|Archive - Jul 1|
Jury clears Abbott of design negligence
A jury recently delivered a verdict in favor of Abbott Laboratories (Abbott Park, IL) in a lawsuit involving the company's Beta hCG pregnancy test. In the lawsuit, the plaintiff claimed that false-positives from Abbott's AxSym Beta hCG blood-serum test, approved only for the early detection of pregnancy, led to an improper diagnosis of cancer and subsequent unnecessary treatment. The jury found that Abbott's test was not defectively designed, but that the University of Washington (Seattle) and various doctors involved were negligent in treating the plaintiff, and that Abbott was negligent in not sufficiently warning users about the possibility of false-positive results.
"We're very gratified that the jury upheld the safety and integrity of the AxSym Beta hCG test," says Brad Keller, trial lawyer for Abbott. "Used properly, this test is highly reliable and a very valuable asset in protecting the health of patients."
Abbott disagrees with the jury's finding regarding its negligent conduct in issuing warnings, and will appeal the verdict.
Copyright ©2001 IVD Technology
Researchers at the Serono Pharmaceutical Research Institute (Geneva) have developed a procedure that they believe will greatly improve the sensitivity of current tests used to detect abnormal prion proteins. These prions are thought to be the cause of such diseases as bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and new-variant Creutzfeldt-Jakob disease in humans.
The technology, termed protein cyclic amplification, makes it possible to amplify abnormal prion proteins up to several hundred times a day. The amplification enables a more-sensitive test for BSE and other prion diseases.
"The procedure mimics the replication of abnormal proteins in the body in 'fast-forward' mode, compressing years of real-life time into a few hours in the laboratory," says Silvano Fumero, senior executive vice president for research and pharmaceutical development at Serono.
Minute samples of abnormal prion protein were taken from the brains of scrapie-infected hamsters and mixed with large amounts of normal prion protein from disease-free hamsters. The combination resulted in rapid conversion of the normal prion protein into many aggregates of the abnormal prion protein. The aggregates were then treated with ultrasound. In the laboratory, this cycle of amplification can be repeated many times a day to produce several hundred times the quantity of abnormal prion protein currently available in the brain tissue of dead animals or humans.
The work completed by the scientists at Serono marks the first time that the abnormal prion protein has been cultivated in vitro. The result is a sensitive in vitro model to help researchers understand the underlying biology of prions, identify other factors that may be responsible for abnormal prion protein conversion, and locate potential novel drug targets for prion diseases.
According to the company, the research has a number of significant potential applications in the diagnosis of protein misfolding diseases. One of the most important is the identification of abnormal prion proteins in live cattle using tissues or biological fluids such as blood and in humans using spinal fluid or blood. The company also believes that the technology may be applicable to other diseases caused by protein misfolding, such as Alzheimer's disease.
Serono is currently exploring opportunities to license its technology to diagnostics companies. The licensing agreements would cover both animal and human tests using brain tissues, other tissue types, and biological fluids. The company has already filed a patent application for its technology
Copyright ©2001 IVD Technology
Three of the proposed projects are of particular interest to IVD manufacturers.
The other two projects are of interest to both manufacturers and their laboratorian customers.
"IVD manufacturers have been pushing NCCLS to do more for them," says Donald Powers, president of Powers Consulting Services (Rochester, NY) and a former member of the NCCLS board of directors. "And FDA has been asking NCCLS to develop standards that manufacturers could use in the abbreviated 510(k) process."
In fact, according to Louise Ciccarelli at NCCLS, FDA suggested the over-the-counter (OTC) pregnancy test kit and the immunochemical reagents and products projects. The quality assessment project was proposed by a member of the industry.
The OTC pregnancy kit project will address false-positive and false-negative test results, as well as issues that affect lay-user testing and interpretation. The consensus standard developed by this group will explain how manufacturers should document consumer complaints and it will also dictate what information should be included in package inserts to help minimize user error.
The quality assessment project will provide manufacturers of analyte-specific reagents with guidelines for production and offer recommendations for design control. It will also provide guidance for professionals who conduct these tests and recommendations for fair assessment of organizations that conduct quality programs.
The proposed project on specifications for immunochemical reagents will provide guidance for establishing reagent and product specifications, and for applying appropriate methods to maintain process control during the manufacture of direct antigen test products. According to NCCLS, the document will provide a framework and details useful for developing applicable quality systems for manufacturing these products using specifications and objective evidence consistent with user needs and intended use.
The purpose of the project on determining the clinical utility of genetic tests is to clearly define "clinical utility" in reference to genetic testing. The need for such a definition is in response to recent statements by several government agencies that genetic testing should not be performed unless a clinical utility has been clearly demonstrated.
"A definition will also determine whether a manufacturer decides to commercialize a test, whether FDA will approve it, and whether the Centers for Medicare and Medicaid Services will cover and reimburse for it," says Powers.
NCCLS says that a consensus guideline developed by this group will provide a means by which users can evaluate potential clinical utility in all phases of genetic testing.
Subcommittee members of the cellular immune functional assay group will craft a consensus guideline for performing assays for cellular immune function. Applications for these tests include HIV, cancer, transplantation autoimmune disease, and allergy. Assays that will be addressed are those to evaluate antigen-presenting cells, CD4T-cells, and CD8T-cell immune function.
Members of the subcommittees will have primary responsibility for drafting individual standards and guidelines, and for evaluating comments received during each phase of the consensus process prior to final approval by the NCCLS board of directors.
Copyright ©2001 IVD Technology
New and better diagnostic tests must be developed in order for the United States to be prepared for an act of bioterrorism. So says the American Academy of Microbiology (Washington, DC) in "Bioterrorism Threats to Our Future," a report published in February of this year.
"Rapid and accurate identification of the biological agent will be critical to implementation of control measures to treat those already infected and to contain the spread to additional individuals," says the report.
The report goes on to say that the optimal methods for detection include rapid immunodiagnostic assays (those that have a turnaround time of less than 24 hours). The assays should also be easy to perform and accurate for pre- and postcultural analysis of specimens. According to the academy, nucleic acid assays should also be developed.
Another method, analysis of the fatty acid composition of cell membranes, has shown promise in differentiating microorganisms that could be used in a bioterrorism attack, says the report. The specificity of this technique may enable rapid recognition of the various strains of possible bioterrorism agents.
Experts in the field of bioterrorism agree that the threat of a bioterrorist attack is not unthinkable in the United States. Signs indicating an increase in bioterrorist activity are on the rise. According to the Centers for Disease Control and Prevention (Atlanta) hoaxes involving the supposed release of anthrax have increased dramatically in this country over the past two years.
In other parts of the world, contemplation of a resort to bioterrorism or biowarfare is becoming more commonplace. Iraq acknowledges having mounted an ambitious and sophisticated bioweapons program. The Japanese terrorist group, Aum Shinrikyo, made at least nine attempts to aerosolize anthrax and botulism throughout central Tokyo before unleashing the nerve gas Sarin in that city's subway system. Reportedly, the group had previously tried unsuccessfully to obtain the Ebola virus to use as a bioweapon. And Russian defectors have documented, and former president Yeltsin has confirmed, the existence of an extensive Soviet bioweapons research, development, and production program.
"If a bioterrorism attack occurs, few if any diagnostic laboratories are prepared to promptly confirm diagnoses," says Donald A. Henderson, director of the Johns Hopkins Center for Biodefense Studies.
One manufacturer has developed a weapon in this battle against bioterrorism in the form of a rapid DNA analyzer. According to Cepheid Inc. (Sunnyvale, CA), its Microfluidic Integrated DNA Analysis System (MIDAS) can identify biowarfare agents in less than 30 minutes. Along with unprecedented processing speed, MIDAS is totally automated, requiring the operator only to inject a specimen into the machine.
MIDAS uses rapid polymer chain reaction (PCR) technology and a fluorescent detection method in a computer-controlled microfluidics circuit. Normally, PCR requires repeated cycles of heating and cooling to amplify the DNA reaction mixture. With microscale volumes, thermal cycling can be accomplished much more rapidly because of the reduced thermal inertia of the sample. In MIDAS, enough DNA for testing can be generated in 15 to 25 minutes, compared with approximately 90 minutes using conventional PCR instruments.
MIDAS performs all the complicated handling of reagents and samples; amplifies the DNA; and detects specific sequences of viruses, bacteria, or spores. The system then cleans and decontaminates itself and is ready for another assay. —Susan Wallace
Illustration By Marco Aguilera
Copyright ©2001 IVD Technology
For IVD manufacturers, this year's annual meeting of the American Association for Clinical Chemistry (AACC) promises to be even more interesting than usual. Already a key venue for manufacturers to meet with their laboratorian customers (and potential new customers), this year's gathering will feature a number of extraordinary industry-related events.
One such event will be the annual meeting of the AACC industry division, which this year launches its push to become a permanent division of the organization. The origins of the division are described by Mary Lou Gantzer, director of clinical and scientific affairs in the chemistry/
immunochemistry group at Dade Behring Inc. (Newark, DE) and president-elect of AACC, in this month's In Person column.
According to Gantzer, the high level of industry involvement in AACC is a rarity among similar professional societies outside the United States. Such involvement leads to useful interaction among AACC's traditional constituency of clinical laboratorians and the manufacturers that supply them. At the annual meeting, for instance, the industry division will sponsor seminars to train laboratorians on how to manage product evaluations and clinical trials on behalf of manufacturers—a key phase in the process of developing a new IVD product.
It's no accident that AACC is developing an industry division now. In recent years, industry researchers have taken an ever-greater role in elaborating and automating clinical laboratory tests, thereby creating a generation of products that are more reliable, less costly, and simpler to use. Without a doubt, the technologies being developed by IVD manufacturers are having a greater impact on the practice of clinical laboratory medicine than ever before.
For laboratorians, such industry involvement may represent a two-edged sword. Take, for example, another event anticipated for the AACC annual meeting—the announcement of new connectivity standards for point-of-care (POC) devices. As described in this issue's article by Ray Jones, "Connectivity of diagnostic systems: A better future is in sight", the standards that will be announced at AACC are the result of a year-long program undertaken by the Connectivity Industry Consortium, a group that involved dozens of manufacturers and suppliers to the industry. The new standards will make it easier for manufacturers to develop the next generation of near-patient testing devices, whose avowed purpose is to move testing out of the clinical laboratory and closer to the site of clinical activity.
Patients, physicians, and even hospital administrators may appreciate the benefits of such POC technologies, but it's questionable whether those who now perform testing in clinical laboratories see the world the same way. Convincing them to do so could be this year's most difficult sales job.
Copyright ©2001 IVD Technology
AACC's industry division looks to make it official
Mary Lou Gantzer, PhD, is director of clinical and scientific affairs in the chemistry/immunochemistry group at Dade Behring Inc. (Newark, DE) and president-elect of the American Association for Clinical Chemistry. She can be reached at firstname.lastname@example.org.
