Hemolysis testing is a core biocompatibility evaluation required for medical devices that contact blood directly or indirectly, quantifying the degree of red blood cell damage caused by a device or its leachables. Conducted in accordance with ISO 10993-4, ASTM F756, and GB/T 16886.4, the test supports regulatory submissions to FDA, EU MDR, and NMPA markets. This article explains the principle, sample requirements, test methods, acceptance criteria, and typical applications of hemolysis testing for medical devices.
Technical Overview
Hemolysis testing is an in vitro biocompatibility test that measures the extent to which a medical device, biomaterial, or its extract damages red blood cells (erythrocytes) and releases hemoglobin into the surrounding medium. If a device surface, residual process chemical, or leachable substance disrupts erythrocyte membranes, free hemoglobin is liberated into plasma, which can clinically manifest as anemia, hemoglobinemia, and kidney injury. Hemolysis assessment is therefore a fundamental safety endpoint for blood-contacting devices.
The test is governed primarily by ISO 10993-4, Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood, which classifies hemolysis within the hematology category of blood/device interactions, and by ASTM F756, Standard Practice for Assessment of Hemolytic Properties of Materials. In China, the harmonized standard GB/T 16886.4 and the product-level method standard GB/T 14233.1 are commonly referenced by NMPA reviewers for disposable infusion, transfusion, and injection equipment.
ISO 10993-4 assigns hemocompatibility test requirements according to the blood contact category of the device, including:
- External communicating devices with an indirect blood path, such as IV catheters, syringes, and extension sets
- External communicating devices with circulating blood, such as hemodialysis circuits, ECMO systems, and blood oxygenators
- Blood-contacting implant devices, such as stents, vascular grafts, heart valves, and ventricular assist devices
Based on the hemolytic index defined in ASTM F756, materials are typically classified as nonhemolytic (0–2%), slightly hemolytic (2–5%), or hemolytic (greater than 5%). For most regulatory submissions, a hemolysis rate of 5% or less is widely regarded as the acceptance threshold.
Testing Samples
Blood-Contacting Medical Devices
Finished, sterilized devices intended for direct or indirect blood contact are the primary test articles, including intravenous and central venous catheters, hemodialyzers, blood tubing sets, blood collection bags, oxygenators, autotransfusion systems, infusion and transfusion sets, and perfusion cannulae.
Cardiovascular Implants
Stents, stent grafts, vascular grafts, heart valves, septal occluders, pacemaker and defibrillator leads, and ventricular assist device blood-wetted components are evaluated because they reside in contact with blood for extended durations.
Raw Materials and Components
During design and development, polymers such as PVC, polyurethane, and silicone; metals such as stainless steel, nitinol, cobalt-chromium, and titanium; surface coatings; adhesives; and 3D-printed materials are screened for hemolytic potential before device integration.
Sample Preparation Requirements
Samples should represent the final product in its sterilized, packaged condition whenever possible. Extraction media, surface-area-to-volume ratios, and extraction conditions follow ISO 10993-12, for example 3 cm²/mL or 0.2 g/mL ratios, with common extraction conditions such as 37 °C for 72 hours, 50 °C for 72 hours, 70 °C for 24 hours, or 121 °C for 1 hour depending on the device and justification.
Testing Items
The principal test item is the hemolysis rate (hemolytic index), determined by spectrophotometric quantification of free hemoglobin released from diluted anticoagulated blood after contact with the test article or its extract. Typical deliverables include:
- Hemolysis rate by the direct contact method
- Hemolysis rate by the extraction (indirect) method
- Free hemoglobin concentration in the supernatant measured at 540–545 nm
- Complementary hemocompatibility endpoints under ISO 10993-4, including thrombosis, coagulation (PT, APTT, thrombin time, fibrinogen), platelet count and activation, leukocyte and erythrocyte counts, and complement activation markers such as C3a and SC5b-9
Many biological evaluation programs combine hemolysis testing with these related endpoints to build a complete ISO 10993-4 hemocompatibility data set in a single study at an accredited third-party laboratory.
