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Xyntha

Manufactured by Pfizer
Sourced in United States

Xyntha is a laboratory equipment product manufactured by Pfizer. It is a device used for the purification and concentration of recombinant factor VIII, a critical component in the blood clotting process. The core function of Xyntha is to enable the efficient extraction and preparation of factor VIII for further medical applications.

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8 protocols using xyntha

1

Comparative Study of FVIII and VWF-FVIII Products

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Plasma-derived VWF-FVIII (2.4:1, Humate-P) was from CSL Behring (King of Prussia, USA), and (1:1, Wilate) was from Octapharma, Lachen, Switzerland. Human recombinant FVIII (rFVIII) products included Advate (Baxter, Deerfield, USA), Kogenate-FS and Kovaltry (Bayer, Leverkusen, Germany), Xyntha (Pfizer, New York, USA), and Nuwiq (Octapharma). Plasma-derived FVIII (pdFVIII) (Haemocetin) (Biotest, Dreieich, Germay) had been purified to contain 1% VWF. All experiments unless otherwise specified were performed with Advate (rFVIII) or Humate P (pdVWF-FVIII).
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2

Murine Liver Laceration Bleeding Model

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Mice were anesthetized with Isofluorane 3% and the abdomen was opened by substernal blunt midline dissection. The liver was mobilized and externalized onto sterile gauze, followed by a defined 10 mm scalpel cut through the left liver lobe, which resulted in complete ventral and dorsal laceration. Immediately after laceration, mice were positioned prone into a small weighing dish (8 cm diameter) filled with saline (37°C, 13 mL) and transferred into the anesthesia chamber which rested on a heating pad (37°C). Anesthesia was maintained at 3% Isofluorane and dishes were changed after 10 minutes to collect blood for the first and second 10 minutes separately. Blood loss was determined as described for the tail clip model. Groups of BALB/c mice were injected intravenously (tail vein) with equal volumes (200 µL) of superFVa or saline 2 minutes prior to intravenous injection of plasma-derived human APC, followed immediately by liver laceration. All agents were diluted in sterile sodium chloride 0.9% for injection (Hospira Inc). In some experiments rhFVIII (Xyntha, Pfizer) was injected intravenously at 200 U/kg.
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3

Chromogenic FVIII Activity Assay

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FVIII activity (FVIII:C) in platelet lysates was determined with a modified chromogenic assay using Coatest SP4 FVIII kit (DiaPharma Group, West Chester, OH, USA) as previously described [7 (link)]. Dilutions of hBDDFVIII (rhFVIII, Xyntha, Pfizer Inc, New York, NY, USA) were used to create a standard curve for measuring FVIII:C levels.
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4

Inhibitor Models for Hemophilia A

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Six to ten-week-old F8−/−VWF+/+ or F8−/− VWF−/− mice were immunized with recombinant human B-domain deleted FVIII (rhF8, Xyntha, Pfizer Inc, New York, NY, USA) at a dose of 50 IU kg−1 via the retro-orbital vein injection weekly for 4 weeks total. One week after the last immunization, plasmas were collected and inhibitor titers were determined by Bethesda assay as described in our previous report.[2 (link)] For 2bF8tg+/− mice, six to ten-week-old animals were immunized with rhF8 at a dose of 600 U kg−1 in the presence of adjuvant (Sigma, St. Louis, MO, USA) twice with a 3-week interval. One week after the second immunization, blood samples were collected and inhibitor titers were determined.
For inhibitor model studies, both chronic and acute models were used. In the chronic inhibitor model, rhF8-immunized F8−/− mice received bone marrow transplantation (BMT) from 2bF8tg+/− mice or rhF8-immunized 2bF8tg+/− mice were used. In the acute model, inhibitory plasmas from rhF8-immunized F8−/−VWF−/− mice with high-titer of inhibitors were infused into unimmunized 2bF8 mice with varying VWF conditions to a level of inhibitor titers of 2.5, 25, or 250 BU mL−1 followed by phenotypic correction assessment.
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5

