In vitro evaluation of a new viscoelastometry-based point-of-care analyzer.

INTRODUCTION: The VCM is a point-of-care analyzer using a new viscoelastometry technique for rapid assessment of hemostasis on fresh whole blood. Its characteristics would make it suitable for use in austere environments. The purpose of this study was to evaluate the VCM in terms of repeatability, reproducibility and interanalyzer correlation, reference values in our population, correlation with standard coagulation assays and platelet count, correlation with the TEG5000 analyzer and resistance to stress conditions mimicking an austere environment. METHODS: Repeatability, reproducibility, and interanalyzer correlation were performed on quality control samples (n = 10). Reference values were determined from blood donor samples (n = 60). Correlations with standard biological assays were assessed from ICU patients (n = 30) and blood donors (n = 60) samples. Correlation with the TEG5000 was assessed from blood donor samples. Evaluation of vibration resistance was performed on blood donor (n = 5) and quality control (n = 5) samples. RESULTS: The CVs for repeatability and reproducibility ranged from 0% to 11%. Interanalyzer correlation found correlation coefficients (r2) ranging from 0.927 to 0.997. Our reference values were consistent with those provided by the manufacturer. No robust correlation was found with conventional coagulation tests. The correlation with the TEG5000 was excellent with r2 ranging from 0.75 to 0.92. Resistance to stress conditions was excellent. CONCLUSION: The VCM analyzer is a reliable, easy-to-use instrument that correlates well with the TEG5000. Despite some logistical constraints, the results suggest that it can be used in austere environments. Further studies are required before its implementation.

Challenges to Laboratory Monitoring of Direct Oral Anticoagulants.

Direct oral anticoagulants (DOACs) exert anticoagulation effect by directly inhibiting Factor Xa (rivaroxaban, apixaban, and edoxaban) or thrombin (dabigatran). Though DOACs are characterized by fixed-dose prescribing and generally do not require routine laboratory drug-level monitoring (DLM), circumstances may arise where the DLM may aid in clinical decision-making, including DOAC dose adjustment, anticoagulant class change, or decisions to withhold or administer reversal agents. We review the current literature that describes high-risk patient groups in which DLM may be beneficial for improved patient anticoagulation management and stewardship. The review also summarizes the limitations of conventional coagulation testing and discuss the emerging utility of quantitative methods for routine and rapid emergent evaluation of DOAC drug levels-in particular, the Anti-Xa activity to detect Factor Xa Inhibitors (rivaroxaban, apixaban, and edoxaban). Both technical and regulatory barriers to widespread DLM implementation are limiting factors to further clinical research that must be overcome, in order to propose universal DOAC DLM strategies and provide clinical-laboratory correlation to formally classify high-risk patient groups.

Investigation of thrombin concentration at the time of clot formation in simultaneous thrombin and fibrin generation assays.

Thrombin generation (TG) and fibrin clot formation represent the central process of blood coagulation. Up to 95% of thrombin is considered to be generated after the clot is formed. However, this was not investigated in depth. In this study, we conducted a quantitative analysis of the Thrombin at Clot Time (TCT) parameter in 5758 simultaneously recorded TG and clot formation assays using frozen plasma samples from commercial sources under various conditions of activation. These samples were supplemented with clotting factor concentrates, procoagulant lipid vesicles and a fluorogenic substrate and triggered with tissue factor (TF). We found that TCT is often close to a 10% of thrombin peak height (TPH) yet it can be larger or smaller depending on whether the sample has low or high TPH value. In general, the samples with high TPH are associated with elevated TCT. TCT appeared more sensitive to some procoagulant phenotypes than other commonly used parameters such as clotting time, TPH or Thrombin Production Rate (TPR). In a minority of cases, TCT were not predicted from TG parameters. For example, elevated TCT (above 15% of TPH) was associated with either very low or very high TPR values. We conclude that clotting and TG assays may provide complementary information about the plasma sample, and that the TCT parameter may serve as an additional marker for the procoagulant potential in plasma sample.

