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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

This protocol describes a comprehensive hemocompatibility evaluation of blood-contacting devices using laser-cut neurovascular implants. A flow loop model with fresh, heparinized human blood is applied to mimic blood flow. After perfusion, various hematologic markers are analyzed and compared to the values gained directly after blood collection for hemocompatibility evaluation of the tested devices.

Abstract

The growing use of medical devices (e.g., vascular grafts, stents, and cardiac catheters) for temporary or permanent purposes that remain in the body's circulatory system demands a reliable and multiparametric approach that evaluates the possible hematologic complications caused by these devices (i.e., activation and destruction of blood components). Comprehensive in vitro hemocompatibility testing of blood-contacting implants is the first step towards successful in vivo implementation. Therefore, extensive analysis according to the International Organization for Standardization 10993-4 (ISO 10993-4) is mandatory prior to clinical application. The presented flow loop describes a sensitive model to analyze the hemostatic performance of stents (in this case, neurovascular) and reveal adverse effects. The use of fresh human whole blood and gentle blood sampling are essential to avoid the preactivation of blood. The blood is perfused through a heparinized tubing containing the test specimen by using a peristaltic pump at a rate of 150 mL/min at 37 °C for 60 min. Before and after perfusion, hematologic markers (i.e., blood cell count, hemoglobin, hematocrit, and plasmatic markers) indicating the activation of leukocytes (polymorphonuclear [PMN]-elastase), platelets (β-thromboglobulin [β-TG]), the coagulation system (thombin-antithrombin III [TAT]), and the complement cascade (SC5b-9) are analyzed. In conclusion, we present an essential and reliable model for extensive hemocompatibility testing of stents and other blood-contacting devices prior to clinical application.

Introduction

The in vivo application of implants and biomaterials, which interact with human blood, requires intense preclinical testing focusing on the investigation of various markers of the hemostatic system. The International Organization for Standardization 10993-4 (ISO 10993-4) specifies the central principles for the evaluation of blood-contacting devices (i.e., stents and vascular grafts) and considers the device design, clinical utility, and materials needed1.

Human blood is a fluid that contains various plasma proteins and cells, including leukocytes (white blood cells [WBCs]), erythrocytes (red blood cells [RBCs]), and....

Protocol

The blood sampling procedure was approved by the Ethics Committee of the medical faculty at the University of Tuebingen (project identification code: 270/2010BO1). All subjects provided written, informed consent for inclusion before participation.

1. Preparation of Heparin-loaded Monovettes

  1. Mix the undiluted heparin (5,000 IU/mL) with sodium chloride (NaCl, 0.9%) solution and prepare a solution with a resulting concentration of 15 IU/mL of heparin.
  2. Add 900 µL of the .......

Representative Results

Briefly summarized, human whole blood was collected in heparin-loaded monovettes then pooled and used to evaluate the baseline levels of cell counts as well as plasmatic hemocompatibility markers.

Subsequently, the tubing containing the neurovascular implant samples was filled, and the blood was perfused for 60 min at 150 mL/min and 37 °C using a peristaltic pump. Again, the number of cells was analyzed in all groups, and the plasma samples were prepared for ELISA analyses (

Discussion

The presented protocol describes a comprehensive and reliable method for the hemocompatibility testing of blood-contacting implants in accordance with ISO 10993-4 in a shear flow model imitating human blood flow. This study is based on the testing of laser-cut neurovascular implants but can be performed with a variety of samples. The results demonstrate that this method enables the broad analysis of various parameters such as the blood cell count, prevalence of several hemocompatibility markers, and microscopic visualiza.......

Acknowledgements

For the performance of scanning electron microscopy, we thank Ernst Schweizer from the section of Medical Materials Science and Technology of the University Hospital Tuebingen. The research was supported by the Ministry of Education, Youth and Sports of the CR within National Sustainability Program II (Project BIOCEV-FAR LQ1604) and by Czech Science Foundation project No. 18-01163S.

....

Materials

NameCompanyCatalog NumberComments
aqua ad iniectabiliaFresenius-Kabi, Bad-Homburg, Germany1088813
beta-TG ELISADiagnostica Stago, Duesseldorf, Germany00950
Centrifuge Rotana 460 RAndreas Hettich, Tuttlingen, Germany-
Citrat monovettes (1.4 mL)Sarstedt, Nümbrecht, Germany6,16,68,001
CTAD monovettes (2.7 mL)BD Biosciences, Heidelberg, Germany367562
EDTA monovettes (1.2 mL)Sarstedt, Nümbrecht, Germany6,16,62,001
Ethanol p.A. (1000 mL)AppliChem, Darmstadt, Germany1,31,08,61,611
Glutaraldehyde (25 % in water)SERVA Electrophoresis, Heidelberg, Germany23114.01
Heparin coating for tubesEnsion, Pittsburgh, USA-
Heparin-Natrium (25.000 IE/ 5 mL)LEO Pharma, Neu-Isenburg, GermanyPZN 15261203
Multiplate Reader Mithras LB 940Berthold, Bad Wildbad, Germany-
NaCl 0,9%Fresenius-Kabi, Bad-Homburg, Germany1312813
Neutral monovettes (9 mL)Sarstedt, Nümbrecht, Germany2,10,63,001
PBS buffer (w/o Ca2+/Mg2+)Thermo Fisher Scientific, Darmstadt, Germany70011044
Peristaltic pump ISM444BCole Parmer, Wertheim, Germany3475
Pipette (100 µL)Eppendorf, Wesseling-Berzdorf, Germany3124000075
Pipette (1000 µL)Eppendorf, Wesseling-Berzdorf, Germany3123000063
Plastic container (100 mL)Sarstedt, Nümbrecht, Germany7,55,62,300
PMN-Elastase ELISADemeditec Diagnostics, Kiel GermanyDEH3311
Polyvinyl chloride tubeSaint-Gobain Performance Plastics Inc., Courbevoie France-
Reaction Tubes (1.5 mL)Eppendorf, Wesseling-Berzdorf, Germany30123328
neurovascular laser-cut implantsAcandis GmbH, Pforzheim01-0011x
SC5b-9 ELISATECOmedical, Buende, GermanyA029
Scanning electron microscopeCambridge Instruments, Cambridge, UK-
Sealing tape (96 well plate)Thermo Fisher Scientific, Darmstadt, Germany15036
Syringe 10/12 mL Norm-JectHenke-Sass-Wolf, Tuttlingen, Germany10080010
TAT micro kitSiemens Healthcare, Marburg, GermanyOWMG15
Waterbath Type 1083Gesellschaft für Labortechnik, Burgwedel, Germany-

References

  1. ISO. . Biological evaluation of medical devices. , (2002).
  2. Weber, M., et al. Blood-Contacting Biomaterials: In Vitro Evaluation of the Hemocompatibility. Frontiers in Bioengineering and Biotechnology. 6, 99 (2018).
  3. Li, Y., Boras....

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Hemocompatibility TestingBlood contacting ImplantsFlow LoopHuman Blood FlowInternational Standard OrganizationThrombotic EventsAnticoagulantsBiomaterial ResearchHeparin LoadingNeurovascular Laser Cut ImplantPeristaltic PumpWhole Blood Count AnalysisEDTA

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