on blood coagulation and platelet function.

The objective of this study was to characterize the heparin-binding properties of a protein secreted by mouse myeloma cells. The characterization was performed using clinical assays, such as heparin activity assays and heparin-induced thrombocytopenia (HIT) platelet activation assays. The tests were performed in the presence of heparin, low-molecular-weight heparins (LMWH), or heparinoids and either heparin-binding protein (HBP) or saline to determine whether the HBP affects the activity of heparins. The characterization of the HBP using heparin activity assays showed that the HBP shortened the prolonged clotting times of the activated partial thromboplastin time (aPTT) and thrombin clotting time induced by high concentrations of unfractionated heparin. The chromogenic assays for antithrombin (AT), thrombin inhibition, and factor Xa inhibition demonstrated that this effect is related to heparin concentrations below 0.5 IU/ml. The Heptest assay did not detect these differences. The HBP did not modify the anticoagulant effect of any LMWH or low- or high-sulfated glycosaminoglycans in the aPTT assay. Activation of donor platelets in the presence of unfractionated heparin, platelet factor 4 (PF4), and HIT-serum was not counteracted by the HBP in any of the assays. The characterization of the HBP using a PF4-enzyme-linked immunosorbent assay (ELISA) confirmed the lack of structural identity with PF4. However, the optical density data indicated that the protein structure may be similar to PF4 by binding to a PF4 antibody. These data suggest that the HBP isolated from mouse myeloma cells has a low affinity to heparin and interacts with the secondary binding site to AT and also perhaps to PF4.     

Shifting landscape of hemophilia therapy: Implications for current clinical laboratory coagulation assays

Clinical coagulation assays are an integral part of diagnosing and managing patients with hemophilia; however, in this new era of bioengineered factor products and nonfactor therapeutics, problems have arisen with use of traditional coagulation tests for the quantification of several of these new products. Discussion over the use of one‐stage clotting assays versus chromogenic substrate assays for clinical decision making and potency labeling has been ongoing for many years. Emerging factor concentrates have heightened concern over assay selection and availability. Emicizumab interferes with all aPTT based assays, rendering them unreliable and potentially falsely reassuring to the unaware provider. This review explores considerations for coagulation assays in the clinical setting and highlights how awareness of institutional coagulation assays and potential limitations have never been more critical for providers caring for patients with bleeding disorders.     

FIXing postinfusion monitoring: Assay experiences with N9‐GP (nonacog beta pegol; Refixia®; Rebinyn®)

N9‐GP (nonacog beta pegol; Refixia^®; Rebinyn^®, Novo Nordisk A/S, Bagsværd, Denmark) is a glycoPEGylated extended half‐life recombinant factor IX (rFIX) that exhibits efficacy and potency comparable to unmodified FIX molecules in non‐clinical models. Phase 3 clinical trials have confirmed the efficacy and tolerability of N9‐GP for the prevention and on‐demand treatment of bleeding episodes in patients with haemophilia B. Recent studies have shown that PEGylation affects clotting times in activated partial thromboplastin time (aPTT)‐based one‐stage activity assays due to interaction between the FIX molecule and certain aPTT reagents. In recognition of the challenges surrounding FIX activity assessment, the identification of consistent, reproducible and accurate assays to measure FIX activity has been a priority for Novo Nordisk, running in parallel to the clinical development program for N9‐GP. N9‐GP activity can be reliably measured using chromogenic substrate assays and specific aPTT reagents. The conjugation of the PEG moiety to the FIX molecule may affect one‐stage aPTT‐based clotting assays in a reagent‐dependent manner. Many aPTT reagents that use silica as the contact activator dramatically overestimate N9‐GP activity due to premature activation. On the other hand, the contact activator in some other aPTT reagents negatively affects the enzymatic activity of FXIa, causing the underestimation of N9‐GP activity. While N9‐GP activity cannot be measured consistently with all available aPTT reagents, accurate N9‐GP measurements can be achieved with certain aPTT reagents. Here, we review the studies that led to these findings and summarize the current options for accurate measurement of N9‐GP in patient 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.     

