We have implemented a simple, inexpensive and rapid procedure for the validation and performance verification of pipettes mounted on automatic liquid handlers (ALH) as required for ISO 17025 accredited laboratories. A serial dilution of six or seven passages of OrangeG in quadruplicate in a flat-bottom 96-well microtiter plate, manually using calibrated pipettes. Each liquid handler pipettor (1–8) dispensed a selected volume (1–200 μL) of OrangeG eight times into the wells of the microtiter plate. All wells contained a total of 200 µl of liquid. The absorbance was read and the volume dispensed from each pipet was calculated based on a plot of volume and absorbance of a known set of OrangeG dilutions.

Finally, the percentage imprecision (%d) and imprecision (%CV) of each pipette were calculated. Using predefined acceptance criteria, each pipette passed or failed. Defective pipettes were repaired or the volume offset was compensated for by applying a calibration curve in the liquid controller software. We have implemented the procedure on one Sias Xantus, one MWGt The ONYX, four Tecan Freedom EVO, one Biomek NX Span-8, and four Biomek 3000 robots, and the methods are freely available.

In conclusion, we have put in place a simple, cheap and fast solution for the continuous validation of HLA used for work accredited according to ISO 17025. The method is easy to use for aqueous solutions but requires a spectrophotometer that can read microtiter plates.


laboratory automation, automated liquid handling, forensic genetics, volume verification


  • Solution Preparation

A 10 mg/ml stock solution of OrangeG (O7252, Sigma) in MilliQ water was prepared in a 500 ml blue top flask wrapped in aluminium foil to avoid photo-induced decomposition. The stock solution was vigorously stirred for 24 h on a magnetic stirrer. Two working solutions were prepared from the stock solution. Working Solution A was a 1:7.5 dilution of the stock solution used for volumes less than or equal to 10 μl. Working Solution B was a 1:75 dilution of the stock solution used for volumes greater than 10 μl.

  • Standard row preparation

For volumes less than or equal to 10 μL, a six-step standard row was prepared in 1.5 mL Eppendorf tubes using working solution A. For volumes greater than 10 μL, a standard row was prepared. of seven steps in 1.5 mL Eppendorf tubes using working solution B. Standard rows were prepared by manual pipetting or by means of a calibrated HLA.

The standard rows were distributed into 96-well flat-bottom microtiter plates (BD Falcon Microassay, Franklin Lakes, NJ) according to the design presented in Figure 1. The remaining wells of the microtiter plates were then filled with MilliQ water. . All wells used contained a total of 200 μL after the addition of orange in the ALHs. Therefore, if an HLA was to be tested to pipet a fixed volume of 50 μL, column wells one through eight were filled with 150 μL of MilliQ water.

  • Addition of OrangeG in ALH

In each ALH, we build methods for the different volumes to be tested. Wherever possible, the volume was made a variable allowing the same method to be used for a series of volumes. Volumes less than or equal to 10 μL were dispensed using the same method. Volumes greater than 10 μL were dispensed using a separate method. Whenever appropriate, the methods used the same pipetting techniques, pipetting templates, and liquid classes or types as the methods used for routine work. For liquids with physically different properties than the OrangeG solution, eg, viscous, volatile, or detergent-containing liquids, a more appropriate type or class of liquid was used.

  • Absorbance measurements

After adding OrangeG, plates were removed from the HLA and covered with a plate seal (AB-0558, ABgene, Epsom, UK) and incubated on a shaker at 150 RPM (Labline Orbit Shaker model 3520, Melrose Park, UK). IL) for 1 h before absorbance measurements. The absorbance was read on a Sirio S spectrophotometer (Radim, Pomezia, Italy) at 450 nm using the attached software BrioWin Ver. 2.10 (SEAC, Florence, Italy). The plates were read twice and the results were compared. If the results of the two readings were inconsistent, the plate was read again.

Results and Discussion

HLAs are based on pipetting techniques and/or liquid classes for precise pipetting of various liquids in various volumes. Pipetting techniques in combination with liquid classes are used to compensate for variations in liquid viscosity, vapour pressure, hydrophobicity, and volume dispensed. A general rule of thumb for accurate pipetting dictates slow aspiration and medium-fast to fast dispensing rates in combination with pauses of varying lengths.

These pipetting properties can be fine-tuned in most liquid handling programs using custom pipetting techniques optimized for individual pipetting operations. Consequently, an HLA can use many different kinds of liquids and pipetting techniques during the execution of a single method. Since HLA pipetting performance inevitably changes by altering aspiration or dispense speeds, validated pipetting techniques and/or liquid classes should, in principle, all be routinely verified.

As there is no adequate procedure to verify the volumes dispensed by different HLAs using different pipetting techniques or liquid types, we developed one. Initially, the procedure was tested using manual pipetting with a conventional plunger pipette to determine the usefulness of the technique. The results were 0.3–0.2 μL lower than expected (Fig. 3). This drift may be the result of pipette heating from simple body heat transfer during the pipetting operation or poor manual pipetting performance. Therefore, the procedure was considered acceptable for use in ALH.


Advanced and precise pipetting can be performed using ALH. To ensure the reliability of the systems, HLAs need frequent verification of the actual dispensed volume similar to manually operated pipettes. The technique presented here allows for a rapid and inexpensive test of most HLA pipettes in microtiter plates.

The technique in its current form does not allow for easy verification of dispensed volumes on automated liquid handlers equipped with a 96 or 384 pipettor head. The technique is simple to use and does not require extensive training of the technical staff. The method allows testing laboratories working under ISO 17025 to perform their own calibration of ALHs and calculate the uncertainty of the dispensed volumes.

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