Medical Device Link.

Originally Published IVD Technology June 2004

Processing Technologies

Figure 1. An example of three different degradation reactions taking place within a hypothetical dry reagent diagnostic test strip. At temperatures above the adhesive melting point, the adhesive may migrate into the porous membrane (a). At temperatures below freezing, the bond between the antibody-enzyme conjugate may break (b). At intermediate temperatures, the indicator dye may gradually deteriorate, reducing the high-level range of the diagnostic (c) (click to enlarge).

IVDs originated in the benign environment of the clinical laboratory, where diagnostic reagents were exposed to room temperatures and were used by skilled technicians. However, this is no longer always the case. IVDs are increasingly being used for a broad array of nonclinical purposes, under varying environmental conditions and by a mix of skilled and unskilled users. At the same time, regulators and customers have come to expect IVDs to incorporate automatic fail-safe systems that inform users if there is a likelihood that results may be erroneous. 

Moreover, IVD reagents are temperature sensitive. So when reagents are used in harsh environments, they are likely to experience thermal conditions that render them unfit for use. In order to cope with such harsh environments and high customer fail-safe expectations, IVD manufacturers are using time-temperature indicators (TTIs) and other temperature-monitoring devices. When properly designed, TTIs are packaged with a diagnostic reagent, experience the same thermal history as the reagent, and inform users if the reagent is usable. This article reviews the present state of the art, and new developments in TTIs. This article also discusses the characteristics that should be examined when selecting TTIs. 

Variables of IVD Stability

IVD reagents are perishable products. Such reagents consist of temperature-sensitive components (e.g., antibodies, enzymes, indicator dyes, ancillary materials) that tend to decay when exposed to thermal energy for prolonged time periods. Despite IVD manufacturers’ efforts, such raw-material limitations make it nearly impossible to achieve perfect stability.¹ 

Although IVD stability is determined by a complex function of time, temperature, humidity, light, oxygen, and vibration, proper packaging can control many of these variables. For example, light- and humidity-resistant foil packages can minimize the effects of humidity and light. Such packages may also contain desiccants or oxygen scavengers. While shock-absorbing materials can limit the effects of vibration, temperature is more difficult to control. 

Thermal decay processes typically cannot be detected in other materials and products. However, since IVDs generate precise results, changes caused by thermal decay can be detected. Thermal decay can damage both precalibrated and end-user calibrated IVDs. This decay can damage the calibrators themselves, or cause reagent deterioration that alters or diminishes the dynamic range of the reagent (e.g., degradation of an indicator dye).

In addition, IVDs are rarely composed of a single molecular entity. IVDs are usually composed of different molecular entities, each with a different thermal decay profile. As a result, some IVDs have complex thermal decay profiles. For example, a diagnostic reagent could have three different temperature-sensitive degradation reactions (see Figure 1). 

The stability of most IVDs is similar to reagents that have a single temperature-sensitive component. Other IVDs will have more complex stability profiles. There are numerous examples of IVDs with simple and complex thermal decay profiles (see Figure 2). 

Stability Regulations

Figure 2. This graph shows the differences between a simple IVD with only a single temperature-sensitive component (red line), and a more complex IVD with three components (blue line). The complex IVD is intolerant of freezing and has an additional degradation mechanism that kicks in at higher temperatures. (Higher temperatures are to the left, and lower temperatures are to the right) (click to enlarge).

Regulators are aware that inexperienced IVD development and management teams often handle stability issues inadequately. For this reason, FDA requires detailed stability validation protocols for new or substantially modified 510(k) applications. In some cases, the agency may also require post-approval stability studies.² 

The IVD stability regulations include 21 CFR 809.10 in the United States and EN 13640 in Europe.³  These regulations are based on the idea that product labeling should reflect stability requirements. However, complying with this requirement can be complex. For example, storage temperatures can fluctuate to unpredictable extremes for unpredictable lengths of time. Some labeling for average temperature conditions may not accurately cover such extremes. At the same time, labeling for extreme temperatures may be overly restrictive and vague. At present, the regulations offer little guidance in this area. 

Changing Role of IVDs

In clinical laboratories, IVDs are usually protected from thermal fluctuations and are run by highly skilled technicians. Such technicians maintain a high level of skepticism toward IVD results, and run ancillary controls and system checks when necessary. The benign lab environment minimized the stability requirements imposed on IVD manufacturers. Simply stating how long re- agents could be kept in a refrigerator or at room temperature was adequate. There was little need to validate worst-case conditions.

However, even in the benign lab environment, problems would occur. Technicians would use outdated reagents or fail to perform necessary quality control checks of reagent integrity. Concerns about reliability and human factor issues in the lab led to the establishment of the Clinical Laboratory Improvement Amendments (CLIA).⁴  

Moreover, IVDs are now used in many other areas, including agricultural, biodefense, drugs of abuse, forensic, food safety, point-of-care, toxicology, and veterinary applications. Many of these new applications occur outside of the controlled-temperature environment of the clinical lab, and tests may be conducted by operators who are not skeptical or careful about test results. 

