The danger of relying on the APTT and PT in patients on DOAC therapy, a potential patient safety issue.

Prolongation of the activated partial thromboplastin time (APTT) and prothrombin time/international normalized ratio (PT/INR) correlates poorly with plasma concentrations of direct oral anticoagulant agents (DOACS) including direct thrombin and direct Xa inhibitors. It has been repeatedly demonstrated that patients can have normal APTT and PT/INR with a therapeutic plasma concentration of a DOAC. Clinicians can no longer rely on a normal APTT and PT to determine that an anticoagulated patient is safe to undergo an invasive procedure. Laboratory scientists need to play a key and active role in educating clinicians about the limitations of the APTT and PT in patients on DOAC prophylaxis or therapy.

A single test to assay warfarin, dabigatran, rivaroxaban, apixaban, unfractionated heparin, and enoxaparin in plasma.

UNLABELLED: Essentials Simple and fast assaying of different anticoagulants (ACs) is useful in emergent situations. We used highly diluted prothrombin time (dPT) or highly diluted Fiix-PT (dFiix-PT) to assay ACs. Both tests could quantify target specific anticoagulants and warfarin anticoagulation. Improved results were consistently observed with the dFiix-PT compared with the dPT. SUMMARY: Background Assaying anticoagulants is useful in emergency situations or before surgery. Different specific assays are currently needed depending on the anticoagulant. Objectives We hypothesized that levels of warfarin, dabigatran, rivaroxaban, apixaban, and heparins could be measured with use of the diluted prothrombin time (dPT) and diluted Fiix-PT (dFiix-PT), using highly diluted thromboplastin (TP). The latter test is affected only by reduced levels of active factors II and X but corrects test plasma for other deficiencies Methods Increasing TP dilutions were used to identify suitable dilutions to measure dabigatran, rivaroxaban, apixaban, unfractionated heparin (UFH), and enoxaparin. Calibrators containing known amounts of direct oral anticoagulants (DOACs) were used to make standard curves. Citrated plasma samples were obtained from patients taking warfarin or DOACs with known drug concentrations as determined by specific assays. Results The dFiix-PT at a TP dilution of 1:1156 could be used to measure all of the drugs tested at therapeutic concentrations except for fondaparinux. The dPT achieved the same but required two TP dilutions (1:750 and 1:300). The warfarin effect could be assessed by using dFiix-PT at 1:1156 with a PT ratio identical to the international normalized ratio. Six different TPs yielded similar results, but two were less sensitive. Dabigatran, rivaroxaban, and apixaban could be accurately measured in patient samples using both dilute PT assays, but a better correlation was consistently observed between the dFiix-PT and specific assays than with the dPT. Conclusion The dFiix-PT using a single dilution of TP may be suitable to assess the anticoagulant effects of warfarin, dabigatran, rivaroxaban, apixaban, heparin, and enoxaparin.

Verifying the performance characteristics of the TEG5000 thromboelastogram in the clinical laboratory.

OBJECTIVE: To verify the manufacturer performance claims of the TEG5000 with traditional laboratory methods. MATERIALS AND METHODS: Samples were concurrently measured using the TEG5000 analyzer and either PT, APTT, fibrinogen, factor activities, platelet count, or platelet function testing using whole blood or platelet-rich plasma methods. RESULTS: Within-run imprecision yielded coefficient of variation (CV) of <5%. There was no correlation of PT or APTT with R time. Only Factor VIII and factor XII activity significantly correlated with R time. There was significant correlation between k and angle with FBG, PLT count, and factor levels. There was weak inverse correlation between angle results and measures of platelet function. All laboratory methods were significantly correlated with MA. There were significant differences between citrated whole blood and fresh citrated plasma for angle and MA, and between fresh and frozen plasma for R time and MA. We demonstrated a high % inhibition noted with normal, drug naïve donors, especially with ADP PLT mapping (50% inhibition), but less so with AA PLT mapping (20% inhibition). For TEG platelet mapping, 19/22 (86.3%) and 17/22 (77.3%) results were concordant with traditional aggregation results. CONCLUSION: We demonstrated both the lack of, and strong correlation between laboratory tests and the TEG parameters.

The laboratory's 2015 perspective on direct oral anticoagulant testing.

