Argatroban is stable in citrated whole blood for 24 hours.

INTRODUCTION: Argatroban is a direct thrombin inhibitor used as an anticoagulant for patients who have Heparin induced thrombocytopenia. Quantification can be performed using a dilute thrombin time or anti-iia assay. Our preferred method is Hemoclot Thrombin Inhibitor Assay (HTI).To the best of our knowledge, no one has published on the stability of plasma argatroban in whole citrated blood at room temperature. METHODS: Forty matched samples obtained from 4 patients receiving argatroban. 1 mL of the whole blood was removed from the sample into a labelled plastic tube and left on the laboratory bench at room temperature for 24 hours prior to centrifugation. The remaining sample was spun at 2000 g for 10 minutes and the plasma aliquoted off and labelled 0 hour and stored at -80°C prior to testing. At 24 hours the plastic tube containing whole blood was centrifuged at 2000 g for 10 minutes and the plasma aliquoted off and labelled 24 hours and stored at -80°C prior to testing. HTI assay was used for plasma argatroban determination. RESULTS: The lowest argatroban level obtained was 0.16 µg/mL at 0 hour; 0.18 µg/mL at 24 hours from the same pair, the highest argatroban level obtained was 1.72 µg/mL at 0 hour;1.76 µg/mL for the same pair at 24 hours. The mean result for 0 hour was 0.57 µg/mL and 0.60 µg/mL at 24 hours. CONCLUSION: This study proposes that patients receiving treatment in hospitals who cannot provide a dedicated argatroban plasma concentration method could have their samples sent on whole blood within 24 hours of venepuncture to a laboratory who could provide the test.

Presenting ADAMTS13 antibody and antigen levels predict prognosis in immune-mediated thrombotic thrombocytopenic purpura.

Immune-mediated thrombotic thrombocytopenic purpura (TTP) is a life-threatening disorder caused by antibodies against ADAMTS13. From the United Kingdom TTP registry, we undertook a prospective study investigating the impact of the presenting anti-ADAMTS13 IgG antibody and ADAMTS13 antigen on mortality. A total of 312 episodes involving 292 patients over 87 months were included; 68% were female, median age 46 (range, 11-88 years), and median presenting ADAMTS13 of <5% (range, <5%-18%). The mortality rate was 10.3% (n = 32); 68% of patients had a raised troponin at presentation conferring a sixfold increase in mortality compared with those with normal troponin levels (12.1% vs 2.0%, P = .04). Twenty-four percent had a reduced Glasgow Coma Score (GCS) at presentation with a ninefold increase in mortality (20% vs 2.2% for normal GCS at presentation, P < .0001). Mortality increased with higher anti-ADAMTS13 antibody levels and lower ADAMTS13 antigen levels. Those with antibody levels in the upper quartile (antibody >77%) had a mortality of 16.9% compared with 5.0% for the lowest quartile (antibody <20%) (P = .004). Those with an antigen level in the lowest quartile (antigen <1.5%) had a mortality of 18% compared with 3.8% for the highest quartile (antigen >11%) (P = .005). The synergistic effect of anti-ADAMTS13 IgG antibody in the upper quartile and ADAMTS13 antigen in the lowest quartile had the highest mortality of 27.3%. We conclude that both anti-ADAMTS13 IgG antibody and ADAMTS13 antigen levels correlate with outcome in TTP with increased cardiac and neurological involvement and increased mortality.

Limitation of the activated partial thromboplastin time as a monitoring method of the direct thrombin inhibitor argatroban.

