he pipette isa reliableprecisioninstrument

that has been used and trusted formany years. However, as withmany forms of instrumentation, apipette will perform only as wellas the operator’s technique allows.

Differences in technique –some more than others – can alterdelivery volumes. As yourlaboratory’s demand for accuracyand precision increases, theimportance of understanding anddeveloping optimal pipettingtechnique becomes imperative.Let’s review the results of a studyconducted at ARTEL to determinethese differences and assess theirimpact upon accuracy andprecision.

Method & ProcedureThe reference pipetting method

used in each of these experimentswas as follows: The pipettemechanism was “warmed up” bygently depressing and releasing theplunger 15 to 20 times. The pipettetip was prewet by aspirating anddispensing an aliquot of the samplesolution three times.1 With theplunger depressed to the first stop,the tip was immersedapproximately 1 millimeter intothe sample solution and held therefor a half second. The aliquot wasaspirated from the sample solutionby gently releasing the plunger,keeping the tip in the samplesolution for two seconds beforeremoval. The aliquot was delivered

into the blank solution vial byplacing the pipette tip on the sideof the glass vial at a 45° angle justabove the meniscus and slowlydepressing the plunger past the firststop to deliver the entire aliquot.

Each experiment consisted oftwo runs of ten data points each,using an adjustable 20 µLEppendorf manual action airdisplacement pipette† set at5.00 µL and Eppendorf disposablepipette tips, on the ARTEL PCS®

Pipette Calibration System, anautomated instrument/reagentsystem which verifies thevolumetric delivery of pipettes.Each experiment was performedas a comparison of one pipettingtechnique versus another. Theresults for each pair of techniqueswere compared concerningprecision and accuracy. Themeasurement used for rating theprecision of each technique wasthe coefficient of variance (%CV).Accuracy was defined as thepercent difference in mean deliveryvolumes between the two pipettingtechniques.

Eliminating sourcesof error■ Don’t leave your tipshigh and dry.

The experiment comparing dryand prewetted pipette tips revealedthe greatest discrepancies observedduring this study. Dry pipette tipsconsistently delivered significantlylower volumes than did theprewetted tips, a fact which otherresearchers have noted.2-6 Thoughno difficulty with precision wasobserved using either methodexclusively in a single run,differences in accuracy of up to7% between runs using dry andprewetted tips were noted whileusing the 20 µL pipette set at 5 µL.Additional experiments using a250 µL pipette set at 25, 50, 100and 250 µL consistently showeddifferences in the accuracy of thevolume pipetted of up to 2%.

Prewetting the pipette tipinfluenced accuracy by increasingthe humidity within the tip, thusminimizing evaporation of thesolution. Similarly, increasedambient humidity minimizedevaporation. The beneficial effectof prewetting was less significantwith constant or high ambienthumidity. Ambient humidity forthese runs was 50%.

■ Not too hot... not too cold...

Variation in the temperature ofthe solution being pipetted wasobserved to be the second largestcause of pipetting error in this study.Three sample solutions were

techniqueImpact of pipetting

“...a small mistake in

pipetting can cause a

large error in the final

result. It is, therefore,

of great importance to

evaluate and to reduce,

wherever possible, both

random and systematic

errors in liquid sample

handling.” 7

Sari Ylätupa,PhD,

“Choosing a Pipetting

Technique Affects the

Results of Your Analysis”,

European Clinical

Laboratory

T

† In an air displacement pipette,many sources of imprecision orinaccuracy are magnified by theratio of “dead air” above the liquidlevel to the liquid volume in thetip.2-6 In conducting theseexperiments, a 20 µL pipette wasused to deliver 5 µL, a ratio of 4:1,so that the amount of errorintroduced by technique variationswould be large enough to measurereliably.

19A3227A

What are other techniques which can affect accuracy and precision?

Other error causing techniques noted in this study included:a. prolonged delay between aspiration and removal of the tip fromthe sampleb. dragging the tip along the side of the container when the tip isexiting the samplec. variations in the size and shape of the sample container, andd. rate of plunger depression and release.

What impact will these techniques have upon my results?

