An MD&DI January 1997 Feature Article
TESTING
Hemocompatibility: Not All Devices Are Created Equal
Current standards for hemocompatibility are too broad; they don't take into account the unique demands of the many types of blood-contacting medical devices.
Sharon J. Northup
Blood-contacting devices such as needles, cannulae, blood containers, and dialyzers all have very different usage requirements. Of course, the hemocompatibility concerns for each will differ as well. For example, a needle may reside in the bloodstream for only a short time; a cannula may be implanted for much longer. The primary hemocompatibility problem for a needle would be hemolysis, the destruction of red blood cells as a result of chemical interaction with the needle material. For a cannula, however, a more likely hemocompatibility problem would be thrombogenicity, or clotting, which can be caused not only by chemical interaction, but also by the flow rate of the blood.
There are standards available for testing whether medical devices can be used safely with blood. But these standards are mostly horizontal, addressing broad groups of products rather than specific devices, and many blood-contacting devices are not adequately covered by them.
The few vertical, or device-specific, standards that have been written are not enough. They also often do not take into account variations in the way a particular device is used. For example, if an anticoagulant is used with a device in some procedures and not in others, this will change the testing required to establish hemocompatibility for all possible procedures.
To create specific standards that will ensure the hemocompatibility of all types of medical devices will require concerted effort on the part of biomedical specialists not only to develop appropriate tests, but also to bring the tests into widespread use.
CURRENT HORIZONTAL STANDARDS
The standards developed by Technical Committee 194 of the International Organization for Standardization (ISO) are recognized as the minimum requirements for biocompatibility testing of all medical devices today. After approval at the international level, regulatory authorities in participating nations have adopted the ISO standards as written or with minor modifications. For example, ISO 10993-1, "Guidance on Selection of Tests," was adopted by FDA in 1995 with modifications for intrauterine and some other types of devices.1,2 Both ISO and FDA standards recommend hemocompatibility testing for medical devices intended for direct or indirect blood exposure.
These horizontal guidelines group medical devices for testing according to route of exposure. For example, percutaneous circulatory support systems, extracorporeal oxygenators, and apheresis equipment are all classified as externally communicating devices, and are therefore given the same testing recommendations. But because they are used for very different lengths of time, they present different risks for air emboli at the blood-air interface and protein denaturation from foaming. Apheresis equipment, for example, is used for hours, whereas circulatory support systems must be designed for days of use. Also, anticoagulants are used for only part of the treatment with circulatory assist devices, but are used throughout therapy with oxygenators or apheresis equipment. Obviously, anticoagulant use will dramatically affect measurement of thrombogenicity.
The current standards describe some hemocompatibility tests in detail. For example, the assay for hemolysis has been described in the research and clinical literature and has been developed as a standard method for medical device testing by the American Society for Testing and Materials (ASTM) in a draft annex to ISO 10993-4, "Selection of Tests for Interactions with Blood" and in several vertical standards for specific medical devices.
But depending on the application, various factors can affect the results of hemolysis testing, and the standards do not account for these possible cases. For example, all the standards that describe hemolysis recommend measuring hemoglobin in a spectrophotometric assay using absorbency at 540 nm; none mentions that the absorption spectrum may shift in the presence of various chemicals.3 Ethanol, propylene glycol, polyethylene glycol 400, dimethylsulfoxide, and dimethylacetamide will shift the absorption spectrum of hemoglobin. The hemolysis standards are also silent about possible interference from fixatives. In the presence of small amounts of formaldehyde or glutaraldehyde, the red blood cell membranes are cross-linked (tanned) and thus are less easily ruptured, making the test results misleading.
Future revisions of the standards for hemolysis and other types of hemocompatibility testing should include sources of variation and methodologies for ascertaining their effect on the interpretation of the assays.
One of the most important factors affecting the results of hemocompatibility testing is how the device contacts the blood. For example, although needles and cannulae serve similar functions, their methods of exposure to blood are quite distinct.
Needles are designed for fast penetration and short exposure time. Most often metallic, they require sharp points and lubricious barrels for nearly painless entry into the vessel. The points may be beveled concavely to increase sharpness. The barrels are lubricated and, in some variations, wall thickness is reduced to enhance tissue penetration.
In-dwelling cannulae, by contrast, are designed to reside in the body for much longer. Made of plastic materials, they have blunt tips that minimize intravascular irritation. Cannulae generally have thick walls to prevent kinking and occlusion through muscular contraction. Their barrels are seldom lubricated because this would lessen skin adherence and increase the likelihood of microbial infection.
