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Dabigatran etexilate is a new oral anticoagulant that functions as a direct thrombin inhibitor. An inhibitor of thrombin has the potential to interfere with essentially all clot-based coagulation assays and select chromogenic assays, whereas the drug would not be expected to interfere in antigen-based assays. The purpose of this study was to evaluate the effect of dabigatran on various specialized coagulation assays using normal plasma specimens with varying concentrations of dabigatran (the active form of dabigatran etexilate). We have demonstrated that samples containing therapeutic levels of dabigatran may lead to underestimation of intrinsic factor activities with abnormal activated partial thromboplastin time (aPTT) mixing study results and a false-positive factor VIII Bethesda titer; overestimation of protein C and protein S activity and activated protein C resistance ratio when determined using aPTT-based methods; and overestimation of results based on chromogenic anti-IIa assays but no effect on antigen assays and select chromogenic assays.

Hemophilia A is caused by mutations within the Factor VIII (FVIII) gene that lead to depleted protein production and inefficient blood clotting. Several attempts at gene therapy have failed for various reasons--including immune rejection. The recent generation of induced pluripotent stem (iPS) cells from somatic cells by the ectopic expression of 3 transcription factors, Oct4, Sox2, and Klf4, provides a means of circumventing the immune rejection barrier. To date, iPS cells appear to be indistinguishable from ES cells and thus provide tremendous therapeutic potential. Here we prepared murine iPS cells from tail-tip fibroblasts and differentiated them to both endothelial cells and endothelial progenitor cells by using the embryoid body differentiation method. These iPS cells express major ES cell markers such as Oct4, Nanog, SSEA-1, alkaline phosphatase, and SALL4. Endothelial/endothelial progenitor cells derived from iPS cells expressed cell-specific markers such as CD31, CD34, and Flk1 and secreted FVIII protein. These iPS-derived cells were injected directly into the liver of irradiated hemophilia A mice. At various times after transplantation (7-90 days) hemophilia A mice and their control mice counterparts were challenged by a tail-clip bleeding assay. Nontransplanted hemophilia A mice died within a few hours, whereas transplanted mice survived for more than 3 months. Plasma FVIII levels increased in transplanted hemophilia A mice during this period to 8% to 12% of wild type and corrected the hemophilia A phenotype. Our studies provide additional evidence that iPS cell therapy may be able to treat human monogenetic disorders in the future.

Rivaroxaban is a new oral anticoagulant that functions as a direct anti-Xa inhibitor. Although routine monitoring is not required, measurement of plasma concentrations may be necessary in certain clinical situations. Routine coagulation assays, such as the prothrombin time and, to a lesser degree, activated partial thromboplastin time, correlate with drug concentration, but because of reagent variability, these methods are not reliable for determining rivaroxaban anticoagulation. To compare different methods and calibrators for measuring rivaroxaban, including the chromogenic anti-Xa assay, which, when calibrated with a rivaroxaban standard, may be more appropriate for determining anticoagulation. We compared measured rivaroxaban concentrations with the same anti-Xa kit but used different calibrators, with different anti-Xa kits but the same calibrators, with antithrombin-supplemented anti-Xa kit versus nonsupplemented kits, and with 2 methods based on rivaroxaban-calibrated, high-phospholipid, dilute Russell viper venom time. Regression and paired t test statistics were used to determine correlation and significant differences among methods and calibrator sources. Although there was strong correlation, statistically significant biases existed among methods that report rivaroxaban levels. A single-source calibrator did not alleviate those differences among methods. High-phospholipid Russell viper venom reagents correlated with rivaroxaban concentration but were not better than chromogenic anti-Xa methods. Rivaroxaban-calibrated, anti-Xa measurements correlate well, but the clinical significance of the variation with rivaroxaban measurements is uncertain. The antithrombin-supplemented, anti-Xa method should be avoided for measuring rivaroxaban.

The use of modern laboratory instrumentation with high levels of test reliability and appropriate quality assurance measures will lead to very few analytical errors within hemostasis testing. Nevertheless, incorrect or inappropriate test results are still reported, often due to events outside the control of the laboratories performing the tests. This is due primarily to pre-analytical events associated with sample collection and processing, as well as post-analytical events related to the reporting and interpretation of test results. This review focuses on the pre-analytical phase, highlighting contributory elements and providing suggestions on how problems can be minimized or prevented, thereby improving the likelihood that reported test results actually represent the true clinical status of the patient rather than that of an inappropriate sample. This review should be of value to both laboratory personnel and clinicians because an appreciation of these issues will enable the optimal clinical management of patients. [PUBLICATION ABSTRACT]

The prothrombin time (PT) and activated partial thromboplastin time (APTT) are screening tests used to detect congenital or acquired bleeding disorders. An unexpected PT and/or APTT prolongation is often evaluated using a mixing test with normal plasma. Failure to correct (\"noncorrection\") prolongation upon mixing is attributed to an inhibitor, whereas \"correction\" points to factor deficiency(ies). To define an optimal method for determining correction or noncorrection of plasma mixing tests through an international, multisite study that used multiple PT and APTT reagents and well-characterized plasma samples. Each testing site was provided 22 abnormal and 25 normal donor plasma samples, and mixing studies were performed using local PT and APTT reagents. Mixing study results were evaluated using 11 different calculation methods to assess the optimal method based on the expected interpretation for factor deficiencies (correction) and noncorrection (inhibitor effect). Misprediction, which represents the failure of a mixing study interpretation method, was assessed. Percentage correction was the most suitable calculation method for interpreting PT mixing test results for nearly all reagents evaluated. Incubated PT mixing tests should not be performed. For APTT mixing tests, percentage correction should be performed, and if the result indicates a factor deficiency, this should be confirmed with the subtraction III calculation where the normal pooled plasma result (run concurrently) is subtracted from the mixing test result with correction indicated by a result of 0 or less. In general, other calculation methods evaluated that performed well in the identification of factor deficiency tended to have high misprediction rates for inhibitors and vice versa. No single method of mixing test result calculation was consistently successful in accurately distinguishing factor deficiencies from inhibitors, with between-reagent and between-site variability also identified.

