Bender MedSystems Troubleshooting Guide for FlowCytomix™ Products

This FlowCytomix troubleshooting guide has been specifically designed to provide a convenient and time-saving optimisation procedure for Bender MedSystems FlowCytomix™ products.
The Bender MedSystems Technical Service is always happy to answer any questions you may have about either the protocol of our FlowCytomix™ products or the information given in this troubleshooting guide.

Contact: tech@ebioscience.com phone +43 1 796 40 40-120

1. 2D Cluster of 2 bead sizes in FS/SS dot plot

1) No events on the screen

Setup the FS/SS voltages: Adjust voltage of FS/SS
(see FlowCytomix manual Chapter 11 Cytometer Setup, examples of final settings are given for BC FC500, BD FACS Calibur and BD FACScan).

Ensure the discriminator is not eliminating the populations by being set too high: Set a discriminator only on the FS – only signals with an amplitude greater than or equal to the discriminator channel value will be processed and sent to the computer. (BD Calibur: FS  52, FC500 FS → 100)

Prime the fluidics to remove air bubbles that may be trapped in the flow cell. These trapped air bubbles can deflect the sample stream away from the laser beam, (disrupting the hydrodynamic focusing) causing no events to be detected.

Mixing:
Inadequate mixing can lead to little or no beads being detected. We recommend vortexing the FlowCytomix beads immediately before mixing with the standards or samples and again before analysis on the flow cytometer. Vortex each sample tube for 3-5 seconds before placing the tube on the flow cytometer as this will yield better discrimination of the bead populations in the FL3/FL4 channel.

2) Only 1 bead population can be found - the 2nd bead populations cannot be distinguished

The discriminator for the SS should be 0. (applicable only on instruments capable of assigning 2 discriminators.)

If you run a FlowCytomix assay on a FC500 Instrument from Beckman Coulter you must ensure that the Forward Scatter (FS) measurements are collected at 1-8°. To accomplish this you must insert the FS 1-8° Field Stop into place: lift the front cover to locate the FS 1-8° Field Stop. Slide the knob from the right ( default 1-19° position) to the left and push to lock into place.

On the BC XL Series systems the EPICS XL/XL-MCL FS Low Angle Collection Kit is required to accomplish this.
Please contact the BMS team for further information.

In case you use FlowCytomix™ Simplex Kits, check the bead populations (see manual chapter 4 "Reagents Provided") - for some combinations only small or large beads are used. Therefore just one bead population is seen.

3) Additional dots can be seen in the bottom left corner of the dot plot

Occurs only in samples (eg. serum, plasma):
The additional dots are due to components in the sample.
The measurement is still valid, as long as the two bead populations can be clearly distinguished and enough beads can be detected in each of the gates.

Occurs in standards and samples:
The FlowCytomix™ assay buffer concentrate (10x) contains BSA, which may form crystals during storage at 2-8°C. If crystals are not dissolved completely during preparation of assay buffer, they are detected in the bottom left corner of the dot plot. The measurement is still valid, as long as the two bead populations can be clearly distinguished and enough beads can be detected in the two gates.
To avoid this, put the assay buffer concentrate into a 37°C water bath until the crystals are fully dissolved.

4) Additional dots can be seen on the whole dot plot

Samples might be lipaemic, which causes agglutination of beads. Centrifugation of lipaemic samples (about 16,000 rcf for 5min) before analysis will help. The sample has to be removed carefully with a pipette from the middle of the centrifuged vial, without disturbing the lipaemic layer on top of the liquid nor the pellet on the bottom.
The measurement is still valid, as long as enough beads can be detected in the two gates.
FlowCytomix™ values of lipaemic samples are always critical using immuno- assays. Lipids may affect antibody binding. We recommend to measure these samples in duplicates.

5) During measurement of the standard curve/samples, the beads start to change their FS/SS position and fall outside of the gates.

