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EN ISO 10993-4

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EN ISO 10993-4

EN ISO 10993-4 is a standard published by the International Organisation for Standardisation (ISO) and adopted by the European Committee for Standardisation (CEN), entitled ‘Biological evaluation of medical devices – Part 4: Selection of tests for interactions with blood”, i.e. “Biological evaluation of medical devices – Part 4: Selection of tests for interactions with blood”. It is generally referred to as the haemocompatibility standard.

Regulatory status and version note: The current base text of the standard is ISO 10993-4:2017 (fourth edition) and has been adopted at European level as EN ISO 10993-4:2017. As work is currently underway on an amendment to the standard, the valid consolidated version and the status of national adoption must be checked before work commences. The standard has been recognised as a harmonised standard under the EU Medical Devices Regulation (MDR 2017/745); its recognition status and scope as determined by the FDA (particularly with regard to certain sections) should be checked before submitting an application.

The key feature of the standard is that it does not prescribe specific test methods, but instead provides a framework and a selection matrix to help determine which test categories should be chosen depending on the nature and duration of the product’s contact with blood. Consequently, ISO 10993-4 is not a ‘method standard’ but a standard for test selection; the specific methods to be applied (e.g. ELISA, flow cytometry, SEM, flow-loop) are determined on the basis of the relevant literature and validation studies.

Device categories

The standard classifies devices that come into contact with blood according to their contact characteristics:

  • Indirect blood path: Devices that do not come into direct contact with blood, but through which blood or blood products flow (e.g. solution administration sets, transfer sets)
  • Direct contact with blood – indirect contact: devices that come into contact with blood but through which blood does not circulate (e.g. cannulae, catheters, blood collection tubes)
  • Direct blood contact – contact with circulating blood: devices in which blood circulates continuously (e.g. extracorporeal circulation systems, haemodialysis machines, blood pumps)
  • Implanted devices: Implanted devices that come into contact with blood (e.g. vascular grafts, heart valves, stents, ventricular assist devices)

Duration of contact

  • Limited: contact lasting less than 24 hours
  • Long-term (prolonged): duration of contact between 24 hours and 30 days
  • Long-term (permanent): contact lasting more than 30 days

Categories of haemocompatibility tests

The standard defines five main test categories and lists typical endpoints for each category:

1. Thrombosis

  • Thrombus formation and accumulation (assessment by macroscopic examination and by SEM)
  • Fibrin(ogen) accumulation and fibrinopeptide A levels
  • Platelet adhesion and aggregation
  • Thrombus assessment in flow-loop or animal models

2. Coagulation

  • Prothrombin time (PT), activated partial thromboplastin time (aPTT)
  • Fibrinogen levels, thrombin time
  • Thrombin-antithrombin complex (TAT), prothrombin fragment F1+2
  • Further markers of coagulation activation

3. Thrombocytes (platelets)

  • Platelet count and platelet aggregation
  • Markers of platelet activation (P-selectin/CD62P, β-thromboglobulin, PF4)
  • Phenotyping of platelet activation using flow cytometry
  • Platelet adhesion and spreading (microscopic assessment)

4. Haematology

  • Complete blood count (red blood cell, white blood cell and platelet counts, haematocrit, haemoglobin)
  • Leucocyte differentiation
  • Haemolysis indicators (free haemoglobin in plasma, haptoglobin; can also be assessed using the ASTM F756 haemolysis standard)

5. Complement system

  • C3a and C5a levels
  • SC5b-9 (terminal complement complex, TCC)
  • Other markers of complement activation, such as CH50, Bb and iC3b

Test environments

  • In vitro testing: Direct contact between the device or material and blood; this is the preferred approach. The test conditions (blood source, anticoagulant, temperature, contact time, surface-to-volume ratio) should be selected to reflect clinical use.
  • Ex vivo models: Controlled blood flow through the device using flow-loop systems; this enables the evaluation, in particular, of devices that come into contact with circulating blood.
  • In vivo models: Assessment of the device’s interaction with blood in animal models (e.g. pig, dog, sheep); this may be required for implants and extracorporeal devices.

