In vitro hemolysis rate testing is an essential biocompatibility test that evaluates the degree of red blood cell damage caused by medical devices and biomaterials that contact blood. Performed in accordance with ISO 10993-4, ASTM F756, and GB/T 16886.4, this assay provides critical safety evidence for regulatory submissions in global markets. This article explains the testing principles, applicable sample types, methodologies, calculation formulas, and acceptance criteria involved in in vitro hemolysis rate determination.
Technical Overview
In vitro hemolysis rate determination (in vitro simulated hemolysis testing) is a hemocompatibility assay designed to quantify the destruction of red blood cells (erythrocytes) caused by a medical device or biomaterial, either through direct mechanical interaction or through the leaching of soluble chemical substances. Hemolysis releases free hemoglobin into the surrounding medium, and the concentration of this free hemoglobin is measured spectrophotometrically to calculate the hemolysis rate.
Hemolysis testing is a fundamental component of the biological evaluation of blood-contacting medical devices, as required by ISO 10993-4 (Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood) and its Chinese counterpart GB/T 16886.4. The detailed test methodology is commonly executed in accordance with ASTM F756 (Standard Practice for Assessment of Hemolytic Properties of Materials), while sample preparation and extraction conditions follow ISO 10993-12. Manufacturers typically engage accredited third-party laboratories to perform this testing under GLP-compliant conditions to support product registration and design verification.
The test involves incubating the test sample, or an extract of the sample, with diluted fresh anticoagulated blood at physiological temperature. After incubation and centrifugation, the absorbance of the supernatant is measured at approximately 540–545 nm, corresponding to the hemoglobin absorption band. The hemolysis rate is then calculated by comparing the sample absorbance against positive and negative controls.
Testing Samples
Blood-Contacting and External Communicating Devices
Devices that directly contact circulating blood or serve as conduits for blood or infusion fluids are the primary candidates for hemolysis testing. Typical examples include:
- Intravenous catheters, central venous catheters, and peripherally inserted catheters
- Infusion sets, transfusion sets, syringes, and needles
- Hemodialyzers, extracorporeal circulation circuits, and membrane oxygenators
- Blood collection bags, blood filters, and cardiotomy reservoirs
- Arterial and venous cannulae used in cardiac surgery
Implantable Cardiovascular Devices
Long-term implants placed within the vascular system are subjected to hemolysis evaluation because sustained erythrocyte trauma can lead to anemia and hemoglobinuria. Representative products include vascular stents, heart valve prostheses, vascular grafts, septal occluders, inferior vena cava filters, and components of ventricular assist devices.
Raw Materials and Biomaterials
Polymers, metals, ceramics, coatings, adhesives, and surface-modified materials intended for blood-contacting applications are frequently screened for hemolytic potential during material selection and research and development, before device-level testing is performed.
Testing Items
The core testing item is the determination of the hemolysis rate (expressed as a percentage), which may be conducted using either the direct contact method or the indirect extraction method depending on the nature and intended use of the device. The results are typically classified as follows, based on ASTM F756 conventions:
- Non-hemolytic: hemolysis rate between 0% and 2%
- Slightly hemolytic: hemolysis rate between 2% and 5%
- Hemolytic: hemolysis rate greater than 5%
In routine regulatory practice, a hemolysis rate below 5% is widely regarded as acceptable evidence that the device or material does not exert a significant hemolytic effect.
Testing Methods
Sample Preparation
Test samples are prepared in their final, sterilized form whenever possible. For the indirect method, extracts are prepared using a polar vehicle such as sodium chloride injection (0.9% saline) or phosphate-buffered saline, with extraction ratios selected according to ISO 10993-12 (for example, 3 cm²/mL for surface-contacting materials or appropriate mass-to-volume ratios for irregular samples). Extraction is commonly performed at 37°C for 24 hours, although alternative validated conditions may be justified.
Blood Preparation
Fresh whole blood is collected from healthy animals (most commonly rabbits) or human donors into anticoagulant-containing tubes. The blood is diluted with phosphate-buffered saline or normal saline immediately before use to obtain a standardized erythrocyte suspension suitable for optical measurement.
