VIRCLIA® VALIDATION METHOD

Each monotest contains ready to use reagents including a Calibrator and a Negative Control to
enable validation and interpretation for each individual sample and for each test.

 The NEGATIVE CONTROL ensures:

o that wells and probe are washed adequately
o that no light enters into the system
o the proper functioning of the luminescence reader

 The CALIBRATOR, set in a range, enables the interpretation of the sample and acts as an
intra-assay positive control, monitoring that:
o the washing is correct
o there is no interference of light in the reader
o there is no over-incubation (through the upper limit)
o reagents have performed correctly (through lower limit)

 Highly efficient method based on an internal calibration, not on stored curves

VirClia® uses a highly effective control method since the sample and the control are processed
together and under the same conditions, ensuring an individual quality control for each test.
For this reason, the results traceability is excellent because each determination incorporates
its own controls for every sample. Processing the samples in parallel with the controls
eliminates the inter-run variation.

 Optimized control system

 With VirClia®, every sample is analyzed together with its two controls, Negative Control
and Calibrator, unlike other individual processing systems.
 The dual function of the Calibrator makes unnecessary a Positive Control in VirClia®.
 The Calibrator is involved in the validation of the test and enables the interpretation of the
sample. The key factor is the Cut-off value to determine if the sample is positive or
negative.
 VirClia® guarantees the price per reportable test
VirClia® kits have no hidden costs since no extra consumption of reagents is required to
perform controls or calibrations.

This is crucial, especially considering the range of infectious diseases products we offer and
because low-number parameters processed with other systems require frequent calibrations
which quickly expire, increasing the final price of the product.

Other manufacturers of monotest kits often set a price per test that does not correspond to
the actual cost per determination as they incur in additional costs such as:
- the need to run additional controls in parallel to samples
- regular calibrations
- repetition of incorrectly performed calibration
- recalibration due to software updates

OTHER SYSTEMS NEED EXTERNAL CALIBRATION

Similar kits do not include a control or calibrator within the cartridge to be processed at the
same time than the sample but, instead, they need external Calibrators that refer to a
standard curve where these values are extrapolated.

When performing frequent calibrations, the correlation with the sample is lost because they
are run as independent assays. In addition, there is no guarantee that these calibrations are
performed when appropriate.

See these examples:

Vidas, from bioMerieux:

 VIDAS strip consists of 10 wells. The first well is for the sample, then the diluent (x1), the
wash buffer (x2), the conjugate (x1), the wash buffer (x3) and the substrate in the last one.
 The control is not included in the strip which makes it necessary to work with stored
controls (Positive Control, Negative Control and Standard are included separately in the
kit).
 A calibration is required with every new lot and following calibrations are necessary every
14 days. The laboratory will incur in waste of reagents when using low-number tests.
Liaison, from Diasorin:

 LIAISON strip consists of 6 wells: one for the magnetic particles coated with the antigen,
then Calibrator 1 (low), Calibrator 2 (high), Diluent x 2 and Conjugate.
 In most of the parameters the calibrator is included but not in all of them.
 Not included material: LIAISON® Controls (negative and positive).
 The reagent integral is stable for eight weeks refrigerated either at 2-8 °C or on board the
instrument.
 The analyzer should be calibrated in triplicate whenever one of the following conditions
occurs:
– A new lot of reagent integral of the Starter Kit is used.
– The previous calibration was performed more than four weeks before.
– The analyzer has been serviced.
– Control values lie outside the expected ranges.

Elecsys & Cobas, from Roche

 The reagent rackpack consists of 3 bottles (M, R1, R2):

o The white bottle with a transparent lid contains suspended magnetic
microparticles that act as the carrier material of the ruthenium-labeled complex
during measurement.
o The black bottle with a grey lid contains reagent 1 (R1).
o The black bottle with a black lid contains reagent 2 (R2).
 Calibration mode: 2-point calibration.
 In most cases, calibrators and controls are not included in the rackpack, they come
packaged separately.
 Lyophilized controls and some calibrators must be prepared and transferred into the
appropriate container before use, as they are lyophilized and must be reconstituted.
 The diluent is also required but not provided.
 Calibration frequency:
o Calibration must be performed once per reagent lot
o Renewed calibration is recommended as follows:
 after 1 month (28 days) when using the same reagent lot
 after 7 days (when using the same reagent kit on the analyzer)
 as required: e.g. quality control findings with PreciControl CMV IgG outside
the defined limits
 Controls for the various concentration ranges should be run individually at least once every
24 hours when the test is in use, once per reagent kit, and following each calibration.
 The rackpack is stable for 12 weeks refrigerated at 2-8 °C after opening or for 3 weeks on
the analyzer.
 The calibrators are stable for 8 weeks refrigerated at 2-8 °C or up to 5 hours on the
analyzer.

Immulite, from Siemens

 The reagent wedge consists of 3 reagent compartments.

 Each kit contains one or two Adjustors. Kits with two Adjustors have a LOW and a HIGH
Adjustor, which contain different concentrations of analyte. The Adjustor can be in either
liquid or lyophilized form
 The recommended calibration interval is two weeks
 The infectious diseases kits include the following: (e.g. CMV IgM assay)
o CMV IgM Bead Pack
o Reagent wedge:
 A: 17.5 mL of a buffer solution with polyclonal goat anti-human IgG
 B: 11.5 mL of a buffer solution with polyclonal goat anti-human IgG
 C: 11.5 mL of alkaline phosphatase (bovine calf intestine) conjugated to
polyclonal goat antihuman Ig antibody (in buffer)
o CMV IgM Adjustor
o One vial of lyophilized human serum with IgM reactive to CMV (with preservative)
o CMV IgM Controls (positive and negative controls with preservative) - Bi-level
controls intended as an aid in monitoring day-to-day assay performance.
 Required but not provided material: CLIA substrate, probe wash, probe cleaning kit and
disposable reaction tubes.
Architect, from Abbott

 Calibrator and Controls are required but not provided

 To perform a calibration, test Calibrator 1 in replicates of three. Once calibration is
accepted and stored, all subsequent samples may be tested without further calibration
unless one or both of the following occur:
o A reagent kit with a new lot number is used.
o Controls are out of range.
 The recommended control requirement for the ARCHITECT assays is that a single sample of
each control be tested once every 24 hours each day of use for each reagent lot.

SUMMARY AND RECOMMENDATIONS

VirClia® is a highly reliable monotest assay simplified to the maximum, self-contained and at
the lowest possible cost without compromising its reliability. Therefore it was decided to:

- use a calibrator per sample to avoid periodical calibrations

- combine the role of the Positive Control and the Calibrator
- simplify the Negative Control

However, although Vircell does not consider it necessary, if local regulations or the institution
foresee additional quality control measures, it is possible to periodically run a known positive
sample as a positive control or an external control. The frequency will be determined by the
norms.