Exploring coagulation parameters as predictive biomarkers of Plasmodium infection: A comprehensive analysis of coagulation parameters.

BACKGROUND: Malaria affects the intravascular environment, leading to abnormal coagulation activation, prolonged prothrombin time, and activated partial thromboplastin time. Despite the high prevalence of malaria in the study area, there has been little published research on the effects of Plasmodium infection on coagulation parameters. OBJECTIVE: The aim was to assess the effect of malaria on basic coagulation parameters among patients attending Dembia Primary Hospital and Makisegnit Health Center. METHODS: A cross-sectional study was carried out from January to March 2020. The study involved 120 participants. Blood specimens were collected, which were analyzed using a Huma Clot Due Plus analyzer. The collected data were entered into EpiData and exported to SPSS version 21 for analysis. Non-parametric statistical methods were employed to analyze the data. The results were considered statistically significant if the p-value was less than 0.05. RESULTS: Individuals infected with Plasmodium exhibit coagulation disorders with elevated levels of PT (Prothrombin Time), APTT (Activated Partial Thromboplastin Time), and INR (International Normalization Ratio) in comparison to healthy controls. The median PT, APTT, and INR values for infected cases were measured at 20.5 [8.6], 39.5 [17.9], and 1.8 [0.9], respectively, while healthy controls had measurements of 15.1 [2.5], 28.8 [8.3], and 1.3 [0.2] (p ≤ 0.001). The severity of coagulation disorders increased with an increase in parasitemia levels. The type of Plasmodium species present had a significant impact on PT and INR values (p ≤ 0.001), whereas APTT did not show any significant impact across the Plasmodium species (p > 0.05). CONCLUSION: The results of this study found that malaria has a substantial impact on various blood clotting parameters, including PT, APTT, and INR. Parasitemia severity is significantly associated with extended PT and INR, implying that the higher the parasitemia, the longer it takes for blood to clot. Furthermore, the study discovered that the PT and INR levels differed based on the type of Plasmodium species responsible for the infection.

Characteristics of factor V and protein C based on results from Korean testing centers.

OBJECTIVE: The global incidence of thrombosis is increasing. However, research on thrombosis in the context of Korea is scarce. We aimed to analyze the relationship between factor V and protein C test results and thrombosis in Koreans through a domestic commissioned testing institution conducting mass examinations. METHODS: Results of factor V and protein C tests of 1386 individuals referred simultaneously to EONE Laboratories (Incheon, Republic of Korea) from January 2017 to July 2023 were analyzed retrospectively to identify the association with thrombotic disease. The tests were performed using a STAR MAX (Diagnostica Stago, Asnieres, France) automatic blood coagulation analyzer. The results were analyzed by age and sex. RESULTS: The inspection rate increased gradually from 2017 to 2022. Women (70.0%) demonstrated a higher test rate than did men (30.0%). Young women reported high test rates; the test rate and age distribution differed by sex. Women aged between 20 and 49 years reported lower factor V and higher protein C concentrations than did men between 20 and 49 years of age. CONCLUSIONS: The tests were more commonly performed in women than in men. Women aged between 20 and 49 years reported lower factor V concentrations and higher protein C concentrations than men between 20 and 49 years of age. This study will facilitate recognizing and preventing thrombotic diseases in women.

In vitro differential inhibition of the factor XI activity assay in the setting of a lupus anticoagulant.

Acquired factor XI deficiencies due to factor-specific inhibitors are rare and may be associated with lupus anticoagulant. We report a 63-year-old male with suspected postsurgical bleeding, prior surgical site infection, an isolated prolonged activated partial thromboplastin time, and a positive lupus anticoagulant. Although the factor II assay was normal, factor VIII and IX assays initially demonstrated nonparallelism with factor activity that consistently increased to normal reference ranges with serial dilutions. A discrepancy in factor XI activity results was discovered when the in-house method demonstrated undetectable activity (<3%); send-out testing using different instrument/reagent combinations revealed the presence of factor XI activity between 70% and 76%. The patient received surgical follow-up and was subsequently discharged home. Given the differential in vitro inhibition of factor XI activity on our initial in-house testing, this case highlights the importance of recognizing factor assay interference in the presence of a known lupus anticoagulant inhibitor, with strategies to mitigate potentially erroneous results.

