In my previous blog post I discussed sample representativeness and provided recommendations on how test articles should be prepared, outlining details that may be overlooked in the process. With various considerations that need to be accounted for, it is difficult to cover all topics in one blog post. As such, this post will focus on another aspect of biocompatibility testing: ensuring that a sufficient number of test articles are provided to the testing facility. This post will focus on the extraction-based tests.
To determine the necessary number of test articles, we can turn to ISO 10993-12. Section 10 of the standard contains a table outlining the different extraction ratios depending on the size and geometry of the test article. The table provides different ratios, using surface area or weight to determine extraction volume. Selecting the most appropriate ratio is the first step in determining the quantity of devices needed for testing.
Many biological tests require a minimum volume of test extract to ensure the appropriate dosing for the given study. Note that the amount necessary for dosing is not dependent on the device, but rather on the specific test itself. Therefore, submitted samples must meet a minimum surface area—or in some cases weight—requirement, as the extraction volume is determined through this parameter. Certain tests also require multiple extracts (e.g., polar and non-polar) in order to account for all possible residuals released from the test article. Furthermore, some tests require multiple exposures/dosing steps for the study, each requiring fresh extracts to be prepared. Therefore, even if one device were large enough to fulfill the surface area requirement, multiple devices/test articles would still need to be provided, as one cannot be used for multiple extracts. This is because a full extractable profile is needed in the test system to mimic patient exposure.
For example, testing for material-mediated pyrogenicity requires only a polar extract. For this study, if the test article is large/heavy enough, one may be sufficient for testing. However, the extraction-based sensitization study typically utilizes both a polar and non-polar extract. Moreover, the study requires multiple exposures that each need a separate, unused test article for each extraction. Thus, while one complete test article may satisfy the requested surface area requirement, in the case of sensitization testing with a polar and non-polar extract, each of which requires three separate extractions, a minimum of six test articles would need to be provided to the testing facility. For instances where acquiring necessary quantities of test articles is exceptionally difficult, there may be the possibility of sharing extracts between tests (e.g., between irritation and sensitization tests, since they both have a polar and non-polar extract). However, this option is reviewed by the testing facility on a case-by-case basis, as additional coordination is typically necessary.
As noted in ISO 10993-12 and the FDA guidance document on the use of ISO 10993-1, the surface-area-based approach to sample preparation is recommended. However, if there is an instance where determining surface area would be incredibly difficult or impossible, a weight-based approach can be applied. Non-solid (e.g., gels) or irregularly shaped samples (e.g., sponges) are examples of instances where the use of weight could be acceptable. Using weight as the basis for extraction volume calculation also has its own minimum requirements for the same reason that ensuring enough test extract is available for dosing for the studies. However, since surface area is the recommended unit of measurement, weight should only be used in the event that surface area is impossible to calculate or a rationale can be provided showing how the use of weight in determining extract volume can be considered worst case.
An example would be when determining extract volume for a braided suture, where using surface area may provide a much higher volume than its weight. It should be highlighted that it is not uncommon for regulatory bodies to request further clarification in the event the weight-based approach is used. Weight may seem like an acceptable alternative if a sample is irregularly shaped, but with the technology available today, such as with the assistance of computer-aided design (CAD) or other software, determining accurate surface area measurements of even irregular devices can be possible. Therefore, it is recommended to use the necessary resources to define and provide the surface area wherever feasible.
Another important aspect to emphasize in sample preparation is that if the sample has areas that are considered non-contacting, then the surface area (or weight) of these portions should not be included in the total calculations and should be excluded from the extractions. Including non-contacting measurements would increase the extract volume, thereby potentially diluting the extract and underchallenging the relevant portions of the test article. Additionally, potential extracts of the non-contacting portions may cause unforeseen failures, providing false positives.
The accurate surface areas or weights of the samples help define the quantities needed for testing. Additionally, they also allow for accurate testing per ISO 10993-12, making sure the samples are properly prepared and tests are conducted with appropriate extracts. It should be noted that while it is not required, providing more samples than the calculated minimum is generally a good practice. It allows the test facility to use some samples in determining ideal preparation methods for complex samples while ensuring there are still sufficient quantities left for testing. Furthermore, on the rare occasion when a retest is required or complications occur during sample preparation, having extra will help avoid potentially long delays associated with having to manufacture, process, and send more samples. It is important to remember that when additional test articles are needed, the testing is paused with no guarantee that it will resume immediately when the test articles are received, potentially resulting in further delays.
Sample preparation can be one of the more complicated parts of the biological testing process. Therefore, taking time to address these considerations preemptively can ultimately save time and resources. With such a complex topic, it is impossible to cover all the nuances of determining sample size. Should any uncertainties or questions arise during the process of determining necessary samples for testing, contact the Nelson Labs Expert Advisory Services. They can be reached at [email protected] and will be happy to assist.
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