The purpose of this phase I study was to evaluate the toxicity and biological activity of autologous blood-derived macrophages activated ex-vivo with recombinant human interferon gamma (rhuIFN gamma) ...monokine-activated killer (MAK) cells and administered intravenously to 11 lung cancer patients once a week for 6 consecutive weeks. Peripheral blood monocytes were collected by leukapheresis and then purified by counterflow elutriation. The MAK cells were generated by culturing the purified monocytes in Teflon bags for 7 days and adding rhuIFN gamma to the cultured cells for the last 18 h. These MAK cells expressed differentiation-associated surface antigen MAX1, and were cytotoxic in vitro against tumour cell line U937. The MAK cells were infused at dose levels from 1 x 10(7) to 5 x 10(8) on an intrapatient dose-escalating schedule. No severe adverse side-effects occurred. Toxicity was mild to moderate primarly fever (75%) and chills (32%), non-dose-dependent, and non-cumulative. No consistent change in haemostatic function, or liver or renal function was observed. Dose-limiting toxicity was not reached at 5 x 10(8) cells (optimal dose reproduced for each patient). The maximum tolerated dose was not determined. The immunomodulatory activity of i.v. infused MAK cells was demonstrated both in vivo by significant increases in granulocyte count and neopterin level in the patients' peripheral blood post-infusion and in vitro by secretory products (IL-1. TNF alpha, neopterin, and thromboplastin-like substance) in the culture supernatants. The in vivo traffic patterns of autologous MAK cells labelled ex-vivo with 111In oxine were studied in 7 patients. Gamma imaging showed an immediate but transient lung uptake (less than 24 h), and a progressive uptake of radioactivity in the liver and spleen was seen from 6 h to 72 h post-infusion. Our results indicate that the preparation of high numbers of autologous, blood-derived MAK cells is a feasible procedure, and their transfusion is safe for patients. This immunotherapeutic approach seems to be encouraging from the point of view of establishing an adjuvant therapeutic modality in cancer patients with minimal residual disease.
Objectives We sought to determine whether low platelet response to the P2Y12receptor antagonist clopidogrel as assessed by vasodilator-stimulated phosphoprotein flow cytometry test (VASP-FCT) ...differentially affects outcomes in patients with or without chronic kidney disease (CKD) undergoing percutaneous coronary intervention (PCI). Background Although both CKD and impaired platelet responsiveness to clopidogrel are strong predictors of unfavorable outcome after PCI, the impact of their association is unknown. The platelet VASP-FCT assay is specific for the P2Y12ADP receptor pathway. In this test, platelet activation is expressed as the platelet reactivity index (PRI). Methods Four-hundred forty unselected patients (CKD: 126, estimated glomerular filtration rate eGFR <60 ml/min/1.73 m2), no-CKD: 314 eGFR >60 ml/min/1.73 m2) undergoing urgent (n = 336) or planned (n = 104) PCI were prospectively enrolled. In each subgroup, patients were classified as low-responders (LR: PRI >=61%) or responders (R: PRI <61%) to clopidogrel. Results At a mean follow-up of 9 ± 2 months, all-cause mortality, cardiac death, and possible stent thrombosis were higher in CKD than in no-CKD patients. Within the CKD group, the LR status was associated with higher rates of all-cause mortality (25.5% vs. 2.8%, p < 0.001), cardiac death (23.5% vs. 2.8%, p < 0.001), all stent thrombosis (19.6% vs. 2.7%, p = 0.003), and MACE (33.3% vs. 12.3%, p = 0.007). Conversely, in no-CKD patients, the LR status did not affect outcomes. Multivariate analysis identified Killip class >=3, drug-eluting stent implantation, and the interaction between LR and CKD (hazard ratio: 11.96, 95% confidence interval: 1.22 to 116.82; p = 0.033) as independent predictors of cardiac death. Conclusions In CKD patients, the presence of low platelet response to clopidogrel is associated with worse outcomes after PCI.
