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The immuassay handbook parte58



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  • 1. 561© 2013 David G. Wild. Published by Elsevier Ltd. All rights reserved. The Abbott ARCHITECT instruments are a family of immunoassay and clinical chemistry analyzers that can be operated either as stand-alone systems, or integrated sys- tems in specific combinations to consolidate clinical chem- istry and immunoassay testing on one platform, operated through a single computer interface. The ARCHITECT family of analyzers is composed of two stand-alone immu- noassay analyzers (i-series, Fig. 1), the ARCHITECT i2000SR and i1000SR, three stand-alone chemistry analyzers (c-series, Fig. 2), the ARCHITECT c4000, c8000, and c16000, three integrated chemistry/immunoassay analyzers (ci-series Fig. 3), the ci4100, ci8200, and ci16200, and one integrated immunoassay analyzer, the i4000SR (Fig. 3). Each of these instruments has different assay throughputs and reagent load capacities (described in the Product Features section) that address specific laboratory testing volume and workflow needs. All instruments in the ARCHITECT fam- ily use a common robotic sample handler (RSH), and have the ability to perform STAT testing, auto-dilution, and auto-retest. The RSH also ensures STAT samples are pro- cessed first, ahead of any routine samples already loaded in the sample bays. All ARCHITECT instruments utilize the same system software, a common sample Abbott ARCHITECT® Family of Analyzers Frank A. Quinn ( David A. Armbruster ( C H A P T E R 7.9 FIGURE 1 ARCHITECT stand-alone immunoassay analyzers (ARCHITECT i-series). (The color version of this figure may be viewed at FIGURE 2 ARCHITECT stand-alone chemistry analyzers (c-series). (The color version of this figure may be viewed at
  • 2. 562 The Immunoassay Handbook carrier, and identical assay reagents across c-series or i-series instruments. These design features result in system com- monality that allows ARCHITECT c-series and i-series instruments to be connected in specific combinations to form integrated systems operated through a single user interface. An ARCHITECT i2000SR may be integrated with an ARCHITECT c8000 or c16000 to form a single instrument, the ARCHITECT ci8200 or ARCHITECT ci16200, respectively. In addition, two ARCHITECT i2000SR instruments may be integrated to form an ARCHI- TECT i4000SR. Similarly, an ARCHITECT i1000SR may be combined with an ARCHITECT c4000 to form an ARCHITECT ci4100. Integrated systems (ci-series) retain the same sample throughput, reagent capacity, and sample handling capabilities of the stand-alone systems. The ability to use stand-alone and/or integrated systems provides labo- ratories with the flexibility to address complex workflow needs, both within a single laboratory, as well as across mul- tiple laboratories and sites. ARCHITECT family common- ality ensures equivalent patient results are obtained, regardless of which instrument configurations are used. Across the ARCHITECT family, reagents are available to perform a wide variety of assays including thyroid, fertility, routine chemistry panels, specific proteins, enzymes, elec- trolytes, oncology, cardiac, metabolic, and infectious dis- ease analytes. All assays on the ARCHITECT i-series analyzers use chemiluminescent immunoassay technology. Multiple immunometric assay technologies are available on the ARCHITECT c-series analyzers. Typical Assay Protocols The ARCHITECT i-series analyzers are capable of simultaneously performing two-step, one-step, delayed one-step, STAT, auto-retest, and automated pre-treatment protocols. The availability of multiple protocols provides the flexibility required to address analyte-specific assay development needs. For illustration purposes, typical i-series assay protocols are described below using the i2000SR and its process path (Fig. 4) as an example. On the ARCHITECT i-series, the majority of assays use a two- step protocol. In two-step routine assays, specimen and microparticles are added to a reaction vessel at positions 1 and 2 of the process path, respectively. Reaction compo- nents are then thoroughly mixed by a pop-up mixer (or, in-track vortexer, ‘ITV’) at position 3. Incubation contin- ues until the reaction vessel reaches wash zone 1. In wash zone 1, magnets attract the microparticles to the inner wall of the reaction vessel, and unbound material is washed away in a series of three washes. After wash zone 1, conjugate or tracer is added to the reaction vessel, and reaction compo- nents are mixed using a pop-up mixer. Incubation contin- ues until the reaction vessel reaches wash zone 2, where the microparticles undergo a second series of three washes to remove unbound material. After the reaction vessel leaves the second wash zone, a pre-trigger reagent is added. This reagent releases the conjugate or tracer into solution, and prepares the label for the light-generating reaction. Mic- roparticles are then magnetically attracted to the inner wall of the reaction vessel, separating them from the label, and