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BEYOND CLINICAL DIAGNOSTICS

The RAMP reader and clinical test kit.

Point-of-care (POC) testing has become a standard element of patient care in a wide variety of clinical applications. However, the concepts that distinguish POC technology—operation simple enough for nonlaboratory users; little or no maintenance requirement; and rapid, reliable results—mean that it can be applied equally well in many nonclinical settings.

First responders, for example, when confronted with an unknown substance suspected of being a biological agent, need tools to determine at the site whether the substance is harmless or contains biohazardous material such as anthrax or botulinum toxin. They quickly have to acquire reliable information that can aid in assessing the severity of the situation and determining the actions to be taken. Also, field epidemiologists evaluating, say, a mosquito population for the presence of West Nile virus need to be able to process a large number of samples and to make decisions as to which preventive measures should be implemented—in a matter of days, not weeks. Practitioners in both of these nonclinical settings can benefit from a POC testing platform that can help them make decisions that affect the safety of the public by providing reliable information rapidly and on-site.

Despite the diverse testing environments of hospital, field, and office, and their wide variety of operators and operating conditions, clinical, biodefense, and field epidemiology settings all have test requirements in common. Chief among these are accuracy, speed, and reliability. POC tests in all these contexts should provide justification for definitive decisions on how to proceed. The extension of clinical POC technology to biodefense, epidemiological, and, potentially, many other fields of application is a natural progression and may eventually ensure that efficient, reliable, laboratory-quality testing can be performed in all critical on-site situations.

This article discusses the potential for such an extension of POC test technology in terms of issues and requirements, systems that are already available commercially, and the utility of a technology that can be adapted to any type of POC testing.

POC Testing in Nonclinical Settings

As part of the continual effort to optimize patient care, clinical POC testing systems have proliferated. This bedside technology, when used appropriately, can improve patient outcomes by reducing the time between the ordering of the diagnostic test and the administration of the most appropriate therapeutic intervention.

POC test systems are designed to be operated by healthcare providers who have not been trained in laboratory practices. For this reason, processes of sample preparation, manipulation, and application must be straightforward and robust. Such system features suggest that people other than healthcare personnel could benefit from portable testing instruments that yield rapid, accurate results for a variety of indications. Expansion of POC technology to nonclinical settings requires a flexible system that has minimal requirements for calibration and quality control (QC) testing, yet is able to maintain expected accuracy and precision under a wide range of conditions.

Various Purposes. While the general requirements of clinical and nonclinical on-site test systems are similar, there are also key differences between them. In the clinical setting, POC testing is performed at or near the patient’s bedside, under controlled environmental conditions, in order to enable the healthcare provider to treat the patient as soon as the rapid-test result is available. The primary focus of these testers is patient care; they cannot be expected to employ complicated equipment that would require shifting their focus from the patient to test methodology.

Biodefense first responders, who need to ascertain fully the threat level of an unknown substance, bring their system directly to the location of the potential threat. The performance of their job requires a testing system that is fully portable, battery operated, easy for someone in protective equipment to use, and rugged enough to be moved around. Environmental conditions—temperature, humidity, vibration, dust level—may vary considerably. As is the case in the clinical setting, the primary focus of first responders to a possible biological threat cannot be performing an analytical test, but rather must be the elimination of any potential danger. Thus, they need a test system that operates quickly with minimal operator intervention.

Mosquito control technicians, for their part, generally wait until a large number of samples have been collected in the field and they have returned to their base of operations before performing testing. Their decision-making time frame can be as long as hours or even days, rather than the scale of minutes requisite for clinical and biodefense applications. In the field epidemiology arena, a testing system that allows delays between sample acquisition, processing, and the running of the test is optimal.

Sample Handling. Each different professional group of users of POC test systems will have different sample-handling requirements and issues. The use of collections of mosquitoes or oral-cavity swabs from birds in epidemiology applications means that there are no problems with sample size or the use of destructive analysis. In the biodefense field, on the other hand, systems must be sensitive enough to identify a possible biological threat with the use of as little sample material as possible.