Leading up to this year's annual meeting of the American Association for Clinical Chemistry (AACC), which will be held in Chicago from July 29 through August 2, some of the association's members will be putting in a little overtime.
That's because this year marks the first official elections for officers of the association's industry division, a group that has had provisional status for the past three years and is now beginning its move to become a permanent division of the association. Ballots for the slate of candidates were expected to be sent to the division members in late June, and results of the election should be known this fall.
To find out more about the purposes of the AACC industry division, IVD Technology editor Steve Halasey talked with Mary Lou Gantzer, director of clinical and scientific affairs in the chemistry/immunochemistry group at Dade Behring Inc. (Newark, DE) and current chair of the provisional division. Gantzer has a long history of service to AACC in a variety of capacities, and is currently president-elect of the association.
IVD Technology: Where did the idea for an industry division in AACC originate?
Mary Lou Gantzer: A bunch of us in industry were sitting around at one of AACC's annual meetings and the idea just popped into our heads, "Gee, maybe we should have an industry division." Then it took probably another year and a half to two years of thinking and talking about it before we finally said, "Okay, we're really going to do this."
Was it hard to convince AACC that having an industry division was a good idea?
It wasn't hard. But the other AACC divisions are based on topics such as point-of-care diagnostics or therapeutic drug monitoring rather than on where the member works, so it was a unique concept that people had to think about. As they thought about it and talked with us, they became aware that industry-based members have concerns that nonindustry members don't have.
When I mentioned ISO 9000 several years ago, for example, other AACC members had no idea what I was talking about. Industry members knew because it was what they were embroiled in; every manufacturer was scrambling to get its quality systems in place and get its ISO certification.
Today, of course, people in clinical labs are very aware of ISO 9000. But it affected industry long before it affected them. So they gradually became aware that there are concerns that affect industry members that hospital-based people wouldn't appear to have any interest in.
The industry division changes AACC's constituency somewhat, doesn't it?
Compared to the traditional constituency, the membership that the organization has responded to primarily in the past, yes. You're correct.
How many members does the division have now?
It changes throughout the year, of course, as people join. The last I heard it was approximately 380.
Which is still a fairly small percentage of AACC's total membership?
Is that company membership or individual membership?
How many companies are represented in that group?
I'm not entirely sure. I would guess that the big six are definitely represented, and probably most of the major 25, and then a number of smaller companies also.
There are some nonindustry members, and it's interesting to try to figure out why. Last year, at least one physician from outside the United States attended the division meeting as a member. And yet he wasn't in industry. It might be that he was just exploring.
Education for Industry
What does having a division within AACC do for industry that wouldn't be or isn't being accomplished by other industry associations?
We're currently focused on educational programs that involve topics such as regulatory or test standardization issues. The IVD Task Force at AdvaMed would be interested in many of the same things. But there are certain programs that we may be able to mobilize faster because we don't have the same hierarchy to go through.
The major program that we organized last year was an audioconference on informed consent, in response to changes made at FDA that virtually none of us were aware of. That would also have been an excellent topic for AdvaMed to cover, but we were able to pull that program together very, very quickly.
We work with AdvaMed because it has a similar but somewhat different constituency. And a lot of times we'll partner and both support the same thing.
AACC has traditionally been very strong in providing educational activities for its laboratorian members. Is the organization equally committed to providing that for its industry members?
The impetus has to come from the industry members themselves. They have to identify what programs are important to them and let the division know what they want.
But because we have an industry division now, people in AACC are much more likely to know what concerns industry-based members might have. And that makes it more likely that they'll pass along information to the industry division and ask the division how it wants to proceed.
Who are the people within IVD manufacturing companies that are the most logical members of the industry division?
The people who are most likely to belong to the division would be R&D staff, regulatory affairs staff, clinical affairs specialists, quality control staff, manufacturing personnel, and possibly some people from marketing. And of course one of the requirements for membership in any of AACC's divisions is that you first have to be an AACC member.
Would the industry division appeal to medical professionals who are working with manufacturers in an R&D capacity, or laboratorians who are actually employed by industry?
Off the top of my head, the people I can think of who are most involved as consultants to industry are not members of the division. But if you were relatively new in the field and wanted to make contacts to look for that type of involvement with industry, the division might be a good way to do it.
Does having an industry division provide an interface between the professional community and industry that isn't available through other trade or professional associations?
Nobody's really approached us from that point of view. And maybe it's just that nobody's thought about it. Outside the United States, in organiza- tions similar to AACC, they're very sur- prised at the involvement of industry in AACC because it's something that does not typically happen in their professional societies.
Many, many years ago there was a sentiment that industry members weren't real members, but I think that has undergone a tremendous change. Everybody is very comfortable these days with the notion that people from industry are also valid scientists with a lot to contribute, and that industry and the laboratorian community share many of the same concerns.
Are there particular issues—such as the regulation of genetic testing—where there is likely to be a great difference of opinion between the professional laboratorian community and the manufacturing community?
The major conflicts are currently in how to regulate point-of-care testing and genetic testing. Many of our customers indicate that they want us to get CLIA-waived status for our technologies. But on the laboratorian side there's great concern about more and more tests becoming CLIA waived.
This is one of those areas that still requires a lot of discussion. Initially, in many people's minds, CLIA-waived status meant that the product would be approved for home use. But clearly there are many products that are truly simple to use and unlikely to result in wrong answers—which are the essential criteria for waived status—and yet they would never be suitable for home use. They're still professional-use products.
Even so, because people tend to think that as soon as something has waived status it's going to be used in the home, the trend toward creating more waived products still causes a lot of concern and conflict.
A Process for Permanence
To initiate the industry division you first had to get it established as a provisional division within AACC, is that correct?
Right, and there is a limit on how long a provisional division can exist. During that period the group has to demonstrate a number of things so that the organization knows it has the likelihood of being a viable division. Having bylaws and having had officers and some turnover in the leadership of the division are important considerations.
We're now in the process of sending the division's initial bylaws and slate of officers for a vote by the division's members. The division has to have those in place and functioning in order to apply for full status.
What is AACC's formal process for making a provisional division into a permanent one?
The division submits its application to become a permanent division to the divisions management group, which determines whether all the requirements for obtaining permanent status have been met. Then the AACC house of delegates and board of directors both look at the application. Ultimately, it is the house of delegates that gives final approval for a division to transfer from provisional to permanent status.
Is all of that procedure accomplished at the annual meeting or is it done in steps beforehand?
It's done in steps. The final step is the vote by the house of delegates, which only comes together at the annual meeting. For the industry division, we're anticipating that vote would take place at next year's annual meeting, because we have to have gone through our first elections and had our bylaws in place for a year.
Are the industry division members strongly committed to the division, or are they more standoffish at this point?
As with most divisions throughout the entire association, we have a small group of people who are very committed to the industry division and are willing to step up anytime we need something done. The majority of members participate in some programs, but otherwise have a wait-and-see attitude. And then there are some members we never hear from. In this respect, I don't think this division differs from other divisions.
Is there a formal program committee within the division?
Yes, but so far this year we have been concentrating on getting the bylaws written and the slate of officers elected.
Last year, we had a division workshop in conjunction with the Oak Ridge Conference, but we didn't have quite the turnout we had hoped for. We've also tried to do programs on AACC's "Division Focus Day," which is the Saturday before the annual meeting, but we discovered that a lot of industry members don't come in until Monday.
So we're still trying to find a time slot that would be acceptable within the annual meeting structure, and when we could have a division meeting in which more of the members would be able to participate.
When is the industry division meeting scheduled at this year's meeting?
It's on Monday at 10:00. That seems to be the day that is most open for industry members.
Of course IVD Technology is a sponsor of the Oak Ridge Conference, which has a focus on emerging technologies that seems especially appropriate for industry members.
I agree, and in the minds of AACC's meeting developers the Oak Ridge Conference is the meeting for industry. And yet, that's not where you see a lot of the industry members coming. Part of that may be related to the fact that in today's environment there is very limited funding for attending meetings.
But there is much more of an interest in going to the annual meeting, because it combines a large number of scientific and clinical presentations with an exhibit hall. And that's really important for industry members also; it's their chance to see competitors' offerings and to interact with the supplier companies in the OEM section.
How can AACC members get involved in the industry division?
For members of the association, the additional fee for any division membership is $15. Each of the divisions has a home page on the AACC Web site (http://www.aacc.org), and the officers and contact information are listed there.
The industry division home page lists the goals and objectives of the division. It also includes a survey of members asking what sector of industry they work in and what activities they're interested in. That's one way we're continuing to get more information about what our members are interested in.
Copyright ©2001 IVD Technology
Donald M. Powers, PhD, is president of Powers Consulting Services (Rochester, NY), an independent IVD regulatory and quality consulting firm, and convener of ISO/TC212/WG3. He can be reached via e-mail at email@example.com.
At its annual meeting, held last month in Dublin, Ireland, the International Organization for Standardization (ISO) technical committee on clinical laboratory testing and IVD test systems, ISO/TC212, continued to advance 15 important IVD standards and technical reports.
Eight of the standards will be harmonized with the Brussels-based European Committee for Standardization (CEN) standards in support of the European Union's IVD Directive. Seven documents approved by ISO member countries as draft international standards (DIS) will be circulated for a final DIS vote after one last revision to address voting comments. Once approved, these standards may begin to influence how the IVD industry is regulated.
This article looks at the major issues facing each of the working groups, and mentions some of the issues related to the ISO/TC212 standardization process. More countries seem to be leaning to the European standards-based regulatory model than to the more prescriptive U.S. regulatory model, so having a nucleus of international IVD standards in place might help tip the balance in favor of standards-based regulations. FDA is also moving toward recognizing international standards, albeit cautiously, in the interest of global harmonization.
Calibration Traceability. This series of standards includes ISO 17511, ISO 15193, ISO 15194, ISO 15195, and ISO 18153. Mandated by the IVD Directive, the series is intended to ensure that calibrators used in IVD systems are accurate. The directive requires that calibrators must be traceable to a higher-order reference material or reference method, if one exists.1 This requirement, the traceability and reference system standards, and the activities they have spawned at the National Institute of Standards and Technology (NIST) and other metrology institutes, have been discussed in detail in recent articles in IVD Technology. 2–6
The problem with the metrological concept of traceability is its focus on the accuracy of calibrator values, rather than on the accuracy of patient sample results. Accurate calibrators do not guarantee accurate patient results, and may even introduce bias into the patients' results. This phenomenon is termed a matrix effect and is caused by the influence of the sample and/or calibrator matrix on the measurement. The logic flaw in the directive cannot be corrected by a standard. However, constructive participation by a few manufacturers resulted in significant improvements in the traceability standard that will allow manufacturers some flexibility to avoid this pitfall. This is a good example of the critical importance of ensuring constructive participation by those who will be affected by a standard.
