Dorothy M. Adcock

Phenotypic correction of murine hemophilia A using an iPS cell-based therapy

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.

Thrombotic Events in Patients With Cancer Receiving Antiangiogenesis Agents

Tumor-associated neoangiogenesis has recently become a suitable target for antineoplastic drug development. In this overview, we discuss specific drug-associated hemostatic complications, the already known pathogenetic mechanisms involved, and the effect of varying antithrombotic strategies. Multiple agents with angiogenic inhibitory capacity (thalidomide, lenalidomide, bevacizumab, sunitinib, sorafenib, and sirolimus) have obtained US Food and Drug Administration approval, and many others have entered clinical trials. Arterial and venous thromboembolism and hemorrhage have emerged as significant toxicities associated with the use of angiogenesis inhibitors. We present a detailed analysis of the literature on thrombotic complication of antiangiogenic drugs. Close attention to hemostatic complications during antiangiogenic treatment is warranted. Further studies are required to better understand the pathophysiologic mechanisms involved and to define a safe prophylactic strategy.

The effect of time and temperature variables on routine coagulation tests.

This study evaluates the effects of time and temperature variables on routine coagulation assays [Prothrombin Time test and Activated Partial Thromboplastin Time (APTT) test]. Four different groups were studied: healthy volunteers, hospitalized patients not receiving anticoagulants, patients receiving oral anticoagulant therapy and patients receiving unfractionated heparin therapy. Samples were subjected to one of four conditions: (1) centrifuged immediately and stored at room temperature (20–22°C); (2) centrifuged immediately and stored on ice (4°C); (3) stored as whole blood without centrifugation, at room temperature and (4) stored without centrifugation, on ice. Coagulation tests were performed as soon as possible after phlebotomy and at specified times up to 24 h. Our data demonstrate that prothrombin time results are stable for up to 24 h, remaining constant regardless of storage conditions. APTT assays are stable for up to 8 h, except for patients receiving unfractionated heparin therapy. Heparinized samples, when stored uncentrifuged at room temperature, demonstrate a clinically significant shortening of the APTT and individual samples demonstrate a greater than 50% decrease in ex-vivo heparin levels at 4 h.

The effect of Dabigatran on select specialty coagulation assays

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.

Heparin-Calibrated Chromogenic Anti-Xa Activity Measurements in Patients Receiving Rivaroxaban: Can This Test Be Used to Quantify Drug Level?

Background: Determination of plasma rivaroxaban concentration may be necessary in certain clinical situations. Rivaroxaban concentration can be accurately and rapidly determined using a chromogenic anti–activated factor X (factor Xa) assay with specific drug calibrator material. However, there are currently no Food and Drug Administration (FDA)-approved rivaroxaban calibrators available in the United States. Objective: To determine whether FDA-approved commercial kits for measuring heparin anti–factor Xa activity can be used to assess rivaroxaban concentrations when calibrated for unfractionated heparin or low-molecular-weight heparins. Methods: Trough and peak samples were taken from 30 patients taking rivaroxaban as part of their routine care for atrial fibrillation or venous thromboembolism. The samples were tested using 3 different FDA-approved commercial kits for measuring heparin anti–factor Xa activity. Results: There was acceptable correlation between rivaroxaban levels and heparin anti–factor Xa activity using Berichrom and COAMATIC heparin kits. The STA liquid heparin method was the most sensitive to presence of rivaroxaban. Conclusion: This study demonstrates a strong correlation, but variability between kits, for assessing rivaroxaban concentrations using heparin anti–factor Xa assays. The extent of the heparin calibration curve significantly limits the measurable rivaroxaban range, and this application may be useful only for trough samples. The STA liquid heparin, being exquisitely sensitive to rivaroxaban, may be suitable for ruling out presence of the drug. The routine use of heparin-calibrated anti–factor Xa assays to quantify rivaroxaban is not advocated, and when applied, it must be used with caution and limitations clearly understood.

Performance of Various Laboratory Assays in the Measurement of Dabigatran in Patients Receiving Therapeutic Doses

OBJECTIVES To study dabigatran etexilate, a new oral anticoagulant that functions as a direct thrombin inhibitor. METHODS This study evaluates four methods, one of which is performed in three different laboratories, and compares results against dabigatran levels measured by Boehringer-Ingelheim (Ingelheim, Germany) using mass spectrometry. RESULTS 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, activated partial thromboplastin time, and thrombin time do not reliably determine levels of dabigatran anticoagulation. Alternative assays, when calibrated with a dabigatran standard, such as the modified dilute thrombin time, ecarin clotting time, and ecarin chromogenic assay, may be appropriate, although a comparison of these methods using samples from patients taking dabigatran has not been performed. CONCLUSIONS Although results using all methods in this study demonstrate adequate correlation, measured dabigatran levels varied in a statistically significant manner, even when the same method was used by different laboratories. The clinical significance of this variation in dabigatran concentrations is uncertain.

