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Pipette Calibration System Strengthens Data Integrity
by Richard Curtis, Ph.D., Chairman and Technical Director, ARTEL
This article presents data showing the frequency and magnitude of pipetting error in laboratories.
Experiments
Air displacement pipettes are complex precision instruments. The accuracy and precision of a properly functioning pipette in the hand of a trained operator are exceptional, even for volumes as low as 5 microliters. Unfortunately, in day-to-day operations, pipettes fail due to wear and tear, unexpected mishaps and operator technique variation. These sources of error impact liquid delivery volumes and, consequently, data integrity. To demonstrate the origin and prevalence of pipetting error in laboratories, three data sets were collected. First, baseline data were collected using trained operators and a properly functioning pipette. Next, pipette malfunction was evaluated, and lastly, operator technique errors were measured.
Baseline with Error Sources Eliminated
To establish baseline conditions, two trained pipette operators used a properly functioning 20-microliter pipette set at 5 microliters to pipette 10 sets of five deliveries. The pipettings were done under laboratory conditions at normal speed (see Table 1). In all cases, the accuracy was better than 2% and the precision better than 1%.
Table 1: Accuracy and Precision Results from Trained Operators using Properly Functioning Pipette
OPERATOR 1 |
|
|
OPERATOR 2 |
Imprecision (% CV) |
Inaccuracy (% absolute) |
Imprecision (% CV) |
Inaccuracy (% absolute) |
1.16 |
2.08 |
.80 |
0.00 |
0.93 |
0.52 |
0.39 |
0.16 |
0.52 |
0.32 |
0.41 |
0.28 |
1.32 |
0.28 |
0.64 |
0.12 |
0.55 |
0.20 |
0.88 |
0.32 |
0.65 |
0.24 |
0.63 |
0.60 |
0.59 |
0.44 |
0.38 |
0.64 |
2.16 |
0.08 |
0.49 |
0.60 |
0.63 |
0.84 |
0.75 |
0.28 |
0.90 |
1.68 |
0.82 |
1.36 |
Mean 0.94 |
Mean 0.67 |
Mean 0.62 |
Mean 0.44 |
Error Source: Pipette
To test pipette failure rates, the same trained operators evaluated the performance of pipettes in use in four laboratories at an internationally recognized biomedical research institute. Fifty-four 20-microliter variable volume pipettes were tested at 5 microliters using the PCS, and the inaccuracy and imprecision of each pipette were plotted (see Figure 1).
Pipettes as Sources of Error.
Click to enlarge. |
The data showed that the ordinary laboratory pipettes were not in good condition.
Only 12 of the 54 pipettes (22%) produced accuracy and precision results as good
as the baseline case, and five pipettes (9%) were severely underperforming with
inaccuracy worse than 20%. These data show that the mechanical condition of laboratory
pipettes cannot be taken for granted.
Error Source: Operator Technique
Next, operator skill was tested. Fifty-four technicians working in QC laboratories at four pharmaceutical companies were tested. Each used the same properly functioning 20-microliter pipette set at 5 microliters. Their technique was tested with the PCS under actual laboratory conditions. The percent inaccuracy and imprecision data are shown in Figure 2. Only 19% of the laboratory personnel produced results as good as or better than the trained operators in the baseline case. Six operators (11%) produced errors more than 10 times larger than the baseline case.
Summary of Results
Operator Technique as Source of Error.
Click
to enlarge. |
Properly maintained pipettes in the hands of skilled operators are capable of
reliably delivering 5 microliters of liquid with better than 2% inaccuracy and
1% imprecision. However, few experienced pipette operators can meet this standard
without some training in pipette technique. Moreover, many pipettes in working
laboratories are malfunctioning and in need of repair. The errors contributed
by pipette failure and untrained operators can be very large, and are frequently
more than 10 times the optimal level.
Problem: Questionable Data
Errors of this magnitude are unacceptable. Clearly, diligent maintenance of pipettes and training of operators would increase overall laboratory accuracy and efficiency. Yet, few laboratories have attained this optimal state of pipetting proficiency.
One explanation for this failure is the limitations of the gravimetric pipette
testing method when working with microliter volumes. The gravimetric method, which
weighs liquids on balances to determine volume, was developed when protocols were
at the milliliter scale or higher. At the microliter level, the gravimetric method
is time consuming, technically challenging and subject to human error and variation
caused by environmental conditions. Outsourced pipette calibration services can
repair failed pipettes but not address the issue of operator training.
Table 2. Summary of experiment results
|
|
% Inaccuracy |
|
|
% Imprecision |
|
|
|
# |
Range |
Mean |
Median |
Range |
Mean |
Median |
Baseline |
2 |
0-2 |
<1 |
<1 |
0-2 |
<1 |
<1 |
As-Found Pipettes |
54 |
0-37 |
5 |
2 |
0-11 |
2 |
1 |
Lab Personnel |
54 |
0-34 |
4 |
1 |
1-69 |
9 |
2 |
Solution: PCS® Pipette Calibration System
Faster and more accurate than gravimetry, photometry is becoming the method of choice for pipette calibration. Measuring light absorption to verify volume, photometric pipette calibration systems are generally five to seven times faster than gravimetric methods and have greater accuracy for volumes less than or equal to 200 microliters. This makes in house pipette checking and operator training significantly more practical.
PCS is a rapid, easy to use photometric instrument and reagent system unique for its dual dye Ratiometric Photometry approach resulting in improved accuracy and precision. The system includes a reagent kit and a photometric instrument (Figure 3). The instrument guides the operator through the test plan for the selected pipette and an optional pipette tracking software program is available to simplify scheduling and data management.
PCS® photometric calibration system.
|
Speed, ease of use and accuracy make the PCS well-suited for training technicians
in proper technique as well as for frequently checking to detect pipette failure.
PCS provides an opportunity for all laboratories to increase accuracy and efficiency
by controlling pipetting error and thereby strengthening data integrity.
Richard Curtis, PhD, chairman and chief technology officer at ARTEL, may be contacted
at rcurtis@artel-usa.com
or by phone at 207-854-0860.
AT A GLANCE
• In day-to-day operations, pipettes fail due to wear and tear, unexpected mishaps and operator technique variation
• Laboratories have traditionally used gravimetric calibration or outside calibration services to ensure against pipetting error
• Errors contributed by pipette failure and untrained operators can be more than 10 times the optimal level
• A photometric instrument and reagent system can deliver improved accuracy and precision
ONLINE
For additional information on the companies discussed in this article, see Laboratory Equipment magazine online at www.LaboratoryEquipment.com or the following Web site:
• www.artel-usa.com
Laboratory Equipment
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Rockaway, NJ, 07866
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