Assessing variations in manual pipetting: An under-investigated requirement of good laboratory practice

Abstract

Pipettes are essential tools for biomedical and analytical laboratories, analogous to workstations for computer scientists. Variation in pipetting is a known unknown, as it is generally accepted that variations exist, but thus far, there have been limited studies on the extent of these variations in practice. In this mini-review, we highlight how manual pipetting is a key technique in the laboratory, and, although simple, inaccuracy and imprecision exist. If variations are not adequately addressed, errors can be compounded and consequently compromise data quality. Determination of the accuracy and precision of manual pipetting is straightforward, and here we review two common approaches that use gravimetry and spectrophotometry as readouts. We also provide detailed protocols for determination of accuracy and precision using manual single and multi-channel pipettes. These simple-to-use methods can be used by any laboratory for competency training and regular checks. Having a common protocol for evaluation of variation will also enable cross-laboratory comparison and potentially facilitate establishment of a reference value of acceptable ranges for operator error. Such a value could be of relevance to the scientific community for benchmarking and assuring good laboratory practice.

Fig. 1

Assessing variations in manual pipetting. (A) Factors affecting pipetting accuracy and precision. (B) Two common methods for determination of accuracy and/or precision, gravimetry and spectrophotometry. For gravimetry, the choice of the balance needs to be considered, as measuring the mass of low volumes will be inaccurate, especially if reaching the readability and repeatability limits of the instrument. (C) Formulae for calculation of accuracy and precision. Low coefficient of variance (CV) and error mean high precision and accuracy respectively. (D) Representation of a simplified model of error propagation. In this case, a serial dilution experiment was considered, where the uncertainty of the diluted concentration ($u_{C_2}$) of the reagent is influenced by the uncertainty of the total volume ($u_{V_T}$), the previous concentration ($u_{C_1}$) and volume of the previous concentration of solution added ($u_{V_1}$)¹². If the error increases (0.5% to 5%), it is clearly propagated at every step of the procedure. More specific details and formulae for determination of uncertainty in gravimetric volume is described in the EURAMET guide⁵.

Fig. 2

Variations in manual pipetting of water and chloroform using air displacement pipettes. To determine the accuracy and precision, various volumes of two types of liquids, water and chloroform (CHCl3), were weighed with an analytical balance (readability: 0.1 mg, repeatability, 0.1 mg) by 10 operators (with supervision). The liquids were handled using a range of single channel pipettes, with six repeat measurements. Accuracy (defined by error %, which is calculated based on the deviation from expected mass) (A) and precision (defined by CV %) (B) were calculated and represented as boxplots, with the orange dashed lines representing the error cutoff from the manufacturer, and pink dashed lines representing the error cutoff from ISO8655. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)