Anti-coagulation assessment with prothrombin time and anti-Xa assays in real-world patients on treatment with rivaroxaban.

Monitoring of anti-coagulation with the direct factor Xa inhibitor rivaroxaban is considered unnecessary in a routine clinical setting. However, assessment of its anti-coagulant effect may be desirable in certain clinical situations. We assessed prothrombin time (PT) reagents and commercially available anti-Xa assays (Biophen) calibrated for rivaroxaban and heparin in comparison to liquid chromatography-mass spectrometry (LC-MS/MS) measurements of rivaroxaban concentration in samples from patients on treatment with rivaroxaban for stroke prevention in atrial fibrillation. Citrate plasma samples were obtained from 30 randomly selected patients on uninterrupted treatment with rivaroxaban for a minimum of 1 month. The anti-Xa assays, direct Xa inhibitor (DiXa-I®), and Heparin LRT® were conducted for both wide and low calibrations for rivaroxaban. Measurements were compared to LC-MS/MS using correlation, linear regression, intra-class correlation, and Bland-Altman analysis. In 30 patients (9 female) of median age 71.5 years and BMI 26.5 kg/m(2), rivaroxaban concentrations between 2.4 and 625 ng/ml (median 82 ng/ml) were measured by LC-MS/MS. PT reagents were poorly correlated with rivaroxaban concentrations (r (2) = 0.52 and 0.09). Anti-Xa assays DiXa-I (r (2) = 0.95) and Heparin LRT (r (2) = 0.97) were correlated with rivaroxaban in all concentrations, but especially in low concentrations with low calibrations (r (2) = 0.97 and 0.98, respectively). The highest agreement occurred between Heparin LRT and low rivaroxaban concentrations with a mean difference of -5.3 ng/ml (limits of agreement, 12.9 to 2.4 ng/ml). Anti-Xa assays can indirectly determine the concentration of rivaroxaban for a wide range of concentrations in real-world patients. An interpretation of anti-Xa and PT measurements in treatment with rivaroxaban requires knowledge of the local reagents.

Evaluation of the automated coagulation analyzer CS-5100 and its utility in high throughput laboratories.

BACKGROUND: Automated analyzers are an important component of modern laboratories. As a representative of the newest generation of coagulation analyzers, the CS-5100 features several technical refinements including a pre-analytical assessment unit as well as multi-wavelength optical detection units. Therefore, the CS-5100 is supposed to rapidly and accurately perform a broad panel of coagulation tests. In the current study, the CS-5100 was evaluated regarding its precision and practicability in a clinical laboratory setting. METHODS: The CS-5100 was evaluated regarding its intra- and inter-assay precision using commercially available control samples. RESULTS of patient samples, including hemolytic, icteric and lipemic specimens, measured on the CS-5100 were compared to reference analyzers, which are used in our accredited laboratory. RESULTS: The coefficients of variation, assessed in the intra- and inter-assay precision analyses were below 5% representatively for most parameters. RESULTS, obtained by the CS-5100 showed predominantly a high comparability to used reference analyzers, with correlation coefficients ranging from 0.857 to 0.990. Only minor ranged systemic or proportional differences were found in Passing-Bablok regression between the CS-5100 and reference analyzers regarding most of the tested parameters. Lipemic samples had a tendency to deteriorate correlation coefficients, but an overall effect of the sample's triglyceride level could be ruled out. In a routine setting, the analyzer reached a sample throughput rate of 160 tests per hour. CONCLUSIONS: The CS-5100 is able to rapidly and precisely measure patient samples. No considerable influence on test comparability was found for elevated levels of free hemoglobin, bilirubin or triglycerides.

Limitations of a calibrated, quantitative APC-R assay under routine conditions.