Protocols currently being implemented by first responders require that the majority of the suspicious material be taken away for laboratory confirmation, leaving only a dusting of powder to be tested immediately on-site. The portable system, therefore, must have a high level of sensitivity. More often than not, the unknown material being tested is not a biological agent. It does represent a real threat, however, legally speaking, so there is a need for excess material to be retained as forensic evidence following sample analysis. This excess material may be used in a prosecution of someone alleged to be responsible for a hoax that, while it might not have been an actual biological danger, might nonetheless have had a significant economic impact on society.

In the area of clinical application, patients are commonly subjected to a large number of blood tests for both diagnosis and treatment management. It can thus be vitally important to minimize the volume of blood sample required for each test being performed, especially when the patients in question are very young or critically ill.

Sensitivity. Hand in hand with any requirement that sample volumes be small is the need for a high-sensitivity assay. If the amount of sample available to test is small, or if the concentration of the substance of interest is low, then the test chosen must be sufficiently sensitive to detect an analyte in these small quantities.

This need for high sensitivity holds for all the areas of application being discussed here. Clinically, the appearance of a certain protein in the patient’s blood can be indicative of a particular diagnosis. Detection of low levels of this protein may mean earlier diagnosis and treatment, and can help prevent exacerbation of the disease. In biodefense, the quantities of bioterrorism agents commonly accepted to be fatal doses are extremely minute. Also, the perpetrator of a biological attack may not be able to make a weapon in which the biological agent is in concentrated form; consequently, the agent may be very dilute. The system used to identify the agent must be sufficiently sensitive to detect it when it is present in the sample and sufficiently robust that no interference is generated by accompanying nonthreatening material. Epidemiological technicians performing analyses of field specimens may grind 50 mosquitoes into a single sample for testing. It is likely, however, that not all 50 mosquitoes will carry a high titer of West Nile virus. Again in this case, sensitivity is a concern in that, if virus is present in very few of the mosquitoes in the sample, it is important that the test detect these few virus particles.

Specificity. Specificity is also a critical requirement in all of these applications. Clinical samples can appear to be relatively homogeneous, as they are all whole blood, but interference may occur as a result of unknown pathologies or medications being present in the patient’s blood. In both biodefense and epidemiology, the samples being tested are by their nature impure. There are carrier materials in the powders being evaluated by first responders that could potentially interfere with sample analysis. Mosquito control personnel cannot separate nonviral materials out of a slurry of crushed mosquitoes. Any test system employed must have sufficient specificity to be able to ignore the contributions of such contaminants in a sample.

Ease of Use. Clinical, biodefense, and epidemiology applications differ in the apparel that practitioners wear routinely in order to ensure their personal safety. These differences in protective equipment can make a given diagnostic device easy or hard to use. While laboratory coats and surgical gloves may be the appropriate attire in clinical and epidemiological test environments, first responders are encased in full protective suits that impair their dexterity and visual acuity. Buttons and displays on any test system they use must be easy to see through plastic face guards and to operate while wearing rubber gloves.

No matter the application, these POC-type test systems must give accurate, reliable results. System maintenance and any additional testing beyond the samples of interest (for example, of calibrators and controls) should be minimal so as not to distract from the primary responsibilities of the operators. Ease of use is critical for all applications.

Commercially Available Systems

Table I. (click to enlarge) A selection of the portable instruments available for use in clinical, biodefense, and epidemiology testing applications. Clinical instruments listed all perform tests for cardiac markers. Epidemiology systems listed all test for West Nile virus.

A wide range of portable on-site testing systems are available for each of the settings discussed in this article (see Table I). When considering which system to use, it is important to look at the appropriateness of the test menu and the various user interface features for each specific test application.

Detection methodologies used in commercial systems range from evaluations of pH and protein in a sample to polymerase chain reaction (PCR) analysis of the nucleic acids in a sample. The BioCheck powder-screening test kit from 20/20 GeneSystems Inc. (Rockville, MD), for example, employs a dipstick-type method in which a color change indicates the presence or absence of protein and a pH range (acid, neutral, or base). Negative protein results are confirmed through the use of a control test strip. This outcome indicates the presence of a potentially hazardous substance without specific identification. At the other end of the spectrum, the RAZOR system from Idaho Technology Inc. (Salt Lake City, UT) employs sequence-specific probes to identify each species of interest. This system utilizes instrumentation, software, syringes, buffers, and unique sample pouches to bring PCR, usually a labor-intensive process, to the field.

