All AtheNA Multi-Lyte® assays (except Borrelia) can be multi-batched and processed on the same plate. When doing so, the diluent and AtheNA Wash Buffer can be used as a universal component for all kits being used. However, the controls, bead suspension, and conjugate should still be used with their respective kits and lots when multi-batching.
Unless otherwise specified in the Package Insert, the Numeric Value is reported in arbitrary units. These units are Semi-Quantitative and are not calibrated to any International Standard and should not be evaluated as such.
If a QC Control fails for one analyte, then the entire run is considered invalid. All QC Controls must pass QC Criteria in order for results to be considered valid.
Beads should be sonicated and/or vortexed as per the Package Insert requirements (30 seconds each procedure). If one wishes to go beyond this time setting, then please note that bead performance & efficiency may be affected if beads are sonicated over 8 minutes. Please note that beads can definitely be over-sonicated. Time will depend on the specific antigens on the specific bead set. Some beads will be more affected than others so it is crucial to follow the instructions listed in the package insert.
All reagents should be returned to their original vials and storage conditions immediately after use. To ensure that assay performance and reproducibility is optimal, these guidelines should be followed. Failure to do so can lead to performance variability and reagent instability over time.
Test system performance has been optimized for the incubation time frames provided in the package insert. Please note that there should be consistency between incubation times. Therefore, all subsequent incubation times should be equal or +/- 10 minutes to the original. Performance efficiency has not been established for increased or decreased incubation times from what is instructed and precision may vary.
Previously used filter plates may be reused as long as the unused wells were not exposed to moisture: i.e. AthenNA Wash Buffer, diluted samples, etc. If a plate has only been partially used, the remaining liquid should be vacuumed out of the wells after the assay has been read on the AtheNA Multi-Lyte® instrument. The used wells should be covered with tape or parafilm to prevent re-use of wells and to ensure optimal vacuum pressure.
Every AtheNA Multi-Lyte® Test System utilizes Intra-Well Calibration Technology™. This allows for each sample to have its own unique calibration curve applied under the same conditions as the patient serum. There are parameters set for each variable of the calibration curve and if sample interaction does not fulfill certain variable’s requirements, then an Invalid Result will be generated. Some common causes for Invalids would be: little to no sample added, abnormally high or low levels of IgG, RF positive interference, and low bead count levels. If an Invalid Result is produced, the sample should be re-run. If another Invalid Result is generated, the instrument should be calibrated and a fresh serum draw of that patient should be acquired to run again.
Specimens that exhibit equivocal results may be considered borderline. While it is up to each laboratory to establish its own guidelines for reporting equivocal results, it is suggested when an equivocal result is produced, the laboratory should either rerun the patient sample in duplicate or evaluate using another methodology.
Arbitrary Units were created during the development of the AtheNA Multi-Lyte® Test System. As these are not International Units, and have not been calibrated to any WHO Standard, they should not be used for numeric correlation studies. Instead, the correlations should be performed based on qualitative comparisons.
For optimal performance the plate should be read within 60 minutes after the completion of the conjugate incubation as outlined in the package insert.
If the AtheNA Multi-Lyte® batch name and the AtheNA work list name do not match exactly when entered, the analyze button will not turn green. It is critical that every time a batch name is entered into the AtheNA software, that the exact same name is also used in the AtheNA software. (Please refer to ZEUS Scientific Tech Tip 19)
Dilutions should be utilized immediately after processing. Failure to do so can lead to sample dilution evaporation which can skew the dilution concentration, which in turn affect results.
In order for results to be valid, the dd temp in the AtheNA Multi-Lyte® software must be within the optimal range. This is indicated on the AtheNA “Run Batch Screen” as the area that is between the two yellow arrows and is highlighted in green. The dd temp is determined by the room or instrument temperature the last time the AtheNA was calibrated and if it deviates +/- 2 degrees while reading it will flag as out. Note that this will not be documented on the AtheNA results print out, therefore it is important to view the AtheNA screen while the run is in process.
In order to adjust probe height, the same filter plate type that is used for performing testing must be used with two large, flat, round disks in it.
The AtheNA Multi-Lyte® instrument should be calibrated once a week, as part of the instrument’s weekly maintenance procedure. Additionally, any time the dd temp goes out of range or the instrument is shut off or moved, the instrument should be re-calibrated.
If Calibration fails, repeat again. Things to try are: 1. Check to see if your AtheNA Multi-Lyte® probe is clogged, 2. Verify that the probe is set at the appropriate height, 3. Verify that the calibration beads were properly vortexed and sonicated, 4. Verify that you have placed sufficient volume of the beads in each well, 5. Be sure the correct bead values have been input into the correct locations, 6.Perform a weekly maintenance procedure on the instrument as this may be a sign that the instrument is not being properly maintained. If after all these points, the calibration still fails, then call your AtheNA Multi-Lyte® distributor to have an engineer come to assess your instrument.
All sonicators have different optimal water levels. To determine the optimal water level, water can be continuously added and removed until there is a visible area in the center of the sonicator where the water appears to splash up and be agitated. When a bottle of bead suspension is held in this “sweet spot”, there should be an audible difference in sound intensity. Also, when the bottle is in this area, the laboratory technician should feel that it is the area of highest intensity. Only after verifying all these points can the water level in the sonicator be considered optimal. Note: This optimal level may not be at the line inside the sonicator that has been painted there by the manufacturer. In internal testing at ZEUS Scientific, the optimal water level is normally well below this line.
While washing a filter plate, any unused wells should be covered by either parafilm, tape, or the technician’s glove hand. The vacuum pressure should not be turned off until wells have been completely evacuated of all liquid. Note: the filter paper in the wells may still appear slightly wet, but any actual liquid must be evacuated. After the three washes, the filter plate should be gently blotted on a stack of paper towels until there is no more liquid being blotted out onto the towels. The pate should then be left on a non-absorbent surface for 3-5 minutes to air dry before proceeding to the conjugate step.