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LABORATORY INSTRUMENTATION

Liquid delivery is an essential part of the manufacture of IVDs. Inaccurate volume dispensing can lead to defective diagnostic kits.

The word calibration often conjures up negative images. This is especially true in the development and manufacture of IVDs, where regulations mandate that laboratories and production plants have regular calibration procedures in place for their equipment. Typically, maintenance and calibration are performed using manual methods or by outsourcing to the original equipment manufacturer or to third-party service providers. The expense and downtime needed to accommodate these service schedules are two common calibration burdens. However, failing to perform calibration is not an option. Both regulatory compliance and device quality control depend on accurate measurements, and when liquid handling is involved, accurate and reliable volumetric dispensing is critical.

The consequences of liquid-delivery failure can include noncompliance and poor-quality devices. Therefore, it is essential that liquid-delivery quality assurance (LDQA) be integrated into laboratory and production operations. This can be difficult to accomplish because of the inherent disruption, lack of control, inconvenience, expense, and inefficiency of outsourcing or performing manual calibration. However, because automated calibration technologies can eliminate these issues while reducing risk and controlling costs, it is now possible for LDQA to be more than just a random act of compliance. With the proper integration of real-time measurement verifications and appropriate software, LDQA can become a positive practice, ensuring continual and optimal quality in liquid-handling processes without decreasing productivity.

New, standardized technologies have made automated LDQA methods, such as those that manage equipment inventories, document and control calibration procedures, compute accuracy and precision statistics, generate pass-fail reports, and perform recordkeeping and compliance, easy to integrate and maintain. This article will explore the various automated liquid-delivery calibration methods as well as the resulting improvements to IVD operations.

The Critical Role of Liquid Handling

Sidebar: Is Automated Liquid-Delivery Calibration the Right Choice?

Liquid handling plays a role in a number of phases of IVD development and manufacturing. For example, liquid- delivery accuracy is critical during the manufacture of reagent strips used in test kits. During strip production, dispensers fire tiny droplets of antibodies onto paper continually fed from a reel. This paper is then cut into strips and placed into the IVDs. If the liquid-dispensing equipment malfunctions, the reagent strips might receive inaccurate antibody volumes, or no antibodies at all. This can lead to defective tests that provide false negatives and other inaccurate results. The potential for equipment failure is alarming, especially given a report released earlier this year by FDA’s Center for Devices and Radiological Health stating that laboratory tests guide up to 80% of medical decision making.¹

In addition, clinical analyzers often rely on built-in liquid-handling instrumentation to deliver both samples and reagents. Volumetric accuracy and precision are essential to achieving reliable and repeatable results. Therefore, instrument manufacturers must calibrate and verify liquid-delivery processes, and these verifications should occur during design, development, and qualification. Volumetric calibration should also become a part of the ongoing maintenance, calibration, and requalification of analyzers, which should occur periodically after being installed in clinical laboratories.

To ensure the quality of their operation, liquid-handling systems are regulated by strict rules. As a result, all device manufacturers must adopt and enforce regular calibration protocols. The Code of Federal Regulations (21 CFR 820.72) states that procedures must be established and maintained “to ensure that equipment is routinely calibrated, inspected, checked, and maintained.” This section of the CFR further stipulates that calibration procedures “shall include specific directions and limits for accuracy and precision,” and that “there shall be provisions for remedial action to reestablish the limits and to evaluate whether there was any adverse effect on the device’s quality.”²

Assessing Manual Calibration

To meet calibration requirements, IVD companies have for years relied on manual or outsourced methods for liquid-delivery performance verification. However, with the advent of automated calibration methods, the inefficiencies of these alternatives have become more apparent.

For example, the most common manual calibration method is based on gravimetry, which weighs liquid quantities on analytical balances. Through this method, laboratory personnel calculate volume using Z-factor tables and correcting for evaporative loss and other factors. Next, accuracy and precision are calculated, and the results are recorded.

Such manual methods are time-consuming and prone to human error. While the automated performance verification of a 384-well plate can be accomplished in less than 10 minutes, manual gravimetric calibration can require hours to complete. Manual methods also place a heavy reliance on operator skill, training, and attention to detail to ensure procedural compliance. Although automation can remedy many of these issues, manual calibration may be preferred when calibration is required infrequently or when laboratories are testing new methods. Even so, once a laboratory commits itself to a given calibration protocol, automation can often result in productivity gains.

Outsourcing liquid-delivery calibration can be useful for organizations that lack sufficient personnel to maintain an automated calibration program but have a reliable, well-known partner. However, removing instruments from the laboratory can also lead to scheduling nightmares, downtime, and loss of productivity. Outsourcing can also prove costly, both in terms of the actual fee paid to the vendor as well as the hidden costs associated with qualifying and managing service partners. Another concern is that the quality of the calibration process can vary depending on the technician charged with performing the calibration. Regardless of service partner, the responsibility for properly investigating any irregularities or failed calibrations rests squarely on the IVD manufacturer. For many manufacturers, this is reason enough to take charge of their calibrations, and thereby control their operations.

