FXII cDNA was ligated into pIRES-hrGFP vector to construct the expression plasmid. FXII variants (wild type 1848 bp, c.1681G>A, c.1556T>A, and c.1561T>A) were generated, and the resulting gene fragments were ligated into the pIRES-hrGFP vector. Human embryonic kidney cells (HEK293T) were maintained in Dulbecco’s modified Eagle medium (DMEM; Invitrogen, Paisley, USA), supplemented with 10% fetal calf serum and 2 mM glutamine in a humidified atmosphere of 5% CO2 and 95% air at 37 °C. Cells were evenly split into 10 cm dishes at a density of 2 × 106 cells/dish a day before transfection, and transient transfection of HEK 293 T cells with wild-type or mutant F12 variants were carried out using the jetPRIME® (Polyplus-transfection, Illkirch-Graffenstaden, France) according to the manufacturer’s instructions. The cells were then grown for 48 h in serum-free DMEM then cells were harvested. Cell lysate was analyzed for FXII antigen; medium was analyzed for FXII antigen by ELISA (Innovative Research, Novi, MI, USA) and FXII activity by one stage clotting assay (Actin-FSL, Dade Behring, IL, USA).
Actin fsl
Manufactured by Siemens
Sourced in Germany, United States, Norway
The Actin FSL is a lab equipment product by Siemens. It is designed for fluorescence spectroscopy analysis. The core function of the Actin FSL is to measure fluorescence signals from samples.
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Lab products found in correlation
Tina quant d dimer, by Roche (3 mentions)
Nycotest pt, by Axis-Shield (3 mentions)
Actin fs, by Siemens (3 mentions)
Thromborel s, by Siemens (3 mentions)
Bcs xp analyzer, by Siemens (2 mentions)
Dulbecco s modified eagle medium, by Thermo Fisher Scientific (1 mentions)
Jetprime, by Polyplus Transfection (1 mentions)
Elisa, by Innovative Research (1 mentions)
Ca 50, by Sysmex (1 mentions)
Synthafax, by Werfen (1 mentions)
Multifibren u, by Siemens (1 mentions)
Cs 5100, by Sysmex (1 mentions)
Cacl2 solution, by Siemens (1 mentions)
Pathromtin sl, by Siemens (1 mentions)
Latex enhanced photometric immunoassay, by Siemens (1 mentions)
Ptt la, by Diagnostica Stago (1 mentions)
Synthafax, by Siemens (1 mentions)
Synthasil, by Siemens (1 mentions)
15 protocols using actin fsl
1
Characterization of FXII Variants
2
FIX Clotting Activity Quantification in Mouse Plasma
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FIX clotting activity was determined using the APTT reagent (Actin ® FSL; Dade Behring, Newark, DE, USA) as the activator in a semiautomated blood coagulation analyzer according to the manufacturer's instructions (CA-50; Sysmex, Kobe, Japan). Pooled mouse plasma was obtained from inbred C57BL/6 male mice (n = 18) to generate standard curves for the quantification of test samples. FIX clotting activity in the standard was assumed to be 100%. The test samples were diluted 1:5 in imidazole buffer prior to analysis. A one-stage clotting assay was performed by incubating 50 μl of the test sample with 50 μl of human FIX-deficient plasma (American Diagnostica, Stamford, CT, USA) for 1 min at 37°C, after which 50 μl of activator was added for 3 min at 37°C. Next, 50 μl of 25 mM calcium chloride (CaCl 2 ; Sigma-Aldrich) was added, and the time required for clotting was measured. Each value was reported after subtracting the mean baseline level in HB mice.
3
Pharmacokinetic Assessment of Coagulation Factor IX
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Each PK session was 10 days long and consisted of eight visits (Figure 1 ). Blood samples were collected for PK assessment at 14 time‐points over each 10‐day PK session: predose, 10 and 30 minutes, and 1, 3, 6, 8, 24, 48, 96, 144, 168, 192, and 240 hours postdose.