Tail Bleeding Assay with Prohemostatic Agents

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Tail bleeding assays were performed as described (19 (link)). Mice were placed on temperature controlled heating pads (37°C) and anesthetized by isoflurane inhalation. The distal portion of the tail was cut at 1.5 mm diameter after which the tail was immersed in a predefined volume of 37°C saline (0.9% NaCl) for 20 minutes. Blood loss was determined after red cell lysis with 2% acetic acid by measuring the absorbance of hemoglobin at 490 nm and using a hemoglobin standard curve derived from defined blood volumes. Blood loss was expressed in µL/g body weight assuming a hematocrit of 46%. FVIII-deficient mice were injected retro-orbitally 5 minutes prior to tail cut with vehicle, superFVa, rhFVIIa, mFVIIa, or rhFVIII (200 units/kg; Xyntha®, Pfizer, NY, NY, USA) in an equal volume (200 µL) of sterile saline for injection (0.9%, Hospira Inc.).
For FVIII inhibitor mice, BalbC mice were injected with the GMA-8015 anti-FVIII antibody (100 µL, 0.25 mg/kg in sterile saline) retro-orbitally 2 hours before tail clip. Prohemostatic agents were injected retro-orbitally in the other eye 5 minutes before tail clip in an equal volume (100 µL) of sterile saline.
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6

Tail Bleeding Assay in Mice

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Mice were anesthetized with isoflurane 3%, placed on temperature controlled heating pads (37°C), and the distal portion of the tail was cut at 1.5 mm diameter after which the tail was immersed in a predefined volume of 37°C saline (0.9% NaCl) for 20 minutes. To study effects on bleeding and clot stability, tubes were changed after 10 minutes to collect blood for the first and second 10 minutes separately. Blood loss was determined by the hemoglobin concentration in the saline solution after red cell lysis with 2% acetic acid and measured by absorbance at 490 nm. Using a hemoglobin standard derived from defined blood volumes, blood loss was calculated assuming a hematocrit of 46% and expressed in µL/g body weight. Groups of BALB/c mice were injected intravenously (retroorbital) with superFVa or saline (200 µL) 2 minutes prior to intravenous (retroorbital) injection of rhAPC. Immediately after APC injection tail cut was performed. All agents were diluted in sterile sodium chloride 0.9% for injection (Hospira Inc, San Diego, California, USA). In some experiments rhFVIII (Xyntha, Pfizer) was injected intravenously at 200 U/kg.
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7

Comparative Study of rFVIII Concentrates

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The following human rFVIII concentrates were used: Kogenate FS® (full-length BHK-rFVIII; Bayer, Leverkusen, Germany), Advate® (full-length CHO-rFVIII; Shire, Dublin, Ireland), and Xyntha® (CHO-B-domain deleted (BDD)-rFVIII; Pfizer, New York City, NY, USA). Information on FVIII clearance, antigen/activity assays, deglycosylation, and von Willebrand factor (VWF) binding is available in the Online Supplementary Methods.
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8

Adoptive Transfer of iTregs for FVIII Tolerance

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One million pltLys-iTreg or purTGFβ-iTreg cells were adoptively transferred by IV injection into F8null/B6 mice followed by rhF8 (Xyntha; Pfizer Inc., New York, NY) immunization weekly at 50 U/kg per week IV for a total of 4 weeks. One week after the last immunization, blood samples were collected. The titers of anti-FVIII inhibitory antibodies (inhibitors) and total anti-FVIII antibodies were determined by Bethesda assay and enzyme-linked immunosorbent assay (ELISA), respectively, as described in our previous report.16 (link),19 (link) Six days after the iTreg cell transfer, CD4+ T cells from peripheral blood were analyzed by flow cytometry for enhanced green fluorescent protein (EGFP) expression to assess the recovery of the infused iTregs. CD45.1 and CD45.2 were used as congenic markers.
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