Taipan snake venom time has high sensitivity for lupus anticoagulants in non-anticoagulated, triple positive antiphospholipid syndrome patients.

INTRODUCTION: Dilute Russell's viper venom time (dRVVT) and activated partial thromboplastin time (APTT) are the mainstay assays in lupus anticoagulant (LA) detection yet they have limitations, particularly in relation to interferences and specificity. The recently validated Taipan snake venom time (TSVT) screening with ecarin time (ET) confirmatory assays overcome many of those limitations due to the innate specificity engendered from direct prothrombin activation, and insensitivity to the effects of vitamin K antagonists (VKA). The present study aimed to further evidence diagnostic utility of TSVT/ET by performing them in samples from 116 nonanticoagulated patients with established triple-positive antiphospholipid syndrome (APS). METHODS: Samples were identified in three expert centres who performed dRVVT, APTT and solid phase antiphospholipid antibody assays with reagents from a variety of manufacturers. All samples additionally received TSVT/ET analysis using standardised reagents. RESULTS: Ninety seven of 116 (83.6%) were dRVVT- and APTT-positive, 85/97 (87.6%) of which were TSVT/ET-positive, 9/116 (7.8%) were dRVVT-positive only, 6 of which were TSVT/ET-positive, and 10/116 (8.6%) were APTT-positive only, 5 of which were TSVT/ET-positive. 96/116 TSVT/ET-positivity returned a high sensitivity for LA of 82.8%. Low coefficients of determination revealed weak relationships between LA potency and anticardiolipin and anti-ß2-glycoprotein I antibody titres for all three LA assays. CONCLUSIONS: TSVT/ET has high sensitivity for the clinically significant LA found in triple positive APS patients. TSVT/ET can establish multiple LA assay positivity in nonanticoagulated patients negative for one of dRVVT or APTT, and is the only assay pairing insensitive to VKAs, the recommended anticoagulation for APS.

Mixing studies for lupus anticoagulant: does it matter how we mix?

Although clear and detailed recommendation regarding the lupus anticoagulant mixing test exist, various sources of NPP are used. We decided to inspect the possible differences in mixing studies depending on the mixing media. Four types of mixing media were prepared for 45 random remnant plasma samples: standard human plasma, control plasma N, previously analyzed patient with normal coagulation values, and home-made normal pool plasma (NPP). Samples were analyzed by using Siemens Dade Actin FSL Activated PTT Reagent on BCS XP analyzer. The median aPTT values of mixing studies with commercial lyophilized NPP, with commercial IQC, as well as with a patient did not differ (26.6, 26.3, and 26.8 s, respectively). Median value of a mixing study with home-made NPP was significantly higher from the rest of the group (27.9 s) ( P  < 0.05). According to the obtained results, we decided to employ the commercial lyophilized NPP for future lupus anticoagulant mixing studies.

Coagulation Testing in Real-World Setting: Insights From a Comprehensive Survey.

The objective of this survey was to gain a real-world perspective on coagulation testing by evaluating the availability of various coagulation laboratory tests, assessing specific analytic and postanalytic steps in clinical laboratories in Korea.Participants were surveyed using a 65-question questionnaire specifically focused on their coagulation testing practices related to prothrombin time (PT), activated partial thromboplastin time (aPTT), plasma-mixing studies, lupus anticoagulant (LA) tests, platelet function tests, coagulation factor assays, and the composition of hemostasis and thrombosis test panels. The survey was performed between July and September 2022.The survey achieved a 77.9% (81 of 104) response rate. PT or aPTT tests were performed directly at all participating institutions, followed by D-dimer and fibrinogen tests, platelet function test, and plasma-mixing studies in order of frequency. Variations existed in the performance of mixing test and LA assessment. Patterns of coagulating testing differed depending on the size of the hospital. The survey revealed that most laboratories conducted coagulation tests following the international guidelines such as Clinical Laboratory Standards Institute guidelines and the Korean Laboratory Certification system. However, some coagulation tests, including mixing test and LA tests, are yet to be standardized in Korea.Continuous education on coagulation test methods and internal and external quality control are required to encourage laboratories to enhance the performance of coagulation testing.

