Variance in multiplex suspension array assays: carryover of microspheres between sample wells

Area of Science:

• Biotechnology
• Assay Development
• Analytical Chemistry

Background:

• Accidental observation of bead carryover from unused wells in 96-well plates.
• Unexplained outliers in replicated wells have been previously noted.
• Carryover issues were observed on both older and new suspended microarray instruments.

Purpose of the Study:

• Investigate the nature and extent of bead carryover in suspended microarray instruments.
• Characterize different types of carryover observed during experiments.
• Inform researchers and biostatisticians about potential data integrity issues.

Main Methods:

• Utilized a multiple blank following well experiment design.
• Conducted experiments on two separate suspended microarray instruments.
• Analyzed bead counts from sequential wells to identify carryover patterns.

Main Results:

• Observed significant and unexpected bead carryover between wells.
• Identified two distinct carryover types: small/predictable and large/unpredictable.
• Unpredictable carryover did not consistently decline between wells and poses a significant challenge.

Conclusions:

• Bead carryover significantly impacts suspended microarray instrument data analysis.
• Biostatisticians must be aware of this phenomenon when designing experiments.
• Mitigation strategies include using multiple replicated samples or an intraplex method.
• Applicability to oligonucleotide-based assays remains undetermined.