Testing Methods
Direct Contact Method
Diluted fresh anticoagulated blood, typically rabbit or human blood anticoagulated with sodium citrate, is brought into direct contact with the test article and incubated at 37 °C for a defined period, commonly 2 to 4 hours depending on the protocol applied; GB/T 14233.1 specifies incubation at 37 °C ± 1 °C for 2 hours. After incubation, the blood is centrifuged, and the absorbance of the supernatant is measured at 545 nm to quantify released hemoglobin. This method is preferred when the blood-contacting surface itself is the safety-relevant factor.
Extraction (Indirect) Method
A device extract is first prepared in 0.9% sodium chloride solution or phosphate-buffered saline using the extraction ratios and conditions specified in ISO 10993-12, for example 37 °C for 72 hours. Diluted blood is then incubated with the extract at 37 °C, centrifuged, and the free hemoglobin in the supernatant is quantified spectrophotometrically. This method is suitable when hemolytic risk is driven by leachable substances rather than surface effects.
Controls
Physiological saline or PBS serves as the negative control, producing minimal hemolysis, while sterile distilled or deionized water serves as the positive control, producing complete hemolysis. The difference between positive and negative control absorbance values must meet the validity criteria defined by the test standard before results are accepted.
Calculation and Acceptance Criteria
The hemolysis rate is calculated as: Hemolysis (%) = (Absorbance of sample − Absorbance of negative control) / (Absorbance of positive control − Absorbance of negative control) × 100. A result of 5% or less is generally considered nonhemolytic and acceptable for regulatory purposes, while results above 5% indicate hemolytic potential requiring material or design remediation.
Dynamic Flow Testing
For active blood-handling devices such as blood pumps, oxygenators, and ventricular assist systems, ISO 10993-4 and related ASTM standards for blood pumps describe dynamic in vitro recirculation loop studies in which hemolysis is evaluated under physiologically relevant flow and shear conditions over extended circulation times.
Applications
Hemolysis testing data are used across the entire device lifecycle:
- Regulatory submissions, including FDA 510(k), De Novo, and PMA applications, EU MDR technical documentation and CE marking, NMPA registration dossiers, and submissions in Japan and Canada
- Material screening and selection during research and development of blood-contacting polymers, coatings, and metals
- Design and process change control, such as material supplier changes, sterilization method changes, or manufacturing site transfers
- Batch release and quality control for infusion, transfusion, and injection equipment in accordance with GB/T 14233.1
- Biological evaluation plans and toxicological risk assessments under ISO 10993-1 and ISO 14971
For externally communicating and implantable blood-contacting devices, hemolysis testing is almost always expected by regulators and should be planned early in the development program.
FAQ
What hemolysis rate is considered acceptable?
A hemolysis rate of 5% or less is the widely accepted threshold for most regulatory submissions. Under the ASTM F756 classification scheme, materials scoring 0–2% are nonhemolytic, 2–5% are slightly hemolytic, and above 5% are hemolytic.
Should I choose the direct contact method or the extraction method?
The choice depends on how the device contacts blood. Direct contact is appropriate when the device surface interacts with blood, while the extraction method targets hemolytic leachables. Many submissions include both methods to cover surface and extractable effects comprehensively.
What blood is used for the test?
Fresh rabbit blood is most commonly used and is specified in GB/T 14233.1 and ASTM F756 protocols; fresh human donor blood is also acceptable. The blood is anticoagulated, typically with sodium citrate, and used within a defined short period after collection.
Do devices without blood contact require hemolysis testing?
Generally no. The requirement is determined by the blood contact categorization in ISO 10993-1 and ISO 10993-4. Devices with no direct or indirect blood contact can usually be justified as not requiring hemolysis evaluation in the biological evaluation plan.
Which standards should the test report reference?
Reports typically reference ISO 10993-4 for test selection, ISO 10993-12 for extract preparation, ASTM F756 for the hemolytic assessment practice, and GB/T 16886.4 or GB/T 14233.1 for products registered in China, according to the target market.
How many samples are required?
Protocols usually require triplicate test article replicates together with positive and negative controls, using sterile finished devices in their final packaging. Exact sample quantities depend on the device geometry and the extraction ratio applied by the accredited third-party laboratory.
Can hemolysis testing be combined with other biocompatibility tests?
Yes. Hemolysis is frequently bundled with cytotoxicity (ISO 10993-5), sensitization and irritation (ISO 10993-10 and ISO 10993-23), and other hemocompatibility endpoints to form a complete biocompatibility package aligned with the ISO 10993-1 evaluation plan.