Edelgard Lindhoff-Last

Intraarterial Administration of Bone Marrow Mononuclear Cells in Patients With Critical Limb Ischemia A Randomized-Start, Placebo-Controlled Pilot Trial (PROVASA)

Background—Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia, but double-blind, randomized trials are lacking. Methods and Results—Forty patients with critical limb ischemia were included in a multicenter, phase II, double-blind, randomized-start trial to receive either intraarterial administration of BM-MNC or placebo followed by active treatment with BM-MNC (open label) after 3 months. Intraarterial administration of BM-MNC did not significantly increase ankle-brachial index and, thus, the trial missed its primary end point. However, cell therapy was associated with significantly improved ulcer healing (ulcer area, 3.2±4.7 cm2 to 1.89±3.5 cm2 [P=0.014] versus placebo, 2.92±3.5 cm2 to 2.89±4.1 cm2 [P=0.5]) and reduced rest pain (5.2±1.8 to 2.2±1.3 [P=0.009] versus placebo, 4.5±2.4 to 3.9±2.6 [P=0.3]) within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups.Repeated BM-MNC administration and higher BM-MNC numbers and functionality were the only independent predictors of improved ulcer healing. Ulcer healing induced by repeated BM-MNC administration significantly correlated with limb salvage (r=0.8; P<0.001). Conclusions—Intraarterial administration of BM-MNC is safe and feasible and accelerates wound healing in patients without extensive gangrene and impending amputation. These exploratory findings of this pilot trial need to be confirmed in a larger randomized trial in patients with critical limb ischemia and stable ulcers. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00282646.

Incidence and clinical relevance of heparin-induced antibodies in patients with deep vein thrombosis treated with unfractionated or low-molecular-weight heparin

Summary. The frequency of heparin‐induced thrombocytopenia (HIT) varies between different clinical treatment settings and remains unknown for patients treated with unfractionated (UFH) or low‐molecular‐weight heparin (LMWH) because of deep vein thrombosis. In this multicentre, open‐label study, 1137 patients with deep vein thrombosis were randomly assigned to UFH for 5–7 d, reviparin, a LMWH, for 5–7 d (short‐treated group) or reviparin for 28 d (long‐treated group). Heparin‐platelet factor 4 antibodies (HPF4‐A) were determined on d 5–7 and d 21. Heparin‐induced thrombocytopenia was defined by clinical evaluation. Two patients in the UFH group (incidence: 0·53%, 95% confidence interval (CI): 0·06–1·91) and two patients in the long‐treated LMWH group (incidence: 0·53%, 95% CI: 0·06–1·92) had HIT, while no HIT was observed in the short‐treated LMWH group. Pulmonary embolism developed in one of the HIT‐patients, who had HPF4‐A and was treated with UFH. On d 5–7 the incidence of HPF4‐A was 9·1% in the UFH group, 2·8% in the short‐treated LMWH group and 3·7% in the long‐treated LMWH group, with a significant increase to 20·7% in the UFH group and to 7·5% in the long‐treated LMWH group on d 21. Therefore the incidence of HPF4‐A and heparin‐induced thrombocytopenia was lower in patients treated with LMWH compared with UFH for the same duration of treatment.

Standardization of light transmittance aggregometry for monitoring antiplatelet therapy: an adjustment for platelet count is not necessary.