One warning to IVD manufacturers is in order: If the right steps are not taken to conform to ISO 17511, the traceability requirement could make current assays ineligible for the CE mark, and thus force them off the European market. An excellent guidance document for interpreting the calibration traceability standard is available on the Web site of the European Diagnostic Manufacturers Association (EDMA).7
Global Labeling. An initiative is under way to create an international standard for labeling requirements. Working Group 3 (WG3) has developed a preliminary "gap analysis" to identify conflicts and inconsistencies among existing national regulations. The project team, led by Kay Setzer of the United States, met in Dublin to examine the labeling regulations of the United States, Japan, and the European Union's IVD Directive, as well as the European labeling standards. Their initial review highlighted enough significant differences to conclude that an attempt to develop an international standard would be premature, so WG3 decided that its first priority would be to publish a report on the current state of IVD labeling regulations. The preliminary gap analysis will be shared with the Global Harmonization Task Force (GHTF) at its July meeting in Washington, DC; medical device labeling, including IVDs, is on the agenda.
Once a detailed gap analysis is completed, WG3 will explore ways to create a harmonized international standard, that is, one that can be used by the European Union to support the IVD Directive as well as by other countries around the world.
Blood Glucose Monitors for Self-testing. Since the first DIS missed approval by one vote, ISO 15197 is undergoing a second DIS vote by ISO and CEN-member countries. The negative votes were due to concerns over the minimum accuracy requirement and over the use of terminology inconsistent with the ISO vocabulary. The accuracy requirement was expected to be controversial, but the terminology issue caught the working group by surprise.
The degree of accuracy required for glucose monitoring continues to be debated, with the American Diabetes Association setting an aggressive goal of no more than 5% total error due to bias and imprecision.8 Manufacturers acknowledge this as a goal, but claim that state-of-the-art glucose meter technology cannot do better than ±15–20 mg/dl below glucose concentrations of 100 mg/dl. Manufacturers point to extensive clinical studies that have shown that the current generation of meters is effective in managing diabetes and argue that the role of standards is not to drive product improvements, but to establish minimum criteria for acceptable devices.
The project team, led by Peter Mueller of Germany, sought additional medical input and reached a compromise that tightens the requirement to ±15 mg/dl below 70 mg/dl. The results of the second vote are expected in the fall.
Oral Anticoagulation Monitors for Self-Testing. ISO 17593 is the second vertical product standard being developed by ISO. At the Dublin meeting, TC212 approved circulation of a committee draft—the first stage in the process of producing an ISO standard—based on a consensus of the working group.
The project team, led by Michael Spannagl, a physician from Germany, is attempting to ensure that a broad spectrum of expert medical input is obtained in setting the performance requirements. The participants in Dublin, evenly divided between diverse manufacturers and nonmanufacturers, are addressing technical challenges related to methods of standardizing measurements and evaluating the accuracy, trueness, and precision of self-testing measurements using unstable fresh blood samples. The working group expects to submit a DIS for an ISO vote in 2002.
Biological Stains. Originally developed by CEN, ISO/TC212 submitted EN 12376 to ISO members as a DIS under the terms of the Vienna Agreement between CEN and ISO.9 ISO/DIS 19001 passed without dissent although several suggested improvements were received as voting comments.
A project team led by Hans Lyon of Denmark evaluated the comments and recommended deferring action until the EN standard is up for revision. Otherwise, global manufacturers would have to deal with an ISO and a CEN standard on the same subject.
Performance Goals Based on Medical Needs. Manufacturers have requested a standard describing a systematic process for setting performance goals based on medical needs, which could be used to establish user requirements for IVD assays under design controls. Lawrence Kaplan of the United States was selected to lead the project.
After extensively debating the mer-its of several approaches, the working group decided that a consensus standard was premature. The main approaches were outcome studies, biological variation and physician interviews, each supported by strong advocates as the preferred approach. The working group decided to issue a technical report that would describe each approach and provide guidelines for its application. The report, ISO 15196, is being written for clinical laboratories, which can use its requirements to select IVD systems and for IVD manufacturers, which can use it to establish performance requirements for new assays. The final vote is expected by year end.
Validation of Manufacturers' Recommendations for User Quality Control. Originally proposed by U.S. manufacturers to provide a basis for an exemption from CLIA-mandated traditional quality control (QC) requirements, ISO 15198 is out for its first vote as a DIS. Led by Mario Werner, MD, of the United States, the project team developed a standard with broader application to all IVD systems. The current version incorporates industry input, but may not have sufficient concrete guidance to help a manufacturer decide whether its QC recommendations require formal validation studies and if so, how the studies should be designed. An auditor might conclude that any recommendations would require formal validation studies to demonstrate that the number of levels, the specific concentrations, and the frequency were sufficient to meet user requirements for an effective quality control procedure.
There is an attempt to grandfather existing systems relying on traditional QC, but traditional QC is not defined and each new or modified assay will have to face the question. QC recommendations based on application of the well-understood Levey-Jennings-Shewhart statistical principles should be justified, but explicitly exempted from formal validation.
Until recently the project suffered from lack of attention by manufactur-ers because they felt the standard was no longer needed. However, that realiza-tion came too late to stop its forward progress.
When an international standard comes into existence, especially one addressing a perceived important need, there is a strong possibility that it will become an expectation of customers, professional associations, and regulatory agencies. A possible scenario might be as follows. Once approved, FDA will list ISO 15198 as a recognized standard. Regulatory guidance documents and product standards will begin to reference it, and major laboratory customers, especially outside the United States, will ask to see a declaration of conformity. To lend support to this possible outcome, ISO 15198 has already been incorporated as a normative reference (i.e., an additional required standard) in the draft standard on anticoagulation monitoring devices.
The voting period for ISO 15198 ends September 5, 2001. Those interested in reviewing a copy of the standard should contact their national standards body or trade association. The U.S. technical advisory group (TAG) is developing the U.S. position on ISO 15198 and would welcome constructive input. U.S. TAG members may obtain a copy of the standard from NCCLS.
Labeling Symbols. The IVD Directive encourages manufacturers to use recognized graphical symbols when-ever possible. TC212 acts as a clearinghouse for symbols proposed for use with IVD products. Carolyn Jones of the United States is the liaison to ISO/TC210, the committee that develops standards for medical devices and maintains a compilation of ISO symbols approved for use with medical devices (ISO 15223).10
Figure 1. Symbols recommended by ISO/TC212 for use with IVD product labeling.
TC212 has forwarded three new symbols to ISO/TC210 for inclusion in ISO 15223. They are: "Contains sufficient for <X> tests," "For IVD performance evaluation only," and "Authorized representative in the European Economic Area." The proposed symbols are illustrated in Figure 1.
Companies and organizations that would like to propose a new symbol for IVD labeling should submit it to TC212 through their national ISO member organization or liaison, such as EDMA. U.S. manufacturers should submit candidate symbols to Carolyn Jones at AdvaMed, who will submit these proposals through the U.S. TAG.
Medical Laboratories—Particular Requirements for Quality and Competence. Working Group 1 developed ISO 15189 as an alternative to ISO/IEC 17025, which was developed for calibration and industrial testing laboratories.11 ISO/IEC 17025 has seen increasing use for accrediting clinical laboratories in some countries, and occasionally has caused laboratories to take extraordinary measures to maintain their accreditation. Thus manufacturers and some accreditation agen-cies, such as the College of American Pathologists, have supported an alternative that recognizes the unique nature and special requirements of clinical lab testing.
Originally titled "Quality Management for Medical Laboratories," ISO 15189 has been given a new title and will be revised to avoid confusion with ISO quality management standards. It will be submitted to ISO for final DIS vote after review by ISO/TC176, which oversees ISO quality management standards, and ISO/CASCO, which oversees ISO standards used for accreditation. These committees have to be satisfied that the work done by TC212 fits into the overall ISO strategy. A companion guidance document, ISO 22869, and a similar standard for point-of-care testing, ISO 22870, are also being developed by Working Group 1.
Medical Laboratories—Requirements for Safety. ISO 15190 is a well-written standard on clinical laboratory safety. The DIS was approved without significant controversy and is being revised to address the voting comments. The revised standard will be submitted to ISO as final DIS.
Next Steps. All three working groups have announced plans to meet in the fall to continue work on their projects. Working Group 1 plans to meet to complete the revision of ISO 15189, laboratory quality and competence. Working Group 2 will meet to address comments received on the two traceability standards. Working Group 3 will meet to continue development of the labeling technical report; to address voting comments on ISO 15198, validation of QC recommendations, and ISO 15197, blood glucose monitoring systems; and to move these standards to the final stage in the ISO process. The times and venues will be announced by NCCLS three months before the meeting.
Several standards should be circulated for a vote in the fall. These include ISO/CD 17593, anticoagulation monitoring systems; and ISO/TR 15196, performance goals. Manufacturers and other stakeholders are advised to obtain copies, review them carefully, and provide comments through one of the many routes available—such as via their national standards organization (e.g., ANSI/ NCCLS, DIN, AFNOR) or through a liaison organization (e.g., IFCC, WASP, EDMA), or a member of a liaison organization (e.g., AACC, CAP, BIVDA). U.S. manufacturers can also provide comments through AdvaMed, which is a member of the U.S. TAG.
1."Council Directive 98/79/EC of the European Parliament and of the Council of 27 October 1998 on In Vitro Diagnostic Medical Devices," Official Journal of the European Communities L331 [7 December 1998]. (Also available on the Internet at http:// www.edma-ivd.be.)
2.Donald M Powers, "Traceability of Assay Calibrators: The EU's IVD Directive Raises the Bar," IVD Technology 6, no. 4 (2000): 26–33.
3.Neil Greenberg, "Calibrator Traceability: The Industry Impact of the IVD Directive's New Requirements," IVD Technology 7, no. 2 (2001): 18–27.
4.Donald M Powers, "Calibration Traceability: NIST Workshop Takes a Major Step Forward," IVD Technology 7, no. 1 (2001): 26–30.
5.Hratch G Semerjian and Ellyn S Beary, "Mutual Recognition of Measurements: Its Potential Impact on International Trade in IVDs" IVD Technology 7, no. 4 (2001): 47–54.
6.Lothar Siekmann, "Measurement Traceability in Clinical Chemistry Analyses," IVD Technology 7, no. 1 (2001): 41–50.
7.European Diagnostic Manufacturers Association, "Interpretation of the CEN/ISO Standards prEN ISO 17511 and prEN ISO 18153 on Metrological Traceability of Values Assigned to Calibrators and Control Materials."
8.American Diabetes Association, "Self-Monitoring of Blood Glucose," Diabetes Care 1994; 17(1): 81–86.
9.In Vitro Diagnostic Medical Devices—Information Supplied by the Manufacturer with In Vitro Diagnostic Reagents for Staining in Biology, EN 12376 (Brussels: European Committee for Standardization, 1999).
