Validation of Automated Liquid-Handling Methods on Caliper Liquid Handlers using the Artel MVS

®

RP-POS-01 08/06

Geoffrey Grove - Caliper Life Sciences, Hopkinton, MATanya Knaide - Artel, Westbrook, ME

AbstractValidation of automated liquid-handling methods requires an evaluation of technique as well as liquid handler performance. Precision is often the only performance parameter measured, while accuracy is commonly overlooked. Demand for method validation is becoming more acute with increasing regulatory requirements, requiring an easy-to-use system to evaluate both precision and accuracy. This study used the Artel Multichannel Verification System (MVS) to rapidly evalu-ate and assess precision and accuracy simultaneously. Three different Caliper liquid handlers, the RapidPlate, Sciclone ALH, and Sciclone inL10; and two different liquid transfer techniques were evaluated. This study demonstrates the application of the Artel MVS as a cross-platform tool for method validation.

IntroductionIn this study we used the Artel MVS on three systems and in three different capacities. First, with the RapidPlate Liquid Handler, to study the effects of different liquid transfer techniques on accu-racy and precision. Second, with the Sciclone ALH 3000, to compare pipetting tools for method transfer. Third, with the Sciclone inL10, to evaluate sub-microliter liquid-handling precision.

ConclusionsAs expected, pipetting techniques can have a significant impact on accuracy. The data shows that although accu-racy is technique dependent, precision is relatively technique independent. It is only possible to make this compari-son when both accuracy and precision are being simultaneously determined. Although additional calibration of liquid classes could further improve the accuracy, our findings suggest that for validating liquid transfer methods, a consid-eration of pipetting technique, when coupled with liquid class calibration, will yield the best liquid handling results.We also found that the performance of the RapidPlate used exceeded the manufacturing specification. Although this was a single unit, it may be inferred from the data that the manufacturing specifications could be safely re-evaluated and lowered to include both a wider range (2-200 μL rather than 5-200 μL) and a tighter precision specification (5% rather than 10%).

The results of our comparison demonstrate that there is not a discernable difference in the accuracy or precision of the 2nd generation disposable tips, when compared to the 1st generation tips. The primary advantage of the 2nd generation tips is therefore the ability to load tips without the need for a tip-loading station. If more time was spent on the calibration of the instrument, and in establishing the optimal aspirating and dispense speeds, the precision and accuracy could be further improved. However, we believe that this quick calibration (<1day) is sufficient for many applications.

Using the Sciclone inL10, we found the precision data for each of the MVS dye ranges (B, C and D), were 1-4% higher than historical results generated using tartrazine-water solutions 2. We also have preliminary data showing that the height of dispensing (non-contact), may have a significant affect on the precision [data not shown]. We plan to do further work to measure the magnitude of the effect of dispensing height on the precision. We have not completed our analysis of the accuracy data at this time.

In all of our work, on each of the three platforms, we found the use of the MVS facilitated rapid, simultaneous generation of the precision and accuracy data, necessary for validation of liquid-handling technique (Part I), valida-tion of method transfer to new equipment (Part II), and evaluation of sub-microliter liquid handling (Part III).

[1] Bradshaw, J.T.; Knaide, T.; Rogers, A. and Curtis, R. Multichannel Verification System (MVS™): A Dual-Dye Ratiometric Photometry System for Performance Verification of Multichannel Liquid Delivery Devices, JALA 2005; 10(1): 35-42.

[2] Wendel, G.J. Liquid Handling With Adaptive Real-Time Validation, JALA 2006, 11(2): 88-91.

Figure 1: RapidPlate Liquid Handler.

ResultsMethods and Materials

Figure 2: 96-mandrel tip array

Figure 3: Caliper Sciclone inL10

Part I:MVS dyes were transferred from an open reservoir to a MVS 96-well “characterized” microtiter plate1 using the RapidPlate (Figure 1).

The two liquid transfer techniques used were run at a variety of volumes, including volumes below the 5 μL manufacturing specification limit. The methods consisted of the following steps:

Method “A” (one-to-one transfer) 1) Aspirate a pre-air gap. 2) Aspirate the desired amount of dye from the dye reservoir. 3) Dispense dye into MVS characterized microtiter plate containing MVS Diluent solution.

Volumes were run in triplicate (three plates per volume). Tips were new for the first dispense and reused for each subsequent dispense. For the 200 μL dispense 200 μL tips were used. All other dispenses were completed using 100 μL tips. All dispenses were completed by aspirating the desired volume and dispensing the contents of the tips with a 5 μL air gap. Parameters included: Diluent aspirate height = 0.7 cm, aspirate speed = 10 μL/sec, air gap = 5 μL, dispense height = 0.7 cm, dispense speed = 8 μL/sec. Sample aspirate height = 0.7 cm, aspirate speed = 7 μL/sec, air gap = 5 μL, dispense height = 0.7 cm and dispense speed = 8 μL/sec.