Methods for the monitoring of direct thrombin inhibitors

Direct thrombin inhibitors are available for prophylactic as well as therapeutic purposes. Application of hirudin in therapeutic doses has been shown to require drug monitoring. Currently, most experience is available for recombinant hirudin, but the principle aspects of drug monitoring are the same for all direct thrombin inhibitors. Most frequently, activated partial thromboplastin time (aPTT) and modifications of the activated clotting time (ACT) have been used for the monitoring of hirudin therapy. However, these methods are insensitive at plasma levels higher than 0.6 mg/L of hirudin, so that overdoses may be missed despite monitoring. Correlations between ecarin clotting time (ECT), enzyme immunoassays, and chromogenic substrate assays on one side and global tests on the other side are poor. Fully automated chromogenic substrate-based assays, also available as point-of-care tests (POCT), are more precise and sensitive and are not disturbed by interferents such as heparin and antithrombin. Good correlations can be observed between chromogenic assays and the ECT performed in plasma or whole blood samples. ECT can also be determined with POCT systems. Test characteristics such as imprecision and measuring range are comparable to those of the chromogenic assays. In conclusion, therapy with direct thrombin inhibitors should be monitored with chromogenic assays or ECT.     

Multilaboratory testing of thrombophilia: current and past practice in Australasia as assessed through the Royal College of Pathologists of Australasia Quality Assurance Program for Hematology

We have conducted a series of multilaboratory surveys during the last 6 years to evaluate testing proficiency in the detection of congenital and acquired thrombophilia. For lupus anticoagulant (LA) testing, participant laboratories used a panel of tests, including activated partial thromboplastin time (aPTT; 100% of laboratories), kaolin clotting time (26 to 70%), and Russell's viper venom time (RVVT; 75 to 100%). Coefficients of variation (CVs) for assays ranged from 5 to 40%. RVVT assays appeared to be most sensitive and specific for detection of LA (fewer false-negatives or -positives), although laboratories performed best when they used a panel of tests. For congenital thrombophilia, tests evaluated comprised protein C (PC), protein S (PS), antithrombin (AT), and activated protein C resistance (APCR). Most participant laboratories performed PC using chromogenic (approximately 75%), or clot based (approximately 15%) assays, with few (< 10%) performing antigenic assessments. PS was most often assessed (approximately 60%) by immunological or antigenic assays, usually of free PS, or by functional or clot-based assays (approximately 40%). AT is usually assessed by functional chromogenic assays (approximately 95%). APCR was assessed using aPTT (approximately 50%) or RVVT (approximately 50%) clot-based assays, with the aPTT APCR typically performed using factor V-deficient plasma predilution, but the RVVT APCR typically performed without. Laboratories using the RVVT APCR generally performed better in detection of factor V Leiden-associated APCR, with the aPTT method group yielding higher false-negative and/or false-positive findings (approximately 5% of occasions). Some clot-based PC and PS assays appeared to be influenced by APCR status, and yielded lower apparent PC and PS levels with positive APC resistance. The overall error rate for PC, PS, and AT was approximately 2 to 8% (i.e., false-normal interpretations for deficient plasma or false-abnormal interpretations for normal plasma). The CVs for these assays ranged from 5 to 40%, with highest CVs typically obtained with PS assays.     

Specific and global coagulation assays in the diagnosis of discrepant mild hemophilia A

The activity of the factor VIII coagulation protein can be measured by three methods: a one or two-stage clotting assay and a chromogenic assay. The factor VIII activity of most individuals with mild hemophilia A is the same regardless of which method is employed. However, approximately 30% of patients show marked discrepancies in factor VIII activity measured with the different methods. The objective of this study was to investigate the incidence of assay discrepancy in our center, assess the impact of alternative reagents on factor VIII activity assays and determine the usefulness of global assays of hemostasis in mild hemophilia A. Factor VIII activity was measured in 84 individuals with mild hemophilia A using different reagents. Assay discrepancy was defined as a two-fold or greater difference between the results of the one-stage and two-stage clotting assays. Rotational thromboelastometry and calibrated automated thrombography were performed. Assay discrepancy was observed in 31% of individuals; 12% with lower activity in the two-stage assay and 19% with lower activity in the one-stage assay. The phenotype could not always be predicted from the individual’s genotype. Chromogenic assays were shown to be a suitable alternative to the two-stage clotting assay. Thromboelastometry was found to have poor sensitivity in hemophilia. Calibrated automated thrombography supported the results obtained by the two-stage and chromogenic assays. The current international guidelines do not define the type of assay to be used in the diagnosis of mild hemophilia A and some patients could be misclassified as normal. In our study, 4% of patients would not have been diagnosed on the basis of the one-stage factor VIII assay. Laboratories should use both one stage and chromogenic (or two-stage) assays in the diagnosis of patients with possible hemophilia A.     

