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Verifying Multichannel Pipettor Performance with Standard Dispense Solutions in the SpectraMax Plus (MaxLine Application Note #28)


INTRODUCTION
Pipettors often fail to meet the manufacturers stated specifications1-5 and pipetting is a major source of laboratory error even when the pipettor is operating properly. Accuracy and precision specifications typically provided by pipettor manufacturers are obtained under ideal conditions. In the laboratory, performance may be very different due to the varying technical skills of the operators, environmental factors and the nature of the solutions being dispensed.1,6 - 8 In order to reduce pipetting errors, an integral part of the QC system in every laboratory should be verification of pipettor calibration. Pipettor manufacturers do not generally specify the frequency of calibration because it depends on usage. The College of American Pathologists, the National Committee for Clinical Laboratory Standards (NCCLS) and the American Society for Testing and Materials (ASTM) suggest a quarterly check and monthly quick checks, or more frequently as indicated by the physical condition or extent of use of the apparatus. 6,7,9

Two common methods for checking precision and accuracy of pipettors are gravimetric and spectrophotometric.1,6-8 The gravimetric method is based on the weight of water dispensed from the pipettor and is considered the primary method.6-9 The water is weighed using a balance calibrated with NIST-traceable weights. The actual dispensed volume is calculated from the measured weight and the density, taking into account temperature and evaporation rate. The gravimetric method is useful for dispense volumes of 10 L and above.

The spectrophotometric method employs a solution containing a known concentration of a highly-colored chromogen. Aliquots of the sample solution are dispensed into a known volume of diluent and the absorbance measured. The actual dispensed volume is then calculated from the absorbance, the light path, the extinction coefficient and the diluent volume. Various chromogens have been used, including Ponceau S dye, methyl red, Evans Blue dye and nicotinamide.1 ,2,10,11 The College of American Pathologists and the NCCLS recommend potassium dichromate because it can be obtained as a primary standard.1,2

The NCCLS and the ASTM recommend that for every pipettor, 10-replicate tests for calibration verification, measuring both inaccuracy and imprecision, be performed at least quarterly and after any maintenance.7,9 For the monthly quick check they recommend 4-replicate tests. If a laboratory has only a few single-channel pipettors, these guidelines can be reasonably accommodated using the gravimetric method. However, in laboratories having numerous pipettors, including multi-channel devices and automated pipetting stations, the testing requirement becomes extremely tedious at best, and impractical at worst. A much more practical approach for multichannel pipettors would be a spectrophotometric procedure in a 96-well format such as that suggested by Kaufman and Wobig.10 The delivery volume of one channel is checked gravimetrically, and the calibration adjusted if necessary. The remaining channels are then compared to the calibrated channel by dispensing a dye solution into the wells of a 96-well plate and measuring the absorbance in a microplate reader. Accuracy is calculated relative to the first channel with a separately-determined calibration factor of absorbance change per microliter.

An important (though often ignored) consideration in pipettor calibration is the fact that variations in surface tension, viscosity, density and temperature of the dispense reagent will lead to differences in dispensed volumes.1,4,7,8,12,13 Addition of a solute (whether buffer, preservative, protein or chromogen) to water will lower the surface tension and tend to increase the dispensed volume.1 Thus the volumes of reagents dispensed during routine laboratory procedures may differ from the dispense volume of pure water.

Ideally, pipettor performance should be checked using the actual dispense reagent as the calibrator solution. However, most dispense reagents are colorless and an absorbance measurement cannot be obtained in the visible range (400750 nm) without the addition of a dye. The SPECTRAmax PLUS microplate spectrophotometer overcomes this limitation by using the near infra-red absorbance of water to determine the pathlength in each well, which is proportional to volume. The dispensed volumes are calculated by reference to a pathlength/volume standard curve (prepared separately with a certified pipettor). The standard curve can be prepared once for a given dispense reagent and batch of microplates and stored for subsequent use.

This application note describes how to check the performance of multichannel pipettors with the SPECTRAmax PLUS. Pathlengths in the wells are measured directly and volumes are calculated automatically. The performance can be checked using the same buffers or reagents that are being dispensed by the pipettor, eliminating possible discrepancies in pipettor performance between normal dispense reagent and calibrator solution. Because initial separate calibration of one channel is not necessary (e.g., as in reference 10), the method is suitable for checking performance of automated pipetting stations, including 96channel pipettors. The spreadsheet capability of SOFTmax PRO software allows the data to be calculated automatically and final results to be displayed as overall Pass/Fail for each pipettor.