Thrombin Generation Assay in Antiphospholipid Antibodies Positive Subjects as a Personalized Thrombotic Risk Assessment: State of the Art and Perspectives.

PURPOSE OF THE REVIEW: Thrombotic risk assessment in antiphospholipid positive (aPL +) subjects is a major challenge, and the study of in vitro thrombin generation (thrombin generation assays (TGA)) could provide useful information. Activated protein C (APC) sensitivity is involved in thrombotic events in antiphospholipid syndrome patients. We summarized methods used to assess APC sensitivity with TGA and evaluated the prognostic role of APC resistance through literature search. RECENT FINDINGS: APC resistance induced by aPL is a complex pathway. Several cross-sectional studies assessed APC sensitivity to understand thrombotic event mechanisms in aPL + subjects. Only one prospective cohort had investigated the prognostic impact of APC resistance in aPL + subjects, with a positive and significant correlation between APC sensitivity and the risk of thrombosis during the follow up (hazard ratio, 6.07 [95% CI, 1.69-21.87]). APC resistance assessed with TGA could be associated with thrombotic events in aPL + subjects.

The Acute Effect of Chamomile Intake on Blood Coagulation Tests in Healthy Volunteers: A Randomized Trial.

BACKGROUND: Chamomile administration may have desirable effects in the perioperative setting. Current practice, however, discourages perioperative chamomile use due to a theoretical increase in bleeding. Therefore, we evaluated if chamomile acutely (within 4 h of ingestion) prolongs coagulation assays. METHODS: Eight healthy volunteers were randomized to receive 2 interventions in a crossover design: (a) single dose of chamomile extract capsule (500 mg) and (b) single dose of chamomile tea (3 g in 150 mL water). Interventions were separated at least 3 days apart from each other. Blood was sampled pre-ingestion, 2 h post-ingestion, and 4 h post-ingestion for each intervention. The primary outcome was the maximal change in prothrombin time (PT) before vs after each intervention. Secondary outcomes included changes in international normalized ratio, activated partial thromboplastin time, thrombin time, reptilase time, and fibrinogen levels. RESULTS: All 8 subjects completed the study. The average pre-ingestion PT values for tea and capsules were 11.9 (1.1) s and 12.0 (0.9) s, respectively. Tea significantly increased the average maximum PT by 0.7 (0.2) s (P = 0.0078). Extract capsules increased the maximum PT by 0.3 (0.2) s (P = 0.06). Neither PT prolongation met the predefined 10% threshold for clinical significance. No significant changes in secondary outcomes were observed. CONCLUSIONS: Chamomile tea ingestion prolongs PT. However, the clinical significance of this is unclear at this time and warrants further investigation. ClinicalTrials.gov Registration Number: NCT05272475.

Magnetic coagulometry: towards a new nanotechnological tool for ex vivo monitoring coagulation in human whole blood.

