Hemolysis Testing of Medical Devices – Evaluation of Hemocompatibility in Accordance with EN ISO 10993-4
Medical devices that come into contact with blood encompass a wide range of products, including blood bags, infusion sets, catheters, stents, heart-lung machine circuits, dialyzers, and artificial blood vessels. The interaction of these products with blood can lead to the breakdown of red blood cells (erythrocytes). This phenomenon is known as hemolysis and is characterized by the release of free hemoglobin into the plasma. Hemolysis can lead to anemia, jaundice, kidney damage, and even life-threatening complications in patients. Therefore, the hemolysis test—EN ISO 10993-4— is an indispensable part of the biocompatibility assessment of medical devices that come into contact with blood.
EN ISO 10993-4 is the internationally recognized standard for the hemocompatibility evaluation of medical devices. This standard defines test categories, methods, and acceptance criteria for evaluating the effects of products that come into contact with blood on blood components. The hemolysis test is one of the most commonly performed tests under this standard.
What is hemolysis, and why is it tested for?
Hemolysis is the release of hemoglobin from erythrocytes (red blood cells) as a result of a disruption in membrane integrity. Under in vivo conditions, hemolysis can be caused by mechanical trauma, osmotic stress, chemical substances, immunological reactions, or microbial infections.
With regard to medical devices, the risk of hemolysis stems in particular from the following factors:
- Surface properties: The roughness, chemical composition, and coatings on the device surface can damage the erythrocyte membrane.
- Mechanical factors: Pumps, circulatory systems with continuous blood flow, and catheters with narrow lumens can exert mechanical stress on the cells.
- Chemical Release: Plasticizers, monomer residues, or solvents released from the device may have a hemolytic effect.
- Osmotic imbalance: The solutions contained in the device or its surface properties can create an osmotic gradient.
The hemolysis test quantitatively determines the extent to which the device causes damage to red blood cells upon contact with blood. The hemolysis percentage determined from the test result indicates whether the device falls within the limits acceptable for safe use.
Hemolysis Test Procedures in Accordance with EN ISO 10993-4
EN ISO 10993-4 specifies various test methods for evaluating hemolysis. The choice of method depends on the intended use of the device, the duration of contact with blood, and the physical form of the device.
1. In-vitro hemolysis test (direct contact method)
This is the most commonly used method. The device, or a sample taken from the device, is incubated with fresh human or animal blood for a specific period of time (usually 4 hours). After incubation, the amount of free hemoglobin in the plasma is measured spectrophotometrically.
The test is conducted using three parallel samples:
- Test sample: Blood that has come into contact with the device.
- Negative control: Blood alone without the device (spontaneous hemolysis).
- Positive control: Complete hemolysis using distilled water or a suitable hemolytic agent.
The hemolysis percentage is calculated using the following formula:
Hemolysis (%) = [(Absorbance of the test sample − Absorbance of the negative control) / (Absorbance of the positive control − Absorbance of the negative control)] × 100
2. In-vitro hemolysis test (extract method)
In cases where direct contact between the device and blood is not appropriate, extracts produced by the device and manufactured in accordance with ISO 10993-12 are used. The extract is mixed with the blood sample, incubated, and the hemolysis rate is determined.
3. In-vivo hemolysis assessment
In certain special cases (e.g., implantable devices or devices that require long-term contact with blood), an in vivo hemolysis assessment may be conducted using animal models. This method demonstrates the product’s effect under real physiological conditions.
Comparison of Hemolysis Test Methods
The following table summarizes the hemolysis test methods used in accordance with EN ISO 10993-4:
| Test Method | principle | Suitable Device Types | Evaluation criterion | Advantages |
|---|---|---|---|---|
| In vitro (direct contact) | The product is incubated with fresh blood; the hemoglobin in the plasma is measured. | Catheters, blood bags, infusion sets, stents | Hemolysis rate ≤ 5% (acceptable) | Fast, repeatable, officially recognized |
| In vitro (extract) | The device extract is incubated with blood; hemolysis is measured. | Devices for which direct contact is not appropriate | Hemolysis rate ≤ 5% | Standardized Extract Preparation |
| In vivo | In the animal model, the device is brought into contact with blood; hemolysis is observed. | Implantable Devices, Long-Term Contact | Physiological Assessment | Real-world physiological conditions |
Inclusion Criteria and Interpretation of Results
The results of hemolysis tests conducted in accordance with EN ISO 10993-4 are generally evaluated based on the following criteria:
- Hemolysis rate ≤ 5%: The product exhibits an acceptable level of hemolysis upon contact with blood (safe).
- Hemolysis rate > 5%: The product has hemolytic potential; further evaluation or a change in formulation is required.
However, this threshold may vary depending on the product’s intended use and the duration of contact. For example, stricter criteria may apply to products that require prolonged contact with blood. The results should be evaluated in light of the product’s clinical conditions of use and data from the scientific literature.
GLP-Compliant Hemolysis Testing Services from Nano-Test
The hemocompatibility evaluation of medical devices that come into contact with blood is only possible by selecting the correct test method and presenting the results in a format acceptable to regulatory authorities. With its GLP-compliant hemolysis tests in accordance with EN ISO 10993-4, Nano-Test is your reliable partner in this process.
Nano-Test – Statement of Work
Nano-Test offers hemolysis testing services for medical devices in accordance with EN ISO 10993-4. In our laboratories, which are accredited according to GLP (Good Laboratory Practice) and ISO/IEC 17025, we conduct in vitro hemolysis tests (direct contact and extraction methods) as well as in vivo hemolysis evaluations.
Our service process:
- Sample Preparation: Extraction of the product in accordance with ISO 10993-12 and preparation of the test samples.
- Blood collection: The collection and use of fresh human or animal blood under standardized conditions.
- Incubation: Incubation at a specified duration and temperature (usually 4 hours at 37 °C).
- Spectrophotometric measurement: Measurement of the absorption of free hemoglobin in plasma at 540 nm.
- Calculation and Evaluation: Calculation of the hemolysis percentage and its evaluation based on the acceptance criteria.
- Reporting: GLP-compliant reports with study approval that can be used for MDR and FDA 510(k) submissions.
All of our studies are conducted in accordance with data integrity standards (ALCOA+ principles).
Advantages of the Nano-Test hemolysis test:
- GLP-certified infrastructure: All studies are conducted in accordance with GLP principles and ALCOA+ requirements.
- Compliance with Standards: Full compliance with EN ISO 10993-4, ASTM F756, and the relevant standards.
- Expertise in both methods: Both direct contact and extraction methods under one roof.
- Fast results: Reports for standard hemolysis tests are available within 5–7 business days.
- Specialized Staff: An experienced team in the fields of hemocompatibility and biocompatibility.
- Regulatory Compliance: Comprehensive support in preparing technical documentation for MDR and FDA 510(k) submissions.
Hemolysis caused by medical devices that come into contact with blood poses a serious risk that directly jeopardizes patient safety. The hemolysis test—EN ISO 10993-4 —is a crucial step in scientifically demonstrating your product’s effect on red blood cells. Selecting the correct test method, conducting the test under standardized conditions, and reporting the results in accordance with GLP are prerequisites for the smooth progression of the regulatory approval process.
With its accredited laboratory infrastructure, expert staff, and GLP-compliant processes, Nano-Test fully meets the requirements of medical device manufacturers for hemolysis testing. Partner with Nano-Test to enhance the safety of your product and accelerate market access.