INTRODUCTION: The aim of this study was to evaluate the Hemoclot Quanti. V-L assay in various clinical conditions. METHODS: We compared the Hemoclot Quanti.V-L assay with DNA testing and with the Pefakit assay in 60 normal (no mutation) vs carriers of the factor V (FV) Leiden mutation (56 heterozygous and three homozygous). We further investigated the interference of lupus anticoagulant on test results in normal and heterozygous individuals and of direct oral anticoagulants (DOACs) at trough and peak levels. Additionally, DOAC-Remove was tested in samples containing DOACs at peak levels. We further evaluated the influence of FV deficiency on this quantitative assay. RESULTS: There was a 100% agreement between the Quant. V-L assay and DNA testing in 60 normal individuals. However, 1.85% of heterozygous and 33% of homozygous samples were falsely classified with the quantitative assay, and no misclassification was observed with the Pefakit assay. Lupus anticoagulant did not influence the test results of the quantitative assay. DOACs also interfered with test results in heterozygous patients, but this effect was prevented with the DOAC-Remove procedure. Even mild FV deficiency affected the test results of the quantitative assay in heterozygous patients leading either to misclassification or the need for subsequent PCR testing. CONCLUSION: The quantitative FV-L assay has several limitations, especially FV deficiency and the presence of DOACs have to be ruled out before running this quantitative assay.

Interference in specialized coagulation assays affecting the protein C pathway: Effects of marked haemolysis, hyperbilirubinaemia and lipaemia on chromogenic and clotting tests on two coagulation platforms.

BACKGROUND: Haemolysis, lipaemia and hyperbilirubinaemia represent important challenges in the coagulation laboratory. Test results are influenced not only by the degree of the interfering substance but also by the detection system. METHODS: We investigated the interference of free haemoglobin, triglycerides and bilirubin on a "modified activated protein C (APC) resistance test," protein C activity and protein S (antigen and activity) with two coagulation analysers, the STA-R Evolution and the ACL TOP. RESULTS: Haemolysis interfered with all assays on the STA-R Evolution resulting in higher levels of protein C activity and lower levels of protein S and a decreased APC ratio compared with baseline levels. On the ACL TOP, haemolysis only diminished protein S antigen levels and the APC ratio. Lipaemia increased protein C activity and protein S activity levels on the STA-R Evolution, whereas APC-R decreased on the ACL TOP and protein S antigen could not be measured in any lipaemic samples. Hyperbilirubinaemia caused an increase in protein C activity and in protein S antigen and a decrease in APC-R on the STA-R Evolution, whereas a decline of protein C activity, of protein S antigen and of the APC-R could be observed in icteric samples on the ACL TOP. CONCLUSIONS: Our data show that the degree of interference associated with haemolysis, lipaemia and hyperbilirubinaemia is different in several assays. Some assay limitations were not reproduced, and limitations stated in kit inserts cannot be assumed to apply to all analysers.

The Effect of 3.2% and 3.8% Sodium Citrate on Specialized Coagulation Tests.

CONTEXT: - Coagulation testing is challenging and depends on preanalytic factors, including the citrate buffer concentration used. OBJECTIVE: - To better estimate preanalytic effects of the citrate buffer concentration in use, the difference between results obtained by samples with 3.2% and 3.8% citrate was evaluated. DESIGN: - In a prospective observational study with 76 volunteers, differences related to the citrate concentration were evaluated. For both buffer concentrations, reference range intervals were established according to the recommendations of the C28-A3 guideline published by the Clinical and Laboratory Standards Institute. RESULTS: - In our reagent-analyzer settings, most parameters evaluated presented good comparability between citrated samples taken with 3.2% and 3.8% trisodium buffer. The ellagic acid containing activated partial thromboplastin time reagent (aPTT-FS) indicated a systemic and proportional difference between both buffer concentrations, leading to an alteration in its reference ranges. Further, a confirmation test for lupus anticoagulant assessment (Staclot LA) showed only a moderate correlation ( rρ = 0.511) with a proportional deviation between both citrate concentrations. Further, a statistically significant difference was found in the diluted Russell viper venom time confirmation testing, coagulation factors V and VIII, and the protein C activity, which was found to be of minor clinical relevance. CONCLUSIONS: - With caution regarding the potential impact of the reagent-analyzer combination, our findings demonstrate the comparability of data assessed with 3.2% and 3.8% buffered citrated plasma. As an exception, the aPTT-FS and the Staclot LA assay were considerably affected by the citrate concentration used. Further studies are required to confirm our finding using different reagent-analyzer combinations.