Oral anticoagulant control with a chromogenic substrate: calibration,precision and cost

Prothrombin time ratios, and bioassays and chromogenic assays for factors VII and X, were obtained on 23 newly anticoagulated patients and 23 patients on long term oral anticoagulants. Factor levels were plotted against prothrombin time ratios. For factor VII, a different relationship was apparent for newly anticoagulated and for stabilized patients by both assay methods, but for factor X the same relationship was found. Chromogenic factor-X levels were calibrated against prothrombin time, and a prescribing exercise showed reasonable agreement between doses based on prothrombin time ratios and on assays for both clinical groups; factor-VII levels could not be used because of the different relationship between newly anti-coagulated and long-term patients. Despite the high precision of reading the chromogenic end point, chromogenic assays were not more precise than bioassays. With S2222, the cost of automated anticoagulant control would be more expensive than control by manual prothrombin times.     

Clotting alterations in primary systemic amyloidosis

BACKGROUND AND OBJECTIVE: The bleeding manifestations frequently observed in patients with immunoglobulin light chain amyloidosis (AL) have been attributed to different pathogenetic factors: amyloid deposits in several organs and systems leading to failures of these latter, the affinity of amyloid for some clotting factors, and the presence of plasma components interfering with fibrin formation could all induce alterations of clotting tests. This investigation was aimed at defining the prevalence of clotting abnormalities and their clinical manifestations in patients with AL. DESIGN AND METHODS: Thirty-six consecutive patients with biopsy proven amyloidosis and documented monoclonal gammapathy were enrolled within one year. The following clotting tests were considered in the study: activated partial thromboplastin time (aPTT), prothrombin time (PT), thrombin time (TT), reptilase time (RT), Russell's viper venom time (RVTT), fibrinogen, factor X and alpha-2 antiplasmin. RESULTS: Hemorrhagic manifestations were mild to moderate in nine patients, but severe and untractable in one. The most frequent clotting anomaly was defective fibrinogen conversion to fibrin, as demonstrated by prolongation of both TT (85% of cases) and RT (90% of cases). Low levels of factor X activity were observed in about 1 out of 4 samples, while fibrinogen and alpha2 antiplasmin levels were distributed over a wide range of values. PT was prolonged in 8 and aPTT in 25 patients. The search for lupus anticoagulant was negative in samples showing a prolongation of aPTT and/or RVVT. INTERPRETATION AND CONCLUSIONS: The prolongation of TT and RT is not dependent on either the presence of a heparin-like substance in the plasma or on fibrinogen levels; furthermore, the prolongation of RVVT is not related to factor X level. The hypothesized presence in the plasma of an inhibitor of fibrin formation could also affect factor X activation by Russell viper venom. The prolongation of TT and RT represents a peculiar feature of amyloidosis. The variability in the behavior of the other clotting times and hemostatic factors studied is mirrored in the heterogeneity of the clinical features observed in this disease.     

Factor VIII inhibitors. Laboratory diagnosis of inhibitors

The diagnosis of inhibitors of blood coagulation is often the most challenging problem in the clinical laboratory. Immediate attention must be given to the following patient groups whose principal laboratory abnormality is the prolonged activated partial thromboplastin time (aPTT): the patient with (1) hemophilia who previously responded to an adequate dose of clotting factor product and now fails to show effective clinical response to the same replacement concentrate; (2) previously benign clinical history who now presents with soft tissue bleeding or emergent internal hemorrhaging; (3) sudden onset of generalized ecchymoses who was previously well; (4) postpartum state; (5) malignancy, lymphoma, rheumatoid arthritis, or other autoimmune disorders; and (6) drug reactions. Immediate attention must be given to the prolonged prothrombin time (PT), aPTT, and thrombin time (TT) in order to respond to urgent queries from a perplexed internist, hematologist, intensivist, or surgeon caring for a patient with unexpected bleeding. Sometimes the problem of a prolonged "clotting time" arises preoperatively, causing unanticipated delay in operative procedures. For this reason, the laboratory support, usually in the coagulation section of a clinical laboratory or reference laboratory, must be quick, unequivocal and precise. The most common finding is an isolated mild, moderate, or severe prolongation of the aPTT with a normal PT, TT, and platelet count. The aPTT mixing study (The Mix), usually modified for time and temperature, along with appropriate controls, is the seminal test. This is the basis for all further testing. It may be supported by direct factor assays, and, therefore, the laboratory must know the reagent responsiveness and sensitivity for each clotting factor. By definition, complete correction of the aPTT in a 1:1 mix of patient and reference plasma is a factor deficiency. In this article, incomplete or minimal correction of The Mix will be characterized with particular attention to the various inhibitor assays, in other words, Oxford, Bethesda, and Nijmegen assays and the enzyme-linked immunosorbent assay (ELISA). An investigative approach to final characterization of the intensity (quantification) of the inhibitor and the exclusion of a lupus anticoagulant (LA) will be discussed.     