the trigger reagent is added. The trigger reagent initiates the final phase of the light-generating reaction, and the resulting chemiluminescence is measured using a photo- multiplier tube. The measured relative light units (RLUs) are processed by the data management software to yield the assay result. Finally, the reaction mixture is aspirated, and the empty reaction vessel is transferred to a waste con- tainer. In routine one-step assays, specimen is added to the reaction vessel at position 1. Particles and conjugate or tracer are added at position 2. Reaction components are then thoroughly mixed by the pop-up mixer at position 3. As the reaction vessel approaches wash zone 1, it is diverted to the outer lane of the process path, allowing the reaction vessel to by-pass wash zone 1 and continue incubation without interruption. Incubation continues until FIGURE 3 ARCHITECT integrated analyzers (ci-series, and i4000). (The color version of this figure may be viewed at
  • 3. 563CHAPTER 7.9 Abbott ARCHITECT® Family of Analyzers the reaction vessel reaches wash zone 2. In wash zone 2, microparticles are magnetically attracted to the inner wall of the reaction vessel, and unbound material is washed away in a series of three washes. From this point, reaction vessel processing is as described for the two-step protocol. For fully automated pre-treatment protocols, specimen and pre-treatment reagent are combined in a reaction vessel at positions 1 and 2 of the process path, respectively. After an incubation of approximately 7min, an aliquot of the pre- treated specimen is removed by the sample pipetter and reintroduced into a new reaction vessel at position 1 of the process path. From this point, the assay runs in either a two-step or one-step protocol as described above. STAT protocols are made possible by the addition of a second sample pipetter and pop-up mixer to the system hardware. These hardware modifications allow for shorter assay incu- bation times by providing for STAT sample introduction and mixing at a point farther along in the system sample processing path. Reagents for STAT assays are optimized to allow for the shorter incubation time without compro- mising assay performance. STAT assay results are available within 15.6min for the ARCHITECT i-series. The ARCHITECT c-series analyzers are capable of performing a variety of different homogeneous assay pro- tocols, as well as STAT assay protocols (quickest results within 2.6min and no longer than 10min). A schematic of the ARCHITECT c-series process path is shown in Fig. 5. Some assays on the ARCHITECT c-series analyz- ers are based on immunoturbidimetric methodologies like particle enhanced turbidimetric inhibition immuno- assay (PETINIA) and particle enhanced turbidimetric immunoassay (PETIA). In PETINIA, latex microparti- cles are coated with small-molecule analytes (antigens). Unbound microparticles suspended in solution are too small to block the passage of light through a reaction cuvette. However, when antibodies to the antigen on the microparticles are added, the particles aggregate and form lattices (large complexes) that block the passage of a light beam. As in many competitive immunoassay methodolo- gies, free antigen present in a patient sample competes with the antigen-coated microparticles for antibody. The greater the concentration of analyte in the patient sample, the less aggregation and lattice formation and the smaller the decrease in light transmission through the cuvette. The smaller the concentration of analyte in the patient sample, the more aggregation and lattice formation and the larger the decrease in light transmission. When the microparticles are coated with antibody instead of anti- gen, the methodology is known as PETIA. Depending on whether the assay is designed to use PETINIA or PETIA, the inhibition of lattice formation or the formation of lat- tices may be measured as a rate reaction. The rate of increase or decrease in light transmission is directly pro- portional to analyte concentration. Enzyme multiplied immunoassay technique (EMIT®) is also used on the ARCHITECT c-series to perform homo- geneous competitive assays. In these assays, analyte (antigen) in the patient sample competes for antibody binding sites with an enzyme conjugate labeled with a modified version of the analyte. The enzyme conjugate is active unless the analyte portion of the conjugate is bound by the antibody, blocking the enzyme’s active site. The enzyme used in EMIT is glucose-6-phosphate dehydrogenase (G6PDH). When the concentration of analyte in the patient sample is high, more antibodies are bound to analyte and less to enzyme conjugate. Conversely, when the concentration of analyte in the patient sample is lower, more enzyme conju- gates is bound by antibody. Free (unbound) enzyme conju- gate converts substrate to product nicotinamide adenine dinucleotide phosphate (NAPDH), and the rate of the FIGURE 4 ARCHITECT i2000SR process path. (The color version of this figure may be viewed at