Increases in Quantitative D-Dimer Levels Correlate With Progressive Disease Better Than Circulating Tumor Cell Counts in Patients With Refractory Prostate Cancer

Changes in quantitative D-dimer levels, circulating tumor cell (CTC) counts, and prostate-specific antigen (PSA) levels were measured in 28 patients with refractory castration-resistant prostate cancer to assess their concordance during the course of therapy and their relationship with risk of progressive disease. A significant correlation was identified between changes in PSA and both CTC counts and D-dimer levels (r = 0.67 and 0.58, respectively; P < .001). In addition, there was a significant correlation between changes in CTC count and D-dimer level (r = 0.62; P < .001). A significantly stronger concordance between these biomarkers was noted for increasing values (sensitivity, 72%-77.8%) compared with decreasing values (specificity, 43.8%-71.4%). Notably, increases in PSA and D-dimer levels, not CTC counts, were associated with increased risks for progressive disease (P < .024). Increases in quantitative D-dimer levels correlate with progressive disease better than CTC counts in patients with refractory prostate cancer.

Evaluating the use of commercial drug-specific calibrators for determining PT and APTT reagent sensitivity to dabigatran and rivaroxaban

Suitable laboratory methodologies for quantifying the non-vitamin K oral anticoagulants (NOAC) include liquid chromatography-tandem mass spectrometry (LC-MS/MS) or drug-calibrated assays such as the dilute thrombin time for dabigatran or anti-Xa measurements for rivaroxaban. In situations when these tests are unavailable, it has been suggested that using commercial drug calibrators on APTT and PT assays would theoretically provide reagent sensitivity to these drugs. The purpose of this study was to determine whether commercial drug calibrators deliver similar reagent sensitivity information as samples from patients receiving dabigatran or rivaroxaban as part of their routine care. Two laboratory sites tested commercial calibrator material for dabigatran and rivaroxaban (Hyphen Biomedical) using PT and APTT reagents and data was compared to samples collected from patients taking NOACs that were quantified by LC-MS/MS. Correlation statistics and calculating the amount of drug required to double the clotting time of normal plasma were performed. All drug calibrator material correlated more strongly (R²> 0.95) for any reagent/drug combination than patient samples (R² ranged from 0.29-0.86). Dabigatran calibrator results and patient data were equivalent for SynthASil and PTT-A APTT reagents. The dabigatran and rivaroxaban calibrator material over-estimated drug sensitivity for all PT reagents when compared to sensitivity data calculated based on drug levels obtained by LC-MS/MS from patient samples. In conclusion, drug-specific calibrators overestimated reagent sensitivity which may underestimate in vivo drug concentration in a given patient. Further studies are required to assess whether this method of determining relative sensitivity of NOAC on routine coagulation assays should be recommended.

The Hemostatic System and Malignancy

There is an intimate relationship between the processes involved in malignancy and hemostasis. The mechanisms by which malignancy promotes thrombosis are reviewed herein. Emphasis, however, is placed upon the potential mechanisms by which the hemostatic system modulates malignant potential of tumors. Both thrombotic and nonthrombotic processes related to the enhancement of tumor growth, angiogenesis, and metastases are presented. Also reviewed are the effects of anticoagulants such as vitamin K antagonists, heparin, and other glycosaminoglycans on survival of patients with cancer. As new anticoagulants and targeted anticancer therapies are developed, we can expect the interactions between coagulation and tumor biology to be an evolving and productive area for breakthroughs in basic science and clinical applications.

Critical pre-examination variables in the hemostasis laboratory and their quality indicators

The total testing process comprises a number of phases of laboratory testing, which can be broadly considered as comprising pre-examination, examination and post-examination activities. Although each phase is crucial to providing accurate and meaningful laboratory results, the pre-examination phase of testing is where most laboratory errors currently occur, and thus requires special attention. The activities in this phase include sample collection, handling, transportation, processing and storage, which are frequently outside the control of the laboratory performing the tests. Samples for hemostasis testing are particularly vulnerable to pre-analytical variables, which may ultimately lead to inappropriate test results. We outline here several strategies to mitigate potential problems in the pre-examination phase. We also recommend the implementation of several processes to reduce errors.