Method “B” (over-aspiration transfer) 1) Aspirate a pre-air gap. 2) Aspirate more than the desired amount of dye from the dye reservoir. 3) Dispense a portion of dye back to the dye reservoir. 4) Dispense dye into MVS characterized microtiter plate containing MVS Diluent solution. 5) Dispense any remaining dye to waste.

The pipetting speed, pre-air gap, number of plates, and height settings were the same as for Method “A”, however, an over-aspiration protocol was employed during which as much as 20 μL was aspirated, 5 μL was then returned to the source, then the desired volume was transferred to the MVS plate. Any dye remaining in the tip was discarded.After the methods were run, plates were analyzed using the Artel MVS system.

Part II:MVS dyes were transferred from an open reservoir to a MVS 96-well "characterized" microtiter plate using the Sciclone ALH 3000 1st and 2nd generation 96-mandrel tip arrays (see Figure 2). The same liquid transfer protocol was used for both 1st and 2nd generation calibrations. The method consisted of the following steps: 1) Aspirate a pre-air gap. 2) Aspirate the desired amount of dye from the dye reservoir. 3) Dispense dye into MVS characterized microtiter plate containing MVS Diluent solution. 4) Mix.

Part III:MVS dyes (Range B, C, and D) were transferred from an open reservoir to a low volume 384-well microtiter plate using the Sciclone inL10 (see Figure 3). The method consisted of the following steps: 1) Aspirate more than the desired transfer amount of dye from an open reservoir. 2) Dip tips in a reservoir containing water to remove any dye remaining on the surface. 3) Dispense the dye one quadrant at a time into a low volume 384-well microtiter plate containing MVS Diluent solution. 4) WashAny dye remaining in the tip was discarded. After the methods were run, plates were analyzed using the Artel MVS system.

Part I: Target Method A Method B Method A Method B Method A Method B Spec (μL) Actual (μL) Actual (μL) % CV % CV % Inacc % Inacc 200 191.84 0.44% -4.08% 10% 100 96.18 0.47% -3.82% 10% 50 48.22 0.64% -3.57% 10% 40 38.22 0.37% -4.46% 10% 25 24.00 0.49% -4.01% 10% 10 9.47 9.80 0.69% 1.01% -5.34% -1.95% 10% 8 7.22 8.13 0.61% 0.92% -9.79% 1.59% 10% 5 4.46 5.23 0.60% 1.06% -10.74% 4.64% 10% 3 2.55 3.13 1.61% 1.65% -14.89% 4.45% 10% 2 1.50 2.15 2.39% 1.97% -25.17% 7.43% 10% 1 0.33 0.99 43.72% 7.68% -66.70% -1.49% 10% 0.5 0.51 24.83% 2.22% 10%

Table 1. Comparison of Method A and B, Accuracy and Precision.

Part II: 1st Generation Tips 2nd Generation Tips Requested Mean CV Inaccuracy Mean Inaccuracy Mean CV Inaccuracy pre-corr. post-corr. 0.10 0.12 25.0% 20.0% 0.07 -30.00% 0.08 25.0% -20.0% 0.15 0.15 20.0% 0.0% 0.20 0.21 14.3% 5.0% 0.25 0.24 8.3% -4.0% 0.30 0.29 6.9% -3.3% 0.26 -13.33% 0.29 10.3% -3.3% 0.35 0.34 5.9% -2.9% 0.50 0.50 6.0% 0.0% 0.48 -4.00% 0.50 6.0% 0.0% 0.70 0.72 4.2% 2.9% 0.59 -15.71% 0.72 4.2% 2.9% 0.90 0.92 3.3% 2.2% 0.75 -16.67% 0.88 3.4% -2.2% 1.10 1.09 2.7% 0.0% 1.30 1.29 2.3% -0.8% 1.50 1.48 2.7% -0.7% 1.40 -6.67% 1.51 2.7% 0.7% 1.90 1.84 1.6% -3.2%

Table 2: Comparison of 1st and 2nd Generation Tip Accuracy and Precision.

Part III: Target (μL) Actual (μL) CV Specification 0.500 0.518 3.27% 5% 0.120 0.119 5.28% 5% 0.050 0.049 10.65% 10%Table 3: Each volume is the average of 4 dispenses.

