Hemolysis testing is an essential biocompatibility study that evaluates whether medical materials and blood-contacting devices damage red blood cells and release hemoglobin into plasma. Performed in accordance with ISO 10993-4, ASTM F756, and GB/T 16886.4, the test quantifies the hemolytic index of materials and is a mandatory requirement for regulatory submissions covering catheters, implants, extracorporeal circuits, and infusion devices. This article explains the test principles, sample requirements, methodology, calculation of hemolysis rate, acceptance criteria, and its role in medical device evaluation.
Technical Overview
Hemolysis testing for medical materials is an in vitro blood-compatibility assay that measures the degree of red blood cell (erythrocyte) membrane disruption caused by a medical device or biomaterial. When a material comes into contact with blood, surface chemistry, leachable substances, residual processing agents, or mechanical shear forces may rupture erythrocyte membranes, releasing intracellular hemoglobin into the surrounding plasma or saline medium. The concentration of free hemoglobin in the supernatant, measured spectrophotometrically, provides a direct and sensitive indicator of hemolytic activity.
Hemolysis assessment is a core component of the hemocompatibility evaluation framework defined in ISO 10993-4 (Biological evaluation of medical devices — Part 4: Selection of tests for interactions with blood) and is further specified in ASTM F756 (Standard Practice for Assessment of Hemolytic Properties of Materials). In China, the equivalent national standard GB/T 16886.4 governs the test and is routinely requested by the National Medical Products Administration (NMPA) for registration dossiers of blood-contacting devices.
Because even mild hemolytic activity can cause anemia, elevated plasma free hemoglobin, renal burden, and thrombotic complications in clinical use, a hemolysis rate of five percent or less is the widely accepted threshold for classifying a material as non-hemolytic. The test is generally conducted by accredited third-party laboratories or in-house biocompatibility facilities using anticoagulated fresh rabbit blood or healthy human donor blood under strictly controlled incubation conditions.
Testing Samples
Finished Blood-Contacting Devices
Testing is typically performed on the final product in its sterilized, clinically representative condition. Common device categories submitted for hemolysis testing include:
- Intravascular catheters, peripherally inserted central catheters (PICC), and cannulae
- Infusion sets, syringes, stopcocks, and IV administration tubing
- Extracorporeal circulation components such as hemodialyzers, blood oxygenators, and blood filters
- Blood collection bags, blood storage containers, and transfusion sets
- Vascular stents, vascular grafts, heart valve prostheses, and inferior vena cava filters
- Ventricular assist device components and artificial heart surfaces
Raw and Candidate Materials
During research and development, hemolysis testing is also applied to candidate materials prior to device fabrication, enabling early screening of blood compatibility. Typical material classes include:
- Polymers: PVC, polyurethane, silicone elastomers, PTFE, PE, PP, and biodegradable polyesters such as PLA and PLGA
- Metals: 316L stainless steel, titanium and titanium alloys, cobalt-chromium alloys, and nitinol
- Surface coatings, hydrogels, drug-eluting layers, adhesives, and packaging materials that contact blood or infusion fluids
- Nanomaterials, ceramic composites, and novel biomaterials under preclinical evaluation
Sample Preparation Requirements
Samples must be cleaned, processed, and sterilized by the same method intended for the final product, since sterilization residues and extractables can significantly influence hemolytic behavior. Test portions are quantified either by mass or by surface area in accordance with ISO 10993-12 extraction ratios, for example 3 cm² per milliliter for film-like samples or 0.2 g per milliliter for irregular solids, and each test should include a minimum of three replicate sample units together with positive and negative controls.
Testing Items
The scope of a standard hemolysis study typically covers the following measurable endpoints:
- Hemolysis rate (hemolytic index) by the direct contact method
- Hemolysis rate by the indirect contact (material extract) method
- Absorbance of the supernatant at the hemoglobin Soret absorption band, conventionally measured at 545 nm or 540 nm using a UV-Vis spectrophotometer
- Concentration of plasma free hemoglobin released from the lysed erythrocytes
- Comparison of test samples against positive control (distilled or deionized water causing complete hemolysis) and negative control (phosphate-buffered saline or 0.9 percent sodium chloride)
Depending on the risk profile of the device, hemolysis data are frequently combined with complementary ISO 10993-4 endpoints such as thrombogenicity, platelet adhesion, coagulation assays (PT, APTT), complement activation, and leukocyte response to build a complete hemocompatibility profile.
Testing Methods
Direct Contact Method
In the direct contact method, the test material is placed in direct contact with diluted, anticoagulated fresh blood. A typical protocol involves diluting fresh rabbit blood with phosphate-buffered saline, adding the material to the diluted blood, and incubating the mixture at 37 ± 1 °C for a defined period, commonly two to four hours with gentle inversion to ensure uniform contact. After incubation, the samples are centrifuged to pellet intact erythrocytes, and the absorbance of the supernatant is measured to quantify released hemoglobin. This method best represents devices whose surfaces are in prolonged direct contact with circulating blood.