  • 4. 564 The Immunoassay Handbook reaction is measured spectrophotometrically at 340nm. In this format, the rate of increase in product is directly proportional to the concentration of analyte in the patient sample. EMIT is routinely used for both drugs of abuse and therapeutic drugs. Product Features G The Abbott ARCHITECT series is a family of modular, stand-alone and integrated chemistry and immunoassay analyzers. Instruments may be combined in specific ways to address a variety of workflow and testing needs. G All instruments in the ARCHITECT family use a robotic sample handler (RSH), identical software, a universal sample carrier, and identical assay-specific reagents and protocols. G ARCHITECT family commonality ensures that equiv- alent patient results are obtained across all ARCHI- TECT chemistry and immunoassay instruments. G Common software ensures a consistent user experience across all instruments in the ARCHITECT family. Integrated systems are operated through a single soft- ware user interface. G The RSH allows prioritization of STAT tests to ensure they are processed first. All ARCHITECT family instruments provide STAT assay results between 2.6 and 10min for c-series tests, and less than 15.6min for i-series tests. STAT assay timing is the same for both stand-alone and integrated systems. G Pressure differential monitoring technology reduces analytical error by detecting clots, bubbles, foam, and insufficient sample volume. SmartWash technology effectively controls sample-to-sample carryover for the integrated systems (<0.1ppm) and reaction vessel-to- reaction vessel carryover for the iSystems (<0.35ppm). G All ARCHITECT i-series assays utilize CHEMIFLEX technology, a combination of flexible assay protocols and Abbott-developed chemiluminescent detection technology. G ARCHITECT i2000SR. Stand-alone immunoassay analyzer with a maximum throughput of 200 tests per hour. Continuous sample access with 25 refrigerated reagent storage positions and sample load-up capacity of 135 via priority (35) and routine (100) areas. Reagent kit sizes of 100 or 500 tests provide high reagent test capacity to maximize walk-away time. May be inte- grated with another ARCHITECT i2000SR, c8000 or c16000 to form an ARCHITECT i4000SR, ci8200, or ci16200, respectively. G ARCHITECT i1000SR. Stand-alone immunoassay analyzer with maximum throughput of 100 tests per hour. “Load on the fly” access to reagents and samples. May be integrated with an ARCHITECT c4000 chem- istry analyzer to form an ARCHITECT ci4100. G ARCHITECT i4000SR. Integrated immunoassay ana- lyzer with maximum throughput of 400 tests per hour. Continuous sample access with 50 refrigerated reagent positions and sample load-up capacity of 285 samples via priority (35) and routine (250) areas. Flexible reagent kit sizes of 100 or 500 tests allow optimization of system capacity for maximum “walk-away” time. Single user interface. G ARCHITECT c8000 and c16000. Stand-alone chemistry analyzers with maximum throughputs of 1200 tests per hour for the c8000 and 1800 tests per hour for the c16000. Load capacity of 215 samples (including 35 priority posi- tions) and 65 refrigerated reagent positions plus inte- grated chip technology (ICT) modules for Na+, K−, and Cl−. May be integrated with an ARCHITECT i2000SR analyzer to form a ci8200 or ci16200, respectively. G ARCHITECT c4000. Stand-alone chemistry platform with maximum throughput of 800 chemistry tests per hour. Load capacity of 100 samples with up to 35 sam- ple priority positions. May be integrated with an FIGURE 5 ARCHITECT c-series process path. (The color version of this figure may be viewed at Position Description 1 Sample pipettor dispenses sample. 2 Reaction carousel rotates approximately 1/4 turns. Reagent pipettor 1 dispenses reagent 1 in the cuvette containing sample. 4 The reaction carousel rotates one cycle to the first mixing position where the mixer unit (mixer 1) mixes the sample and reagent 1. Between 4 and 5 As the reaction carousel rotates from position 4 to position 5, the cuvette passes the photometric position where the lamp is located and the photometer measures the absorbance. 5 Reaction carousel has completed 4 cycles. If onboard dilution is required, the sample pipettor aspirates the diluted sample and dispenses the sample into the new cuvette that is currently at position 1. 31 For an ICT sample, the ICT probe aspirates the diluted sample into the ICT unit. 67 If the reaction requires a second reagent, reagent pipettor 2 dispenses reagent 2 into the cuvette. 68 The mixer unit (mixer 2) mixes the second reagent with the sample and reagent mixture. Note: The reaction carousel continues to rotate and the reaction mixture incubates. The photometer takes absorbance readings every time the cuvette passes the photometric position for a total of up to 33 read times. The cuvette washer removes the reaction mixture to waste and cleans the cuvette with Alkaline Wash, Acid Wash, and DI water. Then the cuvette washer dispenses DI water into the cuvette for a water blank measurement to ensure cuvette integrity. Finally, the cuvette washer aspirates the water and dries the cuvette.
