Sources of Error: Pipettes by Richard Curtis, Chairman and Technical Director, ARTEL
Figure 1: Today’s electronic pipettescontain many internal components andfailures are often not visible to the eye. | Liquid delivery is one of the most common processes in life science laboratories, from drug discovery and compound management laboratories to analytical chemistry and genomics/proteomics facilities. These laboratories use liquid delivery for processes including sample preparation, dilution, standards preparation and reagent addition.
However, the means for delivering liquid samples have advanced drastically over time, from the traditional glass micropipette to today’s electronic, variable volume pipettes and automated liquid handlers. Liquid delivery processes are further complicated by a radical reduction in the average volumes handled. Combining these trends with the potentially significant consequences of liquid delivery error, such as non-compliance, wasted time and money, inefficient use of scarce samples and compounds, and false data, it is clear that liquid delivery can be a major source of risk. Processes must be put in place to monitor, manage and minimize this risk, making the need for liquid delivery quality assurance (LDQA) urgent.
From improper operator technique to fluid viscosity issues, to variable environmental factors and internal pipette component damage, the sources of error are many and the potential for failure is real. Given the numerous factors that influence the accuracy and precision of volumes dispensed from devices, laboratories must first understand how liquid delivery processes can fail and the effects of such failures before they can implement optimal LDQA programs.
The magnitude of risk caused by liquid delivery devices themselves is significant. Research shows that up to 30 percent of pipettes and other liquid delivery devices currently in service are not performing within expected tolerances at any given moment. The risk of non-performing liquid delivery devices is compounded by the smaller volumes handled in today’s laboratories. This means that volumes that are inaccurate by just a few microliters can have significant effects.
Consequences of failure
In the best-case scenario, using a pipette that is not performing accurately results in the need to retest samples or reevaluate data. While this does waste time, resources and money, the consequences are not as severe as when a malfunctioning pipette generates inaccurate test results used for treatment, or when samples cannot be retested. This has a far greater impact with much higher costs of failure.
The worst-case scenario is the failure to identify in a timely fashion a pipette’s performance as being out of tolerance, leading to continued use of the pipette, and consequent reporting of inaccurate data and inaccurate results. Failure not only requires costly and time-consuming remedial action, but also puts patients and research at risk. This is one of the reasons why the FDA has embraced the concept of Process Analytical Technology, whereby quality assurance is built into a process to detect and correct potential problems.
Fortunately, careful examination often shows evidence of the source of failure, and many of these causes are preventable. Understanding pipette failure and preventing recurrence may be the most cost effective means of reducing costs and risk while improving quality and compliance.
How pipettes fail
First, it is important that laboratories define device failure. During calibration, the liquid volumes dispensed by the pipette being tested are compared against a standard and the deviation from this standard is measured. Performance outside of acceptable limits is defined as failure.
As today’s electronic pipettes are complex, relying on a number of components to function, damage is often not visible to the eye or evident by the feel of the pipette action. This is called a silent pipette failure. Silent mechanical failures can take many forms, from improper lubrication, to seal or O-ring leakage, to damage to the shaft where it seals with the tip.
While pipette maintenance should be performed on a periodic schedule and can correct the aforementioned causes of mechanical failure, data collected in a laboratory where pipettes were heavily used showed that the time elapsed since a pipette’s last maintenance did not influence the probability of it failing in the next time period. There are two important conclusions to be drawn from this data. First, the failure rate is relatively constant: it does not increase every month as one might expect. Second, in aggregate terms, approximately 27 percent of the monitored pipettes failed at some point during this six-month cycle. Even recent maintenance cannot guarantee that all pipettes will perform satisfactorily, and laboratories should expect some pipettes to fail during the time between calibrations.
Even more alarming is that only 10 percent of pipette failures are due to normal wear factors such as frequency of use and time since last maintenance. On the other hand, 90 percent of failures are random and unpredictable, caused by incidents such as accidents or misuse.
Verifications of performance
To offset the risk and impact of out of tolerance pipettes and to quickly identify those that are failing, regular calibration programs and verification checks must be implemented.
Critical to an effective calibration program is the frequency at which calibrations are conducted, and the optimal frequency depends on the following factors:
• Mean Time Before Failure:
The average rate at which failures occur can be expressed as Mean Time Before Failure (MTBF). As opposed to the failure rate, measuring the number of failures per unit of time, the MTBF measures the cumulative number of failures in a group of pipettes over a period of time to determine the average time elapsed between failures. Therefore, the MTBF and failure rate are inverses to one another: a high MTBF is based on a low failure rate. A high MTBF is desirable because it means that the chance of any given pipette having failed is small.
Using the MTBF, one can predict how long a pipette can be expected to maintain accuracy and precision. The MTBF for individual pipettes can vary significantly, depending on a number of factors. For example, a pipette used daily will fail more quickly than a pipette used less frequently.
One way to determine MTBF, is to track a group of pipettes until each one fails, determining how long it takes each pipette to fail.
• Target reliability level:
Current best practices for pipette management are to establish target reliability levels at 95 percent or higher. This means that on recalibration or verification, at least 95 percent of the pipettes will be found to be operating within established tolerances, with only 5 percent generating incorrect results. When determining a desired target reliability level, factors such as assay precision, the impacts of failed pipettes on test results, legal defensibility of results and production batch release decisions are important.
Once the established target reliability level for a laboratory and the MTBF for the pipette population are determined, the the optimal calibration frequency can be found.
• Quality control principles:
Like all precision laboratory instruments, mechanical action pipettes should be subject to quality control principles and should be calibrated on a regular basis.
The more frequently pipette checks (calibration or verification) are performed, the sooner malfunctioning pipettes will be detected and taken out of service. Conversely, the longer a defective pipette remains in service, the greater the liability it presents.
• Regulations:
Regulations and standards published by organizations such as the ISO, the FDA, Clinical Laboratory Improvement Amendments and ASTM provide minimum requirements with varying degrees of specificity that help ensure the quality of lab results. These form the groundwork upon which a lab should establish the frequency of pipette calibration.
It is important to note that documentation is critical to developing and implementing a regular calibration program, as well as complying with regulations. If it is not documented, it did not occur in the eyes of regulatory bodies.
For more information, contact: ARTEL, 207-854-0860, www.artel-usa.com
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