One example is a soldier under fire in the desert running a biodefense diagnostic. In this situation, the IVD may have been subjected to extreme environmental conditions and is being used by an inexperienced and distracted user. If the results cannot be trusted due to severe thermal stress, the user needs to know this. Although this is an extreme example, the user profiles of forensic, agricultural, drugs of abuse, food safety, or toxicology diagnostic tests are not all that different. 

Consequently, IVD manufacturers are expected to perform worst-case scenario testing and human factors analysis to ensure that their IVDs will be stable and fail-safe in all situations. 

Time-Temperature Indicators

IVD manufacturers can address some of the more stringent stability requirements by using TTIs. TTIs are small devices that are packaged with an IVD and experience the same thermal history as an IVD. Compared with temperature loggers, which record thermal history but do not interpret it, TTIs contain temperature-sensitive chemical or electronic components that integrate the net amount of thermal energy over time and visually display the IVD’s condition. An optimally designed TTI is tuned to the same thermal sensitivity as the IVD it is monitoring, and gives a clear warning when the IVD’s thermal tolerance has been exceeded. 

Two types of TTIs are available: chemical and electronic. Each type has certain advantages and disadvantages. 

In chemical TTIs, a chemical indicator component undergoes a transition from a first to a second state. The activation energy for the TTI’s transition is roughly the same as the IVD’s deterioration reaction. Chemical indicators used in these types of TTIs include acrylic polymers that react with oxygen and heat to produce a colored reaction product, and lipase enzymes that react in chemical solutions with triglycerides and also generate a colored reaction product. In an alternative design, colored waxlike chemicals with defined melting points migrate into wicks and generate a colored path. 

Chemical TTIs use chemical indicator components with a single transition temperature profile. Such TTIs are most appropriate for IVDs that have a simple temperature sensitivity profile and can tolerate subfreezing temperatures. For safety reasons, TTIs should always expire before an IVD does, so a TTI’s expiration time is slightly below an IVD’s (see Figure 3).

Electronic TTIs (eTTIs) use thermistors, or microprocessor electronic circuits. A new development, these electronic devices had been used only for temperature logging. Such electronic loggers saved temperature data in their memory, but did not interpret it. Programmable eTTIs have recently been developed, which integrate and interpret temperature history and provide supporting statistical data.

Each type of TTI has certain strengths and limitations, and no one product is best for all situations. The factors that should be considered when choosing a TTI are accuracy, readability, cost, reliability, and the ability to supply supplemental statistical data.

TTI Accuracy

Figure 3. This diagram shows the fit between an IVD with a simple one-component degradation profile (solid red line), and a simple chemical TTI (dotted red line) that has a one-component degradation profile. The diagram also shows the fit between an IVD with a more complex three-component degradation profile (solid blue line), and a more sophisticated electronic TTI programmed to follow this IVD’s complex degradation curve (dotted blue line) (click to enlarge).

A TTI’s accuracy is determined by three factors: the fit between the TTI’s temperature sensitivity characteristics and the IVD in question; the accuracy of the TTI’s thermal integration methods; and the user’s ability to read the TTI accurately.

While the safety of TTIs is important, a TTI does not always have to be completely accurate, as long as the device indicates a problem before an IVD becomes unfit for use. Using inaccurate but conservative TTIs can also be justified, as long as the false alarms and waste caused by prematurely expired TTIs are kept to a minimum.

In addition, end-users often read TTIs under suboptimal conditions. Although chemical TTIs are quality control validated using automated readers, the readability of TTIs should also be considered. In some cases, IVD manufacturers should validate the readability of candidate TTIs using focus groups. If the observed reading error rate is too high, the TTI’s parameters should be adjusted.

Choosing TTIs

Many different TTIs are available on the market. Depending on the stability profiles, certain TTIs could be used with certain IVDs.

Chemical TTIs are adequate and cost- effective for IVDs with simple degradation profiles. One such chemical TTI is the Heatmarker by TempTime Corp. (Morris Plains, NJ) (see Figure 4). This TTI is available as an inexpensive label. It has a bull’s-eye design with a central circle consisting of a heat-sensitive acrylic chemical and a surrounding pre-printed reference color circle. TempTime can customize the temperature sensitivity of the acrylic chemical and the color of the reference color circle, allowing users to precisely tune the Heatmarker’s characteristics. 

The enzymatic TTI by Vitsab Inc. (Copenhagen, Denmark) could be considered for very unstable IVDs with short shelf lives. This TTI uses a highly sensitive enzymatic indicator system that operates best with IVDs that last for a few weeks under refrigerated conditions. Vitsab can tune this device by varying the type of substrate and the amount of enzyme. The TTI changes color as the reaction progresses. 