  • 5. 565CHAPTER 7.9 Abbott ARCHITECT® Family of Analyzers ARCHITECT i1000SR analyzer to form an ARCHI- TECT ci4100. G ARCHITECT ci4100. Consolidated clinical chemistry and immunoassay testing on a single platform with maximum throughput of up to 800 chemistry and 100 immunoassay tests per hour. On-board reagent capac- ity of approximately 55 chemistry kits and 25 immuno- assay kits. Load capacity of up to 180 samples with 35 sample priority positions. Single user interface. G ARCHITECT ci8200 and ci16200. Consolidated chemistry and immunoassay testing platform with maximum throughput of 1200 chemistry and 200 immunoassay tests per hour, and 1800 chemistry and 200 immunoassay tests per hour, respectively. Load-up capacity of 365 samples (including 35 priority posi- tions) and up to 90 refrigerated reagent positions plus ICT for Na+, K−, and Cl−. Single user interface. Assay Principle On the ARCHITECT i-series instruments, all assays use chemiluminescent magnetic microparticle immuno- assay (CMIA) technology. The majority of assays are two-step, immunometric assays with antibody attached to paramagnetic microparticles, and an Abbott-devel- oped acridinium-derivative-labeled antibody/small molecule as conjugate or tracer. Some assays have anti- gens or other proteins coated on the microparticles. Using two-step formats wherever appropriate reduces assay non-specific binding (NSB), eliminates exposure of the conjugate/tracer to potential interferents in the specimen (e.g., human anti-mouse antibodies, ‘HAMA’), and minimizes the potential for high-dose hook effects. Low molecular weight analytes are predominantly mea- sured using a two-step ‘competitive’ protocol. In this format, analyte is first extracted from specimen on to the paramagnetic microparticles. In step 2, tracer binds to unoccupied binding sites on the particles. A small number of assays use a one-step or delayed-one step protocol. Pre-treatment assays utilize the same capture and signal-generating principles as one- or two-step assays, except that the specimen is incubated with pre- treatment reagent prior to the capture and detection phases of the assay. STAT assay protocols operate under the same assay principles described above, but have a shorter first incubation time. ARCHITECT c-series instruments use several different homogeneous immunoassay methodologies. These include immunoturbidimetry (e.g., PETIA and PETINIA), and EMIT®. The principles of these assay formats are described above. Calibration Assay calibration is dependent on the particular assay and instrument system. For the ARCHITECT i-series instru- ments, some assays utilize a six-point calibration, while oth- ers utilize two-point adjustment of a master calibration curve. For the latter method, the master calibration curve is established during reagent manufacturing using six calibra- tors, and is stored within the two-dimensional barcode asso- ciated with each reagent lot. The two-dimensional barcode associated with reagent and calibrator lots also contains expi- ration date and reagent inventory information. For the ARCHITECT c-series instruments, the assays are calibrated by the user following assay-specific methods utilizing a mini- mum of two calibrators. The statistical methodology for establishing calibration curves, as well as the process for cali- bration curve adjustment (if applicable), depends on the assay and instrument system. Where available, assays are standard- ized versus accepted international reference preparations (e.g., World Health Organization International Reference Preparations, National Institute of Standards and Technology (NIST) or Institute for Reference Materials and Measurements (IRMM) standard reference materials (SRMs), or certified ref- erence materials (CRMs), highly purified commercially avail- able material (e.g., US Pharmacopeia grade)), or internationally recognized reference measurement procedures. The Joint Committee for Traceability in Laboratory Medicine (JCTLM) maintains a database of internationally recognized reference materials and methods but immunoassays pose a special chal- lenge because in many cases neither a material nor a method may be available. In these cases, a manufacturer’s in-house stan- dard and/or the best available method is the basis for calibrator traceability and assay standardization. Antibodies Immunoassays on the ARCHITECT family of analyzers, both c-series and i-series, may utilize monoclonal or poly- clonal antibodies, or antibody fragments. The choice of antibody is assay specific, and is based on analytical and clinical performance requirements established during assay development. Where applicable, reagent storage buffers contain blocking agents to minimize potential interfer- ences caused by heterophilic antibodies and HAMA. Separation For the ARCHITECT i-series instruments, all assays use paramagnetic microparticles. Magnetic separation of the solid phase from unbound materials occurs in the wash zones. An optimized saline/surfactant buffer is used to per- form the washing. Within each wash zone, the washing event is composed of three distinct dispense/aspirate cycles of this system wash buffer. For the ARCHITECT c-series instruments, immunoassays are homogeneous, and may involve either one or two reagent additions. Immunoassay reactions take place in standard spectrophotometer glass cuvettes and a separation mechanism is not available, so assay methodologies that do not require a separation of bound and unbound reaction components are used. Signal Generation and Detection For ARCHITECT i-series instruments, signal generation is based on an Abbott-developed acridinium derivative with