Blood clotting disorders consisting of unwanted blood clot formation or excessive bleeding are some of the main causes of death worldwide. However, there are significant limitations in the current methods used to clinically monitor the dynamics of clot formation in human whole blood ex vivo. Here a new magnetic coagulometry platform for testing ex vivo coagulation is described. This platform exploits the sensitivity of the out-of-phase component of alternating current (AC) magnetic susceptibility (χ'') to variations in mobility and agglomeration of magnetic nanoparticles when trapped during blood clot formation. By labelling human whole blood with magnetic nanoparticles, the out-of-phase component of AC magnetic susceptibility shows that the dynamics of blood clot formation correlates with a decrease in the out-of-phase component χ'' over time activation of coagulation. This is caused by a rapid immobilisation of nanoparticles upon blood coagulation and compaction. In contrast, this rapid fall in the out-of-phase component χ'' is significantly slowed down when blood is pre-treated with three different anticoagulant drugs. Remarkably, the system showed sensitivity towards the effect of clinically used direct oral anticoagulation (DOAC) drugs in whole blood coagulation, in contrast to the inability of clinical routine tests prothrombin time (PT) and partial thromboplastin time (PTT) to efficiently monitor this effect. Translation of this nanomagnetic approach into clinic can provide a superior method for monitoring blood coagulation and improve the efficiency of the current diagnostic techniques.

Clinical Implications of Discrepancy between One-Stage Clotting and Chromogenic Factor IX Activity in Hemophilia B.

BACKGROUND: Discrepancy in factor IX activity (FIX:C) between one-stage assay (OSA) and chromogenic substrate assay (CSA) in patients with hemophilia B (PwHB) introduces challenges for clinical management. AIM: To study the differences in FIX:C using OSA and CSA in moderate and mild hemophilia B (HB), their impact on classification of severity, and correlation with genotype. METHODS: Single-center study including 21 genotyped and clinically characterized PwHB. FIX:C by OSA was measured using ActinFSL (Siemens) and CSA by Biophen (Hyphen). In addition, in vitro experiments with wild-type FIX were performed. Reproducibility of CSA was assessed between three European coagulation laboratories. RESULTS: FIX:C by CSA was consistently lower than by OSA, with 10/17 PwHB having a more severe hemophilia type by CSA. OSA displayed a more accurate description of the clinical bleeding severity, compared with CSA. A twofold difference between OSA:CSA FIX:C was present in 12/17 PwHB; all patients had genetic missense variants in the FIX serine protease domain. Discrepancy was also observed with diluted normal plasma, most significant for values below 0.10 IU/mL. Assessment of samples with low FIX:C showed excellent reproducibility of the CSA results between the laboratories. CONCLUSION: FIX:C was consistently higher by OSA compared with the CSA. Assessing FIX:C by CSA alone would have led to diagnosis of a more severe hemophilia type in a significant proportion of patients. Our study suggests using both OSA and CSA FIX:C together with genotyping to classify HB severity and provide essential information for clinical management.

Comparisons between diluted thrombin time, ecarin chromogenic assays, and UPLC-MS for plasma level dabigatran quantification: Results from DRIVING study.

INTRODUCTION: The knowledge of dabigatran levels is helpful for decision-making in specific situations such as urgent surgery or when the question of reversal arises (uncontrolled bleeding, eligibility for thrombolysis). However, a limited number of observational studies are available regarding comparisons between quantification methods. The objective of the study was to compare dabigatran plasma levels using three assays including the reference method (high-performance liquid chromatography coupled with mass spectrometry), focusing on the agreement around the 30-50 ng/mL clinically relevant thresholds. METHODS: Sixty healthy volunteers from DRIVING trial (NCT01627665) were given a single 300-mg dabigatran etexilate dose. Serial blood samplings were performed at pre-defined time points (0 to 24 h). We analyzed plasma samples using ultra-performance-liquid chromatography coupled with tandem mass spectrometry (UPLC-MS) (dabigatran reference method); ii/diluted thrombin time (dTT) (Hemoclot-DTI-Hyphen-Biomed); iii/ecarin-based chromogenic assay (ECA-II-Stago). RESULTS: Nine hundred sixty samples were analyzed using the three assays (2759 values). dTT and ECA-II values were highly correlated with those of UPLC-MS (Deming regression). Most values >50 ng/mL were higher using dTT and ECA-II compared to UPLC-MS: biases were constant, +14% and +16% with dTT and ECA-II, respectively (Bland-Altman plots), suggesting that active metabolites accounted for ~15% of thrombin inhibition. Regarding values <30 ng/mL, 30-50 ng/mL, or ≥50 ng/mL, the agreement probability between dTT and ECA-II was of 90.6% [88.4-92.5] (Cohen's kappa coefficient 0.84). CONCLUSION: dTT and ECA-II assays rapidly provide accurate dabigatran-level results for clinical practice, both assays being suitable in emergency, taking into account the thrombin inhibitory effect of dabigatran metabolites.