Testing lupus anticoagulants in a real-life scenario - a retrospective cohort study.

INTRODUCTION: Lupus anticoagulant (LAC) testing is challenging. Most data are derived from a well-controlled study environment with potential alterations to daily routines. The aim of this retrospective cohort study was to assess the capacity of various LAC screening tests and derived mixing tests to predict a positive result in subsequent confirmation tests in a large cohort of patients. MATERIALS AND METHODS: In 5832 individuals, we retrospectively evaluated the accuracy of the aPTT-A, aPTT-LAscreen, aPTT-FS and dRVVTscreen and of their derived mixing tests in detecting a positive confirmation test result within the same blood specimen. The group differences, degree of correlation and the predictive accuracy of LAC coagulation tests were analysed using the Mann-Whitney U test, the Spearman-rank-correlation and by area under the receiver operating characteristic curve (ROC-AUC) analysis. ROC-AUCs were compared with the Venkatraman´s permutation test. RESULTS: The pre-test probability of patients with clinically suspected LAC was 36% in patients without factor deficiency or anticoagulation therapy. The aPTT-LAscreen showed the best diagnostic accuracy with a ROC-AUC of 0.84 (95% CI: 0.82 - 0.86). No clear advantage of the dRVVT-derived mixing test was detectable when compared to the dRVVTscreen (P = 0.829). Usage of the index of circulating anticoagulant (ICA) did not improve the diagnostic power of respective mixing tests. CONCLUSIONS: Among the parameters evaluated, aPTT-LAscreen and derived mixing test parameters were the most accurate tests. In our study cohort, neither other mixing test nor the ICA presented any further advantage in LAC diagnostics.

Lupus-anticoagulant testing at NOAC trough levels.

Non-vitamin K antagonist oral anticoagulants (NOAC), including rivaroxaban, apixaban or dabigatran, regularly show relevant effects on coagulation tests, making the interpretation of results difficult. The aim of this study was to evaluate possible interferences of NOACs in trough level concentrations in lupus anticoagulant (LA) testing. Citrate plasma specimens of 30 healthy volunteers were spiked with rivaroxaban, apixaban or dabigatran in four plasma concentration levels at or below trough NOAC levels. The NOAC concentration was measured using dedicated surrogate concentration tests and a stepwise diagnostic procedure for LA-testing was applied using screening, mixing and confirmatory testing. Results were compared to NOAC-free specimens. Starting with a plasma concentration of 12.5 ng/ml, dabigatran-spiked specimens showed significant prolongations in the lupus anticoagulant-sensitive activated partial thromboplastin time (aPTT-LA) as well as in the Dilute Russell viper venom time (dRVVT), leading to 43.3 % false positives in confirmatory testing in the dRVVT. In contrast, rivaroxaban, beginning with 7.5 ng/ml, exclusively affected dRVVT-based tests. In confirmatory tests, 30.0 % of rivaroxaban-spiked specimens showed false positive results. Starting with 18.75 ng/ml apixaban, a significant prolongation of the dRVVT and up to 20.7 % false positives in confirmatory tests were found. In contrast to other NOACs tested, apixaban did not present with a dose-dependent increase of the dRVVT ratio. In conclusion, the rate of false positive results in LA-testing is unacceptably high at expected trough levels of NOACs. Even at plasma concentrations below the LLOQ of commercially available surrogate tests, LA testing is best avoided in patients with NOAC therapy.