*3.4.2. Tip contamination*

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 transfers in smaller volume ranges (Felton 2003).

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 influenced by the tip shape and the sample-polypropylene interactions, the removal speed should be slow enough to diminish droplet generation.

speed, a "tip touch" function is a useful feature offered in some automated pipettors, where the tips contact the well wall at the end of a dispense step to force the release of the droplet. The path of the moving pipetting arm across the deck should be carefully determined to re‐

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Proper mixing of solutions in the source reservoir before aspiration and in the destination reservoir after dispensing may greatly affect the final assay quality due to the necessity of uniform sample concentrations. To avoid the formation of concentration gradient in wells, mixing can be performed by repetitive pipetting cycles. Mixing of the well contents by pi‐ petting up and down is proven to be a quicker and more efficient method compared to free diffusion or shaking, which are not as successful due to the correlations between well size, content volume and the exerted capillary forces (Berg et al. 2001; Shieh et al. 2010; Travis et al. 2010). Mixing is necessary when dealing with suspensions (cells, beads, etc.). For in‐ stance, cell settling creates uneven cell density in the source reservoir, which would lead to

Verification of transferred volumes and routine quality control (QC) are the most important and inevitable processes when working with liquid handling devices. While the verification method should be reliable enough to quantify the pipettor performance, it should also be easy and fast to be applied routinely. The performance assessment described for bulk liquid dispensers (section 3.2.7.) can also be applied to pipettors as long as the same volume is dis‐

As mentioned previously, liquid handlers are heavily used to perform serial dilutions, and suitable QC techniques should be employed to validate dilution performance, particularly when accurate compound potency is directly dependent on concentration accuracy. Dilution ratio, accuracy, precision and outlier distribution constitute the four major criteria that should be evaluated (Popa-Burke et al. 2009). Artel developed an approach to determine di‐ lution and transferred volume accuracy by using dual-wavelength photometry, where two absorbance dyes with baseline resolved spectra are mixed at various ratios using a liquid handler (Albert 2007; Dong et al. 2007). This dual-dye ratiometric method can be applied by using a multichannel verification system (MVS) equipped with the necessary instrumenta‐ tion and analysis (Bradshaw et al. 2005). Dual-dye photometry is also proven to be suitable to measure the efficiency of different mixing methods (Spaulding et al. 2006) and when pi‐

One of the major concerns with any high throughput microplate handler device is its com‐ patibility with plates of various types and sizes. While most high throughput instruments

duce the chance of contaminating other labware by hanging droplets.

aspiration of decreasing number of cells over time (Fig. 2C)

tributed throughout the plate for %CV and %bias' calculations.

petting non-aqueous solutions (Bradshaw et al. 2007).

**3.5. Considerations for using microplate washers**

*3.5.1. General considerations*

*3.4.5. Routine quality assessment*

Impurities can also leach out of the disposable tips when in contact with solvents such as DMSO. Studies have shown that bioactive compounds released from plastic labware may interfere with assay readouts causing misleading experimental results (McDonald et al. 2008; Niles and Coassin 2008; Watson et al. 2009). Consumable materials, especially polypro‐ pylene tips, tend to adsorb certain compounds, leading to unreliable concentrations in the destination plates (Harris et al. 2010). Therefore, it is recommended to test and validate the influence of consumables on an assay during assay development and whenever there is a change in labware.

### *3.4.3. Foaming*

Pipetting viscous and "sticky" samples is challenging due to bubble formation. Among the most important parameters to consider in avoiding these issues are the speed that the tips exit the sample fluid and the aspirate/dispense rates; they should be slow enough to avoid residu‐ als at the inside and outside of the tips. Pre- and post-air pipetting options should be avoided.
