Reducing the effect of DOAC interference in laboratory testing for factor VIII and factor IX: A comparative study using DOAC Stop and andexanet alfa to neutralize rivaroxaban effects.

INTRODUCTION: Investigation of factors (F) VIII and IX is common, with testing important for diagnosis or exclusion of haemophilia A or B, associated acquired conditions and factor inhibitors. Rivaroxaban, a common direct anti-Xa agent, causes significant interference in clotting assays, including substantial false reduction of factor levels. AIM: To assess whether rivaroxaban-induced interference of FVIII and FIX testing could be neutralized. MATERIALS AND METHODS: An international, cross-laboratory exercise for FVIII (n = 84) and FIX (n = 74), using four samples: (A) pool of normal plasma; (B) pool spiked with rivaroxaban (200 ng/mL); (C) rivaroxaban sample subsequently treated with 'DOAC Stop' and; (D) rivaroxaban sample treated with andexanet alfa (200 µg/mL). Testing performed blind to sample type. RESULTS: All laboratories reported normal FIX and 94% reported normal FVIII in the pool sample. Instead, 55% and 95%, respectively, reported abnormal FIX and FVIII levels for the rivaroxaban sample. DOAC Stop treatment evidenced a correction in most laboratories (100% reported normal FIX and 86% normal FVIII). Andexanet alfa provided intermediate results, with many laboratories still reporting abnormal results (59% for FVIII, 18% for FIX). We also identified reagent-specific issues. CONCLUSIONS: As expected, rivaroxaban caused false low values of FVIII and FIX. This might lead to increased testing to identify the cause of low factor levels and potentially lead to false identification of (mild) haemophilia A or B if unrecognized by clinicians/laboratories. DOAC Stop effectively neutralized the rivaroxaban effect, but andexanet alfa less so, with reagent-related effects evident, and thus, false low values sometimes persisted.

Current and Emerging Direct Oral Anticoagulants: State-of-the-Art.

Anticoagulant drugs comprise a specific subcategory of antithrombotic agents that act to inhibit blood coagulation at various stages, reducing clot development and ultimately lowering the risk of developing new-onset or recurrent thrombosis. Although the long history of anticoagulant drugs has been characteristically shaped by coumarin and heparin derivatives, a new generation of direct oral anticoagulants (DOACs), which specifically inhibit thrombin or activated factor X, combine many advantages of their progenitor drugs, and hence are prepotently revolutionizing the landscape of antithrombotic therapy. Several drugs (apixaban [BMS-562247], dabigatran [BIBR953], edoxaban [DU-1766], rivaroxaban [BAY 59-7939]) have already received widespread approval by national or supranational medicinal agencies. This narrative article provides a state-of-the-art for these and for several other DOACs at different stages of clinical evaluation (betrixaban, darexaban, eribaxaban, letaxaban, nokxaban), and certain others whose development has been discontinued (AZD-0837, fidexaban, LY517717, odiparcil, otamixaban, TTP889, and ximelagatran). What clearly emerges from our analysis is that DOACs sharing very similar mechanisms of action are still characterized by different efficacy and safety profiles. This not only depends on biochemical, biological, and pharmacokinetic characteristics, but also on lack of standardization between different clinical trials in terms of targeted disease, patient recruitment, sample size, duration and endpoints, as well as lack of harmonization around procedures used for drug licensing. These factors contribute to challenging the minds of physicians, who may find difficulty navigating their way through multiple indications, different pharmacological profiles, various side effects, and specific drug-to-drug interactions. Such considerations also burden laboratory professionals, who may face organizational and economic challenges in developing and/or implementing multiple assays to assess the pharmacodynamics (effect on coagulation) or pharmacokinetics (drug levels) of DOACs.

Impact of experimental hypercalcemia on routine haemostasis testing.