BACKGROUND: Argatroban is licensed for patients with heparin-induced thrombocytopenia and monitoring is conventionally by activated partial thromboplastin time (APTT) ratio with a target of 1.5-3.0 and not exceeding 100 s. The APTT may be influenced by coagulopathies, lupus anticoagulant and raised FVIII levels. Variable but not clinically significant sensitivity of APTT reagents to argatroban has been highlighted in other studies. METHODS: Residual plasma of 15 patients (n = 124 samples) was tested on Sysmex(™) CS series using the Hemoclot(®) thrombin inhibitor assay (HTI) and APTT ratio (Actin FS and SynthASil). A subgroup from four patients (n = 31) were tested on ACL TOP(™) to compare the different platforms with the HTI. Spiked normal pooled plasma was tested with Actin FS, Actin FSL, SynthASil and APTT-SP on their respective platforms (CS5100(™) and ACL TOP(™)) to assess reagent sensitivity. RESULTS: Mean concentration of argatroban by HTI assay for patient plasma was 0.47 µg/mL; the mean APTT ratio using Actin FS was 1.89 and for SynthASil 1.56. There was a poor correlation between APTT and the HTI. In the spiked normal pooled plasma, Actin FS gave a significantly higher APTT ratio than the other three reagents for the various argatroban concentrations. CONCLUSIONS: Hemoclot(®) thrombin inhibitor assay should be considered in patients on argatroban, particularly if there is concern the APTT may not be reflective of the degree of anticoagulation with argatroban due to other factors including coagulopathies in critically ill patients, the presence of a lupus anticoagulant or very high FVIII levels.

Management of peri-operative anti-thrombotic therapy.

The management of antithrombotic therapy in the peri-operative setting is a common problem, balancing haemorrhagic risk with continued treatment and thrombotic risk when discontinued. High-quality evidence is lacking regarding the optimal approach for patients on oral anticoagulants or antiplatelet agents. This review discusses the available evidence for the management of patients on warfarin, non-vitamin K antagonist oral anticoagulant drugs, and antiplatelet therapy in the peri-operative setting. Bridging therapy for patients on warfarin should be considered for those at highest risk of thrombosis, whereas it may not be necessary for those on non-vitamin K antagonist oral anticoagulant drugs given the reduced time off anticoagulation and their more predictable pharmacokinetics. Aspirin can be continued for most procedures. Dual antiplatelet agents for patients with a recently inserted coronary artery stent should be continued if possible but decisions should be individualised and taken after multidisciplinary discussion.

Major surgery in severe haemophilia A with inhibitors using a recombinant factor VIIa and activated prothrombin complex concentrate hybrid regimen.

Major surgery in persons with haemophilia A and inhibitors is increasingly being performed. Both recombinant activated factor VII (rFVIIa) and activated prothrombin complex concentrate (APCC) are used to cover surgery but it remains unclear what the optimal dosing schedules are. We describe the use of a hybrid regimen in four inhibitor patients undergoing eight major surgical procedures using rFVIIa in the initial 2-6 postoperative days followed by FEIBA for the remaining period. All patients were also treated with tranexamic acid while receiving rFVIIa. We performed six major orthopaedic procedures, one emergency orchidectomy and one open appendectomy. The dosing schedules were at the higher end of those described in the literature but within the recommendations of the summary of product characteristics. Despite this, we encountered non-surgical bleeding in four of eight episodes. Three of these occurred in one individual suggesting a patient factor. The overall outcome was good for all episodes. The hybrid regimen combines flexibility of dose and dosing frequency of rFVIIa in the immediate postoperative setting with the advantage of a reduced dosing frequency with FEIBA in the subsequent days. This study also emphasizes that surgical procedures in this patient group remain a challenge.

The investigation of a prolonged APTT with specific clotting factor assays is unnecessary if an APTT with Actin FS is normal.

INTRODUCTION: An isolated prolongation to the activated partial thromboplastin time (APTT) can be caused by the presence of the lupus anticoagulant or an intrinsic or contact factor deficiency, of which only deficiencies of factors VIII, IX or XI are associated with bleeding. Our local protocol states that further investigation of a prolonged APTT by specific assays of FVIII, FIX and FXI should only be undertaken where the APTT with one reagent (Synthasil) is more than 3 s prolonged, and further investigation by an APTT with a second reagent (Actin FS) is also prolonged, unless there is a history of bleeding in the patient, in which case assays are indicated irrespective of the APTT. METHODS: We retrospectively reviewed the results of all APTTs performed over a 36-month period to evaluate whether strictly applying our protocol would reduce the number of unnecessary clotting factor assays performed, without leaving patients with potentially significant bleeding disorders undiagnosed. RESULTS: Of a total number of 587 samples tested for coagulation factors VIII, IX and XI, only 117 samples yielded an abnormal result. Thus, 80% of all the assays requested in the 3-year period audited gave a result within the reference range for factors VIII, FIX and XI. Three quarters of the abnormal results revealed mild FXI deficiency. CONCLUSION: This review has demonstrated that no significant coagulation factor deficiency would be left undiagnosed if the protocol was followed. This would have considerably reduced the cost and time spent performing these assays.