  • 6. 566 The Immunoassay Handbook chemical properties optimized for use in immunoassays. This class of compounds (sulfopropyl acridinium carbox- amides) has better aqueous solubility and stability than tra- ditional N10-methylacridinium-9-carboxylic acid phenyl esters. The light-generating reaction is initiated by the addition of a pre-trigger reagent containing acid and hydro- gen peroxide. This pre-trigger reagent causes the label to be released from the solid phase and into solution. The microparticles are then magnetically attracted to the inner wall of the reaction vessel, separating them from the label, which remains in solution. In the final phase of the reac- tion, a trigger reagent-containing base is added. The resul- tant chemiluminescence is measured by a photomultiplier tube (as relative light units, “RLU”) and translated by the data management software into the assay result. The light generating reaction is shown in Fig. 6. On the ARCHITECT c-series instruments, all immuno- assay formats utilize spectrophotometric detection. In some cases, for example EMIT assays, analytical signal is gener- ated by an enzymatic reaction and the rate of the reaction is measured by monitoring absorbance changes over time. For the immunoturbidimetric assays, absorbance change is monitored, either as an increase in absorbance through an antibody-mediated formation of microparticle lattices or the inhibition of formation of microparticle lattices. Informatics, Data Processing, and Laboratory Information Systems (LIS) The ARCHITECT clinical chemistry and immunoassay analyzers have on-board software that performs many functions. The RSH is controlled by the software, which ensures that STAT samples are assigned the highest test- ing priority, being moved to the front of the queue over routine samples already loaded in the sample bays. For the integrated systems, the software shuttles the STAT samples to whichever module, clinical chemistry or immunoassay, will produce a test result in the least amount of time. The system software also tracks routine operational information such as reagent usage and reagent volume, displays the current and a limited num- ber of calibration curves, and can provide graphical dis- plays of long-term historical quality control data. The analyzer software also recognizes typical analytical prob- lems such as the prozone (pre-zone) effect for immunoas- says and rejects results generated under prozone conditions and automatically orders a dilution and retest of the sample. Middleware is available to provide more sophisticated data manipulation. Available capabilities include auto- verification of patient test results, estimation of Six Sigma metrics for all assays (an objective, quantitative measure of analytical quality), formulation of quality control rules based on Sigma metrics (high quality assays can employ simple QC algorithms such as accepting test results if two controls fall within three standard devia- tions with a 90% probability of error detection), the abil- ity to automatically enable or disable individual assays and analyzers based on QC results, and the capability of holding patient results from release to the LIS or hospi- tal information system (HIS) until QC is approved (thus assuring patient safety), sample tracking and delta checks for individual patients, moving averages calculation, and continuous monitoring of test turnaround time. Labora- tory operations “dashboard” displays can also be pro- vided to display the key performance indicators desired by a laboratory. Middleware also offers constant reagent inventory management. Further Reading Adamczyk, M., Chen, Y.Y., Mattingly, P.G., Pan, Y. and Rege, S. Neopentyl 3-trifloxypropanesulfonate. A reactive sulfopropylation reagent for the prepa- ration of chemiluminescent labels. J. Org. Chem. 63, 5636–5639 (1998). Armbruster, D.A. and Alexander, D.B. Sample to sample carryover: a source of analytical laboratory error and its relevance to integrated clinical chemistry/ immunoassay systems. Clin. Chim. Acta 373, 37–43 (2006). Hubl, W., Zogbaum, M., Boyd, J.C., Savory, J., Schubert, M., Meyer, D. and Demant, T. Evaluation of analytical methods and workflow performance of the ARCHITECT ci8200 integrated serum/plasma analyzer system. Clin. Chim. Acta 357, 5–54 (2005). Mali, B., Armbruster, D., Serediak, E. and Ottenbreit, T. Comparison of immuno- turbidimetric and immunonephelometric assays for specific proteins. Clin. Biochem. 42, 1568–1571 (2009). Rukhsana, J., Perotta, P.L., Okorodudu, A.O., Petersen, J.R. and Mohammad, A.A. Fit-for-purpose evaluation of the ARCHITECT i1000SR analyzer. Clin. Chim. Acta 411, 798–801 (2010). FIGURE 6 ARCHITECT i-series light-generating reaction.