MATERIALS
1.
SPECTRAmax PLUS microplate spectrophotometer

2. High-quality microplates; e.g.:
For dispense volumes >150 L
Greiner flat bottom plates (E & K Products, catalog #565101)
Nunc flat bottom plates (Fisher Scientific, catalog #12565226)

For dispense volumes 40 L - 150 L
Costar half area EIA/RIA plates (Costar, catalog #3690)

3. Certified pipettor (previously calibrated with dispense reagent) and tips for obtaining pathlength-vs.-volume calibration curve. (References 3,4 & 7-9 all contain procedures for gravimetric calibration of single pipettors taking into account the density of the liquid.)


OBTAINING A CALIBRATION CURVE
(Note: this procedure may be omitted if you have already generated a calibration curve)

Creating Plate sections for the calibration curve
A certified pipettor is used to dispense defined volumes of reagent (at least 8 replicates per volume) into the wells of a microplate. You can use a single microplate for the entire calibration curve. However, you should pipet, then immediately read, the set of wells for each given dispense volume, in order to minimize evaporation errors. You will therefore need to create a separate Plate section for each dispense volume, with the appropriate strip (i.e., the column in the microplate corresponding to the dispense volumes location) selected to be read.

Step 1 Select an empty Plate section, or create a new Plate section.

Step 2 Set up the Instrument Settings dialog box for the Plate section as shown in Figure 1. Select to perform an endpoint read at any wavelength. (SOFTmax PRO requires that you enter a UV/Vis wavelength at which to read, even though this measurement will not be used in calculating pathlength. Select PathCheck with Water Constant and deselect Pre-Read Plate. (Pre-reading is unnecessary because the UV/VIS measurement is not used in calculating pathlength.) Double click the Strips... button and select to read only the first column of the microplate.


Step 3 Click the Reduction button in the Plate Sections tool bar to display the Reduction dialog box (Figure 2 ). Set the Data Mode to Absorbance. Select Custom from the Wavelength Combination pop-up menu, then click the formula button to display the Calculation dialog box. Type !Pathlength in the Formula field of the dialog box, then click the OK button to return to the Reduction dialog box.


Note: the Apply Pathcheck box can be ignored for this application (it refers to pathlength-correction of the UV-Vis wavelength that you had to select in Step 2, but will not be using).

Step 4 Click the Display button in the Plate sections toolbar to bring up the Display dialog box (Figure 3). Click the Number button under the Reduction: Custom heading to set the pathlength data to be displayed as a number. Do not check the Number box under the Raw OD heading.


Note: if the Number button under the Raw OD heading is selected, the data displayed will be the OD values at the UV/Vis wavelength that you selected in Step 2, not pathlength, which could cause confusion.

Step 5 With the first Plate section selected, create additional Plate sections. Because the first Plate section is selected, its Instrument, Reduction and Display settings will be duplicated in each of the new Plate sections. Create one Plate section for each intended dispense volume in your calibration curve (this example uses 10 dispense volumes, and therefore 10 Plate sections).


Defining the strips and templates for the calibration curve Plate sections
Step 1 In Plate#1s Template Editor, select to create a new group (doing so will cause a Group Settings dialog box will appear). Enter a name for the group, (BufferA in this example) and select Standards from the Column Format pop-up menu. Type Volume into the Sample Descriptor field and L into the Units field (Figure 4). Click OK to return to the Template Editor.


Step 2 Select the first column of wells in the template. Type a sample name into the Sample field (e.g. the intended dispense volume), then type the intended dispense volume into the Volume field. In the example, the group was named BufferA and wells A1-H1 of Plate #1 were named 300ul and assigned a volume of 300 L (Figure 5).


Step 3 Open Plate #2 and change the designated strip to column #2 in the Instrument Setup dialog box. In the Template Editor, assign the wells in the second column to the same Group as in Plate#1, then type in the appropriate sample name and volume. In this example, wells A2 through H2 were assigned to BufferA and named 275ul.

Step 4 Repeat Step 3 for the remaining Plate sections, selecting the appropriate column of wells to be read, assigning them to the same group, then giving them sample names and volumes that correspond to their intended dispense volumes.


Preparing and reading the calibration curve plates
Step 1 Select a clean, dry microplate for the calibration. If the calibration curve uses volumes> 150 L, use a regular 96-well plate. For dispense volumes of 40 L to 150 L, use a Costar half-area plate (to maximize the NIR absorbance values and increase sensitivity.