Optimizing warfarin reversal--an ex vivo study.

BACKGROUND: Warfarin reversal is a common clinical situation. This is commonly performed using vitamin K and, depending on the urgency, fresh frozen plasma (FFP), prothrombin complex concentrates (PCCs), or activated factor VII. Even though PCCs are widely used, the ideal dosing regimen is far from established. OBJECTIVES: To verify differences in warfarin reversal patterns using FFP, recombinant FVIIa (rFVIIa), and PCC; and to test the hypothesis that supratherapeutic International Normalized Ratios (INRs) might not correlate with thrombin generation (TG) and identify the ideal concentrations of PCC required to reverse various INR thresholds. METHODS: We studied the effects of FFP, rFVIIa and Beriplex P/N on the INR and TG, using the calibrated automated thrombography assay in ex vivo warfarinized plasma. Plasmas with different INRs were spiked with different concentrations of Beriplex P/N. RESULTS: Beriplex P/N was the only agent that completely normalized TG and the INR. The endogenous thrombin potential (ETP) and the peak thrombin showed a significant negative correlation with all INRs. The ETP and velocity of TG reached a plateau at an INR of approximately 4.0. A concentration equivalent to a dose of 30 IU kg(-1) Beriplex P/N normalized the ETP, the INR, FII, FVII, FIX and FX of samples with INRs > or = 4.0. Higher doses resulted in hypercoagulable TG patterns. A concentration equivalent to a dose of 20 IU kg(-1) was sufficient to reverse warfarin at an INR range of 2.0-3.9, as judged by the same tests. CONCLUSIONS: Warfarin reversal algorithms could be simplified with the adoption of this strategy utilizing two doses of PCC, depending on the INR of the patient. This would also lead to cost reductions and, possibly, a reduction in thrombotic risk.

The effect of tissue factor concentration on calibrated automated thrombography in the presence of inhibitor bypass agents.

Thrombin generation has been suggested as a method to monitor treatment with factor eight inhibitor bypassing activity (FEIBA) or recombinant FVIIa (rFVIIa). The sensitivity of the assay for individual coagulation factors is dependent on the tissue factor (TF) concentration. An inverse relation between the rFVIIa concentration needed to shorten the clotting time and TF concentration has been shown but the data on thrombin generation are inconsistent. Information on TF concentration in measurements with FEIBA is limited. We studied the influence of TF concentration (1 and 5 pM) on thrombin generation through spiking experiments with rFVIIa and/or FEIBA in the plasma of severe haemophilia A patients and after four and three treatment episodes, respectively, using the calibrated automated thrombin generation assay (CAT) in platelet poor plasma. Spiking with FEIBA showed a linear relation with the endogenous thrombin potential (ETP)/peak at 1 pM but substrate depletion at 5 pM. Spiking with rFVIIa showed a near linear dose-response relation with the ETP/peak at 1 pm but only a shortening of the initiation phase at 5 pM. Similar effects were present in post-treatment samples. FEIBA acted synergistically with rFVIIa. This suggest a role for CAT in monitoring inhibitor bypass treatment but low TF concentrations are required to avoid substrate depletion with FEIBA and to demonstrate the effect of rFVIIa.

Thrombin generation testing in routine clinical practice: are we there yet?

Thrombin is the central enzyme in the coagulation cascade. Estimation of an individual's potential to generate thrombin may correlate more closely with a hyper- or hypo-coagulable phenotype, compared to traditional coagulation tests. The possible correlation and recent technical advances in thrombin generation measurement has caused a significant interest in the method and the development of commercial assays. Several variations of the assay exist depending on the defect to be investigated. Fluorogenic thrombin generation assays have acceptable intra-laboratory variation but a higher inter-laboratory variation. Variation in preanalytical variables makes comparisons between studies difficult. Thrombin generation is highly variable between individuals and there are suggestions that this may allow individualized treatment based on global haemostatic response in patients with bleeding disorders or on anticoagulant therapy. In patients with thrombotic disorders it may be possible to identify those at higher risk of recurrent thrombosis. For both scenarios, however, data from large prospective studies are lacking or inconclusive and a good relationship between thrombin generation and phenotype remains to be established. Further standardization of the assay is needed before large multicentre studies can be conducted and until then thrombin generation in routine clinical practice is not yet a reality.

