Troubleshooting Tactics
Turbidimetric Immunoassay (TIA) Test Format
- Calibration Error or Alarm
Possible cause Resolution High CV between replicates Repeat Calibration Calibrators are not in the correct position in the stat wheel Check Calibration Install window to make sure spaces are assigned to each calibrator and that they are placed in the corresponding # in the stat wheel - Calibrator Abs are too high or low
Possible cause Resolution R1 and R2 buffer addition orders are reversed –results in erratic Abs Make sure R1/R2 buffers are in the correct reagent carrousel Different lots of R1 and R2 buffers are on board Check to ensure the same lot of R1/R2 buffers are used Incorrect absorbance wavelengths Correct wavelengths are: 800/570 (In the Maint/Utility, Application window, under Analyze) Incorrect Assay/Time/Point Correct time points are 10/18/34 (In the Maint/Utility, Application window, under Analyze) Incorrect Calibrator Sample volume Use 6µL for all serum and calibrators (In the Maint/Utility, Application window, under Others) - Controls are out of range
Possible cause Resolution Incorrect Mean and SD In the QC Install window, make sure the right QC mean and SD are typed in the boxes Controls are not in the correct position in the wheel Check Calibration Install window to make sure spaces are assigned to each control and that they are placed in the corresponding # in the stat wheel Reagents have been left out in the reagent disk for an extended amount of time Use fresh or capped reagents - Poor Precision
Possible cause Resolution Sample probe is dirty Perform weekly cleaning Cells are scratched Perform cell clean or cell blank to determine if the cells are scratched. If they are, replace with new cells Cleaning solution is empty or incorrect solution is used Check to make sure enough cleaning solution is in the container and the correct detergent is used Reagents have been left out in the reagent disk for an extended amount of time Use fresh or capped reagents - Sample Quantitation over the limit
Possible cause Resolution R1 and R2 buffer addition orders are reversed –results in erratic quantitation Make sure R1/R2 buffers are in the correct reagent carrousel Sample has high PLAC Use auto-dilute function - No results reported for sample
Possible cause Resolution Not enough sample in the cup for sampling There need to be at least 150µL of samples in the cup Sample too clotty Remove the clotty sample and repeat the sample again There are bubbles in the reagents, or in the sample cup Remove bubbles from reagents or samples Bad calibration Repeat calibration with the correct reagents
ELISA Test Format
- Low ODs for the Calibrators
Possible cause Resolution Reagent temperature Use reagents at room temperature. Use of plate sealers Do not use during any of the incubations Wells overdrying Reduce time between wash step and addition of next reagent or use a 96-well washer instead of a strip washer. Incorrect substrate volume Use correct volume of substrate. Plate reading Read plate immediately after adding stop solution.Confirm that the plate reader is set up to the correct wavelength. Incubation time Incubate for the recommended times. Reagent order Ensure that the reagents are added in the order indicated in the package insert Calibrator volume Ensure that the correct volume of calibrators was used - Controls Out of Range
Possible cause Resolution Control or calibrator storage Store controls or calibrators in ice during use. Incubation times or temperatures Follow protocol for precise incubation times. Incubation steps Do not skip the sample incubation step Plate washing and drying Do not allow plate to overdry after wash step. Assay temperature Ensure that the assay is performed at the recommended temperatures Curve fit Ensure that the plate reader is set to use a point-to-point calibration fit - Results Drift Across the Plate
Possible cause Resolution Time elapsed while loading the plate Load samples within 20 minutes. Use a transfer plate to minimize delays. Have all reagents ready in advance to avoid interruptions. Do not skip the 10 minute sample incubation step. - Poor Precision of Replicates
Possible cause Resolution Plate washing step Ensure that plate is washed 4 times per step and that the plate washer is functioning properly. Do not use DI water or tap water to wash plates. Use the wash buffer included with the kit Pipetting Verify that volumes and pipette calibrations are correct. Contamination Ensure that proper pipetting technique is used to avoid cross contaminating wells while pipetting - General Precaution
Ensure that all automated ELISA instrumentation is adequately validated to run the PLAC assay prior to use.