BACKGROUND: The blood to anticoagulant ratio is standardized according to the physiological calcium concentration in blood samples conventionally used for hemostasis testing. Specifically, one fixed volume of 0.109 mmol/L sodium citrate is added to 9 volumes of blood. Since little is known about the impact of hypercalcemia on the calcium-binding capacity of citrate, this study was planned to investigate the effect of experimental hypercalcemia on routine hemostasis testing. METHODS: Fifteen pooled citrated plasmas with matching lithium-heparin pooled plasma from patients with different values of prothrombin time (PT) were divided in three aliquots of 0.6mL each. The first paired aliquots of both citrate and lithium-heparin plasma were supplemented with 60µL of saline, the second paired aliquots with 30µL of saline and 30µL of calcium chloride and the third paired aliquots with 60µL of calcium chloride. Total and ionized calcium was measured in all aliquots of citrate and lithium-heparin plasma, whereas PT, activated partial thromboplastin time (APTT) and fibrinogen were measured in citrate plasma aliquots. RESULTS: Total calcium concentration gradually increased in both lithium-heparin and citrate plasma aliquots 2 and 3 compared to baseline aliquot 1. The concentration of ionized calcium also gradually increased in lithium-heparin plasma aliquots 2 and 3, whereas it remained immeasurable (i.e., <0.10 mmol/L) in all citrate plasma aliquots. No significant differences were observed for values of PT, APTT and fibrinogen in citrate plasma aliquots 2 and 3 compared to the baseline aliquot 1, with a mean bias was always comprised within the desirable quality specifications derived from biological variability data. CONCLUSION: Hypercalcemia, up to severe hypercalcemia does not generate significant bias in results of first-line coagulations tests, so that hypothetical consideration of adjusting citrate-blood ratio is unjustified in hypercalcemic patients.

The new oral anticoagulants and the future of haemostasis laboratory testing.

The tests currently employed within most haemostasis laboratories to monitor anticoagulant therapy largely comprise the prothrombin time (PT)/ International Normalised Ratio (INR) and the activated partial thromboplastin time (APTT). These are respectively used to monitor Vitamin K antagonists (VKAs) such as warfarin, and unfractionated heparin. Additional tests that laboratories may also employ for assessing or monitoring unfractionated heparin include thrombin time (TT) and the anti-Xa assay, which can also be used to monitor low molecular weight heparin. Several new anti-thrombotic agents have recently emerged, or are in the final process of clinical evaluation. These novel drugs that include Dabigatran etexilate and Rivaroxaban would not theoretically require monitoring; however, testing is useful in specific situations. The tests currently used to monitor VKAs and heparin are typically either too sensitive or too insensitive to the new drugs to be used as 'typically performed in laboratories', and may thus require some methodological adjustments to increase or decrease their sensitivity. Alternately, different tests may be better employed in these assessments. Whatever the case, laboratories may soon be performing a reduced or possibly increased number of tests, the same kind of tests but perhaps differently, or conceivably different assay panels. Specific laboratory guidance on the choice of the appropriate test to be ordered according to the drug being administered, as well as on appropriate interpretation of test results, will also be necessary. The current report reviews the current state of play and provides a glimpse to the possible future of the coagulation laboratory.

Coffee intake and cardiovascular disease: virtue does not take center stage.

Coffee is one of the most popular and heavily consumed beverages worldwide, despite the many different methods of preparation and presentation. The results of several epidemiological studies are suggestive for the existence of a U-shaped relationship between coffee consumption and both cardiovascular events and mortality, whereby a lower risk seems associated with low (i.e., less than one cup per day) or high (i.e., more than or equal to four cups per day) coffee intake, whereas a higher risk is reported for intermediate consumption (i.e., two to four cups per day). Most benefits are evident in individuals with a rapid caffeine metabolizer genotype and a low baseline cardiovascular risk. Benefits have also been differentially associated with consumption of decaffeinated coffee, filtered coffee, coffee consumption during lunchtime or dinner, and when coffee is produced in the Italian style (i.e., by espresso or moka). The leading favorable effects have been attributed to various compounds present in coffee. Thus, chlorogenic acids would be effective in decreasing blood pressure, systemic inflammation, risk of type 2 diabetes, and platelet aggregation, whereas caffeine intake has instead been associated with decreased body weight, as well as with increased flow-mediated dilatation and fibrinolysis.

