Avoiding Cross Talk In 96-well Based Sample Preparation

Importance of the Topic

High throughput 96-well sample preparation is widely used in clinical and industrial laboratories. Preventing cross-well contamination or cross talk is critical to avoid false positive results, ensure data integrity and maintain quality control.

Understanding how hardware design, processing conditions and method parameters interact to cause or prevent cross talk can lead to more reliable analytical workflows and cost savings through fewer re-runs.

Objectives and Study Overview

The primary aim was to identify simple practical strategies to mitigate or eliminate cross-well contamination in 96-well sample preparation. The study reviewed plate design, processing equipment modes (vacuum versus positive pressure), evaporation techniques and mixing steps. An additional focus was placed on handling very high analyte concentrations to prevent evaporative cross-talk in urine drug testing.

Methodology

Sample preparation used 96-well SPE, SLE and protein precipitation plates. Eluates were collected in square or round 96-well collection plates. Processing included automated positive pressure on the Extrahera system and manual positive pressure (PRESSURE+ 96), as well as vacuum processing on the VacMaster-96. Evaporation was performed using a controlled gas-flow evaporator, and mixing steps were carried out on a plate shaker. Solvent volume and additives such as HCl in methanol were varied to study their impact on cross talk.

Instrumentation Used

• Biotage Extrahera automation system with positive pressure head
• Biotage PRESSURE+ 96 positive pressure manifold
• Biotage VacMaster-96 vacuum manifold with adjustable spacers
• Biotage SPE Dry evaporator with temperature and flow controls
• Biotage ACT Plate Adapter for evaporation stages

Main Results and Discussion

Plate Design and Luer Penetration

Optimal tip length and penetration depth of the extraction plate outlet nozzles into collection wells were shown to be key factors in reducing aerosol formation and droplet splatter during elution. Proper spacers ensured uniform Luer fit across different plate types.

Processing Mode Effects

Positive pressure processing consistently reduced sputtering compared to vacuum. Automated control of flow rate in the Extrahera system minimized sudden bursts of solvent. Manual positive pressure also offered improved consistency over vacuum when combined with correct tip alignment.

Evaporation and Adapter Performance

The ACT Plate Adapter significantly curtailed evaporative cross talk by directing gas flow through individual plate chimneys and reducing open well surface area. Without the adapter, high concentration samples created hot-spot carryover into surrounding wells.

Solvent Volume and Additives

Exceeding recommended fill volumes in square wells led to corner creeping and cross well contamination during evaporation and mixing. Switching to round wells or limiting volumes prevented lateral overflow. For volatile analytes at very high concentration, adding 10 µL of 0.5% HCl in methanol to form salt reduced evaporative crosstalk below clinical cutoffs.

Benefits and Practical Applications

• Significant reduction in false positives and reruns
• Improved method robustness for routine drug screening and quantitative assays
• Enhanced throughput by combining optimal plate hardware and automated positive pressure
• Scalable solutions for clinical, forensic and industrial QA/QC laboratories

Future Trends and Applications

Advances in plate materials and geometry may further limit aerosol and evaporation effects. Integration of real-time flow sensors in positive pressure heads could automate tip penetration checks. Novel plate adapter designs and selective well sealing approaches will likely emerge to support ultra-high throughput and extremely sensitive assays.

Conclusion

Strategic selection of plate design, processing mode and evaporation accessories can effectively eliminate cross-well contamination in 96-well sample preparation. Positive pressure systems, optimized Luer penetration and the use of the ACT Plate Adapter together with controlled solvent volumes and targeted additives form a comprehensive toolkit for reliable, high throughput workflows.

References

• Marshall L et al. Effects of Plate Type on Cross-well Contamination in Automated SPE. Pittcon 2011.
• Biotage ISOLUTE SLE+ User Guide: Supported Liquid Extraction. Part UI304.V.3, 2016.
• Biotage SPE Dry 96 and SPE Dry 96 Dual User Manual. Part 415970-B, 2017.
• Biotage White Paper: Current Methodologies for Drugs of Abuse Urine Testing. PPS443, 2019.