Data Analysis Tips
We recommend counting 50 beads when running MILLIPLEX® assays.
- According to Luminex® experts, a minimum of 35 beads per region need to be counted.
- Fewer than 35 beads could cause a shift in the MFI (Median Fluorescence Intensity) value of the bead population.
- MFI will not change for bead counts ≥35.
- Don’t worry if there is a 35-bead count on one bead region and 400 for others. MFIs will not be affected.
Percent Coefficient of Variation (%CV)
- High %CVs for standards or samples can be due to low bead count.
- Our target intra-assay %CV is <15% and our target inter-assay %CV is < 20%.
Calculating Lower Limit of Quantification (LLOQ) and Upper Limit of Quantification (ULOQ)
- Defining LLOQ and ULOQ requires a tight standard curve (e.g., a 1:2 or 1:3 serial dilution to the point that you achieve saturation at both ends).
- Choose the lowest and highest standard curve points that have a recovery of +/- 20%.
- Verify that this is the LLOQ and ULOQ by running 5 assays with the LLOQ and ULOQ as samples against a curve using the assay serial dilution factor where the lowest standard is below LLOQ and the highest standard is above ULOQ.
- Inter-assay precision should be within 20% for LLOQ and ULOQ samples.
- Standard point %CVs should be <15%.
- High %CVs here indicate improper technique was used when making standard curve dilutions. Examples of poor technique include:
- Not vortexing between tubes.
- Not vortexing while loading the plate.
- Not pipetting equal amounts into the plate.
- The lower the concentrations of analytes, the higher the %CVs tend to be. With new users, this improves with time and practice.
- For any standard points that have high %CVs, samples in that range of the curve should be interpreted with caution.
- Alternatively, a standard point or one of the replicate wells can be flagged/masked, although it can be difficult to decide which well to flag if only duplicates are run.
- Percent recovery should be 100% +/- 30% (industry standard), although some researchers will have their own acceptance criteria.
- Percent recovery is usually worse at either extreme of the curve, but this also improves with time and practice.
- For curve statistics, focus on the R2 value, which approaches, but never equals unity. (Note that an R2 value of “1” is seen with software rounding of 0.9999).
Minimum/Maximum Detectable Concentration (minDC/maxDC)
- For many assays, the minDC/maxDC will be outside the standard points (extrapolated) due to good curve performance and fit.
- To avoid seeing extrapolated data, set the desired range of detection in data analysis software.
- Deciding whether to use the “Best Fit” vs. 5-parameter lot option depends on your comfort level to determine how appropriate it is to “play” with curve fit to find the best one.
- If samples fall above the dynamic range of the assay, dilute the samples further with the appropriate matrices/media and repeat the assay.
How To Monitor/Avoid Lot-to-Lot Drift
- MILLIPLEX® standard points maintain consistent values from lot to lot.
- Lot-to-lot drift is monitored and mitigated using full-curve comparison and comparing the relative potency of each analyte against a reference lot.
- All data are compiled in a single database, and trend charts are maintained in our records.
Easy Multiplex Data Analysis with Belysa® Immunoassay Curve Fitting Software
We offer a powerful combination for analyzing multiplex immunoassay data, coupling our Belysa® Immunoassay Curve Fitting software with data acquired using the Luminex® xPONENT® software. Belysa® software enables you to manage, track, and analyze your multiplex assays rapidly and efficiently, giving you more time to focus on advancing your research. Data acquisition and analysis integrate seamlessly with all Luminex® xMAP® instruments.
- Step 1: Drag and drop your .csv file obtained from the xPONENT® acquisition software.
- Belysa® software will accept acquisition files from all RUO Luminex® instruments. Files can be dragged into the open browser to be opened in the Belysa® platform.
- Step 2: Examine and automatically optimize the curve fit for your data.
- Belysa® tools will parse out the raw data and present it to the user, annotating the curve with Standard, Control, and Sample points. Through a simple wizard function, the software will provide the best fit for the acquired data. A manual curve fit is also an available option.
- Step 3: Scan data to ensure replicate hygiene.
- Belysa® software notifies the user in two ways:
- Belysa® tools alert the user to data that is at the low end of the assay range (e.g., below the limit of quantitation or non-detectable).
- Belysa® tools also flag technical errors within an assay, whether at the raw data level (such as bead count) or in calculated parameters (such as recovery or %CV).
- Step 4: Compare standard curves against a previous experiment to confirm mathematical similarity.
- Comparing standard curves’ slopes between batches or runs ensures that different kits perform equally, either within a run or through the course of a longitudinal study. The user first selects a curve to establish as a reference against which subsequent assay curves are compared. If the calculated slope ratio is equal to 1, then the curves are statistically similar.
- Step 5: Export your data.
- Each experimental mode in the software offers a slightly different report structure, single analyte, and multi-analyte. These are available in .csv, .txt, Excel, and PDF for future analysis or record keeping.
Learn more about Belysa® software in our Monitoring Immunoassay Method Reproducibility article.