The standards recommend only hemolysis testing for both needles and cannulae. But because thrombogenicity is a common failure mode for cannulae, these devices should undergo an implant test for thromboresistance as well. Measurements might include blood flow rate, duration of flow, and cellular deposits on the surface or downstream from the cannulae.
Using a thrombogenicity test for cannulae would have the added benefit of taking into account the reaction of muscles to the devices. When the vascular system becomes irritated by a foreign object, the smooth muscles contract vigorously. This constriction could result in collapsing or kinking of a cannula, which would be evident by the loss of blood flow through it. The strength of the cannula must match or exceed that of the muscular constrictions at the vascular access site.
CURRENT VERTICAL STANDARDS
As noted above, there are some vertical standards for device hemocompatibility: blood container standards are good examples. Blood collection sets are covered in ISO 1135-3, "Blood-Taking Set," and ISO 3826-4, "Plastics Collapsible Containers for Human Blood and Blood Components." 4,5 Both documents list requirements for cell culture cytotoxicity, short-term intramuscular implantation, hemolysis in vitro, delayed contact sensitization, intracutaneous irritation, pyrogenicity, and sterility.
The customary measurements for a whole-blood container are total hemoglobin, hematocrit, and cell counts. The preferred hemolysis test is a static assay occurring under the usage conditions of 21 days of storage at 4°–8°C with citrate phosphate dextrose solution or 42 days with citrate phosphate dextrose adenine solution. Common measurements on containers for red cell concentrate are erythrocyte adenosine phosphate (ATP), lactate, and glucose as indices of an energy source and utilization. Red cells may also be evaluated microscopically for morphological changes.
Depending on whether the patient whose blood is in the container has been treated with an anticoagulant, these tests may not go far enough to ensure device safety. If an anticoagulant has been used, measurements of the stability of the anticoagulant over the product's shelf life—for example, the pH and concentration of each additive—should also be made.
Containers for platelets would also require assays that are specific to the products they hold, such as pH, aggregation, morphology, glucose consumption, lactate accumulation, and cell counts.
COMPLEX DEVICES
Standards for complex or invasive devices are particularly in need of development. For some of these devices, there is no consensus among existing standards on what are appropriate tests. For example, there is disagreement on the testing of hemodialyzers. The French and German standards require hemolysis testing on an eluate from the hemodialyzer,6,7 whereas FDA guidelines require the following tests:
- Cytotoxicity in vitro.
- Hemolysis.
- Complement activation.
- Cell adhesion
- Protein adsorption.
- Whole-blood clotting time for thrombogenicity.
- Pyrogenicity.
- Genotoxicity.
- Acute systemic toxicity.
- Intracutaneous injection.
- Implantation.
- Guinea pig maximization for delayed sensitization.
- Subchronic toxicity.
- Thrombogenicity by examining platelet and fibrinogen turnover, thrombus formation, and resulting emboli.8
The FDA requirements do not delineate the biological system and exposure protocol that are necessary for interpretation of the measurements. A whole-blood clotting time assay, for example, may not be meaningful because heparin anticoagulants are used during hemodialysis procedures.
Complement activation has been included in the guidelines to lessen the potential for dialysis-induced chronic lung disease.9 Yet measuring only complement activation as a potential source of inflammation ignores the role of platelet activation in the initiation of free radicals that could contribute to chronic lung disease. Also, some studies have shown that some membrane materials used for hemodialyzers actually absorb complements, making the measurement irrelevant for those materials.
IMPLANTS
Medical devices that are implanted in the vascular system offer an even more challenging task for creating specific testing standards. Hemocompatibility for implantable devices is highly dependent on the material, shape, function, and location of the implant. Many of the currently marketed vascular graft materials are hemolytic. Although it is not clear whether this is due to the specific material or the air-blood interface, clinicians have used this property to seal the grafts before implantation.10 The hemolytic property rapidly creates a tribological surface between the materials of construction and the biological environment by deposition of fibrin and other proteins and globulins, thereby enhancing the biocompatibility of the device and preventing seepage of blood immediately after surgery. For grafts, then, the hemolysis assay has little predictive value for problems encountered in clinical use.
