1、TECHNICAL INFORMATIONIntroductionFor any given assay, many laboratory and environ-mental conditions can contribute to the accuracyof dispensing with an automated liquid handler.Among the obvious are liquid type, technique, solu-tion retention in the tips, laboratory humidity, andlaboratory temperatu
2、re. When determining the accu-racy of a liquid handler, it may not be suitable tocheck accuracy gravimetrically. For volumes lessthan 10 L, difficulties with gravimetric measure-ments are encountered due to volume loss throughevaporation. As a result, it is desirable to use anindirect, spectrophotom
3、etric method to determinethe accuracy of low-volume dispenses. This methodcan be performed for any volume and in any labwith access to a spectrophotometer. Assuming thatthe precision of a particular technique has beenoptimized to meet the requirements of an assay,then the accuracy of the dispense wi
4、ll be a matterof calibration. The BiomekFX software offers afeature to manipulate the Scaling Factor and Offsetof the volume displaced inside the mandrels for anyparticular technique, thus giving the user a methodfor adjusting the accuracy of the dispensed volume. This Technical Bulletin will show t
5、he stepsinvolved in improving the accuracy of thedispensed volume by using the calibration featureof the Biomek FX software. To accomplish this, anintroduction to Techniques and the TechniqueBrowser will be presented, followed by a more in-depth look at the Calibration tab within theTechnique Editor
6、. A detailed description of theprocess used to calibrate a pipetting technique willthen be shown by example. Briefly, the process of calibration involves:1.The creation of a standard curve with a calibratedhand pipettor. 2.The Biomek FX is used to pipette the desired vol-ume using the selected techn
7、ique with the defaultScaling Factor and Offset values of 1 and 0,respectively.3.The actual volume dispensed is calculated bycomparison of the A510/650from the platedispensed with the Biomek FX to the standardcurve. 4.A graph of the desired dispense volume vs. theactual volume dispensed is created an
8、d the slopeand Y-intercept are determined. 5.These calculated values replace the default valuesfor the Scaling Factor and Offset, respectively, inthe technique. 6.Another dispense is performed with the BiomekFX using the same technique with the newScaling Factor and Offset and determine if theaccura
9、cy requirement of the assay has been met.Techniques and Technique BrowserBiomek FX makes use of Techniques within itssoftware to control all the functions associated withpipetting. This use of techniques to control pipettingallows frequently used settings to be saved andreused in other methods rathe
10、r than having to makethe same modifications to each pipetting step withina method. The Biomek FX software, by default,selects the most appropriate technique to use foreach pipetting step when a step is created. If neces-sary, changes can be made to a selected techniqueBioRoboticsT-1915AIMPROVING ACC
11、URACY BY USE OF TECHNIQUE CALIBRATIONLisa Knapp and Curtis FarleyBeckman Coulter, Inc.2in an individual step of a method by selecting theappropriate technique and then using the Customizebutton. This ability to use saved techniques, yet stillmodify parameters in a step or select a differenttechnique
12、 altogether, provides the maximum flexi-bility and precise control of the pipetting processwith the BiomekFX. Some basic techniques andpipetting templates are provided with the Biomek FXsoftware; however, users have the flexibility ofusing them as provided, modifying them, or creat-ing new technique
13、s and pipetting templates to meetthe individual needs of any application or assay.All criteria associated with the selection of theappropriate pipetting technique are set within theTechnique Browser which can be accessed from theTools menu of the Biomek FX software. When theTechnique Browser is sele
14、cted, a list of all availabletechniques, predefined and custom, that have beencreated are listed. Highlighting the name of a tech-nique and selecting the Properties button will showwhich parameters have been designated as selectioncriteria for that technique. A technique may be cre-ated specifically
15、 for a single piece of labware or itmay be used for any pipetting with the multichannelhead. Double-clicking on a technique name or high-lighting the technique and selecting the Edit buttonaccesses the Technique Editor. In the TechniqueEditor, a drop-down list is used to select a pipettingtemplate a
16、nd six or seven tabs are displayed that areused to set values for the aspiration, dispense, mix,liquid level sensing, for Span-8 pod only, liquidtype, and calibration. For this bulletin, we are con-cerned only with the Calibration tab. Additionalinformation on the topic of creating and modifyingtech