Objectives We sought to determine whether low platelet response to the P2Y12 receptor antagonist clopidogrel as assessed by vasodilator-stimulated phosphoprotein flow cytometry test (VASP-FCT) ...differentially affects outcomes in patients with or without chronic kidney disease (CKD) undergoing percutaneous coronary intervention (PCI). Background Although both CKD and impaired platelet responsiveness to clopidogrel are strong predictors of unfavorable outcome after PCI, the impact of their association is unknown. The platelet VASP-FCT assay is specific for the P2Y12 ADP receptor pathway. In this test, platelet activation is expressed as the platelet reactivity index (PRI). Methods Four-hundred forty unselected patients (CKD: 126, estimated glomerular filtration rate eGFR <60 ml/min/1.73 m2), no-CKD: 314 eGFR >60 ml/min/1.73 m2) undergoing urgent (n = 336) or planned (n = 104) PCI were prospectively enrolled. In each subgroup, patients were classified as low-responders (LR: PRI greater than or equal to 61%) or responders (R: PRI <61%) to clopidogrel. Results At a mean follow-up of 9 plus or minus 2 months, all-cause mortality, cardiac death, and possible stent thrombosis were higher in CKD than in no-CKD patients. Within the CKD group, the LR status was associated with higher rates of all-cause mortality (25.5% vs. 2.8%, p < 0.001), cardiac death (23.5% vs. 2.8%, p < 0.001), all stent thrombosis (19.6% vs. 2.7%, p = 0.003), and MACE (33.3% vs. 12.3%, p = 0.007). Conversely, in no-CKD patients, the LR status did not affect outcomes. Multivariate analysis identified Killip class greater than or equal to 3, drug-eluting stent implantation, and the interaction between LR and CKD (hazard ratio: 11.96, 95% confidence interval: 1.22 to 116.82; p = 0.033) as independent predictors of cardiac death. Conclusions In CKD patients, the presence of low platelet response to clopidogrel is associated with worse outcomes after PCI.
Background. The hemostatic activity of plasma is due to thrombin generation (TG) provided by the interrelated activation of platelets and the plasma coagulation system. The global capacity for plasma ...TG can be assessed in the presence of procoagulant phospholipids (PL) as a substitute for activated platelets initiation of the reaction by tissue factor (TF). Photochemical treatment (PCT) of plasma with amotosalen and UVA light (INTERCEPT Blood System, Cerus Europe, Leusden, The Netherlands) retains adequate levels of procoagulant and antithrombotic factors according to European standards and is clinically effective. We have assessed the impact of PCT on TG using the calibrated automated thrombogram (CAT).
Methods. Plasma (650mL) was collected by apheresis from 90 donors (MCS+, Haemonetics, Braintree, MA). Plasma was held at 20°–24°C before PCT and flash frozen at −80°C within 8 hrs of collection. Plasma units of 200mL were stored at −30°C for up to 1 year. TG was continuously measured in platelet poor plasma with a multiwell-plate reader using CAT and reagents (Thrombinoscope BV, Biodis, Signes, France). 20 μL PPP- reagent Low or High (1 or 20 pM TF and 4μM PL) were added at 37°C to triplicates of 80 μL PPP. TG measures were started by addition of 20 μL thrombin fluorescent substrate Z-GGR-AMC (2.5 mM) and CaCl2 (17 mM). Fluorescence accumulation from cleaved AMC was measured at 390 nm and 460 nm wavelengths and converted into nanomolar thrombin using a calibrator. The area under the curve corresponds to the total potential amount of endogenous thrombin formed (ETP) and the height of the peak to the maximum concentration of thrombin (MT).
Results. Plasma from 3 A, 1 AB and 3 O donors (Exp.1, Table) were tested for FVIII: before PCT, 1.08 ± 0.33 U/mL and after, 0.63 ± 0.20 U/mL (p < 0.01). TG was compared before and after PCT (Exp.1) at TF 1pM (control: ETP 1,906 ± 129 nM.min and MT 183–327 nM) and TF 20pM (control: ETP 2,026 ± 151 nM.min and MT 416–474 nM). Low TF (1pM) was further used to discriminate the effect of PCT on TG. In experiment 2, 12 donors (6 O, 1 AB and 5 A) tested after at day T0, before PCT (ETP 1,597 ± 205 nM.min) and after (ETP 1,680 ± 254 nM.min) were not different. Results were similar after day T30 storage (PCT: ETP 1,703 ± 174 nM.min). Results were identical for QC (Exp.3) of 12 pools of PCT of 6 units (3O and 3A donors): mean FVIII (0.69 ± 0.10 U/mL) and ETP (1,704 ± 93 nM.min).
Conclusions. Traditional standards have focused on FVIII levels, but TG, not modified by PCT of apheresis plasma, is of far greater relevance as it measures the efficacy of the entire hemostatic system, regardless of FVIII.