Individually none of these factors resulted in an error greater than 2%.Cumulatively, however, two or more of these sources of error (e.g.,prolonged delay and rate of plunger depression) could affect deliveredvolume significantly.

What factors other than technique differences can affect my results?

Component failure (e.g., a plunger seal or corroded piston), incorrectpipette tip, or incorrect installation of the tip can also affect your results.

References:1 Zeman GH, Mathewson NS. “Necessity of prerinsing disposable polypropylenepipet tips”, Clin Chem 1974; 20(4)497-8.

2 Sternberg JC. “Sampling with air-piston pipettes – a critical study”, Clin Chem1975; 21(7):1037.

3 Ellis KJ. “Errors inherent in the use of piston activated pipettes”, Anal Biochem1973; 55:609-14.

4 Wenk RE, Lustgarten JA. “Technology of manually operated sampler pipettes”,Clin Chem 1973; 20(3):320-3.

5 Joyce DN, Tyler JPP. “Accuracy, precision and temperature dependence ofdisposable tip pipettes”, Med Lab Technol 1973; 30:331-4.

6 Lochner KH, Ballwegt, Fahrenkrog HH. “Factors influencing the measuringaccuracy of piston pipettes with air interface (German)”, J. Lab Med 1996;20(7/8)430-440.

7 Ylätupa Dr. S. “Choosing a Pipetting Technique Affects the Results of YourAnalysis”, European Clinical Laboratory 1996. 10:14.

&Answers

lab report © is published by Artel, Inc. for laboratory professionalsand all pipette users.

For more information, please contact Artel, Inc.

Tel: 207-854-0860 Email: post@artel-usa.com

Fax: 207-854-0867 Web: www.artel-usa.com

25 Bradley Drive, Westbrook, Maine 04092-2013

brought to three differenttemperatures: 8.5 °C, 25.0 °C,and 30.0 °C. Solutions that werewarmed to 30.0 °C consistentlydelivered lower volumes thanroom temperature samples.Similarly, solutions that werecooled to 8.5 °C delivered highervolumes than the ambient(25.0 °C) sample. The accuracydifferences observed in this studywere significant, ranging from3 to 7%. The graph below showsthe effect of sample temperatureon pipetting accuracy.

In addition to accuracyproblems seen with samples thatwere not at room temperature,there was some difficulty inobtaining good precision. Runstypically produced between0.50 % and 1.00 %CV, althoughthe %CV went as high as 3.60.As the solution was allowed toapproach room temperature, theprecision of the results improved.Similarly, if the pipette end or tipwas warmed, even just by casualhandling, differences in deliveryvolumes were observed. Thesedifferences and inconsistencieswere smaller and less significantthan those resulting fromvariations in solution temperature.

■ Shift out of Reverse

Reverse mode pipetting is amethod of pipetting commonlyused with viscous liquids. This

is a technique in which theplunger is depressed past the firststop to aspirate the aliquot fromthe sample, and depressed onlyto the first stop to deliver thealiquot. Reverse mode pipettingcan make obtaining accurate andprecise results (for the pipettingof solutions similar to water interms of density and surfacetension) more difficult. The studyshowed that a typical precisionfor this method was 1.4 %CV.Differences in volumes deliveredby standard and reverse modetechniques ranged up to 5%. Thereverse mode consistentlydelivered a higher volume thanthe standard method of pipetting.

■ Conclusions

Techniques among pipetteusers vary with background,personal preferences, andtraining. These differences inexecution can affect the accuracyand/or the precision of resultsbeing released from the clinicalor research lab. To ensureaccuracy and consistency,facilities should adopt standardoperating procedures for pipettingtechniques and ensure that alloperators are trained to anadequate level of proficiency. Byincreasing the level of consistencyin results obtained, the level ofquality and credibility of thefacility will be enhanced.

Effects of Sample Temperature on Pipetting Accuracy

Experiment showing the effect of sample temperature on the accuracy of pipettingresults, using a 20 µL adjustable air displacement pipette set at 5.00 µL. Ambienttemperature 25.0 °C.