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The Star automated workstation by Hamilton Co. (Reno, NV). (Photo courtesy Hamilton Co.)
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The selection of electronic and mechanical components and software for an automated IVD system is a complex undertaking with major technical, regulatory, and financial implications for the IVD instrument manufacturer. Careful study of critical assemblies and modules and how they would work in concert enables the manufacturer’s designers and engineers, or the contract engineering firm, to make intelligent decisions that will result in developing a system that accurately embodies the manufacturer’s goals and expectations. As the complexity of the application increases, so does the need to communicate clearly in order to control the project scope and costs. At stake are system performance, quality, reliability, service, efficiency, and satisfaction, as well as cost.
Just as discoveries resulting from scientific research translate to practical diagnostics and therapeutics, so also do advancements in instrumentation technology. Automated fluidic transport systems are a classic example, being adopted into systems that facilitate a broad spectrum of IVD tests, including immunoassays, nucleic acid–based diagnostics, and clinical chemistry testing. Manual pipetting of chemistries, reagents, buffers, and samples for diagnostic assays is one of the most labor-intensive and error-prone processes in the laboratory. Thus, selection of a sound automated fluidics system is of prime importance. Of special interest to many IVD instrument manufacturers are considerations regarding automated liquid-handling systems used in preanalytical processing and sample preparation, for which mitigation of risk is a central driver.
Total Process Control
With regard to automated pipetting workstations, the concept of in-process, or total process, control refers to the ability to track, trace, and monitor all chemistry, reagents, buffers, and assay kits used in the IVD application; patient samples and corresponding aliquots; and pipetting processes, specifically all aspiration and dispense steps. Many fluidic handling systems offer mechanisms for tracking and tracing the materials, but few provide capability for monitoring the pipetting process itself. The motivation for in-process control is twofold: first, to identify and address problems early in the process, and second, to meet regulatory requirements for auditing and documentation.
No matter how robust the system, pipetting errors and tracking discrepancies will happen in today’s high-throughput diagnostic laboratory. Thus, a key element of total process control is an effective means of error recovery. Engineers and IVD instrument manufacturers should investigate automated pipetting system capabilities for total process control, error recovery, and error avoidance.
Preserving Fluidics Integrity
Maintaining the integrity of all pipetted fluids in an automated application to ensure that there is no dilution or contamination is obviously important for the diagnostic assay.
Before the advent of automated liquid handling and computer microprocessor technology, manually operated pipettors represented the only means for pipetting fluids. Modern electronic handheld pipettors, still widely used for manual applications, utilize a glass- or plastic-barreled syringe with a fitted plunger. Between the plunger and the surface of the liquid to be pipetted is air. Movement of the plunger displaces air to create negative or positive pressure, facilitating aspiration or dispensing of liquids, respectively. Notwithstanding human error, when calibrated correctly, electronic hand pipettors offer a very high degree of volumetric accuracy and precision, albeit at very low throughput.
First-generation automated pipetting systems—and some even on the market today—take a different approach, employing syringes, diluters, pumps, valves, and complex tubing to effect fluid transport. These systems typically use a system liquid rather than air to create the necessary negative and positive pipetting pressures. While they can offer the flexibility to pipette a wide range of volumes, their use of a system liquid inherently risks mixing that fluid with assay materials and contaminating or diluting reagents, buffers, or, worse, the sample itself.
Functioning similarly to electronic hand pipettes, next-generation automated pipetting systems that utilize air-displacement technology offer especially good performance, reliability, and safety. Automated liquid-handling systems based on air displacement typically do not employ pumps, diluters, valves, or complex tubing, so the pipetting platform is more robust and reliable. Importantly, this technology involves no system liquid. If disposable pipette tips are used, these systems essentially eliminate the risk of sample or reagent dilution or contamination.
Pipetting Performance
High consistency and reproducibility in performing automated pipetting steps is critical to the validity of IVD assays. Samples often arrive in the lab in different types of tubes, in different volumes, and perhaps with variable quality, so the automated liquid-handling system has to be able to accommodate this variability.
Capacitance Sensing. A reliable mechanism for liquid-level detection (LLD) in the vertical axis helps address variable-volume issues and eliminates the need to program specific pipetting heights. Most commercial automated pipetting systems employ a methodology based on electrical capacitance for conductive-fluid LLD and the recognition of troublesome fibrin clots, foam, and bubbles in the sample. Addressing these problems is especially important for short-volume samples, although it can be difficult to do with samples below 10 µl.
While a viable method, electrical capacitance does have limitations. It does not work with nonconductive fluids or nonpolar solutions, can give false readings when there is liquid on the side of the tube or static-charge buildup, and can be affected by interference from externally generated radio frequencies. Furthermore, although capacitance-based LLD was designed to help identify and address issues with bubbles, foam, or clots in the sample, these phenomena can sometimes defeat this method of detection.
IVD manufacturers should seek out automated platforms whose specification for mean time between pipetting failures approaches 500,000 steps. This specification refers to discrepancies due to system hardware or software failure. However, nonsystemic problems can arise with capacitance-based LLD.
Pressure Sensing. Automated liquid-handling technology evolves quickly along with user needs and clinical process control requirements. To identify and resolve aspiration and dispense discrepancies beyond the capabilities of traditional liquid-level capacitance often requires a more reliable and comprehensive means of monitoring the pipetting process. One recent advance involves using in-line or in-channel pressure sensors to monitor the entire process.
IVD manufacturers should consider automated pipetting systems with pressure-based LLD technology that works independently of fluid conductivity and tube geometry or diameter and provides reliable detection of leaky or clogged tips, blood clots, and foam. Pressure-based parameters can be set up to monitor both aspiration and dispensing steps in real time.