The introduction of direct oral anticoagulant (DOAC) therapy into clinical use in the past 5 years has had significant impact on the clinical laboratory. Clinicians' desire to determine plasma drug presence or measure drug concentration, and more recent observations regarding the limitations and utility of coagulation testing in the setting of DOAC treatment, suggest that early published recommendations regarding laboratory testing should be reassessed. These initial recommendations, furthermore, were often based on drug-spiked plasma studies, rather than samples from patients receiving DOAC therapy. We have demonstrated that reagent sensitivity varies significantly whether drug-spiked samples or samples from DOAC-treated patients are tested. Data from drug-enriched samples must therefore be interpreted with caution or be used as a guide only. We present laboratory assays that can be used to determine drug presence and to measure drug concentration, and provide recommended testing algorithms. As DOAC therapy may significantly impact on specialty coagulation assays, we review those tests with the potential to give false-positive and false-negative results.

Assessing nonvitamin K antagonist oral anticoagulants (NOACs) in the laboratory.

Nonvitamin K antagonist oral anticoagulants (NOACs) are being used with increasing frequency due to their safety profile, ease of use, and given that therapeutic monitoring is not required. As these agents have only recently been FDA approved, their effect on routine and specialty coagulation assays is not well appreciated. This article discusses NOAC effect on routine coagulation assays and whether these assays can be used to estimate drug concentration as well as which assays are suitable to quantitate drug concentration in plasma. Also reviewed is the use of manufactured drug calibrators to determine reagent responsiveness and the effect of these agents on various special coagulation assays.

Effects of storage and thawing conditions on coagulation testing.

INTRODUCTION: Current recommendations for coagulation testing storage and thawing are based on historical studies that were performed using unbuffered 3.8% sodium citrate. We sought to measure the effects of freezing and thawing conditions 3.2% buffered sodium citrate plasma samples that have been stored in vials with either snap or sealed screw tops, frozen in -70 °C freezer or dry ice and thawed either capped or uncapped. METHODS: Shed blood samples were pooled and then aliquoted into four snap top and four screw tops vials. Half the vials were stored in a -70 °C freezer, and half on dry ice for at least 16 h. Afterwards, half the frozen samples were thawed in 37 °C waterbath capped, and other half were thawed capped. After thawing cycles, samples were tested for PT, activated partial thromboplastin time (APTT), fibrinogen, D-dimer, factor assays, von Willebrand factor activity, plasminogen, antithrombin, protein C and lupus anticoagulant. RESULTS: Prothrombin time, APTT, factor X, and lupus anticoagulant testing were affected by all vials, freezing and thawing conditions, whereas fibrinogen, D-dimer, von Willebrand activity or protein C were not affected by any vial, freezing or storage condition. CONCLUSIONS: Storage vials, freezing and thawing condition affect coagulation testing, although these differences may not be clinically significant.

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.

Serial haemostatic monitoring of dogs with multicentric lymphoma.

Lymphoma is the most common haematopoietic malignancy in dogs and it has been associated with hypercoagulability and subsequent thromboembolism. The objectives of this study were to serially characterize the haemostatic status of dogs with multicentric lymphoma. Thromboelastography, thrombin-antithrombin complex concentration and routine haematology and coagulation panels were measured. Twenty-seven dogs were included in the study and 15 completed the study in remission. At presentation, 81% (22/27) of dogs with multicentric lymphoma had altered haemostatic profiles consistent with hypercoagulability. Laboratory evidence of hypercoagulability did not resolve during treatment or for up to 1 month following attainment of clinical remission. Accelerated rate of clot formation at the time of chemotherapeutic protocol completion was associated with decreased survival time. We concluded that dogs with multicentric lymphoma were frequently hypercoagulable from presentation through 4 weeks after the completion of chemotherapy. Increased angle and shortened K in dogs that have successfully completed their chemotherapeutic protocol may be associated with shorter survival times.

Characterization of thrombelastography over time in dogs with hyperadrenocorticism.