Indirect Contact (Extraction) Method
In the indirect method, an extract of the test material is first prepared according to ISO 10993-12 using physiological saline or another appropriate vehicle at 37 °C for a standardized duration. The resulting extract is then incubated with diluted blood, and hemoglobin release is measured as in the direct method. This approach evaluates hemolytic effects caused by soluble leachables, residual monomers, plasticizers, sterilant residues, and degradation products, and is particularly relevant for polymers, coatings, and adhesive-containing assemblies.
Calculation and Acceptance Criteria
The hemolysis rate is calculated from the measured absorbance values as follows: the absorbance of the test sample is corrected by subtracting the absorbance of the negative control, divided by the difference between the positive control absorbance and the negative control absorbance, and multiplied by 100 to express the result as a percentage. Under ASTM F756 and GB/T 16886.4 conventions, a hemolysis rate of five percent or less is generally interpreted as non-hemolytic, values between five and ten percent are considered marginally hemolytic and warrant further investigation, and values above ten percent indicate a hemolytic material requiring design or material modification.
Quality Controls and Laboratory Conditions
- Blood is drawn from healthy New Zealand white rabbits or informed human donors and anticoagulated with heparin or acid-citrate-dextrose, then used within a short defined window to preserve erythrocyte integrity
- Positive control (distilled water) must produce near-complete hemolysis, and negative control (saline or PBS) must remain essentially free of hemolysis for the test run to be considered valid
- Incubation temperature, contact time, centrifugation speed, and spectrophotometer wavelength are controlled and documented to ensure reproducibility
- All testing is performed under Good Laboratory Practice principles with calibrated equipment and traceable reference procedures
Applications
Hemolysis testing serves multiple functions across the medical device lifecycle:
- Regulatory submission: hemolysis reports generated per ISO 10993-4 and GB/T 16886.4 are required for NMPA registration in China, FDA 510(k) and PMA submissions in the United States, and CE marking technical documentation under the EU Medical Device Regulation for blood-contacting devices
- Material selection and screening: researchers compare candidate polymers, metals, coatings, and surface treatments during early design to eliminate hemolytic materials before investment in device development
- Change control: when manufacturers alter materials, suppliers, processing parameters, or sterilization methods, repeat hemolysis testing verifies that blood compatibility is maintained
- Batch quality control and supplier verification for tubing, membranes, and polymer granules used in blood-contacting assemblies
- Supporting documentation for biological safety evaluations, risk management files, and ISO 10993-1 biological evaluation plans
- Publication-grade data for academic studies of novel biomaterials, nanomaterials, and hemocompatible surface modifications
FAQ
What is a hemolysis test for medical materials?
It is an in vitro assay that measures the extent to which a medical material or device ruptures red blood cells and releases hemoglobin. The test result, expressed as a hemolysis rate or hemolytic index, indicates whether the material is blood compatible.
Which standards govern hemolysis testing?
The principal standards are ISO 10993-4 for the selection of blood interaction tests, ASTM F756 for assessing the hemolytic properties of materials, and GB/T 16886.4, the Chinese national adoption used for NMPA submissions. ISO 10993-12 governs sample preparation and extraction conditions.
What is the acceptance criterion for a non-hemolytic material?
A hemolysis rate of five percent or less is widely accepted as non-hemolytic. Results between five and ten percent are usually classified as marginally hemolytic and may require additional testing, while results above ten percent indicate hemolytic activity.
What blood source is used in the test?
Fresh anticoagulated rabbit blood is most commonly used because rabbit erythrocytes show sensitivity comparable to human cells and the source is well standardized. Human donor blood of a compatible type may also be used with appropriate ethical approval.
How much sample is required for testing?
Sample requirements depend on the test design. As a general guideline, material portions are prepared at extraction ratios defined by ISO 10993-12, such as 3 cm²/mL by surface area or 0.2 g/mL by mass, and at least three replicate units plus retention samples should be submitted in the final sterilized condition.
How long does the test take?
The laboratory incubation phase typically lasts two to four hours at 37 °C, while extraction-based studies may involve additional extraction time. Including blood collection, replicate analysis, control validation, and reporting, a complete hemolysis study is usually completed within one to two weeks after sample receipt.
What causes a material to fail the hemolysis test?
Common causes include leachable additives such as plasticizers or residual monomers, acidic or alkaline surface degradation products, improper cleaning, sterilant residues such as ethylene oxide, sharp geometric features causing mechanical cell damage, and oxidative corrosion products from metallic surfaces. Failure typically triggers formulation review, improved washing or sterilization validation, or re-testing after design modification.
Can hemolysis testing replace full hemocompatibility evaluation?
No. Hemolysis addresses only one blood interaction category. Depending on device contact category and duration, ISO 10993-4 may also require thrombosis, coagulation, platelet, hematology, and complement activation testing to complete the hemocompatibility assessment.