Figure 1: RapidPlate Liquid Handler.

ResultsMethods and Materials

Figure 2: 96-mandrel tip array

Figure 3: Caliper Sciclone inL10

Part I:MVS dyes were transferred from an open reservoir to a MVS 96-well “characterized” microtiter plate1 using the RapidPlate (Figure 1).

The two liquid transfer techniques used were run at a variety of volumes, including volumes below the 5 μL manufacturing specification limit. The methods consisted of the following steps:

Method “A” (one-to-one transfer) 1) Aspirate a pre-air gap. 2) Aspirate the desired amount of dye from the dye reservoir. 3) Dispense dye into MVS characterized microtiter plate containing MVS Diluent solution.

Volumes were run in triplicate (three plates per volume). Tips were new for the first dispense and reused for each subsequent dispense. For the 200 μL dispense 200 μL tips were used. All other dispenses were completed using 100 μL tips. All dispenses were completed by aspirating the desired volume and dispensing the contents of the tips with a 5 μL air gap. Parameters included: Diluent aspirate height = 0.7 cm, aspirate speed = 10 μL/sec, air gap = 5 μL, dispense height = 0.7 cm, dispense speed = 8 μL/sec. Sample aspirate height = 0.7 cm, aspirate speed = 7 μL/sec, air gap = 5 μL, dispense height = 0.7 cm and dispense speed = 8 μL/sec.

Method “B” (over-aspiration transfer) 1) Aspirate a pre-air gap. 2) Aspirate more than the desired amount of dye from the dye reservoir. 3) Dispense a portion of dye back to the dye reservoir. 4) Dispense dye into MVS characterized microtiter plate containing MVS Diluent solution. 5) Dispense any remaining dye to waste.

The pipetting speed, pre-air gap, number of plates, and height settings were the same as for Method “A”, however, an over-aspiration protocol was employed during which as much as 20 μL was aspirated, 5 μL was then returned to the source, then the desired volume was transferred to the MVS plate. Any dye remaining in the tip was discarded.After the methods were run, plates were analyzed using the Artel MVS system.

Part II:MVS dyes were transferred from an open reservoir to a MVS 96-well "characterized" microtiter plate using the Sciclone ALH 3000 1st and 2nd generation 96-mandrel tip arrays (see Figure 2). The same liquid transfer protocol was used for both 1st and 2nd generation calibrations. The method consisted of the following steps: 1) Aspirate a pre-air gap. 2) Aspirate the desired amount of dye from the dye reservoir. 3) Dispense dye into MVS characterized microtiter plate containing MVS Diluent solution. 4) Mix.

Part III:MVS dyes (Range B, C, and D) were transferred from an open reservoir to a low volume 384-well microtiter plate using the Sciclone inL10 (see Figure 3). The method consisted of the following steps: 1) Aspirate more than the desired transfer amount of dye from an open reservoir. 2) Dip tips in a reservoir containing water to remove any dye remaining on the surface. 3) Dispense the dye one quadrant at a time into a low volume 384-well microtiter plate containing MVS Diluent solution. 4) WashAny dye remaining in the tip was discarded. After the methods were run, plates were analyzed using the Artel MVS system.

Part I: Target Method A Method B Method A Method B Method A Method B Spec (μL) Actual (μL) Actual (μL) % CV % CV % Inacc % Inacc 200 191.84 0.44% -4.08% 10% 100 96.18 0.47% -3.82% 10% 50 48.22 0.64% -3.57% 10% 40 38.22 0.37% -4.46% 10% 25 24.00 0.49% -4.01% 10% 10 9.47 9.80 0.69% 1.01% -5.34% -1.95% 10% 8 7.22 8.13 0.61% 0.92% -9.79% 1.59% 10% 5 4.46 5.23 0.60% 1.06% -10.74% 4.64% 10% 3 2.55 3.13 1.61% 1.65% -14.89% 4.45% 10% 2 1.50 2.15 2.39% 1.97% -25.17% 7.43% 10% 1 0.33 0.99 43.72% 7.68% -66.70% -1.49% 10% 0.5 0.51 24.83% 2.22% 10%

Table 1. Comparison of Method A and B, Accuracy and Precision.