Canine hyperadrenocorticism (HAC) is a common endocrinopathy often associated with hypercoagulability, thrombosis and thromboembolism and it can contribute to increased morbidity and mortality. The condition results in increased, unregulated secretion of glucocorticoids (GCs). While prospective identification of hypercoagulability is challenging, thrombelastography (TEG) is a diagnostic tool that enables the detection of hypercoagulability in a clinical setting. The objective of this prospective cohort study was to serially assess coagulation in dogs with HAC using TEG to test the hypothesis that dogs with HAC have increased TEG maximal amplitude (MA) and that the MA would normalize once clinical control was achieved. Twenty-three dogs with naturally occurring HAC were enrolled and hemostatic (including TEG, platelet function, thrombin-antithrombin complexes and coagulation panel) and hematological variables were measured at presentation. TEG was serially monitored until clinical resolution of HAC was attained. At presentation, most dogs with HAC had increased MA values, increased thrombin-antithrombin complexes and many were hyperfibrinogenemic. Platelet function analyzer-100 (PFA-100) closure times were significantly prolonged. TEG tracings did not normalize in either medically- or surgically-managed dogs, but fibrinogen concentrations decreased. It seems that dogs with HAC have a complex coagulopathy in which hypercoagulability and platelet hyporeactivity or dysfunction might occur simultaneously. As TEG tracings did not normalize in well-controlled dogs, it is unlikely that increased blood GCs are solely responsible for TEG alterations seen in dogs with HAC.

Comparison of the BioRad Variant and Primus Ultra2 high-pressure liquid chromatography (HPLC) instruments for the detection of variant hemoglobins.

INTRODUCTION: Hemoglobin variants are a result of genetic changes resulting in abnormal or dys-synchronous hemoglobin chain production (thalassemia) or the generation of hemoglobin chain variants such as hemoglobin S. Automated high-pressure liquid chromatography (HPLC) systems have become the method of choice for the evaluation of patients suspected with hemoglobinopathies. METHODS: In this study, we evaluated the performance of two HPLC methods used in the detection of common hemoglobin variants: Variant and Ultra2. RESULTS: There were 377 samples tested, 26% (99/377) with HbS, 8.5% (32/377) with HbC, 20.7% (78/377) with other hemoglobin variant or thalassemia, and 2.9% with increased hemoglobin A(1) c. The interpretations of each chromatograph were compared. There were no differences noted for hemoglobins A(0), S, or C. There were significant differences between HPLC methods for hemoglobins F, A(2), and A(1) c. However, there was good concordance between normal and abnormal interpretations (97.9% and 96.2%, respectively). CONCLUSION: Both Variant and Ultra2 HPLC methods were able to detect most common hemoglobin variants. There was better discrimination for fast hemoglobins, between hemoglobins E and A(2), and between hemoglobins S and F using the Ultra2 HPLC method.

Evaluation of a new automated quantitative d-dimer, Advanced D-Dimer, in patients suspected of venous thromboembolism.

The objective of our study was to evaluate the performance characteristics of a new automated d-dimer, the Advanced D-Dimer (Dade Behring Inc., Deerfield, IL) for use in the diagnosis of venous thromboembolism (VTE). To do this we compared the Advanced D-Dimer to existing d-dimer methods using established target cut-off values in patients suspected of VTE who were to undergo definitive radiographic studies for VTE. We studied hospitalized patients and outpatients who were suspected of having VTE and who had whole blood d-dimer performed. The patients who underwent a diagnostic study for VTE had their D-dimer results used to determine sensitivity, specificity and negative predictive values. There was relatively poor correlation between the Advanced D-Dimer and D-Dimer Gold (r = 0.63; t-test: P < 0.005) and Asserachrome D-Di (r = 0.58; t-test: P < 0.005). The Advanced D-Dimer target cutoff values for excluding VTE in hospitalized and outpatients were < or = 1800 microg/L and < or = 1500 microg/l respectively. There were 139 patients suspected with pulmonary embolism (PE) and 328 evaluated for deep vein thrombosis (DVT). There were 24 patients with PE, and 43 with DVT. The Advanced D-Dimer had comparable sensitivity, specificity and negative predictive values (96, 43, 98% for PE and 96, 48, 99% for DVT respectively) to other d-dimer methods used for that purpose. We conclude that the Advanced D-Dimer correlates relatively poorly with enzyme-linked immunosorbent assay methods. This poor correlation is likely due to incorrect reporting units and concentration. When these factors are corrected correlations improved. Compared to existing d-dimer methods used for VTE exclusion, the high sensitivity and negative predictive value would suggest that this method can be used as part of a diagnostic algorithm for the exclusion of PE and DVT.

Platelet activation and function after trauma.