ETP and MT in control and PCT plasmaExperiment NbPlasma (n)F VIII (U/mL)TF (pM)ETP (nM.min)MT (nM)Exp.1.Control (6)1.08 ± 0.33Low 11,906 ± 129183–327Exp.1.PCT (6)0.63 ± 0.20Low 11,683 ± 172134–223Exp.1.Control (6)1.08 ± 0.33High 202,026 ± 151416–474Exp.1.PCT (6)0.63 ± 0.20High 201947 ± 151383 ± 440Exp.2. T0 dayControl (12)0.82 ± 0.17Low 11,597 ± 20584–273Exp.2. T0 dayPCT (12)0.56 ± 0.13Low 11,680 ± 25494–233Exp.2. T30 daysControl (12)0.78 ± 0.17Low 11,530 ± 128Exp.2. T30 daysPCT (12)0.51 ± 0.09Low 11,703± 174Exp.3.QCPCT (12.6=72)0.69 ± 0.10Low 11,704 ± 93162–226
We have constructed new B domain deletion derivatives of human factor VIII (FVIII) by manipulating the cDNA using recombinant DNA techniques. One of these new derivatives, FVIII delta II, in which ...amino acids 771(pro)-1666(asp) have been deleted, no longer contains the protease cleavage site at amino acid position 1648(arg)-1649(glu) known to be involved in the initial step of FVIII processing. We have expressed this molecule in both baby hamster kidney (BHK) 21 cells using the vaccinia virus (VV) expression system and have established Chinese hamster ovary (CHO) derived permanent cell lines expressing either recombinant (r)FVIII or FVIII delta II. The characteristics of FVIII delta II have been compared to those of rFVIII and/or plasma derived (pd) FVIII. FVIII delta II has the following properties: (i) it exhibits FVIII procoagulant activity; (ii) it is expressed at 5-fold higher levels than is the complete molecule in comparable systems; (iii) it migrates for the most part as a single major band on SDS-PAGE, in contrast to the complete molecule; (iv) it is activated to a greater extent by thrombin than is either rFVIII or pdFVIII; and (v) it retains the ability to bind von Willebrand factor (vWf).
Background. A photochemical treatment (PCT) using amotosalen HCl (S-59) and UVA light was developed to inactivate pathogens and leukocytes in therapeutic plasma (INTERCEPT™, I-FFP) frozen within 8 hr ...of collection. Previous studies demonstrated a broad spectrum of pathogen inactivation (Transfusion 2006; 46:1168) and clinical efficacy of I-FFP for support of coagulopathies (Transfusion 2005; 45:1362; Blood 2006; 107:3753), and plasma exchange of TTP (Transfusion 2006;46). Preparation of therapeutic plasma from whole blood would complement blood center logistics and reduce the cost of therapeutic frozen plasma provided sufficient coagulation factors were retained.
Aims. We measured coagulation factors in plasma isolated from whole blood held overnight at controlled temperature (21 ± 3°C), processed with pathogen inactivation, and frozen within 18 hr of blood collection.
Methods. Whole blood units, approximately 460 mL, anticoagulated with CPD (Baxter, La Chatre, France) were drawn from group A, O, B and AB donors. Units were processed after 16 hr storage, and plasma was isolated by centrifugation. Two to 3 plasma units of matched blood group were pooled (n = 30: A = 14, O = 14, B = 1, AB =1) to a final volume of 635 mL. Baseline samples for assay of coagulation factors were withdrawn. Each of 30 pools was mixed with 15 mL of 6 mM amotosalen (150 uM: final concentration) and illuminated with a 3 J/cm2 UVA treatment. Following illumination (~ 8 min) and passage through a flow compound adsorption device (~20 min) to reduce levels of residual S-59, treated plasma units (650 mL) were divided into 3 equal storage units of ≥ 200 mL. Before freezing, post-treatment samples for assay of coagulation factors were withdrawn for assay of coagulation factors. Treated plasma units were flash frozen at -80°C, and transferred to −30°C for 12-month storage. Treated units were withdrawn after 1 month to measure total protein, albumin, IgG, IgM, IgA, fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, VIII-vWF, Proteins C and S, AT III, plasminogen, alpha-2 antiplasmin, D-dimers, PT, and APTT.
Results. Baseline coagulation factor levels (Mean ± SD) were in suitable therapeutic ranges. After PCT, all units had residual platelets < 1×109/L, WBC < 1×104/L, and RBC < 1 × 109/L. After PCT and frozen storage for 1 month, total protein (59 ± 2 g/L), albumin (38 ± 1 g/L), IgG (8.9 ± 1.1g/L), IgA (1.8 ± 0.4 g/L) and IgM (0.9 ± 0.3 g/L) were unchanged from baseline. Mean values for fibrinogen (g/L), coagulation factors (IU/dL), coagulation inhibitors (IU/dL), were variably reduced from baseline, but within ranges defined as suitable for therapeutic plasma (Table). There was no evidence of plasma activation.
Conclusions. Plasma prepared from whole blood after storage on cooling plates before processing with the INTERCEPT system for pathogen inactivation retained coagulation factor activity levels after frozen storage (−30°C) in conformance with French national standards for therapeutic frozen plasma (FP). Approximately 36 units (200 mL) could be prepared per hr of illumination time with this system.
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Background. A photochemical treatment (PCT) using amotosalen HCl (S-59) and UVA light inactivates pathogens and leukocytes in therapeutic single donor apheresis fresh frozen plasma (INTERCEPT™, ...I-FFP) prepared within 8 hr of collection. Previous studies demonstrated a broad spectrum of pathogen inactivation (Transfusion 2006; 46:1168) and clinical efficacy of I-FFP for support of coagulopathies (Transfusion 2005; 45:1362; Blood 2006; 107:3753), and plasma exchange of TTP (Transfusion 2006;46). Preparation of therapeutic plasma up to 18 hr after collection would improve production logistics of frozen plasma provided sufficient levels of coagulation factors were retained.
