*3.3.2. Carry-over*

pins (Fig. 3C). Coated pins should avoid harsh washing procedures, such as going through

**Figure 2.** A-B) Delivery variation by a bulk reagent dispenser distributing a FITC solution into 384-well plates. Certain dispensing cassette channels display either higher %CV or %bias' values than the anticipated cut-off of 10%. C) Cell settling in the reagent reservoir when transferring to a microtiter plate using an automated pipetting system with an 8-channel head, with 1 min delay between transfers to each column. Cell settling is uneven due to the v-shaped bot‐ tom of the reservoir, causing the intensity pattern observed in the plate. The cells (HEK293T) were incubated with Cell‐

**Figure 3.** Magnified view of FP1NS50H pins (V&P Scientific, Inc.) with A) clean slot B) dirty slot C) damaged slot.

powerful sonication washes.

186 Drug Discovery

Titer-Glo®

for 20 min prior to luminescence reading.

After transferring compounds from one plate to another, the pins are washed in DMSO, al‐ cohol, water or a combination of these solutions. The pintool protocol involves dipping the pins in each solution bath certain number of times, at a particular speed and soaking time. The pins are then dried on lint-free blotting paper. Protocols of pintool devices used on ro‐ botic platforms are optimized for effectiveness in removing previous transfers while spend‐ ing the minimum time between wash cycles. In many cases, the drugging (i.e., addition of compound to assay well) step using pintool becomes the bottleneck in a screening cam‐ paign, and the washing step accounts for most of the time consumed. However, certain as‐ says can be very sensitive to compound carry-over, particularly if the compounds are very potent modulators and bind avidly to the pin surface. In such cases, increasing the number of dips and soaking time can improve cleanliness, albeit at the cost of increasing total trans‐ fer time.

Fig. 4 illustrates the effect of four different wash protocols in a kinase assay using stauro‐ sporine as the inhibitor. After compound transfer by pintool to the first assay plate, the pins are immersed in DMSO and isopropanol reservoirs, followed by drying on blotting paper. Subsequently, the pins are dipped in a second assay plate containing the kinase system. Re‐ sidual staurosporine in the pins increases the signal variation as determined by %CV of a set of multiple wells. Protocol 1 has the least number of dips and soaking time per bath, result‐ ing in the most dramatic signal variation due to carry-over. This general approach is recom‐ mended for detecting carry-over and selecting the appropriate pintool wash.

**Figure 4.** General approach to detect compound carry-over and optimize pintool washing. A single wash cycle con‐ sists of dipping the pins in DMSO and isopropanol baths, followed by blotting on lint-free paper.

#### *3.3.3. Routine quality assessment*

Regular pintool calibration and quality assessment can considerably improve data quality. In screening runs at a single compound concentration, well-maintained pins can lead to a reduction of false negative hits, as damaged or dirty pins would usually deliver lower vol‐ umes than anticipated. In dose-response analysis, the quality of the curve fit is highly de‐ pendent on the variability of the data points.

**3.4. Considerations for using transfer devices: Pipettors**

tial component to accomplish cherry-picking tasks.

transfers in smaller volume ranges (Felton 2003).

The automation station is an integral part of any high throughput pipettor, regardless of the type of tips (fixed or disposable) it employs. It typically consists of ANSI/SBS standard com‐ pliant single or multiple deck positions on a stationary or moving platform to hold the lab‐ ware, with a moving arm situated above the platform containing the single- or multichannel pipette head. A major advantage of automated pipettor devices over manual or electronic multichannel hand-held pipettes is the elimination of inconsistency in the transfer process by minimizing human intervention, which also enables high throughput applica‐ tions that are not otherwise feasible. The three major tasks that can be performed with suita‐

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For plate-to-plate liquid transfers, 96- or 384-well pipette heads are preferred to work with 96-/384-/1536-well microplates to speed up the process and increase the throughput. While 4-/8-/12-/16-pipette heads can also be used for direct transfer applications, they are primarily used to perform serial-dilutions. On the other hand, a single channel pipette tip is an essen‐

The speed of an automated pipettor is important for time-sensitive experiments. Especially when performing small volume transfers into microplates, the amount of time spent to transfer liquids in a column-by-column or row-by-row manner may be problematic due to quick evaporation. If the speed of transfer is too slow, some evaporation in the first column or row may be observed before dispensing to the last column or row, causing inconsistent volume across the plate. To avoid evaporation issues during liquid transfers, deck size, pi‐

Sample carry-over is a common problem in liquid handling tasks requiring sequential dip‐ ping steps into various sample reservoirs. With fixed-tips, an adequate cleaning step is es‐ sential between two transfer operations to prevent sample carry-over. An on-deck cleaning protocol often consists of immersion in a bath (DMSO, alcohol and/or water) with optional sonication step. The tips should be allowed sufficient drying time to prevent sample dilution in the following transfer phase. Appropriate wash solutions should be selected and the opti‐ mum length of washing time should be determined during the assay development stage. Al‐ though fixed-tips may have the risk of carry-over, they enable more accurate and precise

Contamination can also be associated with disposable tips, especially when sterile and nu‐ clease-free assay conditions are required. The speed at which the pipette tips are removed from a sample fluid was found to correlate to the amount of macroscopic droplets stuck to the outer surface of the polypropylene tips, which contributed to cross-contamination (Berg et al. 2001). It was also reported that to decrease this form of cross-contamination, which is

ble hardware settings are liquid transfer, cherry-picking and serial-dilution.

pettor speed, head type and the transfer volume should be considered.

*3.4.1. Pipette stations*

*3.4.2. Tip contamination*

A good quality control procedure should provide the transferred volume and the variation associated with the pin set. We implemented a relatively quick and simple procedure using a fluorescent dye (FITC). Prior to the test, the pins are washed as described above. A calibra‐ tion curve is generated of fluorescence intensity as a function of FITC concentration. Using the pintool, FITC in DMSO is transferred from a source plate to several destination plates containing PBS (the use of 4 plates was shown to be sufficient). The average transferred vol‐ ume per pin is calculated using the fluorescence signal of the destination plates and the cali‐ bration curve. Volume variation across the microtiter plate can be readily appreciated by plotting volume against well position (Fig. 5, top charts). The pink and green solid lines rep‐ resent the upper and lower boundaries within 10% CV of the average volume, where outli‐ ers can be clearly identified. The frequency chart (Fig. 5, bottom chart) displays outliers present in 1, 2, 3 or all of the 4 destination plates, and it can be used to identify pins that consistently provide volumes outside a specified range. In the example shown in Fig. 5, pins corresponding to positions A13, B21, D8, F13, K1, N14 and P20 will have to be replaced. De‐ pending on the need, stringency can be adjusted by changing the boundaries as specified by %CV. It is highly recommended to utilize the same freshly prepared fluorescent dye and buffer solutions in all aspects of the protocol. A template for data analysis can be easily cre‐ ated in conventional software such as MS-Excel.

**Figure 5.** A simple and comprehensive approach to analyze pintool performance. Individual pins can be selected for replacement based on consistent variation across multiple transfers.