17、niques in the Technique Browser and TechniqueEditor can be found in the Biomek Users Manualor the Biomek FX Help which is found by access-ing the Help menu of the Biomek FX software.Calibration TabThe Calibration tab of the Technique Editor(Figure 1) shows the basic formula and the textboxes that al
18、low one to change the Scaling Factorand Offset Volume of the calibration curve. Theability to manipulate these values allows a user toadjust the accuracy of the pipetting operations per-formed by the Biomek FX. If accurate, as well asprecise, pipetting of a solution is critical to an assay,calibrati
19、on may be necessary to compensate forproperties, such as viscosity, of the solution.Additionally, the minimum pipetting height for thetechnique is set within this tab. As shown inFigure 1, the calibration equation is as follows:Vdisplaced=Vdesired* (Scaling Factor) + (Offset Volume)whereVdisplaced=V
20、olume displaced in the mandrel;equal to Vdesiredwith Scaling Factor of 1and Offset of 0Vdesired= Desired Volume(the volume set in the method)Scaling = Slope, multiplier of Vdesiredused achieve anFactor accurate dispense volumeOffset = Y-intercept, a fixed amount of overagewhich is pipettedCalibratio
21、n ProcessIn the following example, the technique was opti-mized with respect to precision for low-volumepipetting (0.25 to 1.0 L) on a Biomek FX96-Channel Disposable Tip Pipetting Head (20 L)using Biomek AP96 P20 tips. Ideally, the Biomek FXshould deliver the volume entered into the softwareaccurate
22、ly as well as precisely. Unfortunately, withlow-volume dispensing, there are many factors suchas viscosity of the solution, laboratory temperature,solution temperature, and laboratory humidity thatmake accurate low-volume pipetting difficult. Eventhough the precision was within specification, thedis
23、pensed volume was inaccurate by as much as50% for a 1.0-L dispense. The requirement for theassay was to achieve accuracy of 10% at 0.25 Land 5% at 1.0 L. Figure 1. The Calibration tab within the TechniqueEditor of Biomek FX v2.1 software.3Standard CurveIn order to determine the actual volume dispens
24、ed bythe BiomekFX with the selected technique, a stan-dard curve was created with a hand pipettor to use asa means of comparison. To create the standard curve,an operator using the Artel PCS*Pipette CalibrationSystem calibrated a hand pipettor for volumes of 5,10, 15, 20, and 30 L. To perform the ha
25、nd pipettingfor the standard curve used in the calibration process,the pipette operator was required to have a coefficientof variation (CV) of 1.5% or less, with regard to pre-cision, at each volume. Each laboratory can establishprecision requirements depending on the assayrequirements, the skill of
26、 the pipette operator, and thevolumes pipetted. The actual volumes pipetted by theoperator with the hand pipettor were found to be4.97, 9.89, 14.67, 19.65, and 29.70 L, respectively.If a pipette calibration system is not available, anassumption can be made that the pipette is deliveringthe volume sp
27、ecified if it has been calibrated andmaintained according to good laboratory practice(GLP) standards and the operator is qualified bymeeting established precision requirements. An aque-ous solution of Eosin Y, disodium salt (Sigma, cat.no. E6003), was made to a concentration that deliv-ered the maxi
28、mum absorbance (A) at a dispense vol-ume of 2 L for the linear range of the plate reader, aSPECTRAmax*PLUS384. A surfactant, EDTA (Sigma,cat. no. E5134) at 5.0 g/L, was added to the solution.This 2 L dye solution, used for pipetting with theBiomek FX, was diluted 1:20 using volumetric flasks,thus ma
29、king a 40 L dye that was used for creation ofthe standard curve. The standard curve can also becreated by pipetting the desired dispense volumes,0.25 to 1.0 L, with a hand pipettor. However, it isdifficult to pipette precisely with a 0.1 to 2.0 L handpipettor and many labs do not have access to this
30、pipettor. The dilution method is presented as an alter-native to pipetting small sample volumes. Thedispensed samples were measured at 510 nm. In addi-tion, a reading is taken at 650 nm to determine back-ground. This background value is subtracted from thevalue obtained at 510 nm for all calculation
31、s. Whenthe dispenses are normalized to the same finalvolume in the well, 100 L, with water, the followingphysical property is true:When A510/650(40 L) = A510/650(2 L)then V40 L= 20 * V2 LFurthermore, the graph of A510/650(40 L) vs. V40 Lis linear (Figure 2), given in the form of A510/650(40 L) = m *
