Minimizing Liquid Delivery Risk: Pipets as Sources of Error

This is the first in a series of articles entitled, “Minimizing Liquid Delivery Risk,” addressing the most common causes of liquid delivery error and providing guidance on how to address and overcome these errors. Sources of error covered in the series will include pipet and automated liquid handler failure; operator technique; environmental factors such as temperature, humidity, and barometric pressure; and selection of liquid delivery equipment.

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 micropipet to today’s mechanical action or variable-volume pipets 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 noncompliance, 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, variable environmental factors, and internal pipet 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% of pipets and other liquid delivery devices currently in service are not performing within expected tolerances at any given moment. The risk of nonperforming liquid delivery devices is compounded by the ever smaller volumes typically handled in today’s laboratories. This means that volumes that are inaccurate by just a few microliters can have significant effects on results. For these reasons, error caused by handheld pipets will be the focus of this inaugural article in the “Minimizing Liquid Delivery Risk” article series, and automated liquid handlers the focus of the next.

Consequences of pipet failure

In the best-case scenario, using a pipet 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 pipet 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 pipet’s performance as being out of tolerance, leading to continued use of the pipet and consequent reporting of inaccurate data and inaccurate results. This is clearly unacceptable for life and health science laboratories. Failure not only requires costly and time-consuming remedial action, but also puts patients and research at risk. (This is one of the reasons the FDA has embraced the concept of Process Analytical Technology, whereby quality assurance is actively built into a process to detect and correct potential problems as they arise.)

Fortunately, careful examination often shows evidence of the source of failure, and many of these causes are preventable. Understanding pipet failure and preventing recurrence may be the most cost-effective means of reducing costs and risk while improving quality and compliance.

How pipets fail

First, it is important that laboratories define device failure. During calibration, the liquid volumes dispensed by the pipet being tested are compared against a standard, and the deviation from this standard is measured. Performance outside of acceptable limits is defined as failure.

Figure 1 - Today’s mechanical action pipets contain many internal components, and failures are often not visible to the eye.

Because today’s mechanical action and electronic pipets are complex, relying on a number of internal components for proper function, damage is often not visible to the eye or evident by the feel of the pipet action. This is called a silent pipet failure. Silent mechanical failures can take many forms, from improper lubrication to seal or O-ring leakage, damage to the shaft where it seals with the tip, corrosion of the piston, and contamination by the materials being pipetted. These errors can be highly detrimental if operators unknowingly use malfunctioning pipets in critical assays, diagnostic tests, and experiments (Figure 1).

Figure 2 - The data presented in this graph show a relatively constant failure rate as months elapse between calibration cycles. This means that even recent maintenance does not guarantee that all pipets will perform satisfactorily and that laboratories should expect some pipets to fail during the time between calibrations.

While pipet maintenance should be performed on a periodic schedule and can correct the above-mentioned causes of mechanical failure, data collected in a laboratory where pipets were heavily used showed that the time elapsed since a pipet’s last maintenance did not influence the probability of it failing in the next time period. Figure 2 shows the percentage of pipets that failed each month for the six months between calibration intervals. There are two important conclusions to be drawn from these data. First, the failure rate is relatively constant: It does not increase every month as one might expect. Second, in aggregate terms, approx. 27% of the monitored pipets failed at some point during this six-month cycle. Even recent maintenance cannot guarantee that all pipets will perform satisfactorily, and laboratories should expect some pipets to fail during the time between calibrations.

Even more alarming is that only 10% of pipet failures are due to normal wear factors such as frequency of use and time since last maintenance. On the other hand, 90% of failures are random and unpredictable, caused by incidents such as accidents or misuse. For example, piston corrosion or premature seal failure may result if an operator accidentally draws liquid into the body of the pipet. Also, if the operator lays the pipet down while some fluid still remains in the tip, the fluid can flow up inside the shaft and contaminate seals and rings.

Solution: Regular calibration and verification of pipet performance

To offset the risk and impact of out-of-tolerance pipets 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:

  • MTBF: The rate at which failures occur is related to the mean time before failure (MTBF). As opposed to the failure rate represented in Figure 2, measuring the number of failures per unit of time, the MTBF measures the cumulative number of failures in a group of pipets over a period of time to determine the average time elapsed between failures. Therefore, the MTBF and failure rate are inverse to one another: A high MTBF results from a low failure rate. A high MTBF is desirable because it means that the chance of any given pipet having failed is small.