FIX activity was measured using the one‐stage clotting and chromogenic assays performed on a Siemens BCS‐XP analyzer (Siemens, Marburg, Germany). Normal human plasma (NHP) standards, calibrated against the World Health Organization international FIX standard, were used to calibrate all assays. For the one‐stage clotting assays, the activated partial thromboplastin time (aPTT) reagents were selected by referring to the product label and/or published literature recommendations for each product;8 , 22 , 23 SynthAFax (Instrumentation Laboratory, Bedford, MA) was used for N9‐GP and Actin FSL (Siemens, Marburg, Germany) for rFIXFc. To date, chromogenic assays had not been used in any clinical trials with rFIXFc; however, given that the ROX factor IX (Rossix AB, Mölndal, Sweden) kit has been previously qualified for measuring N9‐GP,24 it was subsequently qualified for measuring rFIXFc in the current trial and selected as the chromogenic assay for both products.
FIX activity was measured using the one‐stage clotting and chromogenic assays performed on a Siemens BCS‐XP analyzer (Siemens, Marburg, Germany). Normal human plasma (NHP) standards, calibrated against the World Health Organization international FIX standard, were used to calibrate all assays. For the one‐stage clotting assays, the activated partial thromboplastin time (aPTT) reagents were selected by referring to the product label and/or published literature recommendations for each product;
4
Coagulation Profile Assessment Protocol
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Citrated 3.2% plasma was used for coagulation samples. Prothrombin time was analyzed with Nycotest PT (Axis-Shield PoC As, Oslo, Norway) and APTT with Actin FSL (Siemens Healthcare Diagnostics, Marburg, Germany, and all following reagents provided by Siemens are from the same source). Fibrinogen levels were determined using Multifibren U (Siemens and BC Thrombin Reagent (Siemens) was used to determine TT. Factor VIII: C (FVIII) was analyzed with a 1-stage clotting assay (Pathromtin SL and Coagulation Factor VIII Deficient Plasma; Siemens) and AT was measured with a chromogenic assay (Berichrom Antithrombin III; Siemens). D-dimer levels were measured with an immunoturbidimetric assay (Tina-quant D-dimer; Roche Diagnostics, Mannheim, Germany).
5
Antiphospholipid Antibody Detection Protocol
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LACs were detected using functional assays [3 (link), 15 (link)]. For LACs measurement, the dRVVT (screening reagent LA1 and LA2; Siemens, Munich Germany) and the APTT-lupus with Actin FSL and Actin FS (Siemens) on the Sysmex CS5100 (Sysmex, Singapore) were used. dRVVT reagent contained heparin inhibitor. In patients on vitamin K antagonist (VKA), the anticoagulant intensity at the time of testing for LACs was measured using the international normalized ratio (INR). Diagnostic tests were mixed with normal pooled plasma to correct for anticoagulant therapy with VKA (INR 1.5–3). In addition, in case of heparin use, LACs testing was performed after incubation of plasma with heparinase. The plasma glycoprotein β2GPI in complex with the anionic phospholipid cardiolipin is recognized by aCL antibodies [16–18 (link)], whereas anti-β2GPI antibodies recognize β2GPI with or without cardiolipin binding. In our study, the antibodies were determined using the HemoSIL AcuStar aCL IgG and IgM and HemoSIL AcuStar anti-β2GPI IgG and IgM assays on the ACL AcuStar (Werfen, Barcelona, Spain). Cut-off values were determined based on the 99th percentile according to the International Society on Thrombosis and Haemostasis Scientific and Standardization Subcommittee guidelines [19 (link), 20 (link)].