Arguments for Automation

Whether the liquid-delivery process itself is automated or manual, automatic performance verification or calibration can provide several advantages over manual and outsourced service methods. First, software enables electronic calculation and documentation and reduces the risk of error, which in turn enhances efficiency by eliminating retesting. Automatic instrument “trackability,” the ability to know precisely when a piece of equipment was last verified and which manufactured devices were produced since the last successful verification, is another key benefit. Automatic calibration software systems can also improve scheduling and compliance efficiency by notifying users of upcoming scheduled maintenance.

Together, software and automated volume measurement can simplify the calibration process, integrating frequent verifications directly into the manufacturing process. This gives IVD companies better control over their instrumentation and processes and greatly reduces, or even eliminates, the need for out-of-tolerance investigations, corrective actions, or root-cause investigations that would otherwise arise from liquid- delivery failures.

Reducing the labor required for calibration allows for thinly stretched resources to be reallocated to more-complex and more-profitable projects, and can reduce costs over the long run. However, the more important end result of an automated calibration system is its creation of a process that is continually and painlessly controlled, instilling confidence in the quality of operations and manufactured products.

How to Automate Calibration

Figure 1. Equipment management software can help laboratorians oversee the calibration of liquid-delivery systems. Pipette Tracker software by Artel (Westbrook, ME) is incorporated into the company’s pipette calibration system to electronically track and manage pipette calibration processes.

One solution for speeding calibration and enhancing productivity is equipment management software. Using bar coding or radio-frequency identification technologies, these systems track liquid-handling instrumentation, schedule calibration, and, based on management input, control and enforce testing protocols such as frequency of verification and number of data points (see Figure 1).

Table I. (click to enlarge) A comparison of electronic data management and manual data management processes.

Other systems incorporate automated data processing and recording, providing documented calibration results and automatic pass-fail determinations. These technologies can be integrated with equipment management systems for full calibration support (see Table I). To automatically process information, system software enforces testing protocols, computes liquid volume measurements, and calculates summary statistics such as accuracy and precision. These software programs are available for the three most common liquid-delivery calibration methods: gravimetry, single-dye photometry, and ratiometric photometry.

Gravimetry. Gravimetric calibration can be automated by integrating balances with computational software or by building balances into automated liquid handlers. Automating this process eliminates human transcription and calculation error, provides automatic documentation, and helps achieve traceability to national standards. Even so, human involvement is typically necessary to monitor and periodically empty the receiving vessels on the balances to avoid overflow. This method can also be time- consuming. For example, one commonly used gravimetric calibration protocol requires about 90 minutes of downtime to calibrate an eight-probe liquid handler. In addition, to attain accurate gravimetry results, a controlled environment is needed.

Single-Dye Photometry. Photometric calibration, which verifies liquid volume by measuring the absorbance of light by a dye solution at a given wavelength, may be more suitable for automation than gravimetry because it is less affected by environment and evaporation. This also gives photometry a distinct advantage at lower volumes. To date, single-dye photometry has only been incorporated into internally developed automated systems and into a few manual commercial systems. Traceability, therefore, is limited and dependent on user expertise.

Figure 2. The multichannel verification system by Artel incorporates ratiometric photometry to automatically verify liquid-handler accuracy and precision.

Ratiometric Photometry. Relying on two dyes for more-accurate and more-precise volume measurement, ratiometric photometry, a patented technology, has been incorporated into commercially available automated systems. Systems based on this technology not only provide speed and traceability, but can also be used on the benchtop. Because ratiometric photometry is more technically complex than its liquid-calibration counterparts, the calculations are incorporated into the software to enable automatic measurements, as well as proper error percentages versus set tolerances. These systems can calibrate single-channel pipettes in five minutes and automated liquid handlers with up to 384-well plates in less than 10 minutes, and are preferred for low-volume applications (see Figure 2).

Automation and Traceability

The fact that automated calibration aids traceability is a benefit that warrants special attention. According to provisions set out in 21 CFR 820.72, measurements made as part of a calibration program must be traceable to national or international standards to ensure that results are consistent across locations and over time. Full traceability requires an estimate of the uncertainty of the measurement. This can be thought of as a statistical margin of error.

For measuring liquid volumes in the milliliter or high-microliter range, there are two common traceability approaches. The original approach, gravimetry, weighs the liquid to determine the mass of the sample, then converts this calculation to a volume using known traceable values of the liquid density and makes other appropriate corrections. As volume decreases, traceability using the mass of the liquid becomes increasingly problematic. This is because solvents, even water, evaporate while being weighed, and this evaporative error becomes more significant as volumes descend into the lower part of the microliter range.