Summary.  Background: Light transmittance aggregometry (LTA) is considered to be the ‘gold standard’ of platelet function testing. As LTA has been poorly standardized, we analyzed the results of LTA in healthy subjects and patients with antiplatelet therapy using different concentrations of agonists and performing tests in non‐adjusted and platelet count‐adjusted platelet‐rich plasma (PRP). Methods: LTA was performed in 20 healthy subjects and in patients treated with aspirin (n = 30) or clopidogrel (n = 30) monotherapy, as well as in patients on combination therapy (n = 20), using arachidonic acid (ARA 0.25 and 0.5 mg mL−1) and adenosine diphosphate (ADP 2 and 5 μm) as agonists and performing platelet function tests in non‐adjusted and platelet count (250 nL−1 ± 10%)‐adjusted PRP. Results: The overall platelet aggregation response is decreased after adjusting the PRP for platelet count compared with measurements in unadjusted PRP. The variability of aggregation results is high in adjusted PRP in the subgroup of healthy subjects, ranging from 9.2–95.3% (5th–95th percentile) relative to 77.6–95.5% in non‐adjusted PRP when determining maximum aggregation to ARA 0.5 mg mL−1. Late aggregation using ADP 2 μm ranges from 3.8–89.9% in adjusted PRP compared with 42.9–92.5% in non‐adjusted PRP. Maximum aggregation using ARA 0.5 mg mL−1 in non‐adjusted PRP differentiates between aspirin‐treated patients and healthy controls well, whereas late aggregation using ADP 2 μm in non‐adjusted PRP offers the best discrimination between clopidogrel‐treated patients and healthy controls. Conclusion: Adjustment of PRP for platelet count does not provide any advantage and therefore the time‐consuming process of platelet count adjustment is not necessary.

Determination of heparin–platelet factor 4–IgG antibodies improves diagnosis of heparin‐induced thrombocytopenia

Only a few patients with heparin‐induced antibodies develop heparin‐induced thrombocytopenia (HIT). In this study, we investigated whether different immunglobulin classes can be used to differentiate between antibody‐positive patients with and without HIT. Four different patient populations were investigated: 32 patients with the immune type of HIT with thromboembolic complications, 13 patients with HIT without thromboembolism, 24 patients with heparin–platelet factor 4 (PF4) antibodies without clinical symptoms of HIT, and 20 heparin‐treated patients with thrombocytopenia caused by other reasons. In all patients the immunglobulin mixture of IgG, IgM and IgA, and the single immunglobulin classes of heparin–PF4 antibodies, were investigated. No significant differences between HIT patients with thromboembolic complications and patients with isolated HIT were found concerning the different immunglobulin classes. Antibody‐positive patients with HIT had significantly higher levels of IgG antibodies than those without HIT (P < 0·05), while they did not differ concerning IgM and IgA antibodies. By determining IgG antibodies, the specificity of the enzyme‐linked immunosorbent assay (ELISA) system was increased without loss of sensitivity. Heparin–PF4–IgG antibodies can identify patients at risk of developing life‐threatening HIT. Future ELISAs should only include this immunglobulin class, as the determination of the antibody mixture may lead to overestimation of HIT.

Rivaroxaban differentially influences ex vivo global coagulation assays based on the administration time.

It was the objective of this study to quantify the effects of rivaroxaban administration on global coagulation parameters associated with routine clinical procedures, we collected plasma samples from patients undergoing major orthopaedic surgery receiving rivaroxaban at various time points after drug administration. Forty-seven patients received rivaroxaban (10 mg daily) for venous thromboembolism prophylaxis. Blood samples were collected at four different time points: A) before surgery; B) before drug administration at day 4-5 after surgery (steady state of rivaroxaban); C) 2 hours (h) after drug administration and D) 12 h after drug administration. The prothrombin time (PT), activated partial thromboplastin time (aPTT), thrombin time (TT), antithrombin (AT) level, fibrinogen level by Clauss method (FibC), and derived fibrinogen (dFIB) level were assessed with various reagents. At 2 h after rivaroxaban administration, the PT and aPTT clotting times were significantly prolonged to different extents up to 1.4 fold, whereas 12 h after drug administration, no significant effect was observed. Rivaroxaban administration had no influence on the TT or the FibC concentration. The dFIB assay was differentially affected by rivaroxaban when different reagents were tested. The AT assay dependent on thrombin activity was not influenced by rivaroxaban, whereas the AT levels dependent on factor Xa activity were significantly increased by rivaroxaban. Clinicians should be aware of the time-dependent influence of rivaroxaban on factor Xa-dependent routine coagulation assays. Therefore, routine coagulation parameters should be assessed directly before drug administration to keep the interaction of rivaroxaban low.

