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13 Troubleshooting Issue Low event count Low assay signal or sensitivity High Background Low Precision Clogged filter plate Possible cause Bead aggregate Bead settle on the well bottom Vacuum too strong Standard not reconstituted well Incubation time too short Excess exposure to light Well-to-well contamination Poor pipetting precision Contamination from adjacent wells High lipid content in the serum, plasma or bodily fluid samples Recommended actions Vortex stock and working bead suspensions well before pipetting. Shake plates at 1000 rpm for 30 seconds prior to acquisition or transferring to sample tubes. Adjust the vacuum pressure so that 100 µL of 1x Wash Buffer in the wells can be clear in 3-5 second. Standard should be incubated on ice for 5min after the addition of standard diluent Follow recommended incubation time in each step During incubation, cover the plate with aluminum foil to minimize exposure of the beads to light Change pipette tips after every transfer. Remove plate seal carefully and avoid contents from one well to mix with another. Use calibrated pipettes. Avoid well-to-well contamination during pipetting and removal of plate seal Centrifuge the samples at 10,000 x g for 10 min at 28 °C. Collect the serum, plasma or bodily fluid fraction. 21 Rev 1.3.4
