Liquid Handling

Much of laboratory work is simply measuring and mixing small volumes of liquid. The faster and more accurate this liquid handling is done, the faster and more accurate the results.

Multiplexing designs are particularly reliant on liquid handling as they are based on creating precise pooled mixtures of samples.

Broadly there are two choices for liquid handling in a laboratory (1) manual and (2) robotic. Manual liquid handling requires a technician to use a pipette to pull a sample from one container and deposit the sample into another container. Sample transfer takes only a few seconds, but the work can be mind numbing and it can be difficult to remain focused when doing hundreds or thousands of transfers.

Robotic liquid handlers replace the human technician with a robotic arm that does the pipetting to and from samples on a defined stage. These robotic liquid handlers are becoming less expensive and more common. For example, the Opentrons OT-2 costs between $5k-$10k (USD) and fits on a bench top.

Comparing the speed of robotic liquid handlers and vs a human, they are comparable, both taking between 2 to 6 seconds per operation.

To work faster, it is possible to use a multi-channel pipette head that transfers 8 samples simultaneously.

Sample calculations: 270 sample case

Some multiplex designs are more complex and therefore take longer to create. If we take as an example the 270 sample case, and take as a base case that each pipetting operation takes 6 seconds, we can calculate the time required to construct each design. The test time depends on the assay:
Assay or Operation time (min)
qPCR 120
RNA extraction(thermal) 10

Each design has a different number of operations:
Number of liquid
transfer operations
Times (minutes)
270 +27 liquid handling
+10 RNA extraction
+15 (LAMP) or +120 (qPCR)
TOTAL: 52-157 min
810 +81 liquid handling
+10 RNA extraction
+15 (LAMP) or +120 (qPCR)
TOTAL: 106-211 min
1350 +135 liquid handling
+10 RNA extraction
+15 (LAMP) or +120 (qPCR)
TOTAL: 160-265 min
270 + 20(?)(retest) +29 liquid handling
+10 RNA extraction
+15 (LAMP) or +120 (qPCR)
retest: +69 (LAMP) or +279 (qPCR)
TOTAL: 160-265 min
Double pool
540 + 10(?)(retest) +55 liquid handling
+10 RNA extraction
+15 (LAMP) or +120 (qPCR)
retest: +69 (LAMP) or +279 (qPCR)
TOTAL: 95-305 min
Fully adaptive
30x20(?)=600 +60 liquid handling
+10 RNA extraction
retest 20x: +300 (LAMP) )
TOTAL: ~370 min

Note that of these designs, the fully adaptive design will take the longest but is also the most conducive to being done by hand at a small scale. The fully adaptive design should use the least tests, and requires a small single run unit such as the Abbott ID Now unit. In addition, the mixes need to be made on demand, but the load of creating the mixes is distributed throughout the assay process. As such the next mix can be produced while the last one is running.

Economic Comparison

If an assay costs $50 (average reimbursement costs), what kind of payback does multiplexing offer?

Scenario: Assume a case where a lab is tasked to screen 508 samples/day
A) Business as usual:
Total cost: $25,400 ($50 x 508 tests/day)

B) Robotic multiplexing:
  $10k liquid handler (Opentrons OT-2) (one time purchase)
    - tips @ $0.03 each
    - 96 well plate: $1.50

  - samples arrive in 2 ml snap cap tubes (24/rack)
  - 15 ml tubes (12/rack)
  - Layout: 12 bays:
    - 1 trash
    - 1 target 96 well plate
    - 1 tip store
    - 9 sample racks (216 samples for 2ml or 108 for 15 ml)

If we assume a 2% prevalence, could use the XL4 OA design for a 5.4x compression (508 samples in 94 wells). This means that the liquid handler will need to be reloaded multiple times in well creation.

Marginal cost:
    - tips: 508*4*0.03= $60
    - time: ~203 minutes
    - robotics: $27 (assume using once per day for 1 year)
    - Assay cost: $4,700 (94 assays @ $50)
Total cost: $4,787

Cost savings associated with multiplexing: $20,612.60/day

Note that the robotics essentially pays for itself in *the first day* of operation. This robotic platform can be used for RNA prep and other laboratory tasks. Furthermore, assuming a 4 hour total cycle time on the robot, a single unit could be run up to 6 times per day.