*3.2.7. Routine quality assessment*

drop formation at the tip. To prevent clogging, the tubing should be primed with deionized water shortly after use, especially prior to priming with alcohol, as salts in the buffer may precipitate and biological reagents may clump. When working with cells, it is recommended to wet the tubing with buffer or media before dispensing cells, and if possible, not to allow the cells to settle in the tubing by emptying the contents back to the reservoir immediate af‐

Solutions with high protein content can cause frothing, including media containing serum and biochemical buffers with high percentage of BSA (used as blocking protein). To mini‐ mize frothing, it is recommended not to empty the tubing between dispensing (as ordinarily performed in fully automated platforms for large screenings). If tubing emptying is un‐ avoidable, it is advisable to empty a volume smaller than the dead volume. Other means to reduce frothing involve decreasing dispensing speed and applying grease to the cassette

The reservoir container is an important component of a liquid dispenser that is often ne‐ glected in troubleshooting. The material of the container can have a detrimental effect on the assay robustness, such as sticking of proteins to plastic surfaces. For peristaltic pump-based dispensers, we suggest using a jacketed glass flask connected to a water chiller (waterbath with adjustable temperature). Careful monitoring of the temperature in the flask using a thermometer is recommended, as the temperature set in the chiller is not always reflected in the container. Suspensions of cells, beads or nanoparticles have to be constantly stirred to prevent settling, which could result in uneven dispensing or clogging. The stirring speed needs to be optimized, as fast stirring can create bubbles and disturb biological components (cells). When working with large reagent volumes at the start of dispensing, the stirring may have to be reduced as the volume decreases to

Extensions can be implemented when the dispensing tubings cannot be immersed in the reservoir container because of its large dimensions. Some commercially available exten‐ sions allow for the 8 tubings of a standard cartridge to be coupled into single elongated tubing through metallic cannulas sticking out of a joint casing. For viscous solutions, these types of elongations can introduce bubbles due to the joint design, particularly during prime/empty cycles. The metallic cannulas can easily tear the tubing during fit‐ ting, which is ameliorated by using glycerol or alcohol to smoothen the surfaces. A bet‐ ter alternative is to build home-made extensions by attaching each of the new tubings to separate discarded tubings through connectors, which can be made by cutting the end of

ter dispensing (prime/empty cycle).

tips. Torn or cracked tubing can pull air generating bubbles.

*3.2.4. Foaming*

184 Drug Discovery

*3.2.5. Reservoir container*

prevent foaming or bubble formation.

*3.2.6. Tubing extension*

a pipette tip.

During assay development and validation, factors affecting liquid dispenser performance are identified and corrected. However, setbacks can occur randomly regardless of detailed preparations ahead of the screens. For instance, torn tubing, tip blockage or incorrect car‐ tridge setup cannot be prevented a priori. Therefore, it is recommended to rapidly monitor dispensing variations at the start of a screen, where problems encountered at this stage can be usually corrected fairly quickly.

We normally dispense a solution of fluorescein isothiocyanate (FITC) in PBS into a couple of 384-well plates. Fluorescence intensities are analyzed for signal variations corresponding to each cassette channel, as described by %CV and %bias' (Fig.2). Determination of %CV for the entire plate is frequently performed in many laboratories, but this approach cannot dis‐ tinguish issues with individual channels. In addition, a flawed channel does not necessarily change drastically the %CV of the whole plate, as illustrated by Fig. 2A. The types of prob‐ lems commonly associated to high %CV include improper cassette mounting, tubing stretching and damage.

There are instances when the tip is partially obstructed, leading to reduced volume deliv‐ ered. Even when a channel displays low fluorescence counts, the signal can still have small %CV values (Fig. 2B). We have adapted the concept of %bias to detect significant deviations in signal intensity for each row (SR) compared to that of the whole plate (ST), resulting in %bias' (Eq. 4). Values lower than 10 %CV and 10 %bias' are acceptable.

$$\% \text{ bias '=} 100 \times \left(\frac{\text{S}\_{\text{K}}\text{S}\_{\text{I}}}{\text{S}\_{\text{I}}}\right) \tag{4}$$

#### **3.3. Considerations for using transfer devices: Pintool**

The pintool has become a mature technology for transferring nanoliter to sub-microliter vol‐ umes. Even though the system is regarded as fairly simple and robust, there are a number of points to consider for a consistent and reliable performance:
