579© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/B978-0-08-097037-0.00046-4

The VITROS® ECiQ Immunodiagnostic System, VIT-ROS® 3600 Immunodiagnostic System, and VITROS® 5600 Integrated System are fully automated, random access labo-ratory analyzer systems that have the capability to perform immunoassays for a wide range of analytes using VITROS Microwell™ technology. All VITROS® systems are engi-neered and manufactured by Ortho Clinical Diagnostics, part of the Johnson & Johnson Family of Companies. Microwell is a heterogeneous immunoassay technology (originally developed by Amersham International, now part of GE Healthcare), with a signal generation system based on enhanced chemiluminescence. A key feature of the Microwell technology is the ability to accommodate one- and two-step assays, with multiple incubation times, enabling each assay to be separately optimized with respect to performance and speed. The Microwell assay menu includes a broad array of infectious disease, as well as a number of analytes in most other major disease states, including: Cardiology, Oncology, Thyroid, Metabolic, Endocrine, and Anemia.

The VITROS ECiQ (Fig. 1) and VITROS 3600 Immu-nodiagnostic Systems (Fig. 2) and VITROS® 5600 Inte-grated System (Fig. 3) have three main centers in common:

Sampling Center. The sampling center (Fig. 4) accommodates up to 90 samples (60 samples for VIT-ROS ECiQ) of various configurations into the Routine lane (80 samples) or STAT lane (10 samples). Samples

are loaded in Universal Sample Trays that can accom-modate a variety of sample containers, including: pri-mary and secondary tubes, sample cups, and micro-collection containers (using a universal micro-collection adapter). Sample trays may be loaded or unloaded during operation.

Microimmunoassay Center. Assay reagents and wells are supplied in special integrated reagent packs (Fig. 5). Reagents for up to 3100 tests (2000 tests for VITROS ECiQ) may be stored in the temperature and humidity-controlled chamber, with access available at any time. Dispensing, incubation, washing, and signal generation are carried out in a specially designed incubator that allows multiple protocols to run simultaneously.

Command Center. The system is operated by a color-coded graphical user interface, with a touchscreen monitor and a detached keyboard. The user interface includes onboard documentation and help as well as maintenance documentation.

Typical Assay ProtocolTests may be ordered directly or downloaded from a host computer. Assays are processed in coated Microwells (Fig. 6). A well is first dispensed into the circular incubator from the reagent pack. Sample is dispensed into the well, which rotates back to the reagent area for reagent addition. Incubation is at 37 °C. At the end of the incubation, the well is transferred

VITROS® ECiQ Immunodiagnostic System, VITROS® 3600 Immunodiagnostic System, and VITROS® 5600 Integrated SystemJohn W. Backus (jbackus@its.jnj.com)

David Wild (david@davidwild.net)

C H A P T E R

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FIGURE 1 VITROS® ECiQ Immunodiagnostic System (The color version of this figure may be viewed at www.immunoassayhandbook.com).

FIGURE 2 VITROS® 3600 Immunodiagnostic System (The color version of this figure may be viewed at www.immunoassayhandbook.com).

580 The Immunoassay Handbook

from the outer to the inner ring. The contents are aspirated and the well washed. Signal reagent is added and the contents of the well are incubated for 4.5 min. Finally, the lumines-cence is measured in a luminometer prior to the well being discarded to the waste container.

Product Featuresll Broad menu capability. A three-ring incubator design

(two-ring for VITROS® EciQ) allows assays of mixed incubation times to be run together (Fig. 7). In the three-ring design, the center ring is dedicated to per-forming dilutions and pretreatment steps.

ll Fully automated, continuous random access. Samples and reagents may be loaded or removed at any time. STAT tests are prioritized upon order with results within 15 min.

ll Integrated reagent pack. All components necessary to process a Microwell assay (including the Microwell) are integrated into a ready-to-use single universal package, thereby greatly reducing the potential for operator error.

ll Small sample volume requirements. Each Microwell assay requires 10–80 µL of sample.

ll Autodilution and reflex testing, i.e., automatic re-assay at a different dilution or for a different analyte.

ll Universal sample trays that can handle primary and sec-ondary sample tubes and cups. There is no need for adapters. Up to 90 samples can be loaded onboard.

ll Positive sample identification. Barcode readers recog-nize all major symbologies.

ll Intellicheck™ Technology. Features include: liquid level sensing, sample clot and bubble detection, and sample and reagent aspirate and dispense verification. Maintains the integrity of patient sample and mini-mizes sample repeats and redraws.

ll Disposable tips. Avoids the risk of cross-contamination.

ll Onboard capacity for up to 31 barcoded integrated reagent packs (100 tests each). On- and off-board

FIGURE 3 VITROS® 5600 Integrated System (The color version of this figure may be viewed at www.immunoassayhandbook.com).

