An Introduction To The Function Of Diagnostic Reagents For In-Vitro Clinical Applications
The reagents used in diagnostic testing assays are one of the most fundamental and critical components necessary for a medical laboratory to properly function.
Antigen used in immunoassay kit manufacture being shipped on dry ice.
Image: The Binding Site
Without question, the reagents used in diagnostic testing assays are one of the most fundamental and critical components necessary for a medical laboratory to properly function. Without them, a laboratory ceases to operate, as it is unable to generate the necessary tests required for the diagnosis, treatment, and monitoring of a patient’s condition. When one considers that almost 70 percent of the information that a physician requires for the assessment of a patient’s general health comes from the information supplied by the clinical laboratory, the importance of these products, and of the laboratory itself, becomes evident.
Diagnostic reagents, be they chemical, biochemical, or biological/biochemical in design, are dependent upon several different components working together to generate accurate, precise, and reliable patient test results. However, when you examine the basic operational principles these reagents follow, regardless of the format or platform, all of them consist of the following core steps:
1. A volume of patient sample to be analyzed and a volume of one or more diagnostic reagents are placed together in some type of reaction vessel and mixed together, starting a chemical reaction.
2. This test mixture is then incubated at a given temperature, usually 37°C, for a given period of time.
3. The reaction is stopped once the assay reaction time is reached, and some type of quantifiable change is observed.
4. This “change” varies per given procedure but is most often an increase or decrease in a color, an increase or decrease in spectrophotometric absorbance, a change in the intensity of light produced or not produced, or an increase or decrease in the optical density or turbidity of the test mixture.
5. This change is measured and compared against a known change that has a definitive value assigned to it (most frequently coming from calibration material, or a standard material), and it is calculated into a test result.
As mentioned earlier, most diagnostic reagents can be classified as chemical, biochemical, or biological/biochemical in nature.
These are the most basic of the three diagnostic reagents and are designed as single- or two-reagent systems. They incorporate dyes, buffers, surfactants, and basic chemicals to form working reagents. A primary example is the diagnostic reagent used to measure serum albumin concentrations. This common diagnostic reagent routinely measures this serum component based on the binding of bromocresol green dye specifically with albumin to produce a colored complex. The absorbance of the resulting complex is measured spectrophotometrically and is directly proportional to the albumin concentration in the sample. Another classic test procedure is the measurement of serum creatinine concentrations – first described more than 100 years ago and still routinely used in the clinical laboratory. Here, creatinine in the sample reacts with picrate (picric acid, sodium hydroxide, and water) to form a creatinine-picrate complex. The resulting increase in absorbance at a given wavelength resulting from the formation of this complex is directly proportional to the concentration of creatinine in the sample.
Biochemical. Biochemical reagents are a bit more complex in design and require more components working in unison together. Designed to perform alone or as part of a two-reagent system, these reagents are frequently enzyme-driven and incorporate buffers, stabilizers, co-enzymes, indicators, surfactants, and preservatives as part of their working components. The diagnostic reagent used for the determination of glucose, one of the commonly run diagnostic tests in the clinical laboratory, fits well into this category. Here, glucose is acted on by the enzyme hexokinase in the presence of adenosine triphosphate and magnesium to produce glucose-6-phosphate. Glucose-6-phosphate dehydrogenase then specifically reacts with glucose-6-phosphate, with the concurrent reduction of NAD to NADH. The NADH produced absorbs light at a specific wavelength and can be spectrophotometrically detected as an increased absorbance proportional to the glucose in the sample.
Biological/biochemical. The biological/biochemical-based diagnostic assays are frequently composed of two or more reagents. These reagent systems are highly complex, incorporating many steps and numerous working components including buffers, conjugates, wash solutions, detection reagents, and serological markers – namely antibodies and antigens as essential elements. These types of diagnostic reagents are typically referred to as immunoassays and are most often employed to measure analytes found at relatively low concentrations in the body. For example, these would include hormones, vitamins, infectious-disease agents, and specific protein components. However, and as discussed earlier, regardless of their complexity, when you look at the basic operational principles these reagent systems follow, all inherently work exactly the same way.
Manufacturing and Sourcing Considerations
In today’s environment, suppliers of IVD reagents can employ various strategies to achieve their goals. These include sourcing raw materials and components for the in-house development and manufacture of reagents, forming OEM relationships with key trusted suppliers to obtain a finished product, or, in many cases, a combination of both. Supplier expertise can often result in technological advantages, while in-house production of components allows for long-term supply stability of critical components. Regardless of the strategies employed, when considering the sourcing of key diagnostic reagents, be they finished products or raw materials for future reagent test kits, it is important to be aware of the following points when dealing with suppliers and vendors to produce the highly reliable and accurate diagnostic reagents that the industry has come to expect.
Manufacturing Capabilities. IVD makers should consider manufacturing capabilities for their immediate, as well as projected, needs, and make certain that their vendors can address their needs now and into the future. Additional factors to consider are vendors’ flexibility, their ability to handle bulk options, and whether their breadth of product coverage includes other associated assay components that are vital to the IVD company’s operation. An especially important consideration
Control material used in, and for, IVD kit manufacture.
Image: The Binding Site
regarding vendors for biological/biochemical-based diagnostic tests (i.e., immunoassays) is whether antibodies are sourced from trusted suppliers rather than produced in-house. Antibody generation is a complicated and intricate process that takes a great deal of time and expertise. An antibody supplier should be able to offer a full range of custom antibody production services, including polyclonal production, monoclonal production, purification and conjugation, freeze drying, and antibody fragmentation, as well as bulk custom formats, purified antigens, and sera. In addition, since the production of antibodies is complex, and their use in diagnostic assays changes over time, it is critical that an antibody supplier provide a strong quality system, technical support, and industry expertise to keep customers apprised of the latest news, regulatory issues, and potential legislation that can potentially affect key assay components.