Step 2 Using the certified pipettor, fill column 1 of the microplate with the first chosen dispense volume. In this example, 300 L volumes were dispensed into column 1 the microplate.

Step 3 Read Plate#1 in the SPECTRAmax PLUS. To avoid significant evaporation error, read the plate within a few minutes of filling the wells.

Step 4 Repeat Steps 2 3 with the remaining columns of the microplate, reading the corresponding Plate section immediately after filling each column.


Graphing the calibration curve
Step 1 Open the Group section. Double click the Concentration column header and change its name to Volume.

Because you have set the data reduction to pathlength, the Values and MeanValues columns display the pathlength (in centimeters). Change the name of the Values column to Pathlength. Change the Mean-Value column header to MeanPathlength.

An example of a Group Table for a calibration standard curve is shown in Figure 6.


Step 2 Create a new Graph section, then open the Plots dialog box ( Figure 7). In the Plot name: field type STD (note: if you name the plot STD, SOFTmax PRO will automatically refer subsequent unknown samples to the standard curve for interpolation.) Select Volume for the X variable and MeanPathlength for the Y variable. If desired, select StDev for Y-axis error bars.


Click OK to close the dialog box and create the standard curve. An example of a standard curve is shown in Figure 8.


Step 3 Save the data file.


CHECKING MULTICHANNEL PIPETTOR PERFORMANCE
Step 1 Open the calibration curve data file. Use the Save As..... command to create a new file for the current calibration.

Step 2 Create a new Plate section. Set up the Instrument Settings dialog box as described in the preceding section (see Figure 1). Select to perform an endpoint read at any wavelength. (SOFTmax PRO requires that you enter a UV/Vis wavelength at which to read, even though this measurement will not be used in calculating pathlength. Select PathCheck with Water Constant and deselect Pre-Read Plate (pre-reading is unnecessary because the UV/VIS measurement is not used in calculating pathlength).

Step 3 Open the Template Editor, create a new group (doing so will cause a Group Settings dialog box will appear). Enter a name for the group, (Pipettor#1 in this example) and select Unknowns from the Column Format pop-up menu. For example, you may choose a group name that designates the pipettor and/or dispense volume and assign well names that correspond to the pipettor channels. A sample template for an 8-channel pipettor is shown in Figure 9. Rows A through H correspond to pipettor channels 1 through 8.

For a 12-channel pipettor, you can set columns 1 through 12 to correspond to pipettor channels 1 through 12.

Step 4 Set up the Reduction as described in Step 3 of the preceding section ( Creating Plate sections for the calibration curve , page 3) and illustrated in Figure 2. In the Reduction dialog box, select Custom from the Wavelength Combination pop-up menu, then click on the formula button to display the Calculation dialog box and type !Pathlength in the formula field.

Step 5 Click the Grayscale button in the Plate sections Display dialog box (Figure 3 on page 4). Grayscale display is useful for quick visualization of the plate. Check the With reduced number box, so that the pathlength (in cm) will be displayed in each well. You do not need to enter the low and high values; SOFTmax PRO will automatically assign limits when the plate is read.

Note: To avoid confusion, it is important that the Display is NOT set to display Raw OD as a number. The Raw OD is the OD value at the UV/Vis wavelength that you were required to enter, but that value is ignored for Pathlength calculation.

Step 6 Take a clean microplate from the same lot as that used to prepare the calibration curve. Using the multi-channel pipettor, fill the entire microplate with the chosen dispense volume.

Step 7 Read the plate in the SPECTRAmax PLUS . To avoid significant evaporation error, you must make the read within a few minutes of filling the microplate.

Step 8 View the Plate section for a qualitative evaluation of the results. Figure 10 shows an example of results from an 8-channel pipettor in which channels C and G had slightly loose pipet tips. The wells in these channels clearly have slightly shorter pathlengths.


Step 9 Open the Group Table to view the quantitative data ( Figure 11). The Val ues column will contain the individual pathlength measurements in centimeters. The Result column will contain the volumes (in microliters) obtained by interpolation from the standard curve. Delete the R column because you do not need it. If desired, you may change the name of the MeanResult column to MeanVolume.


Step 10 Create a summary at the bottom of the Group Table, then type the nominal dispense volume into the formula field of the Calculation dialog box for the summary. Give the summary a short name to allow easy reference to it in subsequent formulas. Enter a description of what the summary contains in the Description field. The Calculation dialog box for a summary named Sum#1, specifying a nominal volume of 200 L is shown in Figure 12.