To the Editor: The review article on thrombophilia testing by Connors (Sept. 21 issue) 1 is excellent, but clinical laboratory physicians are compelled to clarify certain points regarding thrombophilia testing in patients who are taking anticoagulants. Such testing may lead to either false positive or false negative results. 2,3 For example, therapy with direct oral anticoagulants causes falsely elevated activity in the clot-based protein C and protein S activity assays. Furthermore, these assays are not recommended for screening; rather, chromogenic protein C activity and free protein S antigen assays are recommended, 4 since both of these assays are accurate while patients are receiving . . .

Diagnosis and management of hemophilia require accurate and precise measurements of factor activity levels. Activity is traditionally measured via one-stage (OS) clot-based assay; however, chromogenic substrate (CS) assays may be needed for certain cases. A survey was performed to understand assay-related knowledge gaps among hematologists and laboratory professionals. Separate web-based surveys were administered to hematologists who manage hemophilia and to laboratory professionals and queried practice patterns, knowledge of/attitudes toward CS assays, and interest in continuing education. A total of 51 hematologists participated in this study; 67% managed hemophilia patients for ≥10 years and 24% were affiliated with a hemophilia treatment center (HTC). Most (80%) stated familiarity with general assay interpretation. Majorities of non-HTC and HTC respondents agreed that CS assays are more accurate than OS assays (62%/67%), although non-HTC hematologists indicated less understanding of when to order a CS assay (49%/67%). Fewer non-HTC respondents expressed concerns regarding the reliability of OS assays for diagnosis (38%/67%) and monitoring (38%/75%). Most (80%) expressed an interest in factor assay education, especially on available assays, efficacy, and best practices (39%). A total of 57 laboratory professionals participated, averaging 10 years in their current position; most (88%) were hospital based. More performed OS (72%) than CS (10%) or both (17%) assays; only 11% reported confidence with the interpretation of CS results. Few expressed concerns regarding the reliability of OS for diagnosis (9%) or monitoring (12%). Reported barriers to CS use included infrequent need (68%), lack of US Food and Drug Administration (FDA) approval (61%), and need for validation work (56%). Most (70%) were interested in CS assay education; top interests included advantages over traditional assays, general information on CS assays, and indications for testing (each 18%). Future educational efforts may focus on limitations of OS assays, indications for CS assay diagnosis/monitoring, and support for clinic-laboratory dialog.

Context.—Proper diagnosis and therapy of fibrinogen deficiency requires high-quality fibrinogen assays. Objective.—To assess the interlaboratory bias, precision, and grading of fibrinogen assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in coagulation. Design.—Two identical vials of normal plasma were sent to more than 3500 laboratories. Participants measured fibrinogen levels using local methods. Results.—Fifty different fibrinogen methods were evaluated. All-method bias was 8.3% (range of method-specific biases, 0.0%–27.0%) and all-method coefficient of variation was 7.7% (range of method-specific coefficients of variation, 0.7%–25.8%). After controlling for reagent/instrument type, mean fibrinogen levels were 11.6% higher for prothrombin time–based reagents compared to Clauss (P < .001), and coefficient of variation was 46% lower for mechanical endpoint instruments compared to photo-optical. Most testing events (97.4%) could be reliably graded as pass or fail using a target range of ±20% from the method mean (total pass rate, 98.8%). Total fail rate was 3.0-fold lower for mechanical instruments compared to photo-optical (0.5% versus 1.5%, P  =  .001). Nonetheless many photo-optical methods had very high precision and very low fail rates. Conclusions.—Fibrinogen assays showed highly variable methodology and performance characteristics. Bias, precision, and grading were affected by the type of reagent or instrument used.

Context.—Hereditary and acquired deficiencies of antithrombin (AT), protein C (PC), and protein S (PS) are risk factors for venous thromboembolism. Proper diagnosis requires high-quality assays for these proteins. Objective.—To determine the accuracy and interlaboratory precision of AT, PC, and PS assays used by laboratories participating in the United States College of American Pathologists proficiency testing program in thrombophilia and to grade the performance of laboratories. Design.—Standardized normal plasma with assigned analyte values was sent in 2 separate challenges to participating laboratories. Participants measured AT, PC, and PS levels using local methods. Results.—When compared with the assigned values for the international standard, the order of assay accuracy from highest to lowest was AT activity, PC antigen, AT antigen, total PS antigen, PC activity, PS activity, and free PS antigen (range of assay bias, 2.6%–8.8%). The order of assay precision from highest to lowest was PC activity, AT activity, AT antigen, total PS antigen, PS activity, free PS antigen, and PC antigen (range of assay coefficient of variation, 6.1%–20.0%). Most testing events (87.8%) could be graded as pass or fail using a target range of ±3 standard deviations from the method-specific mean. The pass rate was 98.2% for all AT, PC, and PS testing events combined. Conclusions.—Accuracy and precision were higher for AT assays and lower for PC and PS assays. It was feasible to grade individual laboratory performance.