Intraarterial Administration of Bone Marrow Mononuclear Cells in Patients With Critical Limb IschemiaClinical Perspective

Background—Critical limb ischemia due to peripheral arterial occlusive disease is associated with a severely increased morbidity and mortality. There is no effective pharmacological therapy available. Injection of autologous bone marrow-derived mononuclear cells (BM-MNC) is a promising therapeutic option in patients with critical limb ischemia, but double-blind, randomized trials are lacking. Methods and Results—Forty patients with critical limb ischemia were included in a multicenter, phase II, double-blind, randomized-start trial to receive either intraarterial administration of BM-MNC or placebo followed by active treatment with BM-MNC (open label) after 3 months. Intraarterial administration of BM-MNC did not significantly increase ankle-brachial index and, thus, the trial missed its primary end point. However, cell therapy was associated with significantly improved ulcer healing (ulcer area, 3.2±4.7 cm2 to 1.89±3.5 cm2 [P=0.014] versus placebo, 2.92±3.5 cm2 to 2.89±4.1 cm2 [P=0.5]) and reduced rest pain (5.2±1.8 to 2.2±1.3 [P=0.009] versus placebo, 4.5±2.4 to 3.9±2.6 [P=0.3]) within 3 months. Limb salvage and amputation-free survival rates did not differ between the groups.Repeated BM-MNC administration and higher BM-MNC numbers and functionality were the only independent predictors of improved ulcer healing. Ulcer healing induced by repeated BM-MNC administration significantly correlated with limb salvage (r=0.8; P<0.001). Conclusions—Intraarterial administration of BM-MNC is safe and feasible and accelerates wound healing in patients without extensive gangrene and impending amputation. These exploratory findings of this pilot trial need to be confirmed in a larger randomized trial in patients with critical limb ischemia and stable ulcers. Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00282646.

Accurate determination of rivaroxaban levels requires different calibrator sets but not addition of antithrombin

Rivaroxaban is a direct factor Xa inhibitor, which can be monitored by anti-factor Xa chromogenic assays. This ex vivo study evaluated different assays for accurate determination of rivaroxaban levels. Eighty plasma samples from patients receiving rivaroxaban (Xarelto) 10 mg once daily and 20 plasma samples from healthy volunteers were investigated using one anti-factor Xa assay with the addition of exogenous antithrombin and two assays without the addition of antithrombin. Two different lyophilised rivaroxaban calibration sets were used for each assay (low concentration set: 0, 14.5, 59.6 and 97.1 ng/ml; high concentration set: 0, 48.3, 101.3, 194.2 and 433.3 ng/ml). Using a blinded study design, the rivaroxaban concentrations determined by the assays were compared with concentrations measured by HPLC-MS/MS. All assays showed a linear relationship between the rivaroxaban concentrations measured by HPLC-MS/MS and the optical density of the anti-FXa assays. However, the assay with the addition of exogenous antithrombin detected falsely high concentrations of rivaroxaban even in plasma samples from controls who had not taken rivaroxaban (intercept values using the high calibrator set and the low calibrator set: +26.49 ng/ml and +13.71 ng/ml, respectively). Plasma samples, initially determined by the high calibrator setting and containing rivaroxaban concentrations <25 ng/ml, had to be re-run using the low calibrator setting for precise measurement. In conclusion, anti-factor Xa chromogenic assays that use rivaroxaban calibrators at different concentration levels can be used to measure accurately a wide range of rivaroxaban concentrations ex vivo. Assays including exogenous antithrombin are unsuitable for measurement of rivaroxaban.