FIGURE 4 Sampling center of the VITROS® 5600 Integrated System (The color version of this figure may be viewed at www.immunoassayhandbook.com).

FIGURE 5 Reagent pack. The reagent pack contains 50 or 100 coated Microwells™ and one or two reagents. The reagents are protected from evaporation loss by a closure that swivels to allow access to the reagents. Onboard stability is up to 12 weeks, although recalibration is required every 28 days (The color version of this figure may be viewed at www.immunoassayhandbook.com).

FIGURE 6 Microwell™. The tapered shape of the coated Microwells allows them to be nested at the back of the reagent pack. The shape also improves initial mixing and washing efficiency. The use of coated Microwells avoids the need for separate disposable reaction cuvettes, minimizing wastage and disposal (The color version of this figure may be viewed at www.immunoassayhandbook.com).

581CHAPTER 7.13 VITROS® ECiQ Immunodiagnostic System, VITROS® 3600 Immunodiagnostic System, and VITROS® 5600 Integrated System

reagent inventory control. Throughput is up to 189 tests per hour (up to 90 tests per hour for VITROS® ECiQ). Walkaway time is approximately 1 h. Reagent packs may be loaded and unloaded safely while the sys-tem is running tests.

ll Onboard reagent stability (temperature and humidity-controlled) for up to 84 days.

ll Calibration stability is 28 days. Ability to store multiple calibration curves per analyte.

ll Minimal routine maintenance, guided by the software with an automated procedure via a special maintenance pack.

ll Universal wash reagent for reagent dispensing probe (VITROS® ECiQ only) and well wash.

ll User-friendly operator interface is intuitive, logical, and easy to use.

ll No plumbing required. Connection to a water source or drain is not required to execute testing and manage processing waste.

ll Remote, real-time system diagnostics using e-connec-tivity, which can reduce time spent on maintenance.

VITROS® 3600 Immunodiagnostic System and VITROS 5600® Integrated System also:

ll Utilize MicroSensor™ technology to measure sample quality indices for hemolysis, turbidity, and icterus without using additional samples, reagents, disposables, or slowing system throughput.

ll Employ the single-use VersaTip™ reagent metering system, eliminating the need for fixed probes and reducing universal wash reagent usage by up to 50%.

ll Allow for uninterrupted assay processing while chang-ing signal reagent, universal wash reagent, and solid and liquid waste containers.

Assay PrincipleCompetitive and immunometric formats are used for mea-surement of both antigens and antibodies (see Figs. 8 and 9). Most of the assays are designed for quantitative use except for those in the infectious disease range where most are for qualitative testing.

For qualitative tests, other formats such as antiglobulin and antibody class capture are also used.

CalibrationA calibration pack is supplied with each kit lot and includes lot-specific master curve information (typically based on at least seven reference standard concentrations) and either two or three lot-specific calibrators that are run to adjust the master curve for that system. The calibrators are fac-tory calibrated for each new kit lot (coincident with the generation of the master curve data), thereby ensuring that the master curve and adjusting calibrators are perfectly matched for each kit lot.

Semiquantitative assays use a single calibrator to estab-lish a cutoff value.

Calibrations are stable for up to 28 days.

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FIGURE 7 Journey of the Microwell™. Coated Microwells are dispensed into the incubator ring via a shuttle to keep the cooled reagent pack area separate from the warm incubator (1). Sample is metered into the well (2) followed by the reagents (3). The well is incubated for a fixed time, according to the test protocol (4). The well is then shuttled into the inner processing ring (5) for the longer processes of washing (6), signal reagent addition (7), incubation for 4.5 min (8), and signal reading (9). Finally, the well is discarded into the waste collection box (10) (The color version of this figure may be viewed at www.immunoassayhandbook.com).

582 The Immunoassay Handbook

AntibodiesBoth polyclonal and monoclonal antibodies are used, depending on the assay design. In many assays, the anti-bodies are not immobilized onto the plastic wells but are biotinylated and provided in solution. Streptavidin coated onto wells captures the biotinylated antibodies during incubation. This format prevents the deformation of anti-body, with consequent loss of functionality, that can occur with protein immobilization.

SeparationThe solid phase is a polystyrene-based Microwell, tapered to allow 100 wells to be stacked in the back of a reagent pack. The wells, which have exacting specifications, are manufactured in a purpose-designed facility. For the majority of assays, the wells are coated with streptavidin. In some cases, the coating is donkey anti-sheep second

antibody and various other coatings are used for specific tests.