Development of a simple chromogenic factor VIII assay for clinical use

The aim of this study was the development of a simple chromogenic factor VIII assay for practical clinical use. The criteria that the assay fulfils are: (1) The method is so sensitive that even 1% factor VIII in human plasma is easily detected. (2) The method is linear in the amount of factor VIII from 0 to 200% in plasma. (3) The pipetting scheme is very simple; two reagents are prepared, reagent 1 (factor IXa, thrombin, Ca2+ and phospholipids) and reagent 2 (factor X). Then we pipet at t = 0 s, 100 microliters diluted plasma + 100 microliters reagent 1 in a reaction tube; at t = 30 s, 100 microliters reagent 2 in the same tube and at t = 90 s, 200 microliters of the reaction mixture in a cuvette with 700 microliters EDTA buffer (stop buffer) and the formed factor Xa is measured with a chromogenic substrate. (4) The reaction components are stable during at least a whole working day. Factor VIII was measured in an assay using bovine clotting factors, so one avoids the risk of viral infections, which one might catch by working with clotting factors isolated from human plasma.     

Investigation of genotype-dependent differences in factor V activity as well as response to activated protein C by application of different methods.

Coagulation factor V has been at the centre of investigation for several years. In addition to factor V Leiden, various other polymorphisms are becoming the object of interest. Different results have been published about the association of the HR2 haplotype with decreased factor V levels and with reduced response to activated protein C (APC). Due to the central position of factor V in the clotting process, its activity can be determined in both thromboplastin-based and activated partial thromboplastin time (aPTT)-based assays. A multitude of assays are known for the determination of APC response. The aim of our study was to investigate whether different methods disclose genotype-dependent differences in factor V activity as well as APC response. Three wild-type carriers, three carriers homozygous for the R2 allele (4070G), and three carriers homozygous for the G allele (2391G, 2663G, 2684G, 2863G) were investigated. For each individual plasma sample, the factor V activity was determined using 12 different reagent combinations of three different thromboplastins, three different aPTT reagents, and two different factor V deficient plasma sources. The determination of factor V activity in the thromboplastin system revealed differences between the genotypes. These differences were independent of the thromboplastin reagent and the factor V-deficient plasma. The aPTT system exhibited a dependency on the aPTT reagent and the factor V-deficient plasma. Analysis of APC response disclosed genomic differences in specific test systems only. One type of assay could be more appropriate than other types in dependence of the position of genomic variations. Therefore, the applied assay is an important influential factor in investigations of functional consequences of genomic variations.     

In vitro reversal of supratherapeutic rivaroxaban levels with coagulation factor concentrates

BackgroundA bleeding patient undergoing therapy with new oral anticoagulants is every clinician’s nightmare as no specific reversal agent is available yet. This in vitro study investigated the effect of prothrombin complex concentrate (PCC), recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (aPCC) on supratherapeutic rivaroxaban concentrations using standard laboratory parameters (prothrombin time [PT], activated partial thromboplastin time [aPTT] and PT ratio) and thromboelastometry (clotting time [CT]).ResultsThe addition of the reversal agents had the following statistically significant effects (all p<0.01): +25 IU/kg PCC: CT −15 s, aPTT +5 s; +50 IU/kg PCC: aPTT +11 s; +90 μg rFVIIa: CT −141 s; +25 IU/kg aPCC: CT −142 s, aPTT −9 s, PT ratio +14%, PT −10.5 s; +50 IU/kg aPCC: CT −118 s, aPTT −7 s, PT ratio +17%, PT −12.2 s.DiscussionrFVIIa and aPCC, but not PCC, appear to shorten coagulation times significantly in standard laboratory and thromboelastometry assays. These results need confirmation through evaluation of these agents in the clinical setting.