Aims. We measured coagulation factors in apheresis plasma stored for 18 hr at ambient temperature, processed with pathogen inactivation, and frozen.
Methods. Fifteen jumbo plasma units (650 mL), were collected by apheresis with AB16 anticoagulant from group A, B, AB and O donors (MCS+. Haemonetics, Braintree, MA). Plasma collections were held at ambient blood bank temperature (20 – 24 °C) prior to further processing. After 18 hr, baseline samples for assay of coagulation factors were withdrawn before PCT. Plasma (635 mL plasma) was mixed with 15 mL of 6 mM amotosalen (150 uM: final concentration) and illuminated with a 3 J/cm2 UVA treatment. Following illumination (~ 8 min) and passage through a flow compound adsorption device (~20 min) to reduce levels of residual S-59, treated plasma units (650 mL) were divided into 3 equal storage units of ≥ 200 mL. Before freezing, post-treatment samples were withdrawn for factor assays. Treated plasma units were flash frozen at −80°C, and transferred to −30°C for 12-month storage. Plasma units were withdrawn to measure total protein, albumin, IgG, IgM, IgA, fibrinogen, factors II, V, VII, VIII, IX, X, XI, XII, VIII-vWF, Proteins C and S, AT III, plasminogen, alpha-2 antiplasmin, D-dimers, PT, and APTT.
Results. Baseline coagulation factor levels (Mean ± SD) were in therapeutic ranges after 18 hr storage at ambient temperature. After PCT, all units had residual platelets < 1x109/L, WBC < 1x104/L, and RBC < 1 x 109/L. After PCT, total protein (59 ± 4 g/L), albumin (38 ± 2 g/L), IgG (9.0 ± 1.7g/L), IgA (1.6 ± 0.8 g/L) and IgM (0.9 ± 0.5 g/L) were unchanged from baseline. Mean values for fibrinogen (g/L), coagulation factors (IU/dL), coagulation inhibitors (IU/dL) were variably reduced from baseline, but within the ranges defined for therapeutic plasma (Table). Treated plasma showed no evidence of activation.
Conclusions. Apheresis plasma held for 18 hr before processing with the INTERCEPT system for pathogen inactivation retained coagulation factor activity levels in conformance with French national standards for therapeutic frozen plasma (FP). Approximately 36 units (200 mL) could be prepared per hr with this system. A single UVA platform is compatible with the operational requirements of a regional blood center producing 12,000 doses (200 mL) of therapeutic FP and 12,000 doses of platelets per year.
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Objectives The aim of this study was to determine whether low platelet response to the P2Y12 receptor antagonist clopidogrel as assessed by Vasodilator-stimulated phosphoprotein flow cytometry test ...(VASP- FCT) predicts cardiovascular events in a high-risk population undergoing percutaneous coronary intervention (PCI). Background Impaired platelet responsiveness to clopidogrel is thought to be a determinant of cardiovascular events after PCI. The platelet VASP-FCT is a new assay specific to the P2Y12 adenosine diphosphate receptor-pathway. In this test, platelet activation is expressed as platelet reactivity index (PRI). Methods Four-hundred sixty-one unselected patients undergoing urgent (n = 346) or planned (n = 115) PCI were prospectively enrolled. Patients were classified as low-response (LR) and response (R) to clopidogrel, depending on their PRI. Optimal PRI cutoff was determined by receiver-operator characteristic curve analysis to 61% (LR: PRI ≥61% and R: PRI <61%). Follow-up was obtained at a mean of 9 ± 2 months in 453 patients (98.3%). Results At follow-up, total cardiac mortality rates and possible and total stent thrombosis were higher in LR patients. Multivariate analysis identified creatinine clearance (hazard ratio HR: 0.95; 95% confidence interval CI: 0.93 to 0.98, p < 0.001), drug-eluting stent (HR: 5.73; 95% CI: 1.40 to 23.43, p = 0.015), C-reactive protein (HR: 1.01; 95% CI: 1.001 to 1.019, p = 0.024), and LR to clopidogrel (HR: 4.00; 95% CI: 1.08 to 14.80, p = 0.037) as independent predictors of cardiac death. The deleterious impact of LR to clopidogrel on cardiovascular death was significantly higher in patients implanted with drug-eluting stent. Conclusions In patients undergoing PCI, LR to clopidogrel assessed by VASP-FCT is an independent predictor of cardiovascular death at the PRI cutoff value of ≥61%. The LR clinical impact seems to be dependent on the type of stent implanted.
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