Toward a new paradigm for the identification and functional characterization of von Willebrand disease.

The diagnosis and functional characterization of von Willebrand disease (VWD) is challenging. There are inherent difficulties in both its identification and classification because of clinical uncertainty, the limitations in the test processes and test panels typically used by laboratories, and because the classification scheme does not always allow unequivocal assignment of any subtype. This article reviews current thoughts and alternatives to the classic approach of the classification and functional characterization of VWD. Of particular interest to this author is the utility of an extended core test panel that includes additional functional VWF assays, such as the collagen binding assay, and the potential for desmopressin (DDAVP) challenge to not only provide therapeutic information but also assist in the better characterization of individuals with defects or deficiencies in von Willebrand factor (VWF). The potential use of supplementary assays such as the PFA-100 and the VWF propeptide assay after DDAVP challenge is also worth noting.

High rate of deficiency in the amino acids tryptophan and histidine in people with wounds: implication for nutrient targeting in wound management--a pilot study.

BACKGROUND: Malnutrition resulting from inadequate protein, energy, or micronutrient intake has been identified as an independent risk factor for the development of pressure ulcers in older adult patients and is associated with increased morbidity and death. OBJECTIVE: To assess the relationship between albumin, the standard biochemical marker of nutritional adequacy, and amino acid status in people with wounds. METHODS: The authors performed tests for serum albumin, prealbumin, and amino acid profiles on 18 consecutive hospital patients with wounds and 7 patients without wounds. RESULTS: A low level of the essential amino acids tryptophan and histidine was a common finding in older people with wounds. Of the 18 consecutive wound cases, 16 (88.9%) were found to be deficient in tryptophan, histidine, or both. Moreover, levels were generally found to be lower than those in the group without wounds. The levels of all other amino acids were essentially normal for all patients. Finally, although serum albumin is often used as a surrogate marker of amino acid adequacy or nutritional status, clinically abnormal albumin had poor specificity (63.2%), poor sensitivity (60.7%), and low positive predictive value (70.8%) for the identification of a low tryptophan or histidine level. CONCLUSIONS: People with wounds are a relatively at-risk group and are likely to be overlooked in terms of micronutrient deficiencies, and these findings have important implications in terms of potential specific targeting of nutrient supplementation.

Standardization of the INR: how good is your laboratory's INR and can it be improved?

The prothrombin time (PT) assay is the most clinically ordered coagulation test and is most often used for monitoring of vitamin K antagonist (VKA) therapy (e.g., warfarin), where results are expressed as an international normalized ratio (INR). The INR is in essence the patient's PT "mathematically adjusted" to a standardized value by taking into account the peculiarities of the test system through applying two "correction factors" defined by an international sensitivity index (ISI) and mean normal prothrombin time (MNPT). Although some manufacturers provide assigned ISI values for specific PT reagents and instrumentation, it is still recommended practice that laboratories check or locally validate these ISIs, as well as estimate the MNPT based on the population being tested. Where a manufacturer does not provide an ISI, the laboratory needs to define its own (local ISI) value. Current recommendations suggest the use of commercial reference-plasma calibration sets, but there is limited information to validate their performance in laboratory practice. We report our personal experience with use of some of these materials, as well as review alternate or supplementary procedures for calibration and/or validation of ISI and for determination and validation of MNPT. In brief, our data and experience suggests that further verification checks should be performed prior to acceptance of ISI and MNPT estimates generated from commercial reference-plasma calibration sets. We detail various strategies to ensure that laboratory practices are optimized to provide INRs that accurately reflect a patient's true anticoagulant status and to thus assist their clinical therapeutic management.