Wide variation in thrombin generation in patients with atrial fibrillation and therapeutic International Normalized Ratio is not due to inflammation.

Atrial fibrillation (AF) is a common cardiac arrhythmia with a 5-20% annual risk of stroke. Warfarin reduces this risk by at least 60%. Despite adequate anticoagulation within the target International Normalized Ratio (INR) range of 2.0-3.0, some patients still experience thrombotic and bleeding events. It is now possible to assess the intensity of anticoagulation with automated thrombin generation (TG) tests, such as the calibrated automated thrombogram (CAT). These tests were compared and an inverse relationship was found between the INR and CAT in 143 elderly AF patients. There was equally good correlation between the concentration of factors II, VII, IX and X and the INR and TG parameters. The peak thrombin was most strongly associated with the concentration of prothrombin fragment 1 + 2 in plasma. There was wide variability in TG parameters in patients with identical INR values, sometimes up to a fourfold difference. This TG variability in individuals with the same INR is not due to inflammation, at least when the latter is measured as the concentration of factor VIII coagulant activity, von Willebrand factor antigen, high sensitivity C-reactive protein and fibrinogen. It was concluded that, although the TG and INR were closely correlated there was wide variability in peak thrombin and endogenous thrombin potential in patients within the INR therapeutic range, the cause of which remains unclear.

Antiviral therapy for chronic hepatitis C in patients with inherited bleeding disorders: an international, multicenter cohort study.

BACKGROUND: Hepatitis C is a major co-morbidity in patients with hemophilia. However, there is little information on the efficacy of antiviral therapy and long-term follow-up after treatment. OBJECTIVES: To assess the effect of interferon-based (IFN-based) therapy on hepatitis C virus (HCV) eradication, to identify determinants associated with treatment response, and to assess the occurrence of end-stage liver disease (ESLD) after completing antiviral therapy. PATIENTS AND METHODS: In a multicenter cohort study, 295 treatment-naïve hemophilia patients chronically infected with HCV were included. The effect of therapy was expressed as sustained virological response (SVR). Determinants associated with treatment response were expressed as odds ratios (ORs). Cumulative incidence of ESLD was assessed using a Kaplan-Meier survival table. RESULTS: Among human immunodeficiency virus (HIV) negative patients (n = 235), SVR was 29% (29/101) for IFN monotherapy, 44% (32/72) for IFN with ribavirin, and 63% (39/62) for pegylated IFN (PegIFN) with ribavirin. In patients co-infected with HIV (n = 60), IFN monotherapy, IFN with ribavirin, and PegIFN with ribavirin eradicated HCV in 7/35 (20%), 1/2 (50%), and 11/23 (48%), respectively. SVR increased with genotype 2 and 3 [OR 11.0, 95% CI: 5.8-20.5], and combination therapy (IFN and ribavirin OR 3.7, 95% CI: 1.7-8.4), PegIFN and ribavirin (OR 4.2, 95% CI: 1.8-9.5). Up to 15 years after antiviral treatment, none of the patients with a SVR relapsed and none developed ESLD. In contrast, among unsuccessfully treated patients the cumulative incidence of ESLD after 15 years was 13.0%. CONCLUSIONS: Successful antiviral therapy appears to have a durable effect and reduces the risk of ESLD considerably.

Diagnosis of plasma cell leukaemia: findings of the UK NEQAS for Leucocyte Immunophenotyping scheme.

The diagnosis of plasma cell leukaemia, a rare disorder with an aggressive clinical course and poor prognosis, is not always straightforward and may be dependent on the results of immunophenotyping. Samples from two cases of plasma cell leukaemia have been issued by the UK NEQAS for Leucocyte Immunophenotyping Scheme during the last 5 years and on each occasion a significant number of laboratories failed to make the correct diagnosis. The details of the two samples issued and the results of both surveys are presented. The data highlights the need to adhere to guidelines for immunophenotyping, with respect to using the correct antibody panels, the importance of data interpretation in conjunction with morphological appearance as well as the need to participate in external quality assurance schemes.