Calibrate good times

We find out if light measurements really can be used for volume verification

 Laboratories often assume that their liquid handling instruments, from pipettes to automated liquid handlers, are operating within specifications. However, because data integrity for applications, from drug discovery to molecular diagnostics, relies on accurate and precise liquid delivery, this is a risky assumption with potentially high failure costs.

Liquid-delivery instrumentation can be verified using gravimetry, which measures liquid weight on analytical balances. This method is adequate for larger volumes such as millilitre quantities, but is not as effective at lower volumes such as microliter and nanolitre quantities. However, laboratories are increasingly working with smaller and smaller volumes and this trend is expected to continue.

One reason test volumes are getting smaller is the rising cost of reagents. Laboratories are challenged with using lower volumes of reagents to keep costs under control, while at the same time technologies have been developed to analyse smaller volumes more precisely. This trend requires new calibration methods that can handle low-volume verification.

Indeed, the accuracy of gravimetric processes is affected by a variety of environmental factors, including evaporation, static electricity and vibration. This uncertainty grows as test volumes decrease. Another limitation with gravimetry can be seen with verifying the performance of multichannel liquid handlers, which have become a mainstay of the modern drug discovery lab. Gravimetric methods either weigh volumes dispensed from all channels at once, which only provides an aggregate assessment of all channels in a multichannel array, or proceed with a slow serial weighing of each individual channel, which is time consuming. Either way, this approach does not mimic actual liquid handling processes and therefore limits gravimetry’s applicability.

Other calibration methods, including fluorometry and single-dye absorbance photometry have limitations as well. In single-dye absorbance methods, the dynamic range of the chromophore is too narrow to evaluate all of the dilution steps required to make the dose response curve. In contrast fluorometry provides the required sensitivity, but lacks the traceability associated with absorbance methods. These limitations often force users to make assumptions about the repeatability of the liquid handler over multiple aspiration and dispense cycles.

Highly accurate and precise, even at low volumes, ratiometric photometry provides laboratories with an easy-to-use process to validate assay results quickly and enhance laboratory efficiency

Ratiometric photometry – measuring light absorption to verify volume – has been proven as a reliable solution to the accuracy risks posed by other existing methodologies. Highly accurate and precise, even at low volumes, ratiometric photometry provides laboratories with an easy-to-use process to validate assay results quickly and enhance laboratory efficiency. This translates into greater data confidence, more reliable regulatory compliance, and cost reductions due to elimination of repeated assays and remedial action.

While the science behind ratiometric photometry for volume verification is complex, Maine-based Artel has been using it to lead the way in liquid handling quality assurance technology for the past 30 years. The company has evolved ratiometric photometry into the core of its technological strengths by integrating it into easy-to-use, robust, and rapid measurement systems for liquid handling equipment. Artel’s volume verification systems – the PCS (Pipette Calibration System) and the MVS (Multichannel Verification System) – employ ratiometric photometry through dual-dye, dual-wavelength absorbance measurements to determine a target volume of sample solution dispensed from a pipette or liquid handler.

Two colorimetric dyes, with distinct absorbance maxima at 520 nm (red dye) and 730 nm (blue dye), are used to make this measurement possible. Volume measurements are determined by applying the Beer-Lambert Law, which states that the measured absorbance of a dye is proportional to the concentration of a dye, the molar absorptivity of the dye, and the path length of the sample holder. If both the molar absorptivities and concentrations of the dyes are known and closely controlled, the law can be used to determine an unknown path length traversed by a photometric light beam. By measuring the path length through the solution and by knowing the dye concentrations, the unknown volume can be calculated through a series of equations.

The precision benefits of photometric calibration are numerous. Drug companies have made huge investments in automated lab equipment and need methodologies to verify that equipment is functioning properly, and that it is optimised for maximum productivity. There are increasing regulatory demands on manufacturers to ensure that quality control processes are in place throughout the production cycle. Before, these pressures were felt more on the downstream side, but increasingly there has been greater regulatory scrutiny on upstream R&D functions. Business pressures are also intense. When the clock is ticking on patent coverage for promising new drugs, development delays can literally amount to millions of dollars per day in lost revenue.

In a highly competitive market facing pending patent expirations of mature drugs, everyone is racing to find those next-generation blockbuster drugs. Thus, there are enormous hidden costs if data integrity is compromised. If reagents or samples are handled improperly in high throughput screenings, the potential losses from a “false negative” could be in the billions of dollars, to say nothing of the human costs involved in preventing the next medical breakthrough from being discovered.

Laboratories that apply ratiometric photometry, as implemented in the Artel MVS and PCS, find the technology convenient and easy to use. Rather than develop and validate their own measurement methods, laboratories can take advantage of a standardised product which is traceable to the international measurement system (SI) and is fully compliant with metrology standards such as ISO 17025 and ISO 8655. This allows laboratories to focus on their core work with confidence – knowing that their liquid handling processes are performing properly.

Ratiometric photometry raises the bar, promoting uniform standards of accuracy for laboratories around the world. The International Organisation for Standardisation’s ISO 8655-7 standard formally recognises photometry for calibration. In developing the standard, ISO acknowledged that it could allow laboratories to minimise uncertainty in liquid delivery verification, particularly at the small volumes that are so common in today’s research.

Documentary standards (such as ISO) are important as drug discovery and development continues to globalise because it provides an internationally approved verification method. This facilitates compatibility and harmonisation among laboratories, and it provides for easier method transfer so that projects can move seamlessly from one lab to another, across borders and oceans.

The need for liquid delivery quality assurance is more critical than ever. Even as the regulatory environment in life sciences becomes more stringent, budgets and staff continue to shrink. More than ever, laboratories require a more accurate, precise and convenient methodology to verify the performance of liquid handling instrumentation. By providing labs with a user-friendly approach to ratiometric photometry, Artel’s volume verification systems strengthen confidence in assay results and enhance data quality.