10.Medical Devices—Symbols to Be Used with Medical Device Labels, Labeling, and Information to Be Supplied, ISO 15223 (Geneva: International Organization for Standardization, 2000).
11.General Requirements for the Competence of Testing and Calibration Laboratories, ISO/IEC 17025 (Geneva: International Organization for Standardization, 1999).
Copyright ©2001 IVD Technology
Existing technologies and forthcoming global standards promise to link POC devices, laboratory automation systems, and medical information systems via the Web.
Ray Jones is program director for medical information systems at Colorado MEDtech/RELA (Boulder, CO).
A central issue for point-of-care (POC) diagnostic devices in recent years has been the connectivity of individual devices to other diagnostic systems or information systems. Each individual vendor has generally implemented a connectivity solution specific to its own product. In certain cases, the vendor provides other data management products specific to the same clinical application—however, only with the same use-limited connection modality as the particular POC device. The effect of this rampant vendor specificity has been a quagmire of independent products, independent systems, and disparate interface approaches, which have restricted clinical or business value to the end-user.
The impact of this situation on the healthcare environment is huge, both in terms of high costs to end-users and lack of opportunity for vendors to formulate new products based on the integration of data that can provide better clinical value. The magnitude of the issue is indicated by the recent formation of the Point-of-Care Connectivity Industry Consortium (CIC), consisting of 27 member organizations. It is highly unusual for a new standards organization of this size to emerge in a very short time. Even more unusual is the group's aggressive charter to define appropriate standards particular to connectivity for POC diagnostic devices and to actually produce a final standard within one year. Also noteworthy is the fact that the organization will cease to exist when its standards-setting efforts are completed. The final standards generated will be integrated into the work of other ongoing standards organizations such as HL7. Nothing about this trajectory is typical. It is as if vendors threw up their hands in despair at the incompatibility quagmire and decided to do something about it themselves quickly and effectively.
|Figure 1. Relationships between primary types of POC device interface. Source: Connectivity Industry Consortium.|
An initial result of the CIC enterprise was adoption of a standard nomenclature for global connectivity solutions (see Table I.) As indicated in the table, the three primary types of interfaces identified in the CIC standard are an interface to a single device (the POC device interface—PDI), an interface to multiple devices using a concentrator (the access point interface—API), and an interface to an external data management system (the external data interface—EDI). The standard, now in draft form and subject to ratification by major standards bodies during 2001, defines in detail each type of interface. Interfaces can be connected in a variety of configurations (see Figure 1).
|PD||POC Device that performs blood chemistry and other measurements in the patient-care area.|
|DS||Docking Station, which may be used to provide a mechanical and electrical interface that supports the POC device. The docking station may use legacy mechanical-interface, connector, protocol, and power delivery methods. The docking station is optional.|
|PDI||POC Device Interface, which the POC device or its docking station uses to communicate its data (principally output) to an access point interface.|
|API||Access Point Interface, which specifies the interface (principally input) to an access point or concentrator.|
Access Point (or Concentrator), which consolidates the data from one or more devices onto another communication link, possibly using a different physical layer and transport protocol. This subsystem is optional. Examples of an access point are (other implementations are permitted):
|DMI||Data Manager Interface, which specifies the TCP/IP network interface to a data manager.|
|DM||Data Manager, which performs such functions as device data storage and forwarding, QA/QC, or other vendor-specific functionality.|
|EDI||EDI Interface, typically provided by the data manager, which is used to report results to a laboratory information system (LIS), hospital information system (HIS), or other system that is the final repository for the POC measurement results. The EDI interface typically uses HL7 over a network TCP/IP connection.|
Table I. Nomenclature for global connectivity of POC devices. Source: Connectivity Industry Consortium, "Universal Connectivity Standard for Point-of-Care," draft technical specifications (2001), http://www.poccic.org/documents.shtml.
A variety of technology options are available to implement the functionality described in the CIC standard. They fall into two broad categories—PC-based systems and embedded systems. POC device issues identified in the CIC standard are, in certain cases, similar to those involving larger diagnostic systems. Both PC-based and embedded systems are offered in multiple vendor options, and each category includes systems that can both support a POC diagnostic device and be scaled to support a diagnostic system and interface it optimally with a laboratory information system (LIS).
One approach consists of placing a local PC into the clinical environment. The PC supports integration of data from multiple devices or systems via either a multiport serial interface card or, as the CIC standard delineates, an Infrared Data Association (IrDA) interface. The PC may, if necessary, be operated from a medical isolation terminal in order to satisfy regulatory constraints. Software preloaded on the PC provides support for multiple device or system interfaces. In addition, HL7 interface software can be installed directly on the individual PC. A networked interface is supported by the PC; via Web server software loaded onto the computer, appropriate clinical and service applications can be configured either locally on the PC or remotely via the Web.
It should be noted that more than 60% of commercial Web servers use Apache Web server software running on Linux-based PCs or servers. The PC in such a configuration can run on either a Microsoft operating system (Windows NT) or a Linux-based PC solution. Linux systems are widely available from several vendors. Some vendors have recently introduced low-cost, small-footprint PCs that are especially well suited for the space-restricted applications in clinical environments.
Many diagnostic instrument systems are PC-based, allowing software components to be added to a core PC system that is part of the diagnostic instrument. In such cases, ensuring that the instrument design has adequate throughput and capacity to support the added functionality of the connectivity solutions becomes a critical issue. When those conditions are met, the PC approach is very economical. Plus, it offers the user the possibility of a software upgrade to an existing hardware platform without the need for major system upgrades. However, if the existing PC system is already constrained in supporting the overall diagnostic system, then the addition of new functionality can create problems for base-system performance. To resolve this particular issue, some vendors offer plug-in cards with their own internal processor in order to ensure that performance of the host system is compromised as little as possible.
Regulatory issues are also critical for PC-based systems. Often it is important to isolate the connectivity arrangements and information-system functions from the base diagnostic system, so that FDA approval of the two components with different regulatory requirements can proceed independently. If such isolation is viable, then use of the base PC in the diagnostic instrument is a good solution. However, to retrospectively add functionality in an isolated manner may prove difficult, in which case more-isolated system designs are called for. The PC-based approach has advantages and disadvantages (see Table II).
|Hardware technology is available, as are a growing number of software options.||Because it is not an isolatedapproach, other software loaded on the system by users could degrade performance.|
|Functional requirements of data standardization and remote access are supported.||Space requirements impose a moderate cost in the arena of POC devices.|
|The existing PC on which the diagnostic system is based may be utilized in some cases for the new functions.||A constantly evolving PC marketresults in different configurationsif system components are purchased over years.|
|Regulatory approvals of the connectivity solution may be constrained by use of an existing host PC system.|
Table II. The trade-off of advantages and disadvantages with a PC-based diagnostic instrument system.
Embedded Device Systems
Another approach to connectivity takes the form of a small, low-cost device embedded in the diagnostic instrument. This approach architecturally isolates connectivity from both medical device or system performance and the clinical user. Conceptually, the device supports multiple serial ports, with an Ethernet interface. Data-standardization software is downloaded over the Web and provides both a specific type of device interface software and a customized HL7 messaging engine configuration. A Web server is also provided to enable both clinical applications and service applications to reside either locally on the device or remotely on a browser accessing data gathered by the device. The embedded device functions as a generic platform, allowing the clinical user to upgrade the software over time in order to accommodate various device interface protocols, HL7 messaging-engine configurations, clinical applications, and service applications.
Recent advances in technology have brought technical and economic feasibility to the embedded-device approach. Just as the PC domain evolved in the 1990s from a stand-alone environment into a network-enabled, Web-based environment, so now the technology exists to support a similar trend with embedded systems. In the commercial market, the need for connectivity of RS-232-based devices has resulted in industries devoted to a networked-appliance concept. That is, any device or system with a serial port is enabled to provide connectivity over a network and has the potential to convert each device in a system into its own Web site. This trend has also reached healthcare, but with critical distinctions in use and regulatory requirements and in connectivity standards.
Like PC-based systems, embedded-device solutions to connectivity entail both appealing qualities and drawbacks (see Table III).
|Because it is an isolated solution, local users cannot access and change the configuration of the device.||The technology is conceptually new and unproven.|
|Independent of the PC market, it functions as a true medical device, with associated medical approvals if desired.||In PC-based diagnostic systems, the device may represent an additionalbox that is unnecessary if the host PC can provide the functionality.|
|Space requirements and costare minimized.||
All functional requirements related to data standardization and remote access are supported.
Table III. The trade-off of advantages and disadvantages with an embedded-device approach to connectivity.
PC-Based and Embedded-Device Systems Compared
Specific vendor product examples will illustrate existing technology options in the areas of PC-based and embedded-device solutions (see Tables IV and V). The systems presented in the tables are measured against the functional requirements of a connectivity solution. All three embedded-device vendor options support the concept of a network-based connectivity solution. With embedded-device systems, there are more types of business and technical models in place than with PC-based systems.
Functional or Business Criterion
|Product||Instrument Manager NT||530MPC; ResultNet|
|Business model||Software product (users provide PC hardware [Windows 95/98/NT based] and serial interface hardware).Turnkey systems, both with support and service plans.||Stand-alone hardware product and software product, both with support/service plans.|
|Technical model/functionality||Software supports 128 connections, configured as a Windows MDI application.||530MPC: Plug-in PC card with associated software.ResultNet: Connectivity software.|
|Serial to networked connectivity||Yes—Software interfaces exist for more than 80 vendor products. Separate purchase for hardware interfaces.||Yes—Software interfaces exist for 87 vendor products. Hardware models support one or two instruments per plug-in PC card.|
|Web server with clinical or service applications||No||No|
|HL7/ASTM protocol support, XML support||Yes, for HL7/ASTM; XML support is not discussed.||ResultNet software offers HL7/ASTM interfaces. Internal proprietary interface standard. XML offering pending.|
|Hardware connectivity options||User selects and specifies hardware; turnkey hardware platforms are available.||No; however, users may integrate other third-party hardware solutions into the PC host system and complete hardware/software integration themselves.|
|Software application support||Utilizes provided drivers; language not specified and not user accessible.||Extended Basic. Allows users to access and modify the base source code.|
|Cost||Instrument Manager NT software (three connection licenses): $4200.
Additional connection licenses: $1250.
|Single instrument: $1050.ResultNet: $1295.|
Table IV. PC-based connectivity products from two vendors compared with reference to functional requirements.