Most of the portable systems listed in Table I, however, use some form of immunoassay for rapid detection of the analyte of interest. In these systems, labeled antibodies are bound, directly or indirectly, to the analyte if it is present in the sample. Antibodies may be labeled with colloidal gold or with fluorescent or luminescent dyes. Detection technologies, then, vary depending on the labeling method. The systems designed for clinical application deliver quantitative results, while those for use in biodefense yield qualitative results—that is, a positive or negative indication.

In applications for all three settings of interest, the user has a choice between systems that require instrumentation to read the result from a disposable test strip or cartridge and systems that rely on visual reading of a line on a strip to distinguish a positive from a negative result. One biodefense system, the Guardian Biothreat Alert test strips manufactured by Alexeter Technologies LLC (Wheeling, IL) and Tetracore Inc. (Rockville, MD) offers both options. The decision regarding whether to use an instrument-dependent system can be important. In time-critical situations, such as may be common for first responders and clinicians, ambiguity in results, or misinterpretation of them, could result in increased risk to patient or community in a potentially dangerous situation. When a visually read test system is employed, circumstances must be such that neither color discrimination nor impeded vision can be a factor affecting the correctness of the reported result. The visual cue must be sufficiently unambiguous that all potential operators would report the same result for a low-level positive test.

In evaluating a system for possible adoption, the prospective user should include an assessment of its QC features—both those integral to the system and optional ones for external verification of performance—and also appraise the need for routine calibration. Many of the devices listed in Table I have built-in indicators of system performance. These may provide electronic verification of the performance of instrumentation, or visual positive or negative indication on a test strip. They also include integral control regions in a test cartridge that preclude reporting of a result if specific conditions are not met. While the availability of external control solutions or strips is optimal for troubleshooting, integrated controls are critical for instilling confidence in the user that the local conditions surrounding each test do not affect the quality of the result.

A Universal POC Platform

Table II. (click to enlarge) Assays available on the RAMP system of Response Biomedical Corp. (Burnaby, BC, Canada). Data are from the package inserts, except for sensitivity and specificity as referenced.

One system now on the market, the RAMP from Response Biomedical Corp. (Burnaby, BC, Canada), is distinctive in that it is designed to offer solutions for use in clinical, biodefense, and epidemiology settings (see Table II).

Figure 1. The packaged RAMP system for biodefense applications. A case contains a battery-powered reader, test kit, printer, printer cable, and ac adapters for the reader and printer.

The compact, robust RAMP system comprises a reader and test cartridges along with a bar code wand and a printer as optional equipment. When the system is delivered to first responders, all of the components necessary to begin testing are packaged in a case to facilitate transport (see Figure 1). Manipulations necessary to perform testing are minimal, allowing the system’s use by people wearing protective equipment such as hazmat suits. The system has also been adapted for field epidemiology in that it allows mosquito-control groups to batch-process their specimens upon returning from sample collection in the field.

System performance and the validity of results are ensured through the use of internal electronic and optical tests performed by the reader on a predefined basis. These internal QC features run each time the reader is turned on, verifying that it is in proper working condition. Built-in QC features in each test cartridge further ensure that results are reliable and accurate. This cartridge QC corrects for variations in operator technique, sample handling, and reagent and sample integrity. Because QC is built into each test, users can be confident of the veracity of the data they are obtaining.

Figure 2. (click to enlarge) The steps involved in performing a RAMP test are the same in all applications. Mix the sample in buffer (a). Mix in a pipette (b). Place in a cartridge (c). Insert cartridge into reader (d).

Each disposable test cartridge analyzes a single sample for the chosen analyte (see Figure 2). In all cases, the sample is mixed with supplied buffer, the prepared sample is transferred onto the test cartridge, and the cartridge is then introduced into the read instrument. The reader transports the cartridge into the test slot to begin analysis of the sample. With every cartridge processed, the reader scans the bar code on the bottom to determine the type of test being performed and to ensure that the cartridge is within its expiration dating. Each box of test cartridges is supplied with a lot card containing the calibration information for each manufacturing lot, eliminating the need for user calibration. Results appear on a liquid-crystal display screen and are also stored in memory. In addition, they can be printed or transferred to a computer for long-term storage.

