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11 protocols using hemosil recombiplastin 2g

1

Anticoagulation and Coronary Angiography Protocol

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All measurements of INR, sCr, and TB were performed at the presentation of the patients prior to the initiation of anticoagulant therapy and coronary angiography. The blood-collection tubes contained 3.2% sodium citrate (0.5 ml citrate, 4.5 ml blood) for INR, measurement. Samples were immediately centrifuged for routine testing, and analysis was performed within 1 h after sampling. INR was measured using the reagent HemosIL RecombiPlasTin 2G (Instrumentation Laboratory, Bedford, MA, USA). Complete blood count was determined via an Abbott Cell-Dyn 3700 autoanalyzer using commercial assay kits (Abbott Diagnostic, CA, US). Biochemical measurements were performed using Siemens Healthcare Diagnostic Products kits and calibrators (Marburg, Germany).
The standard MELD score was calculated by using the following formula: 11.2 x (ln INR) + 0.378 x (ln total bilirubin) + 0.957 x (ln creatinine) + 0.643 [6 (link)].
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2

Evaluation of the Q Smart Coagulation Analyzer

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The Q Smart system (Diagnostic Grifols, Barcelona, Spain) comprises the Q Smart analyzer and the following DG reagents (Diagnostic Grifols) for each test: DG-PT RecombiLIQ for prothrombin time (PT), DG-APTT Synth for activated partial thromboplastin time (APTT), DG-TT L Human for thrombin time (TT), DG-APTT Synth/DG-FVIII for FVIII, DG-Latex DDimer for d-dimer, and DG-Chrom AT L for Antithrombin(AT). ACL TOP 500 analyzer and Hemosil reagents from Instrumentation Laboratory (Bedford, Massachusetts) were used for comparison in the following tests: Hemosil Recombiplastin 2G for PT, Hemosil APTT-SP (liquid) for APTT, Hemosil Thrombin Time for TT, and Hemosil APTT-SP (liquid)/Hemosil FVIII Deficient Plasma for FVIII. For comparison in the d-dimer and AT tests, the BCS XP coagulometer and reagents were used (Siemens AG, Munich, Germany). Additionally, the PT assay was also performed with the CoaguChek XS point-of-care_POC (Roche, Indianapolis, Indiana).
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3

Cell-Block Preparation for SN-MM Lines

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For cell-block preparation, at least 1 × 106 cells were harvested and processed as follows. Cell suspensions of SN-MM cell lines were centrifuged for 10 min at 3,000 rpm. A solution of plasma (100 mL, kindly provided by Centro Trasfusionale, ASST Spedali Civili) and HemosIL RecombiPlasTin 2G (200 mL, Instrumentation Laboratory; cat. no. 0020003050; 1:2) were added to cell pellets, mixed until the formation of a clot, then placed into a labeled cassette (Bio-Optica; cat. no. 07-7350). The samples were fixed in 10% formalin (Bio-Optica; cat. no. 05-K01004) for 1 h followed by paraffin inclusion.
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4

Apixaban Pharmacokinetics and Coagulation Markers

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Screening and EOS laboratory analysis were performed at the Clinical Institute of Laboratory Medicine, General Hospital Vienna, Austria, according to standard operating procedures. All other parameters were analyzed by Labcon GmbH (Vienna, Austria) using validated assays: ASSERACHROM® β-TG test kit (Diagnostica Stago, Asniéres, France) for β-TG assessment, TAT was assessed using the Enzygnost® TAT micro kit (Siemens GmbH, Munich, Germany), D-dimer was analyzed with the TECHNOZYM® D-dimer ELISA (Technoclone, Vienna, Austria) and P-selectin was determined using the Human sP-selectin Platinum ELISA (eBioscience, CA). The detections ranges were 117.5 to 1880 U/mL for β-TG, 20 to 600 ng/mL for TAT, 189.7 to 1365 ng/mL for D-dimer, and 0.63 to 40 ng/mL for P-selectin.
Apixaban pharmacokinetics were assessed as anti-Xa activity with an ACL TOP500 coagulation analyser (Instrumentation Laboratory, Vienna, Austria) using apixaban as assay standard with a lower limit of quantification (LLQ) <10 ng/mL.
The ACL TOP500 coagulation analyzer was also used for aPTT and PT measurements using HemosIL® SynthASil (Instrumentation Laboratory, Vienna, Austria; [aPTT]) and HemosIL®RecombiPlasTin 2G (Instrumentation Laboratory, Vienna, Austria; [PT]) as reagents. The normal reference range for venous aPTT was 25.1 to 36.5 s and 70 to 130 % for venous PT.
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5

