Performance characteristics of 5 numerical indexes in mixing test interpretation under coexistence of lupus anticoagulant and coagulation factor deficiency.

Background: The mixing test is useful to investigate the cause of unexpectedly prolonged activated partial thromboplastin time (APTT). Several indexes are available for distinguishing correction from non-correction (ie, factor deficiency from inhibitors), but their performance characteristics may differ because of their different formulas. Furthermore, it is unclear how each index performs under the coexistence of factor deficiency and inhibitors. Objectives: The objective of this study was to examine the differences in indexes, depending on factor VIII activity (FVIII:C) levels and lupus anticoagulant (LA) titers in test samples. Methods: APTT was measured in spiked samples with various FVIII:C levels and LA titers, normal pooled plasma (NPP), and their 4:1, 1:1, and 1:4 mixtures. The following 5 indexes were calculated: index of circulating anticoagulant, mixing test normalized ratio, 4:1 and 1:1 percent corrections, and an APTT difference between the 1:1 mixture and NPP. The samples with LA, showing correction, were measured for FVIII:C in a one-stage assay to check parallelism. Results: All indexes showed correction under FVIII deficiency and non-correction under higher LA titers. However, under lower LA titers, some indexes showed non-correction but others showed correction because of dilution effects and variations in formulas and/or sample mix ratios. The differences among the indexes were more pronounced under coexistent FVIII deficiency and LA, even though LA titers were equal in the tested samples; samples with lower FVIII:C showed correction, whereas those with normal FVIII:C showed non-correction. The samples tested for FVIII:C showed non-parallelism. Conclusion: Each index had different performance characteristics to LA samples, which were pronounced under low FVIII:C levels in test samples.

Inhibitor Index in the Clot Waveform Analysis-Based Mixing Test Differentiates among Hemophilia A without and with Inhibitors, and Lupus Anticoagulant.

BACKGROUND: The mixing test is used to identify the pathway to follow-up testing and is also useful for the investigation of lupus anticoagulant (LA) positivity. "To completely correct" indicates clotting factor deficiency, while "to not correct" indicates the presence of a clotting factor inhibitor including LA. "Index of circulation anticoagulant" and/or "percent correction" is used to interpret the results of mixing studies, but it does not accurately differentiate factor inhibitors from LA. AIM: To precisely differentiate hemophilia A (HA), HA with inhibitor (HA-inh), and LA using the clot waveform analysis (CWA)-based mixing test. METHODS: Plasma samples from HA, LA, and HA-inh including acquired HA were incubated with normal plasma in 9:1, 1:1, and 1:9 mix ratios. From activated partial thromboplastin time CWA at 0-minute (immediately) and 12-minute incubation, the ratios of CWA parameters at 12 minutes/0 minute (inhibitor index) were assessed. RESULTS: The inhibitor index values of CWA parameters obtained using the mixing test in a 1:1 ratio demonstrated a significant difference between HA-inh and LA but could not differentiate LA from HA-inh completely. Plasmas used for the mixing tests in 9:1 and 1:9 ratios were able to fully distinguish between HA-inh (>0.5 BU/mL) and LA. These indices significantly correlated with inhibitor titer below 40 BU/mL (r > 0.90), possibly estimating FVIII inhibitor titer from the inhibitor index. Plasmas in HA and LA could be distinguished by mixing in a 1:1 ratio at 0 minute (immediately). CONCLUSION: The inhibitor index from CWA-based mixing tests with a 12-minute incubation could differentiate among HA, HA-inh, and LA quickly.

Expert consensus regarding standardization of sample preparation for clotting time assays.

Accurate clotting time assay results are vital, as the test is employed to indicate the amount of oral anticoagulant to be prescribed, while it is also used for screening the hemorrhagic and thrombotic diseases. The procedure chosen for preparation of a patient blood sample including centrifugation can contribute to significant differences in the results obtained. Thus, for the purpose of proposing a standardized method to appropriately prepare blood samples prior to assay, the Japanese Society of Laboratory Hematology organized the Working Group for Standardization of Sample Preparation for Clotting Time Assays (WG). Following reviews of previously announced guidelines and original experimental results, consensus was obtained by the WG, with the main findings as follows. (1) The recommended anticoagulant in the blood collection tube is sodium citrate solution at 0.105-0.109 M (3.13-3.2%). (2) Whole blood samples should be stored at room temperature (18-25 ˚C) within 1 h of collection from the patient. (3) For plasma preparation, centrifugation at 1500 × g should be performed for at least 15 min or at 2000 × g for at least 10 min at room temperature. (4) After the plasma sample is prepared, it should be stored at room temperature and assayed within 4 h.

Prothrombin Time Tests for the Monitoring of Direct Oral Anticoagulants and Their Evaluation as Indicators of the Reversal Effect.

INTRODUCTION: The prompt assessment and the reversal of direct oral anticoagulants (DOACs) are urgent matters in the emergency care setting. Thus, we planned to elucidate the adequate prothrombin time (PT) test for the evaluation of the anticoagulant effects of various DOACs. METHODS: The anticoagulant effects of rivaroxaban, apixaban, and edoxaban were measured with 3 PT tests (Triniclot PT Excel S, Neoplastin R, and Thromborel S). Human plasma was spiked with each DOAC at a range of 0 to 1000 ng/mL, and the PT was measured using each PT test. In another series, the reversal effect of either 4-factor prothrombin complex concentrate (PCC) or activated PCC (aPCC) was evaluated with each PT test. RESULTS: All PT reagents correlated with the concentrations of each DOAC, however, the reactivity was considerably different between the DOACs and the PT tests. A prolonged PT with DOACs was reversed both by PCC and aPCC in a dose-dependent manner; however, Triniclot PT Excel S showed reprolongation of the PT with a higher dose of PCC. CONCLUSION: The proper choice of PT test is necessary for the assessments of the anticoagulant activity of DOACs. It is also important to understand the different characteristics of each PT test for the assessment of the reversal effects of PCC.

Comparison of prothrombin time tests used in the monitoring of edoxaban and their evaluation as indicators of the reversal effect.

Clinical demand for the prompt assessment of the activity of direct-acting factor Xa inhibitors in the emergency care setting is increasing. In the present study, we examined whether prothrombin time (PT) tests can serve as a clinically useful indicator of anti-factor Xa activity. In the first series, the in vitro effect of edoxaban on PT was evaluated by spiking human plasma with edoxaban and measuring PT using three different commercial PT tests. In the second series, the reversal effect of prothrombin complex concentrates (PCC) and activated PCC (aPCC) in edoxaban-spiked plasma was evaluated. In the third series, PT of plasma samples from patients administered either 15 or 30 mg/day of edoxaban was assessed, and the results were compared with edoxaban concentrations determined by a calibrated anti-factor Xa activity assay. The spike test revealed that all PT reagents positively correlated with edoxaban. The sensitivity to edoxaban varied among the three reagents and Triniclot(®) Excel S showed the best performance. Prolonged PT by edoxaban was reversed by PCC and aPCC in a dose-dependent manner; however, complete reversal was not achieved. Positive correlation between anti-factor Xa activity and PT was shown in the clinical samples at the edoxaban range from 0 to >300 ng/mL.