Step 11 Create an Outcome column in the Group Table to flag any pipet channel falling outside the acceptable range. To do so you must decide upon a pass/fail criterion. The formula for an Outcome column in which the pass/fail criterion is nominal volume +/- 1% is: If (Abs(MeanVolume-Sum#1)/Sum#1<0.01,,Out), where Abs is absolute value and Sum#1 is the nominal dispense volume that was entered in the summary formula in Step 10 , above. The logic of the formula is: if the absolute value of the difference between the measured volume and the nominal volume is less than 1% of the nominal volume, the outcome is (i.e., left blank) otherwise it is Out.

An example of results obtained with an automated pipettor (Pipettor#1) set to deliver 200 L is shown in Figure 13. In this particular example, two of the eight pipettor channels were slightly outside the acceptable range.

NOTE: If you wish, you can also flag pipettor channels which give a standard deviation above a certain limit. For example, the formula to indicate pass/fail with respect to a standard deviation limit of 2.0 could be: If(Std.Dev<2.0,Pass,Fail).


CREATING A
CUSTOMIZED SUMMARY SECTION
You can create a customized report in a Notes section to report final results. An example of such a report is shown in Figure 14. The Nominal Dispense Volume was brought into the Notes section by creating a summary linked to Sum#1 in thePipettor#1 Group Table. The formula for the linked summary is Sum#1@Pipettor#1.


To obtain a Pass/Fail outcome for the pipettor, a column was created in the Pipettor#1 Group Table in which the results from the Outcome column were converted to numerical values ( Figure 15.) The column is named HiddenColumn, because it is normally hidden from view to simplify the Group Table. The formula for the hidden column is If(Outcome=,1,10).


The OVERALL PIPETTOR OUTCOME in the Notes section was created by adding another summary, linked to the Pipettor#1 Group Table with the formula: if (Max(HiddenColumn@Pipettor#1)>1,Fail,Pass). The formula refers to the numerical values in the hidden column if any one of the numerical values exceeds 1, the pipettor fails the performance check.

The time and date displayed in the Notes section report were accessed from the plate and refer to the time and date the plate was read. In this particular example, the Plate section was Plate#12, so the formula for the summary is: !TimeOfRead@Plate#12. Once a plate is read, the Time/Data stamp cannot be changed (unless the data are deleted and new data acquired).

NOTE: you can report results for each individual channel in the final Summary section, however the custom formulas needed to do so are beyond the scope of this application note. For further information on how to set up reports for individual channels, contact Molecular Devices MAXline Technical Support.


DISCUSSION AND SUMMARY
Most pipettors, given enough use, will eventually require repair, and therefore should be checked regularly to avoid having a defective pipettor in service. In the days when the typical laboratory had only a few single-channel, hand-held pipettors, the traditional gravimetric method for calibration was perfectly adequate to satisfy a requirement of 4 to 10 replicates. Modern laboratories, however, have numerous pipettors and many have several multi-channel pipettors and automated pipetting stations. In such cases, the gravimetric method is tedious or even impractical. Pathlength measurements in a 96-well microplate offer an easy and practical alternative method. The major advantages of using pathlength measurements to calibrate or verify performance of multichannel pipettors include:

1 Efficiency. Multiple measurements can be obtained simultaneously, thus the required replicate measurements can be obtained for each channel much more quickly than by making gravimetric measurements. Pathlength measurements for an entire 96-well microplate can be made in less than a minute. The method is especially applicable to automated liquid handling stations (including 96-channel pipettors), where it is impractical to check the performance of each channel gravimetrically.

2 Reliability. The performance check can be done using the same buffers or reagents that will be dispensed by the pipettor, thus eliminating possible discrepancies in between pipettor performance with calibrator solution and with normal dispense reagent.

In the example presented above, the mean dispense volumes for 2 of the 8 channels fell outside the acceptable range (198 - 202 L). Both mean values are significantly different from the nominal value of 200 L (P<.05, students t-test), however, neither is significantly different from the lower limit (198 L). Thus the channels are probably out of specification, but the ultimate Pass/Fail decision depends upon the laboratorys approach to taking measurement uncertainty into account. In a recent publication,14 Ellison et al nicely summarize and interpret the International Standards Organizations recommended method for quantitative estimation of uncertainty.15 The paper also stresses the importance of correct interpretation of accuracy results, so that they are judged neither overly optimistically, nor unduly pessimistically. In practice, such statistical analyses can be avoided by merely repeating the calibration measurements to ensure that the calibration is within or outside the acceptable range.

