Table 2.

A comparison of approaches used for evaluation of microfluidic devices utilized for particle and cell sorting.

Method	Approach Summary	Comments
Haematocytometer	Measure the total sample volume, collect 10 –20µL of the sample, use a haematocytometer to count the particles or cells, and then characterize the device performance	Advantages: relatively easy; cheap. Disadvantages: Time-consuming; prone to human error; highly dependent on the individual user (although automated counters reduce operator bias); prone to a batch-to-batch difference in the results; not a perfect quantitative approach for device performance; not suitable for rare cells or clusters; not ideal for samples with high concentration or with a mixture of cells or particles.
High-speed camera	Record device perfomracne using a large number of frames per second and visually inspect performance.	Advantages: Can provide the motion of particles or cells passing through the channels; can distinguish between cells and clusters. Disadvantages: Only takes a very short snapshot of device behaviour (in the order of µseconds); not recommended for rare cells; high-speed cameras are rare outside of specialist microfluidic labs; and requires high sampling rates, large storage volumes, and advanced image analysis and devices in which the depth of the channel does not exceed the depth of the focal plane in order to calculate metrics for particle or basic cell separation efficiency or purity. In the case of complex cell mixtures, it would require the development of novel label free cell classification algorithms as well as the above considerations for particles and basic cell separation in order to calculate device performance metrics.
Fluorescent tracing	Evaluate the device performance using fluorescent trace of particles or cells	Advantages: Can illustrate the trace of fluorescent particles passing through the channel. Disadvantages: Restricted for analysis of highly fluorescent microbeads; only applicable for brightly stained cells/particles cells; unable to provide metrics for separation efficiency or purity; cannot distinguish between single or clusters of particles; cannot be used with mixture of particles with different sizes; only takes a snapshot from device performance over a very short period of time.
Current study	The step-by-step protocol has been provided in Section 3.2 of the article	Advantages: Highly reproducible; independent of the user; provide metrics for device performance (i.e., separation efficiency or purity); ideal for rare cells; can be used with mixture of cells or particles of different sizes; applicable even in high concentration mixture of particles; able to identify the behaviour of doublets or cluster of cells; evaluates the average device performance over a any period of time; as the method uses a cytometer, it enables a variety of ways for characterization and illustration of the device performance; independent of the material used for the microchannel fabrication or volume of the sample. Disadvantages: Requires the use of a flow cytometer as opposed to high speed imaging and fluorescent tracing and does add an extra validation step; best suited to conditions in which cell/particle types can be easily distinguished from each other; and in cases where cell types of interest are very similar, may require high dimensional characterisation panels to calculate metrics.