Part II: 1st Generation Tips 2nd Generation Tips Requested Mean CV Inaccuracy Mean Inaccuracy Mean CV Inaccuracy pre-corr. post-corr. 0.10 0.12 25.0% 20.0% 0.07 -30.00% 0.08 25.0% -20.0% 0.15 0.15 20.0% 0.0% 0.20 0.21 14.3% 5.0% 0.25 0.24 8.3% -4.0% 0.30 0.29 6.9% -3.3% 0.26 -13.33% 0.29 10.3% -3.3% 0.35 0.34 5.9% -2.9% 0.50 0.50 6.0% 0.0% 0.48 -4.00% 0.50 6.0% 0.0% 0.70 0.72 4.2% 2.9% 0.59 -15.71% 0.72 4.2% 2.9% 0.90 0.92 3.3% 2.2% 0.75 -16.67% 0.88 3.4% -2.2% 1.10 1.09 2.7% 0.0% 1.30 1.29 2.3% -0.8% 1.50 1.48 2.7% -0.7% 1.40 -6.67% 1.51 2.7% 0.7% 1.90 1.84 1.6% -3.2%

Table 2: Comparison of 1st and 2nd Generation Tip Accuracy and Precision.

Part III: Target (μL) Actual (μL) CV Specification 0.500 0.518 3.27% 5% 0.120 0.119 5.28% 5% 0.050 0.049 10.65% 10%Table 3: Each volume is the average of 4 dispenses.

Validation of Automated Liquid-Handling Methods on Caliper Liquid Handlers using the Artel MVS

®

RP-POS-01 08/06

Geoffrey Grove - Caliper Life Sciences, Hopkinton, MATanya Knaide - Artel, Westbrook, ME

AbstractValidation of automated liquid-handling methods requires an evaluation of technique as well as liquid handler performance. Precision is often the only performance parameter measured, while accuracy is commonly overlooked. Demand for method validation is becoming more acute with increasing regulatory requirements, requiring an easy-to-use system to evaluate both precision and accuracy. This study used the Artel Multichannel Verification System (MVS) to rapidly evalu-ate and assess precision and accuracy simultaneously. Three different Caliper liquid handlers, the RapidPlate, Sciclone ALH, and Sciclone inL10; and two different liquid transfer techniques were evaluated. This study demonstrates the application of the Artel MVS as a cross-platform tool for method validation.

IntroductionIn this study we used the Artel MVS on three systems and in three different capacities. First, with the RapidPlate Liquid Handler, to study the effects of different liquid transfer techniques on accu-racy and precision. Second, with the Sciclone ALH 3000, to compare pipetting tools for method transfer. Third, with the Sciclone inL10, to evaluate sub-microliter liquid-handling precision.

ConclusionsAs expected, pipetting techniques can have a significant impact on accuracy. The data shows that although accu-racy is technique dependent, precision is relatively technique independent. It is only possible to make this compari-son when both accuracy and precision are being simultaneously determined. Although additional calibration of liquid classes could further improve the accuracy, our findings suggest that for validating liquid transfer methods, a consid-eration of pipetting technique, when coupled with liquid class calibration, will yield the best liquid handling results.We also found that the performance of the RapidPlate used exceeded the manufacturing specification. Although this was a single unit, it may be inferred from the data that the manufacturing specifications could be safely re-evaluated and lowered to include both a wider range (2-200 μL rather than 5-200 μL) and a tighter precision specification (5% rather than 10%).

The results of our comparison demonstrate that there is not a discernable difference in the accuracy or precision of the 2nd generation disposable tips, when compared to the 1st generation tips. The primary advantage of the 2nd generation tips is therefore the ability to load tips without the need for a tip-loading station. If more time was spent on the calibration of the instrument, and in establishing the optimal aspirating and dispense speeds, the precision and accuracy could be further improved. However, we believe that this quick calibration (<1day) is sufficient for many applications.

Using the Sciclone inL10, we found the precision data for each of the MVS dye ranges (B, C and D), were 1-4% higher than historical results generated using tartrazine-water solutions 2. We also have preliminary data showing that the height of dispensing (non-contact), may have a significant affect on the precision [data not shown]. We plan to do further work to measure the magnitude of the effect of dispensing height on the precision. We have not completed our analysis of the accuracy data at this time.

In all of our work, on each of the three platforms, we found the use of the MVS facilitated rapid, simultaneous generation of the precision and accuracy data, necessary for validation of liquid-handling technique (Part I), valida-tion of method transfer to new equipment (Part II), and evaluation of sub-microliter liquid handling (Part III).

[1] Bradshaw, J.T.; Knaide, T.; Rogers, A. and Curtis, R. Multichannel Verification System (MVS™): A Dual-Dye Ratiometric Photometry System for Performance Verification of Multichannel Liquid Delivery Devices, JALA 2005; 10(1): 35-42.

[2] Wendel, G.J. Liquid Handling With Adaptive Real-Time Validation, JALA 2006, 11(2): 88-91.