THE WORK AHEAD: VALIDATION
The main difficulty in creating specific standards is the amount of work required to validate tests and thus make them available for widespread use. Numerous assays for hemocompatibility have been and continue to be developed in research laboratories, but widespread adoption of the tests is often stalled because they are not rigorously validated. Validation establishes the credibility of a candidate test through intra- and interlaboratory assessments and database development.
Intra- and interlaboratory assessments are used to determine the sensitivity, selectivity, and predictive value of an assay.11 For a test to be valid, it must be adequate in terms of these three factors. Sensitivity is the percentage of positive results, and selectivity is the percentage of negative results. Predictive value is the percentage of correct test results, and is correlated with prevalence, the ratio of positive results to all substances tested. The predictive value of a test may be correlative or mechanistic.
Validating hemocompatibility assays will require reference materials, databases, and reference laboratories. For a particular test, a reference material is a characterized material or substance that yields a reproducible result when tested. A standard reference material is a universally available material that is characterized in regard to elemental composition, formulation, structure, phase and phase distribution, and impurity level in a prescribed physical form including, but not limited to, shape, surface character, and electrical charge. Biomedical scientists are a long way from developing a consensus on the characteristics of reference materials and establishing a repository of standard reference materials with defined biocompatibility and universal availability. In the early 1990s, the ISO task force on sample preparation and reference materials sought to prepare a list of reference materials and standard reference materials, but could find only a few.12
Other requirements for validation—consensus on what constitutes validation of a new material, a central repository for test performance data, an established network of reference laboratories capable of carrying out interlaboratory assessments, and an understanding of the mechanisms of blood biocompatibility—will also require a committed effort.
CONCLUSION
A review of relevant medical device standards shows that the available hemocompatibility assays are not always predictive for specific devices. In some cases, the assays may not be sensitive or selective enough. The community of biomedical specialists needs to recognize these limitations and work toward creating a framework for validating assays that will establish standards for hemocompatibility that are applicable to all blood-contacting medical devices.
REFERENCES
1. "Biological Evaluation of Medical Devices, Part 1: Guidance on Selection of Tests," ISO 10993-1, EN 30993-1, Geneva, International Organization for Standardization (ISO), 1992.
2. "Required Biocompatibility Training and Toxicology Profiles for Eval-uation of Medical Devices," Blue Book Memorandum G95-1, Rockville, MD, FDA, Center for Devices and Radiological Health (CDRH), Office of Device Evaluation, 1995.
3. Reed KW, and Yalkowsky SH, "Lysis of Human Red Blood Cells in the Presence of Various Cosolvents," J Par Sci Technol, 39(2):64–68, 1985.
4. "Transfusion Equipment for Medical Use, Part 3: Blood Taking Set," ISO 1135-3, Geneva, ISO, 1986.
5. "Plastics Collapsible Containers for Human Blood and Blood Components," ISO 3826-4, Geneva, ISO, 1988.
6. "Medical Surgical Equipment, Single Use Sterile Hemodialyzers and Hemo Filters," French Standard NF S 90–302, Paris, Association Française de Normalisation (AFNOR), 1990.
7. "Extracorporeal Circuit Hemodialysis Dialyzers and Blood-Line Systems Made of Plastics, Requirements and Testing," DIN 58 353, Part 3, Berlin, Deutsches Institut für Normung e.V. (DIN).
8. "Guidelines for Premarket Testing of New Conventional Hemodialyzers, High Permeability Hemodialyzers and Hemofilters," Rockville, MD, FDA, CDRH, Bureau of Medical Devices, March 1992.
9. Moinard J, and Guenard H, "Membrane Diffusion of the Lungs in Patients with Chronic Renal Failure," Eur Respir J, 6(2):225–230, 1993.
10. Vann RD, Ritter EF, Plunkett MD, et al., "Patency and Blood Flow in Gas Denucleated Arterial Prostheses," J Biomed Mat Res, 27:493–498, 1993.
11. Goldberg AM, Frazier JM, Brusick D, et al., "Framework for Validation and Implementation of In Vitro Toxicity Tests: Report of the Validation and Technology Transfer Committee of the Johns Hopkins Center for Alternatives to Animal Testing," J Am Coll Toxicol, 12:23–30, 1993.
12. "Biological Evaluation of Medical Devices, Part 12: Sample Preparation and Reference Materials," ISO 10993-12 (draft 4), Geneva, ISO, 1992.
Sharon Northup, PhD, is a managing associate with the Weinberg
Group, Inc. (Washington, DC).