Figure 3. (click to enlarge) On the RAMP cartridge test strip, antigen-bound particles are captured at the detection zone by specific antibodies, and excess particles are captured at the internal standard zone by antiimmunoglobulins. The arrow indicates the direction of sample flow.

The test cartridges employ fluorescently labeled antibodies that bind the agent of interest. The bound antibody- antigen complex is captured on the test strip in the detection and internal standard zones (see Figure 3). The reader monitors the sample flow across the length of the assay strip.

Should an operator delay for too long between loading the sample and inserting the cartridge into the reader, or insert a cartridge that has already been used, or apply too little sample to the cartridge, or not use the supplied assay tip from the kit pouch, the flow detected by the instrument will not adhere to expected patterns, and an error will be reported. Algorithms in the reader use sample flow and bound fluorescence to determine performance anomalies. In this way, each cartridge gives assurance that operator sample handling and loading technique is appropriate.

Once the cartridge passes these first QC checks, the reader measures the fluorescent signal at the detection and internal standard zones. It also measures the background signal, which here serves the same purpose as a blank, or zero well, in a standard laboratory immunoassay. The background is subtracted from the signal in the detection and internal standard zones. In determining the result, the reader calculates a ratio between the fluorescent signal as measured at the detection zone and at the internal standard zone. The ratio corrects for variations in environmental conditions, sample viscosity, and membrane variability among samples, because any factors that affect binding of the fluorescently labeled antibodies at the detection zone will have the same effect on the internal standard zone. The use of this ratio increases the precision of the measurements and reduces sample-to-sample inconsistencies caused by the variables just named.

The test menus and types of sample accepted are application-specific. For clinical applications, all RAMP assays employ EDTA-anticoagulated whole blood. Biodefense assay samples can be powders, liquids, or surface swabs. Epidemiology tests employ material extracted from mosquitoes or birds.

In the clinical setting, the test operator uses the supplied transfer device and assay tip to transfer some of the anticoagulated whole blood into the premeasured buffer. After mixing, the sample is applied to the cartridge. The same minimal sequence of manipulation applies to liquid samples for biodefense assays. Powders to be analyzed in this application area are sampled by using a supplied microbrush to transfer the powder into the buffer vial. Swabs for sample collection can be moistened in the buffer, used to wipe a suspect surface, and then rinsed in the buffer before sample mixing and transfer to the test cartridge. A negative reading allows field efforts to relax and perhaps a facility to reopen. A positive result, on the other hand, will lead to decontamination and quarantine while a sample is sent to a regional laboratory for confirmation and for evidentiary analysis.

Only the epidemiological testing involves any significant amount of sample manipulation. The technician has to crush mosquito samples and remove the particulate waste before mixing and applying the sample to the test cartridge. Bird samples are swabs from the bird’s oral cavity mixed into the buffer before application.

Marcia L. Zucker, PhD, is the director of clinical support at Response Biomedical Corp. (Burnaby, BC, Canada) She can be reached at mzucker@ responsebio.com.

The dissimilarity between biodefense and epidemiological sample types and the relatively standardized clinical samples could be expected to be technologically challenging to a system that monitors sample integrity. However, this universal system overcomes the challenge by adjusting sample flow requirements according to the specific analyte and by limiting the amount of material that is presented to the test buffer. The use of a microbrush instead of a scoop (the system’s high sensitivity making the latter unnecessary) enhances the functionality of the tests for biodefense, as evidenced by the fact that AOAC International has granted them field-use approval for anthrax testing.⁵

In all environments, local protocols determine the level of treatment or action that the test result suggests is required. The high specificity observed in every setting lends a sense of confidence to the user that a negative result truly rules out the presence of the test substance.

Conclusion

The extension of clinical POC technology to biodefense, field epidemiology, and potentially other nonclinical applications is a natural technological progression. Despite the apparent differences in applications and needs among practitioners in the clinical, biodefense, and epidemiology fields, all can now employ a single technology platform usefully. Each of these end-users requires a system that is fast, easy to use, reliable, accurate, and precise. A system that has been designed for use by personnel not trained in laboratory procedures, whose instrument buttons are large enough to accommodate hazmat gloves, and that requires only minimal sample manipulation seems to have the technical capability to ensure efficient, reliable laboratory-quality testing in numerous situations.

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