While numerous challenges exist in exploring the biology of disease identification and classification via multiplexing approaches, improvements in high-throughput technology bode well for new clinical offerings. This was one of the main take-home messages at two conferences that looked at multiplexing for research in areas such as cancer biomarkers, HLA typing, and genotyping for human papilloma virus (HPV).
Maurits de Koning, Ph.D., a scientist at DDL Diagnostic Laboratory discussed his company’s efforts at validating a human papilloma virus genotyping assay. The work, which he described at the BEBPA “Biological Assays” conference in Barcelona, is supported by GlaxoSmithKline Biologicals.
HPV is the causative agent of cervical cancer, for which two prophylactic vaccines are now available: Cervarix™ (containing HPV 16 and 18 VLPs) and Gardasil™, encompassing HPV 6, 11, 16, and 18 VLPs. The company’s assay is a PCR-based method, in which cervical biopsy material is obtained from patients, amplified with several primer sets covering 16 HPV types, and then measured using a Luminex-based procedure.
The DDL group constructed a prototype of the assay for proof of concept. Then, during the development phase, the team optimized and designed controls using this platform for evaluation of clinical samples. The next step was the validation of the system according to ICH guidelines.
The elements of validation, from the analytical side, include specificity, sensitivity, accuracy, precision, and robustness. Specificity takes into account the homology of various primers and probes for the many different HPV genomes, given that there may be numerous species that share some of these sequences. Dr. de Koning’s research group evaluated panels of samples and observed that the accuracy was quite high, 98%, when the samples were tested in a comparison with a reference assay.
The precision of the assay, i.e., the degree to which repeat tests of the same material were in agreement, was also quite high, 97%. Another property, that of robustness, or the stability of the assay in the face of minor variations in conditions, was found to be excellent at 95%, with both cervical swabs and formalin-fixed tissues.
Non-PCR Point-of-Care System
Vincent Gau, Ph.D., CEO and president of Genefluidics, spoke about diagnostics for urinary tract infections at the recent “Biodetection Technologies” conference in Arlington, VA. Most current tests are based on cultivating microbes on selective agar and using a tedious antibiotic susceptibility determination.
While bacteriological culture has been accepted as the most sensitive and accurate tool, it is hobbled by a number of shortcomings. A laborious procedure like this sets the stage for the emergence of multidrug-resistant pathogens, as the pathogen proliferates in the intervening period (which can run to several days) between appearance of clinical symptoms and application of therapeutic measures.
Dr. Gau’s company has introduced a molecular-analysis platform that seeks to improve upon traditional diagnostic approaches through development of a handheld point-of-care system. It possesses a universal sample-processing cartridge for conducting multiplexed assays, either nucleic acid or immunoassay-based, and is field deployable.
The current unit was introduced in 2010 and is extremely compact. It uses exchangeable cartridges which are constructed with channels through which the reagents are pumped. Detection of the signal is through the use of flow impedance. When antibodies, small molecules, or nucleic acids bind to the surface they affect the current flow, which is recorded as a change in impedence in the chip that carries the sample.
According to Dr. Gau, the device produces highly accurate data with tight error limits associated with the measurements. A variety of biological samples have been evaluated including blood, serum, urine, saliva, cerebrospinal fluid, sputum, milk, and meat juices. A wide range of markers have been controlled using the system—IL-8, tropinin, numerous bacterial species, and multiple drug-resistance markers.
“I believe that these products will enable clinical diagnosis to move to a new level of performance, with attendant cost savings and health benefits for our customers,” said Dr. Gau.