*3.2.3. Clogging*

Particles can obstruct the flow of a dispensing cassette mainly by blocking the tips. Com‐ plete clogging is fairly easy to recognize, as the lack of fluid coming out of the tips can be visibly noticed. Depending on the degree of obstruction, partial clogging may not be easily perceived by the naked eye, and it is detected only by photometric or gravimetric testing. However, there are certain indications of partial clogging, such as slanted fluid spray or 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‐ ter dispensing (prime/empty cycle).

*3.2.7. Routine quality assessment*

be usually corrected fairly quickly.

stretching and damage.

*3.3.1. Volume variation*

properties (e.g. viscosity).

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

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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

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

ST

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

The volume delivered by a pin can change due to a number of factors. To minimize volume variations, there should be consistency in immersion depth (Dunn and Feygin 2000). There is a minimum volume required in the source plate, and the destination plate should not be dry (Rudnicki and Johnston 2009). The dwell time that pins spend in the fluid and with‐ drawal speed from the liquid surface should be optimized for solutions of very different

The slot of a pin can be tainted by compound precipitation or formation of suspension de‐ posits (Fig. 3B). Sufficient and robust washing and drying steps are effective in preventing deposition and being critical to avoid carry-over and cross-contamination. The pins can be physically damaged by dipping in highly uneven surfaces, particularly when using slotted

) (4)

% bias '=100 × ( SR-ST

%bias' (Eq. 4). Values lower than 10 %CV and 10 %bias' are acceptable.

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

points to consider for a consistent and reliable performance:
