Hemolysis rate testing is a fundamental biocompatibility study required for medical devices that contact blood directly or indirectly, quantifying the damage that device materials, extracts, and surfaces cause to red blood cells. Performed in accordance with ISO 10993-4, ASTM F756, and GB/T 16886.4, this test is a mandatory element of regulatory submissions for catheters, hemodialyzers, oxygenators, infusion sets, and blood collection products. This article explains the principles, sample requirements, test methods, calculation formulas, acceptance criteria, and practical applications of medical device hemolysis rate determination.
Technical Overview
Hemolysis is the rupture or destruction of red blood cell (erythrocyte) membranes, resulting in the release of hemoglobin into the surrounding plasma or test medium. When a medical device or its material comes into contact with blood, physical shear forces, surface roughness, residual monomers, plasticizers, sterilant residues, metal ions, or other extractables and leachables can damage erythrocytes and trigger hemolysis. Clinically, significant intravascular hemolysis may lead to hemoglobinemia, hemoglobinuria, renal tubular injury, anemia, and disseminated intravascular coagulation, so the hemolytic potential of blood-contacting devices must be evaluated before market release.
Hemolysis rate determination is one of the core tests within the hemocompatibility evaluation framework of ISO 10993-4 (Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood). ISO 10993-4 divides blood-device interactions into five categories: thrombosis, coagulation, platelet function, hematology (which includes hemolysis), and complement activation. The hemolysis test measures the percentage of red blood cells lysed relative to a fully lysed positive control, providing a quantitative index of erythrocyte damage.
- ISO 10993-4 — Selection of tests for interactions with blood, defining when hemolysis testing is required.
- ASTM F756 — Standard Practice for Assessment of Hemolytic Properties of Materials.
- GB/T 16886.4 — Chinese national standard adopting ISO 10993-4.
- GB/T 14233.1 — Test methods for infusion, transfusion, and injection equipment, including hemolysis procedures for these product families.
In most regulatory jurisdictions, including NMPA registration in China, FDA submissions in the United States, and CE marking under the EU MDR, a hemolysis report issued by accredited third-party laboratories or qualified in-house facilities is a standard component of the biological evaluation documentation for blood-contacting devices.
Testing Samples
Direct Blood-Contact Devices (Circulating Blood Path)
Devices that are intended to contact circulating blood for prolonged or repeated periods require hemolysis evaluation as a priority. Typical samples include intravascular catheters (central venous, arterial, and electrophysiology catheters), hemodialyzers, extracorporeal membrane oxygenation (ECMO) components, cardiopulmonary bypass oxygenators and reservoirs, heart-lung machine tubing, blood pumps, blood bags, and blood filters.
External Communicating Devices (Indirect Blood Path)
Devices through which fluids pass before entering the bloodstream, or that briefly contact blood at the point of access, are also subject to hemolysis testing. Representative samples include infusion sets, transfusion sets, syringes, intravenous cannulae, winged blood collection needles, blood collection tubes, extension lines, stopcocks, and indwelling needles.
Implantable Cardiovascular Devices
Long-term implants exposed to blood, such as vascular stents, stent grafts, artificial heart valves, vascular grafts, septal occluders, and inferior vena cava filters, are evaluated using material-based extract or direct contact protocols because whole-device testing may not be technically feasible.
Biomaterials, Coatings, and Components
Raw polymers, elastomers, hydrophilic or antithrombotic coatings, adhesives, and tubing materials can be screened for hemolytic potential during early-stage research and development, supplier qualification, or change control.
Samples should represent the final product in its sterilized, ready-to-use condition, since sterilization residues such as ethylene oxide can themselves be hemolytic. Best practice is to submit samples from three consecutive production lots with documented batch records. The sample-to-extract-medium ratio is typically defined by mass (for example, 0.1 to 0.2 g of material per mL of medium) or by the contact surface area of the device, in accordance with the applicable standard.
Testing Items
The core deliverable of the study is a quantitative hemolysis result, commonly reported together with supporting hemocompatibility endpoints:
- Hemolysis rate by the extract (indirect) method — evaluates chemical mediators released from the device.
- Hemolysis rate by the direct contact method — evaluates combined chemical and physical surface effects on erythrocytes.
- Hemolytic index — the normalized index described in ASTM F756 for material classification.
- Complementary hemocompatibility items — activated partial thromboplastin time (APTT), prothrombin time (PT), thrombin time (TT), fibrinogen, platelet count and activation markers, leukocyte and erythrocyte counts, and complement activation products (C3a, C5a, SC5b-9), depending on the device contact category defined in ISO 10993-4.
Testing Methods
Indirect Method (Extract Method)
In the extract method, the test article is first incubated in an extraction vehicle — typically 0.9% sodium chloride injection or phosphate buffered saline (PBS), since pure water would itself cause hemolysis — under defined conditions such as 37 °C for 30 to 60 minutes or under conditions simulating clinical use. A measured volume of the extract is then combined with diluted, freshly drawn, anticoagulated rabbit blood (or citrated human blood where justified). The mixture is incubated at 37 °C ± 1 °C for approximately 60 minutes with gentle inversion at defined intervals, centrifuged at approximately 2,500 rpm for about 5 minutes, and the absorbance of the supernatant is measured spectrophotometrically at 545 nm (the oxyhemoglobin absorption peak; some protocols use 540 nm).