32、V40 L+ bwhere m = slope b=Y-interceptInitial Dispense AssayOnce the standard curve has been created, the nextstep of the process is to pipette with the automatedliquid handler to create the curve of Vdisplacedvs.Vactualfor the optimized, with respect to precision,technique using the default calibrat
33、ion settings, aScaling Factor of 1 and an Offset of 0 L. Severalvolumes over the range covered in the calibrationshould be dispensed for the initial assay. Thedesired dispense range for this assay was 0.25 to1.0 L. Previous experimentation established thatthe assay conditions tested here require dis
34、placedvolumes between 0.8 L and 1.4 L to yield actualvolumes in the desired range of 0.25 L to 1.0 L.The assay consisted of three plates pipetted ateach desired volume. As a result of using thedefault calibration settings, the desired volume isequal to the displaced volume. From the A510/650values i
35、n the standard curve and the mathematicalrelationship described previously between theabsorbance of the dye used for the assay and thedye used for the standard curve, one can use theA510/650(40 L) vs. V40 Lcurve to calculate theactual volumes dispensed from the unit. TheA510/650value used in the cal
36、culation of Vactualisthe average A510/650across all wells of the threeplates dispensed on the automated liquid handler. Figure 2. Standard curve made with a handpipettor from 5.00 to 30.00 L.4The calculations are as follows:A510/650(40 L) = 0.0441 * V40 L+ 0.0362And whenA510/650(40 L) = A510/650(2 L
37、)thenV40 L= 20 * V2 LThereforeA510/650(2 L) = 0.0441 * (20 * V2 L) + 0.0362 SoV2 L= (A510/650(2 L) - 0.0362)/0.882Although the standard curve shown onlyincludes volumes from 5 to 30 L using the 40-Ldye, or 0.25 to 1.5 L for the 2-L dye, experimen-tal values for actual volumes falling outside thatran
38、ge can be calculated due to the linearity of thestandard curve. The data from the initial dispense assay withthe BiomekFX using the default settings for theScaling Factor and Offset, 1 and 0, respectively, isshown in Table 1.Slope and Offset CalculationSince the objective is to obtain Vactual= Vdesi
39、red, anew graph is created for Vdisplacedvs. Vactualusingthe data from Table 1. The values from the graphshown in Figure 3 for slope and Y-intercept are usedas the new Scaling Factor and Offset, respectively,in the Technique Editor.Scaling Factor = 0.808 Offset = 0.470Once the default values for Sca
40、ling Factor andOffset are replaced by the experimentally deter-mined values in the Technique Editor, the softwarewill calculate the new values for Vdisplacedautomati-cally. The assay is repeated and Vactualis calculatedin the same manner as previously described usingthe standard curve.Table 1.Actual
41、 Volume Dispensed by the Biomek FX as Determined byComparison of OD Values to the Standard CurveDesired Displaced Average A510/650Actual (V2L) AccuracyVolume Volume (2 L Dye) Volume0.80 L 0.80 L 0.387 0.398 L -50.3%1.00 L 1.00 L 0.625 0.667 L -33.3%1.20 L 1.20 L 0.843 0.915 L -23.8%1.40 L 1.40 L 1.0
42、41 1.139 L -18.6%Figure 3. Graph of displaced volume vs. actual volume as calculated in Table 1.Since the accuracy requirement has beenachieved, further iterations of the calibration processwill not be performed. If the accuracy requirementhad not been met, Vdisplacedvs. Vactualwould begraphed again
43、 using the data from Table 2. A newScaling Factor and Offset would be calculated fromthe graph, Figure 4, and these values, 0.762 and 0.480,would be substituted for the current Scaling Factorand Offset, respectively, in the Technique Editor. Another assay would be performed and the accu-racy would b
44、e determined in the same manner aspreviously described. If, after multiple iterations, theaccuracy requirement cannot be met for all volumesin the dispense range, multiple techniques, with spe-cific volume ranges, can be created in the TechniqueBrowser. One could create several techniques withcontig
45、uous volume ranges such as 0.25 to 0.5 L,0.5 to 1 L, and 1 to 1.5 L. With each technique,the same pipetting template and parameters could beused with individualized calibration numbers,Scaling Factor, and Offset, for each range. These fea-tures offer maximum flexibility for improving theaccuracy of
46、pipetting on the BiomekFX.Table 2.Actual Volume Dispensed after Using Experimentally Determined ScalingFactor and OffsetDesired Displaced Average A510/650Actual (V2L) AccuracyVolume Volume (2 L Dye) Volume0.25 L 0.67 L 0.250 0.242 L -3.1%0.50 L 0.87 L 0.503 0.529 L 5.8%0.75 L 1.08 L 0.734 0.791 L 5.
47、5%1.00 L 1.28 L 0.952 1.037 L 3.7%Figure 4. Graph of displaced volume vs. actual volume as calculated in Table 2.Developing innovative solutions in genetic analysis, drug discovery, and instrument systems.Beckman Coulter, Inc. 4300 N. Harbor Boulevard, Box 3100 Fullerton, California 92834-3100Sales:
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