Functional or Business Criterion
|Product||Web Enabled Medical Gateway||SNI—Secure Network Interface; ResultNet||MSS100 device server|
|Business model||Core technology: customize to application.||Hardware product with ability to modify/augment software||Hardware product and software development kit|
|Technical model/functionality||Serial to Ethernet connectivity; specific device drivers available upon request.Embedded Web server.||Serial to Ethernet connectivity; multiple standard device drivers are available.Embedded Web server.||Serial to Ethernet connectivity; no available standarddevice drivers.Embedded Web server.|
|Serial to networked connectivity||Yes—Base technology with two serial ports, other options.||Yes—Standard product with one serial port.||Yes—Base product with one serial port, other options.|
|Web server with clinical or service applications||Yes—User may develop custom applications using standard C/C++, or CMED develops applications to a user specification.||Yes—User develops applications using Enhanced Basic.||Yes—User develops applications using interpreted C.|
|HL7/ASTM protocol support, XML support||Yes—Configurable toolkit supports site-specific HL7/ASTM messaging configurations.||No—Requires separate PC-based product (ResultNet).||No—Requires separate PC-based product from another vendor.|
|Connectivity options||Yes—Options include modem, wireless (including Bluetooth), A/D and D/A, and others as needed.||No—Users may select other third-party solutions and complete system integration themselves.||No—Users may select other third-party solutions and complete system integration themselves.|
|Custom software application support||ANSI Standard C/C++, CMED develops custom applications or supports user development. 16 MB minimum RAM.||Programmable in an EnhancedBASIC for communicationover the serial port with ananalytical device. Can be customized with DLL modules.||Interpreted C with limitations; software development kit. 512K minimum RAM.|
|Cost||Variable; depends upon hardware and software specification.||SNI: $1400. ResultNet: $1295.||$450 for single port interface; software development kit is free.|
No single technology option is appropriate for all solutions. As the examples in the tables suggest, a given application or particular business need may drive a customer toward either a PC-based approach or an embedded-device approach. Additional factors to consider in selecting connectivity approaches for POC diagnostic devices include the following.
PC-based systems offer the flexibility to choose a full-featured Web server; however, the user then becomes responsible for configuring the Web server and its associated application. Embedded devices offer Web server capability at less cost than PC-based systems, for both software and hardware (i.e., memory and disk requirements). So, for a Web application that is relatively small, an embedded device is a good choice. If the Web application is large, then a PC-based approach will probably be better.
HL7/ASTM solutions are typically offered only in a PC-based mode. Only one embedded-device system offers this capability.
From an overall cost perspective, the least expensive solution depends upon the application. It is important that "least expensive" be defined over the long term as well as in immediate reference. Just as with a POC device or diagnostic system itself, the capability of a connectivity product to be upgraded to enhance functionality should be considered at the time of initial product selection.
Future Market Drivers
In the light of existing product options, it is useful to refer to the original set of functional requirements for POC diagnostic device connectivity solutions and determine what factors will drive this market over the next several years. By assessing these driving forces, both users and vendors can make better technology decisions so that a solution appropriate for particular short-term needs will, even more importantly, remain viable over a long term of application.
Adaptability to Changing Interface Standards. The rapid launch of the CIC reflects the dynamic nature of standards. The search for a healthcare plug-and-play standard continues, with the rise of new standards bodies such as CIC being set against the current reality of many HL7 interfaces. The sardonic saying, "When you've seen one HL7 interface, you've seen one," illustrates the diversity of interfaces in use today. Given that the healthcare world has yet to see a truly plug-and-play solution, it is reasonable to assume that standards will continue to change. This assumption drives selection of the technology platform in the direction of one that is flexible and adaptable to changes in standards.
Support for Local Applications. Connectivity solutions hold promise for providing localized application software specific to the product and its clinical domain, as well as connectivity. The ability to analyze data locally at the device or system and then, following logic contained in the application, to move to a variety of decision support modes offers enormous new potential for healthcare. To be able to dynamically update this local software application in accordance with changing needs is exciting. Selection of a particular technology option thus should involve consideration of the software development environment offered on the connectivity solution. Is it expandable? Can an existing software application be ported to the connectivity solution? Does it follow programming standards? These are all critical questions that must drive vendor selection.
Support for Operation in Connected, versus Disconnected, Mode. In an ideal world there would be a network connection near each device, and that network connection would be fully operational all the time. But in healthcare environments, network accessibility is not always readily available, and it is important for the connectivity system to operate in an isolated mode. The system's ability to reconnect in a seamless manner and to transmit data over the network to the enterprise once the network connection has been restored becomes critical.
Support for Hardware Connectivity Options. In many cases, a network connection is the optimal interface point for connectivity solutions. Yet, in a POC environment with a potential home-healthcare setting, the availability of modem support is also crucial. Other types of wireless interfaces such as IrDA, 802.11, and Bluetooth offer prospective benefits in different product areas as well. Thus, the adaptability of the solution to different connectivity options is important.
Regulatory Constraints. Generic network appliance technology may not work well in a healthcare environment. The appropriate solution is often driven by regulatory issues related either to adding to an existing product or by incorporation into a new system design. Specific requirements for healthcare may be unique. Selection of an appropriate technology can depend upon access to expertise covering the medical and regulatory issues surrounding implementation of a connectivity system for a particular product or application.
The market forces just discussed indicate a need for a generic, expandable, POC-device connectivity solution. Providing a system that works today for one specific application area is not good enough. A more beneficial approach is to supply a Web-enabling platform that can support future connectivity needs, service applications, and clinical data systems.
Whether the connectivity system is PC based or an embedded device, the net result of the technology is a versatile platform for POC diagnostic devices that can be used in conjunction with an existing large-scale diagnostic system. The consistency of the technology base then allows vendors to quickly adopt a solution that applies to all of their products, whether for POC devices or large laboratory automation systems. Devices such as those portrayed in this article, when developed under standards as defined by POCCIC, represent a substantive response to the dismaying current state of device connectivity.
Copyright ©2001 IVD Technology
In membrane-based IVDs, hydrophilic constructions can improve test performance while maintaining manufacturing efficiencies.
|William G. Meathrel, PhD, is group leader for medical research and development, Herbert M. Hand Sr. is a medical product development scientist, and Li-Hung Su is a medical product development chemist at Adhesives Research Inc. (Glen Rock, PA). The authors wish to thank David Schaefer and the Department of Physics, Astronomy, and Geoscience at Towson University (Towson, MD) for technical assistance and for the atomic force microscopic imaging of hydrophilic adhesive coatings.|
Lateral-flow test strips are routinely used in clinical and other applications to provide convenient and simple analysis of many important chemicals.1–5 IVD devices incorporating such strips are used to detect such analytes as nutrients, hormones, therapeutic drugs, drugs of abuse, and environmental contaminates. In clinical test devices, biological fluids such as whole blood, plasma, serum, nasal secretions, sputum, saliva, urine, sweat, transdermal exudates, and cerebrospinal fluid may be analyzed for specific components that are important for diagnosis and monitoring. In addition, microbiological suspensions and tissues may be homogenized in compatible liquids and the fluid analyzed for specific components. Typically, the specimen fluid is deposited at the inlet port of a suitable IVD test strip and the sample fluid is drawn into the device by mechanical means such as vacuum or by capillary flow.
In aqueous biological fluids such as blood, urine, and sputum, strong intermolecular attractive forces create high surface tension.6 By comparison, the surface energy of solid substrates commonly used to make IVD devices is low. To achieve lateral flow and wicking of the liquid, the differential between the surface energies of the biological fluid and the solid substrates needs to be overcome—preferably without any mechanical assistance.
Two approaches can be used to improve the flow of biological fluids through a diagnostic device. One approach is to increase the surface energy of the substrate with various surface treatments. A second approach is to reduce the surface tension of the biological fluid.
This article provides information about novel hydrophilic coatings and adhesive constructions for IVD test devices. Hydrophilic constructions reduce the surface tension of biological fluids, thus enhancing the lateral flow and wicking of such fluids. Adhesives are formulated using polymer resins and surfactants to provide multifunctional bonding properties. Hydrophilic adhesives are formulated to be thermally bonded or pressure sensitive. The hydrophilicity of the surface is controllable through the chemical composition and the structure, concentration, and distribution of the surfactant in the adhesive coating.
The Flow of Sample Fluids
Figure 1. Schematic diagram of a typical lateral-flow diagnostic device.
Lateral-flow devices typically incorporate an inlet for receiving the biological fluid (see Figure 1). The sample inlet area or port may be proximal to a conjugate pad that holds reagents specific to the analytical test method. As the sample specimen flows from the inlet area through a reagent area, specific chemical reactions or a complex formation occur. The reaction product or complex continues to flow to a detection area where the analyte is monitored. Specimen fluids may continue to flow and be collected in an absorbent pad. Adhesive backings are typically used in the construction of such lateral-flow devices to support their various components, including the conjugate pad, the microporous membrane containing specific reagents, and the absorbent pad.
The time required for determining the concentration of a specific analyte is dependent on the flow rate of the fluid and the reaction rate between the analyte and a specific test reagent. The flow rate of the sample fluid is typically controlled by capillary flow through the microporous membrane.
Figure 2. Capillary rise in a cylinder or channel.
Controlling Surface Tension to Affect Flow. The surface tension of a fluid is the energy parallel to its surface that opposes extending that surface. Surface energy is the energy required to wet a surface. To achieve optimum wicking, wetting, and spreading, the surface tension of a fluid must be decreased so that it is less than the surface energy of the surface to be wetted.
The wicking movement of a biological fluid through the channels of a diagnostic device occurs via capillary flow. Achievement of capillary flow is a function of cohesion forces among liquid molecules and forces of adhesion between the liquid and the walls of the channel (see Figure 2). The Young/Laplace equation states that fluids will rise in a channel or column until the pressure differential between the weight of the fluid and the forces pushing it through channel are equal, as follows.7
In the equation, Dp is the pressure differential across the surface, g is the surface tension of the liquid, q is the contact angle between the liquid and the walls of the channel, and r is the radius of the cylinder. If the gravitation force is g, capillary rise is h, and the density of the liquid is r, then the weight of the liquid in the column is pr2ghr or the force per unit area balancing the pressure difference is ghr. Therefore,
For maximum fluid wicking through the channels of a membrane, the radius of the channel r and the contact angle q should be small, and the surface tension of the fluid g should be large.
Figure 3. Wetting of a fluid on a smooth, flat surface.
Wetting is the adhesion on contact between a liquid and solid.8 Figure 3 illustrates surface wetting of a fluid on a flat smooth surface. The theoretical explanation of this phenomenon can be described by the classic model known as Young's equation.9
gSV = gSL + gLV cosq
The diagram illustrates the relationship between the contact angle q and the surface tension of liquid gLV and solid gSV.8 When the contact angle q between liquid and solid approaches zero, the liquid will spread over the solid. For maximum wetting, the surface tension of the liquid must be less than or equal to the surface tension of the solid surface. This is the critical wetting tension of the solid.