Global hemostasis assays in acute myeloid leukemia: results of an observational prospective study.

BACKGROUND: Acute myeloid leukemia (AML) is characterized by a complex spectrum of coagulopathy ranging from hemorrhagic to thrombotic symptoms. To date, platelet count (PLT) and conventional coagulation tests (CCTs) cannot predict hemorrhagic events and thrombotic risk. Thromboelastography (TEG) measures the viscoelastic properties of the clot, thus providing information on the entire process of blood coagulation. The primary aim of the study was to assess the hemostatic balance from AML diagnosis to the end of chemotherapy (CHT) by TEG. MATERIAL AND METHODS: Here we present the results of a prospective study enrolling newly diagnosed AML patients treated with chemotherapy. Patients had complete blood counts (CBCs), TEG and CCTs performed at three time points: 1) diagnosis (T0); 2) during the first cycle of CHT (T1); and 3) at the end of CHT (T2). An algorithm of TEG indirectly calculated thrombin generation (TG). Patients underwent daily follow-up for bleeding and thrombotic episodes up to the time of hospital discharge or death. RESULTS: Eighty consecutive patients were evaluated; forty were eligible for the study, and 21 completed the entire study. At T1, maximum amplitude (MA), TG and K-time were significantly shifted toward a hypocoagulability state compared to T0 (p<0.05), while a hypercoagulable state at T2 was shown by changes in α-angle, MA and TG values. Otherwise, there were no statistically significant differences in CCTs between the evaluated time points. DISCUSSION: Overall, TEG revealed complex and dynamic coagulation abnormalities in patients with AML according to both the course of disease and therapy. Further studies are needed to investigate more fully the role of TEG in defining the hemostatic profile in patients with AML.

A global comparative field study to evaluate the factor VIII activity of efanesoctocog alfa by one-stage clotting and chromogenic substrate assays at clinical haemostasis laboratories.

INTRODUCTION: Structural and chemical modifications of factor VIII (FVIII) products may influence their behaviour in FVIII activity assays. Hence, it is important to assess the performance of FVIII products in these assays. Efanesoctocog alfa is a new class of FVIII replacement therapy designed to provide both high sustained factor activity levels and prolonged plasma half-life. AIM: Evaluate the accuracy of measuring efanesoctocog alfa FVIII activity in one-stage clotting assays (OSAs) and chromogenic substrate assays (CSAs). METHODS: Human plasma with no detectable FVIII activity was spiked with efanesoctocog alfa or a full-length recombinant FVIII product comparator, octocog alfa, at nominal concentrations of 0.80 IU/mL, 0.20 IU/mL, or 0.05 IU/mL, based on labelled potency. Clinical haemostasis laboratories (N = 35) tested blinded samples using in-house assays. Data from 51 OSAs (14 activated partial thromboplastin time [aPTT] reagents) and 42 CSAs (eight kits) were analyzed. RESULTS: Efanesoctocog alfa activity was reliably (±25% of nominal activity) measured across all concentrations using OSAs with Actin FSL and multiple other aPTT reagents. Under- and overestimation of FVIII activity occurred with some reagents. No specific trend was observed for any class of aPTT activators. A two- to three-fold overestimation was consistently observed using CSAs and the OSA with Actin FS as the aPTT reagent across evaluated concentrations. CONCLUSION: Under- or overestimation occurred with some specific OSAs and most CSAs, which has been previously observed with other modified FVIII replacement products. Efanesoctocog alfa FVIII activity was measured with acceptable accuracy and reliability using several OSA methods and commercial plasma standards.