Pressure-based LLD works as follows: At the beginning of an aspiration or dispense step, the environmental absolute pressure is measured and used as a zero baseline. When the liquid level is found, the relative pressure of the air within the pipette tip is measured during subsequent aspiration and dispense steps. The measurement values are checked for whether they lie within a predefined tolerance band created from a series of pressure curves related to the specific tips used and the type of sample or liquid to be pipetted. Many labs run 100 to 200 samples in order to establish a set of mean pressure curves. Pass-fail limits are then implemented so that, when pressure values deviate from established tolerances, the pipetting step halts and the operator receives an error message, allowing for timely identification of and response to pipetting problems.
Error Recovery. All IVD instrument control software should be flexible enough to provide a broad spectrum of error avoidance and recovery options, including reversal of the aspiration motors to dispense mode before retrying aspiration from deeper in the tube, and going into not-process mode to skip a particular sample. This represents a so-called intelligent system. A traceable digital audit trail that verifies successful transfer of a sample is an indispensable tool in the IVD regulatory environment, particularly in areas such as molecular diagnostics that involve a challenging array of different sample types (blood, serum, urine, tissue, sputum, stool), each with its own peculiarities.
Pressure-based complete monitoring of aspiration and dispense coupled with capacitance-based LLD offers the best of both worlds; the user enjoys greater walk-away time, and the false alarms commonly associated with capacitance-only-based systems are minimized. An automated pipetting system that can handle the variability in sample quality and character can facilitate higher sample processing success.
To further minimize risk of contamination or sample carryover, a fluidic system should be designed to resist droplet formation at the end of the pipette tip. Liquids with high vapor pressures, such as acetone, ether, and ethanol, can evaporate quickly while in the tip, leading to droplet formation; laboratories need a mechanism to combat this. The issue is addressed by implementing a series of small aspiration steps, which retains the drop in the tip before dispensing begins.
System Precision and Accuracy
Automated liquid-handling systems with factory-precalibrated, independently operating liquid channels are advantageous for maintaining volumetric precision and accuracy. In these systems, simply swapping out the malfunctioning channel quickly addresses any pipetting volume discrepancy. System manufacturers should offer documented traceability to NIST standards for confirmation of aspirate and dispense accuracy for all factory-calibrated liquid channels.
However, virtually all manufacturers use a specially formulated aqueous calibration fluid for verification. Because certain fluid properties, such as viscosity and surface tension, can influence pipetting performance, it is important to note that calibration and traceability of the calibration fluid do not necessarily translate to other fluids. Differences in delivered volume between common lab fluids and calibration fluids used in manufacturing are inconsequential for most applications, but at sample volumes below 20 µl they may be more apparent. The user should perform the final validation studies.
The use of disposable tips for reduced sample carryover has necessitated a robust and reproducible mechanism for tip handling. Many IVD applications require high precision in tip attachment and positioning; thus, instrumentation manufacturers should make sure their automated pipetting systems afford sufficient positional accuracy and alignment of disposable tips for applications using such high-density formats as 384- and 1536-well microplates. Manufacturers should look for systems capable of delivering at least 0.1-mm positional accuracy in all horizontal and vertical axes, with reproducibility around 0.05 mm.
Other Considerations
Safety. IVD manufacturers examining automated liquid-handling components should also consider pipetting safety. Many automated systems exert a downward vertical force of approximately 8–12 lb of pressure per tip to pick up disposable plastic tips. For multichannel 96-well pipetting blocks, this amounts to almost 1000 lb of pressure, posing risk of serious injury to the careless technician. Mitigating this risk can pay dividends to manufacturer and end-user alike.
Handling of Labware. Automated pipetting workstations under consideration for integration into an IVD system should possess the ability to detect and validate all samples, reagents, and buffers before the sample preparation or assay begins. An efficient means of identifying samples and reagent racks—a bar code verification check—before they are introduced to the workstation is important because it identifies discrepancies upstream of sample processing. Tube- and plate-handling capability is another area of importance that should be included in evaluation of systems for automated fluids processing. Systems should be able to provide confirmation of successful transport of plates to integrated incubators, shakers, and other devices.
Service and Maintenance. IVD equipment manufacturers sourcing automated liquid-handling systems should compare mean time between failure estimates from system suppliers and strive for values below one required field service visit per year. Keeping the system clean and free of contaminants is enough, in some cases, to keep it performing to specification, while other systems may require more diligence. A daily regimen of tip-sealing-integrity verification, positional testing, and both capacitance- and pressure-based verification should ensure optimal performance.
System Software. Liquid-handling-instrument control software is a critical yet often underappreciated component of a clinical IVD system. Key considerations for the manufacturer are many, but all IVD software at least should be in compliance with FDA’s regulation in 21 CFR Part 11 for electronic records and signatures and should have undergone formal validation and risk analysis, substantiated and documented by the supplier or manufacturer.
Overall ease of use and new-user training requirements for the programming software should be carefully evaluated to ensure short learning curves for users and technicians. Also, since many automated pipetting systems are highly flexible to allow various sample-prep routines and assays to be performed, software that provides the ability to program a variety of deck layouts and carrier configurations rapidly with minimal user intervention helps streamline setup. Systems that require a vector-based input format for teaching pipetting positions make the technician’s work tedious and time-consuming; control software that does not require vector assignment is to be preferred. Also desirable are instrument control software that simplifies method creation and editing, and utilities that facilitate kit, reagent, and labware lot tracking for prevalidation of all consumables used.
Considering the back end, IVD system manufacturers should look for software that can integrate cleanly with in-house laboratory information management systems and provide capabilities for handling and tracking large amounts of data that should be readily accessible in a secure yet easy-to-share format. System software should additionally contain sample management and tracking features that enable total sample traceability, from primary accession tube to final process location and result.