Venous Blood Collection and INR Analysis

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The venous blood was drawn from patients who fasted within the first 24 h of admission for routine laboratory measurements including INR. The blood samples were obtained, preserved, and processed in a manner recommended by the clinical site laboratory's policies and procedures. The 3.2%citrate tubes are used and filled properly to maintain a blood/citrate ratio of 9/1. INR was measured on a STA-R EVOLUTION automated coagulation analyzer (Diagnostica Stago, Asnières, France) with an international sensitivity index of approximately 1.0 and using HemosIL RecombiPlasTin 2G as reagent (Instrumentation Laboratory, Bedford, MA, USA). The reference range of INR was roughly 0.9–1.1 in all clinical laboratories.
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6

Blood Coagulation Biomarker Measurements

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All the blood coagulation-related values were measured by LSI Medience Corporation (Tokyo, Japan). APTT, PT, and fibrinogen level were measured with HemosIL SynthASil (Instrumentation Laboratory, Bedford, MA, US); HemosIL RecombiPlasTin 2G (Instrumentation Laboratory); and HemosIL Fibrinogen-C (Instrumentation Laboratory), respectively. These three variables were measured using ACL ELITE PRO (Instrumentation Laboratory). Platelet counts were measured with cellpack (Sysmex, Hyogo, Japan) using XT-2000iV (Sysmex).
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7

Automated Coagulation Analysis System

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The APTT-CWA was performed using APTT-SP®, which uses silica as an activator of FXII and synthetic PLs (Instrumentation Laboratory) and PL, with an ACL-TOP® system (Instrumentation Laboratory) as previously reported.12 (link),16 (link) The CWA-sTF/FIXa was performed using PRP and 2,000-fold diluted HemosIL RecombiPlasTin 2G (TF concentration <0.1 pg/ml; Instrumentation Laboratory). Three types of curves are shown on the monitor of this system.12 (link) One curve shows the changes in the absorbance observed while measuring the APTT, corresponding to fibrin formation (FF). The second (1st DP) corresponds to the coagulation velocity, and the third (2nd DP) corresponds to the coagulation acceleration. The height and time of the 1st DP, 2nd DP and FF are called the 1st DPH and 1st DPT, 2nd DPH and 2nd DPT, and FFH and FFT, respectively (Figure 1). PRP was prepared by centrifugation at 900 rpm for 15 minutes (platelet count, 40 × 1010/L), and PPP was prepared by centrifugation at 3,000 rpm for 15 minutes (platelet count, <0.5 × 1010/L).21 (link)
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8

Procoagulant Activity of MonoMac 1 Cells

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The human monocytic cell line MonoMac 1 (MM1) was exposed to immunoglobulin fractions and procoagulant activity was determined as previously described.14 Based on a standard curve of recombinant tissue factor (TF; hemosIL RecombiPlasTin 2G, #0020003051, Instrumentation Laboratory, Munich, Germany) clotting times were converted into procoagulant activity (PCA) units.
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9

Isolation and Characterization of PBMCs

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Fresh PBMCs were isolated from buffy of healthy donor coat by density centrifugation using cat. no. 307618, Biolegend) and sorted using FACSAria II (BD Biosciences). Accordingly, PBMCs were subjected to doublets exclusion and subsequently gated on lymphocytes negative (CD3, CD19 and CD56) and HLA-DR positive cells, finally monocytes were positively selected as CD14 + CD16 - or CD14 -CD16 + whereas BDCA1 + were negatively selected as CD14 -CD16 -CD141 -and CD303 -.
Fluorescence negative controls were performed using intra-simple negative populations as reference.
For cell block preparation, cell suspensions of sorted cells were centrifuged for 10 minutes at 3,000 rpm. A solution of plasma (100 mL, kindly provided by Centro Trasfusionale, ASST Spedali Civili) and HemosIL RecombiPlasTin 2G (200 mL, Instrumentation Laboratory; cat. no. 0020003050; 1:2) were added to cell pellets, mixed until the formation of a clot, then placed into a bio-cassette (Bio-Optica; cat. no. 07-7350). The specimen was fixed in 10% formalin (Bio-Optica; cat. no. 05-K01004) for 1 hour followed by paraffin inclusion.
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10

Clotting Time Assay for EA.hy926 Cells

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After stimulation with histones for 4 h, EA.hy926 cells were harvested. Cells were resuspended to 5 x 106/mL in PBS and 20 μL of cell suspension was added in a cuvette. The plasma (80 μL; Pool Norm; Diagnostica Stago, Asnieres, France) which was anticoagulated with 3.2% sodium citrate was mixed with the cell suspension and the formation of a clot was induced by the addition of 100 μL of pre-warmed 0.025 M CaCl2. The clotting time was measured by ST Art (Diagnostica Stago). The standard curve was produced using HemosIL RecombiPlasTin 2G (Instrumentation Laboratory, Bedford, MA, USA).
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