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The discovery of specific genes and their link to various disease states is facilitating early diagnosis and timely therapy, and paving the way toward personalized medicine. Traditionally, diseases have been classified based on physiological symptoms or phenotypes. Now with a better understanding of the molecular basis of diseases and the individual inherent differences in disease susceptibility and drug responses, molecular diagnostic tests are opening up new clinical possibilities and emerging as a powerful healthcare tool. The increasing knowledge of genetic differences that take into account both nucleic acids and proteins and their integrated roles in diseases has stimulated the development of new diagnostic technologies. Such new technologies provide a sensitive, accurate, and rapid means of assessing comprehensive disease signatures with genomic and proteomic markers.

Genetic variations in drug metabolizing enzymes can influence the way a patient responds to various medications. As a result, innovative IVD products are being developed through greater understanding of such processes and are revitalizing the role of today’s clinical laboratory by providing critical patient information and improving patient care. Such novel tests can provide key information such as identifying patients who react differently to medications, along with an explanation of why variations exist within particular patient populations. The tests can also predict and screen patients who have a greater chance of benefiting from a particular drug, resulting in improved therapeutic interventions. Such advances are revolutionizing the way diseases are diagnosed, monitored, and managed.

Molecular Testing Methods

Over the years, various technologies designed to address the challenges in molecular diagnostics have been developed. Such technologies include amplification (gene and signal), dot blots, capillary electrophoresis, probe technologies, immunoassays, and polymerase chain reaction (PCR) restriction fragment-length polymorphism analysis. Although useful, these tests take two to three days to complete, and are labor intensive and technique dependent. Furthermore, these tests typically address one specific protein, gene, or DNA/ RNA target sequence at a time. Recently, reverse transcription-polymerase chain reaction (RT-PCR)—a sensitive technique for mRNA detection and quantitation and real-time PCR, which requires an instrument combining a thermal cycler and an optics unit for fluorescence excitation and emission collection—have been developed. While RT-PCR and real-time PCR are practical for individual single nucleotide polymorphism (SNP) determinations, they are more difficult to use when multiple SNPs in a gene are targeted.

The completion of the Human Genome Project has created an increased need for examining the expression of multiple genes simultaneously. This need has led to the development of microarray technologies, in which multiple DNA fragments or capture oligonucleotides identifying specific gene coding regions are spotted or arrayed on a solid surface. Fluorescent-labeled DNA targets are hybridized to a capture oligonucleotide. Unbound DNA is then removed by washing, and the spots are scanned and analyzed.

Arrays for research use are often high-density, based on hybridization to thousands of synthetic oligonucleotides. These arrays are designed based on sequence information alone and are synthesized in situ using a combination of photolithography and oligonucleotide synthesizing chemistry.

Figure 1. The Infiniti BioFilmChip microarray by AutoGenomics Inc. (Carlsbad, CA).

With the clinical necessity to multiplex and determine multiple SNPs simultaneously, the need for automated analysis using low-to medium–density arrays has emerged. Performing a microarray test requires the completion of many complex test procedures, including extraction, amplification, hybridization, stringency optimization, optical detection, data analysis, and reporting of results. The current instruments used for such procedures are individual automated devices performing the appropriate discrete steps in DNA analysis. For example, hybridization is performed in a hybridization oven, while scanning is performed in a separate scanner. This process requires highly skilled operators to manage multiple workstations, resulting in the potential for higher error rates due to manual interventions and complex support service issues in dealing with multiple vendors.

Today, a paradigm shift has ushered in a new era in molecular test processing, data analysis, and patient care. The ability to automate molecular tests has empowered the clinical laboratory to move to the front lines of molecular medicine and enhance productivity and patient care. Developing automated microarrays for clinical use presents a unique challenge to system designers, since they must first design a microarray that can be automatically processed through an integrated test procedure.

Developing Automated Multiplex Platforms

The design goals in developing an automated multiplexing platform include the following:

• A microarray that provides uniform spot morphology and reduces intrinsic fluorescence.
• A test process that minimizes cross hybridization and provides results that are reproducible and accurate.
• Process automation to integrate the various discrete processes in DNA analysis such as sample preparation, reagent management, fluidics, hybridization, stringency optimization, optical detection, and result analysis.
• A self-contained platform that can be used for genomics and proteomics, and is intuitive and easy to use.