The spontaneous process of wetting can also be derived from the differential between the work of adhesion and cohesion by substitution of the Dupré equation, as follows.8
WA – WC = gSV + gLV – gSL – 2gLV = gSV – (gLV + gSL)
This equation, where WA is the work of adhesion and WC is the work of cohesion, implies that spontaneous spreading will occur if the work required to separate the liquid-solid interface is greater than liquid separation itself. The Dupré equation can therefore be further derived by introducing the initial spreading coefficient S defined by Harkins, as follows.10
S = WA – WC = gSV – (gLV + gSL)
Since gSL is relatively small in comparison with gLV, the initial spreading coefficient term becomes:
S = gSV – gLV
Spreading is the movement of liquid across a solid surface. Contact angle is a measure of wettability. Spreading increases as the contact angle decreases until wetting is complete. Hence, spreading will occur spontaneously when S is greater than zero, which also indicates that the surface tension of the solid must be greater than that of the liquid, as shown in the above equation. From this initial spreading coefficient equation, it follows that wettability can be increased either by increasing the surface tension of the solid or decreasing the surface tension of the liquid.
Surface Treatments for Solid Phases
Surface treatments to increase the surface energy of a solid include both physical and chemical methods. Physical treatments used to increase surface energy include corona discharge, mechanical abrasion, flame, and plasma treatment.11 Chemical surface treatments include cleaning, priming, coating, and etching.
Corona discharge is the most widely used technique for surface treatment of plastics. During the treatment, the plastic surfaces are heavily bombarded with oxygen radicals at high-energy radiation levels. Consequently, the plastic surface undergoes either electret formation or chemical structural changes.12–15 Either result will improve the wettability of plastics.
Another commonly used method is wet chemical treatment. This treatment involves oxidizing the plastic surface through exposure to oxidizing acids such as a mixture of chromic acid and sulfuric acid.16
Effects of Corona Discharge and Chromic Acid Treatments. To quantitatively demonstrate the effects of corona discharge and chromic acid treatments on the surface energy of solid substrates, a study was conducted employing these techniques on six commonly used industrial plastics. The corona discharge treatment involved exposing the surface of each plastic to an electric discharge of 10,000–50,000 V at a frequency of approximately 500 kHz for approximately 5 seconds. The chromic acid treatment required the plastic surface to be flooded for 15 seconds with chromic acid, which was then removed by washing with distilled water; the surface was then rinsed with isopropanol and wiped dry. In each case, the contact angle was measured immediately after treatment.
Figure 4. Effect of surface treatments on contact angle.
As measured by the water contact angles for untreated and treated samples, both corona discharge and chromic acid treatments were effective in improving the wettability of the surfaces (see Figure 4). The decreased water contact angles indicate an increase in the surface energies of the treated plastics that would similarly enhance their wettability for biological fluids.
Chromic acid was most effective on plastics with functional groups, such as polycarbonate and polyester panels. Corona discharge was most effective in increasing the surface energy of the polyolefin films (polypropylene and high-density polyethylene). The corona discharge treatment method could improve the water contact angle by orienting surface electrical charges or by introducing oxygen on the surface. Either mechanism will increase the polarity of the plastic and thereby increase its surface tension. Consequently, the contact angle q will be smaller due to reduced difference in surface tension between the plastic gSV and the water gLV. A disadvantage of corona discharge treatments is the instability of the treatment. Corona treatment substrates should be coated soon after treatment.
Surfactants in Adhesives
The use of surfactants to lower the surface tension of a fluid is well known.17–19 A variety of anionic and nonionic surfactants can be used to lower the surface energy of an aqueous fluid (see Table I).
|Sodium 2-ethylhexyl sulfate||Branched||Anionic||232|
|Sodium lauryl sulfate||Linear||Anionic||288|
|Sodium nonylphenol ether sulfate||Aromatic||Anionic||498|
|Polyalkyeneoxide modified heptamethyltrisiloxane||Linear siloxane||Nonionic||600|
Table I. Physical properties of selected surfactants.
To determine the properties needed for an adhesive with both bonding and hydrophilic attributes, a substantial amount of research was conducted to understand the interrelationships between polymers and resins used to make adhesives. As described below, the use of surfactants in coatings and adhesives was studied to determine their effects on wettability, fluid flow rate, and adhesive properties. Each surfactant was formulated into a base adhesive at different concentrations. The water contact angle was measured to determine the effect of the surfactant on reducing the surface tension of the water.
This research has resulted in the development of proprietary technology that meets the need for a hydrophilic adhesive to reduce surface tension of fluids and improve flow rate in diagnostic devices.
Preparation of Hydrophilic Adhesives. Hydrophilic coatings and heat-sealing and pressure-sensitive adhesives were prepared in the laboratory. Dissolution of polymeric resins occurred in organic solvents and was followed by measurement of solution solids and viscosity over a period of several hours of mixing.
The surfactant was introduced into the liquid polymer mixture after dissolution of the resin. Gentle agitation for several minutes was sufficient to achieve homogeneity. Hydrophilic pressure-sensitive formulations were prepared by the introduction of a surfactant into liquid acrylic adhesive solutions and emulsions followed by gently mixing until dispersed or dissolved.
Film Preparation. Hydrophilic films were prepared in the laboratory using coating apparatus. The dried coatings had an approximate thickness of 0.0005 to 0.001 in. The hydrophilic adhesive coatings were protected with a film release liner of low surface energy.
Effect of Surfactant Type on Water Contact Angle
Various surfactants were formulated into emulsion pressure-sensitive adhesives. These hydrophilic coatings were tested for surface wetting using deionized water. The sessile drop method was employed to measure the contact angle of liquid water on the surface of the hydrophilic thin film.
A further study of surface wetting was conducted for two hydrophilic heat seal adhesives, HY-5 and HY-10, which were formulated using polyester resins and the anionic surfactants sodium nonylphenol ether sulfate and sodium dioctylsulfo succinate, respectively. Water was dropped onto the surface of the adhesives and the contact angle was measured as a function of time.
Figure 5. Effect of surfactant concentration on contact angle.
Results. With increasing surfactant concentration, most test samples exhibited a similar trend of decreasing contact angle (see Figure 5).
Sodium nonylphenol ether sulfate exhibited the most effective reduction of water surface tension at all three surfactant concentrations used in this study. Of the surfactants evaluated, it had the highest molecular weight among the anionic surfactants. It is proposed that the lower molecular weight anionic surfactants have better solubility into the adhesive matrix so that there is less surfactant concentrated at the water-adhesive interface.
The nonionic surfactant nonylphenol ethoxylate exhibited little effect on the contact angle of deionized water. This may be due to its higher molecular weight and the lower water affinity of the hydrophilic group compared with anionic surfactants. In addition, the nonylphenol group enhances its absorption onto the polymer surface.
Polyalkyeneoxide-modified heptamethyltrisiloxane (PMHS), also a nonionic surfactant, reduced the water contact angle on the adhesive surface compared with nonylphenol ethoxylate. PMHS has a siloxane polymer backbone rather than a hydrocarbon backbone, which accounts for its lower surface energy. In addition, PMHS also has a lower molecular weight than nonylphenol ethoxylate, which enhances its mobility within the adhesive matrix.
The linear structure of sodium lauryl sulfate would improve its solubility into the adhesive so that its effect on the adhesive surface is less than that of sodium 2-ethylhexyl sulfate.
Figure 6. Plot of water contact angle surfactant concentration for hydrophilic heat-seal systems.
The effect of surfactant concentration on the surface wettability of the coatings prepared using different polymeric resins is shown in Figure 6. Polyamide, ethylene vinyl acetate, and polyester resins were formulated with sodium dioctylsulfo succinate. The contact angle is high when no surfactant is present in the coatings since the polymeric resins are hydrophobic. By increasing the surfactant concentration, the surface becomes more hydrophilic and lower water contact angles are observed, indicating significant surface wetting. At very high surfactant concentrations the wetting effect can be enhanced or attenuated depending on the surfactant and its compatibility with the polymer matrix.
The spreading behavior of water on the surfaces of the HY-5 and HY-10 coatings is as follows. Initially there is rapid spreading of the drop as it contacts the surface of the film. The contact angle decreases quickly to less than 10º. Equilibrium is established within 30 seconds to one minute. This spreading behavior is typical of hydrophilic coatings, heat-seal adhesives, and pressure-sensitive adhesives.
Flow Rate in Microfluidic Channels
Experiments were conducted to determine the effects of hydrophilic coatings and adhesives on the flow rate of distilled water in a microfluidic channel. Following a screening of the effects of different types of surfactants on contact angle, the most effective surfactants were formulated into adhesive tapes used as a cover for a microfluidic device. For this set of experiments, a hydrophilic pressure-sensitive adhesive was formulated using concentrations of sodium nonylphenol ether sulfate ranging from 0 to 6%.
Figure 7. Diagram of test device with a microfluidic channel formed using hydrophilic adhesive tapes.
The microfluidic channel, measuring 20 cm x 10 µm x 30 µm, was molded into a polystyrene device (see Figure 7). The hydrophilic tape was used to close the channel to create the microfluidic device. Distilled water was placed in one of the terminal wells and the time for the water to flow through the channel was measured.
Results. When no surfactant was added to the adhesive, water did not flow through the channel. With increasing concentrations of surfactant, however, the rate of water flow through the microchannels increased while the contact angle decreased.
The increased flow rate of water can be attributed to the reduction of water surface tension, in accordance with the principle of capillary rise. The water will advance farther when its surface tension is close to that of the capillary material, which is now determined by the hydrophilic adhesive cover. At surfactant concentrations greater than 4%, however, the rate of flow ceases to increase because the concentration exceeds the critical level. Additional surfactant on the surface of the adhesive does not further reduce the surface tension of the fluid and may become autophobic.20
To investigate the distribution and mobility of surfactants in hydrophilic coatings, chemical surface analysis of the hydrophilic coatings was performed using infrared spectroscopy via attenuated total reflectance (ATR). The FTIR-ATR spectra of the hydrophilic heat-seal adhesive HY-10, which contains sodium dioctylsulfo succinate, were recorded.
Results. Primarily absorption peaks for sodium dioctylsulfo succinate were observed in the fingerprint region of the spectrum at wavelengths which correspond to the methyl stretch at 2967 cm–1 and sulfur-oxygen vibration at 1049 cm–1 (see Figures 8 and 9).21 The HY-10 film surface was then washed and dried and another spectrum of the washed surface was taken in proximity to the original measurement. The disappearance of the 2967 cm–1 methyl stretch and the S-O vibration at 1047–1049 cm–1 confirmed the loss of surfactant as a result of the washing procedure.
Figure 8. FTIR absorption spectrum of the hydrophilic coating HY-10 (C-H stretch region from 2980 cm–1 to 2840 cm–1).
|Figure 9. FTIR absorption spectrum of HY-10 (S-O region from 1065 cm–1 to 1025 cm–1).|
Infrared spectra of the coatings confirm the increase in surfactant concentration on the surface (see Figure 10). The prominent peak at 2958 cm–1 in the ATR is assigned to the C-H stretch of a CH3 group on the surfactant in the hydrophilic adhesive and is used to monitor surfactant accumulation on the surface. A plot of absorbance of the C-H stretch as a function of concentration of surfactant at 0%, 1%, 5%, and 10% shows a flattening resulting from the surface saturation by the surfactant (see Figure 11).
