Innofluor^® Certican^® Assay
Helpful Hints and Frequently Asked Questions

Helpful Hints:

Storage/Handling conditions for Innofluor Certican Reagent, Calibrators, Controls and patient samples

Innofluor Certican Reagent:
Store the reagents with the vials tightly capped at 2 to 8 °C protected from light when not in use. Do not use reagents beyond the expiration date printed on the package and/or the vials.

Innofluor Certican Calibrators and Controls:
Store the calibrators and controls at < 10 °C prior to opening. Once opened or thawed, store the calibrators and controls at 2 to 8 °C for no longer than six weeks. Do not allow calibrators or controls to remain at room temperature longer than is required to perform the assay. Do not use the calibrators or controls beyond the expiration date printed on the package and/or the vials.

Patient Samples:

Patient samples should be stored at 2 to 8 °C for no longer than three days. If longer storage is needed, whole blood should be stored frozen at –20 °C or colder for up to 28 days. Care should be taken to limit the number of freeze thaw cycles.

Prior to extraction, allow the calibrators, controls, and patient samples to completely thaw to room temperature. After thawing, mix thoroughly but gently to provide a homogeneous mixture and to avoid the formation of bubbles.

Extraction of Innofluor Certican Calibrators, Controls and patient samples

The majority of the sample extract is methanol. It is important to limit the time between extraction and sample analysis to prevent evaporation. Evaporation of the sample may lead to falsely elevated results.

After extraction Certican calibrators and controls will be clear with a slight brownish color. Due to the EDTA, patient samples often/should be clear with a slight/faint greenish color. It is normal for the sides of the centrifuge tubes to have brown patches where the cellular material was not dislodged during extraction. The pellet should be hard and not easily disturbed. A small number of brown precipitate in the supernatant is normal and care should be taken to avoid pipetting this material into the sample cup.

The Innofluor Certican reagents and calibrators must be used to generate the calibration curve on the analyzer prior to running controls and patient samples.

Calibration of Innofluor Certican

Two separate extractions for each Innofluor Certican calibrator must be performed and run to generate a valid calibration curve.

The Innofluor Certican calibrator values are lot specific. Therefore, a calibrator value card is enclosed in each calibrator kit. Verify the correct calibrator value is assigned in the instrument assay parameters prior to each run.

The assay calibration should meet the following criteria: PERR limits between -2.0 and +2.0, RMSE < or = to 1.0, and control ranges within established acceptable ranges. If these criteria are not met, verify that the assay activation and parameter edit are correct and repeat the calibration procedure.

Frequently Asked Questions:

Why is it necessary to run the calibrators in duplicate?

It was decided, due to the inherent imprecision in whole blood immunosuppressant immunoassays, that running calibrators in duplicate would provide improved accuracy and precision of the calibration curve thus providing a robust curve in which clinical sample values can be deduced from. The assay was validated on original sample volume (600µL) and reduced sample volume (300µL) with duplicate calibrator extractions to produce the calibration curves. Single calibrator extraction would increase the PERR error considerably. PERR is the product of an average of the duplicate extracted sample readings. From the average it calculates the curve fit. A customer running on single calibrator extraction would be outside the validation of this assay.

What causes PERR and RMSE errors and how can I prevent them?

PERR, polarization error, and RMSE, root mean square error, are used as guidance for a valid calibration curve. If either value is outside of the range within the package insert, check for air bubbles in samples and tubing, check calibrator values in programming, replace reagent pack, verify probe performance, photo check, temperature check, buffer, etc.
Using good pipetting technique is important with Certican extractions.

What causes samples not monotonic error messages and how can I prevent them?

This occurs when the polarization does not change in a constant direction. Calibrators are often in the incorrect order - fluorescence should be going in the descending order. Following the instructions within the package insert and calibrator lot specific assigned values should prevent this problem.

What causes the RANGE TOO LARGE error message and how can I prevent them?

This error occurs when the net difference between calibrator replicates is >/= to 6mP. It is normal for this to occur occasionally as the result of instrument malfunction or debris interfering with instrument readings of the cuvette. If this occurs repeatedly it can be a sign of systemic instrument problem or improper extraction technique. If instrument malfunction is suspected perform pipette check and calibrations per operators manual.

To determine if extraction technique is the cause, verify by visual check that the supernatant in the sample cups from the same extraction appear identical. If one appears more or less cloudy than the other this is a sign of improper extraction technique and can cause poor precision between replicates. Not changing the recommended parameter settings, following the instructions within the package insert and application sheet and training for technique should resolve any issues relating to RANGE TOO LARGE.

Why are the controls and/or patient samples recovering too high?

There are several possible reasons for this:

• Calibrator used to calibrate instrument have degraded due to improper shipment or storage - Obtain new calibrators and re-calibrate (Remember to update calibrator concentration in the TDx parameters)
• Calibration needs to be repeated - Generally a calibration curve will last at least 2 weeks. However, the control values will increase over time. When they increase beyond the published range (or beyond the labs acceptable limits) the TDx should be re-calibrated. Re-calibration may also be necessary when changing to new boxes of reagent. It should be performed with new lots of reagent.
• Extraction Error - Must perform extraction per package insert instructions
• TDx malfunction - Verify TDx passes all maintenance checks and is performing to specification

Why are the controls and/or patient sample recovering too low?

There are several possible reasons for this:

• Controls have been improperly stored or shipped - Obtain a new box of controls and re-test.
• Calibrator values are incorrect in TDx parameters for lot of calibrator used to calibrate the instrument - Verify by checking assay parameters that the values in the instrument match the assay value card that came with calibrators.
• Once extracted, the supernatant was allowed to remain on the pellet for too long - If the supernatant remains on the pellet for too long, the drug may actually absorb back into the lipophilic cellular material in the sample pellet. Samples should be assay immediately after centrifugation or be pulled from the sample pellet and stored sealed and at cold temperatures.
• Improper Extraction technique - Controls and samples must be extracted exactly per the procedure in the package insert.
• Instrument error or malfunction - Verify that the TDx is properly maintained and passes all routine maintenance checks.

Why am I seeing issues with poor precision?

Poor precision could be due to several possibilities:

• Incomplete thawing and mixing of calibrators/controls prior to extraction.
• Deviations in time periods between extraction and running of contols (and calibrators)
• Instrument - cracked or loosely fitted syringes, cracked or loose tubing, worn belts - instrument performance can be tested by running a different assay and performing maintenance checks.
• Possible carry-over - Dirty or damaged probes can cause carry-over. The TDx manual has a procedure for testing carry-over.