Film-Based Microarrays

Glass surfaces that have been used in microarrays often exhibit high intrinsic background fluorescence and lack uniform spot conformity. In addition, silica-based surfaces can be susceptible to sample evaporation and cross-contamination from nonspecific target hybridization.

Figure 2. (click to enlarge) The structure of the BioFilmChip microarray.

To address such issues, AutoGenomics Inc. (Carlsbad, CA) developed the Infiniti BioFilmChip microarray, consisting of multiple layers of a three-dimensional hydro-gel matrix coated on a polyester film base (see Figure 1). This layered matrix provides an aqueous microwell environment that promotes stability and preserves functionality of biological materials (see Figure 2). An intermediate layer incorporates a composition for removing intrinsic fluorescence and hot spots associated with the solid surface, which improves assay sensitivity. The top layer is designed for immobilizing biological molecules (e.g., oligonucleotides, antibodies, or antigens) to enable both genomic and proteomic analyses. The biological molecules (i.e., capture probes) can be coupled noncovalently using the interaction between streptavidin and biotin.

Microarray Test Methods

The BioFilmChip microarrays have unique capture oligonucleotides (or zipcodes) with sequences derived from a nonhuman genome. Sequence-specific zipcodes are immobilized at each spot on the microarray. The antizipcode (i.e., oligonucleotide flap) that is incorporated into the specific detection primer used to detect PCR amplicon hybridizes with its complementary zipcode. Detecting a specific spot depends on the unique hybridization of zipcode and anti-zipcode.

Figure 3. (click to enlarge) During the detection primer extension, complementary probes are hybridized to the amplicon. Primer extension and labeling occur when complementarity is perfect.

Once multiplexed PCR is complete, the amplicons are subjected to the detection primer extension reaction, which proceeds through 40 cycling steps (see Figure 3). At the beginning of each cycle, a primer anneals to the amplicon and is extended. During extension, Cy5-labeled nucleotide is incorporated.

At the end of a cycle, the extended primer is denatured from its target, which is now available for another cycle in which a new primer anneals. Thus, multiple Cy5-labeled probes are generated, which will hybridize to the microarray.

The 3' ends of the detection primers contain sequences complementary to the amplicon sequence being analyzed. Extension of the primers occurs only when the 3' end of a particular primer precisely matches its complementary amplicon sequence. The requirement for perfect matching confers specificity on the reaction and makes the discrimination between wild type and mutant alleles (or variants) possible. It is important to emphasize that the detection primer extension process involves labeling of complementary probes. However, no amplification of the amplicon occurs during this step. Hence, amplicon cross-contamination should not be a problem in the instrument.

Table I. (click to enlarge) Sample carryover results. Readings in RFU (relative fluoresence unit). W= wild type; M=mutant; Ind=indeterminate.

To confirm that amplicon contamination is not an issue during the detection primer extension process, a Factor II–Factor V sample carryover study was performed in which no sample carryover was observed as indicated by 100% correct results for the genotypes for all the specimens assayed (see Table I). The study included 300 ng of a wild type sample (FV-WT, FII-WT), 10 ng of a positive sample (FV-WT, FII-M), 300 ng of a heterozygous sample (FV-H, FII-H), and a no template control or water.

Hybridization to Microarrays

Figure 4. (click to enlarge) Hybridization of the primer to the BioFilmChip occurs by virtue of the zipcode–antizipcode interaction.

The hybridization module is where the extended detection primers are applied to the microarray, which is positioned in a sealed hybridization chamber maintained at 39° ± 0.1°C (see Figure 4). During this step, anti-zipcodes that are part of the extended detection primers target and hybridize to complementary immobilized zipcode oligonucleotides spotted on the microarray. Only those spots where labeled primers have hybridized to their complementary zipcodes are labeled with a fluorescent molecule and are therefore detected by the optics module. Genotypes specific to each patient are then assigned.

Microarray Scanning and Result Interpretation

The optics module is a lightproof assembly comprised of a three-axis electromechanical stage, camera, lasers, and a photomultiplier tube. Using an excitation wavelength from the laser light source, the camera takes micron-level pictures of reference fluorescent dye spots on the microarrays. The integrated software uses that data to calculate the location of the spots on the specific microarray, and the optics module scans those calculated locations. The optics module scans and analyzes the 240 microspots on the microarray within 3 minutes, and up to 24 microarrays can be processed simultaneously.