BACKGROUND: Abnormal hemostasis is associated with many of the complications of trauma-associated morbidity and mortality. Platelets are integral in the maintenance of hemostasis. METHODS: Samples were obtained from 100 trauma patients on arrival at the emergency room (initial time) and at 24, 48, and 72 hours later. Samples were also obtained from 10 healthy controls at the same time intervals. Using flow cytometry, three parameters were used to measure platelet activation: platelet microparticles, expression of P-selectin (CD62P), and expression of the activated conformation of glycoprotein IIb-IIIa (PAC-1 binding). Platelet function was measured using a platelet function analyzer (PFA-100, Dade International Inc., Miami, FL). RESULTS: One hundred trauma patients were enrolled. The average age was 40 years, 75% were men, and 84% had blunt injuries. The mean Injury Severity Score was 22.3 +/- 10.9 (mean +/- SD) and the average Glasgow Coma Scale score was 11 +/- 4. All three platelet activation parameters were increased in trauma patients versus controls for all time periods (p < 0.001). Trauma patients had a trend toward a shorter initial collagen/epinephrine closure time versus controls (p = 0.096). Compared with the 24-, 48-, and 72-hour time intervals, initial collagen/epinephrine closure times were shortened (p < 0.001, p < 0.001, and p < 0.001). Platelet function returned to normal reference ranges within 24 hours but platelet activation parameters remained elevated at least 72 hours after initial trauma. In contrast, when trauma patients with and without brain injury were compared, brain injury patients had increased platelet activation but decreased platelet function (increased collagen/epinephrine closure times). In addition, there was a significant prolongation in collagen/epinephrine closure times for the 24-, 48-, and 72-hour time points in nonsurviving patients versus survivors. There was no association between platelet activation and function and other adverse outcomes including pulmonary embolism, deep venous thrombosis, and disseminated intravascular coagulation. CONCLUSION: Severe injury usually results in increased platelet activation and function. However, the combination of increased platelet activation with decreased function was associated with increased mortality.

Practical utility of the D-dimer assay for excluding thromboembolism in severely injured trauma patients.

BACKGROUND: We have advocated the use of a D-dimer assay to exclude the diagnosis of pulmonary embolism (PE) and deep venous thrombosis (DVT) in surgical and trauma patients suspected of having these diagnoses. Injury is known to increase D-dimer levels independent of thromboembolism. The purpose of this study was to assess the period after injury over which the D-dimer assay remains positive because of injury exclusive of thromboembolism. METHODS: We prospectively sampled the plasma of severely injured patients for D-dimer using an enzyme-linked immunosorbent assay method at admission; at hours 8, 16, 24, and 48; and at days 3, 4, 5, and 6. Patients were then screened for DVT with a routine duplex Doppler at day 7. Patients were followed for PE, adult respiratory distress syndrome, and disseminated intravascular coagulation. RESULTS: One hundred fifty-four patients (mean Injury Severity Score of 23) underwent a total of 1,230 D-dimer assays. Twenty-six (17%) had thromboembolism. Nine (6%) patients developed DVT, 2 (1%) developed PE, 13 (8%) developed disseminated intravascular coagulation, and 11 (7%) developed severe adult respiratory distress syndrome. None of the trauma patients with thromboembolism had a (false) negative D-dimer at or after the time of their thromboembolic complication. True-negative D-dimer results as a function of time from injury are: 0 hours, 18%; 8 hours, 16%; 16 hours, 17%; 24 hours, 22%; 48 hours, 37%; day 3, 34%; day 4, 32%; day 5, 30%; and day 6, 30%. The negative predictive value of the assay was 100%. D-dimer levels were significantly higher in those who developed a thromboembolic complication than in those who did not (independent of Injury Severity Score). CONCLUSION: These data serve to validate D-dimer as a means of excluding thromboembolism, specifically in patients with severe injury (100% negative predictive value). Before 48 hours after injury, however, the vast majority of these patients without thromboembolism had positive D-dimer assays. Because of the high false-positive rate early after severe injury, the D-dimer assay may be of little value before postinjury hour 48.

Anticoagulation of children undergoing cardiopulmonary bypass is overestimated by current monitoring techniques.