For IVDs with degradation profiles that fall close to normal room temperatures or refrigerated temperature levels, the MonitorMark by 3M Corp. (St. Paul, MN) could be used. The MonitorMark is an alternative chemical TTI, in which the indicator chemical has waxlike properties. Above a preset temperature, the chemical melts and migrates up a linear wick, which produces a clear, easy-to-read signal. Unfortunately, 3M has discontinued its customization program and offers the MonitorMark only in certain preset ranges. Nonetheless, IVDs that fall within these preset ranges may find this TTI to be useful. 3M also offers the Freeze Watch indicator, which detects temperatures below zero.

The LifeTrack by CliniSense Corp. (Los Gatos, CA) could be considered for IVDs with complex stability profiles (see Figure 4). The LifeTrack is an eTTI that can be rapidly configured, has an easy-to-read display, and can monitor IVD stability for up to three years. It also contains a gas-gauge-type lifetime-remaining bar, and an optional temperature logger that can output temperature statistics to a computer via an infrared port for recordkeeping purposes. In addition, the device can be reprogrammed and reused multiple times. 

Electronic Temperature Alarms and Loggers

Figure 4. TTIs can range from simple labels to sophisticated electronic devices, such as the Heatmarker (right) by TempTime Corp. (Morris Plains, NJ), and the LifeTrack (left) by CliniSense Corp. (Los Gatos, CA). When IVDs expire, the center of the Heatmarker turns dark, and the LifeTrack changes from “+” to “-” (click to enlarge).

For situations in which only the shipping conditions of IVDs need to be monitored, electronic temperature alarms and loggers should be used. The devices have been used to monitor shipping conditions for many years. Such alarms and loggers are used for brief time periods to ensure that no temperature excursion has occurred during shipment. Unlike TTIs, these devices do not perform time-temperature integration, or shelf-life monitoring. 

The TagAlert and TempTale monitors are produced by Sensitech Corp. (Beverly, MA). The TagAlert is a temperature alarm that can warn users if a temperature parameter was exceeded during shipping. The TempTale is a temperature logger that has a large internal memory capable of storing thousands of individual data points. It also has a built-in temperature alarm function, as well as a proprietary Internet data manager system. 

The iButton temperature logger system by Dallas Semiconductor Inc. (Dallas) consists of small, coin-sized, disks. These disks contain an embedded battery, microprocessor, and memory that also can store thousands of data points. The units lack a visual display, but are highly robust and can have a life span of up to 10 years. 

The LifeTrack by CliniSense is both a temperature logger and a visual eTTI. The logger can be set to monitor temperatures every 30 minutes. It operates like an airplane black box, continually recording the past 100 temperature readings until an IVD stability failure occurs, as well as the time elapsed since the crash. This feature allows users to download data, document the findings, and understand when and how thermal failures occurred. 

Conclusion

All areas of society have been moving toward decreased tolerance of preventable errors and increased emphasis on design safety measures and human factor analysis. The IVD industry is no exception. Regulatory agencies are aware of this trend, and will respond with more-stringent worst-case testing and validation requirements. 

Since IVD reagent stability problems are inevitable and predictable, TTIs will be used in an increasing number of IVDs. Although TTIs offer the option of temperature monitoring for most IVDs, improvements in TTI technologies are desirable and likely. Chemical TTIs tend to be slow, are expensive to customize, and have suboptimal accuracy and suboptimal displays. While electronic TTIs are accurate and can be quickly customized, they tend to be expensive and are larger than would be ideal. 

In the future, more-sophisticated low-cost chemical TTIs will be developed, which can be more accurately tuned to match particular IVD stability profiles. At the same time, decreasing electronics costs may make it economically feasible to put accurate electronic TTIs and hybrid electronic TTI-loggers into a broader range of IVDs. In addition, both chemical and electronic TTIs may be combined with radio-frequency identification (RFID) technology to produce visual RFID TTIs that can warn both users and automated systems whenever stability problems have occurred. 

As TTI technologies become more widespread, both end-users and the IVD industry will benefit. End-users will be able to use IVDs in a broader variety of environments and will have higher confidence in the reliability of the results. This should stimulate demand for IVDs, particularly for point-of-care IVDs and nonclinical IVDs. 

References

1. P Guire, “Stability Issues for Protein-Based In Vitro Diagnostic Products,” IVD Technology 5, no. 2 (1999): 50. 

2. Section 280.100, “Stability Requirements for Licensed In Vitro Diagnostic Products,” Compliance Policy Guide (Rockville, MD: Office of Regulatory Affairs, U.S. Food and Drug Administration, 2000).

3. Stability Testing of In Vitro Diagnostic Reagents, DIN EN 13640:2002 (Brussels: European Committee for Standardization, 2002). 

4. S Ehrmeyer, “What’s New with CLIA ’88, JCAHO, & CAP,” (2000) [cited 12 May 2004]; available from Internet: www.westgard.com/guest6.htm.  

Stephen E. Zweig, PhD, is the founder and president of CliniSense Corp. (Los Gatos, CA). He can be reached at szweig@clinisense.com. 

Copyright ©2004 IVD Technology