The second approach, chemical traceability, is useful for smaller volumes and can be realized using photometry. Chemical traceability is based on the accurate knowledge of a chemical concentration in the parent sample, followed by a measurement of the amount of chemical present in the dispensed droplet. A proper choice of chemical species is one that does not evaporate or degrade during the measurement. It should also produce a sufficiently strong signal that can be measured with adequate accuracy and precision. In the microliter and nanoliter range, it is important to use dyes that have a strong absorbance response and excellent stability. Several commercially available automated systems include standardized dyes to aid traceability—something not possible with home-brew systems.

Selecting the Right Method

Table II. (click to enlarge) A comparison of gravimetry and ratiometric photometry techniques.

In regulated environments like IVD manufacturing, traceability and accuracy requirements usually narrow the choice of calibration method to one of two options—gravimetry or ratiometric photometry. The four factors that most influence this selection are environmental conditions, measurement rate, volume range, and required uncertainty (see Table II).

Environmental Conditions. Gravimetry requires an environment relatively free of vibration. In addition, the accuracy of the method is improved when temperature is stable and humidity is elevated. Ratiometric photometry, although more tolerant of vibration and lower humidity, requires an environment that is free of excess dust, which could contaminate solutions and alter readings. In the clean environments typical of IVD manufacturing, ratiometric photometry is probably the more forgiving technology, but gravimetry can also be effective if volumes are large and vibration is kept low.

Figure 3. Automated liquid handlers process many samples per minute and can often benefit from measurement systems based on ratiometric photometry.

Measurement Rate. For gravimetry, the number of samples per minute needing measurement depends on balance sensitivity and environmental stability, and is generally limited to a few samples per minute. For single-channel dispensers that operate at a slow rate, either gravimetry or photometry can be appropriate. As measurement rates increase, a higher-density 96- or 384-well format may be necessary to attain sufficient measurement throughput. In these cases, ratiometric photometry holds the advantage (see Figure 3).

Volume Range. Gravimetry works best with higher volumes, while photometry has an advantage at low volumes. Commercially available photometric systems have upper volume limits of 200–1000 µl, and greater volumes may not physically fit in the measurement cells. Fortunately, balances generally work well at volumes of hundreds of microliters or more. Although some overlap in capability exists at this range, as volumes decrease into the mid- and low-microliter range, gravimetry becomes increasingly difficult and expensive. At the extreme low-microliters to nanoliters range, photometry is the only practicable choice.

Measurement Uncertainty. For gravimetric measurements, balance resolution is usually to four or five decimal places on the gram scale (i.e., 0.0001- or 0.00001-g resolution). Balances that measure to six decimal places exist but are fragile and have limited use outside a controlled calibration environment. Measurement uncertainties for four- and five-place balances in liquid-measurement operations usually range from 120 to more than 1000 nl.

For ratiometric photometry, the measurement uncertainty typically depends on the volume being measured. The best measurement capability can be less than 1 nl when measuring a 100-nl sample, or less than 1% of the measured volume. Photometric uncertainty grows with sample size, maintaining roughly a constant percentage, and can reach 1000 nl or more as sample volume increases into the high end of the photometric range.

Other Factors

Although automated technologies do present many benefits, their quality must be ensured and continually verified, like all systems and products used in producing IVDs. Procedures must be established to continually document proper functioning. In addition, although automation does reduce manual labor, some degree of human involvement is often still necessary.

Automation is not the right strategy for all operations. To determine whether automation is appropriate, thorough cost-benefit and return-on-investment analyses should be conducted before committing to a new calibration system. Factors to consider in these analyses include capital and start-up costs, cost of labor, reliability, and availability, as well as error-rate reduction and associated error costs. In automated calibration, improvements in overall labor costs and savings due to error reduction are typically the most significant factors that offset capital and start-up costs.

George Rodrigues, PhD, is senior scientific manager at Artel (Westbrook, ME). He can be reached at grodrigues@ artel-usa.com.

If the benefits of automation outweigh the costs for a specific organization, the next step is to evaluate available calibration systems. User intuitiveness is key. For a system to be widely dispersed and successful, its interface must be easy to understand. In addition, automated systems must be secure and prevent nonproficient users from improperly using the technology. These types of controls are readily available in automated systems that are compliant with FDA software requirements—another feature to consider when evaluating automated options.

Finally, the availability of training and consultation by the system provider should be considered. Each manufacturing or development plant may have different needs, objectives, and tolerances for error. It is beneficial to work with a vendor that can provide on-site support to help get the system running, to instruct users on proper use and tactics, and to troubleshoot as the need arises.

Conclusion

Faced with industrywide pressure to maintain quality and productivity, IVD developers and manufacturers are seeking innovative technologies that can help them concurrently meet both of these objectives. It has long been recognized that liquid-delivery instrumentation plays a critical role in manufacturing and quality control processes, and substantial resources are dedicated to verifying device performance. In light of new technological advancements, automated liquid-delivery calibration procedures are becoming more popular as new systems streamline quality assurance and enhance productivity.

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