Direct Contact Method
In the direct contact method, sterile test samples cut into small sections or the intact device are placed directly into contact with diluted anticoagulated blood and incubated at 37 °C ± 1 °C for a defined period, commonly 3 to 4 hours depending on the protocol. After incubation, the blood is centrifuged under the same conditions and the free hemoglobin concentration in the supernatant is determined from the absorbance at 545 nm. This method captures both chemical leachables and mechanical or surface-induced erythrocyte damage, making it suitable for devices whose geometry may create shear or abrasion.
Controls and Test Validity
- Negative control: 0.9% sodium chloride solution or PBS, which should produce negligible hemolysis.
- Positive control: Triton X-100 surfactant solution or deionized/distilled water, which induces near-complete red blood cell lysis.
- Validity criteria: the negative control absorbance must fall below the threshold specified in the protocol, the positive control must demonstrate essentially complete hemolysis, replicate results must show acceptable precision (typically expressed as relative standard deviation), and testing must be completed within the prescribed time after blood collection.
Calculation and Acceptance Criteria
The hemolysis rate is calculated using the mean absorbance values of the test, negative control, and positive control groups:
Hemolysis Rate (%) = [(Atest − Anegative) / (Apositive − Anegative)] × 100%
Under the evaluation practice aligned with GB/T 16886.4 and widely applied in NMPA registration, a device is generally considered non-hemolytic and acceptable when the hemolysis rate is ≤ 5%. ASTM F756 classifies materials more granularly: hemolysis below 2% is nonhemolytic, 2% to 5% is slightly hemolytic, and above 5% is hemolytic. Slightly or clearly hemolytic results require design modification, material substitution, or scientific justification before regulatory acceptance.
Applications
Hemolysis rate determination supports the medical device lifecycle at multiple stages:
- Regulatory submission: inclusion in biological evaluation reports for NMPA registration, FDA 510(k) and PMA submissions, EU MDR technical documentation, and filings in other markets.
- Design verification and material screening: comparing candidate polymers, coatings, plasticizers, and surface treatments during research and development.
- Sterilization and process validation: confirming that residuals from ethylene oxide sterilization, irradiation-induced degradation products, or manufacturing changes do not increase hemolytic potential.
- Change control: re-qualifying devices after changes to materials, suppliers, processing aids, or sterilization cycles.
- Batch release and supplier management: periodic quality control testing performed by accredited third-party laboratories to demonstrate continued compliance.
FAQ
What hemolysis rate is considered acceptable?
A hemolysis rate of ≤ 5% is the commonly accepted pass criterion in device registration practice, particularly under the framework of GB/T 16886.4. Under ASTM F756, results above 5% are classified as hemolytic, 2% to 5% as slightly hemolytic, and below 2% as nonhemolytic. Regulatory reviewers generally expect blood-contacting devices to demonstrate results well below the hemolytic threshold.
Which devices require hemolysis testing?
ISO 10993-4 assigns hemocompatibility testing obligations based on blood contact category. Devices with an indirect blood path (infusion and transfusion sets, syringes, cannulae), devices in the circulating blood path (catheters, dialyzers, oxygenators, ECMO circuits), and blood-contacting implants (stents, grafts, valves) all typically require hemolysis data. Surface devices contacting intact skin generally do not.
Why is rabbit blood commonly used?
Fresh rabbit blood is widely used because erythrocytes from healthy rabbits show reproducible sensitivity, blood can be drawn immediately before testing to guarantee freshness, and the approach avoids some of the donor variability and ethical constraints associated with human blood. ASTM F756 also permits citrated human blood where scientifically justified and available.
How do I choose between the extract method and the direct contact method?
The extract method is preferred when the primary concern is chemical leachables, and it is the routine approach for infusion, transfusion, and injection equipment under GB/T 14233.1. The direct contact method is appropriate when physical surface effects, shear, or abrasion may contribute to erythrocyte damage, such as catheters and pump tubing. Where feasible, both methods can be run in parallel to capture combined chemical and mechanical risks.
How is hemolysis testing related to the broader hemocompatibility evaluation?
Hemolysis is only one of the five interaction categories defined in ISO 10993-4. Depending on the device and contact duration, a complete hemocompatibility panel may also include thrombosis testing, coagulation assays (APTT, PT), platelet studies, and complement activation testing. Hemolysis results should therefore be interpreted as part of an integrated biological evaluation file.
What should be considered when preparing samples for submission?
Submit the final product in its sterilized commercial packaging, ideally from three production batches, together with product specifications, material composition, intended use, blood contact classification, and sterilization method. The testing laboratory will define the extraction ratio, incubation conditions, and blood source in the protocol and will issue a report with individual and mean absorbance values, calculated hemolysis rates, control results, and a conclusion against the acceptance criterion.