Nikolaus Ingenhoven

Development of Standard Test Procedures for Quantifying Carry over from Fixed Pipetting Tips in Liquid-Handling Systems

Robotic liquid-handling systems using fixed reusable pipetting tips are used not only in fully integrated in vitro clinical diagnostic analyzers, but also in open platform robotic liquid-handling systems. These are not confined to particular diagnostic assays, as customers adapt a large variety of assays on these platforms. One major problem with the use of fixed, reusable tips is the carry over (CO) of analyte from sample to sample. Despite widespread use of fixed tips in open platform systems, systematic studies on procedures to quantify CO for analytes other than nucleic acids are missing. In a consortium with three liquid-handling system suppliers and one coating specialist, we developed test procedures for the quantification of CO. The procedures were standardized and tested with the analytes fluorescein, immunoglobulin G, and hepatitis B surface antigen as model substances for small organic molecules, antibodies, and complex biomolecules. The test procedures allow the reproducible quantification of the CO with intra- and interassay precisions of less than 6% coefficient of variation. They were used to investigate the effect of different tip coatings on the CO of the three analytes. Fluoropolymers, inorganic-organic nanocomposites, sodium-silicate glass, titanium dioxide, and silicone resins, which are used in special applications, showed only small differences in CO. The CO test procedures can be easily transferred to different liquid-handling systems and used with different analytes.

Reduction of Carry over in Liquid-Handling Systems with a Decontamination Step Integrated in the Washing Procedure

Robotic liquid-handling systems can be equipped with disposable pipetting tips or fixed reusable pipetting tips. The use of disposable tips is perceived as the best option to avoid carry over (CO) of analyte from sample to sample. We recently developed standardized CO test procedures that allow precise and reproducible quantification of CO for fixed reusable tips. We used these test procedures to reduce CO of the analytes fluorescein, IgG, and hepatitis B surface antigen (HBsAg) to minimal levels. Variations of washing intensity, using water as a washing solution, did not lower CO below acceptable target levels. These target levels would preclude a false-positive detection of IgG and HBsAg in human serum when a negative sample is measured subsequent to a sample with a high analyte concentration. We therefore integrated a decontamination step into the washing procedure. Screening of 12 decontamination solutions showed that sodium hypochlorite solution was very efficient in reducing CO. Optimization led to a final washing routine in which tips are exposed for 0.2 s to 0.17 M NaOCI and subsequently rinsed with 2 mL of water to remove any remaining decontaminant solution. The washing procedure only takes 15 s and is thus suitable for high-throughput applications. The procedure was able to lower CO of IgG and HBsAg in human sera below relevant levels. The decontamination step with hypochlorite can easily be integrated into different liquid-handling systems and is likely to be effective against CO of most proteins and peptides.