Time to think outside the box? Prothrombin time, international normalised ratio, international sensitivity index, mean normal prothrombin time and measurement of uncertainty: a novel approach to standardisation.

BACKGROUND: The prothrombin time (PT) assay is the most clinically ordered coagulation test, and most often used for monitoring of vitamin K antagonist therapy (e.g., warfarin), where results are expressed as an international normalised ratio (INR). The INR is in essence the patient's PT 'mathematically adjusted' to a standardised value taking into account the peculiarities of the test system as defined by an ISI (international sensitivity index) and MNPT (mean normal prothrombin time). Although some manufacturers provide assigned ISI values for specific PT reagents and instrumentation, it is still recommended practice that laboratories check or validate these ISIs, as well as estimate the MNPT. Where an ISI is not provided by a manufacturer, the laboratory needs to estimate its own value. Current recommendations suggest the use of commercial reference-plasma calibration sets, but there is limited information on the performance of these in the field. RESULTS: We report a comparative study that assessed the utility of three such commercial calibration plasma sets, used as recommended, as well as alternate or supplementary procedures for estimation of ISI and MNPT. The latter included one novel approach using comparative data of 'existing' versus 'replacement' reagent, as well as assessment of external quality assurance data. Although MNPT value estimates were not grossly disparate, a wide variety of ISI values (e.g., 1.12-1.30 for our primary instrument) was obtained with the different plasma sets. CONCLUSION: Because of the above, further verification checks are required prior to acceptance of ISI and MNPT estimates generated from commercial plasma calibration sets. We also provide some recommendations regarding the process of standardisation of INR testing.

A diet rich in high-oleic-acid sunflower oil favorably alters low-density lipoprotein cholesterol, triglycerides, and factor VII coagulant activity.

OBJECTIVE: To compare concentrations of factor VII coagulant activity (factor VIIc), fibrinogen, plasminogen activator inhibitor-1, and blood lipids on a saturated fat-rich diet with one rich in monounsaturated fat. DESIGN: Subjects were randomly allocated to two groups. The study design was an ABB/BAA extra-period crossover. One group consumed a diet rich in saturated fatty acid (SFA) with fat making up 20.8% of total energy, for 5 weeks and then one rich in monounsaturated fatty acid (MUFA), with fat making up 20.3% of total energy for 10 weeks. The other group consumed the MUFA diet for 5 weeks followed by the SFA diet for 10 weeks. SUBJECTS/SETTING: Men and women aged 35 to 69 years who were nonsmokers with no chronic illness and not on any medication were recruited to participate. Eighteen subjects were recruited and 15 (5 men, 10 women) completed the community-based study. INTERVENTION: Blood was sampled at the beginning and end point of each 5-week diet period for analysis of coagulation and fibrinolysis factors and blood lipids. Subjects kept 3-day food diaries twice during each of the three diet periods and were weighed on each visit for blood collection. Analysis of plasma fatty acids was used to indicate dietary compliance. MAIN OUTCOME MEASURES: Differences in fasting factor VIIc, fibrinogen, plasminogen activator inhibitor-1, insulin, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoproteins A-1 and B, and plasma oleic acid levels while receiving the SFA diet vs MUFA diet. STATISTICAL ANALYSIS: A general linear model allowing for the ABB/BAA extra-period crossover, was used for each of the outcome measures. RESULTS: Factor VIIc was lower on the MUFA diet ( P <.05) but fibrinogen and insulin concentrations and plasminogen activator inhibitor-1 activity did not differ between diets. Low-density lipoprotein cholesterol ( P <.001) and triglyceride ( P <.01) levels were lower on the MUFA diet compared with the SFA diet. A significant increase in both plasma phospholipid and neutral lipid oleic acid (P <.0001) occurred on the MUFA diet. CONCLUSIONS: Substitution of foods rich in saturated fat with foods rich in high-oleic-acid sunflower oil and margarine has favorable outcomes on blood lipids and factor VIIc. This oil presents another useful source of MUFA for diets aimed at prevention of heart disease.