Maintaining a balance between bleeding and clotting has always been a challenge in treating coagulation disorders. A perturbation in that balance can be associated with substantial morbidity and mortality. As a result, anticoagulant monitoring is extremely important, and inappropriate testing may lead to complications. There are now a variety of new anticoagulant drugs in clinical use including several direct thrombin inhibitors (DTIs), such as argatroban, bivalirudin, and hirudin, as well as a Factor Xa inhibitor, fondaparinux. There are pitfalls associated with some of the currently used laboratory monitoring tests, and newer alternative laboratory monitoring tests have been investigated (Walenga and Hoppensteadt, Semin Thromb Hemost 2004;30:683-695). In addition, laboratory testing can assist with transitioning patients from DTI to warfarin therapy. Am. J. Hematol. 85:185-187, 2010. V V C 2009 Wiley-Liss, Inc. Direct Thrombin InhibitorsDirect thrombin inhibitors (DTIs) do not bind to plasma protein resulting in a more predictable anticoagulant response than is seen with heparin. These anticoagulants inhibit thrombin, including fibrin-bound thrombin [1,2]. As a result, they interfere in clotting time-based coagulation tests that involve thrombin. Specifically, the prothrombin time (PT)/international normalized ratio (INR) and activated partial thromboplastin time (aPTT) are prolonged, and remain prolonged in mixing studies. DTIs act like an inhibitor in clotting-based factor assays, causing an underestimation of the amount of factor present. Fibrinogen results are falsely low, and clotting-based activity assays for Protein C and Protein S are overestimated. Antithrombin has ''anti-thrombin'' activity and will be falsely increased [3]. Activated Protein C resistance ratios (with an aPTT-based method) are falsely increased [4].For monitoring DTIs in the laboratory, the thrombin time is too sensitive, and the result will be prolonged to >200 sec. Monitoring is typically performed with the aPTT. However, as is the case for heparin, using a target aPTT ratio (aPTT on DTI divided by mean of aPTT normal range) is not ideal. In a study looking at different aPTT reagents and the resulting aPTT values in patients receiving DTIs, measurable differences were observed in the capability of the various reagents to measure the response to DTI doses in the range of 0.1-1.2 lg/mL [5]. For some reagents, the aPTT results would not increase as the dose of the DTI increased, making the reagent nonresponsive at certain levels. Therefore, using the aPTT to measure response to DTIs may not accurately reflect correct doses, and subsequently put a patient at risk for either over-or, in some cases, under-anticoagulation.In addition, the aPTT is not as predictable in patients with lupus anticoagulants or deficiencies of factors VIII, IX, XI, or XII. For these reasons, alternative testing options have been investigated, described later. A small number of reference laboratories offer some of these alternative tests. Some tests are FDA-app...

These data demonstrate the importance of establishing locally relevant reference ranges.

The treatment of a patient with a factor VIII (FVIII) deficiency can be complicated. The mainstay of therapy is factor replacement. Replacement therapy can be given prophylactically, with the goal of decreasing hemarthroses and spontaneous hemorrhage, or on-demand for the bleeding patient. Intra- and interindividual variability in a patient's response to treatment has been well documented by the differences in observed half-lives of infused product. Although weight-based dosing nomograms are most often used, personalized therapies are coming into use to ease the burden of therapy and cost. The most significant complication of treatment is the formation of inhibitors to FVIII. The role of the laboratory is to provide results for FVIII activity that accurately reflects a patient's baseline level and response to treatment. However, factor activity assays have many components that can contribute to result variability. These include the methodology and reagent components used to measure the FVIII activity, the reference standard employed, algorithm used to interpret the dilutions, and the replacement factor being measured. An understanding of assay variables and their impact will assist in providing accurate factor activity results and appropriate patient care.

Summary Introduction Obtaining a reference interval (RI) is a challenge for any laboratory and becomes more complicated in the coagulation laboratory due to testing on samples with limited stability on reagents that are poorly standardized. Reference intervals are required to be able to evaluate results in relation to a patients’ hemostatic disorder. This becomes one of the most important tasks conducted in the coagulation laboratory. However, many laboratories lack the time, finances and in many cases the expertise to conduct this study. Methods Many RI are obtained from package inserts, or from publications written by experts in lieu of laboratories conducting their own studies. An overview of validating reference intervals and options for verifying or transference of reference intervals is discussed. Results Based on the confidence interval and the acceptability of risk laboratories are willing to accept, coagulation laboratories have options to conduct robust studies for their RI. Data mining or global reference studies may help to provide data for age specific ranges. Conclusions Pre‐analytical variables and selection of healthy subjects have the largest impact on coagulation testing outcomes and need to be well controlled during the establishment of reference intervals. Laboratories have options in lieu of conducting a full validation on how to verify RI based on smaller RI studies or transference of RI after determining compatibility of the original RI study.

Quantification of inhibitory antibodies against infused factor VIII (FVIII) has an important role in the management of patients with hemophilia A. This article summarizes results from the largest North American FVIII inhibitor proficiency testing challenge conducted to date. Test samples, 4 negative and 4 positive (1-3 Bethesda units [BU]/mL), were distributed by the ECAT Foundation in conjunction with the North American Specialized Coagulation Laboratory Association and analyzed by 38 to 42 laboratories in 2006 and 2007. Whereas laboratories were able to distinguish between the absence and presence of low-titer FVIII inhibitors, the intralaboratory coefficient of variation was high (30%-42%) for inhibitor-positive samples, and the definition of lower detection limits of the assay was variable (0-1 BU/mL). Most laboratories performed the Bethesda assay with commercially supplied buffered normal pooled plasma in a 1:1 mix with patient plasma. These data provide information for the development of consensus guidelines to improve FVIII inhibitor quantification.