Figure 10. Infrared spectrum of HY-10 (C-H stretch region from 3000 cm–1 to 2800 cm–1).
|Figure 11. Methyl absorption versus surfactant concentration for the hydrophilic coating HY-10.|
Surface Topography by Atomic Force Microscopy
The surface topography of the hydrophilic coatings was observed using atomic force microscopy (AFM). Hydrophilic tapes were mounted onto 1-cm-diam metallic stubs and imaged in the tapping mode. This mode of imaging has several advantages over direct-contact-mode imaging. Lateral forces that are prevalent during contact-mode scans are eliminated. Additionally, the tapping mode provides a nondestructive method for the imaging of soft samples. Importantly, phase images obtained using the tapping mode can give additional information concerning the mechanical and adhesive properties of the sample surface.22
All samples were initially scanned in air. The hydrophilic coating HY-10 was rinsed with deionized water for 10 seconds, then wiped dry with a paper tissue. The sample dried overnight and was imaged the next morning.
Figure 12. Atomic force microscopy images of hydrophilic coatings formulated with various percentages of the surfactant sodium dioctylsulfo succinate. Coatings shown have 0% (a), 5% (b), and 10% (c) surfactant.
Results. The AFM images show enrichment of the film surface at the film/air interface with increasing amount of surfactant introduced to the adhesive formula. The AFM image of the coating containing no surfactant shows a relatively smooth, flat surface (see Figure 12a). Transformation is observed when 1% surfactant has been incorporated into the adhesive coating, where raised features are observed on the film surface. Surface topography is increased with 5% surfactant (see Figure 12b). With 10% surfactant, the surface appears to be smoother due to saturation (see Figure 12c).
Figure 13. Effect of surfactant on surface properties.
A comparison of surface peak height and contact angle as a function of surfactant concentration is shown in Figure 13. When no surfactant is present the surface of the coating appears to be smooth with little topography. At 0% additive, the corresponding effect of surfactant concentration on the water contact angle of the film surface shows that the contact angle is 70–80°, which indicates that the surface is hydrophobic. With increasing surfactant in the adhesive formulation, surface roughening was observed. At 1% surfactant additive, the height of the cross-sectional features increased from 0 nm to approximately 5 nm. The cross-sectional analysis reveals a build-up of the surface features related to increased additive. With the addition of surfactant comes amelioration of surface wetting and the contact angle decreases in a nonlinear fashion until total wetting of the surface is achieved at concentrations of 5% and 10%.
AFM images were recorded before and after water washing of the hydrophilic coating containing 10% surfactant to observe the effect on the surface topography. The images showed the surface topography of the unwashed hydrophilic coating with surfactant concentrated on the surface. After washing with distilled water, surfactant is solubilized and removed from the surface leaving a more rugged topography.
When it comes to the adhesives used in their products, IVD manufacturers are requiring high performance, quick dynamic wettability, and good durability. Adhesive manufacturers must control and balance these factors to achieve hydrophilic adhesives that lend themselves to ease of manufacturing.
However, when choosing an adhesive for use in an IVD device, it is important to consider all of the following: the components to be bonded and the surface energies of those components, the type of fluids to be wicked and the surface tensions of those fluids, chemistry of the reagents, compatibility of adhesive with all components, use conditions, shelf life, and packaging.
Membranes used in lateral-flow devices are typically hydrophobic polymers with low surface energy. Consequently, these components are not compatible with aqueous biological fluids. To overcome the low surface energy of such membranes, surface-active agents are often added to increase their wettability and consequent wicking ability. However, the addition of such surface-active agents may decrease the ability of the membrane to bond or to retain proteins that are critical to the performance of the device. In addition, surfactants added to the membrane can reduce test sensitivity by causing extensive spreading of reagent bands.
The use of hydrophilic coatings formulated by mixing surfactants with a polymer resin can enhance the wicking of biological fluids into or through an IVD device. These constructions bond device components and provide a hydrophilic surface that can reduce the surface tension of a biological fluid. Reduced surface tension allows rapid transfer of the fluid from the inlet area to the reagent area of an IVD, thus reducing the time for analysis. More-efficient transport of fluid to reagent also allows for use of smaller sample volumes, thereby enhancing design flexibility and making it possible to employ alternative collection sites for increased patient comfort. Finally, these products can reduce the risk of chemical interference by providing a wicking surface that allows increased separation between the sampling port and test reagents. All of these benefits allow for more-efficient manufacturing processes with the potential for reduced product cost.
Hydrophilic coatings and pressure-sensitive and heat-sealable adhesives may be used in a variety of IVD products, including capillary-flow, lateral-flow, microfluidic, and electrophoretic devices.
The unique proprietary technology developed for hydrophilic coatings and heat-seal and pressure-sensitive adhesives can be custom-formulated to provide bonding surfaces that enable wetting and spreading of fluids into IVD devices. The selection of adhesive and surfactant additive and its concentration are critical to device performance.
The experimental results provide evidence of hydrophilic enhancement of adhesive tape constructions that can be used for IVD devices. To ensure compatibility, however, different polymer systems will require judicial selection of surfactant. Surfactant properties such as molecular weight, charge type, and chemical structure must be considered in selecting the best hydrophilic adhesive construction.
Hydrophilic coatings formulated with surfactants have proven effective in improving wicking and increasing flow rates in IVD devices. The properties of the adhesive and surfactant are selected to be compatible with the diagnostic device and its reagent chemistry. As with any application, the suitability of a tape construction must be determined by the device manufacturer.
1. SM Rosen, "Biomarkers of Chemical Exposure: A New Frontier in Clinical Chemistry," IVD Technology 2, no. 3 (1996): 22.
2. RA Esposito et al., "The Role of the Activated Clotting Time in Heparin Administration and Neutralization for Cardiopulmonary Bypass," Journal of Thoracic and Cardiac Surgery 85 (1983): 174–185.
3. CA McDonald et al., "A Rapid One-Step Colored Particle Lateral-Flow Immunoassay for the Detection of Group 1 Streptococcal Antigen Extracted Directly from Throat Swabs," in Proceedings of the 93rd General Meeting of the American Society of Microbiology (Washington, DC: American Society of Microbiology, 1993), 507.
4. C Huang and E Fan, "One-Step Immunochromatographic Device and Method of Use," U.S. Pat. 5,712,172.
5. A Pronovost and J Pawlak, "One-Step Urine Creatine Assays," U.S. Pat. 5,804,452.
6. N Vallespi i Salvado, VV Shah, and DA Werkems, "Surfactants in Pressure-Sensitive Adhesives," Surface Coatings International 4 (1999): 181–185.
7. Walter J Moore, Physical Chemistry, 3rd ed. (New York: Prentice-Hall, 1962), 730.
8. WA Zisman, "Influence of Constitution on Adhesion," Handbook of Adhesives, 2nd ed., ed. Irving Skeit (New York: Van Nostrand Reinhold, 1977), 33–64.
9. T Young, Philosophical Transactions of The Royal Society 95 (1805): 65.
10. WD Harkins, The Physical Chemistry of Surface Films (New York: Reinhold, 1952).
11. PH Winfield, AF Harris, and AR Hutchison, "The Use of Flame Ionization Technology to Improve the Wettability and Adhesive Properties of Wood," International Journal of Adhesion and Adhesives 21, no. 2 (2001): 107–114.
12. JM Evans, "Nitrogen Corona Activation of Polyethylene," Journal of Adhesion 5 (1973): 1–7.
13. JM Evans, "Influence of Oxygen on the Nitrogen Corona Treatment of Polyolefins," Journal of Adhesion 5 (1973): 9–16.
14. DK Owens, "Mechanism of Corona-Induced Self-Adhesion of Polyethylene Film," Journal of Applied Polymer Science 19 (1975): 265–271.
15. DK Owens, "Mechanism of Corona- and Ultraviolet-Light-Induced Self-Adhesion of Polyethylene Terephthalate," Journal of Applied Polymer Science 19 (1975): 3315–3326.
16. WP Townsend, "Metallization of Plastics," Handbook of Adhesives, 2nd ed., ed. Irving Skeit (New York: Van Nostrand Reinhold, 1977), 841.
17. MJ Rosen, Surfactant and Interfacial Phenomena (New York: Wiley, 1978).
18. THF Tadros, Surfactants (New York: Academic Press, 1984).
19. AC Clark and J Pialet, "New and Improved Waterborne Systems," Adhesives Age 42, no. 9 (1999): 33–40.
20. WA Zisman, "Influence of Constitution on Adhesion," Handbook of Adhesives, 2nd ed., ed. Irving Skeit (New York: Van Nostrand Reinhold, 1977), 46.
21. TA Thorstenson and MW Urban, "Surface and Interfacial FTIR Spectroscopic Studies of Latexes, IV: The Effect of Surfactant Structure on the Copolymer-Surfactant Interactions," Journal of Applied Polymer Science 47 (1993): 1381–1386.
22. A Doring, J Stähr, and S Zollner, "Atomic Force Microscopy: Micro- and Nano-Mapping of Adhesion, Tack, and Viscosity," in Proceedings of the 23rd Annual Technical Seminar: Pressure-Sensitive Tapes for the New Millennium (Northbrook, IL: Pressure-Sensitive Tape Council, 2000), 213–222.
Copyright ©2001 IVD Technology
A variety of membranes have characteristics that can make them useful immobilization substrates for molecular diagnostic applications.
Kevin D. Jones, PhD, is the manager for diagnostic technology at Whatman International Ltd. (Maidstone, Kent, UK). He can be reached via e-mail at firstname.lastname@example.org.
With the mapping of the human genome, the future of many areas of diagnostics, including rapid testing, appears to be inextricably linked to nucleic acids. The College of American Pathologists designates nucleic acid diagnostics as belonging to the field of molecular pathology, which is defined as covering the diverse areas of disease predisposition, therapeutic suitability, and organism identification. The current market for nucleic acid diagnostics is perhaps more than $2 billion, with a predicted growth rate of better than 20% over the next three years. By 2003, the projected market for nucleic acid diagnostics will approach 20% of all diagnostic tests performed.
Figure 1. Double-stranded DNA immobilized on a glass-fiber matrix. Photo Courtesy Whatman International Ltd.
A significant challenge facing the fledgling nucleic acid diagnostic industry is the transformation of laboratory tests now performed by researchers into commercial diagnostics that can be used by nonspecialist technicians in doctors' offices, and even by consumers at home. This challenge looms especially large for developers of point-of-care diagnostic assays.
A nucleic acid test requires that a series of processes be completed: sample collection, sample preparation, amplification (if required), and detection. Discussion of all aspects of these processes is beyond the scope of a single article. The limited aim of this three-part series therefore is to highlight the materials and methods for nucleic acid immobilization that are available to developers of rapid assays, with a particular focus on how various target materials can be immobilized on a membrane substrate (see Figure 1).