System Overview

Figure 5. The Infiniti analyzer by AutoGenomics.

The Infiniti analyzer is a plug-and-play platform that is network compatible (see Figure 5). To run a test on the analyzer, an operator loads the amplified samples, BioFilmChip microarrays, and reagent modules, and generates a work list (see Figure 6). After the analyzer automatically processes the test and scanning is completed, it ejects the processed microarrays into a waste tray.

Figure 6. Infiniti consumables.

After PCR is complete, the time to first test result is 4 hours, and subsequent results are generated rapidly thereafter. Utilizing a new generation format, four samples can be processed simultaneously on a single multiplexed microarray, and 24 patient samples can be processed in 5.5 hours with a throughput of about 100 tests per day.

The analyzer can detect with high specificity multiple genetic variants simultaneously in a patient’s DNA sample. The system can also genotype an allele that contains multiple polymorphic sites. For example, the system can assay all 31 polymorphic sites of the cystic fibrosis transmembrane conductance regulator gene known to be clinically relevant.

Table II. (click to enlarge) For a given VKORC1 haplotype, the DNA analyzed with the Infiniti system agreed with the results from bidirectional sequencing. H=heterozygote.

The system’s open architecture design enables adaptation of multiple methodologies such as hybridization assay and allele-specific primer extension assay to identify SNPs, short tandem repeats, microsatellite analysis, and gene expression analysis. Without any modification, the same platform can perform protein determinations using sandwich, competitive, and enzymatic methodologies. The analyzer also allows users to develop protocols for their own applications.

The analyzer’s operating software provides flexibility and simplicity for performing both genomic and proteomic analyses. It controls all operations such as data processing, assay protocol, robotics (aspiration and dispensing), fluorescence signal measurement, and generation of test result reports. The multitasking real-time software has a schedule manager subroutine that can control all of the analyzer’s timed operations (e.g., thermal cycling, fluid mixing, incubation, washing, and optical scanning) without manual intervention. In addition, the resource manager converts all logical resources to physical resources and builds an event list.

Table III. (click to enlarge) For a given 2C9 haplotype, the DNA analyzed with the Infiniti system agreed with the results from bidirectional sequencing.

The system’s user interface is enabled by using a wireless keyboard, mouse, and touch pad directly mounted on the analyzer’s LCD panel. The interactive user interface allows inventory management, work list entry, and access to test results. Results are available for review via the LCD screen, and the LIS functionality is available for data warehousing.

Assay Results and Comparison Studies

To demonstrate the reproducibility of the assays run on the Infiniti analyzer, three clinical sites tested 147 samples using the CYP 450 2C9-VKORC1 panel on the system. The results from the analyzer agreed 100% with the bidirectional sequencing data for VKORC1 and CYP 450 2C9 (see Tables II and III).¹

In a study presented at the Clinical Virology Symposium, comparisons between the Infiniti HPV genotyping test and the HC2 test by Digene Corp. (Gaithersburg, MD) showed a concordance of 76% on 79 samples. Sequencing confirmed the genotypes identified with the Infiniti assay.²

Furthermore, a study was performed comparing the Infiniti system assays for Factor II and Factor V and the Factor II and Factor V kits by Roche Diagnostics (Indianapolis) on the Roche LightCycler. The results were 98.6% agreement on 208 samples for the Factor II G20210A mutation, and 100% agreement on 175 samples for the Factor V Leiden G1691A mutation.

Clinical Applications

The BioFilmChip microarray’s versatility enables it to be used to conduct a wide spectrum of tests for genetic disorders, infectious diseases, cancer, pharmacogenetics, and proteomics. Such routine tests for genetic disorders include thrombophilia assessment and cystic fibrosis. In the area of infectious diseases, rapid and sensitive detection of viruses and bacteria is critical, while screening for emerging drug resistance in the treatment of viral and bacterial infections is important. AutoGenomics offers tests such as a respiratory panel with 24 viral types that can be run simultaneously with the MTBDR assay to detect mycobacterium tuberculosis drug- resistance strains.