Instrument failure:
The sample flow is erratic which could be caused by a partial flow cell blockage. In these cases a “prime“ cycle should resolve the problem. A gate should not be changed during measurement!
However, the problem could be due to incorrect or erratic sample or sheath pressure which may require a more detailed inspection of the Flow Cytometer to identify the underlying cause.
The data can be analysed as long as the gates can be adjusted around the small and the large bead population. If the bead populations overlap each other, the data cannot be analysed.

6) Additional bead populations can be found

Doublets and triplets may be seen and are a result of two or more beads passing through the laser beam at the same time
The measurement is valid, as long as enough beads can be detected within gate A and B. Doublet- and triplet populations have to be excluded from the gates A and B!

2. Flourescence Cluster 1 and 2

1) Bead clusters merging

If standards show merging populations an instrument service may be required

2) The bead sets of Standard 1 do not show horizontal dense populations

Incorrect colour compensation:
Adjust the colour compensation according to the manual (see Chapter 11 Cytometer Setup) until the bead clusters are horizontal.

3) The bead population do not move horizontally towards the FL-2 axis.

Incorrect colour compensation:
Problem is seen during setup: Adjust colour compensation according to the FlowCytomix manual (see Chapter 11 Cytometer Setup) until the bead clusters are horizontal. Bead clusters must be horizontal to fall within the gate throughout the whole measurement.
Problem is seen during data anlysis with FlowCytomix Pro software: Try to set the gates in such a way, that the bead clusters can be gated despite the movement throughout the whole measurement. If the bead populations overlap each other or move from one gate into the other, the data cannot be analysed.
Some flow cytometers (eg. BC FC500) allow the adjustment of the colour compensation on saved listmode data. In this case the colour compensation can be adjusted and the data can be re-analysed without problems.

4) Double populations appear within one gate

Preparation of the bead mixture: pipette the bead solution all the way down to the bottom of the tube, so that you do not lose any material on the sides of the tube.

When pipetting the bead solution, make sure that the beads do not stick on the side of the tube/well. Beads which do not come in contact with the detection antibody during the incubation step will show a much lower PE signal. During washing, these beads will be mixed with the incubated beads and will lead to a second, almost "PE-negative" population.
Do not use these standard/sample files for analyses. The double populations cannot be excluded from the calculations when using the FlowCytomix Pro software and may lead to incorrect results.

3. Standard Curves

1) FlowCytomix™ Sensitivity

• Preparation of FlowCytomix™ standards: Centrifuge standard vials before opening
• Reconstitution of FlowCytomix™ Standards: Once reconstituted, do not use standards after 12 hours. Use a fresh standard dilution set for each experiment.
• Strict adherence to FlowCytomix™ kit protocols (i.e. accurate pipetting) is also important, as imprecise bead additions will also change the FlowCytomix™ assay sensitivity by changing the amount of capture surface available.
• The FlowCytomix™ assay utilizes R-phycoerythrin (PE) for it's fluorescent signal. It is critical to the assay sensitivity that all steps after the addition of the detector reagents be protected from light to obtain maximum fluorescence of the PE reporter system.

2) What is the absolute lowest value that can be reliably detected in plasma or serum with FlowCytomix™ assays?

It is not recommended that researchers report values extrapolated below the lowest standard curve point. This can add mathematical variation to results (due to extrapolation) and cause increased CV's.

Cytokines are often present in serum and plasma at levels below the limit of detection for conventional immunoassays. This is due to the fact that most cytokines act locally in the environment around specific sites of immune activity, are highly potent, and, in many cases, have short half-lives. Thus, it is reasonable to assume that only in pathological cases would they be measured in high amounts in systemic fluids like serum and plasma. This is not necessarily the case for broader acting proteins like some of the chemokines, growth factors, etc., which may be produced in higher levels or stabilized in system fluids (longer half-lives) and are more readily measured.

4. Samples

1) FlowCytomix™ values vary from ELISA values

A fluorescence-based reporter system provides a number of advantages including a greater dynamic range than ELISA. Due to the complexity and the kinetics of this bead-based assay, quantitative results obtained by ELISA may differ from those obtained by FlowCytomix™. Therefore, values should only be correlated, rather than comparing absolute values of FlowCytomix™ and ELISA.