The Importance of the Quality Control of Laboratory Automation

Introduction

The use of automation at Cambridge Antibody Technology (CAT) is widespread. Routine Quality Control (QC) and maintenance of laboratory automation plays an important part in the upkeep and performance of instruments. QC is important to check the accuracy and repro-ducibility of liquid handling, and indicates any deterioration in performance, ensuring the equipment is performing to expectations. The routine light maintenance extends the lifetime of the instruments and reduces the likelihood of breakdowns. QC data and reliability of the equipment provides information for the purchasing of equipment in the future. Equipment can also be QC'd before purchase to check the manufacturer's claims, and to compare equipment quantitatively.

Methods for the effectiveness of plate washing and quantitative QC of liquid handling instruments are described here.

QC of Plate Washers

Washing microplates is an important part of the screening process and efficient washing is vital to reduce background in assays. QC of the machines needs to be performed to ensure optimal performance. Weekly QC procedures use Rhodamine 110 (a fluorescent fluorophore), which can be detected with a plate reader. Plates are filled with Rhodamine 110 at 12.5 μg/ml with PBS control wells to show background, and then washed using standard programs. PBS is added to the wells before reading on the plate reader. The results are analysed in MS Excel with a surface area chart. This highlights any blocked or damaged pins and once identified, these problems can be rectified and QC performed again. This procedure also enables testing of the available equipment to ensure the best equipment is bought for use at CAT.

VALUE OF MAINTENANCE

Figure 1 shows washer a's washing pattern before and after the QC and maintenance has been carried out.

Figure 1.

A surface area chart to show the difference in washing through maintenance. (Wash buffer is PBS Tween).

COMPARISON OF PLATE WASHERS FROM DIFFERENT MANUFACTURERS

Two washers are used at CAT: Washer A and Washer B. QC analysis of washing allows comparisons to be made between these washers under the same conditions. (See Figure 2).

Figure 2.

A comparison of microplate washing between Washer A and Washer B before maintenance. (Wash buffer is PBS Tween).

Results

QC results show that plate washers benefit from regular maintenance to maintain optimum washing of microplates. QC results also show that there are differences in the consistency of washing between different plate washers. This helps make decisions on future purchases.

QC of Liquid Handlers

Methods

Liquid handling robots are important for transferring liquids in the screening process. They prevent RSI and will do repetitive tasks without user intervention.

The gravimetric and colourimetric methods are used to obtain an accurate representation of the performance of the dispensing equipment.

Gravimetric Method - this gives the absolute volume dispensed within a plate. This is calculated by weighing a dry plate, dispensing water into it and then reweighing.

Colourimetric Method - uses tartrazine to determine the volume dispensed per well, read as absorbance at 405nm on a plate reader. It shows the individual performance of each tip and the precision (CV) across the plate This method requires a comparison with a standard curve set up by hand, meaning the accuracy of dispense is only comparative to the curve and not absolute. Procedures involve setting up a standard curve with changing amounts of tartrazine and then dispensing specific volumes of a particular tartrazine concentration with the liquid handler into a dry plate. MilliQ water is added to increase the volume for reading by the plate reader. The results are analysed using regression analysis.

ANALYSIS OF DISPENSING

The value of using both gravimetric and colourimetric QC can be shown here looking at a 5μl dispense for a multidispenser using an 8 channel cassette. Figure 3 shows the results using gravimetric QC and Figure 4 shows results using colourimetric QC.

Figure 3.

Gravimetric results for multidispenser.

Figure 4.

Anaylsis of 384 dispensing of an 8 channel multidispenser using Colourimetric QC.

The results from the gravimetric method suggest that the multidispenser dispenses evenly and accurately across the plate (CV=0.55%). The colorimetric method actually exposes the fact that the machine alternates between an over dispense and an under dispense and gives a much greater CV (CV=11.78%). The 5μl dispense suggests that the machine is at its limit as the 10μl dispense is excellent.

The data shown in Figure 5 is for a 5μl transfer using a 96 well pipetting head on a replicator.

Figure 5.

Dispensing accuracy determined by Colourimetric QC with a replicator's 96 well pipetting head.

VALIDATION OF NEW EQUIPMENT

Using colourimetric QC is a good way to validate new pieces of liquid handling equipment. Any problems can be identified after installation. Here a piston (Head A, well A9) on a 96 well pipetting head on a replicator is shown to be faulty.

Replacement of the head was required (Head B). Validation of this head showed all of the pistons to be working.

Results

The gravimetric method gives a true value for the volume dispensed per plate. This method cannot give a value for the dispensed volume per well. The colourimetric method shows well to well variation in volume within a microplate. The calculated volume dispensed is not absolute but relative to the standard curve. The colourimetric method can help pinpoint problems with equipment.

Conclusion

The examples shown in this poster demonstrate the importance of maintenance and QC in maintaining smooth running, accuracy and precision of laboratory automation used in processes at CAT.

QC of equipment provides:

• confidence the equipment will work reproducibly

• greater experimental accuracy

• reduced equipment down time

• helps decisions on purchase of new equipment

• highlights problems at installation that can be corrected by the manufacturer

• helps determine future purchases by evaluating long-term reliability