Pricing and Cost Considerations. With regard to costs, it is important to keep an open mind and not to fixate solely on numbers. Since there are so many components in a typical diagnostic reagent, it is important to consider the individual components and the combination of these working together in a particular test. While each element does contribute to the overall cost, it is possible to achieve significant cost savings by changing one element, such as a detection buffer, which can significantly affect the sensitivity of the test, resulting in reduced volume requirements for key reagents and reduced assay costs.
End-user value of the test versus component cost should also be taken into consideration. For example, a faster test, or one with improved performance, may cost more to buy, but it may be able to save the end-user valuable time and labor expenses.
Industry Experience. Other key elements to consider are the vendor’s experience and knowledge of the IVD market, whether it has a well-established reputation and proven track record within the industry, and its financial soundness. While these factors may appear to be insignificant at first glance, they do carry significant importance in the long run.
Validated Processes and Procedures. A vendor operating in today’s quality-focused environment without certified quality standards (such as ISO 9001 or ISO 13485) and without validated procedures and processes in place should immediately raise a red flag.
Regulatory Compliance. Again, manufacturers should ensure that their vendors are in compliance with any and all associated regulatory organizations, not just FDA. A repeated pattern of noncompliance and ongoing issues with any regulatory body should immediately raise a red flag.
Ability To Automate. Automation continues as a trend in the clinical laboratory, and the ability for reagents to be ready, available, and adaptable for current and future instrumentation, platforms, and procedures is critical for industry and end-user acceptance.
Specific Performance Characteristics. These are routinely found on the package insert for the given test assay and refer to the reagent’s overall performance in routine operation. These typically include tests for interferences, linearity, method comparison, and precision. In biological/biochemical-based diagnostic reagents (i.e., immunoassays), sensitivity and specificity testing are important and also need to be addressed. All performance characteristics must be within limits acceptable to regulators and end-users.
Product Lot-To-Lot Consistency. This point is critical: If at all possible, an IVD manufacturer wants the reagents, or raw materials, it brings in today to perform exactly the same way they did a year ago. Those reagents or raw materials must work and perform exactly the same way a year ahead into the future, too. End-users expect to see this, as do some regulatory bodies.
Technical Support Expertise. Selecting a vendor that offers strong technical support and expertise for quickly and efficiently resolving any questions, concerns, or issues the IVD manufacturer or end-users have will only make things better for all involved in the long run.
Current and Potential Industry Trends
With continued growth predicted globally, the IVD market, as it historically has done, will adapt to address new changes in technology and new testing situations. It is important to be aware of certain trends and plan for their inclusion in future test development and for the modification of existing assays to new platforms and testing conditions.
A common trend that can be observed with all of these types of reagents has been a steady movement away from traditional dry-filled and lyophilized reagent platforms to full liquid-stable formats. Aside from the obvious conveniences that a liquid-stable format features, the inherent benefits this reagent design offers include a savings in time and labor by the removal of routine reagent reconstitution procedures, a savings in cost by the elimination of possible reconstitution errors, and, more often than not, improved test turnaround time and efficiencies due to increased instrument uptime. All of these enhancements combine to result in increased overall laboratory productivity.
A second continuing trend is to bring diagnostic tests closer to the patient and move tests away from the traditional laboratory or hospital. Typically referred to as point-of-care (POC) tests, these assays require enhanced component stability and simplified testing parameters, as well as the ability to produce reliable and consistent test results. This expansion of POC testing will allow patients to identify their illnesses early on and monitor their reaction to various treatments and drugs, with an expectation to reduce healthcare costs while improving patient outcomes.
Another prevailing trend is the drive to reduce costs by reducing the volumes of the reagents used, or the size of components used in diagnostic assays, mirroring a tendency long seen in the computer industry. The trick here, however, will be to have the same type of specific performance characteristics the market has come to expect, as well as the higher sensitivity and specificity requirements necessary to achieve optimal outputs. Since this trend appears to go hand-in-hand with POC testing, this trend can be expected to continue for the conceivable future.
Lastly, a key trend that greatly affects the reagents used in some test assays is multiplexing, a diagnostic application that makes the sourcing and exchange of various test components much more complicated. With multiplex assays, there may be pools of antibodies or detection reactions in use within the same solution at the same time, all of which must be optimized to work together in order to produce a test result that is accurate and reliable across all test channels, or assay parameters. With the obvious rise of molecular diagnostics testing, it is also possible that there may be multiplex testing combinations in the future that include the traditional chemical, biochemical, and biological/biochemical-based diagnostic assays discussed here, along with DNA-based molecular assays, all of which must be optimized to work in unison.
To achieve the prevailing goal of high test performance at the lowest possible cost, the individual test components and the complex chemical, biochemical, and biological interactions of any reagent system must come together to produce a reliable and accurate result. Producing such diagnostic reagents that meet the demands of today, as well as those of the future, requires a knowledge, understanding, and appreciation of the various reagents used for clinical testing applications, as well as the breadth of sourcing options available for assay components. It is possible that the use of key strategic/tactical purchasing and manufacturing decisions, including those that encompass partnering with key suppliers and vendors already using advanced technology to obtain increased value, could achieve pricing goals and cost savings while addressing the current and anticipated directions seen within the IVD industry.
Robert J. Janetschek, MS, MT (ASCP), is OEM Account Manager at The Binding Site, Inc., a supplier to the global OEM IVD industry. Janetschek can be reached via e-mail at firstname.lastname@example.org.
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