Treatment of 51 pregnancies with danaparoid because of heparin intolerance

Pregnant patients with acute venous thrombosis or a history of thrombosis may need alternative anticoagulation, when heparin intolerance occurs. Only limited data on the use of the heparinoid danaparoid are available in literature. We reviewed the use of danaparoid in 51 pregnancies of 49 patients identified in literature between 1981 and 2004. All patients had developed heparin intolerance (32 due to heparin-induced thrombocytopenia, 19 mainly due to heparin-induced skin rashes) and had a current and/or past history of thromboembolic complications. The initial danaparoid dose regimens ranged from 1000 to 7500 U/day administered s.c. or i.v.. The median duration of danaparoid use was 10 weeks. Danaparoid was used until delivery of a healthy infant in 37 pregnancies. In the remaining 14 pregnancies it was stopped earlier, because anticoagulant treatment was no longer required (3/14) or an adverse event led to a treatment discontinuation (11/14). Four maternal bleeding events were recorded during pregnancy, delivery or postpartum, two of them were fatal due to placental problems. Three fetal deaths were recorded, all associated with maternal complications antedating danaparoid use. Danaparoid cross-reactivity was suspected in 4 HIT patients and 5 non-HIT patients with skin reactions and was confirmed serologically in one of the two HIT patients tested. In none of five fetal cord blood- and three maternal breast milksamples anti-Xa activity transfer was observed. In conclusion danaparoid can be used as an alternative antithrombotic agent in pregnant women with high thrombotic risk and intolerance to heparins.

Incidence and causes of heparin-induced skin lesions

Background: Little is known about the incidence and causes of heparin-induced skin lesions. The 2 most commonly reported causes of heparin-induced skin lesions are immune-mediated heparin-induced thrombocytopenia and delayed-type hypersensitivity reactions. Methods: We prospectively examined consecutive patients who received subcutaneous heparin (most often enoxaparin or nadroparin) for the presence of heparin-induced skin lesions. If such lesions were identified, we performed a skin biopsy, platelet count measurements, and antiplatelet-factor 4 antibody and allergy testing. Results: We enrolled 320 patients. In total, 24 patients (7.5%, 95% confidence interval [CI] 4.7%–10.6%) had heparin-induced skin lesions. Delayed-type hypersensitivity reactions were identified as the cause in all 24 patients. One patient with histopathologic evidence of delayed-type hypersensitivity tested positive for antiplatelet-factor 4 antibodies. We identified the following risk factors for heparin-induced skin lesions: a body mass index greater than 25 (odds ratio [OR] 4.6, 95% CI 1.7–15.3), duration of heparin therapy longer than 9 days (OR 5.9, 95% CI 1.9–26.3) and female sex (OR 3.0, 95% CI 1.1–8.8). Interpretation: Heparin-induced skin lesions are relatively common, have identifiable risk factors and are commonly caused by a delayed-type hypersensitivity reaction (type IV allergic response). (ClinicalTrials.gov trial register no. NCT00510432.)

A Prospective Study on the Incidence and Clinical Relevance of Heparin-Induced Antibodies in Patients after Vascular Surgery

The heparin-platelet factor 4-antibody assay, polyanion-platelet factor 4-antibody assay and heparin-induced platelet activation test are used for laboratory diagnosis of the immune form of heparin-induced thrombocytopenia. Fifty consecutive patients receiving heparin treatment for more than 5 days after vascular surgery were prospectively screened for heparin-induced thrombocytopenia antibodies, thrombocytopenia (daily platelet counts), deep-vein thrombosis (color-coded duplex sonography), and arterial reocclusion (clinical assessment). None of the patients developed thrombocytopenia or thrombosis in association with formation of heparin-induced thrombocytopenia antibodies. Despite the absence of clinical evidence of heparin-induced thrombocytopenia, many patients formed heparin-induced thrombocytopenia antibodies: 34% of the patients were positive in the heparin-platelet factor 4-antibody assay, 28% in the polyanion-platelet factor 4-antibody assay, 14% in the heparin-induced platelet activation test, and 54% with any of these tests. Patients predominantly developed IgM (24%) and IgA antibodies (16%), whereas IgG antibodies were found in 12% of patients. Whereas the majority of patients with positive ELISA assays had IgM and IgA antibodies, patients with a positive functional assay (heparin-induced platelet activation test) predominantly had IgG antibodies. We conclude that a high percentage of patients develop heparin-induced antibodies after vascular surgery without any clinical symptoms of heparin-induced thrombocytopenia. None of the assays therefore is predictive of the clinical manifestation of heparin-induced thrombocytopenia in asymptomatic patients. Therefore, the diagnostic specificity of both antigen and activation assays for heparin-induced thrombocytopenia appears to be relatively low in the vascular surgery patient population.