Signal Generation and DetectionThe enzyme label is horseradish peroxidase in all assays. The bound fraction (conjugated antibody or antigen) is quantified by measuring the activity of horseradish peroxi-dase in the well after washing and aspiration. A signal reagent is added to the wells that contains a peracid salt, luminol, and an enhancer. It is supplied in a pack that con-tains two solutions. The peracid salt generates peroxide ions in solution, which oxidizes the luminol, catalyzed by the horseradish peroxidase. In a series of reactions, the oxi-dized form of luminol breaks down, with the generation of light. The enhancer increases the level of light produced several 100-fold and prolongs its emission. The light is

FIGURE 8 Typical competitive immunoassay format.

583CHAPTER 7.13 VITROS® ECiQ Immunodiagnostic System, VITROS® 3600 Immunodiagnostic System, and VITROS® 5600 Integrated System

read in the analyzer 4.5 min after the addition of the signal reagent.

Data ProcessingFor quantitative assays, calibration curves are fitted using a modified four- or five-parameter log-logistic program.

The signal levels from the calibrators are used to adjust the master curve, supplied by the manufacturer either on an Assay Data Disk or ADD (on VITROS® 3600 and VIT-ROS® 5600) or on a magnetic card (on VITROS® EciQ). The system reads the luminescent signal for each sample and determines the analyte concentration from the stored calibration curve. Calibration curves and Levey–Jennings quality control charts may be viewed or printed.

FIGURE 9 Typical immunometric format.

584 The Immunoassay Handbook

Interfacing to Laboratory Information SystemsThe VITROS® ECiQ and VITROS® 3600 Immunodiag-nostic Systems and the VITROS® 5600 Integrated System all have RS232-C serial interfaces with bidirectional capa-bility, along with the software necessary to link these sys-tems to central laboratory information management systems. The instruments can be interfaced with lab auto-mation consisting of a variety of transport systems that automate the pre- and post-analytical processes including centrifugation, aliquoting, sample integrity checking, stopper removal and recapping, sample sorting, and sam-ple storage. VITROS systems can also be connected to a variety of automation systems, including the enGen™ Automation solution from Ortho Clinical Diagnostics. The design of these systems includes metering using dis-posable sampling tips from tubes on the automation sys-tem track, without the need for robotics to move tubes to and from the sample supply or extra aliquot tubes. VITROS Systems retain the capability to process urgent STAT samples in a manual mode.

Further ReadingBlasutig, I.M., Jung, B., Kulasingam, V. et al. Analytical evaluation of the VITROS®

5600 Integrated System in a pediatric setting and determination of pediatric reference intervals. Clin. Biochem. 43, 1039–1044 (2010).

Demers, L.M. Thyroid function testing and automation. J. Clin. Ligand Assay 22, 38–41 (1999).

Januzzi, Jr., J.L., Lewandrowski, K.B., Bashirians, G. et al. Analytical and clinical performance of the Ortho-Clinical Diagnostics VITROS® amino-terminal pro-B type natriuretic peptide assay. Clin. Chim. Acta 387, 48–54 (2008).

Tanhehco, Y.C., Peck Palmer, O.M., Derrico, L.S. and Blair, H.C. Biases in antibody-based single-phase assays for free thyroxine due to protein-bound analyte. Clin. Chim. Acta 406, 176–178 (2009).

Thorpe, G.H.G., Kricka, L.J., Moseley, S.B. and Whitehead, T.P. Phenols as enhancers of the chemiluminescent horseradish peroxidase-luminol-hydrogen peroxide reaction: application in luminescence-monitored enzyme immunoas-says. Clin. Chem. 31, 1335–1341 (1985).

Thorpe, G.H.G. and Kricka, L.J. Enhanced chemiluminescent assays for horserad-ish peroxidase: characteristics and applications. In: Bioluminescence and Chemiluminescence, New perspectives Proceedings of the Fourth International Bioluminescence and Chemiluminescence Symposium, Freiburg, September 1986 (eds Schulmerich, J., Andreesen, R., Kapp, A., Ernst, M. and Woods, W.G.) 199–208 (Wiley, Chichester 1986).

Whitehead, T.P., Thorpe, G.H.G., Carter, T.J.N., Groucult, C. and Kricka, L.J. Enhanced luminescence procedure for sensitive determination of peroxidase-labeled conjugates in immunoassay. Nature 305, 158–159 (1983).

Zaninotto, M., Vernocchi, A., Di Serio, F. et al. Assay performance improved, but which “scorecard” designation for Vitros Troponin I? Clin. Chim. Acta 413, 826–828 (2012).