Wichtiger methodischer Hinweis: Bei Hämokompatibilitätstests ist – anders als bei anderen biologischen Tests – der direkte Kontakt mit Blut anstelle eines Extrakts in der Regel besser geeignet, da das Ziel die Bewertung der Wechselwirkung zwischen Oberfläche und Blut ist. Bei einigen Tests kann jedoch die Herstellung eines Extrakts (ISO 10993-12) zum Einsatz kommen. Die Blutquelle (vorzugsweise menschliches Blut; bei Verwendung von Tierblut sind die Artenunterschiede anzugeben), die Art des Antikoagulans (Heparin, Citrat) und der Zeitpunkt der Prüfung wirken sich direkt auf die Ergebnisse aus.

The key parameters reported in the study are as follows:

  • The category of the device and the contact time; rationale for the test selection matrix
  • The selected test categories and specific endpoints
  • Source of blood (human/animal), anticoagulant and conditions for blood preparation
  • Test conditions: surface-to-volume ratio, contact time, temperature, flow rate (for flow-through models)
  • Analytical methods used and reference ranges
  • Quantitative results for each endpoint and a comparison with the control group
  • Positive and negative controls, and validity criteria
  • Result: haemocompatible / not haemocompatible – assessment and justification
  • Data on measurement uncertainty and repeatability

Why is the test carried out?

EN ISO 10993-4 is carried out for the following scientific and regulatory purposes:

  • Assessment of patient safety: determining the potential of products that come into contact with blood to cause adverse effects such as thrombosis, haemolysis, platelet activation, coagulation and complement activation
  • MDR compliance: Provision of the necessary data for the biological safety assessment under the EU Medical Devices Regulation (2017/745)
  • Risk management: Assessment and reduction of thromboembolic and haemolytic risks associated with blood-contacting products
  • Material and design development: Assessment of the influence of surface properties (roughness, hydrophilicity, coatings) and material selection on haemocompatibility
  • Standardisation of test selection: Scientific justification for the appropriate test series based on the product category and duration of contact
  • Support with regulatory documentation: for use as part of the biocompatibility dossier in applications to the FDA, in accordance with the EU MDR and other regulatory authorities
  • Animal welfare (3Rs): Priority should be given to the use of in vitro and ex vivo models, where appropriate
  • Evidence-based approach: Chemical characterisation (ISO 10993-18), cytotoxicity (ISO 10993-5), irritation (ISO 10993-23), sensitisation (ISO 10993-10) and other biocompatibility data contribute to the holistic assessment of the product

EN ISO 10993-4 alone does not cover the product’s entire biocompatibility profile; furthermore, haemocompatibility tests may not fully reflect the product’s clinical conditions of use. The results should be interpreted in the context of the chemical characterisation, other biological tests, the product design and clinical data.

As part of our nano-testing services, we carry out haemocompatibility assessments in accordance with EN ISO 10993-4, utilising our accredited laboratory facilities and quality management system, and applying scientific rigour. Our scope of services includes the following:

Drawing up a test strategy and a protocol

  • Determination of the category of the medical device (indirect contact with blood, direct contact with blood, implantable) and the duration of contact (limited, long-term, permanent)
  • Development and justification of the appropriate test set in accordance with the ISO 10993-4 test selection matrix
  • Analysis of the available chemical characterisation data (ISO 10993-18) and literature data
  • Determination of the blood source (preferably human blood), the anticoagulant and the blood preparation strategy
  • Design of the test conditions (surface-to-volume ratio, contact time, temperature, flow conditions) in a manner that reflects clinical use
  • Planning of positive and negative controls; compliance with ethical and regulatory requirements

For information on testing strategies, the selection of test kits, planning the blood source and anticoagulant, sample collection conditions, the testing schedule and reporting procedures as part of your haemocompatibility assessments in accordance with EN ISO 10993-4, please feel free to contact Nano-Test.

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