Direct Contact Method
In the direct contact method, the test sample is placed directly into contact with the diluted blood and incubated at 37°C ± 1°C for a defined period, typically several hours. This approach is suitable for evaluating both the surface characteristics and the mechanical effects of the device, and is frequently applied to tubular devices, films, and finished product configurations.
Indirect Extraction Method
In the indirect method, the blood is exposed to the material extract rather than to the material itself. Equal volumes of extract and diluted blood are mixed and incubated at 37°C. This method evaluates hemolysis induced by soluble chemical leachables and is appropriate for materials that cannot be conveniently tested in direct contact, such as molds, pellets, or irregularly shaped components.
Controls
Each test run includes parallel control groups to validate the assay:
- Positive control: distilled or deionized water, which causes complete osmotic hemolysis
- Negative control: normal saline or phosphate-buffered saline, which should produce no significant hemolysis
Measurement and Calculation
Following incubation, all tubes are centrifuged to sediment intact erythrocytes, and the supernatant is collected. The absorbance of free hemoglobin in the supernatant is measured by ultraviolet-visible spectrophotometry at approximately 540–545 nm. The hemolysis rate is calculated using the following formula:
Hemolysis Rate (%) = (A_sample − A_negative) / (A_positive − A_negative) × 100%, where A_sample, A_negative, and A_positive represent the absorbance values of the test sample, negative control, and positive control, respectively. Testing is generally performed in triplicate or with multiple replicates to ensure statistical reliability, and results are compared against the acceptance criterion described above.
Applications
In vitro hemolysis rate determination serves multiple purposes throughout the product lifecycle of blood-contacting medical devices:
- Regulatory registration: providing required biological evaluation data for market submissions in China, the United States, the European Union, and other jurisdictions under frameworks such as NMPA registration, FDA premarket notification, and EU MDR conformity assessment
- Design verification: confirming that device geometry, materials, and surface finishes do not induce unacceptable erythrocyte damage
- Material screening: comparing candidate biomaterials, coatings, and suppliers during early-stage development
- Change control: re-evaluating hemocompatibility following material, process, or supplier modifications
- Quality monitoring: supporting periodic verification of manufacturing consistency for high-risk product lines
FAQ
Which standards govern in vitro hemolysis rate testing?
The principal standards are ISO 10993-4 and GB/T 16886.4 for the selection of blood interaction tests, ASTM F756 for the hemolytic assessment methodology, and ISO 10993-12 for sample preparation and extraction. Accredited third-party laboratories select the applicable protocol based on the device type and target regulatory market.
What is the acceptance criterion for hemolysis rate?
A hemolysis rate below 5% is generally considered acceptable, indicating the material or device is non-hemolytic or only slightly hemolytic. Rates exceeding 5% are considered hemolytic and typically require material or design modification followed by retesting.
How do I choose between the direct contact and indirect extraction methods?
The direct contact method is preferred for finished devices and surface materials where mechanical and surface effects are relevant, whereas the indirect extraction method is suited to evaluating chemical leachables from irregular materials or components. Many registration dossiers include both methods to provide comprehensive coverage.
What type of blood is used in the test?
Fresh anticoagulated rabbit blood is the most commonly used test system, particularly under ASTM F756 and GB/T 16886-based protocols. Human donor blood may be used when scientifically justified, provided ethical and biosafety requirements are met.
What sample quantity is required for testing?
Sample requirements depend on the extraction ratio and the number of replicates. As a general guideline, laboratories request sufficient material to prepare multiple replicate samples for both direct and indirect testing, often corresponding to several grams of material or several complete device units. Exact requirements should be confirmed with the testing laboratory before submission.
How long does hemolysis testing take?
The laboratory phase, including blood preparation, incubation, centrifugation, and measurement, is typically completed within a few days, with full test reports generally issued within two to four weeks, depending on sample queuing and protocol complexity.