Figure 2. Structures of DNA (a); RNA (b); and peptide nucleic acid (PNA) (c).
Four types of nucleic acid probes can be immobilized onto a solid phase for a rapid assay: large sections of DNA, small DNA (including cDNA), RNA, and peptide nucleic acid (see Figure 2). The methods for immobilizing these different molecules have some similarities, but differences in molecular structure and size make the methods substantially different. This series is restricted to outlining typical procedures to immobilize to a variety of substrates the shorter, more commonly used DNA probes. (The immobilization of large sections of DNA onto solid phases is no longer routinely used for diagnostic tests.).
The term membrane encompasses a wide range of potential substrates that can be used for the immobilization of nucleic acids. They include traditional cast membranes such as nitrocellulose and nylon, and also innovations such as ceramic or track-etched membranes and the types of substrates used for microarrays. Whatever the substrate, there are only a limited number of ways that nucleic acid can be attached. Most common are physical adsorptive processes or chemical linking processes (including ultraviolet [UV] or covalent methods).1 In addition, a nucleic acid probe can be assembled on the membrane, a novel technique developed by Affymetrix Inc. (Santa Clara, CA).
Nucleic acid probes can be linked to membranes in several ways. In order to appreciate the potential of the various attachment techniques, it is helpful to be familiar with the characteristics of available membrane materials and their application in nucleic acid testing. Future installments in this series will review the generic linking methods, with a concentration on covalent linkage, and consider their potential manufacturability in process-scale production.
The original membrane used for nucleic acid immobilization was nitrocellulose, selected by E. M. Southern for his Southern blotting method.2 This technique involves transferring DNA fragments, produced by restriction endonuclease digestion of DNA, from an electrophoresis gel to a nitrocellulose membrane. A great deal of published material describes methods to achieve nucleic acid binding to nitrocellulose, usually by means of physical adsorption. Researchers have also developed several newer, more-specific binding techniques that extend the utility of this substrate, including those using aptamers or a poly-T tail.
The principal advantages of nitrocellulose are its ready availability and familiarity. However, it is fragile (unless cast on a polyester backing) and has a lower binding capacity than some other membranes. The use of nitrocellulose membranes with radioactive methods of signal detection is well established, but other materials may offer advantages in applications where the sensitivity of the detection technique is lower than for radiolabels.
Recently, nylon has been promoted as a substrate for nucleic acid binding owing to its greater physical strength and binding capacity, and the wider range of available surface chemistries offered, which optimizes nucleic acid attachment. Immobilization on nylon membranes can be performed via physical adsorption, UV cross-linking, or chemical activation. Immobilization on nylon has been demonstrated to be more durable during repeated probe stripping than immobilization on nitrocellulose.3,4 Nylon membranes have also been used in methods to detect DNA by colorimetry, fluorometry, and chemiluminescence.5
The high background typically observed with nylon membranes is their principal disadvantage. This may be due to a nonspecific binding of the sample or detection system, or to some natural property of the membrane. Nitrocellulose has a lower binding capacity and is weaker than nylon, but it has far lower background for most detection systems.
Other membrane materials have also been used successfully, though they have never achieved significant market penetration. They offer some advantages for particular applications, but their general performance has not been able to match that of nylon or nitrocellulose. These alternative membranes have been investigated for their nucleic acid binding capability. The most widely used are made of charge-modified polyvinylidenedifloride, which binds nucleic acids through interaction of the positively charged surface groups with the phosphate backbone of the acid.
With any membrane type, nucleic acid binding protocols must be optimized for each particular membrane, because various manufacturers will use different formulations and manufacturing processes. This can result in a variety of surface chemistries, for instance, which can lead to different binding and subsequent detection characteristics. Membrane manufacturers often make available protocols to help develop the optimal system for each membrane. The balance of properties between nitrocellulose and nylon means that both products have found widespread use for nucleic acid immobilization within the IVD industry.
In many areas of molecular biology, microparticles are widely used as attachment substrates for nucleic acids. Such particles offer a range of surface chemistries to suit the linking technique required (e.g., silica, oligo dT, agarose, and latex with amine). Often, as in the case of a direct-incubation or direct-reading assay, the microparticles replace the membrane.
However, in some applications the use of microparticles can complement the membrane; the nucleic acid is attached to a large particle that becomes entrapped in the membrane structure or to smaller particles that are trapped by well-defined membranes (e.g., track-etched or ceramic membranes). This use of microparticles is well known in the rapid immunoassay industry, as indicated by the "boulders in a stream" approach with cast membranes and by particle-capture immunoassays defined-pore products such as track-etched membranes (TEMs).6,7
The use of microparticles as the adsorption phase offers significant advantages over direct attachment of the nucleic acid to a membrane. Perhaps foremost, methods for linking nucleic acids to microparticle surfaces have been well characterized, and all reactions can be carried out in solution phase under a wide range of conditions. With membranes, on the other hand, the chemistries or reaction conditions that can be employed without damaging the membrane are often limited. Also, once the nucleic acid is immobilized, the particle can be passivated easily and without much increased likelihood of introducing nonspecific interactions. By contrast, the activation and subsequent surface passivation of membranes can introduce undesirable chemistries across the entire membrane surface. Such surface chemistries can interfere with the sample or detection system.
The initial immobilization of a nucleic acid on a microparticle would therefore enable a more-controlled procedure than is possible with immobilization directly on a membrane. Subsequent entrapment of the microparticle in either a traditional cast membrane or a TEM would then allow the assay to be run and detection to occur.
As array technologies become increasingly important in diagnostics, their price is falling to levels that could make them competitive with other techniques rather than being limited to research laboratories. A number of different techniques and substrates for manufacturing such arrays now exist. No longer limited to the original flat-film arrays such as those assembled on glass slides or plastic films, the technology has been extended to membrane-based, three-dimensional, and flow-through arrays.
Traditional membranes such as nylon and nitrocellulose have been used in the production of macroarrays, but their use in microarrays has been limited because of the problem of spot resolution. Because these membranes exhibit lateral wicking characteristics, the label tends to spread from the point of application. This has been a limitation in the production of very high density arrays. However, some microarray systems use a membrane or porous substance that has been cast onto the surface of a glass slide. And there are certain membranes that have no lateral wicking characteristics. They are typically TEMs or anodic membranes, such as Anopore, whose use in DNA microarrays has been demonstrated by Pamgene (Den Bosch, The Netherlands). Such membranes are not limited with respect to spot resolution.
The use of novel membranes in microarrays presents interesting opportunities. All membranes offer much greater surface area than flat sheets. In traditional cast membranes, the ratio of the within-pore surface area to the top surface area of the membrane is about 200:1.
The most extreme example of this characteristic is an anodically oxidized membrane, one in which almost the entire membrane structure consists of pores, with very little material forming the walls. Anopore has a surface area ratio of approximately 500:1. This means that the amount of probe that can be immobilized per unit area is 500 times greater than would be possible on a glass surface. The benefit is either high sensitivity (more immobilized capture probe results in a higher level of material captured) or a higher-density array (the same amount of capture probe can be immobilized in a smaller area).8
Figure 3. Scanning electron microphotograph of nitrocellulose, a typical cast membrane (a); Cyclopore, a track-etched membrane (b); and Anopore, a ceramic membrane (c).
These membranes could be envisaged as the ultimate multiwell plates, each pore becoming in effect a well in which an individual reaction can be completed. Anopore provides a pore density of 108 pores per square centimeter.
If the application technology and bioinformatics could handle the data, probes for the whole human genome could be laid down in an area covered by a single blood spot from a finger prick. With improved detection technology and the capacity for single-copy detection, the potential of these membranes is enormous (see Figure 3).
As mentioned above, some types of membrane have no lateral-wicking capacity. Another key difference between traditional cast membranes and these novel materials is that pore size distribution for the newer-style membranes is extremely well controlled. Without lateral wicking, the material applied can travel only through the transverse pores. Anodically oxidized alumina (e.g., Anopore) will bind nucleic acids predominantly under chaotropic conditions. The process is similar to that widely used in binding nucleic acids to silica in preparation columns. TEMs, by contrast, have no direct binding capacity. However, their utility in the trapping of microspheres enables a wide range of surface chemistries and binding techniques to be used.
While not strictly speaking a membrane, powdered silica or alumina on a support matrix has been used to bind nucleic acids (e.g., coated microarray slides). These materials will bind nucleic acids very strongly under chaotropic conditions. Through silane modification, covalent linkage would be possible. However, the relative fragility of these ceramic materials may limit their potential for use in certain applications.
No single substrate material is best for all applications. Each offers some advantages. A manufacturer's choice of substrate will most likely be determined by the requirements of the application.
Whatever the nature of the substrate, there are only a few attachment techniques, including covalent linkage (for example, through silanation of the substrate), photolinkage, physical adsorption, and assembly of the probe on the substrate. The manner in which the nucleic acid is bound is critical for the performance of the assay. Binding via the bases is potentially detrimental to performance, as the bases are responsible for any hybridization events that occur. If the bases are immobilized on the membrane surface, or constrained in such a way that free movement during hybridization is restricted, the binding is not optimal. The effect is far more significant for shorter probes than for long sequences. Thus, any linking techniques that result in attaching bases to the membrane surface (e.g., UV cross-linking) or large-scale attachment of the probe to a flat surface (e.g., traditional physical adsorption) may be less than ideal because of the reduction in sensitivity and specificity of the hybridization. In extreme cases, the immobilization technique can render the immobilized nucleic acid useless. The optimal form of binding, therefore, is to end-link the nucleic acid to the solid phase, ideally using a controlled-size spacer to ensure that the nucleic acid is free to interact with the sample.
The next installment of this series presents an overview of immobilization techniques available to developers of nucleic acid tests. The advantages and disadvantages of each are reviewed, with reference to various membrane substrates and assay performance potential.
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2. EM Southern, "Detection of Specific Sequences among DNA Fragments Separated by Gel Electrophoresis," Journal of Molecular Biology 98 (1975): 503–517.
3. A Dubitsky and J Defiglia, "Stripping of Digoxigenin-Labeled Probes from Nylon Membranes," BioTechniques 19, no. 2 (1995): 210–212.
4. K Noppinger et al., "Evaluation of DNA Probe Removal from Nylon Membrane," BioTechniques 13, no. 4 (1992): 572–575.
5. A Dubitsky, "DNA Arrays on Nylon Membranes," Lab Automation (abstract), June 1998.
6. LB Bangs, "The Latest Applications of Microspheres," in The Latex Course 2000 (Indianapolis: Bangs Labs, 2000).
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8. KD Jones et al., "Comparative Study of Glass Slides versus Microporous Ceramic Slides for Nucleic Acid Arrays" (poster presented at the 32nd Oak Ridge Conference, Boston, May 5–6, 2000).
Copyright ©2001 IVD Technology