Assessment of factors impacting material-mediated hemolysis results in the ASTM F756-17 testing standard
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Contributing OfficeCenter for Devices and Radiological Health
Abstract
Blood-contacting medical devices, such as vascular stents, hemodialyzers, and circulatory assist pumps, are vital for patients suffering from a wide variety of disease conditions. To ensure the safety of these patients, FDA collaborates with device manufacturers and testing labs to develop standards for device hemocompatibility testing. The goal of this project is to evaluate and improve testing strategies for assessing material-mediated hemolysis (damage to red blood cells), based on existing procedures detailed in ASTM F756-17 (Standard Practice for the Assessment of Hemolytic Properties of Materials). The presence of hemolytic materials in blood-contacting devices can lead to hemoglobin being released from compromised red blood cell membranes. This free, unbound hemoglobin is vulnerable to oxidation reactions that can cause vascular and renal damage along with multi-organ dysfunction if left untreated. Therefore, a thorough understanding and regulation of the amount of hemolysis generated by blood-contacting medical devices is crucial for continued biomedical progress in this area. The ASTM F756 standard has been used for over 20 years to determine the hemolytic potential (% Hemolysis) of various materials, along with positive and negative control materials. % Hemolysis is calculated as the concentration of free hemoglobin divided by the concentration of total hemoglobin in the exposed blood. The experiments performed for this project are variants of the direct contact test as described by ASTM F756-17, in which the test material is in direct contact with the blood. The materials that were tested include 4 brands of nitrile gloves, Buna-N (nitrile rubber), latex, dimethyl sulfoxide (DMSO) at different concentrations, high-density polyethylene (HDPE), and phosphate buffered saline (PBS). Contract testing laboratories often use nitrile gloves or water as a positive control, and HDPE as a negative control material. Per the standard hemolysis testing protocol, 1 mL of diluted rabbit blood (1 g/dL total hemoglobin, pooled equally from 3 rabbits) was added to 7 mL of PBS, for a total volume of 8 mL in a 16 mL polystyrene round-bottom test tube containing the test material. Each test material was cut to maintain a surface area to PBS volume ratio (e.g. 6 cm2/mL), gently washed, and tested in triplicate. After incubating the samples for 3 hours at 37 °C (with gentle inversions every 30 minutes), the supernatant in each tube was obtained by centrifuging the PBS-diluted blood mixture. The concentration of free hemoglobin in the supernatant was determined using spectrophotometry, wherein cyanmethemoglobin reagent was added to the samples to convert all forms of hemoglobin into a stable form measured at a wavelength of 540 nm. The PBS blank samples were used to correct all % hemolysis calculations to account for background absorbance variability between the different pools of rabbit blood. Based on the data gathered from these tests, it was found that different brands of nitrile gloves produced different levels of hemolysis, thus the glove source should be validated and specified in any reports. In addition, increasing the concentration of DMSO caused corresponding increases in % hemolysis, which lends credence to the possibility of using DMSO as a reliable and versatile positive control in this test. HDPE was a consistent negative control material, as its hemolysis index was below 2%. As device manufacturers and testing labs sometimes deviate from the standard protocol, variations to the testing are being assessed to determine factors that may impact the hemolysis levels or may improve the standard (e.g. PBS/blood volume, incubation time of materials contacting blood, effect of dyes that may affect the measurement of free hemoglobin, and the concentration of red blood cells during the testing). For example, ASTM F756-17 states that the surface area of materials should be 21 cm2 or 42 cm2 (depending on thickness) and covered with 7 mL of PBS. But for testing small devices such as vascular stents, it would be beneficial to scale down the blood volume and use less test materials. Our preliminary data suggest that reducing the PBS/blood volume by a factor of 4 did not significantly affect the hemolysis test results. By measuring hemolysis levels in 45-minute increments up to 3 hours, we found that the rate of hemolysis varied for different materials, which supports the 3-hour study endpoint in the standard. We also determined that hemolysis caused by nitrile gloves was most likely due to water-soluble chemicals on their surfaces, since gloves washed in PBS for 48 hours under agitation at 37 °C had much lower hemolysis when compared to unwashed gloves. An interference test will be conducted by adding dye particles to the samples in order to determine their effect on the absorbance readings at 540 nm that inform % hemolysis calculations. In summary, the results of this study will help to improve the methodology used to test the hemolytic potential of medical device materials and provide the scientific basis for updating the widely used ASTM F756 testing standard.