6
Thromboembolic Events Coagulation Profile
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We recorded thromboembolic events and collected the coagulation and fibrinolysis index profile of these patients. The thromboembolic events were recorded according to vascular ultrasound and computed tomography. Since this was a retrospective analysis, the individual method employed was based solely on the treating physician's choice. The coagulation and fibrinolysis index, including plasma prothrombin time (PT) (Nycotest PT, Axis-Shield Poc As, Oslo, Norway, the normal range of PT is between 9.8 and 12.4 sec), activated partial thromboplastin time (APTT) (Actin FSL Siemens Healthcare Diagnostics, Marburg, Germany, the normal range of APTT is between 26.9 and 37.6 sec), D-dimer (Tina-quant D-dimer, Roche Diagnostics, Mannheim, Germany, the normal range of D-dimer is between 0.1–0.5mg/L) and fibrin degradation products (FDP) (SpliPrest, Diagnostica Stago, the normal range of FDP is between 0 and 5 mg/L), were measure by the Central Laboratory Department of our hospital. The D-dimer was in Fibrinogen units. The International Sensitivity Index (ISI) value for the PT assay ranged from 0.96 to 0.99. During the fibrinolysis, dissolution of crosslinked fibrin leads to formation of specific degradation products, including D-dimer [9] (link). A normal D-dimer value excludes the diagnosis of venous thrombosis, while an elevated value supports it [10] (link).
7
Comprehensive Coagulation Profiling
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Standard laboratory tests were measured by the Ludwig-Maximilians-University institute for laboratory medicine, according to institutional rules and regulations.
Standard coagulation variables including international normalized ratio (INR) (Thromborel S, Siemens Healthcare GmbH, Erlangen, Germany), thrombin time (TT) (Berichrom Thrombinreagenz, Siemens Healthcare GmbH, Erlangen, Germany), activated partial thromboplastin time (aPTT) (Actin FSL, Siemens Healthcare GmbH, Erlangen, Germany) and blood count were performed. Substance specific and calibrated anti-Xa/anti-IIa tests were performed using Hemoclot Thrombin inhibitors test (Hyphen Biomed, Neuville-sur-Oise, France) and Coamatic Heparin test (Haemochrom Diagnostica GmbH, Essen, Germany).
Standard coagulation variables including international normalized ratio (INR) (Thromborel S, Siemens Healthcare GmbH, Erlangen, Germany), thrombin time (TT) (Berichrom Thrombinreagenz, Siemens Healthcare GmbH, Erlangen, Germany), activated partial thromboplastin time (aPTT) (Actin FSL, Siemens Healthcare GmbH, Erlangen, Germany) and blood count were performed. Substance specific and calibrated anti-Xa/anti-IIa tests were performed using Hemoclot Thrombin inhibitors test (Hyphen Biomed, Neuville-sur-Oise, France) and Coamatic Heparin test (Haemochrom Diagnostica GmbH, Essen, Germany).
8
Comprehensive Coagulation Profile Analysis
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Citrated 3.2% plasma was analyzed for PT with Nycotest PT (Axis-Shield PoC As, Oslo, Norway) and APTT with Actin FSL (Siemens Healthcare Diagnostics, Marburg, Germany, also providing the following reagents). Fibrinogen levels were determined using Multifibren U, and BC Thrombin Reagent was used for TT. FVIII was analyzed with a one-stage clotting assay (Pathromtin SL and Coagulation Factor VIII Deficient Plasma), and antithrombin (AT) was measured with a chromogenic assay (Berichrom Antithrombin III). D-dimer levels were captured with an immunoturbidimetric assay (Tina-quant D-dimer; Roche Diagnostics, Mannheim, Germany).
9
aPTT Coagulation Measurement Protocol
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Plasma-aPTT was performed using a BCS-XP analyzer (Siemens Healthcare Diagnostics, Marburg, Germany) with two reagents, Actin FSL (Siemens) with a reference interval of 26–33s and PTT- Automat (Stago, Asnières, France) with a reference interval of 28–45s. For clinical reasons, aPTT’s of more than 150s are recorded as ‘>150s’. Coagulation is initiated with a contact activator and phospholipids in the reagent and after recalcification with CaCl2, the coagulation time is measured in seconds through optical detection.
10
Activated Partial Thromboplastin Time Assay
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