HYPOTHESIS: Children who undergo cardiopulmonary bypass (CPB) are proportionally more hemodiluted than adults who undergo CPB. Current methods of monitoring high-dose heparin sulfate anticoagulation are dependent on fibrinogen level. Because of the decreased fibrinogen levels in children, current methods of monitoring heparin anticoagulation overestimate their level of anticoagulation. DESIGN: Prospective controlled trial. MAIN OUTCOME MEASURE: Production of thrombin (adequacy of anticoagulation). METHODS: Children and adults undergoing cardiac surgery who received CPB were anticoagulated in the standard fashion as directed by activated clotting time (ACT) results. Each subject had blood sampled at baseline; heparinization; start of the CPB; CPB at 30, 60, and 90 minutes; and at termination of CPB. Samples were used to assess anticoagulation with the Heparin Management Test (less dependent on fibrinogen level than ACT). We also assessed 2 subclinical markers of thrombosis, thrombin-antithrombin complexes and prothrombin fragment F1.2; a marker of procoagulant reserve, fibrinogen; the natural antithrombotic, antithrombin; and heparin concentration. RESULTS: Ten children and 10 adults completed the study. Children had lower fibrinogen levels than adults throughout CPB (P<.05). All adults had both therapeutic ACT and Heparin Management Test levels measured throughout CPB. Although children had therapeutic ACT levels, their Heparin Management Test levels were subtherapeutic while undergoing CPB. The children had significantly higher thrombin-antithrombin complexes and prothrombin fragment F1.2 than adults, indicating ongoing thrombin production (P<.01). The increases in thrombin-antithrombin complexes and prothrombin fragment F1.2 in children were inversely proportional to their weight. CONCLUSIONS: Children undergoing CPB with heparin dosing adjusted to optimize the ACT manifest inadequate anticoagulation (ongoing thrombin formation). High-dose heparin anticoagulation therapy in children undergoing CPB should be directed by tests (like the Heparin Management Test) that are less dependent on fibrinogen level than ACT.

Whole blood D-dimer assay: an effective noninvasive method to rule out pulmonary embolism.

BACKGROUND: The whole blood D-dimer assay has gained recognition as a noninvasive test to rule out pulmonary embolism (PE) in medical patients. METHODS: We performed a whole blood D-dimer assay in medical and surgical patients undergoing either pulmonary angiogram or pulmonary ventilation perfusion scan for suspected PE or duplex Doppler or venogram for suspected deep venous thrombosis (DVT). RESULTS: A total of 483 patients were enrolled; 16 were excluded because of an equivocal pulmonary ventilation perfusion scan. The 467 remaining patients had a mean age of 56 +/- 27 years. There were 258 women and 209 men. A total of 353 patients were admitted to a medical service and 114 to surgery/ trauma. A total of 82 patients (18%) developed thromboembolism: 20 had PE, and 62 had DVT. CONCLUSION: No surgical patient with PE or DVT (n = 27) had a negative D-dimer. A negative D-dimer result in a stable surgical patient should be considered conclusive evidence to rule out thromboembolism and, thus, negate the need for further diagnostic studies. In our surgical patients suspected of DVT or PE, had D-dimer been used, one third of the patients would have avoided an expensive or invasive diagnostic test.

A new method for measuring D-dimer using immunoturbidometry: a study of 255 patients with suspected pulmonary embolism and deep vein thrombosis.

D-Dimer testing has been suggested as a non-invasive method for the exclusion of pulmonary embolism (PE) and deep vein thrombosis (DVT). In this study, we compared a new method, the Miniquant D-dimer (Biopool International, Ventura, California, USA) to other previously validated D-dimer methods used for the purpose. Patients who were undergoing a definitive diagnostic study for thromboembolism had a blood sample drawn at that time. A whole-blood D-dimer (SimpliRed; Agen Biomedical Ltd, Brisbane, Australia) test was performed, and residual plasma was frozen and later analyzed using two enzyme-linked immunosorbent assay (ELISA) methods (D-dimer Gold; Agen, and Asserachrome D-Di; Stago International, Parsippany, New Jersey, USA) and the Miniquant D-dimer. Once all samples were analyzed, the correlation and accuracy of the Miniquant was compared with the ELISA method using Spearman's regression and Dunn's multiple paired comparison. All D-dimer methods were compared with radiographic studies. The data was analyzed collectively and segregated into in-patient (n = 112) and out-patient (n = 143) populations. The Miniquant D-dimer sensitivity, specificity and negative predictive value (NPV) for all patients were 95, 21, and 94% for DVT, and 100, 26, and 100% for PE. This new D-dimer method demonstrates acceptable sensitivity in patients with PE and DVT and, based on the high NPV of this method, it can be used for the exclusion of thromboembolism.