Luminex® Troubleshooting Guide

This protocol is intended as a guide only.

Researchers can encounter a variety of problems when running a Luminex Assay. Explore this page to understand the potential source of your issue and the stopes we recomment to correct the problem and run a succesful Luminex Assay.

For further assistance, please contact our technical service department.

Table of Contents:

Observation: Acquisition Problems and Error Messages

Possible sourceSuggestion

Instrument is out of calibration

To obtain accurate measurements, regular calibration of the instrument is required. Best practice is to run assays within one week of calibration. Luminex® recommends running verification the day of the assay to confirm the instrument is functioning properly and is within current calibration settings. Perform instrument calibration and verification per the instrument user’s manual.

Incorrect probe height

Adjust the sample probe vertical height and align to the plate per the instrument user’s manual.

Sample probe is clogged

Clean the sample probe per the instrument user’s manual. Replace the sample probe if necessary. Remember to readjust the vertical height each time the probe has been removed.

Microparticle spectral address is not assigned correctly

Ensure microparticle regions are assigned correctly per the kit insert or the Certificate of Analysis. Microparticle maps are custom created depending upon the analytes selected. In the event of omission or an incorrect assignment of a microparticle region, data will be missing in the CSV file, try the “Replay” function to retrieve the data following selection of the appropriate microparticle regions.

Incorrect instrument settings

Follow the insert instructions on instrument settings.

Observation: Low Microparticle Count

Possible sourceSuggestion

Instrument is out of calibration

Perform instrument calibration and verification. To obtain accurate measurements regular calibration of the instrument is required. Best practice is to run assays within one week of calibration. Luminex® recommend running verification the day of the assay to confirm the instrument is functioning properly with current calibration settings.

Wrong event or microparticle setting

Verify that the events/microparticle region is set at 50. A microparticle count of 50 is enough to produce a statistically accurate result. A microparticle count of 25 may be acceptable with duplicate samples and assay performance parameters within defined limits.

The system is timed-out (Luminex® 100/200™ and FLEXMAP3D®)

If your instrument times out when using flow cytometry-based instruments such as the Luminex® 100/Luminex® 200™, stop the plate run. Check the probe height and confirm the appropriate magnetic microparticle type (magnetic or polystyrene) is selected. Then re-run the plate. As each microparticle has a different rate for acquisition and the instrument is set to collect 50 microparticles in a designated time, a “time-out” may result in insufficient microparticle counts for one or more analytes.

The MagPIX® instrument has a fixed read time for each well and does not have time-out functionality.

Sample contains debris which affects acquisition

Centrifuge samples on the day of the assay at approximately 16,000 x g for 4 minutes immediately before use. In rare cases, an extended centrifugation may be necessary.

Miscalculation of microparticle dilution/lower number of microparticles added per well

Confirm microparticles were diluted according to the kit insert.

Microparticles are clumped or aggregated

Centrifuge the microparticle cocktail concentrate (for 30 seconds at 1,000xg) and gently vortex the concentrated before preparing the 1X diluted microparticle cocktail.

Microparticles not in suspension during acquisition

Immediately before placing the plate on the reader, shake the plate for one additional minute in 1X Wash Buffer to resuspend the microparticles.

Shaker with incorrect settings

Use a horizontal orbital microplate shaker with a 0.12" orbit. Ensure the shaker speed is set per recommendations from the kit insert.

Magnetic microparticles not collected at the bottom of plate during wash steps

Use an appropriate magnetic device designed to accommodate a microplate. Wash by applying the magnet to the bottom of the microplate, allow 1 minute before decanting wash buffer. Do not blot dry as this may cause a loss of microparticles.

Sample was run undiluted

Samples require at least a 2-fold dilution with the appropriate Calibrator Diluent. Mix thoroughly. Samples may require higher than 2-fold dilutions. Review the Product Insert, Certificate of Analysis or R&D Systems® Luminex® Assay Customization Tool for the suggested starting dilution for each sample type.

Blockage of sample probe

See above under acquisition and error messages.

