Troubleshooting Guide: Luminex

Observation : Acquisition Problems and Error Messages

Possible source

Incompatible instrument

Suggestion

Use the instrument that is compatible to the microparticle type. R&D Systems® Luminex® assays are compatible with all Luminex® instruments as well as Bio-Rad® Bio-Plex® dual laser analyzers.

With polystyrene microparticles use one of the following instruments in the MicroPlex® acquisition mode:

With magnetic microparticles use one of the same 3 instruments above or the following instrument in the MagPlex® mode:

Possible source

Instrument is out of calibration

Suggestion

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.

Possible source

Incorrect probe height

Suggestion

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

 

Possible source

Sample probe is clogged

Suggestion

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.

Possible source

Microparticle spectral address is not assigned correctly

Suggestion

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.

Possible source

Incorrect instrument settings

Suggestion

Follow the insert instructions on instrument settings.

Observation: Low Microparticle Count

Possible source

Instrument is out of calibration

Suggestion

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.

Possible source

Wrong event or microparticle setting

Suggestion

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

 

Possible source

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

Suggestion

If your instrument times out when using flow cytometry-based instruments such as the Luminex® 100/200™, stop the plate run. Check the probe height and confirm the appropriate 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.

Possible source

Sample contains debris which affects acquisition

Suggestion

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.

 

Possible source

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

Suggestion

Confirm microparticles were diluted according to the kit insert.

 

Possible source

Microparticles are clumped or aggregated

Suggestion

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

 

Possible source

Microparticles not in suspension during acquisition

Suggestion

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

 

Possible source

Shaker with incorrect settings

Suggestion

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

 

Possible source

Vacuum manifold is too strong for polystyrene microparticles

Suggestion

Adjust the strength on the vacuum manifold plate washer device to between -15 and -40 cm of mercury. Excess vacuum can damage the plate membrane resulting in leaky plates. Excess vacuum can also imbed the microparticles into the membrane resulting in microparticle loss and reduced sensitivity. Note that a vacuum manifold is not a recommended washing device for magnetic assays.

 

Possible source

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

Suggestion

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.

 

Possible source

Sample was run undiluted

Suggestion

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.

Possible source

Blockage of sample probe

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

Possible source

Non-optimal preparation of the standard curve

Suggestion

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.

Possible source

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

Suggestion

Confirm reagent dilutions were performed according to the kit insert.

 

Possible source

Photo-bleaching of the PE signal

Suggestion

Streptavidin-PE is light sensitive. Protect from light.

 

Possible source

Incorrect shaker settings

Suggestion

 

Possible source

Incorrect instrument settings

Suggestion

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

Possible source

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.

Suggestion

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.

 

Possible source

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

Suggestion

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 source

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

Suggestion

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.

 

Possible source

Sample type not validated for the assay

Suggestion

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

 

Possible source

Samples with hemolyzed and hyperlipidemic matrices

Suggestion

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.

 

Possible source

Integrity of the sample is compromised while in storage

Suggestion

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

 

Possible source

Non-optimal pipetting technique

Suggestion

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.

 

Possible source

Assay reagents not equilibrated to room temperature prior to use

Suggestion

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

Observation: High background signals

Possible source

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

Suggestion

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

 

Possible source

Blank wells accidently spiked with standard or samples

Suggestion

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

 

Possible source

Extended incubation with detection antibodies or streptavidin-PE

Suggestion

Follow the kit instructions for incubation times and follow precisely.

Observation: Microparticle Aggregation

Possible source

Samples with hemolyzed and hyperlipidemic matrices

Suggestion

 

Possible source

Microparticles not thoroughly mixed

Suggestion

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.

 

Possible source

Doublet Discriminator gates setting is incorrect

Suggestion

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

Observation: Filter plate leaking (for polystyrene microparticle-based assays only)

Possible source

No pre-wetting step performed

Suggestion

Always pre-wet the filter-bottomed microplate prior to adding the microparticles and samples.

 

Possible source

Inappropriate vacuum pressure

Suggestion

A vacuum strength between -15 to -40 cmHg is recommended (excess strength can damage the filter and result in leaky plates or imbed the microparticles into the filter).

 

Possible source

Liquid filtering out of the plate by capillary effect

Suggestion

Always blot the bottom of the microplate with a paper towel after each final wash cycle to prevent wicking/leaking.

 

Possible source

Filter damage at the bottom of the well

Suggestion

Avoid touching the inside bottom of the wells with the ends of the pipette tips.

 

Possible source

Removal of the plastic support under the plate

Suggestion

Do not remove the plastic support under the plate as this can also lead to wicking/leaking of liquids from the wells.

Luminex