In the field of oncology, a clinical need has emerged to screen for underlying defects in genomic structure that result from the accumulation of mutations due to chromosomal aberrations, inactivation of tumor suppressor genes, DNA repair genes, and oncogenes.³ Each cancer type with its characteristic genetic alterations has a unique molecular signature. For early diagnosis of disease, an in-depth understanding of the disease signature is essential.

For example, recent advances in cervical cancer diagnosis indicate that the human papillomavirus (HPV) found in 99.7% of cervical cancers indicate persistent infection with the virus causing the disease.⁴ Based on epidemiological statistics, types 16 and 18 represent about 70% of all cervical cancers. The other nine types increase the sensitivity to almost 96%. HPV genotyping is therefore expected to play a critical role as infections with multiple HPV genotypes may have important implications as markers of persistent disease, multiple versus single cervical lesions, and progression from low-grade to high-grade cervical precancerous lesions.

Pharmacogenetics involves improving patient management by customizing drug dosages to enhance safety and efficacy. Inherited DNA sequence variations (or polymorphisms) in genes encoding for drug metabolizing enzymes, drug receptors, drug transporters, and molecules that are involved in signal transduction pathways play an integral role in a drug’s efficacy or toxicity. Determining individual pharmacogenetic profiles of genes involved in drug metabolism and clearance helps to ensure effective individualized therapy.⁵ The cytochrome P450 (CYP 450) family of drug metabolizing enzymes plays a key role in the metabolism of currently administered drugs.

FDA has so far relied on genotype data to modify doses for patients using the acute lymphatic leukemia drug 6-mercaptopurine, the colorectal cancer agent Camptosar by Pfizer Inc. (New York City), and Coumadin by Bristol-Myers Squibb (New York City). The agency is also seriously considering relabeling of tamoxifen, a drug that has been used for more than 20 years to treat patients with advanced breast cancer.⁶

AutoGenomics has developed a comprehensive portfolio of pharmacogenetic tests for the various CYP 450 enzymes including 2D6 for tamoxifen, 2C9-VKORC1 for Coumadin, 2C19, 3A4, and 3A5. Other such tests include UGT1A1 for Camptosar, N acetyl transferase, and multiple drug resistance.

Protein Applications

Protein microarrays enable simultaneous detection of multiple proteins with minimal sample volume. They can be fabricated by printing biotinylated capture antibodies in a proprietary spotting solution on a streptavidin-coated BioFilmChip microarray. Approximately 500 pl of solution are dispensed per spot on the microarray. The spot diameter is 200 µm, and center-to-center spacing is about 400 µm. After spotting, the microarrays are washed with 100 µl of phosphate wash buffer before performing the immunoassays.

Ramanath Vairavan is senior vice president of sales and marketing at AutoGenomics Inc. (Carlsbad, CA). He can be reached at rvairavan@ autogenomics.com.

Biotinylated mouse alpha-human IL-2 capture antibody was spotted in triplicate onto each microarray. 100 µl of each calibrator solution containing IL-2 antigen (0, 5, 10, 25, 50, 100, and 250 pg/ml) and 25 µl of Cy5-labeled mouse alpha-human IL-2 reporter antibody (500 ng/ml) in MES buffer (containing 4% bovine serum albumin, pH 6.8) were dispensed onto each protein array. The microarrays were incubated for two hours at 37°C, washed three times in phosphate wash buffer, and scanned. The fluorescence signal was proportional to the amount of bound antigen. The detection limit (0+2 standard deviation) of the assay was determined to be 1 pg/ml.

Protein microarrays can be performed based on a competitive or sandwich principle and are rapidly evolving into a powerful technique to detect and multiplex proteins, monitor their expression levels, and investigate protein interactions and functions.

Conclusion

Molecular diagnostics testing is at the forefront of far-reaching changes in healthcare, and has emerged as a field that promises to renew technological progress in the clinical laboratory and vastly improve patient care. The development of new technology platforms that integrate all the complex discrete processes in nucleic acid and protein analysis into a self-contained platform is enabling laboratories to bring in their molecular tests that would have been sent out, generate a new revenue stream, and enhance work flow efficiency and productivity. Activities under development are in the area of automating sample preparation and integrating it with the test processes to realize the full potential of genetic testing and make it an integral part of personalized medicine or individualized therapy.

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