Observation: Low Fluorescence Intensity (FI) signal or Poor Sensitivity

Possible sourceSuggestion

Non-optimal preparation of the standard curve

Confirm the standard reconstitution volume from the standard value card or the Certificate of Analysis. Incorrect reconstitution of the standard will result in inaccurate sample value calculations. Be sure to follow reconstitution instructions for all lyophilized reagents outlined in the kit insert.

Non-optimal dilution of the detection antibodies or streptavidin-PE concentrates

Confirm reagent dilutions were performed according to the kit insert.

Photo-bleaching of the PE signal

Streptavidin-PE is light sensitive. Protect from light.

Incorrect shaker settings

See above.

Incorrect instrument settings

See above.

Observation: Sample Readings are Out of Range (OOR Error Message)

Possible sourceSuggestion

Samples are below assay range (<OOR Error Messages) and contain no analyte, or the analyte level is below the level of detection, or the sample may be too diluted.

Analyte of interest may be undetectable in the assay range due to low abundance of natural protein. Check kit instructions or the R&D Systems® Luminex® Assay Customization Tool for suggested sample dilution. Suggested dilution factors are based on samples from healthy volunteers. Depending on the unique nature of an individual sample, a different dilution factor may be needed to bring the reading within the dynamic range of the assay.

When readings are above assay range (>OOR Error Messages)

Review the Product Insert, Certificate of Analysis or Check kit instructions or the R&D Systems® Luminex® Assay Customization Tool for the suggested initial dilution. Suggested dilution factors are based on samples from healthy volunteers. Depending on the nature of the sample, it may require a further dilution to bring the reading within the assay range.

Note - Samples may require dilution and re-analysis if a specific analyte is out of range.

Observation: Poor Precision with sample measurements

Possible sourceSuggestion

Presence of interfering components in samples, especially samples with complex matrices such as plasma and serum

Check for the presence of interfering components, additives, or if gel separators were introduced into the sample by performing a Spike/Recovery and Linearity test. Contact R&D Systems® Technical Service if you require assistance with this test.

Sample type not validated for the assay

Check the kit insert to confirm if the sample type has been validated for the assay.

Samples with hemolyzed and hyperlipidemic matrices

Avoid the use of samples with hemolyzed or hyperlipidemic matrices. Such samples may disrupt antibody binding or clog the probe. See discussion above on how to clean the sample probe.

Integrity of the sample is compromised while in storage

Follow the kit insert on Sample Collection & Storage. Observe best practices for processing and storing the samples after collection. Avoid repeated freeze-thaw cycles.

Non-optimal pipetting technique

Ensure a consistent and accurate pipetting method. Dispense microparticles, diluents and samples accurately. Change pipette tips between samples and dilutions. Pre-wet tips for sample replicates. Ensure that your pipettes are calibrated regularly.

Assay reagents not equilibrated to room temperature prior to use

All assay components should be equilibrated to room temperature prior to use.

Observation: High background signals

Possible sourceSuggestion

Incorrect buffer used for the dilution of standards and/or samples

Ensure the use of the recommended Calibrator Diluent for the dilution of standards/samples per kit insert.

Blank wells accidentally spiked with standard or samples

Do not add standard or samples to wells designated as blank. Add Calibrator Diluent only.

Extended incubation with detection antibodies or streptavidin-PE

Follow the kit instructions for incubation times and follow precisely.

Observation: Microparticle Aggregation

Possible sourceSuggestion

Samples with hemolyzed and hyperlipidemic matrices

See above.

Microparticles not thoroughly mixed

Follow the kit instructions on the preparation of the diluted microparticle cocktail. Use a plate shaker with appropriate settings for the assay. Shake plate for one additional minute in 1X Wash Buffer immediately before analysing in an appropriate instrument.

Doublet Discriminator gates setting is incorrect

Check the kit insert for the Doublet Discriminator gate settings and adjust settings as needed.

Luminex®, Luminex FLEXMAP 3D®, Luminex® 100/200™, and MAGPIX® are trademarks of Luminex Corporation.


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