The terms accurate and precise are relative. A 5% error may be considered insignificant in one method and totally unacceptable in another. Pipettor manufacturers specifications for dispense volumes of 100 - 200 L are typically less than 1% for both accuracy (+ %) and imprecision (CV%), if obtained under nearly ideal conditions. For clinical analyses, medically-allowable errors (of which pipetting error would be only one of several potential contributors) can be 5%, 10% or 20%.16 Thus many clinical laboratories accept an accuracy requirement of + 3% and a precision requirement of + 1.5% for pipet dispense volumes of <100 L.1

In this application note, aqueous dispense volumes are obtained by measuring pathlength in microplate wells. The volume is calculated from the pathlength-vs.volume calibration curve previously obtained with a calibrated pipettor. Thus an important determinant for accuracy and precision is the reproducibility of the pathlength measurements. Molecular Devices provides a specification for pathlength-corrected absorbance, but not a specification for pathlength itself. During preparation of this application note, several in-house instruments were surveyed for pathlength precision using a 1 cm aqueous pathlength fixture. Within-row and between-row standard deviations were <0.01 mm and <0.04 mm respectively. The standard deviation values for the instrument used to generate the data for this application note were 0.005 mm and 0.025 mm, respectively. Pathlength measurements on microplates filled with 75 to 300 L water with high-quality pipettors typically have standard deviations of 0.03 to 0.04 mm. A pathlength of 0.03 mm corresponds to 1.0 L in a standard microplate and 0.5 L in a half-area microplate.

Well volumes in a microplate can be determined quickly and accurately by using the PathCheck feature of the SPECTRAmax PLUS microplate spectrophotometer. No special colored solutions are needed; pipettor performance can be determined with the same buffers or reagents that are normally dispensed by the pipettor - thus eliminating the uncertainty that its performance differs with a calibrator solution compared to the reagent(s) typically dispensed. Using flexible custom formulas and SOFTmax PROs spreadsheet capabilities, the data are calculated automatically and results are displayed as Pass/Fail for each pipettor. SOFTmax PRO also allows the user great flexibility in creating customized summary sections.


REFERENCES

1. Curtis, R.H. Performance verification of manual action pipets. Part I. American Clinical Laboratory 12(7): 8-9 (1994).

2. Robinson, S.M. And K.R. Johnson. The assessment of the accuracy and precision of semi-automatic pipettes. Medical Laboratory Technology 31: 213-219 (1974).

3. Ryan, W. Titrimetric and gravimetric calibration of pipettors: a survey. Am. J. Med. Technology 48:763-766 (1982).

4. Wenk, R.E. And J.A. Lustgarten. Technology of manually operated sampler pipets. Clin. Chem. 20:320-323 (1974).

5. Loria, A. And R. Salas. Evaluation of pipetting systems. II. Precision and accuracy of precision dispensers. Rev. Invest. Clin. 42(2):157-160 (1990).

6. College of American Pathologists. Laboratory instrument evaluation, verification and maintenance manual, Northfield, IL. Fourth ed., 1989.

7. National Committee for Clinical Laboratory Standards. Determining Performance of Volumetric Equipment. ISSN 0273-3099, NCCLS 4(6) (1984).

8. Devine, J.E. The Oxford Guide to Calibration Validation of Laboratory Pipettes. Oxford Products; St. Louis, MO (no date given).

9. American Society for Testing and Materials. Standard Specifications for Piston or Plunger Operated Volumetric Apparatus. Designation: E 1154-89 (1993).

10. Kaufman, D. And G.H. Wobig. Colorimetric calibration of multichannel pipettes. Clin. Chem. 30: 1885-6 (1984).

11. Dixit, P.K. And A. Lazarow. Calibration of micropipettes. J. Lab. Clin. Med. 58:499-504 (1961).

12. Joyce, D.N. And J.P.P. Tyler. Accuracy, precision and temperature dependence of disposable tip pipettes. Medical Laboratory Technology 30:331-334(1973).

13. Ellis, K.J. Errors inherent in the use of piston activated pipettes. Anal. Biochem. 55:609-614 (1973).

14. Ellison, S., Wegscheider, W. and A. Williams. Measurement of Uncertainty. Analytical Chemistry News and Features. Oct 1, pp. 607A-613A (1997).

15. Guide to the Expression of Uncertainty in Measurement; ISO: Geneva, ISBN 92-67-10188-9 (1993).

16. Kaplan, L.A. And A.J. Pesce, (eds). Clinical Chemistry. Theory, Analysis and Correlation. St. Louis: The C.V. Mosby Company, p. 341 (1984).


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